Inherited Metabolic Diseases Georg F

Inherited Metabolic Diseases Georg F. Hoffmann Johannes Zschocke William L. Nyhan (Eds.)

Inherited Metabolic Diseases

A Clinical Approach Prof. Dr. Georg F. Hoffmann William L. Nyhan, MD, PhD, Professor Ruprecht-Karls-University University of California, San Diego University Children's Hospital School of Medicine Im Neuenheimer Feld 430 Department of Pediatrics 69120 Heidelberg 9500 Gilman Drive Germany La Jolla, CA 92093 [email protected] USA [email protected] Johannes Zschocke, Dr.med.habil., PhD, Professor of Human Genetics Divisions of Human Genetics and Clinical Genetics Medical University Innsbruck Schöpfstr. 41 6020 Innsbruck Austria [email protected]

ISBN: 978-3-540-74722-2 e-ISBN: 978-3-540-74723-9

DOI: 10.1007/978-3-540-74723-9

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v Preface

The fi eld of inherited metabolic diseases has changed from a limited group of rare, untreatable, often fatal disorders to an important cause of acutely life-threatening but increasingly treatable illness. Unchanged is the orphan nature of these disorders with mostly relatively nonspecifi c initial clinical manifestations. The patient does not come to the physician with the diagnosis; the patient comes with a history, symptoms, and signs. This book starts with those and proceeds logically through algorithms from questions to answers. Special emphasis is placed on acutely presenting disorders and emergency situations. The rationale of the approaches presented in this book are based on extensive, collective clinical experience. To utilize as broad an experience as possible, its concept has been extended from a pocket-size book written jointly by fi ve colleagues to a textbook combining the experience of over 20 expert metabolic physicians. It is now imbedded in the environment of Springer Pediatric Metabolic Medicine in addition to the disease-based approach in Inborn Metabolic Diseases edited by John Fernandes and colleagues as well the series edited by Nenad Blau and colleagues on specifi c biochemical diagnostics, laboratory methods, and treatment. A system and symptom-based approach to inherited metabolic diseases should help colleagues from different specialties to diagnose their patients and to come to an optimal program of therapy. For metabolic and genetic specialists, this book is designed as a quick reference for what may be (even for the specialist) infrequently encountered presentations.

Heidelberg, Germany Georg F. Hoffmann Innsbruck, Austria Johannes Zschocke San Diego, California, USA William L. Nyhan

vii Contents

Part A Introduction to Inborn Errors of Metabolism ...... 1

A1 Disorders of Intermediary Metabolism ...... 3 Johannes Zschocke

A2 Disorders of the Biosynthesis and Breakdown of Complex Molecules...... 7 Johannes Zschocke

A3 Neurotransmitter Defects and Related Disorders ...... 11 Georg F. Hoffmann

Part B Approach to the Patient with Metabolic Disease ...... 13

B1 When to Suspect Metabolic Disease ...... 15 William L. Nyhan

B2 Metabolic Emergencies ...... 25 William L. Nyhan

B3 Patient Care and Treatment...... 61 William L. Nyhan and Georg F. Hoffmann

B4 Anesthesia and Metabolic Disease...... 63 William L. Nyhan

Part C Organ Systems in Metabolic Disease ...... 67

C1 Approach to the Patient with Cardiovascular Disease ...... 69 Joachim Kreuder and Stephen G. Kahler

C2 Liver Disease ...... 89 Georg F. Hoffmann and Guido Engelmann

ix x Contents

C3 Gastrointestinal and General Abdominal Symptoms ...... 109 Stephen G. Kahler

C4 Kidney Disease and Electrolyte Disturbances ...... 117 William L. Nyhan

C5 Neurological Disease ...... 127 Angels García-Cazorla, Nicole I. Wolf, and Georg F. Hoffmann

C6 Metabolic Myopathies...... 161 Stephen G. Kahler

C7 Psychiatric Disease ...... 177 Ertan Mayatepek

C8 Eye Disorders ...... 181 Alberto Burlina and Alessandro P. Burlina

C9 Skin and Hair Disorders...... 197 Carlo Dionisi-Vici, May El Hachem, and Enrico Bertini

C10 Physical Abnormalities in Metabolic Diseases ...... 219 Ute Moog, Johannes Zschocke and Stephanie Grünewald

C11 Hematological Disorders ...... 233 Ellen Crushell and Joe T R Clarke

C12 Immunological Problems ...... 243 Ertan Mayatepek

Part D Investigations for Metabolic Diseases ...... 249

D1 Newborn Screening for Inherited Metabolic Disease ...... 251 Piero Rinaldo and Dietrich Matern

D2 Biochemical Studies ...... 263 Kenneth M. Gibson and Cornelis Jakobs

D3 Enzymes, Metabolic Pathways, Flux Control Analysis, and the Enzymology of Speciﬁ c Groups of Inherited Metabolic Diseases ...... 283 Ronald J. A. Wanders, Ben J. H. M. Poorthuis, and Richard J. T. Rodenburg Contents xi

D4 DNA Studies...... 305 Johannes Zschocke

D5 Pathology − Biopsy ...... 311 Hans H. Goebel

D6 Suspected Mitochondrial Disorder ...... 325 Nicole I. Wolf

D7 Postmortem Investigations ...... 335 Piero Rinaldo

D8 Function Tests ...... 339 Johannes Zschocke

D9 Family Issues, Carrier Tests, and Prenatal Diagnosis ...... 357 Johannes Zschocke

Part E Appendix ...... 361

E1 Differential Diagnosis of Clinical and Biochemical Phenotypes...... 363

E2 Reference Books ...... 375

E3 Internet Resources ...... 377

Index ...... 379 Contributors

Enrico Bertini Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome, Italy, [email protected] Alberto Burlina Department of Pediatrics, Division of Metabolic Disorders, University Hospital, Via Giustiniani 3, 35128 Padova, Italy [email protected] Alessandro P. Burlina Department of Neuroscience, Neurological Clinic, University of Padova, Via Giustiniani 5, 35128 Padova, Italy, [email protected] Ellen Crushell 26 Wainsfort Grove, Terenure, Dublin 6 W, Ireland, [email protected] Joe T. R. Clarke Division of Clinical Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada, [email protected] Carlo Dionisi-Vici Division of Metabolism, Bambino Gesu Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy, [email protected] Guido Engelmann University Children’s Hospital, Ruprecht-Karls-UniversityIm , Neuenheimer Feld 430, D-69120 Heidelberg, Germany, e-mail: [email protected] Angels García-Cazorla Servicio de Neurologia, Hospital Sant Joan de Deu, Passeig Sant Joan de Deu 2, 08950 Esplugues, Barcelona, Spain, [email protected] Kenneth M. Gibson Department of Biological Sciences, Michigan Technological University, Dow 740, ESE 1400, Townsend Drive, Houghton, MI 49931, USA [email protected] Hans H. Goebel Department of Neuropathology, Mainz University Medical Center, Langenbeckstraβe 1, 55131 Mainz, Germany, [email protected] Stephanie Grünewald Great Ormond Street Hospital, London WC1N 3JH United Kingdom, [email protected] May El Hachem Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome, Italy, [email protected]

xiii xiv Contributors

Georg F. Hoffmann University Children’s Hospital, Ruprecht-Karls-University, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany, [email protected] Cornelis Jakobs Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands C. [email protected] Stephen G. Kahler Department of Pediatrics, Division of Clinical Genetics, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205, USA, [email protected] Division of Clinical Genetics, Slot 512–22, Arkansas Children‘s Hospital, 800 Marshall Street, Little Rock, AR 72202Ð3591, USA Joachim Kreuder Department Pediatric Cardiology, University Children’s Hospital, Feulgenstraβe 12, 35385 Giessen, Germany, [email protected] Dietrich Matern Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street S. W., Rochester, MN 55905, USA, [email protected] Ertan Mayatepek Department of General Pediatrics, University Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany [email protected] Ute Moog Institute of Human Genetics, Ruprecht-Karls-University, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany, [email protected] William L. Nyhan Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA, [email protected] Ben J. H. M. Poorthuis Department of Medical Biochemistry, K1Ð252, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands, B. J. [email protected] Piero Rinaldo Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, 200 First Street S. W., Rochester, MN 55905, USA, [email protected] Richard J. T. Rodenburg Laboratory for Pediatrics and Neurology, University Medical Centre St Radboud, Nijmegen, The Netherlands, R. [email protected] Ronald J. A. Wanders Laboratory of Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands, [email protected] Nicole I. Wolf Department of Child Neurology, VU Medisch Centrum, 1007 MB Amsterdam, The Netherlands, [email protected] Johannes Zschocke Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria, [email protected] Disorders of Intermediary Metabolism A1 Johannes Zschocke

Key Facts A1.1 Disorders of Amino Acido Metabolism › The classical inborn errors of metabolism are defects in enzymes of the metabolism of amino acids, carbohydrates and fatty acids or in mito- Typical aminoacidopathies result from abnormalities in chondrial energy metabolism (Fig. A1.1). the breakdown of amino acids in the cytosol. In addi- › Disorders of intermediary metabolism are often tion, several disorders involving mitochondrial enzymes dynamic, they fl uctuate with changes in the such as branched-chain ketoacid dehydrogenase (maple metabolic state of the patient and frequently syrup urine disease) or ornithine aminotransferase allow successful therapeutic intervention. (gyrate atrophy of the choroidea) are classifi ed as amin- › Most disorders of intermediary metabolism are oacidopathies as they do not involve CoA-activated readily diagnosed through basic metabolic inves- metabolites. This distinguishes aminoacidopathies from tigations which include blood gases, glucose, the organic acidurias, which are considered a separate lactate, ammonia, plasma amino acids, urinary group of disorders affecting mitochondrial enzymes, organic acids and an acylcarnitine profi le. CoA-activated metabolites, and which have effects on other mitochondrial functions. Clinical symptoms of the aminoacidopathies may be thought of as caused by the accumulation of toxic intermediates that cause specifi c organ damage. Several defects of amino acid metabolism such as histidinemia are benign because The classical inborn errors of metabolism are defects in enzymes of the metabolism of amino acids, carbohydrates, and fatty acids or in mitochondrial energy metab- Carbohydrates Glucose olism (Fig. A1.1). These disorders are often dynamic; Glycogen they fl uctuate with changes in the metabolic state of Glucose-6-P Galactose the patient and frequently allow successful therapeutic Amino Acids Fatty Acids intervention. Most of them are readily diagnosed through Fructose basic metabolic investigations, which include blood NH 3 Urea gases, glucose, lactate, ammonia, plasma amino acids, Pyruvate Lactate Urea urinary organic acids, and an acylcarnitine profi le. Cycle

Acetyl-CoA Beta- Oxidation Krebs Cycle Ketones

ATP ADP J. Zschocke Mitochondrion Respiratory Chain Divisions of Human Genetics and Clinical Genetics, Medical Cytosol University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria e-mail: [email protected] Fig. A1.1 Main pathways of intermediary metabolism

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 3 DOI: 10.1007/978-3-540-74723-9_A1, © Springer-Verlag Berlin Heidelberg 2010 4 J. Zschocke the metabolites that accumulate are not toxic. The fl uctuations occur and complete return to normal inter- pathogenetic relevance of an inborn error of amino acid mediary metabolism is usually impossible. Supplemen- metabolism is not always easy to ascertain as clinical tation with carnitine and sometimes other substances symptoms observed in the child may be coincidental or such as glycine (e.g., to form isovalerylglycine in the reason for performing the analysis in the fi rst place. isovaleric aciduria) are very useful adjuncts to the Aminoacidopathies are diagnosed through the analysis treatment. of plasma (or urinary) concentrations of amino acids Disorders of biotin metabolism are included among and sometimes of urinary organic acids. A majority is the organic acidurias. Biotin is a cofactor of the mito- treatable through dietary restriction of protein and of chondrial carboxylases and a defi ciency of biotini- the amino acid involved in the defective pathway and dase or holocarboxylase synthetase leads to multiple by the avoidance or prompt treatment of catabolic carboxylase defi ciency. It is also usually diagnosed states that lead to the breakdown of large amounts of through urinary organic acid analysis. Biotinidase protein. Another therapeutic strategy that has been suc- enzyme analysis of dried blood spots has been included cessful in hepatorenal tyrosinemia is the inhibition of a into programs of neonatal screening as it is well treated biochemical step before the actual genetic defi ciency, with biotin supplementation. thereby changing a harmful disease into a more benign amino acid accumulation without the accumulation of the more damaging substances downstream. A1.3 Disorders of Ammonia Detoxifi cation A1.2 Organic Acidurias The breakdown of protein produces large amounts of nitrogen in the form of ammonia that is highly The classical organic acidurias are defi ciencies of neurotoxic but is normally converted to urea and enzymes in the mitochondrial metabolism of CoA- excreted in the urine. Defects in enzymes of the urea activated carboxylic acids, most of which are derived cycle and other disorders of ammonia detoxifi ca- from amino acid breakdown. In this way, they are dis- tion present clinically with encephalopathy and tinguished from disorders of fatty acid oxidation, other symptoms of hyperammonemia. Metabolic which not only involve CoA esters but also present dif- investigations should include analysis of the amino ferent diagnostic and therapeutic challenges. The term acids in plasma and urine and urinary orotic acid. organic acidurias is preferred to the alternative term Treatment requires the reduction of protein intake in organic acidemias as they are most often detected by conjunction with the supplementation of essential analysis of the urine. Biochemically, some of the reac- amino acids, the avoidance of catabolic states and tions impaired in the organic acidurias are parallel to the administration of benzoate or phenylacetate/phe- the dehydrogenase, hydratase, or ketothiolase reac- nylbutyrate, which remove nitrogen in the form of tions of the mitochondrial b-oxidation cycle. Clinical alternative conjugates of amino acids such as gly- features are caused not only by the accumulation of cine and glutamine. toxic intermediates but also by a disturbance of mitochondrial energy metabolism and carnitine homeostasis; they may include encephalopathy and episodic metabolic acidosis. Organic acidurias are diagnosed A1.4 Disorders of Fatty Acid Oxidation through the analysis of organic acids in the urine or acylcarnitines in the blood. Treatment is similar to that and Ketogenesis of the aminoacidopathies and involves the dietary restriction of the relevant amino acid(s) and the avoid- Mitochondrial fatty acid oxidation is required for the ance of protein catabolism. However, as the defective provision of energy during fasting, either through com- enzymes are distant (more downstream) from the plete oxidation or through production of ketones in the respective amino acids, restriction may not lead to a liver that then serve as an alternative energy source stoichiometric reduction of pathological metabolites, for the brain. Disorders in this pathway typically although it does in methylmalonic aciduria. Unexpected present as hypoketotic hypoglycemia precipitated by A1 Disorders of Intermediary Metabolism 5 fasting, leading to coma or convulsions. In addition, depending on the tissue localization of the individ- some disorders cause severe hepatopathy and (cardio-) ual defect. Symptoms are frequently gastrointesti- myopathy, probably as results of the accumulation of nal or renal but also include the central nervous toxic metabolites. The diagnosis is best reached in the system (defi cient glucose transport across the blood/ acute situation through the analysis of free fatty acids brain barrier). and the ketone bodies 3-hydroxybutyrate and acetoacetate as well as the acylcarnitine profi le and urinary organic acids. The diagnosis may be missed if samples A1.6 Mitochondrial Disorders are obtained in the normal interval between episodes or after the patient has been treated with intravenous glucose. Treatment consists of avoidance of fasting. Disorders of energy metabolism (usually summarized as Carnitine supplementation is mostly unessessary and mitochondrial disorders although enzymes defi cient, must be carefully balanced in some defects, particu- e.g., in organic acidurias or fatty acid oxidation defects larly those that cause cardiomyopathy or hepatopathy. are also located in the mitochondrion) include genetic defects of the pyruvate dehydrogenase complex, the Krebs cycle and the respiratory electron transport chain, comprising the fi nal pathways of substrate breakdown A1.5 Disorders of Carbohydrate and the production of ATP. Mitochondrial disorders Metabolism and Transport manifest clinically with symptoms and signs of energy defi ciency and a highly variable pattern of organ dysfunctions. In many cases, there are lactic acidemia and The disorders in this group display a relatively wide progressive neurodegenerative disease. Periods of meta- range of clinical features and may cause clinical symp- bolic stress such as intercurrent infections may trigger a toms because of toxicity, defi ciency of energy, hypo- deterioration of the patient’s condition. The diagnostic glycemia, or storage. work-up may be diffi cult and should include frequent • Disorders of Galactose and Fructose Metabolism: measurements of blood lactate levels, CSF lactate, Defects in the cytosolic metabolism of galactose plasma amino acids and alanine, and often a search for and fructose for glycolysis cause disease through mutations in mitochondrial DNA. Repeated, careful accumulation of pathogenic metabolites. Children examinations of organ functions as well as imaging are with galactosemia and fructosemia typically develop essential. Treatment options are limited but usually evidence of severe damage to the liver and/or kid- include various vitamins and cofactors such as ribofl a- ney after dietary intake of lactose (milk, milk prod- vin, coenzyme Q, or thiamine. Heterozygous mutations ucts) or fructose (fruit, sucrose), respectively. in the genes of some Krebs cycle enzymes (e.g., fumarase) Treatment requires the elimination of the intake of cause inherited cancer predisposition syndromes. galactose or fructose. ¥ Disorders of Gluconeogenesis and Glycogen Storage: Typical metabolic features are hypoglyce- A1.7 Disorders of Cobalamine mia after relatively short periods of fasting and lactic acidemia. There may be variable organ dysfunction, and Folate Metabolism most frequently hepatopathy. Glycogen storage leads to hepatic enlargement, which in infancy may Genetically determined or nutritional defi ciencies of be massive. In some disorders such as glycogenosis vitamin cofactors may affect various metabolic path- type III there are elevations of the transaminases and ways and cause a wide range of clinical symptoms. creatine phosphate kinase, and there may be clinical They can frequently be satisfactorily treated by sup- myopathy. Treatment includes frequent meals, corn- plementation of the defi cient substance. Of particular starch supplementation, or continuous overnight importance in intermediary metabolism are cobalamin

tube feeding to avoid hypoglycemia. (vitamin B12) and folate which are essential for cyto- ¥ Disorders of Carbohydrate Transport: There are a solic methyl group transfer. The cellular methylation number of different glucose and other carbohy- reactions require methyl group transfer from serine drate carriers, and clinical symptoms differ greatly to S-adenosylmethionine involving the folate cycle, 6 J. Zschocke

cobalamin (vitamin B12) and the methionineÐhomo- to detoxifi cation of peroxides. Defi ciencies may cysteine cycle. A disturbance in this pathway may be cause neurological and hematological as well as met- caused by methylcobalamin defi ciency, a disturbance abolic problems. Investigations should include the of the folate cycle, or by defi cient remethylation of determination of organic acids in the urine and gluta- homocysteine to methionine. Most disorders of coba- thione in various body fl uids. Treatment is largely lamin metabolism as well as nutritional defi ciency of symptomatic; certain drugs should be avoided. vitamin B12 cause methylmalonic aciduria. Clinically, ¥ Defective breakdown of dipeptides of histidine such disorders of cytosolic methyl group transfer cause an as homocarnosine or carnosine may be found in encephaloneuropathy, often with additional hemato- patients with mental retardation, although the caus- logical problems such as megaloblastic anemia and ative relationship is not always proven. Ulcers of thromb embolic complications of hyperhomocysteine- the skin, particularly of the legs are seen in proli- mia. The diagnosis involves the analysis of urinary dase defi ciency. Investigations should include organic acids, plasma amino acids (homocysteine), amino acid and peptide analysis of the urine. and levels of folate and cobalamin. Treatment includes Treatment is symptomatic; some individuals with supplementation of cobalamin and folate, in some situ- disorders of dipeptide metabolism are asymptom- ations with addition of betain and methionine. atic and do not require treatment.

A1.8 Disorders of Amino Acid Transport A1.10 Disorders of the Transport or Utilization of Copper, Iron, Defi ciencies in the intestinal and/or renal transport of and Zinc amino acids may be nonsymptomatic or cause symptoms because of defi cient absorption of essential amino acids (e.g., tryptophan in Hartnup disease) or because ¥ Disorders of copper metabolism: Wilson disease of increased urinary concentration of unsoluble amino causes a chronic hepatopathy and symptoms of cen- acids which causes nephrolithiasis (e.g., cystein in tral nervous dysfunction, while patients with Menke cystinuria). These disorders are diagnosed by the quan- disease suffer from neurological problems in con- titative analysis of amino acids in plasma and urine. junction with abnormalities of hair, connective tis- Treatment depends on the clinical picture. Defi ciency sue, and bones. Diagnosis involves the analysis of of essential amino acids is treated by supplementation copper and coeruloplasmin in serum, urine, and with large amounts of these compounds, or in the case liver tissue. Treatment in Wilson disease is aimed at of tryptophan defi ciency, supply of the cofactor nico- reducing copper load, while copper should be par- tinic acid that is normally synthesized from tryptophan. enterally substituted in Menke disease. Renal calculi in cystinuria can be prevented by treat- ¥ Disorders of iron metabolism: Patients affected with ment with a chelating agent such as penicillamine, such disorders may present with iron-defi cient ane- which forms mixed disulfi des with cysteine, and cal- mia, e.g., due to insuffi cient intestinal absorption of culi once formed can be resorbed if they have not iron, or with iron overload and liver dysfunction as incorporated too much calcium. in hemochromatosis. Secondary iron overload may be observed in some hemolytic anemias. Treatment is directed at substitution or removal of iron. ¥ Disorders of zinc metabolism: Acrodermatitis A1.9 Disorders of Peptide Metabolism enteropathica is characterized by chronic skin problems, alopecia, and central nervous symptoms. It is ¥ The tripeptide glutathione and the gammaglutamyl diagnosed through reduced levels of zinc and alka- cycle have multiple functions in cellular metabolism, line phosphatase and is treated with supplementa- ranging from amino acid transport across membranes tion of zinc. Disorders of the Biosynthesis and Breakdown of Complex Molecules A2

Johannes Zschocke

Key Facts specifi cally through the analysis of urinary purines and pyrimidines. Some metabolites of pyrimidine break- › Disorders of the biosynthesis and breakdown down are only recognized by urinary organic acid of complex molecules typically show slowly analysis. Nephrolithiasis may be treated or prevented progressive clinical symptoms and are less by allopurinol. A high fl uid intake is helpful. Some likely to cause acute metabolic crises. disorders of pyrimidine metabolism, notably orotic › Disorders in this group are not usually recogn- aciduria and overactivity of 5' nucleotidase (nucleotide ised by basic metabolic analyses but require depletion syndrome), are treatable with uridine or tri- specifi c investigations for their diagnosis. acetyluridine. There is no effective treatment for most of the primarily neurological manifestations of disorders of purine metabolism.

Disorders in this group typically show slowly progressive clinical symptoms and are less likely to cause A2.2 Lysosomal Storage Disorders acute metabolic crises. They are not usually recognized by basic metabolic analyses but require specifi c Lysosomes contain a number of hydrolases required for investigations for their diagnosis. the intracellular breakdown of large lipid and mucopolysaccharide molecules. If one of these enzymes is defi - cient, its substrate accumulates and causes enlargement A2.1 Disorders of Purine and Pyrimidine and/or functional impairment of the organ system. Clinical features include progressive neurological dete- Metabolism rioration, dysmorphic features, and organomegaly. There is usually no metabolic decompensation, although acute Defi ciencies in enzymes required for the biosynthe- symptoms (e.g., severe pain) is a major feature in some sis or breakdown of purines and pyrimidines cause conditions. Investigations include careful roentgeno- neuromuscular abnormalities, nephrolithiasis, gouty graphic examination of the skeleton for dysostosis mul- arthritis, or anemia and immune dysfunction. They tiplex, analysis of leukocytes and other cells for vacuoles, may be recognized through increased or reduced uri- and assessment of parenchymatous organs. The urine nary urea in relation to creatinine, urine microscopy, or may be investigated for abnormal glycosaminoglycans and oligosaccharides; specifi c enzyme studies are usually required to make the exact diagnosis. For most dis- J. Zschocke orders there is no specifi c therapy yet, although enzyme Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria replacement therapy or bone marrow transplantation has e-mail: [email protected] been shown benefi cial in several disorders.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 7 DOI: 10.1007/978-3-540-74723-9_A2, © Springer-Verlag Berlin Heidelberg 2010 8 J. Zschocke

• Mucopolysaccharidoses (MPS), affected children usually recognized through the analysis of very typically develop progressive dysmorphic features, long-chain fatty acids in blood or cultured fi bro- hepatomegaly and psychomotor retardation or blasts. There is no effective treatment. regression. They are usually recognized through the ¥ Refsum disease is a defect in the metabolism of analysis of urine for glycosaminoglycans. exogenous phytanic acid. It causes slowly progres- ¥ Oligosaccharidoses may resemble the MPS, but sive neurological, visual, and auditory abnormali- many show more severe neurological symptoms and ties, and often does not present until adulthood. It is are more frequently symptomatic at birth (nonim- diagnosed through the quantifi cation of serum phy- mune hydrops fetalis). The diagnosis is made through tanic acid and is treatable by a diet restricted of the demonstration of abnormal oligosaccharide pat- phytanic acid. terns in the urine or enzyme analyses. ¥ Defects of ether-phospholipid biosynthesis cause ¥ Sphingolipidoses and lipid storage disorders usu- rhizomelic chondrodysplasia punctata characterized ally present with progressive neurological deterio- by proximal shortening of the limbs in addition to ration. Hepatomegaly may be present, skeletal neurological and other manifestations. It is diag- deformities and dysmorphic features are rare. Other nosed through quantifi cation of plasmalogens in presentation patterns are found particularly in Fabry erythrocytes. There is no effective treatment. disease (pain and paresthesias) and nonneuronop- ¥ Catalase defi ciency is the only known defect of the athic Gaucher disease (hematoma, anemia, massive detoxifi cation of oxygen radicals. It causes chronic splenomegaly, and abdominal/bone pain). The spe- ulcers in the oral mucosal membranes. cifi c diagnosis usually requires enzyme analysis. ¥ Primary hyperoxaluria type I is the only known The neuronal ceroid lipofuscinoses are usually sus- defect of glyoxylate metabolism; it causes nephro- pected by electron microscopy and confi rmed by lithiasis and nephrocalcinosis. It is recognizable by mutation analysis. Mucolipidoses combine clinical organic acid or HPLC analysis for oxalate and gly- features of the mucopolysaccharidoses and sphin- oxylate. It has been treated by transplantation of golipidoses and may refl ect the defi ciency of sev- liver and kidney. eral lysosomal enzymes as a consequence of defective enzyme processing. ¥ Lysosomal transport defects: Cystinosis causes neph- A2.4 Disorders of the Metabolism ropathy and dysfunction of other organs including of Isoprenoids and Sterols the thyroid gland and the eyes; it is diagnosed on the basis of increased cystine content of leucocytes. Isoprenoids and sterols are essential in many cellular Sialic acid storage disease causes progressive and developmental processes. Most defects of their encephaloneuropathy; it is recognized through ele- synthesis are caused by enzyme defi ciencies in the vated free sialic acid in the urine. Both of these disor- postsqualene portion of the pathway. Only mevalonic ders result from defective transport out of lysosomes. aciduria and hyperimmunoglobulinemia D syndrome, Cystinosis is treated with oral cysteamine, cysteam- both due to mevalonate kinase defi ciency, are found in ine eye drops, and renal transplantation. the proximal part of the pathway. ¥ Mevalonate kinase defi ciency is the only known A2.3 Peroxisomal Disorders defect of isoprenoid biosynthesis. It causes dysmorphic features, failure to thrive, mental retardation, and recurrent febrile crises. An attenuated variant causes The biochemical roles of peroxisomes are very diverse. hyper-IgD syndrome. Treatment is symptomatic. Peroxisomal defects usually cause severe, progressive ¥ Defects of sterol biosynthesis cause various struc- multisystem disorders. tural abnormalities including the dysmorphic fea- ¥ Defects of peroxisome biogenesis or the activation tures of the SmithÐLemliÐOpitz syndrome and and b-oxidation of long-chain fatty acids cause pro- mental retardation. Diagnosis involves plasma sterol gressive neurological disease, structural abnormali- analysis. In Smith–Lemli–Opitz syndrome, specifi c ties as in Zellweger syndrome, and abnormalities in treatment by cholesterol supplementation has been hepatic, intestinal, or adrenal function. They are of limited success. A2 Disorders of the Biosynthesis and Breakdown of Complex Molecules 9

A2.5 Disorders of Bile Acid and Bilirubine of one of the more than 40 different enzymes involved Metabolism, Inherited Cholestasis in glycosylation leads to a wide range of structural and Porphyrias abnormalities and disturbances of physiological functions. A disorder from the CDG group should be considered in all patients with unclear multisystem or neurological ¥ Genetic defects of bile acid biosynthesis cause disorder. The diagnosis in N-glycosylation disorders is symptoms either through bile acid defi ciency or usually made by isoelectric focussing of transferrin in through deposition of precursors. The former serum. There is no effective treatment for most disor- causes progressive cholestasis and malabsorption, ders of this group. while the precursors can lead to progressive neurological dysfunction and xanthomas. The bile acid biosynthetic pathway is located partly in the peroxisomes and is affected by peroxisomal disorders. A2.7 Disorders of Lipoprotein Diagnosis involves the analysis of urinary bile Metabolism acids. Treatment with bile acids is effective in the bile acid defi ciency states and to down-regulate Many disorders of lipoprotein metabolism cause clini- bile acid biosynthesis. cal symptoms through the deposition of lipid in tissues ¥ Hem is metabolized to bilirubin and excreted and premature atherosclerosis. Others cause gastroin- together with bile acids in the urine. Genetic defects testinal or peripheral neurological problems. They are may involve specifi c enzymes or mechanisms of recognized by quantifi cation of cholesterol and trig- transport into the bile ducts. They cause indirect or lycerides and through lipoprotein electrophoresis. direct hyperbilirubinemia. Specifi c treatment strate- Many disorders are open to dietary or pharmacological gies have been developed for some disorders. therapy. ¥ Porphyrias are disorders of hem biosynthesis, frequently inherited as autosomal dominant traits. ¥ Elevated blood cholesterol levels in hypercholester- Neurotoxic metabolites accumulate in defi ciencies olemias and mixed hyperlipidemias cause lipid affecting the fi rst few steps of the pathway and typi- deposition in the form of xanthomas and xan- cally cause intermittent acute symptoms such as thelasma. They lead to complications of premature abdominal pain triggered by various factors, in par- atherosclerosis, especially myocardial infarction ticular induction of hem-containing enzymes. and cerebrovascular disease. Therapeutic options Porphyrins accumulating in more distal enzyme defi - include diet, drugs, and lipid apharesis. ciencies are associated with photosensitivity and der- ¥ Hypertriglyceridemia may be caused by genetic matologial symptoms. The diagnosis involves disorders that affect the utilization of chylomicrons analysis of porphyrins and porphyrin precursors in and very low-density lipoproteins. They may cause urine, feces, or erythrocytes. Management entails the failure to thrive and abdominal symptoms, and avoidance of precipitating factors. sometimes severe pancreatitis. These disorders require stringent restriction of dietary fat. ¥ Genetic disorders affecting HDL metabolism cause A2.6 Congenital Disorders a variety of clinical manifestations including premature atherosclerosis, neuropathy, nephropathy, of Glycosylation (CDG) and corneal clouding. Therapy is symptomatic. ¥ Genetic disorders in which there are reduced Many proteins including enzymes, transport and mem- LDL cholesterol and triglycerides lead to symp- brane proteins, as well as hormones require glycosyla- toms of fat malabsorption. They are treated by tion in the Golgi apparatus or endoplasmatic reticulum restriction of fat and supplementation with fat to render them functional glycoproteins. A defi ciency soluble vitamins. Neurotransmitter Defects and Related Disorders A3

Georg F. Hoffmann

Genetic disorders of neurotransmitter metabolism are or serotonine defi ciency, such as infantile parkin- increasingly recognised as causes of severe metabolic sonism, dopa-responsive dystonia, oculogyric crises encephalopathy often starting before birth or soon or disturbed temperature regulation. These diseases thereafter. Diagnosis usually requires investigations of are sometimes recognised by hyperphenylalaninemias the CSF. This group should be considered in children but many exclusively through the analyis of biogenic with neurological problems when basic metabolic amines and pterins in CSF. investigations are normal. Disorders of tetrahydrobiopterin biosynthesis and recycling affect the hydroxylation of phenylalanine and have been called atypical or malignant phenylketonuria. The hydroxylations of tyrosine and tryptophan are A3.1 Disorders of Glycine also affected, leading to defi ciency of both, dopamine and Serine Metabolism and serotonine. Investigations should include the analysis of biogenic amines, pterins and amino acids in the Nonketotic hyperglycinemia is one of the best known CSF as well as amino acids in plasma and pterins in causes of early-onset epileptic encephalopathy. It is urine. The disorders are treated with l-dopa along with recognised via concomitant amino acid analysis of carbidopa and 5-hydroxytryptophan and/or tetrahydro- plasma and CSF. Glycine levels in both are elevated, biopterin and/or tetrahydrobiopterin substitution. and the CSF to plasma ratio is increased. Treatment Disorders of the biosynthesis of biogenic amines with dextrometorphan, benzoate or folate is of limited present similarly with progressive extrapyramidal success. Disorders of serine biosynthesis cause neuro- symptoms and encephalopathy. The defi ciency of biological symptoms. They have been treated with serine genic amines is treated with of l -dopa along with car- and glycine supplementation. bidopa and 5-hydroxytryptophan and/or dopamine agonists.

A3.2 Disorders of the Metabolism of Pterins and Biogenic Amines A3.3 Disorders of Gamma- Aminobutyrate Metabolism Affected children suffer from progressive developmental retardation and epileptic encephalopathy. These disorders cause central nervous dysfunction, There may be specifi c symptoms of dopamine and/ often including seizures and encephalopathy. They are diagnosed through CSF analysis of amino acids and gamma-aminobutyrate (GABA). Urinary organic acid analysis may reveal 4-hydroxybutyric acid indicative G. F. Hoffmann of succinate semialdehyde dehydrogenase defi ciency. University Children’s Hospital, Ruprecht-Karls-University, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany Vigabatrin has been used in the treatment of SSADH e-mail: [email protected] defi ciency.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 11 DOI: 10.1007/978-3-540-74723-9_A3, © Springer-Verlag Berlin Heidelberg 2010 12 G. F. Hoffmann

A3.4 Disorders of Vitamin They are usually diagnosed through the analysis of B Metabolism creatine and guanidinoacetate in urine and serum; 6 treatment centers on creatine supplementation.

Pyridoxal phosphate (PLP, vitamin B6) is a cofactor of all the transamination reactions and some decar- boxylation and deamination reactions of amino acids, and as such is also required for the biosynthesis of A3.6 Other Neurometabolic Disorders several neurotransmitters including dopamine and GABA. Intracellular defi ciency may be caused by pri- ¥ Sulphite oxidase defi ciency is a cause of severe mary or secondary disorders in the biosynthetic path- infantile seizures and encephalopathy. It is recog- way and leads to a neonatal epileptic encephalopathy. nised through a sulphite stix test of the urine. Amino acid analysis of plasma and urine may be In the well-known entity of vitamin B6-dependent seizures, PLP is inactivated by delta 1-piperideine- diagnostic but is less reliable. When it is caused by 6-carboxylate, which accumulates because of an molybdenum cofactor defi ciency there is also xan- enzyme defi ciency in a different pathway. Disorders thine oxidase defi ciency, which may be detected by purine analysis of the urine. There is no specifi c of vitamin B6 metabolism are generally treatable with pyridoxine or PLP. treatment. ¥ Various cerebral organic acidurias including Canavan disease, l-2- and d-2-hydroxyglutaric aciduria, 2-keto- glutaric aciduria, fumaric aciduria and malonic acidu- A3.5 Disorders of Creatine Metabolism ria present with central nervous dysfunction, which is usually progessive. General metabolic abnormali- Creatine is the central compound in cytosolic energy ties are absent, but the specifi c metabolites are found metabolism, and defi ciencies in the biosynthesis or on organic acid analyis of the urine. The molecular transport of creatine manifest as neurometabolic disor- basis of most of the conditions has now been estab- ders with progressive central nervous dysfunction. lished. There is no specifi c treatment. When to Suspect Metabolic Disease B1 William L. Nyhan

Key Facts second opinion should be sought of in case of unexplained symptoms or disease courses. › Careful clinical and family histories, repeated › Metabolic investigations are usually not indi- clinical examinations and a sequential work- cated in children with moderate developmental up by routine laboratory and organ evaluation delay, isolated delay in speech development in remains the best and most often only way to early childhood, moderate failure to thrive, fre- diagnosis. quent infections, occasional seizures, e.g. dur- › While it is imperative to exclude disorders for ing fever, or defi ned epileptic syndromes. which effective treatments are available, in cases of slowly progressive, and by experience often incurable disorders, diagnostic procedures should be performed stepwise. › Unexpected fi ndings in the “routine” laboratory in patients with unusual and unexplained B1.1 History symptoms may be indicative of an inborn error of metabolism. › Every child who is suspected of suffering from B1.1.1 Family History an inborn error of metabolism requires a careful evaluation of organ functions aided by rou- A careful family history may reveal important clues tine laboratory and imaging investigations. that point towards the diagnosis of an inborn error of The involvement of multiple organ systems is metabolism. Most metabolic disorders are inherited as an especially strong indication for an inherited autosomal recessive traits, which may be suspected if metabolic disease. the parents are consanguineous or the family has a con- › As it can be very diffi cult to recognize a constel- fi ned ethnic or geographic background. Carriers for lation which was not personally experiences, a particular disorders, and as a consequence affected children, may be more frequent in remote villages, close- knit communities (such as the Amish in Pennsylvania), certain ethnic groups (such as Ashkenazi Jews), or countries that have seen little immigration over many centuries (such as Finland). Quite often specialist investigations are started only after a second affected child is born into a family. Older W. L. Nyhan siblings may be found to suffer from a similar disorder Department of Pediatrics, University of California, UCSD as the index patient or may have died from an acute School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA unexplained disease classifi ed as “sepsis with uniden- e-mail: [email protected] tifi ed pathogen,” “encephalopathy” or “sudden infant

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 15 DOI: 10.1007/978-3-540-74723-9_B1, © Springer-Verlag Berlin Heidelberg 2010 16 W. L. Nyhan death syndrome.” The latter is a frequent feature in dis- B1.1.2 Prenatal Development orders of intermediary metabolism that may have acute and Complications of Pregnancy lethal presentations such as disorders of ammonia detoxifi cation, organic acidurias, or fatty acid oxidation disorders. Toxic small molecules that accumulate in many disor- In assessing medical records of previously affected ders of intermediary metabolism do not harm the fetus but undiagnosed family members, it should be taken because they are removed via the placenta and meta- into account that the written clinical descriptions of bolized by the mother. Children affected with such dis- complex conditions can be inconsistent and even mis- orders usually have a completely normal intrauterine leading. Depending on the presumptive diagnosis at development and are born with normal birth measure- that time, important clinical clues may be missing. ments at term. In contrast, disorders that interfere with Parents are sometimes more reliable sources of infor- cellular energy metabolism, e.g., mitochondrial disor- mation. On the other hand, the clinical expression of ders, may impair fetal organ development and prena- the same inborn error of metabolism may be variable tal growth, causing structural (in particular cerebral) even within families. Some more common Mendelian abnormalities, dysmorphic features, and dystrophy. disorders are caused by a wide range of different muta- Structural abnormalities and dysmorphic features may tions with different degrees of disease severity. Disease be even more pronounced in disorders of the biosyn- manifestations are especially variable in females with thesis of complex molecules that are necessary for X-linked traits because of differences in the lyoniza- developmental pathways and networks. Notable exam- tion of the X chromosome in carrier females, e.g., orni- ples are the defects of sterol biosynthesis that inter- thine transcarbamylase defi ciency. Similarly, dominant fere with cholesterol-dependent signaling pathways disorders with variable penetrance may cause variable of development and cause, for example, the SmithÐ clinical problems in different members and genera- LemliÐOpitz syndrome. Disorders affecting the break- tions even of one family, such as Segawa syndrome down of complex molecules such as lysosomal storage due to GTP cyclohydrolase defi ciency. disorders cause specifi c dysmorphic characteristics as As a result of the successful treatment of disorders in the Hurler disease, and when severe, may already of intermediary metabolism in which toxic small mol- present at birth. An unusual prenatal disease manifesta- ecules accumulate, an increasing number of relatively tion is found in mothers carrying a fetus affected with healthy affected women are reaching the reproductive long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) age. If they become pregnant, there is a risk for their defi ciency or carnitine palmitoyltransferase II defi - fetuses to be harmed by pathological amounts of toxic ciency, defects of fatty acid b-oxidation. These mothers metabolites from the mother, although the children are have an increased risk of developing acute fatty liver of themselves not affected but heterozygous. Especially pregnancy, preeclampsia, or hemolysis, elevated liver important is maternal phenylketonuria (PKU), which enzymes, and low platelets (the HELLP syndrome). is likely to become a major health problem. Some Systematic studies in mothers showed that fetal women at risk may not even know that they are affected LCHAD defi ciency is present in a signifi cant number with PKU, if they come from countries where newborn of women with acute fatty liver of pregnancy, but only screening did not exist or if they have discontinued in a very small proportion of the far more common dietary treatment and medical follow-up in late child- HELLP syndrome. The neonates of such mothers hood. The latter will however remember that they had should be screened for fatty acid oxidation disorders by followed a special diet, which should be specifi cally acylcarnitine analysis in a dried blood spot. asked for. Several mothers have been found to suffer from mild PKU only after maternal PKU was diagnosed in one of her children. Other maternal condi- B1.1.3 Age of Presentation tions may cause “metabolic” disease in the neonate or infant postnatally, e.g., methylmalonic aciduria and and Precipitating Factors hyperhomocystinemia in fully breastfed children of mothers ingesting a vegan diet, which causes nutri- The “typical” ages of manifestation of different groups tional vitamin B12 defi ciency. of metabolic disorders in the fi rst year of life are depicted B1 When to Suspect Metabolic Disease 17

Fig. B1.1 Typical ages of manifestation of metabolic disorders in the fi rst year of life in Fig. B1.1. Disorders of intermediary metabolism that lactose) in the fi rst week of life, while children with cause symptoms through the accumulation of toxic hereditary fructose intolerance develop symptoms after molecules (“intoxication”) are usually asymptomatic the introduction of fruits, vegetables, and particularly in the fi rst hours of life. They present after exposure to table sugar (the fructoseÐglucose disaccharide sucrose) the respective substrate derived from catabolism or to the diet, often between 4 and 8 months of age. diet. Postnatal protein breakdown requires amino acid Disorders with a reduced fasting tolerance include catabolism and nitrogen detoxifi cation. Patients with genetic defects of fatty acid oxidation and ketogenesis, acute aminoacidopathies (e.g., maple syrup urine disas well as defi ciencies in the production and release of ease (MSUD) ), classical organic acidurias, or urea glucose. They typically present during periods of cycle defects most frequently develop progressive reduced food intake and/or increased energy require- symptoms between days 2 and 5 of life. Subsequent risk ment such as prolonged fasting or metabolic stress, periods include the second half of the fi rst year of life and the age of presentation thus overlaps with the (in particular, age 6Ð8 months) when solid meals with “intoxication” disorders. However, the disorders with higher protein content are introduced and the children reduced fasting tolerance are less frequently or less start to fast overnight, and late puberty when hormonal severely symptomatic in the postnatal period and more changes and a reduced growth rate change the meta- frequently present in association with infections in the bolic state. Important precipitating factors throughout second half of infancy. life are catabolic states caused by infections, fever, vac- Disorders of energy metabolism are frequently cinations, high-dose steroid therapy, surgery and acci- symptomatic at birth, but may essentially present at any dents, as well as prolonged fasting. time of life, depending on the severity of the genetic Of the disorders of carbohydrate metabolism, galac- defect and the organs involved. Acute decompensation tosemia usually presents after the introduction of milk in mitochondrial disorders may specifi cally be trig- (which contains the galactoseÐglucose disaccharide gered by major alterations in carbohydrate intake or the 18 W. L. Nyhan ingestion of large amounts of rapidly absorbed carbo- Generalized organomegaly is often indicative of a (lys- hydrates, while long-chain fatty acids that interfere osomal) storage disorder, while isolated hepatomegaly with energy metabolism in some b-oxidation defects is observed in a great variety of enzyme defects. Urine cause clinical features of a mitochondrial disorder dur- color and body odor can provide diagnostic clues, as ing fasting periods. Another characteristic feature of discussed later. A list of differential diagnoses of char- mitochondrial disorders is a marked and frequently acteristic symptoms and signs is given in the appendix. irreversible deterioration of the clinical condition with intercurrent illnesses. Disorders in the metabolism of complex molecules rarely show acute metabolic crises but present with B1.2.1 Unusual Odor variable and often progressive organ dysfunction throughout childhood. There are usually no precipitat- Unaccustomed odors can serve as alerting signals for ing factors. The clinical presentation of neurotransmit- several metabolic diseases (Table B1.1). The most ter defects and related disorders depends on the commonly encountered is the sweet smell of acetone ontogenetic expression of neurotransmitter systems found in the acute ketoacidosis of diabetes mellitus and receptors. Affected children are often symptom- and the organic acidemias. Other characteristic odors atic immediately after birth, and there may even be are that of MSUD, the acrid smell of isovaleric aci- symptoms of intrauterine epilepsy as evidence of pre- demia and glutaric aciduria type II, and the odor of natal disease manifestations. There are usually no pre- phenylacetic acid in PKU. The phenylacetic acid odor cipitating factors. is much more prominent in patients with urea cycle defects, treated with sodium phenylacetate, or phenylbutyrate. Very prominent unpleasant odors are found in trimethylaminuria and dimethylglycinuria. Odors B1.2 Physical Examination can be very useful in suggesting a diagnosis or an appropriate test. It is also important not to discard a Every child who is suspected of suffering from an potential diagnosis because of the absence of the odor. inborn error of metabolism requires a thorough physi- Some people are simply unable to detect some odors. cal examination and a careful evaluation of organ func- Many physicians have never really been able to smell tions aided by routine laboratory and imaging the ketotic patient. In other conditions, the acute meta- investigations. In addition, hearing and vision should bolic crisis leads to a cessation of oral intake and vig- be examined at specialist appointments. Depending on orous parental fl uid therapy, so that by the time the the presenting symptoms and the clinical course, a reevaluation, especially a detailed physical examination, should be repeated every 6 months. The detection Table B1.1 Diagnostic utility of unusual odors of additional manifestations is of great importance Odor Substance Disorder even if the patient does not complain of them, particu- Animal like Phenylacetate PKU larly if the fi nal diagnosis is still unknown. Maple syrup Sotolone Maple syrup urine The involvement of multiple organ systems is one disease of the strongest arguments in favor of an inherited Acrid, Isovaleric acid Isovaleric aciduria, short-chain glutaric aciduria metabolic disease. This is especially true for defects acid type II of organelle metabolism such as mitochondrial or per- Cabbage 2-Hydroxy- Tyrosinemia type I, oxisomal disorders or the quickly enlarging group of butyric acid Methionine glycosylation defects or CDG syndromes. Structural malabsorption abnormalities such as dysmorphic features or malfor- Rancid butter 2-Keto- Tyrosinemia type I 4-methiolbu- mations may be caused by disorders in the metabolism tyric acid of complex molecules as well as disorders affecting Rotten fi sh Trimethylamine, Trimethlaminuria, mitochondrial energy metabolism, but are not usually dimethylgly- dimethylglycinuria observed in other disorders of intermediary metabolism. cine B1 When to Suspect Metabolic Disease 19 patient reaches the referral hospital the odor has long The classic unpleasant odor is that of patients with since disappeared. trimethylaminuria. Trimethylamine is the odor of fi sh that is not fresh. The compound is a major end product of nitrogen metabolism of teleost fi shes, which convert Remember it to the oxide and employ the resulting compound to Diagnosis by smell is underutilized, but many are balance their osmotic pressure with surrounding sea not good at it. Too, a characteristic odor maybe water. In man, trimethylamine is formed from dietary absent in a severely ill infant partaking nothing by trimethylamine oxide in fi sh and from choline absorbed mouth and receiving parenteral fl uids. from the intestine and transported to the liver, where the trimethylamine oxide is formed and ultimately excreted in the urine. Patients with trimethylaminuria have an inborn error in the metabolism of the oxide, The odor of maple syrup led to the recognition and defective activity of hepatic trimethlyamine N-oxide original description of MSUD, before it was known synthetase. The metabolic abnormality does not appear that this was a disorder in the metabolism of the to produce a disease as we usually know one; its con- branched-chain amino acids. A keen sense of smell sequences are nevertheless terrible. An odor so unpleas- can still be useful in the detection of this disease, but ant leads to social ostracism, poor performance in the seriousness of the presentation of metabolic imbal- school, depression, and loss of employment. Suicide is ance and a readiness to carry out an analysis of amino a possibility. Diagnosis is important because a diet low acids in plasma are such that most patients diagnosed in fi sh, liver, and egg yolks is usually suffi cient to elim- today do not trigger the smell test. This is also true of inate the odor. The diagnosis is made by identifying acute exacerbation in established patients. Testing for the compound by gas chromatography, gas chromatog- urinary ketones with DNPH and organic acid analysis raphyÐmass spectroscopy (GCÐMS), FAB-MS, or of the urine are also useful in the diagnosis of this dis- nuclear magnetic resonance (NMR) spectroscopy. Its ease. The odor of the patient with isovaleric acidemia excretion is increased by loading with choline, and this has been described as like that of sweaty feet, but it may be necessary for the diagnosis in patients who does not smell anything like a locker room. The smell have found dietary ways of minimizing their odor. is penetrating, pervasive, and readily recognized. It is Following a morning specimen of urine, a 5 g oral sup- the odor of a short-chain volatile acid, and the same ply of choline bitartrate in 3 doses over 24 h led to a smell may be appreciated in patients with multiple 44-fold increase in trimethylamine excretion to acyl-CoA dehydrogenase defi ciency (glutaric aciduria 1.098 mmol/mg creatinine. Normal individuals excreted type II) during times of acute illness. 0.0042Ð0.405 mmol/mg creatinine. The activity of the Now that screening of newborns for PKU is univer- enzyme has been measured in biopsied liver. It is a sal in developed countries, patients with this disease fl avin-containing monooxidase, designated FMO3. are not likely to be diagnosed because of the character- Several mutations in the gene have been identifi ed. istic odor, but some of us have made the diagnosis in Patients have been described in whom the odor of this way in patients born prior to the development of trimethylamine is mild or intermittent. Mutations have screening. The odor has variously been described as been identifi ed in the FMO3 gene on chromosome musty, barny, animal-like or wolf-like. It is actually the 1q23Ð25. For instance, the P153L mutation has been odor of phenylacetic acid. Now that patients with identifi ed in patients with severe trimethylaminuria defects in the urea cycle are treated with phenylacetic and no enzyme activity in vitro. The patients with the acid or its precursor phenylbutyric acid, specialists in mild phenotype have had an allele with two common inherited metabolic disease are quite as accustomed to polymorphisms, E158K in which a 472G→A mutation this odor. coded for a lysine instead of a glutamate, and E3086 in Patients with hepatorenal tyrosinemia and other which a 923A→G mutation coded for glycine instead of nonmetabolic patients with hepatic cirrhosis may have glutamate. Patients have generally been heterozygous a very unpleasant odor that results from the accumula- for this allele and a disease-producing mutation, but one tion of methionine. patient has been homozygous for the variant allele. The 20 W. L. Nyhan variant allele is common in Caucasian populations; The diagnosis is confi rmed by quantitative analysis of allele frequency was found to be 20% in Germans. homogentisic acid in the urine. Dimethylglycinuria is a newly recognised inborn error of metabolism that causes a fi shy odor. The Remember defective enzyme is the dimethylglycine dehydrogenase, which catalyzes the conversion of this compound The diagnostic black pigment of alkaptonuria is to sarcosine. A missense mutation in the gene has been often missed. A red color may be seen in plastic identifi ed in an affected patient. Trimethylamine was diaper, or a positive test for reducing substance may absent from the patient’s urine. He also complained of be alerting (See Table B1.2). muscle fatigue and had elevated levels of creatine kinase in the serum. Dimethylglycine is most readily detected by 1H-NMR spectroscopy. Its presence was confi rmed by 13C-NMR spectroscopy and by GCÐMS of nonex- tracted urine, but the compound could not be detected B1.2.2.1 Examination of the Urine by GCÐMS after the usual ethylacetate extraction. for the Signifi cance of Color

Urine has a normal amber color that is the color of the pigment urochrome. Pale, dilute, or watery urine results from a plentiful fl uid intake or diuresis as in diabetes B1.2.2 Color of the Urine or Diaper mellitus or diabetes insipidus, or in the recovery phase of a tubular necrosis. Very dark urine or concentrated Physicians since at least the time of Hippocrates have urine results from dehydration. Pale urine with a high recognized that the color of the urine may be the clue specifi c gravity suggests diabetes mellitus. Dark urine that leads to the diagnosis. It was Garrod’s recognition with a low specifi c gravity suggests the presence of uro- of the signifi cance of the dark urine of patients and bilin or bilirubin and is best checked by analysis of the families with alkaptonuria that led to the conceptual- blood for bilirubin. Very bright yellow urine may be ization of the inborn errors of metabolism. seen in infants who ingest large amounts of carotene, Alkaptonuria is recognized surprisingly infrequently but the skin of such infants is usually carotenemic. Urine in this way, and many patients reach adulthood and may, of course, be red because of hematuria, but this is clinical arthritis before the diagnosis is made. This is readily recognized by microscopic analysis, and such a the result of many factors, among them that the black specimen is not the subject of differential diagnosis by pigment forms with time and oxygen, and that fl ushing color. Free hemoglobin in the urine appears brown or does away with both. In a patient in whom one seeks to black as methemoglobin is formed. The most famous make this visual diagnosis, it is best to alkalinize and example of this is the black water fever of malaria. shake the urine and look with excellent light for the fi ne black precipitate. In times past when infants wore cloth diapers, which were laundered with strong alkaline soap, the conditions were perfect, and the diagno- B1.2.2.2 Dark Brown or Black Urines sis could be made by the appearance of black pigment in the diaper. Now they wear plastic disposable dia- In addition to alkaptonuria, hemoglobinuria and myo- pers, many of which turn pink on contact with alkapto- globinuria both produce brown or dark urine and both nuric urine. So, we can still make the diagnosis early are detected by the dipstix for hemoglobin or by the by examining the diaper. benzidine test. Hemoglobin in the urine is often Alkaptonuric urine also gives a positive test for accompanied by hematuria. Hemoglobinuria in the reducing substance and is glucose-negative, and this absence of red cells in the urine is accompanied by may be an alerting signal for the diagnosis. Homogentisic evidence of hemolysis, such as anemia, reticulocyto- acid also reduces the silver in photographic emulsion, sis, or hyperbilirubinemia, while myoglobinuria is and alkaptonuric urine has been used to develop a pho- often accompanied by muscle pains or cramps and tograph, an interesting qualitative test for the diagnosis. elevation of creatine phosphokinase and uric acid. An B1 When to Suspect Metabolic Disease 21

Table B1.2 Syndromes of abnormally colored urine or diapers myophosphorylase defi ciency (McArdle disease) and Conditions Confi rmation myodenylate deaminase defi ciency. Melaninuria is Dark brown or seen in disseminated melanotic sarcoma. black urine or diapers Alkaptonuria Standing, Homogentisic (Pampers alkaline aciduria B1.2.2.3 Red Urine become red) Clinitest positive Melaninuria Disseminated Porphyrias are the major metabolic cause of red urine. melanotic Congenital erythropoietic porphyria is an autosomal sarcoma recessive disease caused by mutations in the gene for Red urine or uroporphyrinogen synthase. Uroporphyria and copropor- diapers phyria are found in the urine. It manifests a variable Hematuria Microscopic phenotype from nonimmune hydrops fetalis to a mild Hemoglobinuria Guaiac, benzidine History adult-onset form with only photosensitive cutaneous Beets lesions. The disease is often fi rst recognized because of (anthrocyanins) a pink, red, or brown stain in the diapers. These patients Congenital Blood, urine, stool, also develop erythrodontia in which a red fl uorescence erythropoietic uroporphyrin, of the teeth is visible with ultraviolet illumination. porphyria uroporphyrin-III Red urine may also be seen following the ingestion cosynthase (CEP) activity of large quantities of colored foods. The anthrocyani- Red dyes (Monday History nuria of beet ingestion is quite common. Blackberries morning have also been associated with red urine. Red dyes, disorder, such as rhodamine B, used to color foods and cold rhodamine B) drinks have led to red urine of so many children after a Red diaper 24Ð36 h of Culture syndrome oxidation Neomycin Rx weekend party that the condition was termed the (Serratia after Monday morning disorder of children. Phenolphthalein marcescens) passage in laxatives may also cause red urine. In the neonatal Phenolphthalein History period, distinct red spots in the diaper were seen where pH sensitive crystals of ammonium urate dried out. In previous days Green-blue urine when cloth diapers were used and accumulated for a Blue diaper Tryptophan while before laundering, a red diaper syndrome was syndrome malabsorption recognized in which the color developed after 24 h of (indigotin) Indicanuria incubation and came from the growth of the chro- Indole-acetic aciduria mobacterium, Seratia marcescens, which does not Biliverdin Serum bilirubin produce pigment in the infant’s intestine, but only after (obstructive aerobic growth at 25Ð30¡C. Red stools may also be jaundice) seen after the ingestion of red crayons, and in some Methylene blue History patients receiving cefdinir, in most but not all of whom (ingestion, Rx) receive oral iron. Orange sand Urate overproduc- Chemical assay for tion (urates uric acid, blood may stain and urine diaper red Hypoxanthine-guanine B1.2.2.4 Green or Blue Urines in neonatal phosphoribosyl period) transferase (HPRT) Blue pigment in urine containing urochrome usually leads to a green color. Blue color was seen in the attack of myoglobinuria should signal a work-up for a blue diaper syndrome. This disorder of the intestinal disorder of fatty acid oxidation (Chap. C6). It is also absorption of tryptophan was described in two siblings seen in enzyme defects localized to muscle, such as who also had hypercalcemia and nephrocalcinosis. 22 W. L. Nyhan

When tryptophan is not effi ciently absorbed, intesti- Table B1.3 Routine laboratory investigations nal bacteria convert it to indole metabolites that are Finding Indicative of absorbed and excreted in the urine. The blue color Anemia (macrocytic) Disturbances in cobalamin or comes from the oxidative conjugation of two molecules folic acid metabolism or transport of indican to indigotin, or indigo blue, a water insolu- Reticulocytosis Glycolysis defects, disorders of ble dye. The excretion of indole products is increased the g-glutamyl cycle by an oral tryptophan load. The condition must be very Vacuolized lymphocytes Lysosomal storage disorders rare because further patients have not been reported ↑ Alkaline phosphatase Hypoparathyreoidism, bile acid since the initial report in 1964. Indoles including indi- synthesis defects ↓ can are also found in the urine of patients with Hartnup Cholesterol A-, hypobetalipoproteinemia, sterol synthesis defects, disease, in which there is defective renal tubular reab- peroxisomal disorders sorption, as well as intestinal absorption of a number of ↑ Triglycerides Glycogen storage disorders, amino acids including tryptophan, but blue diapers or lipoprotein disorders, e.g., urine have not been observed. lipoprotein lipase defi ciency ↑ Biliverdin, the oxidation product of bilirubin, is CK Mitochondrial disorders, fatty acid oxidation defects, excreted in the urine, and so green urine may be seen glycogen storage disease in jaundiced patients, particularly those with chronic types II, III, and IV, obstructive jaundice. glycolysis defects, muscle- Benign pigments such as methylene blue, found in AMP-deaminase defi ciency, dystrophinopathies some tablets, are excreted in urine, and if a suffi cient ↑ a-Fetoprotein Ataxia telangiectasia, hepator- quantity is taken, will color the urine. Indigo-carmine is enal tyrosinemia another blue dye that may fi nd its way into food stuffs. ↓ Glucose in CSF Mitochondrial disorders, glucose transport protein defi ciency ↑ Uric acid Glycogen storage disorders, disorders of purine metabolism, fatty acid oxidation B1.3 Routine Laboratory Investigations defects, mitochondrial disorders ↓ Uric acid Disorders of purine metabolism, Unexpected fi ndings in the “routine” laboratory require molybdenum cofactor critical evaluation. Particularly in patients with unusual defi ciency and unexplained symptoms they may be indicative of ↓ Creatinine Creatine synthesis defect ↑ an inborn error of metabolism and can help to direct Iron, transferrin Hemochromatosis, peroxisomal disorders specifi c diagnostic investigations. Table B1.3 gives a ↓ Copper (in plasma) Wilson disease, Menkes disease noncomprehensive collection of such sometimes unex- ↑ Copper (in plasma) Peroxisomal disorders pectedly obtained laboratory abnormalities that may ↑ Copper (in urine and Wilson disease, peroxisomal be suggestive of certain metabolic disorders. liver) disorders ↓ Ceruloplasmin Wilson disease, Menkes disease, aceruloplasminemia Hypothyroidism, Mitochondrial disorders, CDG hypoparathyreoidism syndromes B1.4 When Not to Suspect AMP adenosine monophosphate; CDG congenital disorders of a Metabolic Disease glycosylation; CK creatine kinase

Inborn errors of metabolism may be considered in the differential diagnosis of a great variety of clinical prob- test, the likelihood of litigation, and the personal expe- lems, and at times it can be diffi cult to decide that spe- rience of the clinician. It is imperative to exclude disor- cialist metabolic investigations are not warranted. ders for which effective treatments are available, while Whether or not certain specialist investigations are in cases of slowly progressive, and by experience often indicated quite obviously also depends on secondary incurable disorders diagnostic procedures should be factors such as local or national availability, costs of the performed stepwise depending on the results of the fi rst B1 When to Suspect Metabolic Disease 23 investigations and the appearance and development of previously asymptomatic. Key factors in the evalua- signs and symptoms with time. The diagnosis of some tion of symptoms are their isolated appearance vs. the metabolic disorders involves procedures that are stress- presence of additional pathology, however subtle, i.e., ful, such as sedation or lumbar puncture, or potentially the lack or presence of additional neurological and/or dangerous for the child (e.g., fasting or loading stud- systemic abnor malities, and a static vs. a progressive ies), and that are often also stressful for the parents. clinical course. Multisystem or progressive disorders Psychosocial factors should be taken into consideration are much more likely to be caused by inborn errors of when the diagnostic work-up is planned. The families metabolism. need to be guided and supported. In the worst case, a specifi c diagnosis with a doomed prognosis that shat- ters the expectations of the parents can even damage Key References the parentÐchild relationship. On the other hand, in almost all families a specifi c diagnosis no matter how negative will be one of the most important supports for Moolenaar SH, Poggi-Bach J, Engelke UFH, Corstaensen JMB, Heerschap A, De Jong JGN, Binzak BA, Vockley J, Wevers coping, and of course is critical for timely genetic diag- RA (1999) Defect in dimethylglycine dehydrogenase, a new nosis in young families and appropriate counseling. inborn error of metabolism: NMR spectroscopy study. Clin Specialist metabolic investigations are not usually Chem 45:459Ð464 indicated in children with moderate developmental Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic diseases, 2nd edn. Hodder Arnold, London delay, isolated delay in speech development in early Saudubray JM, Charpentier C (2001) Clinical phenotypes: diagno- childhood, moderate failure to thrive, frequent infec- sis/algorithms. In: Scriver CR, Beaudet AL, Valle D, Sly WL tions, occasional seizures, e.g., during fever, or defi ned (eds) The metabolic and molecular bases of inherited disease, epileptic syndromes. An inborn error of metabolism 7th edn, Vol 1 . McGraw-Hill, New York, pp 1327Ð1403 Zschocke J, Kohlmueller D, Quak E, Meissner T, Hoffmann GF, is also unlikely in the healthy sibling of an infant Mayatepek E (1999) Mild trimethylaminuria caused by who died of SIDS, provided that this child had been common variants in FMO3 gene. Lancet 354:834Ð835 Metabolic Emergencies B2 William L. Nyhan

Key Facts this way, but usually the presentation is more indolent. Disorders which present with potentially lethal meta- › The classic presentation of inborn errors of bolic emergencies usually do so fi rst in the neonatal metabolism is with a free period of apparent period or early infancy. In fact, we have felt that prior health that may last days or even years, but it to the advent of programs of expanded neonatal screen- is followed by overwhelming life threatening ing a large number of such infants probably die(d) disease. without the benefi t of diagnosis. › The episode usually follows catabolism intro- Episodes of acute illness and metabolic decompen- duced usually by acute infection; sometimes sation are often precipitated by acute infection and its after surgery. attendant catabolism. Catabolism may also be induced › Initial laboratory evaluation needs only the rou- by surgery or injury. The duress of birth may be suffi - tine clinical laboratory to establish acidosis or ciently catabolic to induce an early neonatal attack. alkaoisis, hyperammonemia, ketosis, hypocly- The diseases in which the fundamental defect is in an cimia, or latic acidemia. enzyme involved in the catabolism of a component of food, such as protein, are often characterized by a period of build-up of body stores of toxic intermediates until the levels are great enough to produce metabolic imbalance. Such a patient may have cycles of B2.1 General Considerations acute illness precipitating admission to the hospital, cessation of feedings and administration of parenteral fl uids and electrolytes with recovery and discharge The most demanding cases in the fi eld of genetic dis- only to repeat the cycle until the diagnosis is made and ease that pose problems for rapid diagnosis and rapid appropriate therapy initiated, or the patient dies in such initiation of effective treatment are patients who pres- an episode. In the disorders of fatty acid oxidation, epi- ent with episodes of acute life-threatening illness. This sodes of metabolic emergency are brought on by fast- is the mode of presentation of a considerable number ing. This can be when the infant begins to sleep longer, of inherited metabolic diseases (Table B2.1.1). It is or more commonly, when intercurrent infection leads particularly characteristic of the organic acidurias, the to vomiting or failure to feed. disorders of the urea cycle, maple syrup urine disease, The classic presentation of the diseases that produce nonketotic hyperglycinemia, and the disorders of fatty metabolic emergencies is, for the initial acute presenta- acid oxidation. The lactic acidemias may present in tion in infancy, often in the neonatal period, followed by recurrent episodes of metabolic decompensation usually with infection. Nevertheless, some patients W. L. Nyhan with these diseases, usually those with variant enzymes Department of Pediatrics, University of California, UCSD in which there is some residual activity, may present School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA fi rst in childhood or even in adulthood. The diseases of e-mail: [email protected] fatty acid oxidation typically require fasting for 16–24 h

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 25 DOI: 10.1007/978-3-540-74723-9_B2, © Springer-Verlag Berlin Heidelberg 2010 26 W. L. Nyhan

Table B2.1.1 Metabolic diseases presenting as acute overwhelming disease Disorder Detect Deﬁ nitive diagnosis Maple syrup urine Urinary 2,4-DNP, plasma Branched-chain amino academia, branched disease amino acids chain ketoacid decarboxylase Organic acidurias, e.g., Smell Isovaleric acid in blood, isovalerylglycinuria isovaleric aciduria methylmalonic aciduria Methymalonic acid in urine Methylmalonic acid in blood and urine, methylmalonyl-CoA mutase, complementation assay propionic aciduria Hyperglycinemia, urinary organic Propionic acidemia, propionyl-CoA carboxylase acid pattern multiple carboxylase deﬁ ciency Urinary organic acid pattern Biotinidase, holocarboxylase synthetase d-2-hydroxyglutaric aciduria Urinary organic acid pattern Isomer differentiation of 2-hydroxyglutaric acid

Urea cycle defects Blood NH3, plasma amino acids, Ornithine transcarbamylase, c carbamoyl urinary orotic acid phosphate synthetase, N-acetylglutamate synthetase, argininosuccinate synthetase, argininosuccinate lyase, HHH syndrome Disorders of fatty acid oxidation Hypoketotic hypoglycemia Acylcarnitine profi le, enzyme assay, MCAD DNA Lactic acidemias Lactate, alanine Enzyme assay; mitochondrial DNA Hyperglycinemia, nonketotic Glycinemia, CSF glycine Glycine cleavage enzyme in liver Methylenetetrahydrofolate Homocysteinemia, Enzyme assay reductase defi ciency hypomethioninemia Sulfi te oxidase defi ciency Sulfi te test, amino acid pattern Sulfi te oxidase Adenylosuccinate lyase defi ciency Succinyladenosine, SAICA riboside Enzyme assay Lysosomal acid phosphatase Lysosomal acid phosphatase Enzyme assay defi ciency Adrenogenital syndrome 17 – ketosteroids Pregnanetriol, testosterone Fructose intolerance Fructosuria Hepatic fructose -1-P-aldolase Galactosemia Urinary reducing substance Galactose-1-phosphate-uridyl transferase CSF cerebrospinal fl uid; 2,4-DNP 2,4-dinitrophenol; HHH hyperammonemia, hyperornithinemia, homocitrullinuria; MCAD medium-chain acyl-CoA dehydrogenase; SAICA succinylaminoimadazole carboxamide before metabolic imbalance ensues. Some people reach metabolic emergency. Acidosis and hyperammonemia adulthood without ever fasting that long. Nevertheless, are indicative of an organic acidemia. Hyperammonemia the fi rst episode may be lethal, regardless of age. and alkalosis are characteristic of urea cycle defects. The initial clinical manifestations of metabolic Hypoglycemia along with elevation of uric acid and emergency are often vomiting and anorexia, or failure creatine kinase (CK) is seen in disorders of fatty acid to take feedings. This may be followed by rapid deep oxidation. If ketones are absent from the urine, this is breathing in the acidotic infant. A characteristically almost certainly the diagnostic category. The presence ketotic odor may be observed. There may be rapid proof ketones in the urine does not rule it out; the blood gression through lethargy to coma, or there may be level of 3-hydroxybutyrate is a better indicator of the convulsions. Hypothermia can be the only manifesta- adequacy of ketogenesis. Hypocalcemia may be a non- tion besides failure to feed and lethargy. Further pro- specifi c harbinger of metabolic disease. gression is to apnea and, in the absence of intubation Elevated levels of lactate in the absence of cardiac and assisted ventilation, death. disease, shock, or hypoxemia are signifi cant, and seen The initial laboratory evaluation (Table B2.1.2) in organic acidemias and even hyperammonemias, as involves tests that are readily available in most hospi- well as in the lactic acidemias of mitochondrial disease tal laboratories, particularly clinical chemistry. Most (Chap. B.2.3). A normal pH in the blood does not rule important in early discrimination are the electrolytes out lactic acidemia; the pH usually remains neutral and the ammonia. Blood gases are listed because they until lactic acid concentrations reach 5 mM. The blood are often the fi rst data available in a very sick infant. count is useful in indicating the presence or absence of They give some of the same information as the elec- infection. More importantly, neutropenia with or with- trolytes on the presence or absence of metabolic acido- out thrombocytopenia or even with pancytopenia is sis. In general, it is advisable to get everything on the characteristic of organic acidemia, while patients with list on admission of the patient suspected of having a mitochondrial disease often develop thrombocytosis. B2 Metabolic Emergencies 27

The dinitrophenylhydrazine (DNPH) test is positive In the presence of acidosis suggesting organic acidu- in any disorder in which there are large amounts of ria the assay of choice is organic acid analysis of the ketones in the urine. It is particularly useful as a spot urine. A positive DNPH in the absence of acidosis or indicator of the presence of maple syrup urine disease. with mild acidosis indicates amino acid analysis of the A positive urine reducing substance may be the fi rst plasma for maple syrup urine disease. Hyperammonemia indicator of galactosemia. Today in most developed without acidosis indicates that a urea cycle defect should countries this disorder is discovered through routine be followed up by amino acid analysis of the plasma and neonatal screening for the defective enzyme. Chemical analysis of the urine for orotic acid. This can be done by testing for blood in the urine is also useful in detecting organic acid analysis or better by a specifi c assay for hemoglobinuria and myoglobinuria, the latter indicat- orotic acid. In an infant found to have an elevated plasma ing a crisis in a disorder of fatty acid oxidation. The concentration of glycine, the availability of simultane- test for sulfi te indicates the presence of sulfi te oxidase ously obtained CSF permits a defi nitive diagnosis of defi ciency, either isolated or as a result of molybde- nonketotic hyperglycinemia. Following initial testing num cofactor defi ciency, which may present with indicating a disorder of fatty acid oxidation, defi nitive intractable seizures shortly after birth. testing requires organic acid analysis of the urine, an It is advisable in a seriously ill infant to save some acylcarnitine pattern of the blood, and a medium-chain blood and urine (Table B2.1.2) while this initial testing acyl-CoA dehydrogenase (MCAD) assay of the DNA. is taking place. Then if the initial indicators point to an In most of these patients, quantifi cation of concentra- area of metabolic disease, these samples can be pro- tions of carnitine in blood and urine is useful. cessed appropriately, obviating such problems as a later Precise molecular diagnosis is made by enzyme lack of availability of urine in an infant in whom dehy- assay, usually of lymphocytes or cultured fi broblasts, dration and shock lead to a renal tubular necrosis. or by determination of the mutation in the DNA. DNA Further testing requires a biochemical genetics labora- analysis is particularly useful in those situations in tory expert in these procedures. Programs of profi ciency which there is a common mutation, such as the A 985G testing indicate that not all laboratories that undertake mutation in the MCAD gene in Caucasians. these specialized procedures do them properly.

Table B2.1.2 Initial laboratory investigations B2.1.1 Neonates Blood Electrolytes: bicarbonate, anion gap The classic presentation of the metabolic disorders that Blood gases: pH, pCO , HCO , pO 2 3 2 lead to medical emergencies is with life-threatening Ammonia Glucose illness in the newborn period. These infants are usually Lactate, pyruvate born healthy, and there is classically a hiatus, or period Calcium of apparent well-being, before the onset of symptoms. 3-Hydroxybutyrate This free period can be as short as 12 h, or even less; it Uric acid is usually at least 48 h; it can be as long as 6–8 months Creatine kinase (Fig. B1.1). Nevertheless, within 24 h of the fi rst symp- Complete blood count tom the infant is usually admitted to the intensive care Urine unit with artifi cial ventilation begun. Ketones Reducing substance Dinitrophenyl hydrazine Remember Sulfi tes (sulfi te test) Acute life-threatening neonatal illness following a Benzidine, guaiac hiatus in which the infant appears well is a strong Store at −20°C (−4°F) indicator of the presence of inherited metabolic Urine disease. Heparinized plasma CSF Store at 4°C (39°F) The most important key to the diagnosis of a metabolic Heparinized whole blood disease in such an infant is a high index of suspicion. 28 W. L. Nyhan

Table B2.1.3 Clinical features suggesting the presence of metabolic disease in an infant Remember Overwhelming illness in the neonatal period An infant who is thought to have pyloric stenosis, Vomiting, pyloric stenosis but is acidotic, must be worked up for metabolic Acute acidosis, anion gap disease. Massive ketosis Hypoglycemia Coagulopathy Electrolyte analysis also serves to indicate the pres- Deep coma ence of adrenal insuffi ciency or the adrenogenital syn- Seizures, especially myoclonic drome, especially in the male, which may present in this Hiccups, chronic way. Some patients present with poor sucking or a com- Unusual odor plete inability to feed. An odd smell may be very help- Extensive dermatosis, especially monilial ful in the diagnosis of metabolic disease (Table B1.1 in Family history of siblings dying early Chap. B1), especially isovaleric acidemia and maple syrup urine disease. There are some alerting signals (Table B2.1.3). The pic- The critically ill infant is often fi rst seen in coma in ture of overwhelming illness in a neonate most often an intensive care unit. An algorithmic approach to the calls to mind a diagnosis of sepsis. Some alert physi- infant in coma is shown in Fig. B2.1.1. Initial evaluation cians have initiated a metabolic work-up in such an of NH3, pH, and electrolytes permits early separation infant once the blood culture is negative. In general, into those with elevated ammonia and no acidosis, most awareness toward metabolic disorders still needs to be of whom have urea cycle defects (Chap. B2.6). Similarly, increased. In a full-term newborn with an uncompli- those in whom the ammonia is elevated or normal, but cated delivery, septicemia is also not as common as it there is metabolic acidosis and usually massive ketosis, is pursued, and a metabolic emergency should be con- commonly have organic aciduria. Patients with lactic sidered and investigated in parallel. This approach may acidemia and pyroglutaric aciduria may present with well provide an earlier diagnosis than is unfortunately neonatal acidosis, but not usually with coma. The patient the standard outcome in the diagnosis of most neonates with maple syrup urine disease may be convulsant or with metabolic disease. However, it should be kept in opisthotonic as well as comatose, but there is usually mind that some patients with metabolic disease may little or no acidosis, and the DNPH test is positive. actually present in the newborn period with septicemia. Comatose patients without hyperammonemia or Prior to the advent of newborn screening for galac- acidosis and a negative DNPH most commonly have tosemia, the earliest diagnoses of this disease were nonketotic hyperglycinemia. The identical presentation often made by physicians who recognized that these can be seen in babies suffering from the treatable disor- patients present with neonatal Escherichia coli sepsis. ders of pyridoxine metabolism or from sulfi te oxidase We have also encountered positive blood cultures and defi ciency, molybdenum cofactor defi ciency, adeny- clinical sepsis in citrullinemia, propionic aciduria, and losuccinase lyase defi ciency, methylenetetrahydrofo- other disorders. Cerebral hemorrhage and pulmonary late reductase defi ciency, or leukotriene C4-synthesis hemorrhage may complicate the initial episode of met- defi ciency (see Chap. C5). A urinary sulfi te test must abolic imbalance. Coagulopathy may be the fi rst sign of therefore be done in every child presenting with cata- the presence of hepatorenal tyrosinemia. strophic encephalopathy, and specifi c tests for homo- Many metabolic diseases and particularly the organic cysteine in blood as well as urinary purine analysis acidurias and the hyperammonemias present fi rst with should be ordered. Leukotrienes are best analyzed in vomiting. This has led frequently to a diagnosis of pylo- CSF, which must be stored at −70°C or, as an interme- ric stenosis or duodenal obstruction, and a number of diate measure, in dry ice or under liquid nitrogen. If pyloromyotomies or other explorations have been car- intractable seizures dominate the clinical picture, foli- ried out. The organic acidurias should not be missed nic acid responsive seizures should be considered, and this way by the alert physician, because for all such so should pyridoxine- (B6-) as well as pyridoxal-phos- patients results of electrolyte analysis are available, and phate-responsive seizures. A therapeutic trial pyridox- pyloric stenosis causes alkalosis. A patient who appears ine and pyridoxal-phosphate is followed, if negative, to have pyloric stenosis and has acidosis has an organic by the administration of folinic acid with 5 mg/kg/day aciduria even if someone can feel an olive. in three doses intravenously or orally (see Chap. C5). B2 Metabolic Emergencies 29

Fig. B2.1.1 The metabolic ALGORITHMIC APPROACH TO THE DIAGNOSIS OF THE diagnosis of a newborn infant NEONATE IN COMA in coma Blood for NH3 , pH, Electrolytes Urine for ketones

NH3 increased NH3 normal NH3 normal NH3 normal No Acidosis No Acidosis or increased Acidosis, No Acidosis anion gap DNPH negative Urine Massive ketonuria DNPH Primary hyperammonemia Amino Acid Organic Analysis Positive Acid Urea cycle defect Analysis Nonketotic hyperglycinemia HHH syndrome Amino (Rarely) Transient hyperammonemia Acid Organic Sulfite oxidase deficiency of the newborn Analysis acidemia ± xanthine oxidase deficiency Cbl C or D methylmalonic Maple syrup acidemia urine disease Disoder of Propionate metabolism lsovaleric acidemia Glutaric acidemia II Oxothiolase deficiency

Severe neonatal/infantile epileptic encephalopathy Remember is one indication for specialized CSF analyses testing metabolic pathways of brain metabolism, especially The urine of the ill newborn should always be tested neurotransmitters (see Chap. C2 – Epilepsy – Neonatal for ketones. Massive ketonuria indicates an organic Period, page 11). Defects in the metabolism of biogenic acidemia, while an absence of ketosis in a hypogly- monoamines are diagnosed this way and so is GABA cemic infant leads to a diagnosis of a fatty acid oxi- transaminase defi ciency. An electroencephalographic dation defect. fi nding of a burst suppression pattern is characteristic of nonketotic hyperglycinemia, but it is also found in other metabolic disorders such as disorders of pyridox- B2.1.2 Infancy ine metabolism, propionic aciduria or molybdenum cofactor defi ciency causing sulfi te oxidase defi ciency. The infant with a metabolic emergency that presents The occurrence of ketosis and acidosis in the neonatal after the neonatal period may have a period of some period is an almost certain indicator of metabolic dis- months of failure to thrive. Such an infant may feed ease. Ketosis is rare in newborns. Even the neonatal dia- poorly and vomit frequently, but the metabolic crisis is betic is not ketotic; so testing of the urine at this time is avoided until the advent of an intercurrent infection often not performed. However, this is a mistake because or a switch from human to cow’s milk. Such an infants with organic acidemia have massive ketosis. A infant may then promptly develop the picture of life- reasonable position might be that any infant admitted to threatening illness just like that of the newborn. The a neonatal intensive care unit with life-threatening non- etiologies are often the same, organic acidurias, hyper- surgical illness should be tested for blood pH, NH3, elec- ammonemias, hepatorenal tyrosinosis, and fructose trolytes, glucose, and for ketonuria. One could argue intolerance. that valuable time would be saved by testing at the out- The disorders of fatty acid oxidation (Chap. B2.5) set for lactate and amino acids in the blood, organic present classically at 7–12 months of age, consistent acids in the urine, and acylcarnitines in blood spots. with the time infants begin to sleep longer or the timing 30 W. L. Nyhan of the fi rst intercurrent infection that leads to prolonged infants with the other metabolic diseases. So a posi- fasting because of anorexia or vomiting. They can of tive liver biopsy will not make the diagnosis. A meta- course present fi rst at any age in which these conditions bolic work-up is required. are met. The infantile presentation of these diseases is with hypoketotic hypoglycemia. Clinically, there may Remember be convulsions or coma. Cardiac arrhythmias are common. It is important to assess the level of glucose in the An infant thought to have Reye syndrome has a blood, and the adequacy of ketogenesis. A urine nega- metabolic disease until proven otherwise. tive for ketones in the presence of hypoglycemia is very helpful, but if there are ketones in the urine, examination of blood concentrations of free fatty acids The differential diagnosis of primary metabolic coma and 3-hydroxybutyrate is necessary to evaluate keto- in infancy overlaps with that of the neonatal period as genesis. Roentgenographic examination of the chest, shown in Fig. B2.1.1 and described earlier. In later electrocardiography, and echocardiography should be infancy patients with defective B12 metabolism, includ- carried out. ing cobalamin C disease, transcobalamin II defi ciency, Muscle tone is affected in a variety of infants with and the breast fed infant of a mother on a vegan diet or metabolic diseases. These include the organic acidu- a mother suffering from sometimes unrecognized per- rias and the disorders of fatty acid oxidation, which nicious anemia may also present in this way. A patient are classically complicated by metabolic emergen- in coma may also have hypoglycemia (Chap. B2.4). A cies. Thus, inclusion of a work-up for metabolic dis- very specifi c cause of hypoglucorrachia is due to the ease in the hypotonic or fl oppy infant may bring to defects of the glucose transporter 1 protein, the facilita- light the more important need to treat an infant prior tive glucose transporter of the brain. This diagnosis, to the onset of that fi rst episode of metabolic decom- also termed De Vivo syndrome, can be anticipated pensation that so often leads to death or mental from a pathologically decreased CSF/blood glucose retardation. ratio <0.45, normal mean 0.8 ± 0.1, in the absence of Neutropenia, thrombocytopenia, and even pancy- pleocytosis or elevated CSF lactate. Care must be taken topenia are concomitants of a number of metabolic to perform the lumbar puncture and determinations of diseases, notably the organic acidurias and the cobal- blood sugar at least 4 h postprandially. Recurrent exag- amin-related disorders. Recognition of this associa- gerations of neurological and psychiatric symptoms tion in infancy may lead to an early diagnosis and often leading to metabolic coma are a major presenting forestall what is usually the most life-threatening cri- feature of several late-onset inborn errors of metabo- sis, the fi rst. lism in older children and adults. Clinical and labora- The infant thought to have Reye syndrome is an tory presentations are especially variable and require a excellent candidate for a diagnosis of an inborn error high index of suspicion on the part of the physician. of metabolism. Single episode Reye syndrome was These constellations and the most important diagnostic once relatively common, but it is not any longer, pre- approaches are discussed in Chaps. B2. 7 and C5. sumably related to the sparing use of salicylates in acute viral illness. Today, most infants with the typical Reye presentation of hypoglycemia, hyperam- B2.1.3 Older Children and Adults monemia, and elevated transaminases have inherited metabolic disease. Most of them have MCAD defi - ciency (Chap. B2.5) or ornithine transcarbamylase Any of the disorders, which present in early infancy, (OTC) defi ciency (Chap. B2.6), but any disorder of may develop repeated attacks of metabolic emergency fatty acid oxidation or of the urea cycle may produce at any age, despite generally successful therapy. Late- this picture. We have diagnosed the hyperammone- onset forms are also seen, albeit uncommonly, in many mia, hyperornithinemia, homocitrullinuria syndrome of the diseases that present classically in infancy. They (Chap. B2.6) in an infant thought to have Reye syn- are more common for the urea cycle defects and deter- drome. The liver biopsy in this infant had the typical mination of ammonia should be tested in every patient Reye picture of microvesicular steatosis, and so have with fl uctuating consciousness or unexplained coma. B2 Metabolic Emergencies 31

The lateness of onset is usually a function of the fact B2.2 Work-up of the Patient with that the variant enzyme resulting from the mutation Metabolic Acidosis and Massive has greater residual activity than that of the patient Ketosis with the early neonatal presentation. Nevertheless, the dangerous nature of these diseases is clearly indicated by the fact that episodes of metabolic imbalance occur- William L. Nyhan ring in childhood, adulthood, or even old age may be quickly fatal. This is particularly true of urea cycle defects, and OTC defi ciency is notoriously unpredict- Key Facts able. A series of readily manageable hyperammonemic episodes may be followed by one that leads promptly › Massive ketosis in a neonate or young infant is to cerebral edema, herniation, and death. We have also a key to the diagnosis of an organic aciduria. observed carbamoyl phosphate synthetase defi ciency Testing of the urine for ketones is a must in all leading to edema in a fi rst episode in adults up to the ill infants. age of 50. Branched-chain ketoaciduria has been › The most frequent organic acidurias are propi- reported in a small number of late-presentation patients. onic aciduria, methylmalonic aciduria, multi- Again, as in the case of urea cycle defects, a late-onset ple carboxylase defi ciency, isovaleric aciduria, patient is still in danger of dying from the fi rst or a and 3-oxothiolase defi ciency. subsequent episode of metabolic imbalance. › Routine clinical chemistry reveals low pH, low The defects in fatty acid oxidation may present late, bicarbonate, and an increased anion gap. The simply because the patient has never before fasted long urine pH is low. Hyperchloremic acidosis and enough to exhaust liver glycogen and call upon oxida- a normal anion gap mean intestinal losses or a tion of fats. In this way, a MCAD defi ciency can pres- renal tubular acidosis, the former with acidic ent fi rst as a fatal episode of hypoketotic hypoglycemia urine and the latter with alkaline urine. in an adult. Other diseases of fatty acid oxidation may › Quantitative organic acid analysis by gas- present later with acute rhabdomyolysis and cardiac chromatography mass-spectrometry is essen- arrhythmias. These patients usually have elevated lev- tial in differential diagnosis. els of CK and uric acid at the time of the crisis. Others › Acylcarnitine (MS/MS) profi le may be a quicker may present with acute cardiac failure, a consequence route to diagnosis. of year after year accumulated cardiomyopathy and the depletion of body stores of carnitine. Mitochondrial diseases (Chaps. B2.3 and D1) may present at any age, but they more commonly present fi rst in childhood or even adulthood. The fi rst episode could be of coma with lactic acidosis and ketoacidosis. There are a number of metabolic diseases that present More commonly, particularly in the MELAS disease with acidosis (Table B2.2.1), most of them for the fi rst (Chap. B2.3), there is a stroke or stroke-like episode. time in the neonatal period. Metabolic acidosis may be Such episodes have also been seen in propionic acidu- caused by the accumulation of the carboxylic acid ria, methylmalonic aciduria, OTC defi ciency, and con- itself, as in the case of lactic acidemia (Chap. B2.3) or genital disorders of glycosylation (CDG). Patients pyroglutamic aciduria, or hawkinsinuria in infancy. with mitochondrial disease often have abnormal neu- However, in most of the severe acidoses, the acidosis is roimaging studies. In addition to CT or MRI evidence caused by a massive ketoacidosis, in which acetoacetic of stroke, there are areas of increased signal in the basal ganglia and elsewhere. Many have the radiological appearance of Leigh syndrome. We have diagnosed NARP mutation in a patient who carried a radiological W. L. Nyhan diagnosis of acute demyelinating encephalomyelitis. Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, Basal ganglia lesions are also characteristic of propi- USA onic acidemia and methylmalonic acidemia. e-mail: [email protected] 32 W. L. Nyhan acid and 3-hydroxybutyric acid accumulate in the Ketosis can be readily quantifi ed by measuring the con- blood, and the urine tests strongly for ketones. These centration of 3-hydroxybutyrate in the blood. Normally are classic metabolic emergencies. It is critical to make this is <1.0 mM in the nonfasting state, and <0.1 mM in the diagnosis as soon as possible, and to get therapy children and adults. After a 24-h fast, levels of 2–3.5 mM started, even before the precise diagnosis is known are achieved. Infants and children with ketoacidosis (Chap. B2.8). It is important that testing of the urine may have levels over 7 mM. for ketones be incorporated into the work-up of a Massive ketosis and metabolic acidosis are the hall- severely ill infant. Until the recognition of the organic mark features of the organic acidurias. Those most acidurias, it was thought that ketonuria did not occur in frequently encountered are propionic aciduria, methyl- the neonatal period, and testing for ketones early in life malonic aciduria, multiple carboxylase defi ciency, isoval- is often neglected. Its presence can signify an underly- eric aciduria, and 3-oxothiolase defi ciency (Table B2.2.1). ing metabolic diagnosis. 3-Hydroxyisobutyric aciduria causes episodic ketoacidosis and may otherwise mimic lactic acidemia. 3-Hydroxyisobutyryl-CoA deacylase defi ciency, meth- acrylic aciduria, may also present with ketoacidosis and Remember 3-hydroxyisobutyric aciduria. The initial episode may begin with vomiting, anorexia, Testing for ketones in the urine is essential in any and lethargy, but progresses rapidly to life-threatening profoundly ill neonate. Massive ketosis is the hall- acidosis, dehydration, coma, and apnea. In the absence mark feature of the organic acidosis. of intubation and assisted ventilation, the infant dies.

Table B2.2.1 Metabolic diseases which may present with acute metabolic acidosis Disorder Detect or suspect Defi nitive diagnosis Propionic aciduria, Ketotic Massive ketosis, propionic acidemia, Methylcitraturia, Propionic acidemia, hyperglycinemia hyperglycinemia Propionyl-CoA carboxylase Methylmalonic aciduria Massive ketosis, Methylmalonic acid in blood and urine, methylmalonic acid in urine methylmalonyl-CoA mutase, Complementation analysis Isovaleric aciduria Acrid smell Isovalerylglycinuria, isovaleric acid in blood, hydroxyisovaleric aciduria Multiple carboxylase Hydroxyisovaleric aciduria, 3-methylcrotonylg- Biotinidase, holocarboxylase synthetase defi ciency lycinuria, methylcitrate Oxothiolase defi ciency Massive ketosis, tiglylglycinuria 3-Oxothiolase Methylcrotonyl-CoA 3-methylcrotonylglycinuria 3-Methylcrotonyl-CoA carboxylase carboxylase defi ciency Maple syrup urine disease Urinary 2,4-DNP Branched-chain amino acidemia, branched- chain keto acid dehydrogenase Lactic acidemia Growth retardation, ataxia, stroke, Defective fructose-1,6- diphosphatase or hyperalaninemia, lactic acid in pyruvate dehydrogenase, mitochondrial blood, CSF DNA, electron transport chain enzymes Lysosomal acid Lysosomal acid phosphatase phosphatase defi ciency Pyroglutamic aciduria Pyroglutamic acid in blood or urine Hawkinsinuria Iodoplatinate Cysteinyl-dihydrocyclohexyl acetic acid Ketolysis defects Ketosis Cytosolic or mitochondrial acetoacetyl-CoA thiolase, succinyl-CoA 3-oxoacid CoA transferase LCHAD defi ciency (Cardio)-Myopathy, hypoglycemia, lactic Acylcarnitine profi le, LCHAD acidemia SCHAD defi ciency Hypoglycemia Acylcarnitine profi le, SCHAD 2,4-DNP 2,4-dinitrophenylhydrazones; LCHAD long-chain hydroxyacyl-CoA dehydrogenase; SCHAD short-chain hydroxyacyl- CoA dehydrogenase B2 Metabolic Emergencies 33

A clinical clue to the diagnosis is acidosis with hematological fi ndings and moniliasis indicate a lack vomiting. Some infants with organic aciduria have of metabolic control. been thought to have pyloric stenosis and some have The defi nitive diagnosis of an organic aciduria been treated surgically; however, pyloric stenosis and is made by gas-chromatography mass-spectrometry its attendant vomiting lead to alkalosis. A patient who (GCMS). In this way, the presence of isovalerylglycine seems to have pyloric stenosis and has paradoxical aci- indicates the diagnosis is isovaleric acidemia; methyl- dosis has an organic aciduria. malonate, methylmalonic aciduria; methylcitrate, These infants are often thought to have sepsis and 3-hydroxypropionate and tiglylglycine, propionic aci- some even have positive blood cultures. Septic infants duria; tiglylglycine and 2-methyl-3-hydroxybutyrate, can certainly be acidotic, but they are not ketotic, at 3-oxothialase defi ciency; and 3-hydroxyisovalerate, least in the neonatal period. So an infant with real or 3-methylcrotonylglycine, 3-hydroxypropionate and apparent sepsis and massive ketonuria should be inves- methylcitrate, multiple carboxylase defi ciency. tigated and treated for an organic aciduria. Patients with these disorders go on to have recurrent episodes of acidosis, always heralded by ketonuria. Remember This is in response to the intake of the usual amounts of If you suspect organic aciduria, order GCMS, organic protein in patients prior to a specifi c diagnosis and the acid analysis of the urine and acylcarnitine profi ling. introduction of dietary therapies, and in response to infection in patients with an established diagnosis and receiving an appropriate therapeutic regimen. The results from the clinical chemistry laboratory Among the organic acidurias, 3-oxothiolase defi ciency indicate severe acidosis. The arterial pH may be 6.9– is the most likely to be cryptic. For reasons that are not 7.2. The serum concentration of bicarbonate is low, clear the key metabolites may not be found in the urine and may be as low as 5 mEq/L or less. The anion gap is at the time of the acute crisis, as they may be masked increased. The pH of the urine is <5.5. An acidosis somehow by the massive quantities of acetoacetate and with a high urinary pH signifi es a renal tubular acido- 3-hydroxybutyrate. On the other hand, when the patient sis, but these disorders are usually chronic problems, is well, the tiglylglycine and 2-methyl-3-hydroxybu- not acute metabolic emergencies and the anion gap is tyrate may be missing from the urine. In such a situation, not increased. Hyperchloremia and a normal anion gap an isoleucine loading test will reveal the characteristic in an acidotic infant indicate renal tubular acidosis or presence of these organic acid products of isoleucine. intestinal losses of electrolyte. In the acute crisis of the Quantifi cation may also be important in diagnosis. organic acidurias, levels of lactic acid in the blood are For instance, the presence of 3-hydroxyisovalerate, also elevated, and this may contribute to the acidosis. 3-hydroxypropionate, and methylcitrate may suggest a There may also be hypoglycemia (Chap. B2.4), diagnosis of multiple carboxylase defi ciency, but these hypocalcemia, and, at least in the neonatal period, compounds are also found in propionic aciduria, because hyperammonemia (Chap. B2.6), and each of these may 3-hydroxyisovalerate increases in any patient with keto- be symptomatic. Hyperammonemia leads to respira- sis. The two are readily distinguished by quantifi cation. tory alkalosis. An elevated ammonia in a patient with In multiple carboxylase defi ciency, the amounts of acidosis indicates that the diagnosis is an organic aci- 3-hydroxyisovalerate are large and those of the other duria. Routine clinical hematology may also indicate compounds small, while in propionic aciduria, the the presence of an organic aciduria, especially in a very reverse is found. The distinction is important, because young infant. These disorders lead regularly to neutro- to send a patient home with biotin and no restriction of penia, often to thrombocytopenia, and sometimes to protein intake, thinking that the diagnosis was multiple anemia. Pancytopenia and acidosis are seen in infants carboxylase, could be lethal in propionic aciduria. with sepsis; they are also seen in infants with organic Most of the organic acidurias may be detected by aciduria. If there are also large amounts of ketones in analysis of the blood or urine for acylcarnitines by tan- the urine, it is an organic aciduria. Chronic moniliasis dem mass spectrometry. Today, an elevated plasma may also indicate the presence of an organic aciduria. C3-carnitine is often the fi rst indication that the diagno- In a known patient with organic aciduria, abnormal sis is propionic acidemia or methylmalonic aciduria. 34 W. L. Nyhan

Once this biochemical genetic diagnosis is made, or hyperammonemia. It is thought that they have defi nitive molecular diagnosis may be undertaken at defective peripheral utilization of acetoacetate and the level of the enzyme or the gene. Enzyme analysis 3-hydroxybutyrate. The prototype condition is cytoso- can often be made by analysis of freshly isolated leu- lic acetoacetyl-CoA thiolase defi ciency. Actually, at kocytes, but for precision in diagnosis, especially at a least one patient with this disorder was reported to distant laboratory, it is usually preferable to establish a have moderate hypoglycemia; and others had elevated fi broblast culture, send a confl uent culture, and ensure concentrations of lactate and pyruvate and a normal enzymatic analysis of viable cells. lactate to pyruvate ratio. Other ketolytic defects include Most of the other disorders listed in Table B2.2.1, the mitochondrial acetoacetyl-CoA thiolase defi ciency while causing acidosis, do not usually present as a met- and succinyl-CoA:3-oxoacid CoA transferase defi - abolic emergency. Pyroglutamic aciduria may rarely ciency. This last enzyme catalyzes the conversion of present with severe acidosis without ketonuria, but acetoacetate to acetyl-CoA. Patients with this disorder more commonly the acidosis is mild or absent. Maple are ketotic in the fed condition. syrup urine disease (MSUD), on the other hand, presents as a metabolic emergency (Chap. B.2.1), but the acidosis is usually absent or minor. In MSUD, the DNPH test is very useful in detecting the presence of Key References the large quantities of the keto acid derivatives of the branched-chain amino acids. GCMS analysis of the Nyhan WL, Barshop BA, Ozand PA (2005) Atlas of metabolic organic acids of the urine will identify these keto acids disease, 2nd edn. Hodder Arnold, London, pp 3–56 Wendel U, Ogier de Baulny H (2006) Branched-chain organic and also their hydroxy acids. The specifi c pattern is acidurias/acidemias. In: Fernandes J, Saudubray JM, van den diagnostic of MSUD; as of course is the analysis of the Berghe G, Walter JH (eds) Inborn metabolic diseases, 4th amino acids of the plasma. edn. Springer, Berlin, pp 245–72 Ketoacidosis occurs, of course, in diabetes mellitus. This diagnosis is readily made clinically. A diabetic infant or child is not likely to be missed if routine clinical chemistry is employed. On the other hand, an occasional organic aciduria has been mistaken for diabetes when an isolated elevation of glucose occurred at the time of presentation in ketoacidosis. In these patients, there may be elevated ammonia or lactic acid in the blood, providing keys to the diagnosis. The hyperglycemia is also transient and responds exaggeratedly quickly to a small amount of insulin. Disorders of carbohydrate metabolism, especially von Gierke glucose-6-phosphatase defi - ciency, but also fructose-1,6-diphosphatase defi ciency and glycogen synthase defi ciency, can have impressive levels of ketones in the blood. However, these disorders seldom present with an acute metabolic acidotic emergency. They present rather with hypoglycemia (Chap. B2.4). Ketosis is seldom considered in the lactic acidoses (Chap. B2.3). However, we have repeatedly observed crises of ketoacidosis in patients with episodic illness in electron transport abnormalities, such as the NARP mutation. These episodes respond to the administration of parenteral glucose and water (Chap. B2.8). The disorders of ketolysis may present with a more or less pure ketoacidosis in which there is no hypoglycemia, hyperglycemia, organic aciduria, lactic acidemia, B2 Metabolic Emergencies 35

B2.3 Workup of the Patient with Lactic Glycogen

Acidemia – Mitochondrial Disease OPO3H2

CH2 = CCOOH William L. Nyhan

NH2 O CH -COOH CH3 CHCOOH 3 C O Key Facts OH COOOH CH CHCOOH › Inborn errors characterized by lactic acidemia 3 CH COOH fall into two categories: abnormalities in gluco- O 2 neogenesis and defects of oxidation. Distinction CH3 C-S-CoA Citrate is important because management and prognosis are different. Fig. B2.3. 1 Pathway of pyruvate metabolism with pyruvate in › As a fi rst step exclude factitious and secondary the center and lactate and alanine sinks to the left elevations of levels of lactic acid in order to focus on specifi cs of work-up. patients in whom a molecular diagnosis remains elu- › Ratios of lactate to pyruvate and 3-hydroxybu- sive. Defi nitive diagnosis documents defi ciencies in tyrate to acetoacetate are useful in elucidating activity of a growing group of enzymes and mutations the area of metabolic defect. in DNA, fi rst in mitochondrial and lately also in nuclear › Postprandial rise or fall in lactate gives impor- DNA (See also Chap. E1). tant information. The fi rst step in investigation of a patient with lactic › A monitored fast may be required to distin- acidemia is the documentation that the level of lactic guish oxidative defects from those of gluco- acid in the blood or cerebrospinal fl uid (CSF) is truly neogenesis. Molecular methods have decreased elevated. The most common situation in which the con- the necessity for this. centration of lactic acid in blood is elevated is factitious, the result of improper technique, the use of a tourniquet, or diffi culty in drawing the blood. It is also true that levels are variable; even in patients with known mitochondrial disease the concentration of lactic acid is not The lactic acidemias represent a family of disorders of always increased. Lactic and pyruvic acids are located pyruvate metabolism. Under these circumstances large distant from many of the enzymatic steps that are defec- elevations of pyruvate concentration might be expected, tive, especially those of the electron transport chain. but are seldom seen. Accumulating pyruvate does not For the evaluation of energy metabolism, lactate lead to large elevations of pyruvate concentration; but should be determined repeatedly throughout the day rather to conversion to its two sinks, lactate and ala- (especially before and after meals). It is useful to also nine (Fig. B2.3.1). determine levels of pyruvic acid and alanine in the Genetically determined causes of lactic acidemia blood, as well as lactic acid. Alanine is not falsely fall into two categories, defects in gluconeogenesis raised by problems of technique. It is important to be and defects in oxidation (Fig. B2.3.2). It is important rigorous about methods of sampling and to obtain in the work-up to distinguish clearly into which of the blood fl owing freely without a tourniquet. The con- two categories each patient falls. The distinction is centration of lactate and alanine in the CSF should useful in determining optimal therapy, even in those also be determined, particularly in those who appear to have mitochondrial disease with neurological symptoms despite normal levels of lactate in the W. L. Nyhan blood. Many have elevated concentrations of lactate Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, in the CSF, while the plasma level is normal, slightly, USA or intermittently elevated. Lactate to creatinine ratios e-mail: [email protected] in urine are less sensitive. Raised urinary lactate does, 36 W. L. Nyhan

ALGORITHMIC WORK UP OF PATIENT WITH LACTIC ACIDMIA Document elevated Lactic Acid and/or Pyruvic Acid and/or Alanine in Blood and/or CSF and Urine

Blood mt DNA Tandem MS

Organic Acid MELAS Analysis Diagnose or MERRF Exclude Kearns-Sayre Glucagon- Fast-Procedure Organic Acidemia Pearson Glucagon after 6 hours fasting depletes hepatic glycogen. Continued fasting for up to 24 hours Elevated [Lactate] Ratio requires gluconeognesis to avoid hypoglycemia. [Pyruvate] Second glucagon test at end of fast- flat in the absence of gluconeognesis Normoglycemia Acetoacetate 3-Hydroxybutyrate

Hypoglycemia [3-Hydroxybutyrate] Ratio Passes Fast Elevated Fails Fast [Acetoacetate] Gluconeo-genesis Defect Oxidation With Lactic Aciduria Defect

Serum Leukocyte Individual Loading Tests Muscle Biopsy Biotinidase Carboxylase with Fructose, Glycerol, Assess D or L-Lactic Acid, Skin Biopsy Galactose or Alanine Threonine Loading Fibroblast Culture Ragged Diagnosis Diagnosis Red Fibers No Ragged Biotinidase Pyruvate Intestinal Red Deficency Caboxylase Liver Biopsy Malabsorption Fibers Deficiency Syndromes with Assay PDHC, PDC, Mitochondrial Absorption of DNA Pyruvate Oxidation, Mitochondrial Assess Specific Enzymatic Diagnosis D-Lactic Acid KGDH,E 3 DNA respiratory Formed by e.g. Fructose-1, 6-diphos- chain complexes phatase Deficiency Intestinal MELAS Treat with Bacteria Initiate High fat MERRF NARP Mitochondrial Biotin or Ketogenic Diet Kearns-Sayre Myopathy, Leigh, Initiate high carbohydrate Pearson Infantile diet and avoidance of fasting Acidosis

Fig. B2.3.2 An approach to the stepwise evaluation of a patient Myoclonus epilepsy with ragged red fi bres; MS mass spectros- with lactic acidemia. E3 estriol; MELAS Mitochondrial encepha- copy; mt mitochondrial; NARP Neuropathy, ataxia, and retinitis lomyelopathy, lactic acidemia and stroke-like episodes; MERRF pigmentosa; PDHC pyruvate dehydrogenase complex however, support the signifi cance of questionable An uncommon cause of lactic acidosis is d-lactic increased lactate levels in blood. If urinary lactate is acidemia, resulting from absorption of d-lactic acid profound more consistently elevated than blood lactate, duced by intestinal bacteria (see also Chap. C3). Most predominant or even isolated disease of the kidney is such patients have obvious malabsorption or short-gut to be considered. At fi rst seemingly unrelated to the syndromes, metabolic acidosis, and massive lactic aci- underlying mitochondrial disorders, patients often duria, found by colorimetric test or by urinary organic show a constant thrombocytosis and hypertrichosis. acid analysis. Testing for lactate in routine clinical Before embarking on a specifi c investigation for lac- chemistry is now usually done in an enzymatic assay, tic acidemia it is important to exclude conditions that which is specifi c for l-lactate, so this situation is often cause secondary lactic acidemia. Patients with hypox- not even recognized. The discrepancy between urine emia, hypoventilation, shock, or hypoperfusion are and blood lactate levels, plus the history, is the key to generally readily recognized as patients with sepsis, diagnosis. A short course of treatment with oral neomy- cardiac, or pulmonary disease. Anaerobic exercise also cin or metronidazole will cause a dramatic fall in d-lac- produces lactic acidemia, but this is seldom clinically tate production, and the lactic acidemia will disappear. relevant except in the convulsing patient. A variety of inherited metabolic diseases produce secondary lactic Remember acidemia including propionic aciduria, methylmalonic Before embarking on a work-up for lactic acidemia aciduria, isovaleric aciduria, 3-hydroxy-3-methylglutaric exclude functions or secondary lactic acidemias, which aciduria, and pyroglutamic aciduria. Each of these con- are even more common, and even d-lactic acidemia. ditions can be excluded by organic acid analysis. B2 Metabolic Emergencies 37

B2.3.1 Workup of a Patient with Lactic storage diseases types 0, III, and VI/IX. In primary Acidemia (Congenital or Late- defects of the respiratory chain, the redox state may Onset) become more abnormal; in addition, and there may even be a rise of total ketone bodies (paradoxical ketonemia). A postprandial fall of lactate occurs in glycogen storage Once it has been decided that a patient has lactic aci- disease type I and defects of gluconeogenesis. demia the redox status and the response to a carbohy- The differentiation between problems in gluconeo- drate load may be evaluated fi rst. Lactate should be genesis or in oxidation (Fig. B2.3.2) can be achieved determined preprandially and postprandially together by evaluating the response to a prolonged fast. Fasting with pyruvate, 3-hydroxybutyrate and acetoacetate pref- studies are not appropriate in the diagnosis of a child erably from the same samples collected in tubes pre- with a defect in fatty acid oxidation. An intravenous fi lled with perchloric acid. Plasma glucose must be catheter is inserted to facilitate the drawing of samples. determined as well. Elevated ratios of the cytosolic Prior to the initiation of fasting, blood is obtained for (lactate:pyruvate >20) as well as of the mitochondrial glucose, lactate, pyruvate, and alanine. In this method, redox status (3-hydroxybutyrate:acetoacetate >3) point 0.5 mg of glucagon is given intramuscularly at 6 h in to a disturbance of oxidative phosphorylation. An ele- order to deplete the liver of glycogen made from glu- vated ratio of lactate:pyruvate without elevation of the cose, and the glucose response is determined at 15, 30, 3-hydroxybutyrate:acetoacetate ratio is indicative of 45, 60, and 90 min. The response to glucagon should severe pyruvate carboxylase defi ciency. In these patients, be a sizeable increase in glucose (>20%) except in gly- elevations of the amino acids citrulline and lysine may cogenosis type I. As the fast is continued for 18–24 h, also be found, as well as hyperammonemia. or until the development of hypoglycemia, the body is A postprandial rise of lactate (≥ twofold) occurs in dependent on gluconeogenesis for the maintenance of pyruvate dehydrogenase defi ciency and also in glycogen normal levels of glucose in the blood. Hypoglycemia

Table B2.3.1 Twenty-four hour fast for lactic acidemia Protocol and specimens Begin fast at time T = 0° at 4 p.m. The ﬁ rst 16 h are the least hazardous; so should happen overnight T(ime) = 0° (4 p.m.): end of last meal with documented intake T = 1° (5 p.m.): serum glucose, electrolytes, phosphate, uric acid, (transaminases, creatine kinase). Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate from perchloric acid tube. Plasma alanine. Plasma for acylcarnitines. Can be spotted on Guthrie card. Blood spot sample should be collected as backup T = 6° (10 p.m.): blood glucose T = 6° (10 p.m.): 1 mg glucagon is given i.m. or i.v. after ﬂ ushing the line with 5 mL 5% albumin T = 6°15′: blood glucose T = 6°30′: blood glucose T = 6°45′: blood glucose T = 6°90′: blood glucose T = 9° (1 a.m.): blood glucose If any glucose level from the 9° and after blood draws is >85 mg/dL (4.7 mM), collect glucose levels q3 h >65 but <85 mg/dL (3.6–4.7 mM), collect glucose levels q2 h >50 but <65 mg/dL (2.8–3.6 mM), collect glucose levels q1 h > 40 but <50 mg/dL (2.2–2.8 mM), collect glucose levels q1/2 h T = 15° (7 a.m.): serum glucose, electrolytes, phosphate, uric acid. Lactate, pyruvate, 3-hydroxybutyrate. Plasma alanine T = 24° (4 p.m.) or at the time of development of hypoglycemia: serum glucose, electrolytes, phosphate, uric acid, creatine kinase, (transaminases). Blood gases. Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate collected in perchloric acid tube. Plasma alanine, free fatty acids. Plasma acylcarnitines. Collect urine for quantitative analysis of organic acids If blood sugar is <40 mg/dL (2.3 mM) draw samples as above and in addition for insulin, growth hormone, and glucagon. Give 1 mg glucagon i.m. or i.v. and collect blood glucose at 15 min. If glucose rises, collect at 30 and 45 min followed by 3–5 mL 10% glucose/kg b.w./h. In the presence of any symptoms or if glucose does not rise, give 2 mL of 20% or 4 mL of 10% glucose/kg b.w. intravenously as a bolus, followed by 3–5 mL 10% glucose/kg b.w./h until normoglycemia is restored, and the patient is able to resume adequate oral intake 38 W. L. Nyhan

(blood glucose <45 mg/dL = 2.6 mM) at any time sig- will be suspected and effective treatment can be insti- nals the conclusion of the fast. If the patient is asymp- tuted on the basis of fasting and loading data. Information tomatic, glucagon is given again. During this time if as to the area of the defect may be obtained by loading there is no rise in glucose, the defect is in gluconeo- tests, with fructose (Chap. D8), alanine, or glycerol. genesis. Glucose is given intravenously to restore nor- Each compound is given by mouth as a 20% solution moglycemia without waiting for the usual interval of a 6–12 h postprandially in a dose of 1 g/kg. glucagon test, and in the presence of any symptoms is Most patients who pass the fasting test have defects given immediately without testing glucagon respon- in oxidation of pyruvate, refl ecting mitochondrial siveness. Concentrations of lactic and pyruvic acids, dysfunction (see also Chap. D6). The elucidation of alanine, acylcarnitines, free fatty acids, and ketone oxidation defects may be initiated by obtaining a skin bodies are determined at the end of the fast. In a hypo- biopsy for fi broblast culture. A diet high in fat and glycemic patient concentrations of insulin, growth low in carbohydrate, with vitamin supplementation hormone and glucagon are also obtained at the time the can be begun while waiting for suffi cient quantities fast is terminated. It must be remembered that lactate of cells for analysis. Fibroblasts may be assayed for will be raised in a struggling child, and as a result of a defects in the pyruvate dehydrogenase complex. convulsion, as might occur with severe hypoglycemia. Defects in the fi rst enzyme of the complex, pyruvate Errors in sample acquisition, technique, and sample decarboxylase or E1a, can also be tested for by muta- handling all raise the lactate level measured by the tional analysis. Measuring respiratory chain complex laboratory. activities, and identifying the cause of the dysfunc- Fed and fasted responses to glucagon can provide a tion, may require lengthy laboratory investigations. A discrimination between glycogenosis type I and gly- more immediate answer may be obtained by the anal- cogenosis type III. In suspected glycogen storage dis- ysis of blood for abnormalities in mitochondrial DNA eases, it is useful to test the response to glucagon in the (Chaps. D4 and D6). Among the newly recognised fed as well as in the fasted state. After a fast of 24-h or mitochondrial diseases of electronic transport are the fasting to mild hypoglycemia, if a fast of 24-h is too mitochondrial DNA depletion syndromes. Among long, administration of glucagon yields a fl at response the causes of mitochondrial/DNA depletion is muta- in glycogen storage diseases type I and type III, while tion in the gene for the mitochondrial DNA poly- blood glucose rises normally in glycogenosis type VI/ merase g. Other patients with this syndrome have had IX. On the other hand, when glucagon is given 2, 3, or defects in deoxyguanosine kinase and in thymidine 6 h after a meal, the blood glucose rises in type III gly- kinase. The syndrome is now known also to be the cogen storage disease, refl ecting glucose molecules on result of mutation in the gene (SUCLA2) for succinyl the outer branches released by phosphorylase. In type CoA ligase. I glycogen storage disease, there is minimal production of glucose in response to glucagon, whereas lactate will increase markedly. In the further work-up of a patient with a defect in gluconeogenesis who fails the fasting test it is conve- Key References nient to assay biotinidase in serum or blood spot, and carboxylase activity in leukocytes or fi broblasts; in this Munnich A (2006) Defects of the respiratory chain. In: way a defi nitive diagnosis of multiple carboxylase defi - Fernandes J, Saudubray JM, van den Berghe G, Walter JH ciency (due to holocarboxylase synthetase or biotini- (eds) Inborn metabolic diseases, 4th edn. Springer, Berlin, dase defi ciency) or pyruvate carboxylase defi ciency can pp 197–209 be made. Patients with disorders of gluconeogenesis in Nyhan WL, Barshop BA, Ozand PA (2005) Atlas of metabolic disease, 2nd edn. Hodder Arnold, London, pp 303–369 whom these are not the diagnoses, such as those with Smeitink J, van den Heuvel L, DiMauro S (2001) The genetics fructose-1,6-diphosphatase defi ciency, require liver and pathology of oxidative phosphorylation. Nat Rev Genet biopsy for defi nitive enzyme assay, but the diagnosis 2:342–352 B2 Metabolic Emergencies 39

B2.4 Work-Up of the Patient the blood. This permits the distinction of ketotic hypo- with Hypoglycemia glycemia, which includes the disorders of carbohydrate metabolism and the transient disorder termed ketotic hypoglycemia, from hypoketotic hypoglycemia, which, William L. Nyhan in the absence of hyperinsulinemia, includes most of the disorders of fatty acid oxidation. Key Facts Acute hypoglycemia is a manifestation of a variety › Timely determination of blood concentrations of different disorders. Its prompt recognition and rever- of insulin, growth hormone, and cortisol at the sal are critical because this absence of substrate for time of hypoglycemia can elucidate endocrino- cerebral metabolism can lead to permanent damage of logic causes of hypoglycemia. The endocrinolo- brain just as surely as can lack of oxygen. The acute gist may be the fi rst consultant to see the patient, episode can also be fatal. Its management requires the and if these determinations have not been made, provision of enough glucose to bring the blood concen- often orders a control LED fast. It is important to tration of glucose to normal and enough on a continuing educate these colleagues of the importance of basis to keep it there. Hypoglycemia is defi ned as a obtaining metabolic testing (Fig. B2.4.1) under serum concentration under 50 mg/dL (3 mmol/L) or a these circumstances. concentration in whole blood under 45 mg/dL. Low concentrations of glucose are so common in the neona- › Liver disease is a common cause of hypogly- tal period that neonatologists (used to) defi ne hypoglycemia even in pediatric patients. cemia as a concentration under 30–35 mg/dL and in › The important distinction of ketotic vs. hypoke- preterm infants <25 mg/dL. However, there is little evi- totic hypoglycemia is best made by determina- dence that the brain of the very young is any more toler- tion of free-fatty acids, acetoacetate, and ant of hypoglycemia, and we prefer to maintain 3-hydroxybutyrate at the time of hypoglycemia. concentrations of glucose >45 mg/dL in any age group. Hypoglycemia after a short fast is the hallmark › The classic symptoms of hypoglycemia are sweat- of a disorder of carbohydrate metabolism; ing, pallor, irritability, and tremulousness, but there may after a long fast, it signifi es a disorder of fatty be considerable variability even in the same individual, acid oxidation. and convulsions or coma may be the initial manifestation, particularly in the neonate. There may be vomiting, but this may be the result of an intercurrent illness that induces the acute hypoglycemic episode. Headache, Hypoglycemia must be recognized promptly and lethargy, altered behavior, or psychosis may be seen in treated effectively, if permanent damage to the brain is older children and adults, while apnea, tachypnea, to be prevented. Treatment means bringing the blood cyanosis, or hypothermia may occur in the newborn. glucose to a normal concentration and maintaining it Some individuals for whom very low levels of glucose there. Rational treatment demands a specifi c diagnosis are a chronic occurrence, for example, patients with von of the disease causing the hypoglycemia. Determination Gierke disease or glycogen synthase defi ciency tolerate of the blood concentrations of insulin, growth hor- surprisingly low levels without symptomatology. Also, mone, and cortisol at the time of hypoglycemia leads sudden drops in glucose levels are more apt to induce to the defi nition of the classic forms of hypoglycemia. symptoms than those achieved slowly. Liver disease must be excluded as a cause. The metabolic causes of hypoglycemia may be elucidated by the response to fasting and determination of the levels of B2.4.1 Algorithmic Approach free-fatty acids, acetoacetate, and 3-hydroxybutyrate in to Diagnosis

W. L. Nyhan Deﬁ nitive diagnosis is the elucidation of the cause of Department of Pediatrics, University of California, UCSD the hypoglycemia. This is an essential feature of the School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA design of therapy. It also permits prognostication of a e-mail: [email protected] transitory or a potentially recurrent nature. 40 W. L. Nyhan

Fig. B2.4.1 An algorithmic HYPOGLYCEMIA approach to the deﬁ nitive diagnosis of the cause of INITIAL LABORATORY EVALUATION OF THE HYPOGLYCEMIC PATIENT hypoglycemia Confimed Hypoglycemia Insulin, Growth Liver Function Tests Hormone, Cortisol Biliriubin Ammonia

Blood BOB, AcOAc Liver Disease (Urinary Ketones) Reye Syndrome Plasma Alanine Ketotic Hypoglycemia Hypoketotic Hypoglycemia

Hyperinsulinemia Insulin Normal Insulin Normal (Insulin > 10 μU, or Elevated, Glucose < 40 mg/ul) Inverted Nipples Cortisol Cortisol and Deficiency Growth Hormone Blood Ketones Diazoxide Normal Carbohydrate (BOB,AcOAc) Deficiency No Response Response Glycoprotein Growth Growth Elevated Low Diease Hormone Hormone Deficiency Normal Islet Cell Islet Cell Ketotic Hypoketotic Adenoma Hyperplasia Hypoglycemia Hypoglycemia Nesidioblastosis Adrenal Insufficiency Elevated Gycerol

Gycerol Congenital Kinase Deficiency, Adrenal Hyperplasia, Contiguous Multiple Gene Deletion Endocrinopathy

The diagnostic work-up is ideally initiated when babies. Ketotic hypoglycemia is a common transient the patient is seen at the time of the acute attack of disease with onset usually between 1 and 2 years of hypoglycemia at which time blood can be obtained age and disappearing by 6–8 years of age. Endocrine for insulin, growth hormone and cortisol to elucidate and metabolic causes may present fi rst of any age, the common endocrine causes of hypoglycemia, tests including the stressful fi rst days of life, but there is a of hepatic function to elucidate disease of the liver, a tendency to onset after 7 months of life when infants very common cause of hypoglycemia, as well as spe- sleep longer and are more likely to acquire infectious cialized tests, such as the blood concentrations of illnesses that lead to anorexia or vomiting and hence free-fatty acids, acetoacetate, and 3-hydroxybutyrate fasting. and plasma alanine to elucidate whether hypoglyce- Patients with disorders of carbohydrate metabo- mia is ketotic or hypoketotic (Fig. B2.4.1). More lism become hypoglycemic after short periods of fast- often, we are called upon to evaluate the patient after ing; 6–8 h leads to hypoglycemia in a patient with treatment of the acute attack when euglycemia has glycogenosis type I or glycogen synthase defi ciency. been restored. In this situation, a controlled moni- On the other hand, even an infant may have to fast tored fast may be required to reproduce the hypogly- 16–24 h before stores of glycogen are exhausted, and cemic state and initiate the work-up. fatty acid oxidation must be carried out to avoid hypo- The clinical history may guide the differential diag- glycemia. Hypoglycemia resulting from the ingestion nosis. The age of the patient may help in certain types of a toxin, such as salicylate or ethanol is usually evi- of hypoglycemia that are commonly encountered at dent from the history, but covert administration of different ages. Transient neonatal hypoglycemia is a insulin in the Munchausen - by - proxy - syndrome is condition of the fi rst days of life and is seen particu- more diffi cult to suspect. Hyperinsulinemia with a larly in preterm and small for gestational age (SGA) normal C-peptide gives this situation away. B2 Metabolic Emergencies 41

Most of either group with persistent hyperinsulinemia Remember require surgical removal of most of the pancreas. In Hypoglycemia after a short fast signifi es a disorder children, many with diffuse hyperinsulinism can be of carbohydrate metabolizing, and after a long fast, managed medically, while those with focal lesions a disorder of fatty acid oxidation. require surgery as do adults with true adenomas. Distinction between focal and diffuse lesions of the pancreas has been diffi cult short of surgery; the use of The physical examination is useful in leading to the [18F] fl uoro-l-dopa positron emission tomography diagnosis of specifi c syndromes, in which hypoglyce- (PET) appears to be useful in making that distinction. mia is common. Macrosomia in an infant with full Hypoglycemia is also seen in patients with a variety of rounded cheeks and a plethoric, edematous appearance tumors that secrete insulin-like material. An interesting is the alerting picture for the danger of hypoglycemia in cause of hyperinsulinemic hypoglycemia is known as the infant of the diabetic mother. These infants also have the Hyperinsulism-Hyperammonemia Syndrome and is a behavioral phenotype of drowsiness, hypotonia, a long caused by mutations in the gene (GLUD1) for gluta- latency for response and only brief periods of alertness. mate dehydrogenase (GDH). Mutations in this gene Macrosomia and hypoglycemia are also seen in the impair sensitivity to its allosteric inhibitor GTP, leading Beckwith–Wiedemann syndrome along with macroglos- to gain of function enzyme activity and increased sensi- sia, omphalocele and hepatomegaly. In both the syn- tivity to its activator leucine. Concentrations of ammo- dromes, hypoglycemia is transitory, lasting 1–3 days; nia in these patients, while clearly elevated are not very other syndromes associated with hyperinsulinism are high and patients have not had symptoms attributable to congential disorders of glycosylation (CDG) and hyperammonemia. Changes in protein intake do not Pearlman, Simpson–Golabi–Behmel, Sotos, and Usher change the hyperammonemia – some of these patients syndromes. A micropenis may be the only external clue have epilepsy without hypoglycemia. Persistent hyper- to the presence of panhypopituitarism. Some such insulinemic hypoglycemia of infants is also caused by infants have midline defects, such as clefts of the lip and mutation in the pancreatic b-cell sulfonylurea receptor palate. Hepatomegaly is characteristic of even very gene (SUR1) and in the inward rectifi er potassium young infants with glycogen storage disease. In the older channels gene (KIR6.2), as well as in the glucokinase child, it can sometimes be differentiated from the hepato- gene (GK). megaly of acute liver disease by its lack of tenderness. SUR1 and KIR6.2 are channelopathy genes. The initial laboratory evaluation of the hypoglyce- Channels are composed of four KIR6.2 subunits and mic patient (Fig. B2.4.1) rests on tests readily available four SUR1 subunits. Dominant mutations may lead to in the clinical laboratory. Hyperinsulinemia is present haploinsuffi ciency in which the channel may be unable when the insulin value is 10 U/mL or more in the pres- to stay open enough to maintain depolarization of the ence of a plasma glucose concentration of 40 mg/dL or membrane, or dominant negative mutation may lead to less. Another level of defi nition is >3 U/L of insulin, destruction of channel structure. Mutations in the same while glucose is <3 mmol/L (54 mg/dL) along with a gene may lead to diabetes or hyperinsulinism. Some positive response to injected glucagon (increase > patients with the same mutations have hyperinsulinism 2 mmol/L after 1.0 mg). Many of the infants labeled as in infancy and diabetes later. having idiopathic hypoglycemia, the infants of diabetic Patients with hypoglycemia and normal levels of mothers, and those with leucine sensitive hypoglyce- insulin may have defi ciency of cortisol with or without mia have hyperinsulinism. Persistent or recurrent growth hormone defi ciency and may be diagnosed hyperinsulinemic hypoglycemia results from hyperpla- as having one of the classic endocrine disorders sia of the b-cells of the pancreatic islets, referred to as (Fig. B2.4.1). nesidioblastosis, or less commonly in older children an Patients in whom endocrine evaluations are normal islet cell adenoma. In both the conditions, there is a may have ketotic or hypoketotic hypoglycemia (Chap. relative absence of ketosis and a substantial risk of B2.5). This distinction can sometimes be made on the damage to the brain. Some patients without adenomas basis of the presence or absence of ketonuria at the respond to diazoxide with a lessening of hypoglycemia. time of hypoglycemia. However, the presence of 42 W. L. Nyhan ketones in the urine may be misleading. Many patients ascertained. Two true inborn errors of metabolism that with documented disorders of fatty acid oxidation have present with this constellation are the defi ciency of been missed because of the presence of positive tests glycogen synthase (see later) or of succinyl-CoA:3- for ketones in the urine. Quantitative assays of acetoac- oxoacid CoA-transferase (SCOT). In the latter, there is etate and 3-hydroxybutyrate in the blood along with usually constant ketonuria even in the fed state. the levels of free-fatty acids clearly show that such a patient is hypoketotic at the time of hypoglycemia. B2.4.3 Disorders of Carbohydrate Metabolism B2.4.2 Ketotic Hypoglycemia Patients with glycogenosis type III, a consequence of Ketotic hypoglycemia is the name given to a very com- defi ciency of the debrancher enzyme, also have low mon disorder of late infancy and childhood which sel- blood concentrations of alanine consistent with their dom begins before 18 months and disappears by 4–9 very active gluconeogenesis. They are distinguished years of age. Actually there are a number of disorders on examination from those with ketotic hypoglycemia in which abundant amounts of ketones accompany because they have quite large livers. They do not hypoglycemia. Massive ketosis is the hallmark of the respond to glucagon after fasting, but they do respond organic acidurias (Chap. B2.2) in which hypoglycemia to the fed glucagon test. sometimes accompanies the acute attack of ketoacido- Patients with glycogenosis type I or von Gierke dis- sis. The other molecularly defi ned conditions in which ease, by contrast, have very high concentrations of ala- ketosis accompanies hypoglycemia are disorders of nine. Hypoglycemia occurs early in life and is carbohydrate metabolism, notably von Gierke disease recurrent. Concentrations of lactate are high, as are (see also Chap. B2.3). those of acetoacetate and 3-hydroxybutyrate, and keto- The syndrome known as ketotic hypoglycemia nuria is frequent. Hypercholesterolemia and hypertrig- presents classically with symptomatic hypoglycemia lyceridemia lead ultimately to cutaneous xanthomata. in the morning after a long fast, often precipitated by Hyperuricemia may lead to gouty arthritis or renal dis- an intercurrent illness that causes the child to miss din- ease. The liver is quite large, stature is short, and there ner. At the time of the hypoglycemia tests of the urine may be a cherubic facial appearance. In these patients, for ketones are positive, and concentrations of acetoac- the glucagon test is fl at under all conditions. The etate and 3-hydroxybutyrate in the blood are elevated. enzyme defi ciency is in glucose-6-phosphatase. Analysis of amino acids indicates the concentration of Patients with disorders of gluconeogenesis, such as alanine to be low. This is often a hallmark of a problem glycerol kinase defi ciency, pyruvate carboxylase defi - with gluconeogenesis. ciency, pyruvate carboxykinase defi ciency, or fructose Physical examination is often unremarkable, but 1,6-diphosphosphatase defi ciency, also tend to have the patient may be short and have diminished subcuta- high concentrations of alanine and lactate in the blood. neous fat, and there may be a history of low birth Defi nitive diagnosis is by enzyme assay in leukocytes weight. Glucagon administered at the time of hypo- or fi broblasts in the case of fructose-1,6-diphosphatase glycemia is followed by little or no increase in glucose defi ciency of biopsied liver. They may be elucidated concentration. by the occurrence of hypoglycemia during a moni- In patients seen after recovery from hypoglycemia, tored fast (see Sect. D8.5.2) especially if glucagon is the syndrome may be reproduced by fasting (see Sect. given after 6 h to deplete the liver of glycogen. The D8.2.1), but in some patients, this test may be nega- glycemic response to glucagon is normal at 6 h. tive. In these patients, a test of a ketogenic diet con- Loading tests with fructose, glycerol, or alanine may taining 67% of the calories as fat initiated after an clarify what enzyme to assay. Patients with glycerol overnight fast may reproduce the syndrome, and again kinase defi ciency may have adrenal insuffi ciency as there is no response to glucagon. Although in more part of a X-chromosomal contiguous gene deletion severe or familial cases a defi nitive inborn error of syndrome. Some may also have ornithine transcarbam- energy homeostasis is suspected this can rarely be ylase defi ciency and Duchene muscular dystrophy. B2 Metabolic Emergencies 43

B2.4.3.1 Glycogen Synthase Defi ciency B2.5 Approach to the Child Suspected of Having a Disorder of Fatty Acid Defi ciency of glycogen synthase is a rare, unique cause Oxidation of hypoglycemia in which there is a distinctive pattern of biochemical abnormality. Patients have fasting William L. Nyhan hypoglycemia, usually without acidosis but with high concentrations of acetoacetate and 3-hydroxybutyrate along with ketonuria. Thus, this is a ketotic hypoglycemia. Concentrations of alanine and lactate are low at Key Facts times of hypoglycemia, but feeding or a glucose toler- › Hypoketotic hypoglycemia signifi es a disorder ance test (see Sect. D8.3.2) leads to hyperglycemia and of fatty acid oxidation. An absence of ketones elevated concentrations of lactate in the blood. in urine at the time of hypoglycemia is an Glucagon during fasting has no effect on blood con- important clue; but the presence of ketonuria centrations of glucose, lactate, or alanine, while the fed may be misleading. Blood levels of free fatty glucagon test yields a glycemic response (see Sect. acids and 3-hydroxybutyrate may be required D8.5.1). Molecular diagnosis by enzyme assay requires to differentiate. biopsied liver, but mutational analysis can defi ne the › The acylcarnitine (MS/MS) profi le is a very defect in the gene. helpful clue to the diagnosis. › Medium-chain acyl-CoA dehydrogenase (MCAD) defi ciency resulting from the A985G Key References mutation is so common that this test should be added to the initial work up of a patient with hypoketotic hypoglycemia. Cornblath M, Schwartz R (1976) Disorders of carbohydrate metabolism in infancy. W.R. Saunders, Philadelphia De Lonlay P, Giuregea I, Sempoux C et al (2005) Dominantly inherited hyperinsulinaemic hypoglycaemia. J Inherit Metab Dis 28:267–276 Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic diseases, 2nd edn. Hodder Arnold, London, pp Initial presentation of disorders of fatty acid oxida- 241–245;303–311 tion is usually with hypoketotic hypoglycemia. Hyper- Orho M, Bosshard NW, Buist NRM et al (1998) Mutations in the ammonemia may suggest Reye syndrome. Sudden liver glycogen synthase gene in children with hypoglycemia due to glycogen storage disease type 0. J Clin Invest 102: infant death syndrome (SIDS) is another acute presen- 507–515 tation. Some patients present more chronically with myopathy or cardiomyopathy. Medium-chain acyl-CoA dehydrogenase (MCAD) defi ciency is the only common disorder, and most patients have the same mutation. So a modern work-up may begin with assay of the DNA for the A-985G mutation. Those not revealed in this way are now assayed by tandem mass spectrometry for the acylcarnitine profi le, and this may indicate the diagnosis and the appropriate enzymatic assay. Organic acid analysis should reveal the diagnosis in those with

W. L. Nyhan Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA e-mail: [email protected] B2 Metabolic Emergencies 47 acetyl-CoA. Whereas the latter usually constantly shows B2.6 Work-up of the Patient with a characteristic organic aciduria, the fi rst one is very dif- Hyperammonemia fi cult to spot as organic acid analysis is unspecifi c and acylcarnitines normal. In this constellation enzyme analysis of the liver or direct mutation analysis will provide William L. Nyhan the diagnosis. All of these patients would be expected to have abnormal ketogenesis following an LCT load. When these patients are tested with MCT, MCAD Key Facts patients display abnormal ketogenesis, while those with LCAD, LCHAD and VLCAD defi ciency have normal › Routine clinical chemistry is very helpful in ketogenesis. The follow-up of this testing is via assay pointing the direction of the work-up of a for the specifi c enzyme or enzymes as suggested in Fig. patient with hyperammonemia. Patients with B2.5.1. urea cycle defects are alkalotic or normal; The specifi c enzyme assays for specifi c disorders those with organic acidemias or disorders of are technically demanding and not generally available. fatty acid oxidation are acidotic. Presence or A reasonable step following the fast, if a specifi c dis- absence of ketosis distinguishes the latter two. ease is not identifi ed, is to pursue a more general study › Urea cycle defects are elucidated by analysis of metabolism in cultured cells in which oxidation of of the amino acids of the plasma (citrullinemia fatty acids of varying chain length is studied in vitro and argininemia) and urine (argininosuccinic and carnitine esters are separated and identifi ed after aciduria). incubation with 14C- or 13 C-labeled long-chain fatty › Urinary orotic aciduria distinguishes ornithine acids such as hexadecanoate. Impaired oxidation of transcarbamylase defi ciency from carbam- long-chain fatty acids such as palmitate in vitro may oylphosphate synthetase and N-acetylglutamate also be seen in patients with mitochondrial disorders synthetase defi ciencies. (see Chap. B2.3). Such patients may also have hypoketotic hypoglycemia with increased levels of 3-hydroxydicarboxylic acids because of failure to oxidise the NADH produced in the 3-hydroxyacyl-CoA dehydrogenase step via the electron transport chain. Such patients usually display lactic acidemia. Elevated concentrations of ammonia occur episodi- cally in a variety of inherited diseases of metabolism. These include not only the disorders of the urea cycle but also organic acidurias and disorders of fatty acid Key References oxidation. Effective management is predicated on a precise diagnosis and understanding of the nature of the pathophysiology. A systematic progression from Aledo R, Zschocke J, Pié J et al (2001) Genetic basis of mito- routine clinical chemistry to more specifi c analyses of chondrial HMG-CoA synthase defi ciency. Hum Genet 109:19–23 amino acids, organic acids, and acylcarnitines will lead Saudubray JM, Martin D, de Lonlay P et al (1999) Recognition the clinician to the diagnosis. Liver biopsy has been and management of fatty acid oxidation defects: a series of required for enzymatic diagnosis of carbamyl phos- 107 patients. J Inherit Metab Dis 22:488–502 phate synthetase defi ciency and ornithine transcarbam- Vreken P, van Lint AEM, Bootsma Ah et al (1999) Rapid diagnosis of organic acidemias and fatty acid oxidation defects ylase (OTC) defi ciency, as well as N-acetylglutamate by quantitative electrospray tandem-MS acyl-carnitine analysis in plasma. In: Quant PA, Eaton S (eds) Current views of fatty acid oxidation of ketogenesis. From organelles to point mutations. Kluwer Academic/Plenum Publishers, New York, pp 327–337 W. L. Nyhan Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA e-mail: [email protected] 48 W. L. Nyhan synthetase defi ciency. Mutational analysis may obvi- concentration of ammonia in the blood. The next step is ate this invasive approach. the quantifi cation of serum concentrations of bicarbon- Defi ciencies of enzymes of the urea cycle and some ate, sodium, chloride, and the anion gap and testing of other disorders, such as the organic acidemias and the the urine for ketones. Acidosis and/or an anion gap indi- disorders of fatty acid oxidation present with hyperam- cate against the disorders of the urea cycle, which tend monemia. Normally, values of NH3 are <110 mmol/L to present with respiratory alkalosis. The acidotic patient (190 mg/dL) in newborns and below 80 mmol/L (140 mg/ with massive ketosis has an organic aciduria, such as dL) in older infants to adults. In the newborn period, a propionic aciduria, methylmalonic aciduria, isovaleric diagnostic work-up for hyperammonemia is warranted aciduria, or multiple carboxylase defi ciency (see Chap. at values >150 mmol/L (260 mg/dL) and in older infants B2.2). A specifi c diagnosis is made by quantitative anal- to adults at values >100 mmol/L (175 mg/dL). The clas- ysis of the organic acids of the urine or of the acylcarni- sic onset of urea cycle defects is with sudden poten- tines from dried blood spots. The disorders of fatty acid tially lethal neonatal coma. The male with OTC oxidation, which may present with hyperammonemia, exemplifi es the classic presentation, but distinct disor- are characteristically hypoketotic (see Chap. B2.5). ders result from defi cient activity of each of the However, testing of the urine for ketones may be mis- enzymes of the urea cycle (Fig. B2.6.1). leading. We have observed impressively positive urinary The work-up of an infant in hyperammonemic coma ketostix tests in patients with disorders of fatty acid oxi- is shown (Fig. B2.6.2). The differential diagnosis is dation. Quantifi cation of concentrations of free fatty very important because different disorders require dif- acids together with acetoacetic and 3-hydroxybutyric ferent treatments. It must proceed with dispatch if the acid in the blood of such patients reliably indicate them correct diagnosis is to be made and appropriate therapy to have defective ketogenesis. The acute crises in these instituted to prevent death or permanent damage to the patients often display hypoglycemia, and diagnoses of brain be done. The initial steps in the algorithm are Reye syndrome have been made. We have reported an available in the routine clinical chemistry laboratory. acute hyperammonemic episode in a teenage girl with Defi nitive diagnosis requires the services of a bio- medium-chain acyl-CoA dehydrogenase defi ciency that chemical genetics laboratory. met all the criteria for a diagnosis of OTC defi ciency The fi rst step in the evaluation of a patient, especially including orotic aciduria; however, when biopsied liver an infant in coma, is the measurement of the was tested for OTC activity, it was normal. A patient

NH O=C 2 NH2 O Urea NH2 Carbamylphosphate OH NH3 + CO2 + 2ATP + H2O C NH2 (CH2)3 H N CH Aspartate 2 2 C=NH CHNH 2ADP+Pi 2 O=C HC-COOH NH o COOH Ornithine N Ornithine NH COPO H (CH2)3 2 3 2 H Carbamylphosphate CHNH2 Carbamyl COOH Aspartate Arginine UTP CTP NH2 H2O C=O O Fumarate Pi NH Orotidylate C NH COOH (CH ) 2 3 HN CH2 C NH CH CHNH2 O O=C HC-COOH NH CH 2 COOH C N Citrulline HN CH (CH2)3 COOH H Dihydroorotate CHNH2 O=C C-COOH COOH Citrulline N Argininousuccinate H Aspartate Orotate

CYTOSAL MITOCHONDRION CYTOSOL

Fig. B2.6.1 Urea cycle B2 Metabolic Emergencies 49

Fig. B2.6. 2 An approach HYPERAMMONEMIA to the stepwise evaluation − HCO3 of a patient with Na, K hyperammonemia Ketones

Acidosis No Acidosis Amino Acid Analysis Urine Ketones Blood 3-hydroxybutyrate Acetoacetate Prominent Hypoketotic Specific No Specific Amino Acid Amino Acid Elevation Organic Acidemia Disorder of Elevation Fatty Acid Oxidation Orotic Acid Organic Acid Esp. MCAD Analysis Argininosuccinic Analysis Deficiency Citrullinemia Aciduria Propionic Acidemia Argininemia Methylmalonic Acidemia Orotic Aciduria No Elevation Multiple Carboxylase Deficiency Urinary Orotic Acid Isovaleric Acidemia Omithine Glutaric Aciduria Type II Assess Plasma Transcarbamylase 3-Hydroxy-3-Methylglutaric Aciduria Citruline Deficiency Absent-Trace Normal or Increased

Carbamyl Phosphate Synthetase Deficiency Transient (N-Acetylglutamate Hyperammonemia Synthetase Deficiency) of the Newborn

with hyperammonemic coma resulting from a urea cycle is found in patients with OTC. It is also found in citrul- defect may develop hypoxia, leading to lactic acidosis. linemia and in argininemia. In a patient without an Adequate oxygenation and perfusion should be assured elevation of a specifi c amino acid and without orotic before a urea cycle defect is conceptually excluded and aciduria, the usual diagnosis is carbamylphosphate a diagnosis of organic acidemia pursued. synthetase (CPS) defi ciency. N-acetylglutamate synthetase defi ciency will present with an indistinguishable clinical and biochemical constellation, but is much Remember rarer. Transient hyperammonemia of the newborn may Hyperammonemia occurs not only in disorders of also present this picture, but for reasons that are not the urea cycle but also in organic acidemias and dis- clear this disorder is nowhere near as commonly orders of fatty acid oxidation. Testing for organic encountered as it was 20 years ago. Failure of immedi- acids in urine and acylcarnitine profi les lead to the ate closure of the ductus Avenosus after birth is thought correct diagnosis. to result in (transient) hyperammonemia of the newborn because portal blood bypasses the liver. The defi nitive diagnoses of CPS, OTC, and N-acetylglutamate The defi nitive diagnosis of a urea cycle abnormality is synthetase defi ciencies are made by assay of enzyme initiated by the quantitative assay of the concentrations activity in biopsied liver or determination of the muta- of amino acids in blood and urine. The plasma concen- tion. If transient hyperammonemia of the newborn is trations of amino acids provide the diagnosis in patients suspected, it is well to wait before performing liver with argininemia and citrullinemia. Study of the urine biopsy because the elevated ammonia in this disorder is required in argininosuccinic aciduria. could resolve within 5 days. Too, this gives time to If hyperammonemic patients are found not to have a bring the patient into control of the blood concentra- diagnostic abnormality in the concentration of an tion of ammonia. The level of citrulline in the blood amino acid, the urine should be tested for the excretion may be helpful in distinguishing CPS defi ciency from of orotic acid. This is not reliably performed as a part transient hyperammonemia of the newborn, in which it of organic acid analysis by GCMS and a specifi c assay is usually normal or elevated. In neonatal CPS, OTC or for the compound should be employed. Orotic aciduria N-acetylglutamate synthetase defi ciencies, citrulline is 50 W. L. Nyhan barely detectable. In citrullinemia, concentrations of investigated in detail even after the patient recovers, citrulline in plasma usually exceed 1,000 mmol/L. They even in adults or aged adults. In those instances, in are elevated to levels of 150–250 mmol/L in arginino- which the amino acids are normal, an allopurinol load- succinic aciduria, and to 54 ± 22 mmol/L in transient ing test may reveal the diagnostic direction. hyperammonemia in the newborn. The normal range is The differential diagnosis of hyperammonemia also 6–20 mmol/L. Persistent hypocitrullinemia can, in gen- includes the HHH syndrome, which results from defi - eral, be viewed as a marker for disorders of mitochon- ciency of the ornithine transporter in the mitochondrial drial urea cycle enzymes (N-acetylglutamate synthetase, membrane, and lysinuric protein intolerance. HHH sig- CPS I, and ornithine carbamoyltransferase) as well as nifi es hyperammonemia, hyperornithinemia, and homoc- for defi cient pyrroline-5-carboxylate synthetase. itrullinuria. It is usually suspected fi rst by the identifi cation Citrulline synthesis is directly coupled to ATP concen- of large amounts of homocitrulline in the urine. The tration. Consequently, hypocitrullinemia can also be diagnosis of lysinuric protein intolerance is best made by observed in patients with respiratory chain disorders, fi nding very low levels of lysine in the blood. These especially as caused by NARP mutation. patients fail to thrive, and when body stores of lysine are Amino acid analysis also reveals concentrations of much depleted, the characteristic amino aciduria may glutamine to be regularly elevated in patients with not be present; it returns when the diagnosis is made, and hyperammonemia. Concentrations of alanine are usu- blood amino acid concentrations are brought to normal. ally elevated, while concentrations of aspartic acid are The metabolic abnormalities become more obvious by elevated in some patients. These are nonspecifi c fi nd- calculating the fractional clearances of lysine and other ings. They are not helpful in the differentiation of the dibasic amino acids. The concentration of citrulline in different causes of hyperammonemia. They are poten- the blood may be high. Symptomatic hyperammonemia tially helpful in diagnosis, as sometimes an elevated may also fi nally result from a urinary tract infection in level of glutamine is found in a patient that had not been which the infecting Proteus mirabilis has urease activity, expected to have hyperammonemia, and while concen- which produces ammonia from urea. trations of ammonia may vary from hour to hour, the elevated concentration of glutamine signifi es a state in which there has been more chronic overabundance of ammonia. The transamination of pyruvic acid to alanine and oxaloacetic acid to aspartic acid, as well as Key References 2-oxoglutaric acid to glutamic acid and its subsequent amidation to glutamine are detoxifi cation responses to Ballard RA, Vinocur B, Reynolds JW Wennberg RP, Merritt A, the presence of excessive quantities of ammonia. Sweetman L, Nyhan WL (1978) Transient hyperammonemia of the preterm infant. N Engl J Med 299:920–925 Amino acid analysis may also reveal elevations of Batshaw ML, Bachmann C, Tuchmann M (eds) (1998) Advances tyrosine, phenylalanine, and the branched-chain amino in inherited urea cycle disorders. J Inher Metab Dis 1(21): acids. If these are substantial, a primary liver disease 1–159 should be carefully sought. Batshaw ML, Brusilow S, Waber L, et al Treatment of inborn errors of urea synthesis: activation of alternative pathways of Some patients with defects of urea cycle and resid- waste nitrogen synthesis and excretion. N Engl J Med ual enzyme activity, especially many females with OTC 1982:306:1387 defi ciency, display completely unremarkable values of Nyhan WL, Barshop BA, Ozand PA (2005) Ornithine transcar- NH , amino acids, and orotate in between the crises. It bamylase defi ciency. In: Atlas of metabolic diseases, 2nd 3 edn. Hodder Arnold, London, pp 199–205 is indispensable that the cause of an unexplained symp- Thoene JG (1999) Treatment of urea cycle disorders. J Pediatr tomatic episode of hyperammonemia should always be 134:255–256 B2 Metabolic Emergencies 51

B2.7 Work-Up of the Patient urea cycle defects and MSUD do not directly interfere with Acute Neurological with mitochondrial energy production, and the typi- or Psychiatric Manifestations cal signs of acute mitochondrial decompensation such as lactic acidosis may be lacking. They may develop later as the condition of the patient deteriorates. Acute Georg F. Hoffmann hemiplegia may be a presenting symptom; it has also been reported in patients with organic acidurias, in Acute or recurrent attacks of neurological or psychiat- particular propionic aciduria and methylmalonic aci- ric features such as coma, ataxia, or abnormal behav- duria as well as phosphoglycerate kinase defi ciency ior are major presenting features especially of several and mitochondriopthies. late-onset, inborn errors of metabolism. The initial An acute onset of extrapyramidal signs during the diagnostic approach to these disorders is based on a few metabolic screening tests. It is important that the Remember biologic fl uids are collected during the acute attack, Acute or recurrent attacks of neurological or psychi- and at the same time it is also useful to take specimens atric symptoms such as coma, ataxia, or abnormal both before and after treatment. Some of the most sig- behavior are major presenting features of several nifi cant metabolic manifestations, such as acidosis and late-onset, inborn errors of metabolism. The initial ketosis, may be moderate or transient, dependant on diagnostic approach to these disorders is based on a symptomatic treatment. On the other hand, at an few metabolic screening tests, in particular ammo- advanced state of organ dysfunction, many laboratory nia, lactate, amino acids, and organic acids. It is abnormalities such as metabolic acidosis, hyperlactic important that the biologic fl uids are collected acidemia, hyperammonemia, and signs of liver failure simultaneously at the time of the acute attack. Flow may be secondary consequences of hemodynamic charts for the differential diagnosis and diagnostic shock and multisystem failure. Flow charts for the dif- approach to acute neurological manifestations such ferential diagnosis and diagnostic approach to acute as coma, ataxia, and psychiatric aberrations are pre- neurological manifestations such as ataxia and psychi- sented in Figs. B2.7.1–B2.7.3. atric aberrations are presented in Figs. B2.7.1–B2.7.3. Acute attacks of neurological or psychiatric manifestations, such as coma, intractable seizures, stroke, course of a nonspecifi c intercurrent illness, minor sur- ataxia, or abnormal behavior, resulting from inherited gery, accident, or even immunization may initially be metabolic diseases require prompt and appropriate misinterpreted as encephalitis, but represent a conspic- diagnostic and therapeutic measures (see also Chap. uous feature of several metabolic disorders. In glutaric C5). Acute metabolic cerebral edema must be espe- aciduria type I (GA1), a dystonic dyskinetic movement cially quickly recognized and treated in order to prevent disorder is caused by the acute destruction of the basal herniation and death. All too often, only a diagnosis ganglia, specifi cally the striatum. The acute encephalo- of encephalitis is initially pursued. Cerebral edema is pathic crisis in GA1 typically occurs between the ages observed particularly in the acute hyperammonemias 6 and 18 months; affected children may have had mild (most frequently ornithine transcarbamylase (OTC) neurological abnormalities prior to the acute episode defi ciency) and maple syrup urine disease (MSUD). and frequently are macrocephalic. Almost half of the In contrast to respiratory chain defects or organic aci- patients with Wilson disease present with neurological durias that involve the mitochondrial metabolism of symptoms, usually after the age of 6 years. Dysarthria, CoA-activated compounds, the metabolic blocks in incoordination of voluntary movements, and tremor are the most common signs. There may be involuntary choreiform movements, and gait may be affected. Acute hemiplegia may be a presenting symptom; it has been reported in patients with organic acidurias, in G. F. Hoffmann University Children’s Hospital, Ruprecht-Karls-University, particular propionic aciduria and methylmalonic aci- Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany duria, as well as phosphoglycerate kinase defi ciency. e-mail: [email protected] Some patients with propionic acidemia present a basal 52 G. F. Hoffmann

Differential Diagnosis of Metabolic Coma

Metabolic Coma

Cerebral Edema Hemiplegia Extrapyramidal Stroke-like Thromboembolic (Hemianopsia) Signs Episodes Incidences, Abnormal Coagulation, Hemolytic Anemia

MSUD, MSUD, MMA, Urea Cycle AT III / Protein C / MCAD, OTC Deficiency, GA I, Disorders, Protein S Deficiencies, Urea Cycle MMA, Wilson Disease, MMA, PA, IVA, Homocystinuria Disorders PA, Classical Respiratory Chain (Classical and (OTC, ASS, ASL) Phosphoglycerate Homocystinuria Defects (MELAS), Variants), Kinase Deficiency Homocystinurias, Sickle Cell Anemia CDG-Syndromes, Fabry Disease

Fig. B2.7.1 Differential diagnosis of metabolic coma. ASS medium-chain acyl-CoA dehydrogenase; MELAS (mitochon- argininosuccinate synthetase defi ciency, citrullinemia; ASL drial encephalopathy, lactic acidosis and stroke-like episodes); argininosuccinate lyase defi ciency, argininosuccinic aciduria; MMA methylmalonic aciduria; MSUD maple syrup urine dis- AT antithrombin; CDG congenital disorders of glycasylation; ease; OTC ornithine transcarbamylase defi ciency; PA propionic GAI glutaric aciduria type I; IVA isovaleric aciduria; MCAD aciduria

Differential Diagnosis of Metabolic Causes of Acute Ataxia ? Coma

Acute Ataxia/Coma

Ketosis/ Acidosis Hyperammonemia Hyperlactacidemia No Metabolic Disturbance Fig. B2.7.2 Differential diagnosis of metabolic causes of acute ataxia (coma). ASS Special Neutropenia, Resp. Alkalosis, L/P-Ratio L/P-Ratio High, Dermatosis, argininosuccinate synthatase Odor Thrombocytopenia, Hepatomegaly Normal, Ketosis, Pellagra, deﬁ ciency, citrullinemia; IVA Hyperglycinemia Ketosis, Thrombocytosis, Sun Intolerance isovaleric aciduria; L/P Peripheral Cutaneous Signs lactate pyruvate ratio; MMA Neuropathy methylmalonic aciduria; MSUD maple syrup urine disease; OTC ornithine Respiratory transcarbamylase deﬁ ciency; MSUD MMA Urea Cycle PDHC Chain Disorders; Hartnup PA propionic aciduria; PDHC (Including Late PA Disorders Multiple Disease pyruvate dehydrogenase Onset) IVA (OTC, ASS) Carboxylase complex Deficiency ganglia picture indistinguishable from those of GAI or episodes may present initially with vomiting, head- Lesch–Nyhan disease. ache, convulsions, or visual abnormalities and may Acute stroke-like episodes and strokes occur in sev- be followed by hemiplegia or hemianopia. The mor- eral metabolic disorders (Table B2.7.1). They are the phological correlates are true cerebral infarctions, but hallmark feature of the mitochondrial encephalopathy, there is no evidence of vascular obstruction or athero- lactic acidosis, and stroke-like episodes, (MELAS) sclerosis and they do not correspond to vascular terri- syndrome, a disorder caused by mutations in the tories. Stroke-like episodes (metabolic stroke) are also tRNALeu(UUR) gene in the mitochondrial DNA. In total, observed in other metabolic disorders such as the con- 80% of patients carry the mutation 3243A > G. Acute genital disorders of glycosylation, and methylmalonic B2 Metabolic Emergencies 53

Fig. B2.7.3 Differential Differential Diagnosis of Metabolic Causes of diagnosis of metabolic causes Acute Psychiatric Manifestations ? Coma of acute psychiatric manifestations (coma). ASS argininosuccinate synthatase Psychiatric Manifestations/ Coma defi ciency, citrullinemia; ASL argininosuccinate lyase defi ciency, argininosuccinic aciduria; LPI lysinuric Portwine Urine protein intolerance; MSUD Hyperammonemia Ketoacidosis Positive Brand Reaction Mild Liver Dysfunction Ataxia Abdominal Pain Stroke-like Episodes maple syrup urine disease; Vomiting Neutropenia Neuropathy Seizures OTC ornithine transcarbamy- Failure to Thrive Vomiting Myelopathy lase defi ciency

Urea Cycle Defects Organic Acid Disorders, Acute Intermittent Methylene- (OTC, ASS, ASL, MSUD Porphyria, tetrahydrofolate Arginase Deficiency, Hereditary Reductase Deficiency LPI) Coproporphyria

Table B2.7.1 Metabolic causes of stroke and stroke-like fi bers are the cause of ischemic cerebral infarctions in episodes Menke disease. True thromboembolic events may be Homocystinuria caused by defects in the anticoagulant systems, such as Mitochondrial encephalomyopathy antithrombin III, protein C, or protein S defi ciencies. Pyruvate dehydrogenase defi ciency Acute ataxia (Fig. B2.7.2) or psychiatric manifesta- Pyruvate carboxylase defi ciency Respiratory chain disorders tions (Fig. B2.7.3) can be the leading signs of several Leigh disease organic acidurias and late-onset MSUD. In these Urea cycle disorders instances, the metabolic derangement is most often Carbamoylphosphate synthetase defi ciency associated with ketoacidosis (see also Chap. B2.2). A Ornithine carbamoyltransferase defi ciency rather confusing fi nding in some patients with organic Arginosuccinate synthase defi ciency acidurias is the presence of ketoacidosis in combina- Arginosuccinate lyase defi ciency tion with hyperglycemia and glycosuria mimicking Arginase defi ciency Congenital disorders of glycosylation diabetic ketoacidotic coma. In late-onset MSUD, a Fabry disease special odor may be noticed. In organic acidurias such Menkes disease as methylmalonic aciduria, propionic aciduria, or Sulfi te oxidase defi ciency isovaleric aciduria, moderate to severe hematological Purine nucleotide phosphorylase defi cency manifestations are common, and have led astute Vanishing white matter disease observers to suspect an organic acidemia. These disorders are usually characterized by neutropenia and, especially in infancy, thrombocytopenia. Recurrent and propionic acidurias. Strokes may of course be absent infections, particularly mucocutaneous candidiasis, in affected members of families with the MELAS syn- may be a common fi nding. The specifi c diagnosis may drome; some have migraine-like headaches as the only be obtained through the analysis of amino acids in manifestation of the disease, while others manifest only plasma and organic acids in urine. diabetes or are asymptomatic. Classic cerebral strokes Urea cycle disorders, such as OTC defi ciency, as well as cardiovascular accidents may also be caused argininosuccinic aciduria, citrullinemia, arginase defi - by various metabolic disorders that cause vascular dis- ciency, or lysinuric protein intolerance may present in ease such as classical homocystinuria, the thiamine- childhood or adolescence with acute or recurrent epi- responsive megaloblastic anemia syndrome, and Fabry sodes of hyperammonemia (Chap. B2.6). Clinical fea- disease. Vascular changes resulting from altered elastic tures may include acute ataxia or psychiatric symptoms 54 G. F. Hoffmann such as hallucinations, delirium, dizziness, aggressive- contrast to the thrombocytopenia associated with ness, anxiety, or schizophrenia-like behavior. In addi- organic acidurias. tion there may be hepatomegaly, liver dysfunction, and Some inborn errors of metabolism, like Hartnup failure to thrive. The correct diagnosis will be missed disease, may present with clinical symptoms of recur- unless ammonia levels are determined in plasma at rent acute ataxia without causing general metabolic the time of acute symptoms. Hyperammonemia may disturbances. Typical additional symptoms like skin be moderate or mild (150–250 mmol/L) even when the rashes, pellagra, and sun intolerance may lead to anal- child is in deep coma, and especially the late-onset ysis of the urinary amino acid which provides the spe- forms of urea cycle disorder such as OTC defi ciency cifi c diagnosis. The characteristic pattern is of an can be easily overlooked and misdiagnosed as schizo- excess of neutral monoamino-monocarboxylic acids in phrenia, encephalitis, or even intoxication. Diagnostic urine with (low) normal concentrations in plasma. procedures should include analyses of plasma amino Acute intermittent porphyria and hereditary copropor- acids, urinary organic acids, and orotic acid. phyria usually present with recurrent attacks of vomit- Mitochondrial diseases manifest in the CNS pri- ing, abdominal pain, unspecifi c neuropathy, and marily in high energy consuming structures such as psychiatric symptoms. These disorders have to be basal ganglia, capillary endothelium, and the cere- excluded in the differential diagnosis of suspected psy- bellum. Whereas disturbed function of the cerebel- chogenic complaints and hysteria. Diagnosis can be lum and the basal ganglia leads to characteristic made by specifi c analyses of porphyrins. Patients disorders of movement, disturbed capillary function affected with disorders of the cellular methylation path- results in fl uctuating neurological signs from stroke- way such as methylenetetrahydrofolate reductase defi - like episodes to nonspecifi c mental deterioration and ciency may also present with psychiatric symptoms. progressively accumulating damage of both grey These often resemble acute schizophrenic episodes, but and white matter. Acute ataxia in association with they may respond to folate therapy. Homocysteine is peripheral neuropathy is frequently found in patients elevated in plasma, and a positive Brand reaction in the with pyruvate dehydrogenase defi ciency. Moderate urine may be the fi rst abnormal laboratory fi nding. Other or substantial elevation of lactate with a normal lac- neurological features include stroke-like episodes, sei- tate/pyruvate-ratio and absence of ketosis supports zures and myelopathy. Diagnosis is made by analysis of this diagnosis. Acute ataxia associated with a high amino acids and homocysteine in plasma and CSF. lactate/pyruvate-ratio is suggestive of multiple Methyltetrahydrofolate in CSF is greatly reduced. carboxylase defi ciency or respiratory chain defects. In the latter, thrombocytosis is often observed in B2 Metabolic Emergencies 55

B2.8 Emergency Treatment of Inherited • Disorders in which there is reduced fasting Metabolic Diseases tolerance. • Disorders in which there is disturbed energy metabolism. Georg F. Hoffmann and William L. Nyhan A preliminary differentiation of these three groups should be possible with the help of basic investigations, available in every hospital setting, namely the Key Facts determination of acid–base balance, glucose, lactate, › Timely and correct intervention during the ini- ammonia, and ketones, as discussed in previous chap- tial presentation of metabolic imbalance and ters. With this information, appropriate therapy can be during later episodes precipitated by dietary initiated even before a precise diagnosis is known. In indiscretion or intercurrent illnesses is the instances, especially during the initial manifestation of most important determinant of outcome in a metabolic disease, i.e., when the exact diagnosis is inherited metabolic diseases at risk for acute not yet known, measures must be quick, precise and metabolic decompensation. not halfhearted, and every effort must be undertaken to › Patients should be supplied with an emergency obtain the relevant diagnostic information within 24 h letter, card, or bracelet containing instructions or sooner, i.e., results of acylcarnitines in dried blood for emergency measures and phone numbers. spots or plasma, amino acids in plasma, and organic › Logistics of therapeutic measures should be acids in urine. repeatedly evaluated by the specialist team with In all instances, provision of ample quantities of ﬂ uid the family and the primary care physician(s). and electrolytes is indispensible. Differences in the therapeutic approach relate primarily to energy requirements and methods of detoxiﬁ cation (Table B2.8.1).

Remember The most critical challenge in many inherited metabolic Emergency treatment must start without delay. diseases is the timely and correct intervention during acute metabolic decompensation in the neonatal period or in later recurrent episodes. Fortunately, there is only Table B2.8.1 Emergency treatment: energy needs in Infants a limited repertoire of pathophysiological sequences in Disorders requiring anabolism (acute intoxication): the response of infant to illness, and consequently, a 60–100 kcal/kg/day Organic acidurias, maple syrup urine disease, urea cycle limited number of therapeutic measures are to be taken. disorders⇒ glucose 15–20 g/kg/day + fat 2 g/kg/day Three major groups of disorders at risk for acute meta- always: + insulin, starting with 0.05 U/kg/h, Adjustments are bolic decompensation that require speciﬁ c therapeutic made dependent on blood glucose (useful combination is approaches in emergency situations can be delineated 1 U/8g glucose) (see Fig. B1.1). early: central venous catheterization Disorders requiring glucose stabilization (reduced fasting • Disorders of intermediary metabolism that cause tolerance): acute intoxication through the accumulation of Fatty acid oxidation defects, glycogen storage disease type I, disorders of gluconeogenesis, galactosemia, fructose toxic molecules. intolerance, tyrosinemia I ⇒ glucose 7–10 mg/kg/ min» 10 g/kg/day Disorders requiring restriction of energy turn-over (disturbed energy metabolism): PDHC deﬁ ciency⇒ reduce glucose supply: 2–3 mg/kg/min» 3 g/kg/day G. F. Hoffmann () Add fat: 2–3 g/kg/day University Children’s Hospital, Ruprecht-Karls-University, ⇒ Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany Electron transport chain (oxphos) disorders glucose e-mail: [email protected] 10–15 g/kg/day 56 G. F. Hoffmann and W. L. Nyhan

B2.8.1 Acute Intoxication (Fig. B1.1) In the hospital, therapy must be continued without interruption. In most instances, an intravenous line is essential, but nasogastric administration, for instance, of In diseases, in which symptoms develop because of amino acid mixtures in maple syrup urine disease is “acute intoxication,” rapid reduction of toxic molecules very useful. The management of many of these infants is a cornerstone of treatment. In disorders of amino acid is greatly simplifi ed by the early placement of a gastros- catabolism, such as maple syrup urine disease, the classi- tomy. This can usually be discontinued after infancy. cal organic acidurias, or the urea cycle defects, the toxic compounds may be derived from exogenous as well as endogenous sources. In addition, to stopping the intake Remember of natural protein until the crisis is over but no longer Calculations for maltodextran/dextrose, fl uid and than (12 to) 24 (to 48) h, reversal of catabolism and the protein intake should be based on the expected and promotion of anabolism and consequently reversal of the not on the actual weight! breakdown of endogenous protein is a major goal. In patients, known to have a disorder of amino acid Large amounts of energy are needed to achieve anabo- catabolism, who develop an intercurrent illness and lism, e.g., in neonates >100 kcal/kg b.w./day. In a sick manifestations of metabolic imbalance, such as keto- baby, this can usually be accomplished only by hyper- nuria in a patient with an organic aciduria, primary osmolar infusions of glucose together with fat through emergency measures at home for 24–48 h consist of a central venous line. Insulin should be started early, frequent feedings with a high carbohydrate content especially in the presence of signifi cant ketosis or in and some salt (Table B2.8.2), and reduction, even to maple syrup urine disease, to enhance anabolism. One zero, of the intake of natural protein. In diseases such approach is to use a fi xed ratio of insulin to glucose as maple syrup urine disease, the individual amino (Table B2.8.1). The administration of lipids intrave- acid mixture devoid of the amino acids of the defective nously can often be increased to 3 g/kg, if serum levels pathway is continued. Detoxifying medication such as of triglycerides are monitored. carnitine in the organic acidurias, benzoate, phenylac- Acute episodes of massive ketoacidosis, as seen in etate, and arginine or citrulline in the hyperammone- methylmalonic or propionic aciduria, require espe- mias is employed. During this time period, patients cially vigorous supportive therapy. Large amounts of should be reassessed regularly regarding state of con- water, electrolytes, bicarbonate, and high doses of sciousness, fever, and food tolerance. If the intercur- intravenous carnitine must be infused together with rent illness continues into the third day, symptomatology glucose. Blood concentrations of electrolytes and worsens, or if vomiting compromises external feed- bicarbonate are determined and an intravenous infu- ings, admission to hospital is mandatory. sion started before taking time for history and physical examination. Following a bolus of 20 mL/kg of ringer Table B2.8.2 Oral administration of fl uid and energy for lactate or normal saline, intravenous fl uid should episodic acute intercurrent illness in patients with metabolic contain 75–150 mEq/L of isotonic NaHCO (75 with disorders 3 massive ketosis, 150, if coma or HCO <10 mEq/L). Age Glucose polymer/maltodextrin solution 3 (years) Carnitine supplementation should be given at 300 mg/ (%) (kcal/100 mL) Daily amount kg intravenously. Electrolytes and acid–base balance are checked q6h. Serum sodium should be ≥138 mmol/L. 0–1 10 40 150–200 mL/kg The NaHCO content of the infusion is reduced, after 1–2 15 60 95 mL/kg 3 2–6 20 80 1,200–1,500 mL the serum HCO3 has become normal, but the same rate 6–10 20 80 1,500–2,000 mL of infusion is continued until ketonuria has subsided. >10 25 100 2,000 mL Some children with these disorders benefi t from Adapted from Dixon and Leonard (1992). Oral substitution implantation of a venous port to facilitate blood sam- should only be given during minor intercurrent illnesses and for pling and intravenous infusions. a limited time of 2–3 days. Intake of some salt should also be provided. The actual amounts given have to be individually Forced diuresis with large amounts of fl uids and adjusted, e.g., according to a reduced body weight in an older furosemide is especially useful in the methylmalonic patient with failure to thrive acidurias in removing methylmalonate from the body. B2 Metabolic Emergencies 57

Oxo-acid   Glutamate NH + 4 Phenylbutyrate CoA

  2-Oxo-glutarate Benzoate CoA Glycine Amino acid

 Glutamine Phenylbutyryl-CoA

Benzoyl-CoA Phenylacetyl-CoA

 Hippuric acid  Phenylacetyl-glutamine

Fig. B2.8.1 Pharmacological detoxiﬁ cation of ammonia. The glutamine yields phenylacetylglutamine and these are the end apple symbols represent nitrogens. Conjugation of glycine with products excreted. One nitrogen is excreted with each molecule benzoate yields hippurate; conjugation of phenylacetate with of hippuric acid and two with phenylacetylglutamine

Detoxifi cation in the organic acidurias depends of organic acidurias that present with hyperammonemia is the ability to form esters such as propionylcarnitine, the provision of alternative methods of waste nitrogen which are preferentially excreted in the urine, thus excretion (Fig. B2.8.1). Benzoate is effectively conju- removing toxic intermediates. In these disorders, tis- gated with glycine to form hippurate, which is then sue stores of carnitine are depleted. Carnitine is given excreted in the urine. Similarly, phenylacetate is conju- to restore tissue supply, but its major utility is to pro- gated with glutamine to form phenylacetylglutamine, mote detoxifi cation by the formation and excretion of which is effi ciently excreted. Administered orally, phe- carnitine esters. It is given intravenously in doses of nylbutyrate is converted to phenylacetate. In a metabolic 200–300 mg/kg. Carnitine is less well tolerated enter- emergency with an as yet unknown diagnosis and a doc- ally. Oral doses of >100 mg/kg are employed, but the umented hyperammonemia ≥200 mmol/L (350 mg/dL) dose may have to be reduced in some patients. and in relapses of known patients benzoate and pheny- In isovaleric aciduria it is useful to add glycine to lacetate should be given intravenously (Table B2.8.3), the regimen to promote the excretion of isovalerylgly- along with arginine. Especially during combined intra- cine (500 mg/kg). venous supplementation of benzoate and phenylacetate In maple syrup urine disease, the major element of electrolytes must be checked regularly to avoid hyper- therapy is to harness the forces of anabolism to lay natremia. A dose of 400 mg sodium benzoate corre- down accumulated leucine and other branched-chain sponds to 2.77 mmol and therefore to 2.77 mmol of amino acids into protein. This is done by the provision sodium. In some situations, enteral phenylbutyrate or of mixtures of amino acids lacking leucine, isoleucine benzoate along with arginine may be adequate. In urea and valine. The use of intravenous mixtures is very effi - cycle defects, carnitine administration is also recom- cient, and essential in a patient with intractable vomit- mended (50–100 mg/kg). Zofran (0.15 mg/kg i.v.) ing, but these are expensive and not generally available. appears to be especially effective against hyperemesis, Enteral mixtures are mixed in minimal volume and which can accompany hyperammonemia. dripped over 24 h in doses of 2 g/kg of amino acids. Hyperammonemias may require extracorporal dial- Often even a vomiting patient will tolerate a slow drip. ysis for detoxifi cation. This should be considered at Since concentrations of isoleucine are much lower than ammonia levels > 600 mmol/L (1000 mg/dL). Hemo- those of leucine, concentrations of amino acids must be dialysis has been repeatedly shown to be more effective measured at least daily and when the concentrations of than exchange transfusions, peritoneal dialysis, or arte- isoleucine become low isoleucine is added to the enteral riovenous hemofi ltration. This is an argument for the mixture. In many patients valine must also be added transport of such an infant, if the necessary logistics before the leucine concentration is lowered adequately. cannot be promptly organized locally. The pharmacological approach to the detoxifi cation In aminoacidopathies, other than maple syrup urine of ammonia in urea cycle defects, and also in those disease, such as phenylketonuria, the homocystinurias 58 G. F. Hoffmann and W. L. Nyhan

Table B2.8.3 Emergency treatment of hyperammonemic crises Drug Loading dose over Intravenous daily dose Preparation 1–2 h (mg/kg) Na-benzoate 250 250(–500) mg/kg 1 g in 50 mL 5–10% of glucose Na-phenylacetatea 250 250(–600) mg/kg 1 g in 50 mL 5–10% of glucose l-arginine 250 250 mg/kg in OTC, CPS, and in as yet unknown 1 g in 50 mL 5–10% of glucose disease – 500 mg/kg in ASS, ASL Folic acid 0.1 mg/kg q.day Pyridoxine 5 mg q.day OTC ornithine transcarbamylase defi ciency; CPS carbamylphosphate synthetase defi ciency; ASS argininosuccinate synthetase defi - ciency; ASL argininsuccinate lyase defi ciency aIn a known patient, not vomiting, it may be possible to employ oral or gastric Na-phenylbutyrate or the tyrosinemias, the toxic metabolites lead primar- require vigorous administration of glucose in amounts ily to chronic organ damage rather than a metabolic suffi cient to restore and maintain euglycemia. Frequent emergency. Hepatorenal tyrosinemia may lead to a cri- monitoring of blood glucose is essential if symptom- sis of hepatic insuffi ciency. The rationale and princi- atic hypoglycemia is to be avoided. The patient seen ples of therapy remain the same as described earlier in fi rst in an emergency room following a convulsion and the metabolic emergencies, but hypercaloric treatment found to have little or no measurable glucose in the through central catheters is seldom indicated. Glucose blood is usually treated fi rst with enough hypertonic should be administered in accordance with endogenous glucose to restore euglycemia. It is acceptable to fol- glucose production rates together with fat. Oral ther- low that with infusion of 5% glucose and water, but apy should be resumed as soon as possible including glucose levels should be obtained promptly and the medication and the appropriate amino acid mixture. concentration changed to 10% or higher, as required to Patients with tyrosinemia type I should be treated with keep the sugar elevated. nitisinone (NTBC) as soon as possible. NTBC is a Supplementation of carnitine in suspected or proven potent inhibitor of p-hydroxyphenylpyruvate dioxyge- defects of fatty acid oxidation is currently controver- nase; it blocks the genesis of the highly toxic fumary- sial. At least, restoration of levels of free carnitine lacetoacetate and its derivatives (see Chap. C2). appears indicated. A dose of 100 mg/kg is usually ade- In patients with galactosemia or fructose intoler- quate. Disorders of carbohydrate metabolism do not ance, toxic metabolites derive predominantly from need detoxifying treatment. exogenous sources. Once the diagnosis is suspected or Long-term management of patients with disorders of made, therapy consists of the elimination of the intake fatty acid oxidation is best served by avoidance of fast- of galactose and fructose. However, if intravenous ali- ing. Diagnosed patients should have written instructions mentation devoid of galactose and fructose is begun that if anorexia or vomiting precludes oral intake, the without suspicion of the underlying metabolic disorder, patient must be brought to hospital for the intravenous gradual reintroduction of oral feeding will lead to pro- administration of glucose. Supplemental cornstarch is a tracted disease courses with varying and complicated useful adjunct to chronic therapy in disorders of carbo- symptomatology until the diagnosis is made. Patients hydrate metabolism and of fatty acid oxidation. with galactosemia or fructose intolerance require energy to stabilize and maintain blood glucose. B2.8.3 Disturbed Energy Metabolism (Fig. B1.1) B2.8.2 Reduced Fasting Tolerance (Fig. B1.1) The “disorders of disturbed energy metabolism” include defects of the pyruvate dehydrogenase com- Patients with defects of fatty acid oxidation and gluco- plex (PDHC), the Krebs cycle, and the respiratory neogenesis, such as medium chain acyl-CoA dehydro- electron transport chain. These disorders are character- genase defi ciency and glycogen storage disease type I, ized by chronic multisystemic disease rather than acute B2 Metabolic Emergencies 59 metabolic emergency. The situation calling for emer- damage to the patient´s brain. The diseases leading to gency treatment is the occasional occurrence of life acute intoxication, such as maple syrup urine disease, threatening acidosis and lactic acidemia. Therapy in the classical organic acidurias, and the urea cycle this situation calls for vigorous treatment of the acid– defects, carry the greatest risk of major sequelae. base balance as outlined earlier. An issue is the fact that Additional supportive therapeutic measures are used patients with PDHC defi ciency are glucose sensitive. informally in some centers to enhance this goal. These Glucose infusions can result in a further increase in include mannitol for the treatment of cerebral edema, lactate. In fact it is advisable to test all patients with which may also enhance detoxifi cation through lactic acidemia for the lactate response of lactate to increased diuresis. Increased intracranial pressure may glucose. In sensitive patients, intravenous glucose be monitored neurosurgically. Overall supportive care may be employed at rates well below the endogenous is critical in patients in intensive care units, with spe- glucose production rate (Table B2.8.1). The correc- cial vigilance for the detection and prompt treatment tion of metabolic acidosis may require high amounts of infection. of sodium bicarbonate. In as many as 20% of children In summary, the metabolic emergency calls for with mitochondrial disease the acute decompensation prompt diagnostic and therapeutic measures, which fol- may be complicated by renal tubular acidosis, and this low the principles of adequate energy supply, the pro- may increase the requirement for sodium bicarbonate. motion of anabolism and the use of pharmacological, Regardless of the cause, levels of lactate can be lowered and if necessary extracorporal detoxifi cation. These are by dialysis or the administration of dichloroacetate. the determinants of success in handling the metabolic Dichloroacetate activates the PDHC in brain, liver, and emergencies of inborn errors of metabolism. muscle. Although levels of lactate have been shown to improve, the overall outcome may not be altered. In patients with mitochondrial disease, replacement of cofactors is commonly undertaken. Evidence in Key References support of the argument is a positive response to biotin in multiple carboxylase defi ciency and to ribofl avin in Dixon MA, Leonard JV (1992) Intercurrent illness in inborn some patients with multiple acyl-CoA dehydrogenase errors of intermediary metabolism. Arch Dis Child 67:1387–1391 defi ciency. It is common practice to prescribe a combi- Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic nation of coenzyme Q10, vitamin E, and a balanced diseases, 2nd edn. Hodder Arnold, London B-vitamin supplement called “B50.” B50 contains a Nyhan WL, Rice-Kelts M, Klein J, Barshop BA (1999) Treatment combination of thiamine, ribofl avin, niacin, pyridox- of the acute crisis in maple syrup urine disease. Arch Pediatr Adolesc Med 152:593–598 ine, biotin, folate, B12, and pantothenic acid. If a mea- Prietsch V, Ogier de Baulny H, Saudubray JM (2006) Emergency sured defi ciency in blood carnitine is found, or if treatments. In: Fernandes J, Saudubray JM, van den Berghe urinary excretion of carnitine esters is high, the patient G, Walter JH (eds) Inborn metabolic diseases – diagnosis is treated with l-carnitine. and treatment, 4th edn. Springer, Berlin, pp 71–79 Prietsch V, Lindner M, Zschocke J, Nyhan WL, Hoffmann GF The ultimate goal of therapy is not simply to reverse (2002) Emergency management of inherited metabolic dis- the metabolic emergency but to prevent irreversible ease. J Inherit Metab Dis 25:531–546 Patient Care and Treatment B3 William L. Nyhan and Georg F. Hoffmann

Care and treatment of patients with an inherited meta- adolescent and adult. This goal can only be achieved by bolic disease require both a detailed knowledge of the a multidisciplinary approach. Care and treatment of the natural history of the diseases and a comprehensive patient and the family should involve different medical understanding of the molecular basis and the pathophys- specialties as well as associated professions such as iological consequences of gene defects. Continuous dieticians, nurses, psychologists, physiotherapists, sympathetic company and guidance of patients and their social workers, speech therapists, and teachers. Families families are essential for optimal outcome. Inherited may gain valuable emotional support and much practi- metabolic diseases are chronic conditions that involve cal advice by meeting other affected families. Ideally, a various different organ systems and often show progres- specialist in inherited metabolic diseases coordinates all sive pathology. In addition, several genetic aspects such aspects of care and treatment of the patient in close col- as passing on a disease to one’s children, implications laboration with the local family doctor or pediatrician. of consanguinity, the possibility of carrier detection, The objective of this book is not to provide a detailed and prenatal or preimplantation diagnosis, can create a coverage of the diverse issues of the long-term care and severe psychosocial burden for individuals and families treatment of inherited metabolic diseases. Treatment as a whole. This implies the need for an equally diverse is discussed in detail where it is practically relevant multidisciplinary approach to care and treatment. for physicians particularly the emergency situation in Primary correction of the genetic defect, i.e., gene or which there is an acute presentation of a metabolic dis- molecular therapy, has not yet been established for any order. There is also a section on anesthesia, a subject inherited metabolic disease. Treatment is usually aimed seldom considered, but of major importance for patients at circumventing or neutralizing the genetic block, with a variety of inherited metabolic diseases. e.g., through the reduction of dietary phenylalanine in phenylketonuria. In addition, symptomatic treatment of the disease, such as medication for seizures or a por- table electric wheel chair, is essential for outcome and Key Reference improved quality of life of the patient and the family. The aim is to help the affected individual to achieve Blau N, Hoffmann GF, Leonard J, Clarke JTR eds (2005) optimal development during childhood and maximal Physician’s Guide to the Treatment and Follow-up of independence, social integration, and self-esteem as an Metabolic Diseases. Springer, Berlin Heidelberg New York

W. L. Nyhan Department of Pediatrics, University of California, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 61 DOI: 10.1007/978-3-540-74723-9_B3, © Springer-Verlag Berlin Heidelberg 2010 Anesthesia and Metabolic Disease B4 William L. Nyhan

Key Facts › Malignant hyperthermia is seen in response to inhalation anesthetics and succinylcholine. It is › Succinylcholine persistence in patients with usually the result of mutations in the RYR1 butyrylcholinesterase variants; they may fail gene. Rhabdomyolysis in disorders of fatty acid to breathe long after the surgery is completed. oxidation may mimic malignant hyperthermia. Artifi cial ventilation must continue. Malignant hyperthermia may follow anesthesia › Instability of the atlantoaxial joint in patients in a variety of myopathies, especially central with mucopolysaccharidoses may lead to disas- core disease and KingÐDenborough syndrome. ter during anesthesia. These patients should have surgical procedures in centers with anesthesiologists who have experience in dealing with these patients, and preoperative evaluation B4.1 General Remarks of the cervical spine and cord is mandatory. › (Long) fasting must be avoided in disorders of Special considerations for anesthesia and surgery were fatty acid oxidation and in Refsum syndrome. dramatized by the recognition, more than 50 years ago, The provision of ample amounts of glucose in that genetically determined variants in butyrylcholin- these disorders prevents hypoglycemia, rhab- esterase, the cholinesterase found in serum leads to domyolysis, and the crises of Refsum syndrome. prolongation of the action of succinylcholine, the agent Catabolism is inherent in surgery; it is magni- › used in surgery for relaxation of muscle. Patients with fi ed by fasting. It can be minimized in patients defi cient activity of this enzyme remain paralyzed and with organic acidemia or urea cycle defects by unable to breathe for long after the surgery is com- the provision of parenteral glucose. pleted. Testing for these variants, of which there are a Urea cycle defects may also be amenable to › number, is done spectrophotometrically in the presence the provision of intravenous arginine during of the inhibitor, dibucaine, and the percentage of inhibi- the procedure. Intravenous benzoate/pheny- tion is called the dibucaine number. Management of the lacetate provides an alternate mode of waste patient with this problem is simply to continue assisted nitrogen excretion. ventilation until the succinylcholine is broken down. › Preoperative preparation of patients with All carboxylic acid esters will ultimately be hydrolyzed homocystinuria, especially those who are B 6 despite the absence of specifi c esterase activity. responsive, is designed to minimize levels of homocystine during the procedure. Remember Ideal presurgical procedure is to obtain the butyl- W. L. Nyhan cholinesterase dibucaine number. Then the anesthe- Department of Pediatrics, University of California, UCSD siologist can plan to continue ventilation until the School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, succinylcholine has broken down as a result of the USA action of other esterases. e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 63 DOI: 10.1007/978-3-540-74723-9_B4, © Springer-Verlag Berlin Heidelberg 2010 64 W. L. Nyhan

B4.2 Mucopolysaccharidoses B4.3 Avoidance of Hypoglycemia

The great risk of anesthesia in mucopolysaccharidosis Patients with many metabolic diseases are at risk for the (MPS) is the result of instability of the atlantoaxial development of hypoglycemia. For these patients, the joint. This is a particular problem in Morquio disease, usual fasting prior to general anesthesia and surgery but patients with MPS II and VI are also at risk. Deaths could be disastrous. The objective of management is the have been recorded as complications of anesthesia. maintenance of euglycemia: a concentration of glucose Careful positioning is required and hyperextension of in the blood above 4 mmol/L. Relevant disorders include the neck must be avoided. General anesthesia should the disorders of fatty acid oxidation (Chap. B2.5), gly- be undertaken in these patients only in centers in which cogen storage diseases, disorders of gluconeogenesis, anesthesiologists have had experience with patients such as fructose 1,5-diphosphatase defi ciency, hyperin- with these diseases. In preparation for surgery the sulinism, and ketotic hypoglycemia (Chap. B2.4). In patient or parents should be asked about previous prob- each instance, the patient’s history and tolerance of fast- lems with anesthesia, obstructive sleep apnea, or tran- ing should be known prior to making plans for surgery. sient paralysis that might be an index of cervical Most patients with disorders of fatty acid oxidation do instability. The patient should be examined for evi- not become hypoglycemic until they have fasted more dence of cord compression, kyphoscoliosis, and exces- than 12 h, while some patients with glycogenosis or sive upper respiratory secretions. The blood pressure hyperinsulinism cannot tolerate a 4-h fast. should be determined, and an EKG and echocardio- Patients undergoing short or minor procedures can gram. Recent roentgenograms of the chest and of be scheduled for noon or later and given glucose. the cervical spine should be reviewed. Those with Patients receiving overnight nasogastric glucose should kyphoscoliosis should have pulmonary function stud- have intravenous glucose started before the nasogastric ies. Sleep studies may be useful. Those with evidence administration is discontinued. Every patient should or history of cord compression should have an MRI of be receiving 10% glucose intravenously well prior to the spine. Intubation and induction of anesthesia may the time that hypoglycemia would be expected to be diffi cult because of limited space; smaller tubes begin; and intravenous glucose should be discontinued than usual may be required. Visualization may be lim- only after the patient has demonstrated an ability to eat ited by macroglossia, micrognathia, and immobility of and retain sources of oral sugar. The canula should not the neck. It may be necessary to immobilize the neck be removed until the possibility of vomiting has been with a halo brace or plaster to avoid damage to the cer- excluded. In general, 10% glucose should be employed vical cord. Thick secretions may lead to postoperative at rates approximating 2,500 mL/m2/24 h. This would pulmonary problems. Recovery from anesthesia may be equivalent to 150 mL/kg in infants under 1 year, be slow, and postoperative obstruction of the airway 100 mL/kg, 1Ð2 years; 1,200Ð1,500 mL, 2Ð6 years; has been observed. Wherever possible, local anesthe- and 1,500Ð2,000 mL, over 6 years of age. Rates must sia is preferable, but in young or uncooperative patients be readjusted on the basis of determined levels of glu- such as those with Hunter or Sanfi lippo diseases, this cose in the blood. may not be possible. General anesthesia is preferable to sedation, because of the need to control the airway. B4.4 Rhabdomyolysis and Myoglobinuria in Disorders of Fatty Acid Oxidation/ Refsum Remember Disease The instability of the atlantoaxial joint is the major anesthetic issue in MPS, but macroglossia and General anesthesia and the stress of surgery have each micrognathia, may interfere with intubation. Thick been thought responsible for the acute breakdown secretions may cause postoperative problems. of muscle that has been observed in patients with B4 Anesthesia and Metabolic Disease 65 abnormalities of fatty acid oxidation. These triggers of intravenous preparations of amino acids designed for the acute attack are particularly notable for the myo- the treatment of MSUD. Following the procedure, pathic form of carnitine palmitoyl transferase (CPT) II intravenous glucose should be continued until the oral defi ciency. They may do the same in any disorders of route is clearly feasible and the electrolytes are stable. fatty acid oxidation, especially long-chain hydroxya- A patient with MSUD may be maintained for a while cyl-CoA dehydrogenase (LCHAD) defi ciency (Chap. with intravenous amino acids or in their absence with a C6). Renal failure may be a complication of myoglobi- nasogastric drip of a mixture of amino acids containing nuria. The best answer to preventive anesthesia and no isoleucine, leucine, or valine; blood concentrations surgery in these patients is an ample supply of glucose of amino acids should be monitored. Mixtures of amino and water and the avoidance of fasting. This is accom- acids containing no sugar, fat, or minerals that can be plished by early placement of an intravenous line so made in minimal volume and dripped so slowly that that the patient is fasting not more than 6 h. In the pres- they are tolerated by patients usually thought of as ence of myoglobinuria, intravenous glucose should be requiring nothing by mouth are available. 10% or higher; adjunctive insulin may be helpful in maintaining euglycemia. Surgery and anesthesia may also induce a metabolic crisis in Refsum disease via Remember mobilization of phytanic acid in fat stores. The same Catabolism can be minimized by the provision of preventive approaches apply. parenteral glucose. Patients with MSUD can best avoid catabolic elevation of leucine as a consequence Remember of surgery by the provision of mixture of amino acids, which do not contain the offending branched- The avoidance of fasting and the provision of par- chain amino acids. This is quite successfully done enteral glucose are essential for the prevention of with intravenous mixtures, but these are seldom myoglobinuria in disorders of fatty acid oxidation. widely available. These precepts will also prevent crises in Refsum syndrome.

B4.6 Urea Cycle Defects B4.5 Organic Acidemias: Maple Syrup In disorders of the urea cycle, the objective is the Urine Disease avoidance of hyperammonemia by the minimization of catabolism. The approach is as outlined for organic The objective in the management of anesthesia and sur- acidemias, except that it is the ammonia that must be gery in patients with organic acidemia is the minimiza- carefully monitored. tion of catabolism. This objective is met best by avoiding In addition, patients whose usual medication includes anesthesia and surgery, if at all possible, until the patient arginine or citrulline should be given intravenous argin- is in an optimal metabolic state and well over any infec- ine. The patient’s usual dose is employed, diluted 2.5 g tions. In preparation for the procedure, metabolic bal- in 50 mL 10% glucose, and piggybacked via syringe ance should be ascertained by checking the urine for pump to the glucose infusion. In patients receiving ketones, the blood for ammonia, pH and electrolytes, sodium benzoate or phenylbutyrate or both, the intrave- and in the case of maple syrup urine disease (MSUD), nous mixture of benzoateÐphenylacetate is employed, the plasma concentrations of amino acids. Catabolism again in a dilution of at least 2.5 g of each per 50 mL is minimized by the administration of glucose and and given by piggyback pump. For short procedures, water in the regimen employed for hypoglycemia. In these medications can be begun in the postoperative MSUD, a dose of the amino acid supplement, either 1/3 period. For a longer procedure or one particularly likely of his/her daily dose or at least 0.25 g/kg, is given as late to induce catabolism, and certainly in the presence of prior to anesthesia as feasible. This would be a place for hyperammonemia, they can be given intraoperatively. 66 W. L. Nyhan

B4.7 Homocystinuria Lactic acid accumulates and there is a mixed metabolic and respiratory acidosis. Hypoxia, hypercarbia, and metabolic acidosis accompany rhabdomyolysis. Patients with cystathionine synthase defi ciency are pre- Ventricular tachycardia, pulmonary edema, or dissem- disposed to the development of thrombosis. This may inated intravascular coagulation may ensue; as well as create an added risk for general anesthesia. Those who cerebral edema and renal failure. are pyridoxine responsive can be prepared for surgery The RYR1 gene on chromosome 19q 13.1 is mutated by titrating values for homocysteine and homocystine in heterozygous fashion in a majority of patients. Very with added B until an optimal preoperative level is 6 many different mutations have been identifi ed over the achieved. Nonresponders may be treated with betaine 106 exons. The RYR1 protein is an integral part of the in quantities to achieve minimal concentrations. structure in the sarcolema/reticulum involved in the voltage dependent Ca++ channel. Rows of RYR1 bind to tetrads of the dihydropyridine receptor (DHPR) and mutations in DHPR genes have also been found in B4.8 Malignant Hyperthermia patients with malignant hyperthermia. Malignant hyperthermia has also been encountered Malignant hyperthermia is a genetically determined following anesthesia in a variety of muscular dystro- response to inhalation anesthetics or succinylcholine in phies, including Duchenne. It is particularly found in which rapidly escalating fever and generalized muscle central core disease and KingÐDenborough syndrome spasm may be fatal. Prognosis has improved with pre- (KDS). Central core disease gene mutations have been anesthetic identifi cation and the discovery that dant- linked to RYR1. rolene is specifi cally therapeutic. Hyperthermia is often a late sign. The earliest manifestation is an increase in end-tidal CO2. Extreme spasm of the masseter may Reference make insertion of a laryngoscope impossible (jaws of steel). Muscle rigidity is generalized. The skin becomes Cole CJ, Lerman J, Todres ID (2009) A practice of anesthesia for hot to the touch. infants and children. Elsevier, Philadelphia, pp 847Ð866 Approach to the Patient with Cardiovascular Disease C1

Joachim Kreuder and Stephen G. Kahler

Key Facts › Peripheral vascular disease is prominent in homocystinuria. Disorders of lipoproteins meta- › Metabolic disorders are associated with a wide bolism possess a signifi cant long-term, but variety of cardiovascular manifestations, includ- modifi able burden of premature atherosclerosis ing cardiomyopathy, dysrhythmias and conduc- to a large number of children. tion disturbances, valvular heart disease, vascular › Pulmonary arterial hypertension may be a quite disorders, and pulmonary hypertension. rare complication in a few metabolic diseases, › Most metabolic cardiomyopathies result from especially glycogen storage disease I. disorders of energy production, mostly involving other organs, particularly skeletal muscle and liver. › Taken as a group, the disorders of fatty acid oxidation and of oxidative phosphorylation are the most common causes of metabolic cardiomyopathies; another important group is disor- C1.1 General Remarks ders of glycogen metabolism, especially Pompe disease. A substantial number of metabolic disorders signifi - › In some metabolic disorders, the cardiac man- cantly contribute to cardiovascular morbidity because ifestations may be late, subtle, or secondary to of a direct relationship between the inborn error and metabolic derangements in other organs. outcome or a genetic predisposition resulting from › Valvular dysfunction and infi ltrative cardio- interrelations between gene variants and environmen- myopathy occur as a late complication in many tal factors. lysosomal storage disorders. Clinical manifestations of metabolic cardiovascular › Myocardial dysfunction is common in hemo- disease are mainly determined by the site of involve- chromatosis, a metabolic cardiomyopathy most ment. Cardiomyopathies may present with reduced easily prevented. exercise capacity, edema, tachypnea and dyspnea, or › Symptomatic coronary heart and cerebrovas- increased frequency of respiratory infections. Palpi- cular disease during childhood is restricted tations and syncopes are typical clinical features of to severe defects of low-density lipoprotein cardiac rhythm disorders. In some cases, sudden car- metabolism. diac death due to ventricular tachycardia or fi brillation may be the fi rst manifestation. Metabolic vascular disorders may present as stroke-like episodes, coronary heart disease with angina pectoris or myocardial infarction, peripheral thrombembolism, or tuberous xan- J. Kreuder ( ) thoma. In pulmonary hypertension, reduced exercise Department Pediatric Cardiology, University Children’s Hospital, Feulgenstrabe 12, 35385 Giessen, Germany capacity, exercise-induced cyanosis, or syncope are e-mail: [email protected] the main clinical features.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 69 DOI: 10.1007/978-3-540-74723-9_C1, © Springer-Verlag Berlin Heidelberg 2010 70 J. Kreuder and S. G. Kahler

As in all metabolic disorders the family history C1.2.1 Special Aspects of Cardiac refl ects the various patterns of inheritance, including Metabolism differing penetrance, maternal inheritance of some mitochondrial disorders, and acceleration from one generation to the other. Family examination by echocar- The major fuels for the heart are glucose and fatty diography and electrocardiography is especially impor- acids. The heart, like skeletal muscle, maintains a tant in cardiomyopathies and cardiac channellopathies. reserve of high-energy phosphate compounds (e.g., The age at presentation may be another important phosphocreatine) as well as glycogen. Before birth the key to diagnosis in cardiac metabolic disorders. Overt heart uses less fatty acids, more glycolysis, and toler- cardiomyopathy within the fi rst year of life has a much ates anaerobic metabolism more readily than after the higher association with inborn metabolic diseases than neonatal period. This transition period after birth in older age groups. sometimes leads to the appearance of a cardiomyopathy that was clinically silent before then. After a few weeks, the metabolism of the heart relies on both the fatty acids and glucose for fuel; if glucose becomes limiting (during hypoglycemia) the heart can function C1.2 Cardiomyopathy and Cardiac well, whereas hypoglycemia in the newborn period Failure may lead to signifi cant impairment of cardiac function and dilatation of the heart. During times of increased In epidemiological studies, 5–10% of cardiomyopa- energy demand and greater cardiac output there is thies in children result from identifi ed or suspected increased utilization of fatty acids and glucose. Long- inborn errors of metabolism. However, a comprehen- chain fatty acids, which require carnitine for transport sive diagnostic approach including biochemical analy- into the mitochondria, are the usual forms present in sis of cardiac biopsies revealed a metabolic disease in the blood. Medium-chain fatty acids, which enter the up to 22% of all the affected children. These conditions mitochondria directly, can be used to provide fuel to constitute a less frequent proportion of cardiomyopa- the heart and other organs, if there is a problem with thy among adults, where ischemic heart disease and long-chain fatty acid oxidation. Furthermore, during diabetic cardiomyopathy have become two of the major times of metabolic stress the myocardium switches to causes in the developed countries. During infancy and use more glucose than at other times, and so provision early childhood, metabolic disorders represent a more of continuous glucose during times of cardiac dysfunc- frequent cause of cardiomyopathy than in the older tion can be benefi cial. children and adolescents. The heart, because it relies more than the other tis- Approximately, 5% of the inborn metabolic disor- sues on fatty acid oxidation, may be particularly vul- ders are associated with cardiomyopathy. These disor- nerable to disturbances of this major energy source. ders can be conveniently divided into those in which Disturbances of cellular function affecting a small the cardiac manifestations are primary or prominent group of cells can have a devastating effect if the region (Table C1.1) and those in which they are less common or affected is part of the conduction pathways or is able to less signifi cant (Table C1.2). Rarely, the heart is the only infl uence them. affected organ, as described in cardiac phosphorylase kinase defi ciency and certain mitochondrial disorders. The type of metabolic involvement may determine C1.2.2 Patterns and Pathophysiology the age at clinical presentation. Pompe disease [severe lysosomal type II glycogen storage disease (GSD)] of Metabolic Cardiac Disease usually presents in infancy, whereas cardiomyopathy in other lysosomal storage diseases becomes symp- From a functional point of view, cardiomyopathies tomatic during later childhood. Defects of fatty acid during infancy and childhood may be categorized as a oxidation are likely to present in infancy; mitochon- hypertrophic, dilated, hypertrophic–hypocontractile drial disorders may manifest at any age. (mixed type), restrictive, or noncompaction type. C1 Approach to the Patient with Cardiovascular Disease 71

Table C1.1 Metabolic disorders with cardiomyopathy as a presenting or early symptom Disorder Comments le c treatment c Hypertrophic type Dilated type type Mixed Noncompaction type Tachyarrhythnias Conduction disorders Acids Organic Carnitine Level Profi Acylcarnitine Diagnostic Tissues Specifi Disorders of fatty acid oxidation and the carnitine cycle Carnitine transporter ++++ + + ¯¯ + F, M, W, D Carnitine defi ciency Carnitine– + + ++ ++ + ¯ ++ F, W Low fat, high acylcarnitine carbohydrate translocase diet, MCT. defi ciency Carnitine, if low Carnitine palmitoyl- ++ + ++ ++ + ¯ ++ F, M, L, W, D Low fat, high Heterozygote may have CoA transferase II carbohydrate symptoms, be defi ciency diet, MCT. vulnerable to malignant Carnitine, if low hyperthermia Very-long-chain ++ + + + ++ ¯ ++ F, W, D Low fat, high acyl-CoA carbohydrate dehydrogenase diet, MCT. defi ciency Carnitine, if low Long-chain ++ + ++ ++ ¯ ++ F, W, D Low fat, high HELLP syndrome hydroxyacyl-CoA carbohydrate in pregnant dehydrogenase/ diet, MCT. heterozygotes trifunctional enzyme Carnitine, if low defi ciency Multiple acyl-CoA ++ + ++ ¯ ++ F and M Low fat, high dehydrogenase carbohydrate defi ciency diet, Ribofl avin, d,l-3-hydroxybutyrate, carnitine Mitochondrial disorders Complex I–V ++ + ++ + + + + F, H, L, M, D High fat, low Heart block or sudden defi ciencies carbohydrate death may occur. diet, vitamins, May have lactic antioxidants, acidosis and increased cofactors lactate/pyruvate ratio Kearns-Sayre + + ++ F, M, D Typical heteroplasmic syndrome deletions of mitochondrial DNA Barth syndrome + + ++ + ++ B, D Pantothenic acid Specifi c isolated left ventricular noncompaction. Increased monolysocardiolipin as a biochemical marker suitable for screening Disorders of amino and organic acid metabolism Propionic aciduria, ++ + ++¯¯ ++ F, L, W Protein-modifi ed Cardiomyopathy unrelated Methylmalonic diet carnitine, to metabolic decom- aciduria, Cbl C OH-cobalamin pensation or nutritional defect status; may be presenting symptom. QT prolongation may be the fi rst sign (continued ) 72 J. Kreuder and S. G. Kahler

Table C1.1 (continued ) Disorder Comments le c treatment c Hypertrophic type Dilated type type Mixed Noncompaction type Tachyarrhythnias Conduction disorders Acids Organic Carnitine Level Proﬁ Acylcarnitine Diagnostic Tissues Speciﬁ

b-Ketothiolase ++ ++¯ ++ F, W Protein-modifi ed defi ciency diet, carnitine, malonic aciduria bicarbonate. High carbohydrate, low fat diet, carnitine Lysosomal storage disorders Lysosomal glycogen ++ ++ + W, F, M, D Enzyme replacement Macroglossia, severe storage disease cardiomyopathy, and (severe form – skeletal myopathy. Pompe) Characteristic ECG MPS I (Hurler type) + + W, F, U, D Enzyme replace- Early cardiac manifestation ment, due to coronary hematopoietic vascular involvement stem cell transplantation Disorders of glycogen metabolism GSD type III ++ + F, M, L (debrancher defi ciency) GSD type IV ++ + W, F, M, L Severe liver disease. (brancher Neuropathy, dementia defi ciency) in adult form Phosphorylase b ++ + H Rapidly fatal. Very rare kinase defi ciency cardiac glycogenosis Disorders of + + + B, F Abnormal subcutaneous glycoprotein fat distribution, metabolism psychomotor retardation, pericardial effusion Hemochromatosis + + + + + P, D Phlebotomy Restrictive cardiomyopathy occasionally Nutrient defi ciency Secondary ++ + + ¯ ¯ P, M, and U Carnitine Underlying causes should carnitine be clarifi ed defi ciency Selenium + + + E Selen Additional pancreatic defi ciency insuffi ciency Thiamine defi ciency/ + P, E, and U Thiamine Lactic acidosis dependency Diagnostic tissues commonly used. D DNA (leukocytes and other tissues); E erythrocytes; F fi broblasts; H heart; L liver; M skeletal muscle; P plasma; U urine; W white blood cells (leukocytes) ++ Usually and prominently abnormal; + mildly abnormal

With the exception of mitochondrial disorders, each Hypertrophy can be the result of accumulated material metabolic disease is preferentially associated with a stored in the myocardium (e.g., glycogen), the heart’s functional type of cardiomyopathy by echocardiogra- response to poor functioning of the contractile appara- phy, which helps to focus the differential diagnosis. tus (abnormalities of structural proteins), or impaired C1 Approach to the Patient with Cardiovascular Disease 73

Table C1.2 Disorders in which cardiovascular involvement is of disorders of fatty acid oxidation, oxidative phospho- usually mild or late – symptoms are recognized after systemic rylation, and storage disorders. illness or involvement of other organs Disorder Comments and special issues MAD defi ciency (mild, Biochemically similar to severe including SCHAD forms, but onset later and Remember defi ciency) symptoms milder or intermittent. Cardiomyopathy In general, the most adaptive response of the heart similar to fatty acid oxidation in metabolic disorders is hypertrophy, with or with- defects (Table C1.1) out dilation and systolic dysfunction. Friedreich ataxia Mitochondrial damage may be due to iron accumulation. Diabetes common Mucopolysaccharidoses – Myocardial thickening, Pure restrictive cardiomyopathy is extremely rare in chil- general pathology especially septum and LV dren with metabolic cardiomyopathy, but restrictive ven- wall. Aortic and mitral valve thickening and regurgitation, tricular performance is observed in endomyocardial narrowing of aorta, and fi broelastosis. The noncompaction type mimicking the coronary, pulmonary, renal morphological appearance of the embryonic myocar- arteries dium represents a nonspecifi c myocardial response to a MPS I-S, I-H Infi ltration, thickening of septum, LV wall, severe variety of stimuli. From a metabolic view, noncompac- systolic dysfunction tion appearance is suggestive for Barth syndrome or (10% of patients) defects of oxidative phosphorylation. MPS II EFE, MS, AS, AR, MR, Beyond direct damage of myocardial cells, coro- infarction nary vasculopathy due to mucopolysaccharidoses, oli- MPS VI MV, AV calcifi cation, stenosis Gaucher disease Myocardial thickening due gosaccharidoses, or lipoprotein disorders may induce to infi ltration; pulmonary dilated cardiomyopathy. arterial hypertension Fabry disease Infi ltration leading to LVH; MV prolapse, thickening Fucosidosis Myocardial thickening due to C1.2.2.1 Endocardial Fibroelastosis infi ltration Sialidosis MR, CHF, pericardial effusion Mucolipidosis II, III Aortic regurgitation, myocardial Endocardial fi broelastosis describes a condition peculiar thickening due to infi ltration, to infants and young children, in which there is signifi - CHF cant thickening and stiffening of the endocardium. The Aspartylglycosaminuria MR, aortic thickening basis for this response is not yet known. It can occur in Homocystinuria Hypercoagulability, strokes. disparate conditions ranging from viral myocarditis to MV prolapse carnitine transporter defi ciency, Barth syndrome, severe AR AS CHF aortic regurgitation; aortic stenosis; congestive mucopolysaccharidosis I (Hurler syndrome), and muco- heart failure; EFE endocardial fi broelastosis; LV left ventricle; LVH left ventricular hypertrophy; MR mitral regurgitation; MS polysaccharidosis VI (Maroteaux–Lamy syndrome). It mitral stenosis, MV mitral valve is a predictor of poor outcome. energetics (mitochondrial disorders). Dilatation results when the abnormal or poorly functioning heart is C1.2.3 Inborn Errors of Metabolism unable to contract adequately due to impaired energy production or toxic intermediates like acids or oxidiz- that Cause Cardiomyopathy ing mitochondrial components, and begins to stretch out of shape. This type preferentially occurs in the dis- C1.2.3.1 Disorders of Fatty Acid Oxidation orders of carnitine availability and in some of the and the Carnitine Cycle organic acidurias. The hypertrophic–hypocontractile (mixed) type presents the transition from the hypertro- Disorders of fatty acid oxidation involving long-chain phic to the dilated type and may occur in the late stages fatty acids often present as cardiomyopathy. Sometimes 74 J. Kreuder and S. G. Kahler the onset is abrupt or overwhelming particularly in the LCHAD/Trifunctional Enzyme Defi ciency newborn period. Sudden death may occur, presumably refl ecting arrhythmia or apnea. Liver involvement Many infants with long-chain 3-hydroxyacyl-CoA (manifest as fasting hypoketotic hypoglycemia, hyper- dehydrogenase defi ciency present with cardiomyo- ammonemia, or a Reye-like syndrome) and skeletal pathy, sometimes in the newborn period. This is one muscle involvement are common. Very-long-chain of the common disorders of cardiac fatty acid oxida- acyl-CoA dehydrogenase (VLCAD) and carnitine tion. Liver dysfunction may be severe, including cir- palmitoyl-CoA transferase II (CPT II) defi ciencies are rhosis, and there may be signifi cant skeletal muscle probably the most common; long-chain hydroxyacyl- involvement. Urine organic acids reveal increased CoA dehydrogenase (LCHAD)/trifunctional protein saturated and unsaturated dicarboxylic and hydroxyl and carnitine–acylcarnitine translocase (CACT) defi - species. Plasma carnitine level may be low; acylcar- ciencies present similarly. The metabolic pathways nitine analysis shows increased hydroxy forms of and skeletal muscle aspects of these conditions are dis- C16:0, C18:1, and C18:2. Additional fi ndings that cussed in Chap. C6. MCAD defi ciency, the most com- are unusual for other disorders of fatty acid oxida- mon disorder of fatty acid oxidation (and a cause of tion are lactic acidosis, pigmentary retinopathy (in carnitine depletion), is unusual for a fatty acid oxida- older children and adults), and peripheral neuropa- tion disorder, in that cardiomyopathy is an extremely thy. Absence of deep tendon refl exes and toe-walk- uncommon feature, but dysrhythmias and sudden death ing have been reported. are real concerns. In the late-onset form, dominated by intermittent muscle symptoms, there may be asymptomatic ventricular hypertrophy. Treatment is similar to that in VLCAD defi ciency. Disease Info: Defects of Long-Chain Fatty Acid Oxidation VLCAD Defi ciency Very-long-chain acyl-CoA dehydrogenation is the Multiple Acyl-CoA Dehydrogenase Defi ciency fi rst step of b-oxidation (Figure in Chap. C6) in the mitochondria, after synthesis of the acyl-CoA (e.g., Infants with the severe form of multiple acyl-CoA palmitoyl-CoA) catalyzed by CPT II. It is easy to dehydrogenase defi ciency (MADD), especially the understand why impairment of this step can lead to subgroup without malformations, often have cardio- severe organ dysfunction, especially in heart, liver, myopathy. Hypotonia, encephalopathy, overwhelm- and skeletal muscle. Cardiac symptoms (congestive ing acidosis, and metabolic derangements resulting failure with feeding diffi culties and tachypnea), if from impairment of multiple pathways for the degra- they occur, are likely to occur in infants and young dation of fatty acids and amino acids are common children, and there may be concurrent liver dys- features. The diagnosis is usually established by function. Cardiac hypertrophy, especially of the left analysis of urinary organic acids (dicarboxylic acidu- ventricle, is common. ECG may show increased ria, including ethymalonic, adipic, and glutaric acids) voltages. Urinary organic acids may show increased or plasma/blood acylcarnitines (increased median- dicarboxylic acids; plasma or blood spot acylcarni- and long-chain species), and confi rmed by enzyme tine analysis shows elevation in C14:1 species. analysis, western blot in fi broblasts, or mutation Enzyme assay can be done on fi broblasts and lym- analysis. Carnitine depletion is common. Severely phocytes. Treatment involves avoiding fasting, lim- affected patients are treated with a low-fat, high-car- iting essential long-chain fats to the amount needed bohydrate diet, avoidance of fasting, provision of for growth, and providing medium-chain lipids and abundant carnitine, and ribofl avin (50–100 mg/days), other foods as calorie sources in place of long-chain but response to therapy is often poor. Administration fatty acids. Although the total plasma carnitine level of d,l-3-hydroxybutyrate (100–800 mg/kg/days) has is often low, chronic carnitine supplementation has been shown to induce regression of myocardial sys- not led to documented improvement. tolic dysfunction and hypertrophy. C1 Approach to the Patient with Cardiovascular Disease 75

Disease Info: Defects of the Carnitine Cycle effective in the infantile form, whereas patients with CACT Defi ciency the neonatal type responds poorly to therapy. Mild CPT II defi ciency, among the most com- Most patients with translocase defi ciency have had mon fatty acid oxidation disorders is typifi ed by severe disease in early infancy, including acute car- recurrent rhabdomyolysis and does not present with diac events (shock, heart failure, and cardiac arrest). signifi cant cardiac involvement. Most of these Arrhythmias can be prominent. There may be ven- patients harbor at least one copy of a mild CPT II tricular hypertrophy. There is signifi cant hepatic and mutation allowing higher residual activity than in skeletal muscle dysfunction (hyperammonemia and the severe infantile form. weakness). Total blood carnitine level may be low, especially the free fraction. Acylcarnitine analysis shows mainly long-chain species C16:0, C18:1, and C18:2, refl ecting their synthesis by CPT I, and accumulation. Emergency management includes provi- Disease Info: Carnitine Transporter Defi ciency sion of glucose, restriction of long-chain fats, and A defi ciency of the plasma membrane carnitine trans- supplementation with medium-chain lipids and car- porter leads to severe carnitine depletion by a few nitine (see also Chap. B2.8). Despite this, neonatal months to several years of age. In the most extreme fatality is common. cases it can present as neonatal hydrops. There is hypertrophy of the left ventricle, hepatomegaly with hepatic dysfunction (hypoglycemia, hyperammonemia and increased transaminases), hypotonia, and CPT II Defi ciency developmental delay. The ECG may show high, The severe neonatal/infantile form of CPT II defi - peaked T waves and left ventricular hypertrophy. ciency leads to severe cardiac, skeletal muscle, and Total plasma carnitine is exceedingly low, often less liver dysfunction, resulting in circulatory shock, than 5 mmol/L, and the acylcarnitine profi le shows no heart failure, hypoketotic hypoglycemia, hyperam- abnormal species. In contrast to specifi c elevations in monemia, and coma. Dysmorphic features and renal other disorders of fatty acid oxidation the quantities dysgenesis may be present, reminiscent of the cystic of all acylcarnitines may be very low, suggesting the malformations that occur in glutaric aciduria type II diagnosis. Dicarboxylic aciduria is rarely observed. or pyruvate dehydrogenase defi ciency (see also Endomyocardial biopsy (if done – not needed for Chap. D2). diagnosis) can show lipid infi ltration and, in some Plasma and tissue carnitine levels are low, espe- cases, endocardial fi broelastosis. cially free carnitine. Acylcarnitine analysis shows The milder, later onset form may present with long-chain species, as in translocase defi ciency. cardiac dilatation, abnormal ECG, and hepatomeg- Arrhythmias represent a common feature of CPT aly. Skeletal muscle strength may appear to be nor- II defi ciency. Long-chain acylcarnitines, long-chain mal on static testing, but endurance is poor. Muscle acyl-CoAs, and long-chain free fatty acids all have biopsy shows lipid storage. Urinary organic acids are detergent effects on membranes, so an accumulation usually unremarkable. Diagnosis is confi rmed by might have disruptive effects. On the other hand, the demonstrating markedly reduced (<10% of controls) relative defi ciency of free carnitine adversely affects carnitine transport in fi broblasts. Heterogeneous the ratio of free CoASH to acyl-CoA, and the plasma mutations have been found in the SLC22A5 gene and tissue defi ciency of carnitine would impair fatty encoding the OCTN2 carnitine transporter. acid oxidation. The true magnitude of the contribu- In this autosomal recessive condition, the fraction of long-chain acylcarnitines to arrhythmias in tional excretion of carnitine in the urine approaches CPT II defi ciency (and related disorders) is neither 100%, as the renal carnitine reabsorptive transport not known nor is the true risk of carnitine adminis- system is the same as that for most other tissues. tration in a desperately ill child with this disorder. Accordingly, carnitine must be provided frequently. Therapy like this for CACT defi ciency is somehow Intravenous carnitine (during an acute crisis) can be 76 J. Kreuder and S. G. Kahler

are found for a short time during episodes of illness given in a dose of 300 mg/kg/days, as a continuous with MCAD defi ciency and glutaric aciduria type I, infusion. Oral carnitine should be given four times they are not usually associated with cardiomyopathy. daily. The oral dose for children is 100–200 mg/kg/ days and for adults 2–4 gm/days, but some patients have required much more to maintain satisfactory C1.2.3.2 Mitochondrial Disorders plasma levels. The maximal oral dose is usually set by intestinal tolerance, while diarrhea does not occur The mitochondrial disorders are discussed in general with parenteral use. If treatment started before irre- terms in the Chaps. B2.3 and D1. About 40% of patients versible organ damage occurs, the cardiomyopathy with mitochondrial disorders suffer from cardiac improves dramatically, and the heart size returns involvement in terms of cardiomyopathy or dysrhyth- to normal. Skeletal weakness may also improve, mias, leading to earlier presentation and much worse although muscle carnitine levels remain low (2–4% prognosis for survival compared to noncardiac affected of normal). The long-term prognosis is favorable as patients. Involvement of other organs is common, espe- long as children remain on carnitine supplements. cially the brain, eye (and eye movements), skeletal However, recurrence of cardiomyopathy or sudden muscle, liver, and kidney. However, isolated cardiac death from arrhythmia even without cardiomyopa- defi ciency of oxidative phosphorylation represents a thy have been reported in patients discontinuing car- substantial part of mitochondrial cardiomyopathies. In nitine supplementation. about 50% of cardiac manifestations, cardiomyopathy shows the hypertrophic pattern, followed by the mixed hypertrophic-dilated, the dilated, and the noncompac- Secondary Carnitine Defi ciency tion pattern. Disturbances of cardiac rhythm such as complete heart block or bundle branch block affect Secondary carnitine defi ciency occurs in a large num- about 15–25% of these patients and sudden death may ber of settings, discussed in Chap. C6. Even with low occur, sometimes as the fi rst indication of a mitochon- plasma levels the cardiac uptake of carnitine is usually drial disorder. A diagnosis in this situation may be pos- suffi cient to avoid symptoms. However, there are occa- sible if tissues for mitochondrial studies (especially sional premature infants receiving long-term total par- heart, skeletal muscle, and skin) are acquired rapidly enteral nutrition (TPN) without carnitine supplementation after death. who become profoundly depleted after a few months. The most common defects of the respiratory chain The need for carnitine in infants may exceed their syn- complexes were complex I, complex IV, and combined thetic capability, as an infant needs suffi cient carnitine defi ciencies. An apparent correlation between specifi c for the increasing mass of muscle and other tissues, defects of the respiratory chain complexes and cardiac whereas adults need only to replace what is lost. A involvement is not possible. Lactic acidosis with an lower renal threshold for carnitine in a sick infant may increased ratio of lactate/pyruvate is common, but not also become pathophysiologically relevant. obligatory. Mutations of mitochondrial protein-encod- Infants with severe carnitine depletion may have ing genes, tRNA and rRNA, and deletions/duplications poor cardiac output and dilated cardiomyopathy. The are only found in a minority of pediatric patients with plasma carnitine level may be less than 10 mmol/L. mitochondrial disorders suggesting that mutations of One would expect hypoglycemia from liver carnitine nuclear genes are the leading cause for these diseases depletion, but this does not occur because of the con- (for details of distinct genetic lesions in mitochondrial tinuous high-dose glucose of the TPN. Carnitine sup- disorders, see www.mitomap.org). plementation (15–30 mg/kg/days IV, given with the The Kearns–Sayre syndrome is the prototypical TPN infusion) leads to rapid improvement in blood mitochondrial disorder in which there is progressive levels and cardiac function. disturbance of conduction. Most patients represent non- Severe carnitine depletion can occur in the setting of familial heteroplasmic deletions of mitochondrial DNA. renal Fanconi syndrome (as in cystinosis and mitochon- Cardiac symptoms include hypertrophic or dilated car- drial dysfunction) or treatment with the antibiotic piv- diomyopathy, atrial or ventricular arrhythmias, Wolf– ampicillin. Although very low plasma carnitine levels Parkinson–White syndrome, and other preexcitation C1 Approach to the Patient with Cardiovascular Disease 77 syndromes. Progressive heart block may necessitate In some cases, metabolic therapy can be directed to the pacemaker placement. Major noncardiac symptoms pathobiochemical substrate like inherited primary include retinitis pigmentosa, ophthalmoplegia, ataxia, coenzyme Q defi ciency or copper-histidine supple- myopathy, and increased CSF protein, with onset before mentation in SCO2 defi ciency. age 20. Many other manifestations may occur, including deafness, seizures, diabetes or other endocrinopathy, renal and gastrointestinal symptoms, and lactic acidosis. Similar manifestations with later onset are Disease Info: Barth Syndrome typical of chronic progressive external ophthalmople- Barth syndrome is an X-linked recessive condition gia-plus (CPEO-plus). that is similar to many mitochondrial oxidative Other clinical entities of mitochondrial DNA disor- phosphorylation diseases. Neutropenia, cardiomyo- ders are likely to have cardiac involvement including pathy, and increased urinary excretion of 3-methyl- Leigh syndrome, MERFF, and MELAS. Mutations in glutaconic acid, 3-methylglutarate, and 2-ethylhy- nuclear genes which are also associated with the dracrylate – compounds suggestive of mitochondrial appearance of cardiomyopathy may affect both the dysfunction – occur. Noncompaction pattern is typi- structural proteins (part of the respiratory chain com- cal, but not exclusive for the cardiac involvement in plexes, e.g., NDUFS2 and NDUFV2 in complex I) as Barth syndrome may occur independent of the other well as nonstructural proteins responsible for the features. The onset is typically in infancy, but there assembly of respiratory chain complexes (e.g., SCO2 may be spontaneous improvement in later child- and SURF1), mtDNA stability (e.g., POLG), iron hood. Biopsy of skeletal muscle or heart shows homeostasis (e.g., frataxin), mitochondrial integrity abnormal mitochondria, which may be abnormal in (e.g., tafazzin = G4.5), or mitochondrial metabolism shape and contain inclusion bodies and dense, tightly (e.g., PDH E1-a-subunit). packed concentric cristae. Lactic acidosis may be Sengers syndrome, Barth syndrome, and Friedreich prominent. The molecular defect in Barth syndrome ataxia represent other nuclear encoded diseases of is attributed to the G4.5 or tafazzin (TAZ) gene, the oxidative energy production with neuromuscular which encodes a mitochondrial transacylase involved involvement. Sengers syndrome (autosomal recessive) is in the remodeling of cardiolipin. Defi ciency of the characterized by hypertrophic cardiomyopathy, congenital TAZ protein results in reduced levels of cardiolipin cataracts, and exercise-induced lactic academia. Defi ciency and increased monolysocardiolipin content, which of adenine-nucleotide-translocator ANT1 most probably can serve as a biochemical marker for this disease. due to transcriptional or posttranscriptional lesions was At least one patient has had a dramatic and found to be associated with Sengers syndrome. sustained response to treatment with pantothenic Disorders of oxidative phosphorylation are often acid, a precursor of coenzyme A. Pantothenol was attempted before treating with a high-fat, low- ineffective. carbohydrate diet and supplemental vitamins (especially coenzyme Q, carnitine, and ribofl avin in complex 1 defi ciency), antioxidants, electron acceptors, cofactors, and metabolic intermediates (e.g., creatine, argin- Disease Info: Friedreich Ataxia ine in acute MELAS manifestation, see Chap. D1). This disorder presents with slowly progressive spi- Response to metabolic therapy is highly variable and a nocerebellar dysfunction in children and young matter of continuing debate. adults. Cardiomyopathy, cardiac dysrhythmias, and diabetes are common. The genetic defect is usually a trinucleotide expansion in the frataxin gene. The mechanism of cellular injury appears to be related Remember to iron accumulation within mitochondria. Beyond Serial echocardiography, 24 h-ECG, and measure- defi cient energy production, cellular damage lead- ment of BNP/NT-proBNP are valuable tools to ing to apoptosis may be due to peroxidative damage supervise metabolic therapy. to mitochondrial membranes. 78 J. Kreuder and S. G. Kahler

accumulation initially results in isolated diastolic dys- In roughly half the patients idebenone, a coen- function, but may progress to severe systolic failure. In zyme Q analog, is beneﬁ cial to reduce myocardial most cases, echocardiography shows global concentric hypertrophy and rhythm disturbances. ventricular hypertrophy.

Disease Info: GSD II (Pompe Disease and C1.2.3.3 Disorders of Amino and Organic Acid Lysosomal Acid a-Glucosidase Defi ciency) Metabolism Pompe disease is among the most common primary Two common organic acid disorders, propionic aciduria metabolic cardiomyopathies and the leading lyso- and methylmalonic aciduria, in the branched-chain amino somal disorder in which there are severe cardiac man- acid catabolic pathway, may present with a cardiomyopa- ifestations. In the classical infantile form, symptoms thy, from birth to a few years of age. The onset may be appear in early infancy and the disorder progresses subtle or sudden. Cardiac failure with dilatation, poor rapidly. Weakness, fl oppiness, and macroglossia may contractility, and rhythm disturbances including ventric- be apparent within a few months. Cardiac manifesta- ular electric instability or bradycardia, may occur. tions include shortness of breath and poor feeding. Chest X-ray shows cardiomegaly, echocardiography concentric symmetric hypertrophy with or without Remember outfl ow tract obstruction, ECG left axis deviation, short PR interval, high QRS voltages, and inverted T Prolongation of normalized QT (QTc) time may be waves. Peripheral blood leukocytes may show vacu- the fi rst sign of cardiac involvement in organic aci- oles and there is often pathological excretion of oli- durias and should initiate regular cardiac surveil- gosaccharides in the urine. Enzyme assay can be lance by echocardiography and measurement of performed on mixed leukocytes and purifi ed lym- BNP/NT-proBNP blood levels phoytes. Dried blood spot on Guthrie cards is available in some laboratories and may be applied to newborn screening programs. Reduced a-glucosidase Cardiomyopathy may also occur in b-ketothiolase activity in a blood sample should be confi rmed by defi ciency (isoleucine degradation), methylhydroxy- enzyme measurement in fi broblasts, muscle biopsy, butyryl-CoA dehydrogenase defi ciency (isoleucine or by DNA analysis. Muscle biopsy shows a major degradation), and malonic aciduria. accumulation of PAS-positive material and mem- The basis for this cardiomyopathy is not known. In brane-bound (i.e., in lysosomes) accumulation of gly- some cases it has occurred in children being treated suc- cogen in electron microscopy. cessfully for the metabolic disorder and in a few it has Infants with infantile Pompe disease often suc- been the presenting symptom. Suggested causes include cumb in a few months due to cardiac failure or primary toxicity of metabolites accumulating as a result arrhythmias, but may survive for 2 or 3 years. of the underlying enzymopathy and nutritional defi - Enzyme replacement therapy has recently become ciency of carnitine or of some other nutrients. available with encouraging results. Treatment is directed at correcting the metabolic Patients with the late-onset form have higher derangement (e.g., diet and carnitine) and providing residual activity of a-glucosidase. They present appropriate supportive care. beyond the fi rst year of life with progressive weakness, whereas cardiomyopathy is rare and much less severe (see Chap. C6). C1.2.3.4 Lysosomal Storage Disorders (Infi ltrative Cardiomyopathies) Cardiomyopathy with lysosomal glycogen storage, but In this subgroup, the myocardium may become thick- normal a-glucosidase activity is observed in X-linked ened because of accumulated material, particularly Danon disease, which is caused by mutations of the gene within lysosomes. Because of mechanical interference, encoding lysosome-associated membrane protein 2. C1 Approach to the Patient with Cardiovascular Disease 79

Other Inﬁ ltrative Cardiomyopathies C1.2.3.6 Disorder of Glycoprotein Metabolism

In men with Fabry disease, there may be thickening of The heart is quiet frequently involved in organ dys- the left ventricle. This can also occur in the heterozy- function in patients with congenital diseases of glyco- gous women of this X-linked disorder, perhaps refl ect- sylation (CDG) leading to early death within the fi rst ing skewed lyonization. Coronary artery disease is also year of life in a substantial number of patients. common in Fabry disease. The mucopolysaccharidoses Hypertrophic cardiomyopathy is associated with type and fucosidosis may be complicated by storage in the Ia CDG disorder (phosphomannomutase defi ciency), septum and posterior wall of the left ventricle. dilated appearance was found in nonclassifi ed forms. Lysosomal storage in the heart may also occur in Specifi c therapy is not available. Gaucher disease.

Disease Info: Hemochromatosis C1.2.3.5 Disorders of Glycogen Metabolism Hereditary hemochromatosis (HH) characterized by iron deposition and tissue injury in multiple Glycogen Debrancher Defi ciency (Cori or Forbes organs is among the most common genetic meta- Disease, GSD III) bolic diseases in many parts of the world, occurring as often as in 1 in 300 persons. In all types of HH, In type IIIa GSD there is involvement of liver, and iron overload results from impairment of the hepci- skeletal or cardiac muscle; in the less common type din–ferroportin regulatory pathway. The most com- IIIb there is only hepatic disease, manifest as fasting mon of the inherited forms (>95%) is autosomal hypoglycemia, increased transaminases, and rarely recessive type 1 or HFE-related HH. Two mutant mild lactic acidosis. The cardiomyopathy in type IIIa alleles of the HFE gene that regulates the expres- usually does not cause symptoms before adulthood, sion of the iron-regulatory hormone hepcidin but outfl ow tract obstruction and heart failure occur account for essentially all cases. Homozygotes for occasionally. During childhood, there may be ventric- C282Y are at highest risk; compound heterozygotes ular hypertrophy on ECG and echocardiography C282Y/H63D are at less risk; and homozygotes for H63D are at minimal risk for iron overload. The clinical penetrance of C282Y homozygosity for iron overload-related diseases is up to 30% in men Glycogen Brancher Defi ciency (Anderson Disease, and 2% in women in fourth to sixth decade and very GSD IV) rare before adulthood. In total, 25–35% of C282Y homozygotes may not develop iron overload. The usual form of this disorder manifests hepatomeg- HH is diffi cult to recognize clinically, because aly and cirrhosis. The defi ciency of the branching its early symptoms of fatigue and depression do not enzyme activity leads to the accumulation of an amyl- point to iron overload. Hepatic dysfunction and opectin-like glycogen (also known as polyglucosan hepatomegaly are common, manifested by mild bodies), which incites an infl ammatory response. Rare, elevation of transaminases (see also Chap. C2). severely affected patients may have heart involvement Pancreatic dysfunction may lead to diabetes and in childhood with ventricular hypertrophy and second- adrenal dysfunction to Addison disease. Darkening ary dilatation. Liver transplantation is curative for the of the skin from iron may be mistaken for the effects liver disease. The heart condition may be ameliorated of Addison disease. Arthropathy is common. as well, as shown by improved cardiac function and Cardiomyopathy has an insidious onset and may the lessening of storage. be attributed to diabetes. Compared to the normal Intramyocardial polyglucosan bodies may also be population, cardiomyopathy is 306-fold more fre- observed in phosphofructokinase defi ciency (GSD7), quent in patients with HH. The cardiomyopathy mutations of the PRKAG2 gene encoding the a-2 sub- usually results in biventricular dilatation. Ventricular unit of AMP-activated protein kinase, and unclassifi ed thickening is an early fi nding; a restrictive form polysaccharidoses of the heart and skeletal muscle. 80 J. Kreuder and S. G. Kahler

receiving limited diets. In this setting, lactic acidosis has occurs occasionally. Arrhythmias (ventricular ecto- been the presenting feature, refl ecting impaired activity pic beats, tachycardias, ventricular fi brillation, and of pyruvate decarboxylase, the fi rst step in the pyruvate heart block) may occur. dehydrogenase complex. Defi ciency of thiamine result- Raised transferrin-iron saturation (>45%) and ing from thiamine transport defects (autosomal reces- ferritin levels are the usual biochemical markers for sive) typically results in anemia (usually megaloblastic, iron overload; defi nitive diagnosis, formerly done but may be sideroblastic or aplastic), sensorineural deaf- by liver biopsy, is now generally achieved by ness, and diabetes; tachycardia and edema may occur in molecular testing, although biopsy may be needed the most severely affected infants. Diagnosis is made by to assess the degree of liver injury. measuring blood or urine thiamine content. As hemochromatosis is a recessive disease, parents and all siblings must be tested. Molecular genetic testing to identify C282Y (and H63D) Selenium mutants is the fi rst step of cascade testing, followed by iron studies in C282Y homozygotes and C282Y/ Selenium in the amino acid selenomethionine is a H63D heterozygotes. component of several proteins, and cofactor, particu- A distinct, but rare disorder is juvenile or type 2 larly for glutathione peroxidase. Selenium defi ciency, hemochromatosis (JH), in which there are similar especially when it occurs with vitamin E defi ciency, fi ndings, but the onset is in childhood. Autosomal impairs the antioxidant function of various pathways. recessive JH is caused by mutations in the gene of There may also be increased vulnerability to cardiotro- hepcidin-regulating protein hemojuvelin or the hep- pic enteroviruses. Selenium defi ciency is a common cidin gene itself. cause of cardiomyopathy in the Keshan district of Type 3 and 4 HH are caused by mutations in the China, where there is insuffi cient selenium in soil and transferring receptor 2 gene or the ferroportin gene. foods. Manifestations include cardiac dilatation, dys- Symptoms in these subtypes also manifest in the rhythmias, and ultrastructural changes. The condition fourth to fi fth decade. may be fatal unless treated. Pancreatic insuffi ciency HH is treated by phlebotomies whose frequency may also develop. is targeted by serum ferritin levels (<300 ng/mL in Selenium defi ciency may also occur in developed men und 200 ng/mL in women). Tissue damage, countries. It has been observed following prolonged such as cardiomyopathy and hepatic fi brosis, may parenteral nutrition and in patients with small bowel not be reversible, so early diagnosis is essential. disease. Patients with many forms of heart disease, including ischemic heart disease and HIV cardiomyopathy, have lower selenium levels than controls, rais- C1.2.3.7 Nutrient-Defi ciency Cardiomyopathies ing the possibility that selenium depletion may play a role. Selenium defi ciency is diagnosed by the measure- Thiamine ment of erythrocyte selenium, along with glutathione (total and reduced), and glutathione peroxidase; it is treated with sodium selenite, 60–100 mg/days. Thiamine (vitamin B1), as thiamine pyrophosphate, is essential for the function of several decarboxylases of 2-oxoacids, including pyruvate, the branched-chain oxoacids, and 2-oxoglutarate. Severe thiamine defi - C1.2.4 Investigations for Metabolic ciency leads to beriberi, a form of cardiac and peripheral vessel dysfunction characterized by loss of peripheral Cardiomyopathies vascular tone, edema, tachycardia, cardiac dilatation, and heart failure. Brain damage (Wernicke’s encephal- Laboratory tests to investigate a suspected metabolic opathy) reminiscent of Leigh disease may occur, and cardiomyopathy should focus initially on obtaining other organs may become damaged. Thiamine defi - information rapidly to determine if there is a specifi c ciency can occur in patients with organic acidurias treatment for the disorder. At the same time, supportive C1 Approach to the Patient with Cardiovascular Disease 81 care to ease myocardial workload should be instituted liver dysfunction. In inborn errors of metabolism that and preparations made for invasive testing is needed. impair energy production or produce toxic metabolites, congestive heart failure often occurs in the setting of an acute metabolic decompensation triggered by intercur- Remember rent infections, surgery, fasting, or physical exertion. Since these inciting events may suggest an alternative Because of complex etiology, diagnosis of cardio- etiology like e.g., viral myocarditis; it is important to myopathy requires a systematic and integrated include laboratory testing of blood and urine as part of approach including careful clinical examination, the initial evaluation. echocardiography, laboratory investigations, and Initial laboratory tests include complete blood count, search for additional organ dysfunctions ( Fig. C1.1). glucose, blood gas analysis, plasma lactate, amino acids, carnitine, and acylcarnitine profi le, electrolytes, transaminases, creatine kinase, urea, creatinine, and uric Cardiomyopathy may be the presenting or dominant acid. Troponin I and BNP or proNT-BNP levels refl ect clinical feature, but careful searching often reveals the extent of myocardial damage, ischemia and over- other signs of a multisystemic disease as well as abnor- laod, independent from their etiology (Table C1.3). mal metabolites in blood or urine. Clinical features Lactate is often elevated in patients with cardiac fail- may include dysmorphic features, neurological and ure simply refl ecting poor perfusion. In such a setting muscular symptoms, and skeletal fi ndings and signs of the lactate/pyruvate ratio is likely to be elevated and not

Echocardiography: morphology+ function Metab-1, Cardiolipin in thrombocytes (boys) , skeletal muscle DCM LVNC Barth syndrome biopsy. If negative -> Metab-2 OXPHOS disorders

HCM+LV-Dysfunction Metab-1. TNI, CK-MB Ý ? FAO, OXPHOS, storage, Consider glycogen, organic acid disorders HCM metab-2 Yes no RCM

Associated symptoms? Defined syndrome Targeted metabolic / DNA analysis

Dysmorphic signs Not-defined syndrome CGH, metab-1+2 Yes Developmental delay, seizures Metab-1+2, brainMRI

Ataxia Friedreich/Refsum disease, OXPHOS disorders Targeted Metab-1+2

Myopathy, < 1 y congenital myopathy EMG, skeletal muscle biopsy

Myopathy, > 1 y muscular dystrophy EMG, DNA analysis, muscle biopsy

Myotonia myotonic dystrophy EMG, DNA analysis no Cataract, abnormal fundus, hepato(spleno)megaly OXPHOS, Metab-1, storage disorders metab-2 Isolated Isolated DCM * HCM* Familial CM Consider DNA analysis (structural proteins)

Ischemic/inflammatoryCM Coronary angiography, endomyocardial biopsy

Fig. C1.1 Algorithm for the diagnostic approach to cardiomyo- examinations; Green background potentially associated symp- pathies. DCM dilated cardiomyopathy; HCM hypertrophic car- toms; Red frame suspected etiology; Yellow background recom- diomyopathy; HDCM hypertrophic–hypocontractile (mixed mended investigations. *In the case of isolated CM, complete type) cardiomyopathy; LVNC left ventricular noncompaction; fi rst-line metabolic investigations should be performed in all OXPHOS mitochondrial; RCM restrictive cardiomyopathy; TNI children younger than 3 years. Otherwise well-defi ned second- troponin I. Metab-1 fi rst-line investigations (Table C1.3); ary cardiomyopathies with recognizable etiology (e.g., hyper- Metab-2 second-line investigations (see Table C1.4); Blue type tensive CM and anthracycline-induced CM) were not included echocardiographic types of CM; Blue background early routine in this algorithm 82 J. Kreuder and S. G. Kahler

Table C1.3 First-line investigations in suspected metabolic per se suggestive of disturbed oxidative phosphoryla- cardiomyopathy tion (see Chap. B2.3). Renal tubular function can be Examination Criteria/suspected disorder assessed by checking for wasting of amino acids, phos- (examples) phate, and bicarbonate, resulting in a Fanconi syndrome Additional clinical symptoms or isolated renal tubular acidosis. Urinary organic acids Dysmorphic features Coarse facial appearance, cloudy corneas, slow with increased Krebs cycle intermediates and lactate growth may be suggestive of mitochondrial dysfunction, while Muscular symptoms Weakness, hypotonia, prominent 3-methylglutaconic acid suggests Barth syn- cramps, myotonia drome (type II) or defective oxidative phosphorylation, Neurological fi ndings Encephalopathy, develop- especially complex V defi ciencies (type IV). Distinct mental delay, seizures Liver and spleen Organ size, ascites, organic acidurias may present with their typical excre- involvement liver skin signs tion pattern. Hepatic dysfunction may be refl ected by Skeletal fi ndings Dysostosis multiplex increases in transaminases, bilirubin and ammonia, Blood hypoglycemia, an abnormal amino acid profi le, and Complete blood count Vacuoles, lysosomal reduced coagulation factors. Consideration should be storage disease given to determine thiamine, selenium, vitamin E, and Blood gas analysis, Defects in oxidative glutathione levels when nutritional defi ciencies are lactate phosphorylation Sodium, potassium, Renal involvement (tubular) suspected.. calcium, phorphorus Abdominal ultrasound and eye examination should Urea, creatinine, uric Renal involvement be included in the initial diagnostic workup. acid (glomerular) Encephalopathy, hypoglycemia, metabolic acidosis, Creatine kinase Myopathy and neuromuscular symptoms have been proposed as Alanine and aspartate Liver involvement key entry points to diagnostic algorithms and second- aminotransferases Carnitine (free Carnitine transporter line tests in suspected metabolic cardiomyopathy [for and total) defi ciency detailed algorithms see Cox (2007)]. Acylcarnitine profi le Defects in fatty acid Second-order investigation (Table C1.4) include oxidation analysis of additional metabolites, targeted genetic Amino acids Organic aciduria, analyses, and enzyme studies in fi broblasts, lympho- tyrosinemia Transferrin isoelectric CDG syndromes cytes, or tissue (e.g., fatty acid oxidation and lysosomal focusing enzymes). The echocardiographic type of cardiomyo- Acid a-glucosidase Pompe disease pathy and some ECG fi ndings may allow to near the Cholesterol, Lipoprotein disorders diagnostic approach. However, the substantial hetero- triglycerides geneity of echocardiographic and ECG fi ndings has to Troponin I Myocardial ischemia be considered. BNP/pro-NT-BNP Ventricular overload/ dysfunction Morphological studies including ultrastructural exam- Urine ination as well as biochemical investigations of endomyo- Protein, electrolytes Proteinuria, electrolyte loss cardial and skeletal muscle biopsies should be performed (renal involvement) in all cases, which unresolved etiology after the noninva- Organic acids Organic acidurias sive work up. Glycosaminoglycanes Mucopolysaccharidoses Oligosaccharides Oligosaccharidoses Eye Lens Cataract, defects in oxidative Remember phosphorylation Retina Storage disease, defects Skeletal biopsy should be performed in all the infants in oxidative with hypertrophic or hypertrophic–hypocontractile phosphorylation phenotype due to the high probability of defective Other organs oxidative phosphorylation, even in the absence of Abdominal ultrasound Hepatic or renal involvement lactic acidemia BNP, brain-type natriuretic peptide; NT, N-terminal C1 Approach to the Patient with Cardiovascular Disease 83

Cardiac restricted defects of glycogen metabolism or morphologic pattern, effi cient reduction of b-adrener- oxidative phosphorylation will only be detected by gic sympathetic activity may be crucial for the long- direct endomyocardial analysis. (See section Chaps. term prognosis especially in cardiomyopathies due to B2.3 and D1). defective energy production. In hypertrophic cardiomyopathy with isolated diastolic dysfunction, b-block- ing agents or calcium antagonists should be at least C1.2.5 Principles of Treatment introduced when outfl ow tract obstruction exists. In the case of severe myocardial failure, extracorpo- in Metabolic Cardiomyopathy ral membrane oxygenation or ventricular assist devices offer the potential for patient’s survival during diagnos- Treatment should be directed to the specifi c metabolic tic workup and in initiating a specifi c metabolic ther- defect as well as to the functional state of the myocar- apy. Unloading conditions largely facilitate myocardial dium. Signs of acute metabolic disturbance like hypo- recovering and bridge the time until specifi c and non- glycemia and metabolic acidosis should be corrected as specifi c treatment works effi ciently. If advanced heart soon as possible. In metabolic acidosis, carnitine supple- failure is accompanied by ventricular dysynchronia, mentation may help to restore intermediate metabolism implantation of a biventricular pacemaker may be con- by replenishing low free carnitine levels, binding acid sidered. Heart transplantation may only be an option in compounds, and liberating acyl coenzyme A from CoA- cardiac restricted metabolic cardiomyopathies or well- species. For disorders of the intermediary metabolism, controlled generalized metabolic diseases. there are several guiding principles for treatment. Diet should reduce the intake of nonmetabolizable substrates and offer alternative, metabolizable energy sources and C1.3 Dysrhythmias and Conduction nutrients (e.g., substitution of long-chain by medium- chain fatty acids in VLCAD defi ciency, d,l-3-hydroxy- Disturbances butyrate in MADD). Fasting should be avoided by use of night feeding or continuous tube feedings during Disturbances of the cardiac rhythm due to metabolic infancy, supplemented cornstarch at bedtime may pro- disorders are often combined with overt cardiomyopa- vide suffi cient carbohydrate supply through the night in thy, but may also proceed morphological and functional older children. Dietary supplements can reduce second- abnormalities of the myocardium and be the fi rst, in ary toxicity (e.g., carnitine in organic acidurias and anti- some cases even fatal clinical manifestation. Routine oxidants in defects of oxidative phosphorylation). In ECG may show preexcitation in respiratory chain dis- some cases, residual enzyme activity can be enhanced orders, Pompe and Danon disease. Prolongation of QTc by the use of pharmacologic doses of vitamin cofactors interval which increased with age has been observed in (e.g., ribofl avin in MADD). Enzyme replacement ther- about 70% with propionic acidemia. Prolonged repo- apy has emerged as a new therapeutic option, at least in larization of the myocardium may predispose propionic terms of myocardial function and morphology, in sev- acidemia patients to ventricular fi brillation or torsades eral lysosomal storage diseases like Gaucher disease, de pointes and be the cause of the increased rate of sud- Pompe disease, Fabry disease, and MPS I, II, and VI. den death in these patients. Benefi cial effects of hematopoetic stem cell transplanta- All FAO disorders that affect cardiac metabolism are tion have been observed in MPS I and VI. In certain another disease group in which rapid ventricular dys- cases of severe cardiomyopathy, preceding enzyme rhythmias (e.g., fi brillation, torsades de pointes) can replacement is an essential requisite for succeeding occur. LCHAD, VLCAD, and CPTII defi ciencies pos- hematopoetic stem cell transplantation. sess the greatest risk for ventricular tachycardia, proba- In systolic myocardial failure, conventional therapy bly due to the accumulation of potentially arrhythmogenic with ACE inhibitor, b-adrenergic receptor blockers, long-chain acylcarnitines. Even in MCAD defi ciency spironolactone, and other diuretics should be instituted that usually does not affect the myocardium, ventricular as soon as possible. Digoxin may be introduced in the tachycardia has been observed in adulthood. presence of atrial fl utter or fi brillation, but may also be In addition to the specifi c metabolic therapy, tach- benefi cial in pure dilated forms. Independent from the yarrhythmias should be treated by b-receptor blockers, 84 J. Kreuder and S. G. Kahler

Table C1.4 Second-line investigations in suspected metabolic cardiomyopathy Abnormalities Second-line investigation Clinical symptoms Dysmorphic features Blood Acylcarnitines (if not done before), chromosome analysis, CGH array Urine Organic acids, mucopolysaccharides, oligosaccharides (if not done before), free sialic acid Ataxia Blood Phytanic acid DNA Frataxin gene mutations Developmental delay

Blood Lactate/pyruvate ratio (L/P) (preferentially after glucose load), NH3 CSF Lactate + pyruvate, amino acids Urine Free sialic acid Cranial MRI, MR spectrosopy Muscular symptoms See below ECG AV block mtDNA analysis Pre-excitation mtDNA analysis Blood High lactate + hypoglycemia

Blood Lactate/pyruvate ratio (L/P), NH3 During acute hypoglycemia Blood Insulin, GH, free fatty acids, b-OH-butyrate, amino acids, acylcarnitines Urine Ketones, organic acids With high ketones and uric acid Amylo-1,6-glucosidase (debrancher enzyme) (GSD III) (liver, muscle, fi broblasts) Persistent high lactate Blood Lactate/pyruvate ratio (L/P), acetoacetate/b-OH-butyrate ratio L/P >25:1: Assays for respiratory chain enzymes (skeletal muscle biopsy, fi broblasts) mtDNA analysis (blood, fi broblasts) L/P < 15:1 Pyruvate dehydrogenase activity (fi broblasts) CSF Lactate + pyruvate, amino acids, protein Cranial MRI, MR spectrosopy Moderate creatine kinase Ý (<10× of normal), proximal or generalized muscle weakness Blood Lactate + pyruvate + L/P ratio (preferentially after glucose load) Urine Lactate, organic acids, oligosaccharides (if not done before) Electrophysiology EMG, NCV, EEG Skeletal muscle biopsy (Ultrastructural) morphology, histochemistry, enzyme studies DNA According to biopsy studies (mtDNA, nDNA) High creatine kinase Ý (>10× normal), proximal and limb girdle muscle weakness Man: DNA if negative: skeletal muscle biopsy Dystrophin gene mutation analysis (ultrastructural) morphology, immunhistology, histochemistry, enzyme studies Woman: skeletal muscle biopsy See above Moderate creatine kinase Ý (<10× normal), scapulo-peroneal muscle weakness + contractures (w/o AV block) DNA Lamin A/C (autosomal dominant), Emerin (X-chromosomal) If negative: skeletal muscle biopsy See above Moderate creatine kinase Ý (<10× normal), myotonia DNA Number of CTG repeats in DMPK gene Liver enzymes Ý (w/o hepatomegaly) Blood Lactate/pyruvate ratio (L/P) (preferentially after glucose load),

NH3, ferritin Liver biopsy (Ultrastructural) morphology, immunhistology, histochemistry, enzyme studies CGH comparative genomic hydridization; mtDNA mitochondrial DNA; nDNA nuclear DNA; EMG electromyogram; NCV nerve conduction velocity; EEG electroencephalogram; w/o with or without C1 Approach to the Patient with Cardiovascular Disease 85 propafenone, sotalol, or amiodarone as indicated. In Remember some cases, implantable converters may be indicated due to sustained ventricular tachycardia or symptom- In contrast to their beneﬁ t for ventricular function, atic arrhythmias. long-term enzyme replacement therapy, now avail- AV and ventricular conduction disturbances and able in MPS I, MPS II, and MPS VI, and allogeneic bradycardia are typical sign of the Kearns–Sayre syn- hematopoetic stem cell transplantation seem to have drome and other mitochondrial disorders with cardiac no or low beneﬁ t for the preexisting valvular lesions manifestation. and may only result in abbreviated progression of valvular changes.

Remember The mucolipidoses, especially mucolipidosis I (siali- Because of the potentially rapid progression, pace- dosis), can be complicated by mitral regurgitation. The maker implantation should be considered early in lipidoses, especially Gaucher disease and Farber dis- patients with neuromuscular disorders such as ease, may infrequently show storage in the connective Kearns–Sayre syndrome who have second degree tissues of the valves, causing thickening and subse- AV block or fascicular block, even when they are quent calciﬁ cation of the aortic and mitral valves, and asymptomatic. the development of nodules. The chordae tendineae may develop similar deposits. Functionally, there may be aortic or mitral stenosis or regurgitation. Stiffening and calciﬁ cation of the aortic and mitral valves occurs AV block may also occur in MPS II and VI and Fabry in alkaptonuria, a disorder of tyrosine catabolism that disease. causes darkening (ochronosis) and stiffening of cartilaginous tissues.

C1.4 Metabolic Disorders and Valvular C1.5 Thrombembolic and Vascular Abnormalities Disorders

Several metabolic disorders are associated with valvu- Morphological and functional abnormalities of blood lar dysfunction late in the course of the disorder. vessels occur in several metabolic disorders. Tortuosity Thickening of the valves, sometimes with calcifi ca- of vessels is particularly prominent in Menkes syn- tion, especially the aortic and mitral, occurs in several drome. Angiokeratomata of the umbilicus, buttocks, lysosomal storage disorders, including Hurler (MPS and genitalia are a feature of X-linked Fabry disease, I-H), Scheie (MPS I-S), Hunter (MPS II), Sanfi lippo where they appear in adolescence. In fucosidosis (auto- (MPS III), Morquio (MPS IV), and Maroteaux–Lamy somal recessive) they appear in the fi rst few years. (MPS VI) syndromes. Mitral and aortic regurgitation Hypercoagulability is a prominent aspect of homo- can be observed in more than 50% of MPS patients, cystinuria and the CDG syndromes which can lead to especially in MPS I, II, and VI: aortic and mitral steno- major occlusions of cerebral vessels or multiple small sis and mitral valve prolapse are less frequent. As a cerebral infarcts. Strokes are also a frequent feature in rule, prevalence of valvular abnormalities largely mitochondrial disorders, perhaps as a consequence of depends on the age of the patients. In some cases, post- endothelial dysfunction and gradual vascular occlusion. capillary pulmonary hypertension may also develop. Migraine with stroke and stroke-like episodes without Therefore, serial color Doppler echocardiography in residua or with persistent hemiplegia may all occur in regular intervals is required in patients with MPS to mitochondrial disorders, particularly MELAS. assess ventricular function and the progression of car- Beyond lipoprotein disorders, coronary atheroscle- diac abnormalities with age. rosis is accelerated in Fabry disease (including some 86 J. Kreuder and S. G. Kahler heterozygous females), and in some mucopolysaccha- hypercholesterolemia) or by mutations in the receptor ridoses. In MPS I, infi ltration of small and precapillary that recognizes apolipoprotein B100 protein on the arteries by storage material may result in severe isch- surface of LDL particles (familial defective apo emic cardiomyopathy in infants and young children. B-100), which may present with myocardial infarction and cerebrovascular disease even during the second decade in untreated homozygotes or severe compound heterozygotes. Therefore, screening and treatment of C1.5.1 Lipoprotein Disorders affected children have to take into account the severity of the phenotype, the long-term risk of developing vas- The long-term sequelae of lipoprotein disorders cular disease and available evidence of clinical benefi t (Table C1.5) lead to atherosclerotic vascular disease in in a group of diseases that are mostly asymptomatic all the arterial beds. Plasma elevation of low-density within childhood and adolescence. lipoprotein cholesterol (LDL-C), very low-density lipo- Fasting LDL-C and HDL-C levels should at least be proteins, lipoprotein(a), and reduced levels of high- determined in children with a positive family history density lipoprotein cholesterol (HDL-C) are well-known for premature cardiovascular events in parents or grand- risk factors for coronary heart and cerebrovascular dis- parents below the age of 60 years or with risk-burden ease. LDL-metabolism may be impaired by mutations diseases like diabetes mellitus, chronic kidney disease, of the hepatic cell-surface LDL-receptor (familial after orthotopic heart transplantation, or after Kawasaki

Table C1.5 Genetic lipoprotein disorders with signifi cantly increased cardiovascular risk Disorder Inheritance Prevalence LDL-C (mg/dL) HDL-C TG (mg/ Genetic Risk of (mg/dL) dL) lesion atherosclerosis (age at clinical presentation) Familial hypercholesterolemia Heterozygous AD 1:200–1:500 >135 n-↓ Normal LDL- ↑–↑↑. (Early receptor to mid adulthood) Homozygous AD 1:1,000,000 >425 <35 Normal LDL- ↑↑↑. (Starting in receptor fi rst decade) Familial defective apo B-100 Heterozygous AD 1:500–1:700 >135 n-↓ Normal Apo B-100 ↑ (Mid to late adulthood) Homozygous AD 1:1,000,000 >320 <35 Normal Apo B-100 ↑↑–↑↑↑ (Starting in second decade) Familial combined AD 1:20–1:50 150–250 n-↓ 150–400 Polygenic ↑–↑↑ (Mid hyperlipidemiaa to late adulthood) Familial dysbeta- AR 1:10,000 >130 n-↓ > 270 apoE-2/-2 ↑↑ (Early to lipo-proteinemia mid adulthood) Apolipoprotein A–I AR <1: 100,000 N <10 n Apo A-I ↑–↑↑. (Early defi ciency to mid adulthood) Sitosterolemia AR 1:1,000,000 Sitosterol↑. , N Normal ABCG5, ↑↑–↑↑↑ (homozy- campesterol↑ ABCG8 (Starting in gous) fi rst decade) AD autosomal dominant; AR autosomal recessive; LDL-C LDL-cholesterol; TG triglycerides; HDL-C HDL-cholesterol aPrevalence is age dependent C1 Approach to the Patient with Cardiovascular Disease 87 disease. Age-dependent normal levels of LDL-C and C1.6 Pulmonary Hypertension HDL-C, stratifi cation by underlying diseases and the assessment of additional risk factors [hypertension, Pulmonary arterial hypertension (PAH), a chronic vascu- overweight, impaired glucose tolerance, smoking, loproliferative disorder of the pulmonary arterial vascu- reduced HDL-C, and elevated lipoprotein(a)] may pro- lature, has been associated with a few inborn metabolic mote gradual treatment goals. disorders. PAH is defi ned as a mean arterial pressure ≥25 mmHg at rest, pressure values between 21 and 24 mmHg were recently classifi ed as borderline PAH. Remember In GSD Ia, PAH may be a late complication observed in about 10% of these patients during adolescence and Lifestyle modifi cation like diet and exercise and a adulthood. Abnormal production of serotonin and its lipid-lowering medication should aim for levels of release from thrombocytes has been suggested to be the LDL-C ≤100 mg/dL in the high-risk group, ≤130 mg/ most probable cause for pulmonary vasoconstriction and dL in patients with moderate risk, and ≤160 mg/dL remodeling of the vessel wall in GSD Ia. Several cases of in patients with a low, but increased risk of epide- unexplained PAH have been reported in association with miological evidence. Gaucher disease. Liver disease, splenectomy, capillary plugging by Gaucher cells, and enzyme replacement therapy could all play a role in the development of PAH. PAH may also be a rare manifestion of multisystemic Statins as HMG-CoA-reductase inhibitors are the fi rst- mitochondrial respiratory chain disorders. Preceding line drugs to lower increased LDL-C to target levels in liver disease and young age have been consistently found children ≥10 years. Ezetimibe inhibiting intestinal in these patients. Recurrent episodes of cyanosis or oxy- cholesterol absorption via the membranous Niemann– gen dependency due to pulmonary vasoconstriction result Pick C1-like 1 protein and LDL apheresis are addi- from the marked muscular thickening and intimal prolif- tional options in severe hyper lipoproteinemia. eration of the medium and small pulmonary arteries.

Table C1.6 Investigation in pulmonary arterial hypertension Aim/methods Parameter Value

Screening of PH Echocardiography Tricuspid valve regurgitation Normal <2.5 m/s Borderline 2.5–2.8 m/s Abnormal >2.8 m/s Veriﬁ cation of suspected pulmonary hypertension Right heart catherization Pulmonary artery pressure pulmonary Baseline hemodynamics changes of vasodilator studies including oxygen, pulmonary arterial pressure and nitric oxide, inhaled iloprost resistance (especially Rp/Rs ratio) Main differential diagnosis Disorders of the respiratory system Spirometry, oxygen diffusion capacity Chest HR-CT, sleep studies Grading of pulmonary hypertension Echocardiography Right atrium area, TAPSE, Tei-index, left ventricular eccentricity index Exercise testing 6-Min walk test, spiroergometry Assessment of additional risk factors Homocysteine >12 mmol/L Lipoprotein (a) >30 mg/dL Genetic thrombophilic factors FV-Leiden mutation (1691G > A), Prothrombin variation 20210G > A, MTHFR variation 677C > T Rp/Rs ratio pulmonary arterial resistance/systemic arterial resistance; HR-CT high-resolution computer tomography; TAPSE tricuspid annular plane systolic excursion 88 J. Kreuder and S. G. Kahler

Early detection of increased pulmonary pressure is pulmonary function and additional risk factors as well crucial to initiate medical therapy (e.g., endothelin as exercise testing and echocardiographic grading of antagonists, phosphodiesterase inhibitors, systemic, or pulmonary pressures and right ventricular function inhaled prostacyclin analogs). complete the diagnostic spectrum (Table C1.6).

Remember Routine echocardiographic assessment of right ven- Key References tricular performance and pulmonary pressure should be included in the follow-up of patients with meta- Böhles H, Sewell AC (eds) (2004) Metabolic cardiomyopathy. bolic disorders at risk for PAH. Medpharm Scientiﬁ c Publishers, Stuttgart Cox GF (2007) Diagnostic approaches to pediatric cardiomyopathy of metabolic genetic etiologies and their relation to Suspicion for PAH should be veriﬁ ed by right heart therapy. Prog Pediatr Cardiol 24(1):15–25 Lipshultz SE, Colan SD, Towbin JA, Wilkinson JD (eds) (2007) catherization and measurement of pulmonary artery Idiopathic and primary cardiomyopathy in children. Progress pressures including vasodilator studies. Assessment of in pediatric cardiology, vol 23 and 24/1. Elsevier, Amsterdam Liver Disease C2 Georg F. Hoffmann and Guido Engelmann

Key Facts and initiated early. Successful outcome depends › Liver disease is a common and important very much on early institution of specifi c ther- sequel of inherited metabolic diseases. apy. In addition to the increasing therapeutic › Defects in the degradation of fatty acids, fruc- options for individual disorders, pediatric liver tose, galactose, and glycogen as well as of gluco- transplantation has developed into a well-estab- neogenesis, ketogenesis, ammonia detoxifi cation, lished procedure, with the best outcome rates or oxidative phosphorylation can lead to signifi - of ³90% in liver-based metabolic disorders. cant liver disease. › Specifi c symptoms of these disorders refl ect response to the demand on the pathway affected; it includes pathological alterations of blood ammonia, glucose, lactate, ketone bodies, and pH. C2.1 General Remarks › Specifi c symptoms are often obscured by consequences of rather nonspecifi c responses of Despite the complex interrelated functions of the liver in the liver to injuries that lead to hepatocellular intermediary metabolism, the hepatic clinical response damage, decreased liver function, cholestasis, to inherited metabolic disease is limited and often indis- and hepatomegaly. tinguishable from that resulting from acquired causes, › Other inherited diseases interfere primarily such as infections or intoxications. The diagnostic labo- with hepatic cell integrity; these are Wilson ratory evaluation of liver disease must therefore be broad. disease, tyrosinemia type I, cystic fi brosis, Careful evaluation of the history and clinical presenta- a1-antitrypsin defi ciency, and defi ciencies of tion should include a detailed dietary history as well as biosynthetic pathways, such as in cholesterol a list of all medications, the general appearance of the biosynthesis, peroxisomal disorders, CDG patient, somatic and psychomotor development, signs syndromes, and defects in bile acids synthesis. of organomegaly, neurological signs, and an ophthal- Very striking physical involvement of the liver mologic examination. In combination with the results with relatively little functional derangement is of routine clinical chemical investigations (Table C2.1), seen in lysosomal storage disorders. this should lead to the suspicion of an inborn error of › The diagnostic laboratory evaluation of liver metabolism, the initiation of specifi c metabolic tests disease must be broad, especially in neonates, (Table C2.2), and to a provisional grouping into one of the four main clinical presentations (A – jaundice/ cholestatic liver disease, B – liver failure/hepatocellular necrosis, C – cirrhosis, or D – hepatomegaly). The family and personal history of the patient can G. F. Hoffmann ( ) especially be helpful in acquired as well as inherited University Children’s Hospital, Ruprecht-Karls-University, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany liver disease. Routes of infection may become obvious. e-mail: [email protected] Linkage of symptoms to oral intake of food or drugs

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 89 DOI: 10.1007/978-3-540-74723-9_2, © Springer-Verlag Berlin Heidelberg 2009 90 G. F. Hoffmann and G. Engelmann

Table C2.1 First line investigations in disease of the liver Table C2.2 Second line investigations in suspected metabolic Alanine and aspartate aminotransferases (transaminases) liver disease Lactate dehydrogenase Plasma/serum Cholinesterase Copper, ceruloplasmin Alkaline phosphatase a-Fetoprotein g-Glutamyltranspeptidase Cholesterol, triglycerides Serum iron and ferritin, transferrin, and transferrin Bilirubin, conjugated and unconjugated saturation Bile acids Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate Coagulation studies: PT, PTT, factors V, VII and XI Free fatty acids Albumin, prealbumin Amino acids Urea nitrogen, creatinine, uric acid, CK Free and total carnitine, acylcarnitine profi le Glucose a1-Antitrypsin activity and PI phenotyping Ammonia Lysosomal enzymes Hepatitis A, B, C Urine Cytomegalovirus, EBV, herpes simplex, toxoplasmosis, HIV Amino acids Viral cultures of stool and urine Ketones Reducing substances/ sugars (galactosemia, fructose Abdominal ultrasound (gall bladder before and after meal, intolerance) hepatic tumor) Organic acids (incl. specifi c assays for orotic acid and Ultrasound of the heart (vitium cordis, peripheral pulmonary succinylacetone) stenosis) Individual bile acids (bile acid synthesis defects) In infancy Copper in 24 h urine Rubella, parvovirus B19, echovirus and a variety of Sweat chloride (cystic fi brosis) enterovirus subtypes In infancy Bacterial cultures of blood and urine Galactose-1-phosphate uridyl transferase (galactosemia) PT prothrombin time; PTT partial thromboplastin time; EBV Chromosomes (especially when liver disease is accompa- Epstein–Barr virus; HIV human immunodefi ciency virus nied by malformations suggesting trisomy 13 or 18)

Free T4 and TSH (hypothyroidism) Transferrin isoelectric focusing (CDG syndromes) may almost be diagnostic in fructose intolerance, parac- Very long-chain fatty acids (peroxisomal disorders) etamol intoxication, or accidental poisoning. Another important key to diagnosis is the age at presentation. During the fi rst 3 months of life, the that does not react on phenobarbitone therapy, leads to majority of patients with liver disease, including those severe nonhemolytic jaundice. Severe neurological with inherited metabolic diseases present with conju- damage and death due to kernicterus is a common gated hyperbilirubinemia. Later in infancy, the presen- sequel. Patients with Crigler–Najjar type 2 have biliru- tation becomes broader (Fig. C2.1). bin levels of up to 20 mg/dL. Liver transplant has proven Only a few well-defi ned inherited diseases cause successful in a number of patients, and in future, unconjugated hyperbilirubinemia: hemolytic anemias Crigler–Najjar syndrome type 1 will certainly be an or impaired conjugation of bilirubin resulting from a attractive candidate for hepatocyte transplantation and recessive defi ciency of UDP-glucuronyl transferase in gene therapy (Table C2.3). Crigler–Najjar syndrome types 1 and 2 or due to a dom- Pediatric liver transplantation has developed into a inantly inherited mild reduction of UDP-glucuronyl well-established procedure. Patients with cholestatic transferase in Gilbert syndrome (bilirubin, 1–6 mg/dL). or liver-based metabolic diseases have the best out- The latter is a benign condition manifesting in neonates come rates of ³90% without signifi cant decline after only, if they are affl icted by a second hemolytic disor- the fi rst year after transplantation. Further progress can der, such as glucose-6-phosphatase defi ciency. In later be expected from auxilary partial orthotopic transplan- life, mild jaundice aggravated by fasting or intercurrent tation, when there is no signifi cant fi brosis and the illness is the only symptom, and the condition is often objective of treatment is the replacement of the miss- uncovered accidentally. Crigler–Najjar syndrome type 1, ing enzyme, e.g., in Crigler–Najjar syndrome type 1 or usually defi ned as bilirubin > 20 mg/dL or >360 mmol/L urea cycle disorders. C2 Liver Disease 91

!Investigate and exclude extra-hepatic biliary atresia and known infectious diseases!

Acutely ill Cholestasis Hepatomegaly Neurological symptoms

Tyrosinemia type I Glycogen storage diseases Urea cycle defects Wolman/cholesteryl ester storage Fatty acid oxidation defects disease Neonatal hemochromatosis Lysosomal storage diseases Glycogenosis type IV (mucopolysaccharidoses, Fructose intolerance mucolipidoses, etc.) Galactosemia Respiratory chain defects (Mitochondrial DNA depletion syndromes) Phosphomannose isomerase deficiency (CDG Ib) Niemann-Pick types A, B, C

Alpha 1-antitrypsin deficiency Urea cycle defects Tyrosinemia type 1 Peroxisomal defects Niemann Pick type C CDG syndromes Bile acid synthesis defects Progressive Familial Intrahepatic Cholestasis I-III Peroxisomal disorders Respiratory chain defects (Mitochondrial DNA depletion syndromes)

Fig. C2.1 Differential diagnosis of metabolic liver diseases in infants

Table C2.3 Hepatic diseases that may be cured by liver transplantation Disease Timing Acute Wilson disease If no response to therapy Neonatal hemochromatosis Soon if no response to antioxidant cocktail a-1 Antitrypsin deﬁ ciency In early infancy if cholestasis deteriorates Urea cycle disorders Early in the course to prevent further crises Tyrosinemia Type I Early in the course if no response to therapy PFIC I-III If pruritus and portal hypertension develops Primary hyperoxaluria Liver transplantation before development of renal impairment Combined LTX/NTX when CCR < 20 Crigler–Najjar Type 1 Before school age, consider liver cell or auxiliary transplantation Cystic ﬁ brosis If cholestasis deteriorates and portal hypertension develops Glucogenosis Type I In adolescents when risk of malignancy Questionable: organoacidurias, glucogenosis IV, mitochondri opathies

C2.2 Cholestatic Liver Disease (conjugated bilirubin > 15% of total). An important early sign is a change in the color of the urine from colorless C2.2.1 Cholestatic Liver Disease or only faintly yellow to distinctly yellow or even brown. The signiﬁ cance of this ﬁ nding is often missed, as this in Early Infancy color is similar to adult urine. Of course, by this time the sclerae are yellow – with chronic direct hyperbilirubine- Cholestatic liver disease may aggravate or prolong physi- mia, the skin becomes yellow and may have a greenish ological neonatal jaundice. It becomes obviously patholog- hue. Cholestasis frequently causes pale or acholic stool. ical when conjugated hyperbilirubinemia is recognized A colored stool, however, does not exclude cholestasis. 92 G. F. Hoffmann and G. Engelmann

Remember Disease Info: a1-Antitrypsin Defi ciency A stool specimen should always be looked at in the a1–Antitrypsin is one of the most important inhibi- fi rst visit of jaundiced neonates with conjugated tors of proteases (e.g., elastase, trypsin, chymotrypsin, hyperbilirubinemia. thrombin, and bacterial proteases) in plasma. Different protein variants of a1–antitrypsin are differentiated by isoelectric focusing. The Z variant is characterized Transient conjugated hyperbilirubinemia can be by a glutamine to lysine exchange at position 342 in observed in neonates, especially premature infants, the protein. It alters the charge and tertiary structure after moderate perinatal asphyxia. It is associated with of the molecule and is associated with reduced a high hematocrit and a tendency to hypoglycemia and enzyme activity. The frequency of this particular has an excellent prognosis, if the pathological condi- allele is very high in Caucasians; 5% of the populations described later have been excluded. tion of Sweden is MZ heterozygotes and 2% of the United States. The frequency of the homozygous Pi-ZZ phenotype is 1 in 1,600 in Sweden and 1 in Remember 6,000 in the United States. Normal serum levels of Cholestatic liver disease in infancy may be the ini- a1-antitrypsin are from 20 to 50 mmol/L, and in the tial presentation of cystic fi brosis, Niemann–Pick ZZ phenotype, 3–6 mmol/L. Patients with values disease type C and tyrosinemia type I. below 20 mmol/L should have PI phenotyping. Cholestatic liver disease occurs in 10–20% of infants with the Pi-ZZ a1-antitrypsin phenotype, which in turn accounts for 14–29% of the neonatal Biliary obstruction. Infants with cholestatic liver dis- hepatitis syndrome, once infectious and toxic causes ease often appear well. Early differentiation between have been excluded. Bleeding may occur as a result biliary atresia, a choledochal cyst, and a “neonatal hep- of defi ciency of vitamin K with prompt response to atitis syndrome” is most important. Biliary atresia intravenous vitamin K. The stool is acholic. Low accounts for 20–30% of neonatal cholestasis syndromes. serum a1-antitrypsin and genetic phenotyping usu- Structural cholestasis can also be due to intrahepatic ally leads to the diagnosis. In the acute stages, liver bile duct paucity. The syndromal form is Alagille syn- biopsies of infants with a1-antitrypsin defi ciency drome, in which a dominantly inherited dysplasia of the may show giant cell hepatitis with PAS-positive pulmonary artery occurs with distinctive dysmorphic diastase-resistant granules. features and paucity of intrahepatic bile ducts resulting Some patients with a1-antitrypsin defi ciency, in cholestatic liver disease. Intrahepatic bile duct pau- who had no history of neonatal cholestasis, develop city can appear without any other dysmorphies. Routine cirrhosis eventually and may present with unex- clinical chemical investigations can seldom differenti- plained liver failure. These patients have had the ate between biliary obstruction and neonatal hepatitis Pi-types ZZ, MZ, and S. In addition, hepatomas and syndrome, although low albumin concentrations and hepatocellular carcinomas have been described in decreased coagulation unresponsive to vitamin K point ZZ and MZ phenotypes. to a longer disease course such as in prenatal infections A major proportion of patients with a1-antitrypsin or inherited disorders. defi ciency remain free of liver disease throughout their life. In adulthood, 80–90% of patients with the Pi-ZZ phenotype develop destructive pulmonary emphy- Remember sema. a1-Antitrypsin is protective of the lung, because The differentiation between biliary obstruction and it is an effective inhibitor of elastase and other prote- neonatal hepatitis syndrome must be vigorously olytic enzymes, which are released from neutrophils pursued by ultrasonographic, possibly radioisotopic, and macrophages during infl ammatory processes. studies and by liver biopsy, as surgical correction of Children with the rare Pi null variant may have severe extrahepatic biliary atresia or a choledochal cyst dis- emphysema early in their life. Intravenous infusions of orders must be performed as early as possible to a1-antitrypsin have been shown to impede the devel- minimize intrahepatic biliary disease. opment of emphysema in the affected individuals. C2 Liver Disease 93

In dysmorphic infants with cholestasis, very long-chain Table C2.4 Differential diagnosis of metabolic cholestatic liver fatty acids, as well as chromosomes, should be investi- disease (conjugated hyperbilirubinemia) gated as peroxisomal diseases, and trisomies 13 and 18 Age of Diseases to be Additional fi ndings presentation considered are associated with the neonatal hepatitis syndrome <3 Months and biliary atresia (trisomie 18). Alagille syndrome a1-Antitrypsin ß a1-Globulin, defi ciency ß a1-antitrypsin has been discussed earlier. Cystic fi brosis Ý Sweat chloride A group of inherited metabolic diseases character- Tyrosinemia type I Ý AFP ized by progressive intrahepatic cholestasis starting Niemann–Pick type C Foam cells in marrow in infancy is becoming more recognized. These are Peroxisomal diseases Encephalopathy the defects in bile acid biosynthesis. Unfortunately Bile acid synthesis Prominent the necessary laboratory facilities to diagnose these defects malabsorption >3 Months Progressive familial Progressive cirrhosis treatable disorders are only available in a few labora- intrahepatic tories worldwide. Four different enzyme defects in the cholestasis (e.g., modifi cation of the steroid nucleus of bile acids have Byler disease) been elucidated. Malabsorption of fat-soluble vita- Rotor syndrome Normal liver function mins is pronounced and results in spontaneous bleed- Dubin–Johnson Normal liver function syndrome ing and rickets. The level of alkaline phosphatase is Known infectious diseases should have been ruled out by labo- often highly elevated, but this feature is not diagnos- ratory tests listed in Table C2.1 and extrahepatic biliary disease tic. Determination of total bile acids is not helpful. by imaging techniques More widespread availability of fast atom bombardment mass spectrometry for rapid analysis of urine samples will hopefully result in a quicker recognition of these disorders in the future. After diagnosis, there is a good clinical and biochemical response to supple- Disease Info: Progressive Familial Intrahepatic mentation with bile acids, such as cholic acid in com- Cholestasis (Byler Disease) bination with chenodesoxycholic or ursodeoxycholic acids. Progressive familial intrahepatic cholestasis (PFIC) is a genetically heterogeneous group of autosomal recessive liver disorders, characterized by cholestasis that progresses to cirrhosis and liver failure Remember before adulthood. Symptoms start later in infancy with jaundice, pruritus, growth failure, and conju- After exclusion of other diseases, infants with chole- gated hyperbilirubinemia. Liver function slowly static liver disease should be examined for the treat- deteriorates, and terminal liver failure usually occurs able defects of bile acid biosynthesis, which requires before 15 years. Liver transplantation has become determination of individual bile acid metabolites. the treatment of choice, but may be complicated by postoperative intractable diarrhea and pancreatitis. PFIC may be caused by a defi ciency of several hepa- tobiliary transporters. The term Byler disease is gen- C2.2.2 Cholestatic Liver Disease erally used for PFIC type 1 caused by a defi ciency of in Later Infancy and Childhood a P-type ATPase that is required for ATP-dependent amino phospholipid transport and encoded by the ATP8B1 gene on chromosome 18q21. Other PFIC After 3 months of age, the initial clinical and biochem- types are linked to the ABCB11 gene on chromosome ical presentation usually allows a clearer suspicion 2q24 (PFIC2) and the ABCB4 gene on chromosome and differentiation of inherited metabolic liver dis- 7q21.1 (PFIC3). PFIC4 is sometimes used as a ease than in neonates. In patients with a1-antitrypsin term for 3b-hydroxy-D5-C27-steroid oxidoreductase defi ciency, cholestatic liver disease gradually subsides defi ciency, a bile acid synthesis defect (see earlier) before 6 months of age. In later infancy, three distinct caused by mutations in the HSD3B7 gene on chro- metabolic diseases present with cholestatic liver disease mosome 16p12-p11.2, which has a similar pheno- (Table C2.4). 94 G. F. Hoffmann and G. Engelmann

The age of presentation (Table C2.6) as well as associ- type. Patients with PFIC or the clinically similar bile ated features (Table C2.7) may be helpful in directing acid synthesis defects have normal serum levels of g- the investigations. Jaundice is usually present, but glutamyltransferase (GGT). more important and characteristic features are elevated A different type of PFIC can be distinguished from liver transaminases and markers of hepatic insufﬁ - the defects of bile acid synthesis or secretion by high ciency, such as hypoglycemia, hyperammonemia, serum GGT activity and liver histology that shows hypoalbuminemia, decrease of vitamin K dependent, portal inﬂ ammation and ductular proliferation in an and other liver-produced coagulation factors. Failure early stage. The histologic and biochemical character- to thrive is mostly present. Deranged liver function istics of this subtype resemble those of the mdr2−/− may result in spontaneous bleeding or neonatal ascites, knockout mice very closely. Lack of MDR3 mRNA indicating end-stage liver disease. negative canalicular staining for MDR3 p-glycoprotein was demonstrated in the liver of patients. One patient was found to be homozygous for a frameshift deletion; another patient was homozygous for a nonsense Remember arg957-to-ter mutation. Interestingly, a heterozygous Encephalopathy associated with severe liver failure mother of an affected child experienced recurrent epi- is not necessarily obvious, especially, not in neonates sodes of intrahepatic cholestasis of pregnancy. and young infants.

Acute disease may progress rapidly to hepatic failure. Disease Info: Dubin–Johnson and Rotor In severely compromised infants, routine clinical syndromes chemical measurements do not help to differentiate acutely presenting inherited metabolic diseases from Dubin–Johnson and Rotor syndromes are both severe viral hepatitis or septicemia, although dispro- autosomal recessively inherited disorders charac- portionate hypoglycemia, lactic acidosis, and/or hyper- terized by isolated conjugated hyperbilirubinemia. ammonemia all point to a primary metabolic disease. Patients are usually asymptomatic except for jaun- Furthermore, it is not uncommon for septicemia to dice. Bilirubin levels can range from 2 to 25 mg/dL complicate and aggravate inherited metabolic diseases. (34–428 mmol/L). Both the conditions are rare and Help in the differential diagnosis may come from the can be differentiated by differences in urinary por- judgment of liver size. Decompensated inherited meta- phyrins and appearance of the liver, which is deeply bolic diseases are often accompanied by signifi cant pigmented in Dubin–Johnson syndrome and unre- hepatomegaly due to edema, whereas rapid atrophy markable in Rotor syndrome. Excretion of conju- can develop in fulminant viral hepatitis or toxic injury. gated bilirubin is impaired in both the disorders. In babies with acute hepatocellular necrosis, rapid The gene involved in Dubin–Johnson syndrome is diagnosis of the inherited metabolic diseases listed in the canalicular multispecifi c organic anion trans- Tables C2.5 and C2.7 is essential as specifi c therapy porter on chromosome 10. The gene defect in Rotor is available for most and must be initiated as soon as syndrome has not yet been identifi ed. possible. Metabolites accumulating in galactosemia (galactose-1-phosphate) and hereditary fructose intolerance (fructose-1-phosphate) have a similar toxicity C2.3 Fulminant Liver Failure particularly for the liver, kidneys, and brain but are usually differentiated by different clinical settings C2.3.1 Fulminant Liver Failure (different age groups) in which fi rst symptoms occur. Determination of amino acids in plasma and urine in Early Infancy and analysis of organic acids in urine (particularly succinylacetone, dicarboxylic acids, and orotic acid) The differential diagnosis in this age group is wide, should elucidate the presence of hepatorenal tyrosine- ranging from toxic or infectious causes to several inher- mia, fatty acid oxidation defects, and urea cycle ited metabolic diseases (Table C2.5). Mortality is high. disorders. C2 Liver Disease 95

Table C2.5 Differential diagnosis of fulminant liver failure (acute or subacute hepatocellular necrosis) Age of Diseases to be considered Additional fi ndings presentation <3 Months Neonatal hemochromatosis Ý Ý Ý Ferritin, Ý Ý Ý AFP Galactosemia Cataracts, urinary reducing substance Tyrosinemia type I Ý Ý AFP Urea cycle defects Ý Ý Ý Ammonia Respiratory chain defects Ý Ý Lactate Long-chain fatty acid oxidation defects Ý Lactate, Ý urate, Ý CK, Ý ammonia, (cardio-) myopathy, myoglobinuria Niemann–Pick types A, B, C Foam cells in marrow Phosphomannose isomerase defi ciency (CDG Ib) ß Transferrin isoforms 3 Months– Fructose intolerance ß Glucose 2 years Tyrosinemia type I Ý Ý AFP Fatty acid oxidation defects ß Glycose, Ý uric acid, Ý CK, ß ketones, Ý lactate, Ý ammonia Respiratory chain defects Ý Lactate Urea cycle defects Ý Ý Ý Ammonia >2 Years Wilson disease Corneal ring, hemolysis, renal tubular abnormalities, neurologic degeneration a1-Antitrypsin defi ciency ß a1-Globulin, ß a1-antitrypsin Fatty acid oxidation defects ß Glucose, Ý uric acid, Ý CK, ß ketones, Ý lactate, Ý ammonia Glycogenoses type VI/IX ± ß Glucose, Ý transaminases, Ý lactate, Ý CK Urea cycle defects Ý Ý Ý Ammonia Known infectious diseases should have been ruled out by laboratory tests listed in Table C2.1 and extrahepatic biliary disease by imaging techniques

Table C2.6 Age at presentation as a clue to the cause of hepatic failure in infancy from hepatic and renal failure. Whenever galac- 0–7 days Herpes simplex type 1 and 2 tosemia is suspected, adequate blood and urine tests mtDNA depletion should be initiated (galactose and galactose-1-phos- Neonatal hemochromatosis phate in serum, erythrocytes, or dried blood spots; 1–4 weeks Infections enzyme studies in erythrocytes) and a lactose-free Galactosemia Tyrosinemia diet should be started immediately. Galactose in 4–8 weeks Hepatitis B (vertical transmission) urine is not detected by standard stix tests based on 2–6 months Bile acid synthesis defects the glucose oxidase method (Clinistix® and Tes-tape®) 0.5–1 year Hereditary fructose intolerance and there is a strong argument for the continued use of mtDNA depletion the older methods of screening urine for reducing Wolcott Rallison syndrome substances (Benedict or Fehling test and Clinitest®), Viral hepatitis which also detect galactose. Urinary excretion of galac- Autoimmune tose depends on the dietary intake and will not be detectable 24–48 h after discontinuation of milk feed- Disease Info: Galactosemia ings. On the other hand, babies with severe liver disease from any cause may have impaired galactose metabo- Galactosemia is caused by a deﬁ ciency of galactose- lism and gross secondary galactosuria. After discontin- 1-phosphate uridyltransferase (GALT). Clinical uing galactose for 2–3 days, a baby with galactosemia symptoms usually start after the onset of milk feeds begins to recover. Cataracts may have developed in on the third or fourth day of life and include vomit- only a few days (Fig. C8.2, Chap. C8) and slowly clear ing, diarrhea, jaundice, disturbances of liver func- after removal of the toxic sugar. tion, or sepsis and may progress untreated to death 96 G. F. Hoffmann and G. Engelmann

Table C2.7 Neonatal liver failure Disorder Additional clinical features Mitochondrial hepatopathy, often mtDNA depletion Muscular hypotonia, multi-system disease, encephalopathy, lactate Neonatal hemochromatosis Hepatocellular necrosis, cirrhosis, ferritin, AFP, transaminases may be low Galactosemia Onset after milk feeds, jaundice, renal disease, cataracts Fatty acid oxidation disorders (Cardio)myopathy, hypoketotic, hypoglycemia, lactate Urea cycle disorders Ammonia, encephalopathy Niemann–Pick type C Jaundice, hypotonia, hepatosplenomegaly Glycosylation disorders (CDG, especially type Ib) Hepatomegaly, hepatocellular dysfunction, protein losing enteropathy, multi-system disease

Rarely: a1-Antitrypsin deﬁ ciency, bile acid synthesis disorders

Remember free and total carnitine, and analysis of acylcarnitines in addition to determinations of amino acids in In any baby who has received milk and developed blood and organic acids in urine should therefore liver disease, the investigation should include determi- be performed in any baby with progressive hepato- nation of the enzymatic activity of galactose-1-phos- cellular necrosis. If the clinical and biochemical phate uridyl transferase in erythrocytes, regardless of presentation is suggestive of a defect of fatty acid the results of neonatal screening. oxidation or of the respiratory chain, appropriate enzymatic confirmation in muscle and liver, or molecular studies of nuclear or mitochondrial DNA, Remember should be sought (see also Chaps. B2.3, D6, and Urinary elevations of succinylacetone may be small B2.5). If results are negative, they should be fol- in young infants, and repeated analyses with special lowed up by investigating defects of fatty acid oxi- requests for speciﬁ c determination by stable isotope dation in patients initially thought to have a defect dilution may be warranted in patients in whom clin- of the respiratory chain, and vice versa. Small ical suspicion is strong. infants with primary defects of fatty acid oxidation may present with overwhelming lactic acidosis and infants with severe liver disease due to defects of Genetic defects of fatty acid oxidation and of the the respiratory chain with hypoketotic hypoglyce- respiratory electron transport chain have become mia and dicarboxylic aciduria. recognized as causes of rapidly progressive hepatocellular necrosis in infancy. Defects of fatty acid oxidation are suggested in prolonged intermittent Remember or subacute presentations by myopathy, cardiomyo- When cardiomyopathy is present, lactic acidosis pathy, hypoketotic hypoglycemia, hyperuricemia, may be due to heart failure and poor perfusion. elevation of CK, moderate lactic acidosis, and dicarboxylic aciduria (see also Chap. B2.5). Defects of the respiratory chain causing hepatocellular necrosis are characterized by additional variable Disease Info: Neonatal Hemochromatosis multiorgan involvement, especially, of the bone Neonatal hemochromatosis should be considered in marrow, pancreas and brain, moderate to severe the differential diagnosis of rapidly progressive lactic acidosis, and ketosis (see also Chaps. B2.3 hepatocellular necrosis in infancy. As neonatal and D6). During acute hepatocellular necrosis in hemochromatosis has occurred in several diseases infancy, these differentiating features may be with known origin, such as prenatal infection, masked by generalized metabolic derangement. tyrosinemia type I, congenital hepatic ﬁ brosis, or Repeated determinations of metabolites such as Down syndrome, neonatal hemochromatosis can be lactate, pyruvate, 3-hydroxybutyrate, acetoacetate, C2 Liver Disease 97

The commonest form of acute hepatocellular necro- thought to present a clinicopathological entity sis in older children is acute or decompensated chronic rather than an individual disorder. However, in viral hepatitis. A small or rapidly decreasing liver size approximately 100 reported infants who presented is a strong argument against an inherited metabolic with liver disease from birth, no known cause could disease. Autoimmune disease must also be taken into be defi ned and extrahepatic siderosis and occur- consideration. Autoantibodies are present in the major- rence in siblings suggested a primary genetic disor- ity of affected children, and there usually is an increase der of fetoplacental iron handling. Diagnosis is by in IgG, serum protein, and autoantibodies. exclusion of other causes and demonstration of Disorders of fatty acid oxidation and urea cycle increased concentrations of serum iron, ferritin defects should be high on the list of differential diag- (>2,000 mg/L), and a-fetoprotein as well as nosis of children presenting with acute hepatocellular decreased concentrations of transferrin. Complete necrosis. If disproportionate hyperammonemia or or near complete saturation of iron-binding capac- hypoketotic hypoglycemia has been observed, vigor- ity is suggestive and demonstration of extrahepatic ous emergency measures should be promptly initiated siderosis appears to be especially characteristic. and diagnostic confi rmation sought by specialized The latter can be assessed by minor salivary gland metabolic investigations (Chap. B2.8). Both are poten- biopsy or an MRI of the liver. Successful treatment tially lethal in the acute episode. depends on early recognition and successful man- Tyrosinemia type I and fructose intolerance are usu- agement of hepatic failure, rapid referral to a trans- ally diagnosed in infancy or early childhood; urea plant center, and if the patient does not recover cycle and fatty acid oxidation defects can cause acute quickly, early liver transplantation (Table C2.3). hepatic dysfunction at any age. Some authors recommend an antioxidant cocktail In later childhood, Wilson disease and a1-antitrypsin in patients with neonatal hemochromatosis as well defi ciency are important metabolic causes of severe as Desferrioxamine. The outcome with and without hepatocellular necrosis. In the Asian race, citrullinemia this cocktail is extremely variable. Iron storage is type II (citrin defi ciency) is relatively common. Courses not permanently disturbed as survivors with and may be subacute or chronic, and initial presentation is without transplantation do not develop permanent variable, ranging from isolated hepatomegaly, jaundice iron-storage disease. or ascites to a chronic active hepatitis-like picture or acute liver failure. Hemolysis, when present, may be an important clue to Wilson disease. C2.3.2 Fulminant Hepatic Failure in Later Infancy and Childhood

Fulminant hepatic failure in later infancy or early child- Disease Info: Tyrosinemia Type I hood may present in a similar fashion to that in neonates Tyrosinemia type I (fumarylacetoacetase defi ciency) with elevated transaminases, hypoglycemia, hyperam- usually presents with pronounced acute or subacute monemia, decrease of coagulation factors, spontaneous hepatocellular damage, and only occasionally with bleeding, hypoalbuminemia, and ascites. Mortality is cholestatic liver disease in infancy. Patients who high with liver transplantation sometimes the only thera- have an acute onset of symptoms very quickly peutic option (Table C2.3). On liver biopsy hepatocellu- develop hepatic decompensation. They may have lar necrosis is obvious. Renal tubular dysfunction or jaundice and ascites along with hepatomegaly. There rickets is indicative of an inherited metabolic disease. In may be gastrointestinal bleeding. Several infants combination with early infancy insulin-dependent diabe- have been noted by their mothers to have a peculiar tis mellitus, Wolcott Rallison syndrome (OMIM #226980) sweet cabbage-like odor. Generalized renal tubular should be looked for by mutation analysis. The associa- dysfunction occurs, leading to glucosuria, amino- tion of acute hepatocellular necrosis with a prominent aciduria, and hyperphosphaturia. Very low levels of noninfl ammatory encephalopathy suggests a diagnosis phosphate in serum are common fi ndings, as are of Reye syndrome; many patients with this syndrome are hypoglycemia and hypokalemia. In some patients now found to have inherited metabolic disease. 98 G. F. Hoffmann and G. Engelmann

the diagnosis of tyrosinemia type I can be diffi cult, signs include hypertension, tachycardia, and ileus. as increases of tyrosine and methionine occur in Symptoms can continue for up to a week and many forms of liver disease. A highly elevated slowly resolve. These episodes should be much a-fetoprotein is very suggestive, but again not less frequent, now that there is therapy for this proof. Very high elevations of a-fetoprotein are also disorder. Intellectual function is usually not com- seen in neonatal hemochromatosis, which should promised in tyrosinemia type I. be differentiated on the basis of gross elevations of In the natural disease course, most children with iron and ferritin. Diagnostic proof of tyrosinemia tyrosinemia type I die in their early years, most of type I comes from the demonstration of succinylac- them before 1 year of age. Chronic liver disease is etone in urine and confi rmation of the enzyme that of macronodular cirrhosis. Splenomegaly and defect in fi broblasts or biopsied liver. esophageal varices develop and are complicated by If tyrosinemia type I does not become symptom- bleeding. A common complication is hepatocellular atic until later in infancy, patients usually follow a carcinoma, which may fi rst be suspected because of less rapid course. Vomiting, anorexia, abdominal dis- a further rise in the level of a-fetoprotein. Liver or tension and failure to thrive, rickets, and easy bruis- combined liver–kidney transplantation was the only ing may be the presenting features, and there is promising option of treatment until the advent of usually hepatomegaly. Although transaminases may 2(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclo- be normal or only slightly elevated, prothrombin time hexanedione (NTBC). Nowadays, liver transplanta- and partial thromboplastin time are usually markedly tion in tyrosinemia type 1 is only necessary in elevated, as is a-fetoprotein, which may range from nonresponders to NTBC therapy. The drug is a 100,000 to 400,000 ng/mL. Individual patients may potent inhibitor of p-hydroxyphenylpyruvate dioxy- present with different clinical pictures, such as with genase, thus preventing the formation of the highly acute liver disease and hypoglycemia as a Reye-like toxic fumarylacetoacetate, and its products succiny- syndrome. They may present with isolated bleeding lacetoacetate and succinylacetone. Vigorous com- and undergo initial investigation for coagulation dis- pliance is most important as hepatic malignancy orders before hepatic disease is identifi ed. may develop rapidly after a short interruption of or Renal tubular disease in tyrosinemia type I is that inconsistent treatment. If the treatment is instituted of a renal Fanconi syndrome with phosphaturia, early, hepatic and renal function slowly improve to glucosuria, and aminoaciduria (Chap. C4). There normal, and neurological crises are prevented. may be proteinuria and excessive carnitine loss. Concentrations of succinylacetone, a-fetoprotein, Renal tubular loss of bicarbonate leads to systemic and delta-aminolevulinate gradually decrease to metabolic acidosis. The affected infants have been near normal values. observed to develop vitamin D resistant rickets at less than 4 months of age. The differential diagnosis of the patient with elevated Beyond infancy, neurologic crises very similar levels of tyrosine in the blood include tyrosinemias type to those of acute intermittent porphyria are a more II and III and transient neonatal tyrosinemia. Tyrosinemia common cause of admission to the hospital than type II (tyrosine aminotransferase defi ciency), known hepatic decompensation. Succinylacetone inhibits as the Richner–Hanhart syndrome, results in oculocu- phorphobilinogen synthase (the enzyme affected taneous lesions, including corneal erosion, opacity, and in acute intermittent porphyria); increased urinary plaques. Pruritic or hyperkeratotic lesions may develop excretion of delta-aminolevulinate and porpho- on the palms and soles. About half of the patients bilinogen occurs during neurological crises. described have had low-normal to subnormal levels of About half of the patients experience such crises, intelligence, but this may refl ect bias of ascertainment. starting with pains in the lower extremities, fol- The phenotype of tyrosinemia type III (p-hydroxyphe- lowed by abdominal pains, muscular weakness, nylpyruvate dioxygenase defi ciency) is similar. It may or paresis and paresthesias. Head and trunk may include neurological manifestations such as psychomo- be positioned in extreme hyperextension, sug- tor retardation and ataxia but also remain asymptomatic. gesting opisthotonus or meningismus. Systemic As treatment with NTBC in patients with tyrosinemia C2 Liver Disease 99 type I shifts the metabolic block from fumarylacetoace- Hereditary fructose intolerance deserves consideration tate hydrolase to p-hydroxyphenylpyruvate dioxygenase as a cause of renal calculi, polyuria, and periodic or or from tyrosinemia type I to tyrosinemia type III, treat- progressive weakness or even paralysis. A major clue ment with NTBC must be supplemented by dietary treat- to diagnosis may be an accurate dietary history that ment with a phenylalanine- and tyrosine-reduced diet, will reveal an aversion to fruits and sweets. which is the rational approach to treatment in tyrosine- Characteristic clinical chemical laboratory fea- mias type II and III. tures include elevated transaminases, hyperbiliru- Metabolic investigations or neonatal screening, partic- binemia, hypoalbuminemia, hypocholesterolemia, ularly in premature infants sometimes detects tyrosinemia and a decrease of vitamin K dependent, liver-pro- and hyperphenylalaninemia not due to a defi ned inherited duced coagulation factors. There is an occasional metabolic disease. The protein intake is often excessive, pattern of consumption coagulopathy. In addition, especially, when an evaporated milk formula is being used. patients may have hypoglycemia, hypophosphatemia, This form of tyrosinemia is thought to result from physi- hypomagnesemia, hyperuricemia, and metabolic ological immaturity of p-hydroxyphenylpyruvate dioxy- acidosis in a renal Fanconi syndrome with proteinu- genase and is a warning that protein intake should be ria, glucosuria, aminoaciduria and loss of bicarbon- moderate during the fi rst weeks of life. Relative mater- ate, and high urine pH despite acidosis. In these nal vitamin C defi ciency may play a role. This condi- circumstances detecting fructose in the urine is virtu- tion is sometimes associated with prolonged jaundice ally diagnostic of the disease, but it may be absent. and feeding problems and can cause diagnostic confusion. Elevated plasma levels of tyrosine and methionine in combination with markedly elevated excretion of tyrosine and its metabolites in urine may mislead- Disease Info: Hereditary Fructose Intolerance ingly suggest a diagnosis of tyrosinemia type I. Symptoms develop in hereditary fructose intoler- The prognosis in hereditary fructose intolerance ance when fructose or sucrose is introduced into the depends entirely upon the elimination of fructose from diet. The recessively inherited defi ciency of fruc- the diet. After withdrawal of fructose and sucrose, tose-1-phosphate aldolase results in an inability to clinical symptoms and laboratory fi ndings quickly split fructose-1-phosphate into glyceraldehyde and reverse. Vomiting stops immediately, and the bleeding dihydroxyacetone phosphate. Fructose-1-phosphate tendency stops within 24 h. Most clinical and labora- accumulates in liver, kidney, and intestine. Pathophy- tory fi ndings become normal within 2–3 weeks, but sio logical consequences are hepatocellular necrosis hepatomegaly will take longer time to resolve. and renal tubular dysfunction, similar to what The excellent response to treatment supports a occurs in galactosemia. There is an acute depletion presumptive diagnosis of fructose intolerance. of ATP caused by the sequestration of phosphate Confi rmation of diagnosis should fi rst be attempted and direct toxic effects of fructose-1-phosphate. by molecular analysis. Several frequent mutations Depending on the amount of fructose or sucrose are known, such as A149P in Caucasians. An intra- ingested, infants may present with isolated asymptom- venous fructose tolerance test can usually not be per- atic jaundice or with rapidly progressive liver failure, formed any longer in diagnostically diffi cult cases as jaundice, bleeding tendency, and ascites, suggesting there are no i.v. preparations of fructose available. septicemia or fulminant viral hepatitis; hepatospleno- Demonstration of the enzyme defect in liver tissue megaly, if present, argues against the latter diagnosis. may be necessary in exceptional constellations. Postprandial hypoglycemia develops in 30–50% of the patients affected with fructose intolerance and may progress to coma and sudden death. Most patients Mitochondrial DNA depletion syndromes are increas- present subacutely with vomiting, poor feeding, diar- ingly identifi ed as causes of rapidly progressive liver rhea, or sometimes failure to thrive. Pyloric stenosis disease. Forms known so far are caused by defects in and gastroesophageal refl ux are common initial diag- the deoxyguanosine kinase, polymerase-g (POLG), noses. The clinical picture may be more blurred in MPV17, recessive Twinkle helicase (PEO1) genes, as later life and laboratory fi ndings may be unrevealing. well as in the EIF2AK3 gene. 100 G. F. Hoffmann and G. Engelmann

Disease Info: Alpers Disease episode may subside spontaneously. In the presence of splenomegaly, the diagnosis may appear to be In patients with Alpers disease, liver failure mostly infectious mononucleosis. Recurrent bouts of hepa- occurs later in the course of disease, often triggered titis and a picture of chronic active hepatitis in chil- by the use of valproic acid. Mutation in the POLG dren above the age of 4 years are suggestive of gene results in defects in respiratory chain com- Wilson disease. These patients experience anorexia plexes I and IV. Despite fulminant liver failure, and fatigue. Hepatosplenomegaly is prominent. In aminotransferases typically are only mildly ele- some patients isolated hepatosplenomegaly may be vated. Most patients with Alpers disease present at discovered accidentally. Any hepatic presentation preschool or school age with neurological symp- of Wilson disease leads to cirrhosis; the disease toms like seizures or even epileptic state and epilep- may present as cirrhosis. The histological picture is sia partialis continua. Patients’ history usually indistinguishable from chronic active hepatitis. reveals mild and then progressive psychomotor Terminally there may be hepatic coma or a hepator- development. However, liver failure may develop enal syndrome. before signifi cant neurological disease. If in an Hemolytic anemia may be a prominent feature unclear situation a therapeutic liver transplantation of Wilson disease in children, and its presence is is being considered, mutation analysis of the POLG very suggestive of the diagnosis. Renal tubular dis- gene should be pursued as soon as possible. Alpers ease is subtle; a generalized aminoaciduria often syndrome due to mutations in the POLG gene can displays an unusually high excretion of cystine, be considered a contraindication for transplantation other sulfur-containing amino acids and of tyrosine. as the children will continue to develop a progres- Later there may be a full-blown Fanconi syndrome, sive fatal encephalopathy. and patients may develop renal stones or diffuse nephrocalcinosis. Disease Info: Wilson Disease In adults, the onset of Wilson disease is classically neurological, predominantly with extrapyra- Wilson disease, or hepatolenticular degeneration, is midal signs. Choreoathetoid movements and characterized by the accumulation of copper in var- dystonia refl ect lenticular degeneration and are fre- ious organs and low serum levels of ceruloplasmin. quently associated with hepatic disease. This pic- Clinical manifestations are highly variable, but ture has been associated with poor prognosis. hepatic disease occurs in »80% of the affected indi- Progression tends to be much slower in patients viduals. This is usually manifest at school age, presenting with parkinsonian and pseudosclerotic rarely before 4 years of age. About half of the symptoms, such as drooling, rigidity of the face, patients with hepatic disease, if untreated, will and tremor. Speech or behavior disorders, and develop neurological symptoms in adolescence or sometimes frank psychiatric presentations, can adulthood. In other patients with Wilson disease, occur in children. The lack of overt liver disease can neurological manifestations are the presenting make diagnosis diffi cult. Dementia develops ulti- symptoms. mately in untreated patients. One extreme of the clinical spectrum of Wilson The availability of effective treatment for Wilson disease is a rapid fulminant course in children disease by copper-chelating agents makes early over 4 years of age progressing within weeks to diagnosis crucial. A key fi nding is the demonstra- hepatic insuffi ciency manifested by icterus, ascites, tion of Kayser–Fleischer rings around the outer clotting abnormalities, and disseminated intravas- margin of the cornea as gray–green to red–gold pig- cular coagulation. This is followed by renal insuf- mented rings (Fig. C2.2). Slit lamp examination fi ciency, coma, and death, sometimes without may be required. The rings are diffi cult to be seen in diagnosis. green–brown eyes. They are pathognomonic in neu- Wilson disease can also present with a picture of rological disease, but they take time to develop and acute hepatitis. Nausea, vomiting, anorexia, and are absent in most children who present with hepatic jaundice are common presenting complaints, and the disease. C2 Liver Disease 101

Biochemical diagnosis of Wilson disease may sometimes be diffi cult. The diagnosis is usually made on the basis of an abnormally low serum ceruloplasmin, liver copper content, and urine copper content plus mutation analysis. In 95% of patients, ceruloplasmin is below 20 mg/dL (200 mg/L) (control children 25–45 mg/dL). However, intermediate and even normal values have been reported in patients. Levels of copper in the serum are usually elevated, but measurement of urine copper is more reliable. Urinary excretion of copper is increased to >100 mg/day (1.6 mmol/day in about 65% of patients) (control children < 30 mg/day (0.5 mmol/day) ). Further increase in urinary copper excretion can be provoked by loading with 450 mg of d-penicillamine 12 h apart while collecting urine for 24 h. Controls Fig. C2.2 Kayser–Fleischer ring in Wilson disease. Courtesy of Prof. Dr. Wolfgang Stremmel, Heidelberg, Germany excrete less than 600 mg (9.4 mmol)/day, whereas in Wilson disease excretion ranges from 1,600 mg (25 mmol) to 3,000 mg (47 mmol)/day. The most sen- Remember sitive test is the measurement of the concentration The association of liver disease with intravascular of copper in the liver, and this test may be required hemolysis and renal failure is highly suggestive of for diagnosis. Disposable steel needles or Menghini Wilson disease as are bouts of recurrent hepatitis needles should be used. Patients with Wilson dis- and a picture of chronic active hepatitis in children ease usually have highly elevated concentrations of above the age of 4 years. copper in the liver (>250 mg (4 mmol) of copper per gram of dry weight; heterozygotes 100–200 mg/g, controls < 50 mg (0.8 mmol) ). Elevated concentra- C2.4 Cirrhosis tions of copper in the liver may also be found in children with extrahepatic biliary obstruction or Cirrhosis is the end stage of hepatocellular disease. cholestatic liver disease. The list of diseases that can result in cirrhosis and fail- Liver histology is not specifi c. Rodamin or other ure of the liver is extensive and includes infectious, staining techniques for copper are not sensitive in infl ammatory and vascular diseases, biliary malforma- childhood Wilson and therefore do not contribute tions, as well as toxic and fi nally metabolic disorders. to the diagnosis. As 80% of patients with Wilson’s Cirrhosis can result from most of the diseases dis- disease show at least one of the 200 known muta- cussed in this chapter. Exceptions are primary defects tions in the ATP7B gene, molecular diagnosis of of bilirubin conjugation and some of the storage disor- Wilson disease is a helpful tool in confi rming the ders that lead to hepatomegaly. diagnoses. Glycogenosis type IV, branching enzyme defi ciency, Once the diagnosis of Wilson disease is estab- can cause primarily cirrhotic destruction of the lished, family members (particularly sibs) should be liver. In Wilson disease, and even more frequently in thoroughly examined. Examination of liver tissue a1-antitrypsin defi ciency, a cirrhotic process may be will be necessary in the case where an asymptomatic quiescent for a long period with no evidence of signs or sibling has a low ceruloplasmin level, elevated liver symptoms of liver disease. Patients may be recognized enzymes and high copper urine, which can be con- following family investigations, during investigations of sistent with heterozygosity as well as homozygosity. an unrelated disease, or the disease may remain unrec- Early diagnosis must be vigorously pursued, as the ognized until decompensation leads to full-blown liver best prognosis has been demonstrated following failure. Specifi c metabolic diseases leading to cirrhosis early treatment of asymptomatic patients. are listed in Tables C2.8 and C2.9. 102 G. F. Hoffmann and G. Engelmann

Table C2.8 Differential diagnosis of cirrhosis Age of presentation Diseases to be considered Additional ﬁ ndings <1 Year Glycogenosis type IV Myopathy Galactosemia Cataracts, urinary reducing substance Neonatal hemochromatosis ÝÝÝ iron, ÝÝ ferritin, ÝÝ AFP, ß transferrin Tyrosinemia type I ÝÝ AFP >1 Year a1-Antitrypsin deﬁ ciency ß a1-Globulin, ß a1-antitrypsin Wilson disease Corneal ring, hemolysis, renal tubular abnormalities, neurologic degeneration Tyrosinemia type I Ý AFP Known infectious and autoimmune diseases should have been ruled out by laboratory tests listed in Table C2.1 and extrahepatic biliary disease by imaging techniques

Table C2.9 Chronic hepatitis or cirrhosis in older children Remember Disorder Clinical features Wilson disease Neurological and renal disease, a1-Antitrypsin should be quantiﬁ ed in any child, corneal ring adolescent, or adult in the differential work-up of Hemochromatosis Hepatomegaly, cardiomyopathy, cirrhosis. diabetes mellitus, diabetes insipidus, hypogonadism

a1-Antitrypsin Failure to thrive, ¯ a1-antitrypsin As cirrhosis progresses, signs and symptoms of decom- defi ciency pensation eventually emerge. Regardless of the primary Tyrosinemia type I Coagulopathy, renal disease, failure disease patients develop weight loss, failure to thrive, to thrive, AFP Hereditary fructose Symptoms after fructose intake: muscle weakness, fatigue, pruritus, steatorrhea, ascites, intolerance hypoglycemia, renal disease, or anasarca, as well as chronic jaundice, digital clubbing, failure to thrive, urate spider angiomatoma and epistaxis, or other bleeding Transaldolase Hepatosplenomegaly, cirrhosis (single (Table C2.10). Complications of cirrhosis include por- defi ciency patient) tal hypertension, bleeding varices, splenomegaly, and Cystic fi brosis Failure to thrive, recurrent airway infections encephalopathy. Terminally there may be hepatic coma. Coeliac disease Failure to thrive, diarrhea, small Liver transplantation provides the only realistic therapy. stature In some cases bridging to transplantation with an albumin dialysis or liver cell transplantation may be indicated.

Disease Info: Glycogenosis Type IV Table C2.10 Signs and symptoms of liver cirrhosis Infants with this rare form of glycogen storage General Malnutrition, failure to thrive, muscle disease often present around the fi rst birthday wasting, hypogonadism, elevated with fi ndings of hepatic cirrhosis, an enlarged temperature, frequent infections CNS Lethargy progressing to coma, behavioral nodular liver and splenomegaly. Hypotonia and changes, depression, intellectual muscular atrophy are usually present and may be deterioration, pyramidal tract signs, severe. Treatment is symptomatic and palliative. asterixis Transplantation of the liver is curative in predom- Gastrointestinal Nausea and vomiting, splenomegaly, caput inant liver disease; untransplanted patients usu- tract medusa, hemorrhoids, epistaxis hematemesis, abdominal distension, ally succumb to complications of cirrhosis before steatorrhea, ascites the age of 3 years. Cardiomyopathy and myopa- Kidney Fluid and electrolyte imbalance, progres- thy are often present. sive renal insuffi ciency The disorder is due to a defi ciency of the branch- Skin Jaundice, fl ushing, pruritus, palmar ing enzyme a-1,4-glucan: a-1,4-glucan-6-glucosyl erythema, spider angiomatoma, digital clubbing transferase that leads to a decrease in the number of C2 Liver Disease 103

branch points making for a straight chain of insolu- C2.5 Hepatomegaly ble glycogen like starch or amylopectin. The content of glycogen in liver is not elevated, but the Hepatomegaly is often the fi rst clinical sign of liver abnormal structure appears to act like a foreign disease. Two clinical aspects are helpful to the diag- body causing cirrhosis. The diagnosis can be estab- nostic evaluation of the patient: fi rst, the presence or lished by enzyme assay of leucocytes, cultured absence of splenomegaly and second, the consistency fi broblasts, or liver. and structure of the enlarged liver.

Remember a1-Antitrypsin defi ciency (see textbox chapter C2 Splenomegaly, especially hepatosplenomegaly, is page 4) is an important cause of neonatal cholestasis as the hallmark of storage diseases. well as of chronic active hepatitis (v. r.). In patients with a1-antitrypsin defi ciency manifesting cholestatic liver disease in infancy, cholestasis gradually subsides Functional impairment of the liver, such as decreases before 6 months of age and patients become clinically of coagulation factors and serum albumin or impaired unremarkable. However, 20–40% of these children glucose homeostasis, is usually absent in lysosomal develop hepatic cirrhosis in childhood. storage diseases, and aspects of liver cell integrity are unremarkable. Exceptions are Niemann–Pick diseases, both the types A/B and C (Table C2.11). In lysosomal Remember storage diseases, the liver and spleen are fi rm but not hard on palpation. The surfaces are smooth and the About 50% of apparently healthy children with the edges easily palpated. The liver is not tender. There homozygous Pi-ZZ phenotype have subclinical may be a protuberant abdomen and umbilical hernias. liver disease, as indicated by elevated levels of ami- Hepatosplenomegaly may lead to late hematological notransferases and g-glutamyltranspeptidase. complications of hypersplenism. A presumptive diag- In the presence or absence of a history of neona- nosis of lysosomal storage disease is strengthened by tal cholestasis or hepatits, a1-antitrypsin should be involvement of the nervous system and/or mesenchy- quantifi ed in any child, adolescent, or adult with mal structures resulting in coarsening of facial appear- unexplained liver disease. ance and skeletal abnormalities. Macroglossia makes a

Table C2.11 Differential diagnosis of hepatomegaly Age of presentation Diseases to be considered Additional fi ndings <3 Months Lysosomal storage diseases, specifi cally Splenomegaly Wolman disease Adrenal calcifi cations CDG syndrome type I Lipodystrophy, inverted nipples Defects of gluconeogenesis ß Glucose, Ý lactate Mevalonic aciduria Severe failure to thrive, splenomegaly, anemia 3 Months–2 years Glycogen storage diseases ± ß Glucose, Ý lactate, Ý lipids, myopathy Defects of gluconeogenesis ß Glucose, Ý lactate Lysosomal storage diseases Splenomegaly a1-Antitrypsin defi ciency ß a1-Globulin, ß a1-antitrypsin >2 Years Hemochromatosis Diabetes mellitus, hypogonadism Cystic fi brosis Pulmonary involvement, malnutrition, Ý sweat chloride Lysosomal storage diseases, specifi cally Splenomegaly Niemann–Pick, type B Pulmonary infi ltrates Cholesterol ester storage disease Hypercholesterolemia Glycogenosis type VI/IX ± ß Glucose, Ý lactate, Ý CK Fanconi–Bickel syndrome Fanconi syndrome 104 G. F. Hoffmann and G. Engelmann storage disorder virtually certain. In addition, slow hepatomegaly is discovered during physical exami- gradual progression is evident. nation. On palpation, the liver may feel fi rm but not The diagnostic work-up for lysosomal storage dis- hard and the surface is smooth. The liver may be orders in a patient with hepatosplenomegaly may start tender. Clinical or routine clinical chemical studies with the investigation of mucopolysaccharides and oli- (Table C2.1) are likely to reveal abnormalities which gosaccharides in urine. Positive results are followed up direct further diagnostic evaluation. Inherited meta- with confi rmatory enzymatic studies. If mucopolysac- bolic diseases considered in this category have been charides and oligosaccharides are negative, lympho- discussed under acute or subacute hepatocellular cytes are investigated for vacuoles (D5 – Pathology). If necrosis (Table C2.5). negative, bone marrow is investigated for storage cells. If the enlarged liver feels hard, is not tender, and has If storage cells are found, corneal clouding is sought sharp or even irregular edges, a detailed evaluation of with a slit lamp. If both are present, N-acetylglucosami- causes of cirrhosis should be performed even in the pres- nylphosphotransferase is determined to make a diagno- ence of unremarkable liver function tests. A hard irregu- sis of mucolipidoses II or III. If corneal clouding is lar or nodular surface is virtually pathognomonic of not present, potential enzymes to be determined are cirrhosis. Metabolic causes of silent liver disease associ- sphingomyelinase (Niemann–Pick type I, A and B), ated with hepatomegaly, which may lead to quiescent cir- acid lipase (Wolman), and cholesterol uptake and stor- rhosis are Wilson disease and a1-antitrypsin defi ciency. age (Niemann–Pick type II or C). If there are neither Another important metabolic disorder is hemochromato- pathological urinary screening results nor storage cells sis, in which hepatomegaly may be the only manifesta- but peripheral neuropathy, the activity of ceramidase tion in adolescence and young adulthood. Although this is determined seeking a diagnosis of Farber disease. disease usually does not progress to hepatic failure, as Histological, histochemical, electron microscopical, and does Wilson disease, early recognition and initiation of chemical examinations may be required of biopsied liver treatment allows the prevention of irreversible sequels. (D5 – Pathology). In patients with persistent isolated hepatomegaly, Any acutely developing liver disease due to infec- additional fi ndings are helpful in the differential diag- tious, infl ammatory, toxic, or metabolic origin may nosis and should be specifi cally sought (Table C2.12). cause hepatomegaly as a result of edema and/or Defects of gluconeogenesis result in severe recurrent infl ammation. In these disorders, other manifestations fasting hypoglycemia and lactic acidosis (Table C2.13). of the disease have usually led to consultation, and Of the three enzymatic defects of gluconeogenesis,

Table C2.12 Differential diagnosis of metabolic causes of hepatomegaly Additional fi ndings Suggestive disorder Cardiomyopathy Glycogenosis III, hemochromatosis, phosphoenolpyruvate carboxykinase defi ciency, disorders of fatty acid oxidation and of oxidative phosphorylation Muscular weakness Glycogenoses III, IV, VI, and IX, fructose intolerance, disorders of fatty acid oxidation and of oxidative phosphorylation Enlarged kidneys Glycogenosis I, Fanconi–Bickel syndrome Renal Fanconi syndrome Glycogenoses I and III, Wilson disease, fructose intolerance, tyrosinemia type I, Fanconi–Bickel syndrome, mitochondrial disorders Hemolytic anemia Wilson disease, fructose intolerance Fasting intolerance, hypoglycemia Glycogenoses I and III, fructose-1,6-diphosphatase defi ciency, disorders of fatty acid oxidation and of oxidative phosphorylation Diabetes mellitus, hypogonadism Hemochromatosis Neurologic deterioration (Kayser–Fleischer ring) Wilson disease Early onset emphysema a1-Antitrypsin defi ciency Susceptibility to infections Glycogenoses Ib/c Malnutrition Cystic fi brosis Lipodystrophy, inverted nipples CDG syndrome type I Isolated Glycogenosis Typ IX C2 Liver Disease 105

Table C2.13 Hepatomegaly plus hypoglycemia Disorder Clinical features Glycogen storage disease I Hepatocellular dysfunction, large kidneys, triglycerides, urate, lactate Glycogen storage disease III Short stature, skeletal myopathy Fanconi–Bickel disease Tubulopathy, glucose/galactose intolerance Disorders of gluconeogenesis Lactate Glycosylation disorders (CDG, e.g., type Ib) Hepatomegaly, hepatocellular dysfunction, protein losing enteropathy, multisystem disease hepatomegaly is a consistent fi nding in fructose-1, The value of early diagnosis cannot be overempha- 6-diphosphatase defi ciency, while patients with defi - sized, because optimal outcome can be achieved ciency of pyruvate carboxylase or phosphoenolpyruvate through treatment of pre- or oligo-symptomatic carboxykinase tend to present with lactic acidemia and patients, before the onset of irreversible damage. multisystem disease without hepatomegaly. Laboratory investigations of symptomatic patients Confronted with an infant or young child with a reveal increased concentrations of iron and ferritin moderately enlarged smooth, soft liver and other- in serum, as well as increased saturation of transfer- wise completely unremarkable history and physical rin of 77–100%. The diagnosis of hemochromatosis examination, investigations may be postponed until is made by determination of the iron content of the confi rmation of persistence of hepatomegaly on repeat liver. Molecular methods are now available, as there clinical examinations a few weeks later. If unexplained are only two major mutations that account for nearly hepatomegaly persists or additional indications of liver all the mutant alleles. disease develop, a liver biopsy should be obtained for histological and electron microscopic investigation. Additional tissue should be frozen at −80°C for bio- C2.5.1 The Glycogenoses chemical analyses. Patients with hepatomegalic glycogenoses may present with a moderately enlarged smooth, soft liver and otherwise completely unremark- Isolated hepatomegaly is found in several glycogen able history and physical examination in infancy and storage diseases. The combined frequency varies con- early childhood. Similarly, patients with hemochro- siderably according to the ethnic background and matosis may present with hepatomegaly without addi- approximates 1:20,000–1:25,000 in Europe. The origi- tional symptoms in late childhood and adolescence. nal description was by von Gierke in 1929, and glycogenoses were the fi rst inborn errors of metabolism defi ned enzymatically by Cori and Cori in 1952. The current classifi cation of the glycogenoses has been extended to nine entities. Glycogenosis type 0, also Disease Info: Hemochromatosis referred to as aglycogenosis, is the defi ciency of glycogen synthetase. As the glycogen content in the liver is Recessively inherited hemochromatosis is one of the actually reduced, it is not a storage disorder but a disor- most common genetic disorders in Caucasians; prev- der of gluconeogenesis (see also Chap. B2.4). The alence rates are between 2 and 5/1,000. Hepatomegaly symptoms of glycogenoses types II, V, VII, and some- is the most frequent early manifestation. Usually it times IV are primarily those of muscle disease. develops in adolescence or young adults. Additional Advances in techniques of enzymatic and molecular symptoms that may be present include diabetes mill- diagnosis may provide defi nitive diagnosis without tus, hypogonadism, skin pigmentation, recurrent epi- requiring liver biopsy. However, quantitative determina- gastric pain, cardiac arrhythmias, and congestive tion of the glycogen content of the liver is still necessary heart failure. in the work-up of many patients with suspected glycogen Hemochromatosis is usually considered as an adult storage disease. Glucagon challenges may be helpful in disorder; however, early manifestations are being glycogenoses I and III (see (D8) Function Tests- increasingly recognized in adolescents and children. Monitored Prolonged Fast and Glucagon Stimulation). 106 G. F. Hoffmann and G. Engelmann

Disease Info: Glycogen Storage Disease Type I mal hemostasis and persistent oozing may complicate traumatic injuries or surgery. Glycogen storage disease type I results from a defi - Patients with glycogen storage disease type I ciency of any of the proteins of the microsomal non-a (type Ib/c) develop progressive neutropenia membrane-bound glucose-6-phosphatase complex. and impaired neutrophile functions during the fi rst In classic Ia glycogen storage disease (von Gierke year of life. As a result, recurrent bacterial infec- disease), glucose-6-phosphatase is defi cient. Type tions result including deep skin infections and Ib is due to defective microsomal transport of glu- abscesses, ulcerations of oral and intestinal mucosa, cose-6-phosphate, Ic to defective transport of phos- and diarrhea. In the second or third decade infl am- phate, and Id due to defective transport of glucose. matory bowel disease may develop. Molecular studies contradict types Ib and Ic as sep- In the presence of a suspicion of glycogen stor- arate entities. They are both caused by mutations in age disease type I, diagnosis is ascertained by liver the glucose-6-phosphate translocase and should be biopsy. Hepatocytes are usually swollen because of taken together as glycogen storage disease type I extensive storage of glycogen and lipids. The struc- b/c or type non-a. A variant type Ia results from a ture of the glycogen stored is normal. Care must be defi ciency of the regulatory protein; this has so far taken to obtain enough liver to allow the assay of only been reported in a single patient. glucose-6-phosphatase and, if need be, associated The hallmark of von Gierke disease is severe fast- transport proteins. Primary molecular diagnosis of ing hypoglycemia with concomitant lactic acidosis, glycogen storage disease type Ia and Ib/c is becom- elevation of free fatty acids, hyperlipidemia, elevated ing increasingly available. This is helpful in obviat- transaminases, hyperuricemia, and metabolic acido- ing liver biopsy. sis. Lactic acidosis may be further aggravated by Several late complications have been observed in ingestion of fructose and galactose, as the converted patients with type I glycogen storage diseases despite glucose is again trapped by the metabolic block in treatment. Most patients have osteoporosis and some the liver. Affected patients may be symptomatic in have spontaneous fractures. Hyperuricemia may result the neonatal period, when there may be hepatomeg- in symptomatic gout after adolescence. Xanthomas may aly, hypoglycemic convulsions, and ketonuria. develop. Pancreatitis is another consequence of hyper- However, the condition often remains undiagnosed triglyceridemia. Multiple hepatic adenomas develop, until hypoglycemic symptoms reappear in the course sometimes to sizable tumors. They are usually benign; of intercurrent illnesses or, when the infant begins to however, malignant transformation has occurred. Renal sleep longer at night, at 3–6 months of age. Infants complications include a Fanconi syndrome, hypercalci- are chubby in appearance, but linear growth usually uria, nephrocalcinosis, and calculi. Microalbuminuria lags. The liver progresses slowly in size. An immense may be followed over time by proteinuria, focal seg- liver down to the iliac crest is generally found by the mental glomerulosclerosis, intestitial fi brosis, and renal end of the fi rst year when the serum triglycerides failure. Pulmonary hypertension is a rare, although very reach very high levels. Because of the accumulation serious, complication in adult patients. of lipid the liver is usually soft and the edges may be diffi cult to palpate. With increasing activity of the child at around the fi rst birthday, the frequency of Disease Info: Glycogen Storage Disease hypoglycemic symptoms tends to increase. As in Type III any of the diseases that cause severe hypoglycemia, convulsions and permanent brain injury or even Glycogen storage disease type III results from a defi - death may occur. However, many children are quite ciency of the debranching enzyme, amylo-1,6- adapted to low glucose levels, and in the untreated glucosidase. The physical and metabolic manifestations state, the brain may be fuelled by ketone bodies and of liver disease are usually less severe than in type I lactate. Unusual patients may remain clinically glycogenosis, and fasting intolerance gradually asymptomatic of hypoglycemia until up to 2 years of diminishes over the years. The predominant long- age. Increased bleeding tendency may result in term morbidity of this disease is myopathy. In infancy severe epistaxis and multiple hematomas, and abnor- it may be impossible to distinguish type I and III on C2 Liver Disease 107

Defects of the phosphorylase system are sometimes clinical grounds. Hypoglycemia and convulsions listed as two or even three separate groups of glycogen with fasting, cushingoid appearance, short stature, storage diseases. When done this way, the category and nosebleeds characterize either disease. However, type VI is then restricted to rare primary defects of in contrast to type I glycogenosis, concentrations of hepatic phosphorylase, type VIII to impaired control of uric acid and lactate are usually normal. The transam- phosphorylase activation, and type IX to defi cient activ- inases are elevated. Creatine phosphokinase level is ity of the phosphorylase kinase complex. The phospho- elevated as well. This may be the earliest evidence of rylase kinase complex consists of four different myopathy. tissue-specifi c subunits. By far the most common of these In glycogen storage disease type III, glycogen defects is an X-linked recessive defect of the a-subunit of accumulates in muscle as well as in the liver. In phosphorylase kinase, which affects »75% of all patients approximately 85% of patients, both the liver and mus- with defects of the phosphorylase system (type IXa). cle are affected, and this is referred to as glycogenosis type IIIa. When the defi ciency is only found in the liver, it is referred to as IIIb. With time in many patients the major problem is Disease Info: Glycogen Storage Diseases a slowly progressive distal myopathy. It is charac- Type VI, VIII, and IX terized by hypotonia, weakness, and muscle atrophy. It is often notable in the interossei and over the The clinical symptoms of defects of the phosphory- thumb. Some patients have muscle fasciculations, lase system, types VI, VIII, and IX glycogenoses, suggestive of motor neuron disease, and storage has are similar to, but milder than in, types III or I. been documented in peripheral nerves. Weakness Hepatomegaly is a prominent fi nding, and may be tends to be slowly progressive. Ultimately the the only indication of a glycogen storage disease. patient may be wheel-chair bound. Rarely, the myo- Muscle hypotonia, tendency to fasting hypoglyce- cardium may be involved as well with left ventricu- mia, lactic acidosis, elevation of transaminases, and lar hypertrophy or even clinical cardiomyopathy. hypercholesterolemia are mild and may be normal Several functional tests have been designed to after childhood. differentiate glycogen storage disease type I from Following a glucose or a galactose load in gly- type III. Following a glucose load the initially ele- cogenoses type Vl, VIII, and IX blood lactate will vated blood lactate will decrease in glycogenosis show pathological increase from normal or only type I. In type III, lactate levels are usually normal moderately elevated levels. Overactive gluconeo- but rise postprandially. In type I gluconeogenesis is genesis results in lowered concentrations of plasma blocked and alanine concentration is increased. In alanine. The response to the administration of glu- type III, gluconeogenesis is overactive, resulting in cagon even after a 12–14h fast is usually normal. signifi cantly lowered concentrations of alanine. Diagnosis of glycogenoses type Vl, VIII, and IX One of the most useful tests is a glucagon challenge can be proven by demonstration of the enzyme defi - 2–3 h after a meal, which will yield a good response ciency in the affected tissue, liver, or muscle. in GSD type III (but no increase in glucose in GSD Primary molecular diagnosis has greatly facilitated type I). After a 14-h fast, glucagon will not usually the diagnostic process. provoke a rise in blood glucose in GSD type III, as all the terminal glycogen branches have been catabolized (see E7 – Function Tests, Monitored The rare hepatic glygogenosis with renal Fanconi syn- Prolonged Fast and Glucagon Stimulation). Finally, drome (Fanconi–Bickel syndrome, glycogen storage the diagnosis of glycogenosis type III is proven by disease type XI) was shown to be due to a primary demonstrating the defi ciency of the debranching defect of the liver-type facilitated glucose transport. enzyme amylo-1,6-glucosidase in leukocytes, Hepatomegaly with glycogen storage, intolerance to fi broblasts, liver, or muscle. Prenatal diagnosis is galactose, failure to thrive and consequences of full- possible through enzyme analysis in amniocytes or blown Fanconi syndrome are usually obvious in early chorionic villi. childhood. 108 G. F. Hoffmann and G. Engelmann

Remember Key References

Type I glycogenosis is the most serious of all Green A (ed) (1994) The liver and inherited diseases. J Inher hepatic glycogenoses because it leads to a com- Metab Dis 14(Suppl 1) plete blockage of glucose release from liver, Kelly DA (ed) (2004) Diseases of the liver and biliary system. impairing both glucose production from glycogen Blackwell, UK Suchy FJ (ed) (2007) Liver disease in children, 3rd edn. and gluconeogenesis. Cambridge University Press, Cambridge, MA Gastrointestinal and General Abdominal Symptoms C3

Stephen G. Kahler

Key Facts C3.2 Vomiting › Vomiting is typical of the organic acidurias and hyperammonemic syndromes. Vomiting is a characteristic feature of many disorders › Pain and constipation occur in the porphyrias of intermediary metabolism, including those character- and familial fevers. ized by acidosis (the organic acidurias), hyperammonemia due to disorders of the urea cycle, and fatty acid › Pancreatitis occurs with many organic acidu- oxidation defects (Table C3.1). Initial laboratory inves- rias, lipid and fatty acid disorders, and defects tigations for vomiting, including acid–base balance, of oxidative phosphorylation. lactate, ammonia, and urinary organic acids, can point Malabsorption and diarrhea can be due to dis- › the way to a diagnosis. orders of digestive enzymes, especially disac- Chronic or recurrent vomiting in infancy, leading to charidases, defects of carrier proteins, and formula changes before the underlying acidosis is dis- mitochondrial dysfunction. covered, is a common event in the organic acidurias Ascites occurs in many lysosomal disorders. › due to defects in the metabolism of branched-chain amino acids – propionic, methylmalonic, isovaleric acidurias, etc. (see also Chap. B2.2). The hyperammonemia of disorders of the urea cycle often provokes vomiting. In severe cases, this will be C3.1 General Remarks rapidly followed by deterioration of the level of consciousness, but in milder cases the vomiting can be intermittent. Hyperammonemia can also be prominent Gastrointestinal manifestations of metabolic disorders in the fatty acid oxidation disorders, especially MCAD include vomiting, diffuse abdominal pain, pancreatitis, deﬁ ciency. Symptoms in these disorders occur inter- slowed transit time and constipation, maldigestion and mittently and are triggered by fasting or intercurrent malabsorption (which may result in diarrhea), and infections. The appropriate investigations are discussed ascites. These symptoms may occur as part of a sys- in more detail in Chap. B2.5. temic disorder of intermediary metabolism in which Vomiting will lead to alkalosis, so the discovery of other symptoms predominate, or as the major or exclu- acidosis when investigating a vomiting infant or child sive symptoms. should immediately raise the possibility of an underlying organic aciduria, or loss of base (e.g., renal tubular acidosis).

Remember S. G. Kahler Department of Pediatrics, Division of Clinical Genetics, Hyperammonemia can provoke hyperventilation, University of Arkansas for Medical Sciences, 4301 W. leading to respiratory alkalosis, so the discovery of Markham Street, Little Rock, AR 72205, USA alkalosis when acidosis is expected (i.e., during a e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 109 DOI: 10.1007/978-3-540-74723-9_C3, © Springer-Verlag Berlin Heidelberg 2010 110 S. G. Kahler

explanation in others. However, there remain a sub- work-up for suspected sepsis, especially in an stantial number of children with this syndrome for infant) should immediately lead to measurement of whom a coherent explanation has not yet been found. the blood ammonia level. Impaired uptake of ketones into peripheral tissues has been suspected. This disorder is sometimes confused with ketotic hypoglycemia, but the blood glucose level is not abnormally low, the patients do not have the slight C3.2.1 Cyclic Vomiting of Childhood body build of many children with ketotic hypoglyce- (Ketosis and Vomiting Syndrome) mia, and the treatments for ketotic hypoglycemia (avoiding fasting, cornstarch at bedtime, etc.) do not Vomiting is a prominent feature of a relatively common seem to be particularly benefi cial in cyclic vomiting. and poorly understood condition characterized by ketosis and abdominal pain, and triggered by fasting, often in the setting of infection – otitis, etc. In some children, C3.3 Abdominal Pain episodes can also be provoked by strenuous exercise. Typically episodes begin in the second year and usually end the latest with puberty. The abdominal pain may be Crampy abdominal pain occurs with intestinal dys- intense, similar to that which occurs in diabetic ketoaci- function – malabsorption, infectious diarrhea, etc., or dosis. Urinary organic acid analysis shows prominent mechanical problems, while diffuse pain is often due ketosis, but no pathological metabolites, and acylcarni- to an infl ammatory response. Metabolic disorders are tine analysis shows prominent acetylcarnitine. Treatment usually not considered until several episodes of abdom- with intravenous glucose usually results in rapid resolu- inal pain have occurred without an obvious explanation of symptoms. Phenothiazine antiemetics are of tion. This section will address diffuse abdominal pain; limited use, but ondansetron can be quite benefi cial. pancreatitis is discussed separately. Abdominal pain Reassurance that this condition is diffi cult but not dan- due to the disorders discussed below is often intense, gerous is helpful. Very few patients with this pattern and may lead to surgical exploration for suspected of symptoms have been shown to have defi ciencies appendicitis. Recent advances in imaging may help of b-ketothiolase or succinyl-CoA:3-oxoacid CoA in lessening unnecessary surgery. On the other hand, transferase. Abdominal migraine appears to be the when the patient has a metabolic disorder associated with recurrent abdominal pain, the physician must be careful during each episode that appendicitis or another surgical problem is not being attributed to the meta- Table C3.1 Important causes of vomiting in metabolic disorders bolic disorder. Neonatal/early infancy or later, with or without encephalopathy Organic acidurias Urea cycle defects/hyperammonemia syndromes C3.3.1 The Porphyrias Fatty acid oxidation disorders With severe abdominal pain Porphyrias (acute intermittent porphyria, coproporphyria, The porphyrias are disorders of the synthesis of heme, a variegate porphyria) component of cytochromes as well as hemoglobin. As a symptom of associated pancreatitis Diffuse or colicky abdominal pain and constipation With acidosis/ketoacidosis occur in three of the hepatic porphyrias – acute intermit- Organic acidurias tent, variegate, and hereditary coproporphyria. All three With liver dysfunction show autosomal dominant inheritance, and enzyme Organic acidurias (chronic or recurrent) Urea cycle defects/hyperammonemic syndromes activity is typically ~50% of normal. The dominant Galactosemia porphyrias are among the few dominantly inherited Fructose intolerance enzymopathies. Tyrosinemia type I Symptoms are uncommon in childhood. Episodes Fanconi–Bickel syndrome of illness in all three of the dominant porphyrias with Fatty acid oxidation disorders (acute) abdominal symptoms appear to be related to increased C3 Gastrointestinal and General Abdominal Symptoms 111 activity of the fi rst step of porphyrin synthesis, heterozygote. Photosensitivity is more common in var- d-aminolevulinic acid synthase. Although many of the iegate porphyria than in hereditary coproporphyria. porphyrias intermittently have the characteristic red The diagnosis of any of the porphyrias can be diffi - (or dark) urine, this feature is not always evident, espe- cult. Sometimes a positive family history of porphyria cially in coproporphyria. Many of the porphyrias have or illness suggestive of porphyria (depression, recur- prominent photodermatitis, with reddening and easy rent abdominal pain, etc.) will be present. Dark or red blistering after sun exposure; hypertrichosis can also urine can be a major clue. A positive urine screening occur. In unusual cases, an individual (doubly heterozy- test (the Watson-Schwartz test or similar reaction) may gous) may have more than one form of porphyria and be present only during acute illness and cannot be present with severe symptoms, even in childhood. relied upon at other times. Urine PBG is quite reliable Acute intermittent porphyria (which does not have during times of clinical illness. Comprehensive evalua- skin lesions) is the commonest form in most populations. tion of blood, urine, and stool for porphyrins is the It is due to defi cient activity of porphobilinogen (PBG) most reliable diagnostic approach (see Table C3.2). In deaminase (also called hydroxymethylbilane synthase the conditions with abdominal pain erythrocyte por- and previously known as uroporphyrinogen synthase). phyrins are normal. Determination of enzyme activi- The incidence of carriers (heterozygote frequency) is ties or molecular analyses should not be the primary generally thought to be 5–10/100,000, but it may be as diagnostic measure. high as 1 in 1000 (Northern Sweden). A large proportion Diffuse abdominal pain also occurs in the familial (50–90%) has no symptoms. Episodes of mental depres- fevers. Familial Mediterranean fever (FMF) is an auto- sion, abdominal pain, peripheral neuropathy, or demyeli- somal recessive condition characterized by episodes nation may be triggered by ethanol, barbiturates, oral of noninfective peritonitis, pericarditis, meningitis contraceptives or other drugs, or hormonal changes, (Mollaret meningitis), orchitis, arthritis, and erysipelas- but often no precipitating factor can be identifi ed. like erythroderma. Onset in childhood is common in Hereditary coproporphyria, due to defects in copro- the severe forms. Amyloidosis leading to renal failure porphyrinogen III oxidase (coproporphyrin decarbox- can develop. FMF is so common in some ethnic groups, ylase), is generally milder than acute intermittent including Sephardic and Armenian Jews, and some porphyria, and is less likely to have neurological symp- Arab communities, that pseudodominant pedigrees are toms. Onset is unusual in childhood. The rare homozy- regularly encountered. Defects in pyrin (marenostrin), gote may have the onset of symptoms in infancy, with a protein in the myelomonocytic-specifi c proinfl amma- persistent jaundice and hemolytic anemia. tory pathway, is the underlying cause. Pyrin down- Variegate porphyria is due to protoporphyrinogen regulates several aspects of the infl ammatory response. oxidase defi ciency. The highest incidence is among Diagnosis is based on molecular testing of the FMF Afrikaners in South Africa, where the heterozygote fre- gene called MEVF. Most patients respond well to quency is 3 per 1000; about half will have symptoms, colchicine; interferon-alpha, thalidomide, etanercept, typically triggered by medications, and made worse infl iximab, and anikara have been helpful. by iron overload (e.g., consider concurrent hemochro- Autosomal dominant familial periodic fever is a matosis). Heterozygotes rarely develop manifesta- similar disorder, much less common than FMF. It tions in childhood, but symptoms can begin as early was originally reported in an Irish/Scottish family as in infancy in the rare homozygote or compound and called familial Hibernian fever. It is also called

Table C3.2 Characteristic laboratory ﬁ ndings in porphyrias with abdominal symptoms Disease OMIM Enzyme Urine Stool Acute intermittent porphyria 176000 Hydroxymethylbilane ALA, PBG, ±Protoporphyrin synthase ±coproporphyrin Hereditary coproporphyria 121300 Coproporphyrinogen ALA, PBG, Coproporphyrin oxidase coproporphyrin Variegate porphyria 176200 Protoporphyrinogen ALA, PBG, Protoporphyrin, oxidase ±Coproporphyrin ±Coproporphyrin ALA delta-aminolevulinic acid; PBG porphobilinogen 112 S. G. Kahler

TNF-receptor-associated periodic fever syndrome transferase (CPT) I. Lipid disorders, especially hyper- (TRAPS) and is caused by mutations in the tumor lipidemia due to lipoprotein lipase defi ciency, and necrosis factor receptor-1 gene TNFRSF1A. hypo/abetalipoproteinemia, are also often associated Abdominal pain and fever also occur in mevalonic with pancreatitis. Homocystinuria due to cystathionine aciduria, in the form known as hyper IgD with periodic b-synthase defi ciency is the aminoacidopathy most fever and in hemochromatosis. commonly associated with pancreatitis. Depletion of antioxidants (glutathione, vitamin E, selenium, etc.) may play a role in the pathogenesis of pancreatitis. The contribution of mutations in genes which can contrib- C3.4 Pancreatitis ute to pancreatitis in other situations (e.g., CFTR, SPINK1, and PRSS1) is not yet known. Pancreatitis remains one of the most mysterious of acute life-threatening conditions. Except for mechanical obstruction of pancreatic secretion due to gallstones, C3.5 Constipation/Slowed Transit/ the mechanisms of pancreatitis are not understood, even when a precipitating or proximate cause is known, Pseudoobstruction such as chronic ethanol use. Gallstones in children are usually associated with hemolytic disorders; in adults, Constipation and pseudoobstruction occasionally are there is often no obvious cause. manifestations of systemic metabolic disease, although The organic acidurias primarily associated with pan- in most cases they are due to diet, habit, or intestinal creatitis are in the catabolic pathways for branched- motility abnormalities associated with neuronal dys- chain amino acids. They include maple syrup urine function. The porphyrias are noted for constipation, disease, isovaleric aciduria, 2-methylcrotonyl-CoA car- especially during times of crisis. The syndrome of boxylase defi ciency, propionic aciduria, methylmalonic hyper IgD with fever may have constipation (c.f. FMF, aciduria, and b-ketothiolase defi ciency. Acidosis, keto- in which diarrhea is more likely). Constipation may sis, vomiting, and abdominal pain are common during also be prominent in the Fanconi–Bickel syndrome of episodes of metabolic decompensation in these disor- glycogen storage and renal tubular dysfunction and ders, so pancreatitis may not be suspected. Conversely, malonyl-CoA decarboxylase defi ciency. pancreatitis may be the presenting illness in mild forms In the organic acidurias and hyperammonemias con- of these conditions, especially isovaleric acidemia. A stipation can be troublesome. Altered bowel function search for an underlying organic aciduria (urine organic may lead to decompensation of the primary metabolic acids, plasma or blood acylcarnitines, and plasma disorder, because of the accumulation of intermediate amino acids) should therefore be part of the initial compounds (e.g., propionate and ammonia) produced by investigation of pancreatitis (Table C3.3). gut fl ora. Treatment of the constipation can lead to sig- Pancreatitis also occurs in disorders of oxidative nifi cant improvement in metabolic control. Metronida- phosphorylation, especially cytochrome oxidase defi - zole is often used to alter bowel fl ora, to diminish the ciency, and MELAS syndrome due to mutations in the production of propionate or methylmalonate in patients tRNA leucine gene, and carnitine-palmitoyl-CoA with disorders of propionate metabolism

Table C3.3 First-line investigations for pancreatitis Urinary organic acid analysis C3.6 Ascites Plasma amino acids Plasma total homocysteine Ascites is rarely the sole presenting symptom of meta- Blood spot or plasma acylcarnitine analysis Blood lipid profi le (lipoprotein electrophoresis) bolic disease, but it accompanies several of them. Selenium Ascites or hydrops occurs in many lysosomal diseases, Total glutathione including sialidosis (mucolipidosis I), galactosialidosis, Zinc sialic acid storage disease, Farber lipogranulomatosis, Vitamins A, C, E Gaucher disease, Niemann-Pick disease, mannosidosis, Calcium and mucopolysaccharidoses IV-A, VI. In severe or C3 Gastrointestinal and General Abdominal Symptoms 113 early-onset disorders, the ascites may occur before birth gene leads to impaired mitochondrial DNA synthesis. as nonimmune hydrops fetalis. Hydrops is especially Mutations in POLG (DNA polymerase gamma) are common in MPS VII (Sly disease–b-glucuronidase another cause of MNGIE, but without leukodystrophy. defi ciency). A similar autosomal recessive condition, whose cause is not known, has been called oculogastrointestinal myopathy or familial visceral myopathy with external ophthalmoplegia. There is destruction of the gastrointestinal C3.7 Diarrhea smooth muscle, whereas the myenteric plexus appears normal. Abdominal pain, diarrhea, diverticuli, and dila- Mitochondrial neurogastrointestinal encephalopathy tation of the bowel occur. Patients also suffer from a syndrome (MNGIE) (myo-, neuro-, gastro-intestinal demyelinating and axonal neuropathy, focal spongiform encephalopathy) is a generalized disorder of mitochon- degeneration of the posterior columns, ptosis, and exter- drial dysfunction. Onset of intestinal symptoms is in nal ophthalmoplegia. The onset is in childhood or ado- childhood or early mid adult life, and includes chronic lescence, with death by age 30 in many patients. Most diarrhea, stasis, nausea, and vomiting, resulting in reports of this condition are from the 1980s, and there impaired growth. Wasting and cachexia may occur. may be a considerable overlap with MNGIE, which was Skeletal growth may be retarded. There is eventual loss generally not excluded enzymatically or molecularly. of longitudinal muscle, diverticuli (which may rup- Chronic diarrhea also occurs in Menkes disease, ture), intestinal scleroderma, and pseudoobstruction. perhaps because of autonomic dysfunction. Electrophysiologic studies have shown visceral neuropathy with conduction failure. Prokinetic drugs have been generally ineffective. Lactic acidosis is often C3.8 Maldigestion present. The extraintestinal symptoms are variable but typical of a mitochondrial disorder (see Table C3.4). In vitro analysis of mitochondrial function reveals a Generalized impairment of digestion due to pancreatic variety of impairments, especially defi ciency of com- problems (e.g., cystic fi brosis) or liver disease is well plex I or complex IV, or combined defi cits. Mitochondrial known. Several disorders of digestion involve primary DNA analysis (liver and muscle) shows depletion and enzymes of carbohydrate metabolism (Table C3.5). multiple DNA deletions. Recurrences in sibs, high fre- A defi ciency typically results in severe watery (osmotic) quency of parental consanguinity, and lack of vertical diarrhea when the affected substrate or its precursors are transmission are consistent with an autosomal reces- ingested. There may also be excessive gas production sive inheritance. Mapping of MNGIE to distal 22q has been followed by identifi cation of pathologic mutations Table C3.5 Disorders of carbohydrate digestion in the gene ECGF1 (platelet-derived endothelial growth Disorder OMIM Enzyme Major factor). The gene product is also known as thymidine substrate phosphorylase and gliostatin. Impaired function of this Disaccharide 222900 Sucrase/ Sucrose intolerance I isomaltase Disaccharide 223000 b-Glycosidase Lactose Table C3.4 Extraintestinal manifestations in mitochondrial intolerance complex – neurogastrointestinal encephalopathy syndrome II – congenital lactase, Organ Findings alactasia glycosylce- ramidase Growth Slow; cachexia and wasting Disaccharide 223100 b-Glycosidase Lactose Brain Leukodystrophy intolerance III – complex – Increased CSF protein adult lactase lactase, Ataxia defi ciency glycosylce- Eye Ophthalmoplegia, ptosis ramidase Ear Sensorineural deafness Trehalase 275360 Trehalase Trehalose Cranial nerves Dysarthria, dysphonia, facial palsy defi ciency (mush- Heart Heart block rooms) Skeletal muscle Ragged-red fi bers, weakness Not recognized 154360 Glucoamylase Peripheral nerves Demyelinating neuropathy, axonal (maltase) degeneration I and II 114 S. G. Kahler and bloating. All are autosomal recessive. The Pearson of milk in mammalian nutrition after infancy. In a few marrow-pancreas syndrome, usually caused by deletion regions, including Northern Europe, unfermented milk of mitochondrial DNA, has exocrine pancreatic failure. remains an important dietary component after infancy. The same deletion (and pancreas failure) can be found The ability to digest milk is clearly an advantage, so in Kearns–Sayre syndrome. Failure of the exocrine and/ among individuals from those regions there are a minor- or endocrine pancreas and other endocrine organs may ity with lactose intolerance. (It may be that the custom of occur in a variety of other mitochondrial disorders (see drinking unfermented milk arose because of a chance Chap. D1). mutation that interrupted the usual decline in lactase activity with age.) Intestinal lactase activity depends on the summed expression from both the alleles. Differences in C3.8.1 Disaccharide Intolerance I – lactase expression do not reside in the coding sequence itself but in a cis-acting regulatory element. Sucrase/Isomaltase Defi ciency

This defi ciency is a rare cause of infantile diarrhea, which becomes evident with the introduction of sucrose C3.9 Malabsorption in the diet. There are several different mechanisms for enzyme defi ciency, including impaired secretion, abnor- Malabsorption in metabolic disorders can be due to mal folding, defi cient catalytic activity, and enhanced abnormalities in ion channels, transport molecules, destruction. carrier proteins for lipids, or cotransport molecules. Symptoms are attributable to the defi ciency of the substance not being properly absorbed (i.e., essential fatty C3.8.2 Disaccharide Intolerance II – acids and fat-soluble vitamins), and the effects due to Infantile Lactase Defi ciency and abnormally high amounts of the substance within the gut. Ion-channel defects distort the balance of water Congenital Lactose Intolerance and electrolytes, leading to diarrhea; abnormalities of transport molecules lead to diarrhea; and defi ciency of Congenital lactase defi ciency is extremely rare. In a cotransport molecule (e.g., intrinsic factor) can have nearly all cases, lactase defi ciency in infants and young major consequences because of the resulting defi - children is acquired through infection and loss of the ciency of an essential nutrient. First-line investigations mature brush border. for metabolic causes of malabsorption should include Congenital lactose intolerance appears to be distinct stool pH and analysis of reducing substances in stool. from congenital lactase defi ciency. In the former, there Characterization of stool sugars will confi rm what has is excessive gastric absorption of lactose (leading to been suggested by the history. A breath hydrogen test lactosemia and lactosuria), vomiting, failure to thrive, after dietary challenge can confi rm malabsorption. liver dysfunction, and renal Fanconi syndrome. This condition may be fatal if not recognized and treated by elimination of lactose from the diet. Interestingly, lactose becomes well tolerated after 6 months. The basis C3.9.1 Glucose–Galactose Malabsorption for this condition is not known. Glucose–galactose malabsorption is clinically similar to sucrase/isomaltase defi ciency –severe life-threatening C3.8.3 Disaccharide Intolerance III – watery diarrhea from early infancy. Elimination of glucose and galactose from the diet is necessary. Fortunately, Adult Lactase Defi ciency fructose is well tolerated so a fructose-based formula is effective. This condition is most often recognized in Adult lactase “defi ciency” is a common polymorphism. In middle Eastern Arab populations. The defect in the fact, declining expression of intestinal lactase during child- sodium–glucose cotransporter SGLT1 is due to muta- hood is the usual state, mirroring the declining importance tions in the solute-carrier (SLC) gene SLC5A1. C3 Gastrointestinal and General Abdominal Symptoms 115

C3.9.2 Electrolytes Tryptophan malabsorption (Hartnup disease) occurs as an autosomal recessive disorder that may be clinically silent. The transport of several neutral amino acids in the C3.9.2.1 Chloride Diarrhea intestine and kidney are involved, but tryptophan is the one most likely to be limiting. In the absence of adequate In this recessively inherited form of diarrhea, there is niacin in the diet there will be a defi ciency of nicotinic voluminous watery diarrhea with high chloride content acids and nicotinamide, resulting in a pellagra-like rash, (greater than the sum of sodium and potassium), from light sensitivity, emotional instability, and ataxia. In an early age. Polyhydramnios is common, perhaps uni- severe cases, this can progress to an encephalopathy versal. The defect is in the brush-border chloride/bicar- with delirium and seizures. There may be an increase in bonate exchange mechanism. Treatment with sodium stool indoles and urinary indican, refl ecting the action of and potassium chloride is effective. Mutations have intestinal bacteria on the unabsorbed tryptophan. The been discovered in the gene SLC26A3. diagnosis is suggested by fi nding high urinary levels of Defects of the Na+/H+ exchange mechanism result in the neutral amino acids alanine, serine, threonine, aspar- sodium diarrhea and metabolic acidosis. The presenta- agine, glutamine, valine, leucine, isoleucine, phenylala- tion is similar to congenital chloride diarrhea, but the nine, tyrosine, tryptophan, histidine, and citrulline. stool electrolyte composition shows high sodium and an Plasma levels of these amino acids are normal or slightly alkaline pH. Treatment with oral Na–K–citrate will nor- low. Mutations have been found in the gene SLC6A19. malize the electrolyte status of the patient. Mutations A few infants were reported to have methionine have been found in the serine protease inhibitor gene malabsorption. Clinical manifestations have included SPINT2. white hair, tachypnea, mental retardation, seizures, Microvillus inclusion disease is another recessive diarrhea, and a peculiar odor like that of hops drying in cause of intractable diarrhea in infancy, and perhaps an oast house. The urine may show increased alpha- the most common noninfectious cause. Jejunal biopsy hydroxybutyric acid after a methionine load. The con- shows intracytoplasmic inclusions, consisting of brush- dition appears to be autosomal recessive. No new cases border microvilli, suggesting that this is a disorder of have been reported in several decades. intracellular transport, impairing assembly. Mutations have been found in the myosin type 5 gene (MYO5B).

C3.9.5 Vitamins and Other Small C3.9.3 Protein-Losing Enteropathy Molecules

Protein-losing enteropathy, often due to infection or Vitamin B12 impaired function of intestinal lymphatics, is also a cardinal feature of the congenital disorder of glycosylation The intricate story of vitamin B12 illustrates the inter- CDG 1B due to phosphomannose isomerase deﬁ ciency. face between diet, digestion, absorption, and intermedi-

There may be liver disease and a bleeding diathesis. ary metabolism. Vitamin B12, synthesized by microbes, Unlike the other CDG syndromes, mental retardation and is the precursor of substances known as cobalamins. severe neurological problems do not occur. Diagnosis is Two forms are essential for human metabolism: based on isoelectric focusing of transferrin and enzyme Methylcobalamin is a cofactor for the remethylation of assay. Treatment with oral mannose is effective. The CDG homocysteine to methionine, and adenosylcobalamin is syndromes are discussed in greater detail in Chap. C10. used by methylmalonyl-CoA mutase. Defects of the cobalamin pathway can therefore cause problems with either or both of these reactions.

Vitamin B12 in food (primarily meat and dairy prod- C3.9.4 Amino Acids ucts) is fi rst released by digestion in the stomach. It binds to specifi c glycoproteins (r-proteins), especially The two main disorders of intestinal amino acid trans- transcobalamin I (TC I). In the duodenum, the pancre- port are those involving tryptophan and methionine. atic proteases act to release B12 again, and it now binds 116 S. G. Kahler to intrinsic factor produced by gastric parietal cells. Table C3.6 Disorders of transport molecules Disorder OMIM Gene/ Nutrient The intrinsic factor-B12 complex (B12–IF) binds to a specifi c receptor (cubilin) on ileal enterocytes. The transporter receptors internalize the B –IF, which then dissociates. Glucose/galactose 606824 SCL5A1 Glucose, 12 malabsorption galactose The absorbed B is then transported to the bloodstream, 12 Thiamine- 249270 SCL19A2/ Thiamine coupled to transcobalamin II (TC II). The B12–TC II responsive THTR1 complex is the main way of distributing newly absorbed megaloblastic anemia B12 to the tissues. However, most of the B12 in the Imerslund– 261100 CBN/ B –intrinsic bloodstream is bound to TC I, as methylcobalamin. 12 Grasbeck Cubulin factor Autosomal recessive genetic defects in intrinsic fac- syndrome; or complex tor or the ileal receptor system typically lead to megalo- Norwegian type AMN blastic anemia with neurologic changes (developmental megaloblastic delay, hypotonia, hyporefl exia, and coma). Onset is usu- anemia ally after the fi rst year. Infants in the fi rst year apparently Systemic primary 212140 SLC22A5/ Carnitine carnitine OCTN2 do not depend on the ileal receptor system for B12 uptake, defi ciency; which explains the lack of symptoms in young infants carnitine uptake with problems in this system. The receptor defect is the defect Imerslund–Grasbeck syndrome (megaloblastic anemia I), Acrodermatitis 201100 SLC39A4/ Zinc enteropathica ZIP4 which may have proteinuria as well as anemia. Defects Hereditary folate 229050 SCL46A1/ Folate in TC II, on the other hand, become apparent in the fi rst malabsorption PCFT/ few months with megaloblastic anemia, failure to thrive, HCP1 Unknown hRFT1 Ribofl avin and neurological delay. Congenital B12 defi ciency can occur in the offspring of vegan/vegetarian mothers who Unknown SCL5A6/ Biotin, alpha SMVT lipoate, have not had adequate intake of B . Defi ciency of B 12 12 pantoth- will lead to mild methylmalonic aciduria and hyperho- enic acid mocysteinemia. Infants with congenital B12 defi ciency may be fi rst recognized by increased propionylcarnitine defi ciencies of only a few are known at present. They on newborn screening. Late-onset B12 defi ciency is usu- include SCL19A2 for thiamine, SCL22A5/OCTN2 for ally due to lack of intrinsic factor due to atrophic gastri- carnitine, and SLC39A4 for zinc (Table C3.6). tis, other stomach problem, or an unexplained mechanism.

The defects of B12 metabolism after uptake from the bloodstream lead to variant forms of homocystinuria, methylmalonic acidemia, or both. Key References

Investigation of suspected B12 disorders includes determining erythrocyte indices, plasma homocysteine, Gross U, Hoffmann GF, Doss MO (2000) Erythropoietic and acylcarnitine analysis for propionylcarnitine, and urine hepatic porphyrias. J Inherit Metab Dis 23:641–661 Hediger MA, Romero MF, Peng JB et al (2004) The ABCs methylmalonic acid. Serum cobalamin levels are low of solute carriers: physiological, pathological and therapeu- in defects involving intrinsic factor and gut uptake, but tic implications of human membrane transport proteins. are normal or close to it in TC II deﬁ ciency (see also Introduction. Pﬂ ugers Arch 447:465–468 Chap. D8). Kahler SG, Sherwood WG, Woolf D et al (1994) Pancreatitis in patients with organic acidemias. J Pediatr 124:239–243 Many transporters and their genes are now known – Pfau BT, Li BU, Murray RD et al (1996) Differentiating cyclic there are at least 360 members of the solute-carrier SLC from chronic vomiting patterns in children: quantitative cri- system, organized into more than 40 groups. Clinical teria and diagnostic implications. Pediatrics 97:364–368 Kidney Disease and Electrolyte Disturbances C4

William L. Nyhan

Key Facts tubular dysfunction, and/or urinary tract calculi and crystalluria (v.i). › Hypochloremic metabolic acidosis with Renal pathology may also lead to failure to thrive, increased anion gap points to classic metabolic myoglobinuria (Chap. C6), unusual odor (see B1 Table disorders, e.g. methylmalonic acidemia. B1.1), and abnormal color (see B1 Table B1.2). Renal › Renal tubular acidosis is hyperchloremic and cystic disease is seen in Zellweger syndrome, carnitine there is no increase in anion gap; differential palmitoyltransferase II defi ciency, and multiple acyl- diagnosis is diarrheal disease. CoA dehydrogenase defi ciency (glutaric aciduria type II) › Urinary tract stone disease in pediatric popu- due to the defi ciency of electron transfer fl avoprotein lations should suggest an inborn error of meta- (ETF) and ETF dehydrogenase. Multiple microcysts bolism. lead to congenital nephrotic syndrome in CDG-Ia › Renal Fanconi syndrome is the common result (Table C4.1). of several inherited metabolic disorders that In methylmalonic aciduria, a major subset of patients cause renal tubular dysfunction. develops a variety of renal manifestations leading to › Renal tubular alkalosis is characteristic of the chronic renal failure. Isolated renal tubular dysfunction Bartter and Gitelman syndromes. Calcium may lead to acidosis and hyperchloremia along with excretion is decreased in Gitelman syndrome; it proximal renal tubular bicarbonate wasting. It may also is normal or increased in the presence of defec- be complicated by interstitial nephritis and renal glom- tive genes, which cause Bartter syndrome. erular failure. A hemolytic uremic syndrome has been observed in infants with cobalamin C disease, the combined methylmalonic aciduria and homocystinuria. Renal disease is also a late complication of glycogenosis type I (von Gierke disease). These patients may C4.1 General Remarks develop proteinuria, increased blood pressure, urinary tract calculi, or nephrocalcinosis. Many of these manifestations have been attributed to hyperuricemia. Kidney disease and disturbed fl uid and electrolyte homeo- However, there is also glomerulosclerosis and intersti- stasis are important sequelae of inherited metabolic dis- tial fi brosis leading to glomerular dysfunction that may ease. They may occur as presenting manifestations. end in renal failure. Therapy must be addressed promptly and must be continued. The most important renal manifestations of inherited metabolic disease are (recurrent) dehydration, renal Remember Different types of renal pathology, but especially W. L. Nyhan proximal tubular dysfunction (Fanconi syndrome) Department of Pediatrics, University of California, UCSD School and focal segmental glomerulosclerosis, can be the of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA result of a primary mitochondrial disorder. e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 117 DOI: 10.1007/978-3-540-74723-9_C4, © Springer-Verlag Berlin Heidelberg 2010 118 W. L. Nyhan

Table C4.1 Primary Renal Tubular Disorders Table C4.2 Recurrent Or Episodic Dehydration Cystinuria: amino acid transport defect Hyperchloremic acidosis Diabetes insipidus: usually vasopressin receptor defect Failure to thrive, Renal tubular acidosis Bartter syndrome: electrolyte transport defects in the loop of rickets, polyuria Cystinosis Henle Diarrhea Lactase defi ciency Gitelman syndrome: Na/Cl cotransport defect in the distal Sucrase, isomaltase defi ciency, tubule Glucose galactose Hypophosphatemic rickets: pathological activation of malabsorption fi broblast growth factor 23 Acrodermatitis enteropathica Renal tubular acidosis (RTA) type I: defi cient distal tubular Hypochloremic alkalosis H+ secretion Polyuria Bartter and Gitelman RTA type II: defi cient proximal tubular HCO - secretion 3 syndromes RTA type IV: reduced aldosterone effect, usually drug Diarrhea Congenital chloride diarrhea induced Diarrhea, hypoproteine- Cystic fi brosis mia, anemia Vomiting Pyloric stenosis, bulimia C4.2 Dehydration Hypernatremia Nephrogenic diabetes insipidus Diabetes insipidus Hyponatremia, Congenital adrenal Renal and electrolyte abnormalities present frequently hyperkalemia hyperplasias, with acidosis and dehydration, and this may be indica- adrenal aplasia, hypoplasia, tive of a metabolic disease (Table C4.2). More com- pseudohypoaldosteronism monly, this picture of abnormal clinical chemistry is Hypochloremic acidosis Propionic acidemia caused by infectious diarrhea. Increased anion gap, Methylmalonic aciduria Rarely, chronic diarrhea is caused by an inherited ketoacidosis Diabetic ketoacidosis Isovaleric acidemia disorder of intestinal absorption (C3). Patients with clin- 3-Oxothiolase defi ciency ical manifestations of dehydration, decreased skin turgor, depressed fontanel, sunken eyes, decreased urine output, or documented acute loss of weight are fortu- Acute infectious diarrhea can precipitate an attack of nately regularly assessed by measuring the electrolyte metabolic imbalance in many inborn errors of metabo- concentrations, acid–base balance, urea nitrogen, and lism, but in these situations the metabolic abnormality creatinine in the blood. Among the clinical signs of predominates and there is an anion gap. Dehydration dehydration is poor skin turgor. Not so widely known is resulting from inherited disease is also characterized the fact that turgor is measured by the relative rate that a by recurrent or episodic attacks of dehydration. pinched up piece of skin becomes restored to its resting state. In fact, in rats, measured weight loss in experimentally induced dehydration had a straight-line corre- Remember lation with the stop-watched time of the skin to fl atten. Metabolic acidosis with increased anion gap indi- Electrolyte analysis in the dehydrated patient with cates either ketosis (®check ketostix, measure renal disease often reveals hyperchloremic acidosis. ketones in blood), the presence of pathological The anion gap is not increased. Most commonly, this organic acids (®examine urinary organic acids, picture results from acute diarrhea, although chronic blood lactate), or both. diarrhea can lead to chronic acidosis and chronic dehydration. Hyperchloremic acidosis also results from renal tubular acidosis (RTA) (v-i). A combination of the electrolyte pattern and the clinical manifestations permits a ready dissection of the heritable causes of recurrent dehydration. Actually, Remember many of the chronic diarrheas, such as the disacchari- dase defi ciencies, do not often lead to dehydration. Metabolic acidosis resulting from the classic inborn Patients are so accustomed to their problem that they errors of intermediary metabolism is hypochlore- compensate with ample fl uid intake. The water intake mic, and the anion gap is increased (Chap. B2-2). of infants and children with nephrogenic diabetes C4 Kidney Disease and Electrolyte Disturbances 119 insipidus is enormous. These patients often get into they are rare in children. In adults, the composition of trouble when exogenous forces such as intercurrent ill- the stone is usually a salt or a mixture of salts of cal- ness interfere with their ability to compensate. cium, calcium oxalate, and calcium phosphate. Admission to hospital and a requirement for parenteral fl uids, even with fairly trivial surgery, can lead to major morbidity and mortality if physicians do not recognize Remember and supply large quantities of water necessary to main- In total, 60–70% of calculi found in pediatric popu- tain these patients. lations result from inborn errors of metabolism Hypochloremic alkalosis results from depletion (Table C4.3) (Fig. C4.1). of intracellular potassium resulting from intestinal losses. In small infants, it is the hallmark of pyloric stenosis. Infants with congenital chloride diarrhea and those with cystic fi brosis (CF) both have diarrhea, but Table C4.3 Urinary Tract Calculi those with CF are edematous from hypoproteinemia Stone Disorder Enzyme and pale from anemia. Those with Bartter syndrome Cystine Cystinuria Uric Acid Lesch–Nyhan HPRT and Gitelman syndrome do not have diarrhea. A teen- PRPP synthetase PRPP synthetase ager or adult with bulimia can mimic Bartter syndrome; superactivity so can one with chronic laxative abuse. Glycogenosis I Glucose-6- phosphatase The adrenal hormone defi ciency diseases are readily Renal hypouricemia URAT1 (SLC22A12 recognized in those females with ambiguous genitalia. gene) They are often missed in those without, such as male 2,8-Dihydro- APRT defi ciency APRT xya denine infants with adrenal hyperplasia or infants with absent Xanthine Xanthinuria Xanthine oxidase or hypoplastic adrenals or with pseudohypoaldoster- Oxalate Oxaluria, glycolic Alanine:glyoxylate onism. The hyponatremia and renal salt wasting and the aciduria amino-transferase hyperkalemia should be giveaways for the diagnosis. Oxaluria, glyceric d-Glycerate aciduria dehydrogenase Calcium salts Hypercalciuria + Multifactorial uricosuria C4.3 Urinary Tract Calculi Wilson disease P-type ATPase transporter HPRT hypoxanthine guanine phosphoribosyl transferase; PRPP Kidney stones or calculi occurring anywhere in the uri- phos-phoribosylpyrophosphate; APRT adenine-phosphoribosyl- nary tract represent a common affl iction among adults; transferase; ATPase adenine triphosphatase

Fig. C4.1 Urinary calculus passed by a hyperuricemic 2-year-old boy with Lesch– Nyhan disease. Reprinted with permission from Nyhan et al. (2005) 120 W. L. Nyhan

Lithiasis in the urinary tract may present with pain, day and also at night to keep cystine soluble. Cystine which is referred to as renal colic. This sudden onset crystallizes at concentrations above 1,250 mmol/l at pain is of such extreme intensity that it may lead to pH 7.5. Treatment with penicilliamine is effective by nausea and vomiting. The pain refl ects the movement the formation of mixed disulfi des with cysteine, which of a stone along the ureter, and may be well localized are soluble. by the patient to this curvilinear distribution. Pain may Uric acid stone disease is seen commonly in Lesch– disappear on entry of the calculus into the bladder, Nyhan disease as well as the other variant hypoxan- only to reappear on entry into the urethra. There may thine-guanine phosphoribosyltransferase defi ciencies. be associated frequency or dysuria. Some patients may Patients with phosphoribosyl-pyrophosphate (PRPP) have these symptoms as a result of crystalluria as well synthetase superactivity and with glycogenosis type I as with discrete stones. Hematuria can also result from also overproduce purine and excrete it as uric acid in crystalluria or calculi. large quantities. Deafness is common in patients with PRPP synthetase mutations. Other patients with uric acid calculi have uricosuria as a result of increased Remember urate clearance by the kidney. Some well-defi ned kin- Clinical symptoms in small children with urinary dreds have been reported. Most of them have normal tract calculi are often nonspecifi c (abdominal pain, blood concentrations of uric acid; some are hypourice- nausea, vomiting, and recurrent urinary tract infec- mic and some, like the Dalmatian dog, are hyperurice- tions). Urinary tract ultrasound is indicated in all mic. Mutations in the SLC22A12 gene lead to children with microhematuria. abnormalities in URAT1, the predominant transporter of uric acid at the apical membrane of the renal tubule. Urinary tract infection is a common complication of In interpreting values of uric acid in plasma or urinary tract calculi. It may present with pyuria, dysu- serum, it must be remembered that children have an ria, and fever. Infection will usually not disappear until especially high clearance capacity for uric acid and the stone is removed, by passage, lithotripsy, or sur- consequently can keep serum levels within normal gery. Repeated episodes of pyelonephritis and obstruc- ranges despite a pathologically increased endogenous tion may lead to renal failure. production (Table C4.4). Urinary concentrations of Patients with symptoms should not only be exam- uric acid give more reliable results. For diagnostic pur- ined for the possibility of stones, but also those with poses, a random urine sample should be promptly ana- diseases in which calculi are common should be moni- lyzed for uric acid and creatinine. Age-related control tored. Stones containing calcium are radiopaque, but ranges are summarized in Table C4.5. urate stones (Fig. C4.1) are not; cystine stones are radiopaque but may be diffi cult to visualize roentgeno- graphically. Ultrasonography is useful in detecting Table C4.4 Investigations In Children With Renal Stones hydronephrosis or hydroureter, but may miss small Ultrasound scan of renal tract (stones? dilatation?) stones. Intravenous urography is useful in delineating Determine urinary oxalate, calcium, citrate, cystine (® amino acids), uric acid (® purines) calculi and defi ning the presence or absence and degree Urine microscopy of obstruction. Retrograde pyelography permits visu- Consider intravenous urography, CT alization without intravenous injection of the dye, but it requires cystoscopy. CT scan may permit detection of radiolucent stones not evident with other methods. Table C4.5 Uric Acid Excretion In Control Subjects Cystinuria results from mutations in the gene, which Age (years) <2 2–4 4–8 8–10 10–12 >12 is responsible for the cystine transporter of the kidney upper limit <2a <1.5 <1.3 <1.1 <1.0 <0.8 and intestine. It leads to increased urinary excretion of Ua/Crea a lysine, ornithine and arginine, as well as cystine. Only lower limit <0.5 <0.5 <0.4 <0.3 <0.3 <0.3 Ua/Crea cystinuria causes symptoms, all the consequence of its aIn very young infants, especially, during the fi rst week of life lack of solubility and consequent formation of calculi. the standard deviation is extremely high (99th percentile between Treatment is by ensuring ample fl uid throughout the approximately 0.2 and 2.8) C4 Kidney Disease and Electrolyte Disturbances 121

Fig. C4.2 Massive calcium oxalate material obtained from a 6-month-old patient with hyperoxaluria type II

Table C4.5 provides estimates of uric acid/creati- Adenine phosphoribosyltransferase defi ciency, com- nine ratios in morning urine samples (Kaufman et al. mon in Japanese, leads to increased excretion of the very 1968), which can be taken as a possible hint to either insoluble dioxygenated derivative of adenine. Xanthine elevate or reduce excretion of uric acid. A few facts oxidase defi ciency is associated with stones composed have to be borne in mind when interpreting uric acid of the very insoluble xanthine and with hypouricemia. excretion in spot urine samples in general and using Xanthine stones are also observed in patients with hype- this table in particular. ruricemia treated with allopurinol, but in many it is pos- The fi gures are estimated from a continuously falling sible to fi nd a dosage regimen that minimizes or avoids curve with varying standard deviations at different ages the propensity for stone formation, because hypoxan- (Kaufman et al. 1968). By that they represent “forced thine is very soluble. mean values” for the given age group and give an approx- The two forms of oxaluria, known as type I and imation of the normal range of uric acid excretion. type II, are characterized by very early onset renal Patients with Lesch–Nyhan-Syndrome (HPRT defi ciency) stone disease (Fig. C4.2) and early renal failure. In seem to be always clearly above those upper limits. type I, there is glycolic aciduria as well as hyperoxalu- In patients with either “borderline fi ndings” with this ria, in type 2 there is glyceric aciduria. Successful proposed uric acid/creatinine ratio or high clinical suspi- treatment has combined transplantation of both liver cion of primary or secondary disturbances of uric acid and kidney. Hyperoxaluria and calcium oxalate calcu- metabolism, a determination of the 24 h excretion cor- lus formation has also been reported in cystic fi brosis. rected for body surface is probably more accurate. 520 ± 147/1.73 m2/24 h (mean ± 1 SD) is given by Wilcox (1996) based on several previous publications. As an C4.4 Renal Tubular Dysfunction alternative, correction of uric acid excretion for creatinine clearance gives a constant value between 3 and 40 Fanconi Syndrome years of age: 0.34 ± 0.11 mg/dL of glomerular fi ltrate (mean ± 1 SD) according to Wilcox (1996). Glomerular The Fanconi syndrome represents a generalized dis- fi ltrate was estimated from simultaneous measurement ruption of renal tubular function in which the proximal of serum creatinine (Scr), urinary creatinine (Ucr), and uri- tubular reabsorption of amino acids, glucose, phosphate, nary uric acid (Uua) in overnight fasted patients. All the bicarbonate, and urate is impaired, leading to a general- values are in mg/dL, using the following equation: ized aminoaciduria, glycosuria, phosphaturia, uricosu-

(Ucr) × (Scr)/(Ucr) = uric acid excreted in mg/dL of ria, and increased urinary pH. As a consequence, there glomerular ﬁ ltrate. is vitamin D-resistant rickets and osteomalacia. 122 W. L. Nyhan

tyrosinemia, usually as the major clinical feature, and Remember the diagnosis is made by urinary organic acid analysis Fanconi syndrome is characterized by polyuria and for succinylacetone. Hepatic dysfunction is also seen increased urinary excrection of amino acids, glucose, along with cataracts in galactosemia and with hypogly- and phosphate (frequently also calcium, urea, and cemia in hereditary fructose intolerance. Confi rmation protein). Metabolic acidosis with increased urinary of each is by enzyme assay; in the latter liver is required; pH is common. as an alternative mutational analysis may reveal the common Caucasian mutations. Fanconi syndrome has Obligatory polyuria in this syndrome can lead to clini- been reported in glycogenosis I, III, and XI. Type I can cally important dehydration, especially when fl uid intake be diagnosed clinically by glucagon testing (Chap. D8 – is restricted. Chronic hyperchloremic acidosis may Glucagon Stimulation), by enzyme assay of liver or by worsen in acute situations. Losses of ions and metabo- mutaion analysis. Currently, we reserve liver biopsy for lites in the urine may lead to symptomatic depletion. those in whom mutational analysis does not provide Thus, hypokalemia may lead to muscle weakness or the diagnosis. The clinical chemistry is in charac- even paralysis, constipation, and ileus, as well as distur- terized by hypoglycemia, lactic acidemia, hyperal- bances of cardiac rhythm and function. Excretion of car- aninemia, hyperlipidemia, and hypercholesterolemia. nitine may deplete body stores and lead to disturbed Other renal complications are common in glycogeno- fatty acid oxidation and hypoketotic hypoglycemia sis I, including distal renal tubular disease, amyloido- (Chap. B2-5), muscle weakness, or congestive cardiac sis, nephrocalcinosis, and calculi (v.s.). Renal failure failure. Losses of calcium may lead to tetany or convul- may be a late complication with proteinuria, focal sions. Hypomagnesemia may similarly develop. glomerulosclerosis, and interstitial fi brosis. In Lowe Shortness of stature or failure to thrive is seen regularly. syndrome and Wilson disease, the full Fanconi picture A number of genetically determined diseases lead is often absent from the urine, but a generalized amino- to the Fanconi syndrome (Table C4.6). The most com- aciduria is the rule. In Wilson disease, the pattern also mon of these is cystinosis (v.i). includes increased cystine and methionine, indices of Among these disorders, associated syndromic features the liver dysfunction. In electron transport defects, a lead to the diagnosis and the appropriate confi rmatory variety of patterns of RTA is seen, including the com- test. Hepatic dysfunction is seen in hepatorenal plete Fanconi syndrome. Raised levels of lactate should

Table C4.6 Heritable Causes Of The Fanconi Syndrome Disorder Molecular defect or basis Distinguishing characteristics Cystinosis Lysosomal cystine transporter Corneal deposits, shortness of stature Hepatorenal tyrosinemia Fumarylacetoacetate hydrolase Hepatic dysfunction Galactosemia Galactose-1-phosphate uridyltransferase Hepatic dysfunction, cataracts, mental retardation Hereditary fructose intolerance Fructose-1-phosphate aldolase Hepatic dysfunction, hypoglycemia Glycogenoses Glucose-6-phosphatase, amylo-1,6-glucosidase, Hepatomegaly, hypoglycemia, hyperlipi- phosphorylase-b-kinase demia, hypercholesterolemia, lactic acidemia Fanconi–Bickel syndrome GLUT2 mutations Hepatomegaly Lowe syndrome OCRL-1 gene on Xq25–26, phosphati- Cataracts, glaucoma, hypotonia, dylinositol 4, 5-biphosphate-5-phosphatase developmental retardation Wilson disease P-type ATPase transporter Hepatic dysfunction, Kayser–Fleischer ring, neurologic dysfunction Electron transport defects mtDNA deletions (Pearson, Kearns–Sayre), Lactic acidemia, encephalomyopathy mtDNA depletion (RRM2B), cytochrome c oxidase, complex III or IV Drugs Outdated tetracycline, gentamycin, valproic acid, 6-mercaptopurine, azathioprine Heavy metal poison Cadmium, lead, manganese Idopathic — C4 Kidney Disease and Electrolyte Disturbances 123 give the major clue. An early lethal mitochondrial DNA depletion syndrome caused by defects in the RRM2B gene characteristically results in the Fanconi syndrome. In some clearly genetic kindreds with idiopatwhic Fanconi syndrome, a molecular defect has not been found, and these are classifi ed as idiopathic. In addition, an acquired Fanconi syndrome has been observed with a variety of renal insults, including the ingestion of outdated tetracycline, 6-mercaptopurine, and heavy metal poisoning. Defi ciency of vitamin D leads to a moderate generalized aminoaciduria, but not usually Fig. C4.3 The ATP-dependent lysosomal carrier-mediated effl ux of cystine which is defective in patients with cystinosis. Reprinted to the rest of the Fanconi syndrome. with permission from Nyhan et al. (2005)

C4.4.2 Renal Tubular Acidosis C4.4.1 Cystinosis Renal tubular acidosis (RTA) includes the Fanconi syn- Cystinosis is one of the most important causes of the drome and cystinosis (v.s.) (Table C4.7). A small popu- Fanconi syndrome and as such manifests all the features lation of patients have RTA without any of the features of the syndrome (v.s.). In addition, there are some clini- of the Fanconi syndrome. These patients fail to thrive cal manifestations that are unique to this disease. Patients as infants and display shortness of stature later. Vomiting generally have fair skin, hair, and irises. Ophthalmic is frequently encountered in infants, and many infants abnormalities include photophobia that is caused by are anorexic. Infants are often described as irritable or deposits of cystine in the cornea. These refractile crys- apathetic. Metabolic bone disease manifests itself as talline bodies may be seen early by slit lamp. They may rickets and osteomalacia, particularly, in proximal lead ultimately to thickened, hazy corneas and may be RTA. The diagnosis is often fi rst suggested by roent- complicated by corneal ulcers. Crystalline cystine may genographic examination of the bones of a patient with also be identifi ed in conjunctiva. In addition, there is a failure to thrive. Urolithiasis is sometimes seen in older characteristic peripheral retinopathy visible as pigmen- patients with distal RTA. tation and depigmentation. Some adults with the disease A lowered serum bicarbonate, with hyperchloremia have been legally blind. and a normal anion gap is characteristic. Patients seldom − Hypothyroidism results late from deposition of cys- have an HCO3 over 19 mEq/L, but values are usually tine in the gland. Myopathy may follow cystine deposi- over 10 mEq/L. The serum bicarbonate is the most reli- tion in muscle or carnitine depletion because of losses able indicator, as the pH and pCO2 may change in a cry- in urine. Some patients have developed diabetes melli- ing infant. Relative alkalinity of the urine is often the key tus. Late neuologic abnormalities include tremor, sei- to the diagnosis. In distal RTA, the urine pH remains high, zures, or mental retardation. Hydrocephalus and cerebral and net excretion of acid is low even when the serum atrophy have been documented. The diagnosis is usu- bicarbonate is low. In some patients, it may not be readily ally made by the assay of cystine content in leukocytes evident that in an acidotic patient a urinary pH of six may or cultured fi broblasts. The molecular defect is in the be relative alkalinity. Also, modern clinical chemistry transporter for cystine from lysosomes (Fig. C4.3). laboratories have largely dispensed pH meters to determine urinary pH, and dipstix may be less than precise. Remember Defective acidifi cation of the urine leads to renal losses of sodium and potassium and polyuria. Bone Cystinosis is a lysosomal transporter defect that resorption leads to hypercalciuria. The serum cal- causes increased intracellular deposition of cystine cium is normal. Phosphaturia may lead to hypophos- crystals. Typical clinical features include failure to phatemia. Alkaline phosphatase may be increased. The thrive, short stature, and renal tubular disease. excretion of citrate may be low; with hypercalciuria 124 W. L. Nyhan

Table C4.7 Renal Tubular Acidosis aciduria are associated with proximal RTA. A mixed Type I (distal) Isolated proximal and distal RTA is seen in patients with car- With deafness bonic anhydrase defi ciency in which there is a geneti- Hypergammaglobulinemia (Sjögren) cally determined syndrome of RTA, osteopetrosis, and Nephrocalcinosis (e.g., hyperparathyroidism, vitamin D poisoning) cerebral calcifi cations. A mixed RTA is also seen in Drugs: amphotericin B, lithium carnitine palmitoyltransferase I (CPTI) defi ciency. tubulointerstitial disease, renal transplantation, obstruction Type II Isolated (proximal) Pyruvate carboxylase defi ciency C4.4.3 Bartter Syndrome Methylmalonic acidemia Carbonic anhydrase II defi ciency (with osteopetrosis) Bartter syndrome is an uncommon cause of salt- Drugs (acetazolamide) wasting hypokalemic alkalosis in which calcium excre- Combined Type Isolated tion is normal or increased. It is in this way distinguished IV (distal Carnitine palmitoyl transferase I from Gitelman syndrome, in which hypokalemic alka- hyperkalemic) defi ciency losis is accompanied by hypocalciuria and hypo- Mineralocorticoid defi ciency magnesemia. Pseudohypoaldosteronism Hyporeninemic, hypoaldosteronism Bartter syndrome (type 1) is caused by loss of func- (diabetes, NSAIDs) tion mutation in the Na–K–Cl co-transporter gene SLC Drugs (spironolactone, amiloride, ACE 12A, previously called NKCC2, on chromosome inhibitors) 15q15–21, which codes for a protein that mediates electrolyte transport in the loop of Henle, at the site of leading to nephrocalcinosis and nephrolithiasis. Renal action of the diuretics, furosemide, and butmetanide. ultrasonography is useful for early detection. Mutations in this gene have also been found in antena- The distinction of the two major types of RTA, tal Bartter syndrome, in which ultrasonography has proximal RTA (sometimes called type II) and distal revealed fetal nephrocalcinosis in utero. Bartter syn- RTA (sometimes called type I), is best made by the drome is heterogeneous; patients with the typical syn- assessment of urinary loss of bicarbonate and serum drome have been found in whom the mutation was in concentration during intravenous or oral loading with the inwardly rectifying potassium channel, ROMK + NaHCO3 that causes a progressive increase in the (KCNJ1), which recycles reabsorbed K back into the serum level. This requires the collection of urine tubule (type 2). The classic syndrome as described by under oil and so maintained until analyzed. The frac- Bartter (type 3) is caused by mutations in the renal tional excretion of bicarbonate (FEHCO3%) is calcu- chloride channel B gene CLCNKB. Infantile Bartter lated as UHCO3/UCreatinine(Cr) × SCr/SHCO3 × syndrome with sensorineural deafness (type 4) may be 100, where U and S indicate urine and serum, respec- caused by mutation in the BSND gene or by simultane- tively. The percentage in patients with distal RTA is ous mutations in both the CLCNKA and CLCNKB less than 10, whereas in proximal RTA values of genes. Bartter syndrome has also been reported in 10–15% are seen when the serum level is 20 mEq/L, patients who turned out to have mutations in the and there may be a massive bicarbonaturia, over 15%. calcium-sensing receptor (CASR) gene, in which loss Patients with distal RTA and hyperkalemia may have of function mutations usually lead to hypocalciuria values from 5 to 10%, whereas those with classic and secondary hyperparathyroidism, whereas gain of hypokalemia usually have values less than 5%. The function mutations lead to hypercalciuria and hypocal- hyperkalemic patients may represent a generalized cemia. In Bartter syndrome, presentation is usually tubular dysfunction and are sometimes referred to prior to 5 years of age. The initial presentation may be as type IV. These patients may have hypoaldoster- with dehydration and hypovolemia. Some patients onism, pseudohypoaldosteronism, or hyporeninemic present with cramps or weakness in the muscles result- hypoaldosteronism. ing from the hypokalemia. Some have had convulsions Some discrete syndromes are associated with RTA. or tetany. Chvostek and Trousseau signs may be posi- Pyruvate carboxylase defi ciency and methylmalonic tive. There may be polyuria, nocturia, or enuresis. C4 Kidney Disease and Electrolyte Disturbances 125

A craving for salt is common. Some patients display C4.4.4 Gitelman Syndrome vomiting, and constipation is common. Shortness of stature or failure to thrive is the rule. Gitelman syndrome is a disorder in which depletion of Mental retardation has been observed in about two-thirds magnesium and potassium lead to hypokalemic alka- of the children. Some have had abnormal electroencepha- losis. It was once thought to be a variant of Bartter lography. Ileus has been observed, and attacks of hypoven- syndrome, but it is caused by mutations in the thiazide- tilation, both also concomitants of hypokalemic alkalosis. sensitive Na–Cl cotransporter gene on chromosome Rickets has been reported rarely. Nephrocalcinosis has 16q13, referred to as SLC12A3, which codes for a been observed in many, especially in those with the so- transporter that mediates sodium and cloride reabsorp- called antenatal or infantile forms of Bartter syndrome. tion in the distal convoluted tubule. This transporter is Hypokalemia, alkalosis, hyperaldosteronism, and the target of the thiazide diuretics used to treat hyper- hyperreninemia are regular laboratory fi ndings, as is tension. At least 17 different nonconservative muta- normal blood pressure. Bartter and colleagues noted tions have been found. unresponsiveness of the blood pressure to intravenous Patients with this disorder present later than those angiotensin II, and hypothesized that increased produc- with Bartter syndrome, often in adulthood, and with- tion of renin was a compensatory response to maintain out hypovolemia, but there are patients with fi ndings blood pressure, and that aldosterone production was that overlap both the syndromes. also stimulated. Volume expansion can reduce levels of Episodic muscle weakness has been one presentation. renin and aldosterone. Histologically there is hyperpla- Tetany has been another, often precipitated by intercur- sia of the renal juxtaglomerular apparatus. There is rent infectious illness. There may be paresthesias or car- increased renal prostaglandin production. Large urinary popedal spasm. Patients have been described with a losses of sodium and potassium lead to contraction of chronic dermatosis, in which the skin was thickened and volume. Hypokalemia is usually profound. The concen- had a purple–red hue. Erythema of the skin has been tration is less than 2.5 mEq/L. Patients are unable to observed in experimental magnesium depletion in rats. form a concentrated urine, and this isosthenuria does Prolongation of the QT electraradiographic interval is not respond to administration of antidiuretic hormone. common; ventricular fi brillation is a rare complication. Less common laboratory fi ndings include hypo- In Gitelman syndrome, hypokalemic metabolic magnesemia, hypocalcemia, and glucose intolerance alkalosis is accompanied by hypomagnesemia and without fasting hyperglycemia. Some patients have hypocalciuria. Renal wasting of potassium and magne- hyperuricemia, and clinical gout has been observed. sium is characteristic. There is increased excretion of The differential diagnosis of Bartter syndrome sodium, chloride, and magnesium in response to intra- includes Gitelman syndrome (see below). It also includes venous furosemide, as well as abolition of the hypocal- primary hyperaldosteronism, a renin producing tumor ciuria, consistent with a defect in transport in the distal and renal artery stenosis, all three of which are dif- tubule as opposed to the loop of Henle. ferentiated by the presence of hypertension. Chronic diarrhea, especially laxative abuse, and bulimia may mimic Bartter syndrome, as of course may chronic or surreptitious diuretic use. C4.5 Investigation for Renal Tubular Dysfunction Remember Both Bartter syndrome and Gitelman syndrome are Initial clinical chemistry is undertaken to determine characterized by renal loss of salt with hypokalemic serum values for Na, K, Cl, HCO , and pH. This will alkalosis and hyponatremia. Bartter syndrome is clini- 3 establish the presence or absence of acidosis and cally more severe with hypercalciuria and often neph- whether or not it is hyperchloremic. Other useful blood rocalcinosis, failure to thrive, growth retardation, and chemistry data include Ca, PO , Mg, alkaline phos- muscle weakness. Clinical manifestation of the more 4 phatase, urea, and creatinine. frequent Gitelman syndrome is mild and often nonspe- The urine is studied to assess the presence of a cifi c; electrolyte changes resemble thiazide therapy. Fanconi syndrome by analysis of glucose, amino acids, 126 W. L. Nyhan

Ca, PO4, and creatinine. Proteinuria or increased amount In the presence of acidosis, the pH of urine is of retinol-binding protein or N-acetylglucosaminidase measured. For this purpose it is important to analyze indicate tubular damage and proximal tubular leak. promptly after passage. NaHC03 supplements should Decreased urinary concentrating ability may also indi- have been discontinued. In a patient suspected of hav- cate generalized tubular dysfunction. ing RTA in whom acidosis is not present an ammonium chloride load may be useful (Table C4.8).

Table C4.8 Investigations For The Analysis Of Renal Tubular Dysfunction Initial clinical chemistry Key References

Plasma: Na, K, C1, HCO3, urea, creatinine, urate, Ca, Mg, PO ionized Ca, alkaline phosphatase, parathy- 4, Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic roid hormone diseases, 2nd edn. Hodder-Arnold, London Urine: reducing substances, Kaufman JM, Greene ML, Seegmiller JE (1968) Urine uric acid amino acids, tubular reabsorption of phosphate, Ca/ to creatinine ratio – a screening test for inherited disorders of creatinine ratio, albumin/creatinine ratio, retinol purine metabolism. J Pediatr 73:583–592 binding protein/creatinine ratio, Wilcox WD (1968) Abnormal plasma uric acid levels in children. N-acetyl glucosaminidase/creatinine ratio J Pediatr 128:731–741 Further investigation aimed at a specifi c cause Bartter FC, Pronove P, Gill JR Jr et al (1962) Hyperplasia of the Blood lactate and pyruvate juxtaglomerular complex with hyperaldosteronism and Pyruvate carboxylase-leukocytes or fi broblasts hypokalemic alkalosis: a new syndrome. Am J Med 33: Leukocyte cysteine 811–828 Renal (liver) ultrasound Birkenhäger R, Otto E, Schürmann MJ et al (2001) Mutation of BSND causes Bartter syndrome with sensorineural deafness In the presence of hepatic dysfunction and kidney failure. Nat Genet 29:310–314 Plasma: 1.25 – hydroxylvitamin D Schlingmann KP, Konrad M, Jeck N et al (2004) Salt wasting Erythrocyte: galactose 1-phosphate uridyltransferase and deafness resulting from mutations in two chloride chan- Urine: organic acids for succinylacetone nels. New Engl J Med 350:1314–1319 DNA: common mutations for hereditary fructose Gitelman HJ, Graham JB, Welt LG (1966) A new familial disor- intolerance and glycogenoses Ia, Ib der characterized by hypokalemia and hypomagnesaemia. Plasma: ceruloplasmin, urine, plasma: copper Trans Assoc Am Physicians 79:221–235 Roentgenograms: joints (rickets, osteomalacia) Simon DB, Nelson-Williams C, Bia MJ et al (1996) Gitelman’s Plasma: renin, aldosterone variant of Barter’s syndrome, inherited hypokalaemic alka- Blood or muscle mtDNA losis, is caused by mutations in the thiazide-sensitive Na–Cl Blood acylcarnitine profi le cotransporter. Nat Genet 12:24–30 Scheinman SJ, Guay-Woodford LM, Thakker RV, Warnock DG Urine: toxicology (1999) Genetic disorders of renal electrolyte transport. Muscle biopsy: electron transport chain activity N Engl J Med 340:1177–1187 Liver biopsy: aldolase, GSD I, Fanconi–Bickel Simon DB, Bindra RS, Mansfi eld TA (1997) Mutations in the Skin biopsy: phosphatidylinositol-4, 5-bisphosphate chloride channel gene, CLCNKB, cause Bartter’s syndrome 5-phosphatase (Lowe syndrome) type III. Nat Genet 17:171–178 Neurological Disease C5 Angels García-Cazorla, Nicole I. Wolf, and Georg F. Hoffmann

Remember Key Facts › The correct diagnosis of inherited metabolic Signs and symptoms suggestive of a neurometa- diseases that affect the nervous system primar- bolic disorder ily is a major challenge because the same neuro- Positive family history, consanguinity logical symptoms and often even disease course Unusual age and/of sequence of neurological may be caused by non-metabolic disorders. symptoms Slowing/plateau of development, regression › Neurometabolic diseases often start with com- Progressive loss of hearing and/or vision mon and non-specifi c signs, such as isolated Simultaneous affection of different neurological developmental delay/mental retardation, sei- systems zures, dystonia, or ataxia. They are especially Suggestive neuroimaging fi ndings to be suspected when the course of the disease Involvement of other organs than the CNS is progressive or when additional neurological systems or other organs become involved. An important clue is the co-existence of different › Metabolic investigations are usually not neurological features that cannot be explained indicated in children with moderate static by a “simple” neuroanatomic approach. developmental delay, isolated delay of speech development in early childhood, occasional sei- › Acute or recurrent attacks of neurological zures, e.g. during fever, or well-defi ned epilep- manifestations such as coma, ataxia or abnor- tic syndromes. Other genetic aetiologies outside mal behaviour are major presenting features the metabolic fi eld have also been considered, especially in the late-onset inborn errors of especially as causes of mental retardation, metabolism (see also B2.7). ataxia, dystonia, and spastic paraplegia. › The initial diagnostic approach to these disorders is based on a few metabolic screening tests. It is important that the biologic fl uids are collected at the time of the acute attack. And always consider treatable disorders fi rst when choosing your plan of investigations. C5.1 General Remarks

Neurological disease can be considered as the most common and important consequence of inherited metabolic diseases for three reasons. In building and maintaining the structure and function of the brain many more genes are involved when compared to the other A. García-Cazorla ( ) organs. Also, the brain seems to possess restricted Servicio de Neurologia, Hospital Sant Joan de Deu, Passeig Sant Joan de Deu 2, 08950 Esplugues, Barcelona, Spain capabilities for repair. As a consequence even slowly e-mail: [email protected] but continuously or repeatedly ongoing insults will

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 127 DOI: 10.1007/978-3-540-74723-9_C5, © Springer-Verlag Berlin Heidelberg 2010 128 A. García-Cazorla et al.

result in lasting and often progressive neurological dis- cerebellar ataxia in some forms of coenzyme Q10 ease. Finally, in the development of modern human defi ciency, and isolated mental retardation in cre- societies many physical disabilities from reduced atine transporter (CRTR) defect, some more benign vision or hearing to loss of a limb or even an organ will forms of adenylosuccinate lyase defi ciency, as well still allow relatively independent and unimpaired activ- as in some patients with Sanfi lippo disease, in par- ities and life. On the contrary, mental abilities and ticular, type A. capacities are being required at an ever increasing level 4. Asymmetry of the symptoms: Contrary to perceived and even marginal or specifi c partial inabilities can wisdom, asymmetrical involvement does not argue have profound negative effects on the status and well- against neurometabolic diseases. Although the evo- being of an individual. lution of these disorders tends to result in generalised clinical manifestations, it is common to detect unilateral signs. Examples include unilateral dystonia in Segawa disease, Leigh disease, some cases of C5.2 History and Neurological GLUT1 defi ciency, and unilateral tremor in atypical Examination forms of glutaric aciduria type I. 5. Fluctuation of symptoms: In inborn errors of intermediate metabolism symptoms often fl uctuate. In Family history, including possible consanguinity, and neurotransmitter disorders with dopaminergic dys- age at onset of symptoms are crucial to arrive at a sus- funcion, dystonia and oculogyric crises may worsen picion of a neurometabolic disorder. Five aspects with fatigue and improve with rest; in GLUT1 defi - should be specifi cally kept in mind: ciency, symptoms like epilepsy, ataxia, dysarthria 1. Multi-system involvement: In patients with neuro- and dyskinesia may worsen with fasting and logical diseases of undefi ned origin, searching for improve after eating or while resting; in urea cycle multi-system involvement can be very helpful. This disorders, consciousness, hyperkinesia and other includes special neurological functions, such as symptoms may develop with intercurrent illnesses vision and hearing, as well as overall growth and or fl uctuate depending on protein intake. physical development. Eye, liver, spleen, heart, kidney, skin and the skeletal system are the most prominently affected organs in inborn errors of metabolism C5.3 Neurological Regression/ besides the nervous system. 2. Stability/progression of the disease: Although neu- Deterioration rometabolic disorders classically lead to progressive neurodegenerative disease courses due to organelle Progressive neurologic and mental deterioration is a dysfunction, e.g. lysosomal and peroxisomal disor- very important clinical hallmark of inborn errors of ders and mitochondriopathies, many of them can metabolism. In this context, it is helpful to take into con- present as static or non-progressive neurological sideration the age of onset, presence or absence of epi- dysfunctions. Metabolic disorders affecting small lepsy, other neurologic signs as ataxia or other movement molecules often show a stable neurological course. disorders, and the co-existence of non-neurologic symp- Examples include creatine defi ciency syndromes, toms, especially hepatosplenomegaly, bone or retinal 4-hydroxybutyric aciduria, and some defects of involvement. Altered metabolism of complex mole- purine metabolism. Some CDG disorders have also cules, defects of energy metabolism and organic acidu- been proven to be static encephalopathies. rias are the main pathophysiological groups involved in 3. Multi-neurological involvement: When isolated neurological deterioration (Zschocke et al. 2004). neurological symptoms persist over time, the diag- It is important to analyse the speed of progression in nosis of an inborn error of metabolism is less likely. these disorders. In storage diseases, development usu- Neurometabolic disorders tend to involve more than ally slows down before reaching a plateau and frank one system. However, some exceptions have to be regression starts. Loss of acquired skills follows at kept in mind, including progressive dystonia without variable speed. In slow progressive disorders, it is often additional symptoms in Segawa disease, isolated diffi cult to decide whether there is true regression or C5 Neurological Disease 129 simply a residual syndrome. Old family photographs Table C5.1 Important differential diagnoses in regression or home videos may be helpful. Cerebral imaging may Additional Leading Disease also change only slowly or even be normal, especially Symptom at the onset of symptoms. Organic acidurias can mani- Epilepsy Late-infantile neuronal ceroid lipofuscinosis, Alpers disease, fest with acute and dramatic deterioration, with a pla- MELAS, MERRF and other teau (or even partial recovery) later on. This is especially progressive myoclonus epilepsies true for glutaric aciduria type I or mitochondrial disor- Ataxia Late-infantile neuronal ceroid ders. The latter may display acute deteriorations with lipofuscinosis, GM2- regression, followed by partial recoveries. Over time, gangliosidosis, mitochondrial disorders, cerebrotendinous the course of these disorders is relentlessly downhill. xanthomatosis, Refsum disease, There are several non-metabolic disorders giving vitamin E-responsive ataxias rise to a degenerative disease course. This is especially Neuropathy Metachromatic leukodystrophy, true for disorders from the autistic spectrum and Rett Friedreich ataxia, Refsum disease, vitamin E-responsive ataxias syndrome in girls where early development is usually Retinopathy Infantile and late-infantile neuronal normal, and deterioration may be rapid. Continuous ceroid lipofuscinoses, juvenile spike-waves during sleep (CSWS) is another differen- neuronal ceroid lipofuscinosis, tial diagnosis that is amenable to treatment. Sleep elec- PKAN, Refsum disease, troencephalography (EEG) studies must therefore be mucolipidosis type IV Cataract Cerebrotendinous xanthomatosis, performed in all children with regression in addition to a-mannosidosis, Fabry disease metabolic investigations, regardless of whether seizures Hepatosplenomegaly Sandhoff disease, Gaucher disease, are present or not. Also in other epileptic encephalopa- Niemann–Pick disease, infantile thies, the untreated epilepsy itself, if severe enough, form of Salla disease can lead to regression mimicking a neurodegenerative Spasticity Metachromatic leukodystrophy, adrenoleukodystrophy, Krabbe disorder, e.g. Dravet or Lennox–Gastaut syndrome. disease and many others Differential diagnosis is completely different from a Dystonia Glutaric aciduria type I, Wilson true neurodegenerative disorder. Additional important disease, biotin-responsive basal causes of neurologic and mental deterioration are as ganglia disease follows: side effects of antiepileptic drugs, especially Dysostosis multiplex Mucopolysaccharidoses, oligosaccharidoses, GM -gangliosidosis valproate, intoxication by heavy metal contamination 1 (lead and mercury); and the Münchausen-by-proxy syndrome (Table C5.1).

absent refl ecting polyneuropathy. Cerebrospinal fl uid (CSF) protein is elevated. Cognition remains Remember relatively intact until late in the course of the dis- In the differential diagnosis of neurodegenerative ease, seizures are uncommon. Arylsulphatase A disorders, age of onset, additional (leading) symp- activity is severely impaired; excretion of sulphati- toms and the dynamics of regression are important des is increased. The latter can differentiate between clues for differential diagnosis. true arylsulphatase A defi ciency and pseudodefi - ciency that occurs in 7–15% of the general population. The juvenile form may present with psychiatric symptoms. If done before neurologic symptoms Disease Info: Metachromatic Leukodystrophy develop, bone marrow transplantation is a success- (MLD) ful treatment, at least for some patients. Magnetic The classical late-infantile form of this autosomal resonance imaging (MRI) shows involvement of the recessive disorder usually starts between 10 and 25 posterior central white matter with sparing of the months with fl oppiness or gait disturbance. Severe subcortical areas; the posterior part of the corpus spasticity develops, tendon refl exes are decreased or callosum is also usually involved. 130 A. García-Cazorla et al.

Disease Info: X-linked Adrenoleukodystrophy the fi rst 3 months of life. Some infants go on to (X-ALD) develop severe cholestatic liver disease with impaired liver function; they do not display neuro- This X-chromosomal disorder starts in childhood logic symptoms. Mental retardation and regression with progressive gait disturbance and insidious or initial normal development followed by regres- onset of cognitive decline. The course is relent- sion in childhood are common. Vertical supranu- lessly downhill; spasticity and dementia develop. clear (upward) gaze palsy is a typical symptom, but Adrenocortical involvement is not a regular fi nding is usually absent in small children. Cataplexy and in this form that occurs in about half of the affected epilepsy are common. Hepatosplenomegaly is not individuals. About 25% of the patients show another always present. If disease starts later in childhood or clinical picture with adrenal insuffi ciency and neu- in adulthood, ataxia and slowly progressing demen- ropathy (adrenomyeloneuropathy), 10% have iso- tia are the leading symptoms. There is great clinical lated Addison’s disease. VLCFA are elevated with heterogeneity. In cultured fi broblasts, cholesterol an increased ratio of C26:C22. Bone marrow transesterifi cation is defective, but this test may be nor- plantation in presymptomatic boys at risk for devel- mal. Chitotriosidase activity is moderately increased oping X-ALD is curative but cannot stop the and a useful biochemical marker. Sea-blue histio- progress if done after onset of neurological symp- cytes are found in bone marrow aspirates. toms. Whether Lorenzo’s oil may lower the risk of developing the severe childhood form is still under debate (Moser 1997). Disease Info: Neuronal Ceroid Lipofuscinoses (NCL)

Disease Info: GM2-Gangliosidosis These disorders, all with autosomal recessive inheritance, show storage of autofl uorescent material. The most common form is due to the absence of There are several forms with onset from neonatal hexoaminidase A activity and starts in infancy, also age until adulthood; the retina is involved in almost called Tay–Sachs disease, with regression, acoustic all. Infantile NCL (Santavuori–Haltia disease) is fre- startle reaction and hypotonia. Spasticity, blindness, quent in Finland, but can also be found in other eth- macrocephaly and seizures develop later. A cherry- nic groups. Affected infants show an initially normal red spot in the macular region is almost invariably development, regression becomes apparent at the present. Sandhoff disease shows the same neuro- end of the fi rst year of life with ataxia, loss of vision logical symptoms, but can also lead to liver involve- and myoclonus. EEG shows progressive slowing ment; hexoaminidase A and B activities are absent. and in the later stages of the disease isoelectric trac- The juvenile form of GM2-gangliosidosis can pres- ing. The disease is due to absent activity of palmi- ent with ataxia, action myoclonus, dystonia and toylprotein thioesterase 1 (CLN1). Mutations in the dementia. Cerebellar atrophy is a frequent neurora- same gene can give rise to the juvenile form of the diological fi nding in this form. disease. Late-infantile NCL (Jansky–Bielschowsky disease) is caused by mutations in the gene coding for tripeptidylpeptidase 1 (TPP1) (CLN2). It starts between 2 and 5 years of age with epilepsy, ataxia Disease Info: Niemann–Pick Disease Type C and regression followed by loss of vision and spas- (NPC) ticity. Juvenile NCL (Batten disease) starts with loss In total, 95% of cases in this autosomal recessive of vision due to retinitis pigmentosa around the age disorder are caused by mutations in NPC1, which is of 6. Regression starts thereafter; parkinsonism and important for intracellular cholesterol traffi cking. epilepsy are additional symptoms. There is a com- The remaining 5% are caused by mutations in mon 1.02 kb deletion in the CLN3 gene coding for a NPC2. Half of the patients have a cholestatic neona- membrane protein with palmitoyl-protein D-9 desat- tal icterus, which disappears spontaneously within urase activity. A useful and simple screening test is C5 Neurological Disease 131

looking for lymphocyte vacuolisations in a periph- Skin biopsy shows spheroid inclusions in nerve eral blood smear. This should be done in every child endings. Cerebral MRI is remarkable for T2 hyper- with retinitis pigmentosa. CLN8 shows similar intense cerebellar cortex; signal of globus pallidum symptoms as the late-infantile form is due to CLN2 may be hypointense (Fig. C5.1). EEG displays fast mutations; it is caused by mutations in CLN8 that rhythms (Fig. C5.2). A large proportion of cases also lead to progressive epilepsy with mental retar- have mutations in PLA2G6, a gene coding for a dation (EPMR, Northern epilepsy). There is also an phospholipase A2 (Morgan et al. 2006). The exact adult-onset form of NCL, Kufs disease. The genetic pathogenesis of this disorder is not yet understood. defect(s) is not yet known.

C5.4 Delayed Speech Development Krabbe Disease

Defi ciency of b-galactocerebrosidase gives rise to this Isolated delay in speech development is frequent. Per se disorder. Its infantile form starts around the age of it does not indicate a metabolic disorder and metabolic 6 months with irritability, progressive stiffness and investigations are not warranted. If speech delay is part opisthotonus. Later, infants become hypotonic because of the global developmental delay, investigations should of neuropathy. CSF protein is elevated. Seizures are be performed for mental retardation (see later). There relatively common. The juvenile form has a less are several disorders in which expressive speech is more stereotyped clinical picture. Ataxia, dementia, dys- profoundly affected than overall development. This is tonia and loss of vision are frequent symptoms. the case for creatine defi ciency syndromes, 4-hydroxybutyric aciduria and adenylosuccinate lyase defi ciency, but it may also be found in mitochondrial disorders, Sanfi lippo Disease especially when associated with hearing impairment. It is important to evaluate cognitive abilities indepen- This autosomal recessive disorder (mucopolysaccha- dently. Equally important is a thorough approach of ridosis type III) is caused by mutations in four differ- therapists in order to enable assisted communication or ent genes coding for heparan N-sulphatase (type A), teach simple sign languages. Abnormal speech devel- a-N-acetylglucosaminidase (type B), acetyl CoA:- opment may be seen in galactosemia despite treatment. a-glucosaminide acetyltransferase (type C) and N-acetylglucosamine 6-sulphatase (type D). Mental retardation and insidious regression become apparent between 2 and 6 years of age. Coarse features C5.5 Deafness may be absent or not very prominent. Urine screening for mucopolysaccharides by glycosaminogly- Deafness can be caused by a great variety of different cane electrophoresis should therefore be done in all genetic and environmental causes. Genetic factors children with mental retardation and/or regression. account for at least half of all the cases of congenital deafness. In fact, about 300 independent genes have shown to cause hearing loss. Amongst them, from 77 to Infantile Neuroaxonal Dystrophy (INAD) 88% are transmitted as autosomal recessive traits, 10–20% as dominant and 1–2% as X-linked. In 20–30% This is a very rare neurodegenerative disorder,which of the cases, other associated clinical manifestations starts at the end of the fi rst or in the second year of permit the diagnosis of a specifi c form of syndromic life after normal development. First symptoms are deafness (Nance 2003). Metabolic disorders that exhibit stagnation of development, muscle hypotonia and deafness in variable forms (from profound deafness to truncal ataxia. Nystagmus and further deterioration variable degrees of hypoacusia) belong to this syndro- follow. Children develop spasticity, optic atrophy mic group (Table C5.2). The hearing loss in metabolic and additional evidence of chronic denervation. disorders is mostly sensorineural, symmetrical, and 132 A. García-Cazorla et al.

Fig. C5.1 MRI of a child with INAD. (a), (c) and (d) are axial T2w images. In (a), cerebellar atrophy is evident, (c) demonstrates low pallidal signal, (d) some cortical atrophy. In (b), the axial FLAIR image displays hyperintensity of the cerebellar cortex

manifests a predominant involvement of high frequen- do not divide, and receive poor, indirect metabolic sup- cies, although in advanced stages, all frequencies can be port from Deiter cells, especially those at the basal coil, affected. Moreover, it is usually progressive, and in gen- which are the most metabolically active. It is postulated eral, there is no specifi c medical treatment except pos- that a drop in the level of ATP results in progressive sibly for early therapy in biotinidase defi ciency. Cochlear ionic imbalances in both the outer hair cells and stria implants have been reported to be effective in mitochon- vascularis, leading to cell injury and cell death. drial encephalopathy, lactic acidosis and stroke-like epi- Mitochondrial dysfunction is the most important sodes (MELAS), maternally inherited diabetes and cause of deafness from metabolic disorders. However, deafness (MIDD), Kearns–Sayre (KSS) and chronic the great majority of the mitochondrial causes of deaf- progressive external ophthalmoplegia (CPEO) syn- ness are not included in our conventional classifi cation dromes. Mitochondrial dysfunction is the most impor- of metabolic disorders. That refers to more than 50 tant cause of deafness of metabolic origin (Kokotas nuclear genes involved in non-syndromic hearing loss. et al. 2007). Outer hair cells have a high ATP demand, In addition, several rare mutations in the mitochondrial C5 Neurological Disease 133

Fig. C5.2 Prominent general fast b-activity in this child with INAD during sleep stage II

MTTS1 and MTRNR1 genes have been found to be sensory-motor polyneuropathy, chronic progressive responsible for non-syndromic hearing loss. In contrast, external ophthalmoplegia and mitochondrial myopathy well-known mutations in the mitochondrial DNA such characterised by cytochrome c oxidase negative and as those associated with NARP, Pearson, MELAS, ragged red ﬁ bres. Finally, deafness/dystonia peptide MERFF or Kearns–Sayre syndromes have multi-system (DDP) is the product of a gene on Xq22 that gives rise involvement (varying degrees of myopathy, cardiomyo- to deafness, blindness, retardation and dystonia found pathy and encephalopathy), in addition to, although not in the Mohr–Tranebjaerg syndrome. always, hearing loss. In MIDD, neurosensory hearing loss is a very prominent component of the clinical picture. MIDD syndrome is caused by mitochondrial DNA Fabry Disease rearrangements. Diabetes is non-insulin dependent at Both progressive hearing impairment and sudden onset but progresses to insulin dependency with age. deafness have been reported in this disorder. Hearing Retinopathy, cardiomyopathy, myopathy, encephalopa- loss on high-tone frequencies has also been found on thy and kidney disease are present in many patients audiograms in Fabry patients with clinically “normal” resulting in a phenotypic overlap with MELAS syn- audition. Furthermore, the incidence of hearing loss drome. On the other hand, mutations in the OPA1 gene appeared signiﬁ cantly increased in Fabry patients that encodes a dynamin-related GTPase involved in with kidney failure or cerebrovascular lesions. The mitochondrial fusion, cristae organisation and control origin and mechanisms of deafness probably involve of apoptosis, have been linked to non-syndromic optic the inner ear. Neuropathologic studies have disclosed neuropathy transmitted as an autosomal dominant trait. evidence of glycosphingolipid accumulation in the ear However, it can also be responsible for a syndromic in vascular endothelial cells and in various ganglion association of sensorineural deafness, ataxia, axonal 134 A. García-Cazorla et al.

cells. More specifi cally, sudden deafness could also C5.5.1 Other Lysosomal Disorders be potentially caused by vascular mechanisms due to the accumulation of glycosphingolipids within lyso- Many lysosomal disorders can exhibit hypoacusia or somes of endothelial and smooth muscle cells leading deafness due to abnormal lysosomal storage in the to progressive narrowing, ischemia, and frank occlu- inner ear or bone hearing malformations. Here, we sion in the vessels feeding the cochlea. Middle ear outline some of the most involved disorders. fi ndings of seropurulent effusions and hyperplastic mucosa have been demonstrated in temporal bones of patients. Strial and spiral ligament atrophy in all turns and hair cell loss mainly in the basal turns were also C5.5.2 Mucopolysaccharidoses common fi ndings. Neverthe less, the exact pathophys- iologic mechanisms of the cochlear involvement Hypoacusia and profound deafness are frequent in deserves further studies (Germain et al. 2002). mucopolysaccharidoses, in particular, mucopolysac- The diagnosis is made by enzyme studies (a-galac- charidosis type I (MPS I) and Morquio’s syndrome tosidase) in plasma, serum, leukocytes or fi broblasts. (MPS IV). Clinical fi ndings of otitis media with mixed Enzyme replacement therapy is available. hearing loss are common. Although audition could be

Table C5.2 Differential diagnosis of syndromic deafness Metabolic disorders and syndromic deafness Other genetic syndromic deafness Mitochondrial disorders Pendred syndrome Mutations in MTTS1 and MTRNR1: non-syndromic Sensorineural deafness, malformations of the inner ear and deafness goiter. Is the most common form of syndromic deafness. Autosomal recessive. Mutations SLC26A4 gene Mitochondrial DNA mutations (syndromic deafness): NARP, Pearson, Wolfram, MELAS, MERFF, Kearns–Sayre, MIDD, OPA1, DDP Branchio-oto-renal syndrome Sensorineural or mixed. Branchial cleft fi stulas, pre-helical pits, deformities of the inner ear, dysplastic or polycystic kidneys Autosomal dominant. Mutations EYE 1 gene Biotinidase defi ciency Waardenburg syndrome Lysosomal disorders Sensorineural hearing loss, patches of cutaneous dyspigmenta- Fabry disease tion, blue eyes, heterochromia, synophorys, lateral displace- Mucopolysaccharidoses ment of the inner canthi of the eyes. Genetically Sphingolipidoses heterogeneous, at least eight loci involved Oligossaccharidoses Peroxisome biogenesis disorders Usher syndromes Sensorineural deafness with retinitis pigmentosa. Phenotypically and genetically heterogeneous. Autosomal dominant and recessive forms Purine disorders PRPS (phosphoribosylpyrophosphate defi ciency) Jervell, Lange–Nielsen syndromes Sensorineural deafness and prolongation of the QT interval. Autosomal recessive Mutations KVLQT1 potassium channel gene Alport syndrome Sensorineural deafness, progressive nephritis, ocular abnormalities. Mutations involving tissue specifi c polypeptide subunits of collagen encoded by the COL4A genes. Autosomal recessive or X-linked C5 Neurological Disease 135 preserved at initial stages of the disease, hearing loss C5.6 Global Developmental tends to progress over time from mild and moderate to Delay/Mental Retardation total deafness. In a mouse model of MPS I, cells with lysosomal storage vacuoles were observed in spiral ligament, spiral prominence, spiral limbus, basilar Mental retardation is a frequently occurring condition, membrane, epithelial and mesothelial cells of Reissner’s affecting up to 3% of the population, with a major impact membrane, endothelial cells of vessels and some gan- on the life of the affected individual, the family and soci- glion cells. The number of vacuoles increased with ety. Establishing an etiologic diagnosis is a major chal- aging. Total loss of the organ of Corti appeared also lenge as the spectrum of possible causes is enormous and over time. These age-related changes suggest the the range of diagnostic investigations extensive and costly. necessity of early therapeutic intervention. Although the hope that a specifi c diagnosis will lead to Sphingolipidoses such as Krabbe disease and oligo- a rational therapy is mostly futile, a specifi c diagnosis sacccharidoses such as mannosidoses have also a high by itself is benefi cial by ending the diagnostic odyssey, incidence of profound neurosensorial deafness. and providing knowledge of the short- and long-term prognosis, the recurrence risk, treatment options as well as contact with other families. Often it is a great step towards acceptance of the disability (AACAP, 1999). C5.5.3 Peroxisomal and Purine Disorders There are as yet no guidelines available for investigating mental retardation, which have been established in Perceptive deafness, either congenital or as progressive an evidence-based approach. Studies report the yield of hearing loss, has been reported as a common sequel of different diagnostic approaches between 10 and 81%. peroxisome biogenesis disorders such as Zellweger With regards to inborn errors of metabolism as a cause of syndrome and variants, as well as Refsum disease. mental retardation studies are not comparable, e.g. with Phosphoribosylpyrophosphate (PRPS) defi ciency is a regards of the parameters investigated. Especially those purine disorder characterised in childhood by a variety disorders which could lead to unspecifi c mental retarda- of neurological defi cits, including inability to walk or tion (see Table C5.3) were mostly not investigated even talk, abnormal facies and sometimes inherited nerve in any of the few recent studies. What can be concluded deafness. In early adulthood it gives rise to gout or is that inborn errors of metabolism are not a major cause stones. PRPS defi ciency may also manifest in the car- of isolated mental retardation. In these disorders, mostly rier female and should be suspected in any seemingly additional neurological and/or somatic systems are X-linked defect where the mother presents with gout or involved guiding the diagnostic process. They are eluci- hyperuricemia and may be deaf. Both the complete and dated in other sections of this chapter, e.g. neurodegen- “partial” defects can present as acute renal failure in eration, movement disorders, etc. Nevertheless, inborn infancy. errors of metabolism remain responsible for 1–5% of unspecifi c mental retardation even in countries offering extended neonatal screening. Metabolic disorders have a high recurrence rate and available prenatal diagnosis, Remember

¼ Deafness in metabolic disorders is commonly Table C5.3 Inborn errors of metabolism presenting with neurosensorial, symmetrical, progressive and primary mental retardation syndromic (associated with other clinical signs) Mitochondriopathies ¼ Transmission or mixed hypoacusia correspond Mucopolysaccharidoses normally to muco- or oligo-saccharidoses Creatine defi ciency syndromes ¼ Mitochondrial disorders are the most frequent Defects of purine and pyrimidine metabolism (Maternal) phenylketonuria cause of deafness in metabolic diseases 4-Hydroxybutyric aciduria ¼ Always consider the possibility of biotinidase Fumaric aciduria defi ciency because of its therapeutic possibilities Homocystinuria 136 A. García-Cazorla et al. furthermore some of them are treatable. Therefore, Table C5.4 Checklist of laboratory tests in mental retardation guidelines considering which metabolic tests to be focusing on monogenic disorders employed in mental retardation, are badly needed. Laboratory tests in unspecifi c mental retardation without The assessment of a person with mental retardation dysmorphic features Genetic analyses, e.g. chromosomes, consider fragile X is typically multidisciplinary. Taking a good clinical syndrome history and performing a detailed clinical examination Basic laboratory tests (blood glucose, lactate, ammonia, (including formal testing, neurological and dysmor- acid–base status, blood counts, liver function tests, phological assessment) by a trained specialist remain creatine kinase levels, uric acid) the bases and must come before any laboratory testing Thyroid function Creatine metabolites (urine → creatine transporter (CRTR) (Van Karnebeek et al. 2005). Auditory and visual capa- defi ciency) bilities must also be ascertained (Shevell et al. 2005). Glycosaminoglycanes in urine (by electrophoresis → With the present techniques, routine high-resolution Sanfi lippo disease) chromosome testing is indicated in any child with Consider maternal phenylalanine mental retardation, even in the absence of dysmorphic Consider purines and pyrimidines (urine) features. FISH analysis for subtelomeric arrangements, Additional laboratory tests in mental retardation with neurological abnormalities MECP2 molecular testing for , catch 22, fragile X, Consider additional genetic analyses, e.g. Rett syndrome, Angelman and/or Prader–Willi syndromes should be Angelman syndrome, FISH or microarray analysis initiated using selection criteria, such as clinical check- Urine: Simple tests, organic acids, glycosaminoglycans, lists. Microarray analysis is on the edge of becoming oligosaccharides, sialic acid widely available replacing FISH analysis. Plasma/serum: quantitative amino acids Neuroradiological studies have a high yield for rela- Biotinidase activity, if not included in neonatal screening (dried blood spots) tively unspecifi c brain abnormalities in patients with Consider purines and pyrimidines (urine), glycosylation mental retardation, such as delayed myelination, but a disorders (CDG) low relevance in helping to establish a specifi c diagno- Consider thiamine defi ciency sis. It is debatable whether they should be routinely Additional laboratory tests in mental retardation with performed as they often involve invasive sedation. They dysmorphic features Additional genetic analyses, e.g. microarray analysis have to be initiated in the presence of specifi c symp- Sterols, peroxisomal studies (very long-chain fatty acids, toms such as abnormal brain size, focal neurological phytanic acids, plasmalogens) fi ndings, epilepsy, skin lesions or neurodegeneration. Transferrin isoelectric focusing for glycosylation studies Metabolic studies should also not be initiated in the (CDG) fi rst “diagnostic round”, but in the absence of clues for Consider maternal phenylalanine other causes the yield is still suffi ciently high (at least Psychomotor retardation and… Progressive loss of skills or organomegaly: consider 1% in an average population) to recommend testing (see lysosomal disorders Table C5.4). Neonatal screening programs have Multi-system disorder: consider mitochondrial disorders decreased the number of mentally retarded persons due peroxisomal disorders, glycosylation disorders (CDG) to well treatable metabolic diseases. A special issue in Progressive myopia, dislocated eye lenses: measure total testing for metabolic disorders is whether the disorder homocysteine Abnormal hair: consider Menkes disease thought of is potentially treatable. Doing focused or Macrocephaly: check urinary organic acids (glutaric aciduria sequential metabolic testing (i.e. based on results of type I, Canavan disease), lysosomal disorders. MRI is basic tests) can increase the diagnostic yield up to 14%. recommended as hydrocephalus must be ruled out. One Individual relatively homogeneous populations can of the recently identifi ed leukodystrophies, megalenceph- have a much higher yield of specifi c metabolic disor- alic leukodystrophy with Subcortical cysts can only be diagnosed by MRI ders, e.g. the Finnish or the Ashkenazi Jewish popula- (adapted from: Zschocke and Hoffmann (2004) ) tion. Parental consanguinity, loss of developmental milestones and/or a previously affected sibling point to a monogenic disorder. In these circumstances, com- Remember prehensive metabolic evaluation should be initiated ¼ early together with neuroimaging studies, and genetic Inborn errors of metabolism are rare causes of and ophthalmologic evaluation. isolated non-specifi c mental retardation. C5 Neurological Disease 137

disorders in which epilepsy can start at any age. It is also ¼ In metabolic disorders presenting with mental important to look for other symptoms, such as ataxia or retardation, additional neurological and/or dementia. EEG features are sometime of help, but usu- somatic systems are involved guiding the diag- ally non-speciﬁ c. nostic process. ¼ Doing a focused or sequential metabolic testing can increase the diagnostic yield up to 14%. C5.7.1 Neonatal Period

C5.7 Epilepsy The great majority of vitamin-responsive epilepsies start in the neonatal period or early infancy (Surtees Epilepsy is an important sign of many metabolic disor- et al. 2007). Although rare, they are treatable disorders. ders, although metabolic disorders are rarely the underly- Seizures consist of a mixture of partial, massive myo- ing cause of epilepsy (Wolf et al. 2005). Convulsions are clonus, erratic myoclonus of face and extremities, or considered to refl ect grey matter involvement in neurode- sometimes tonic seizures. If myoclonic seizures domi- generative disorders, but may also be prominent in acute nate the clinical pattern, the epilepsy syndrome is called metabolic decompensation, which may be reversible. “early myoclonic encephalopathy” (EME). EEG often Seizure semiology or EEG patterns mostly do not depend shows a burst-suppression pattern. Myoclonic jerks on the underlying disorder, but are rather determined by may be without EEG equivalent. Among the non-treat- age at presentation, although there are exceptions. able disorders, non-ketotic hyperglycinemia (NKH) is Seizures in metabolic disorders are more often focal than the most frequent. In many cases, the aetiology remains generalised. In most disorders, their treatment is guided unclear despite adequate investigations. In Table C5.5, by seizure semiology and age of onset and requires con- treatable and non-treatable epileptic encephalopathies ventional antiepileptic drugs, as in epilepsy due to other starting in the neonatal period are summarised. causes. Specifi c therapy – mostly cofactors as in biotinidase defi ciency but also dietary treatment as in phenylketonuria – only exists for few metabolic disorders. In these, Pyridoxine-Dependent Seizures (PDS) it must be started early in order to avoid persisting neuro- First described in 1954, the genetic basis of pyridox- logic damage. Therefore, it is of utmost importance to ine (B )-dependent seizures was discovered in 2006. think about metabolic causes of epilepsy early in the 6 Mutations in ALDH7A1 encoding antiquitin, which course of presentation, especially in neonates and has a-aminoadipic semialdehyde dehydrogenase infants, in order to provide adequate treatment. activity, lead to accumulation of a-aminoadipic In otherwise healthy children with febrile seizures semialdehyde and D1-piperideine-6-carboxylate. or clear-cut epileptic syndromes responding promptly The latter metabolite inactivates pyridoxal-5′- to adequate antiepileptic treatment, metabolic investi- phosphate, which is involved as an essential cofactor gations are not warranted. In children with cryptogenic in many enzymatic reactions including neurotrans- diffi cult-to-treat epilepsy, metabolic investigations mitter metabolism. The classical form of PDS starts should be initiated. However, it is important to correctly within the neonatal period with therapy-resistant sei- classify the epileptic syndrome and to consider other zures, especially myoclonic, but also tonic seizures. underlying disorders such as focal cortical dysplasia, Administration of pyridoxine usually has an imme- which might be diffi cult to fi nd even with advanced diate positive effect; children may become apnoeic. MRI technology or other genetic disorders such as chan- Atypical forms of PDS start later in infancy or show nelopathies or mutations in genes like ARX or CDKL5 partial effect of conventional antiepileptic drugs. In instead of repeating or expanding metabolic testing. some children, the response to pyridoxine may ini- One of the most important aspects in the differential tially not occur, and some respond to folinic acid diagnosis of epilepsy caused by metabolic disorders is instead. There is no universal protocol for a pyridox- the age of onset of seizures. Diagnoses to consider in a ine trial (see below). The disorder can now be unequiv- neonate are completely different from possible diagno- ocally diagnosed by measuring a-aminoadipic ses in a school-aged child. Exceptions are mitochondrial 138 A. García-Cazorla et al.

semialdehyde and pipecolic acid, which are diagnos- vitamin B6, in the CNS. Activity of different tically elevated in urine and plasma even under treat- enzymes dependent of PLP is reduced, leading to a ment. Psychomotor development is better the earlier disturbed metabolism of neurotransmitters and the pyridoxine treatment is started, but even with amino acids. Measurement of these metabolites adequate and immediate treatment outcome is mostly often, but not invariably, gives abnormal results, not entirely normal. typically elevations of 3-methoxytyrosine, glycine, threonine as well as lactate, and decreases of homovanillic acid and 5-hydroxyindolacetic acid in CSF. The latter constellation mimics aromatic l-amino Pyridoxal Phosphate-Dependent Seizures acid decaboxylase deﬁ ciency. Affected neonates Mutations in the PNPO gene encoding pyridox(am) are often born prematurely with signs of foetal or ine 5-phosphate oxidase lead to decreased levels neonatal distress. Seizures start early within the ﬁ rst of pyridoxalphosphate (PLP), the active form of day of life and are resistant to therapy. They respond

Table C5.5 Epileptic encephalopathies presenting in the neonatal period Treatable disorders Treatment Diagnostic test Pyridoxine dependency Pyridoxine (or pyridoxal-5-phos- Response to pyridoxine; elevated pipecolic acid phate) 30 mg/kg (usually (CSF, plasma, urine) and a-aminoadipic 100 mg) as starting dose, then semialdehyde (urine) 30 mg/kg/day Folinic acid dependency Folinic acid 2–3 mg/kg/day in 3 Response to folinic acid; elevated pipecolic acid doses (CSF, plasma, urine) and a-aminoadipic semialdehyde (urine); unknown peak in CSF HPLC PNPO defi ciency pyridoxal-5-phosphate 30–40 mg/ Response to pyridoxal 5-phosphate; elevated kg/day in 3–4 single doses glycine, threonine, 3-orthomethyldopa, lactate Phosphoserine aminotransferase Serine supplementation Low CSF and plasma serine and glycine defi ciency Urea cycle defects Appropriate treatment Hyperammonaemia, plasma amino acids, urine orotic acid Organic acidurias Appropriate treatment Urinary organic acids, acylcarnitines Holocarboxylase synthetase High-dose biotin Organic acids defi ciency Maple syrup urine disease Appropriate treatment Plasma amino acids: elevation of leucine, Not treatable disorders Non-ketotic hyperglycinaemia Elevated CSF:plasma glycine ratio Zellweger syndrome Elevated VLCFA Neonatal adrenoleukodystrophy Elevated VLCFA Molybdenum cofactor disease Sulphite test in fresh urine, fi broblast studies, uric acid Sulphite oxidase defi ciency Sulphite test in fresh urine, fi broblast studies GABA transaminase defi ciency GABA in CSF Adenylosuccinate lyase defi ciency Modifi ed Bratton–Marshall test, purines in urine Respiratory chain disorders and Lactate elevation in CSF, plasma and urine, PDHc defi ciency activity of respiratory chain enzymes and PDHc in muscle and fi broblasts Glutamate transporter defi ciency Glutamate oxidation in fi broblasts, genetic studies (sequencing of SLC25A22) Congenital glutamine defi ciency Extremely low levels of plasma, urine and CSF glutamine Neonatal form of neuronal ceroid Cathepsin D activity lipofuscinosis C5 Neurological Disease 139

intravenously. If there is no response within 24 h, the promptly to administration of PLP (30–60 mg pyri- same dose should be repeated (and possibly increased doxal 5′-phosphate/kg b.w./day), which needs to be up to 500 mg total) before excluding pyridoxine given at least three times per day in order to prevent responsiveness. If there is uncertainty about a partial recurrence of seizures before the next dose. If treat- response, pyridoxine should be continued at 30 mg/kg/ ment is not initiated, the disorder leads to severe day for 7 days before fi nal conclusions are drawn. Also neurologic defi cits and is lethal. If treatment is with this medication, CSF, plasma and urine samples started in the neonatal period, children may survive should be acquired before treatment; again, this must without neurologic symptoms. not delay treatment initiation. The use of vitamins does not preclude the introduction of other vitamins/drugs during this period of time if seizures do not stop. The possibility of late-onset pyridoxine-dependent seizures Non-ketotic Hyperglycinemia (NKH) in children up to 3–4 years of age should be considered NKH is caused by impaired function of the glycine (pyridoxal phosphate or pyridoxine may be tried). cleavage system, a multienzyme complex, with the Folinic acid is effective for the extremely rare folinic P protein subunit being the most frequently affected. acid-dependent seizures in a dose of 2–3 mg/kg/day, Most patients suffer from the classical form starting which appear also to be due to mutations in ALDH7A1 in the fi rst day of life with seizures including singul- encoding antiquitin. It is recommended to maintain the tus, lethargy progressing to coma and apnoea neces- treatment for at least 1 month to test effi cacy. If biotini- sitating artifi cial ventilation. Glycine concentration dase cannot be ruled out quickly, e.g. with neonatal in CSF is highly elevated especially in relation to its screening, biotin: 10–100 mg/day can be administered concentration in plasma (quotient > 0.04). EEG as well. Urinary organic acids should be analysed shows a burst-suppression pattern. If children are before starting treatment. Plasma biotinidase activity ventilated, they may survive, but prognosis regard- will remain diagnostic despite therapy. ing the neurologic outcome is extremely poor. Development is virtually absent, and refractory epilepsy persists even with dietary restriction of gly- Remember cine and treatment with sodium benzoate, which lowers also glycine levels in CSF. In neonatal-onset epileptic encephalopathy, results of pending metabolic investigations must not be waited for in order to start treatment which must not be delayed. Every neonate with seizures should C5.7.2 Treatment Protocol in Neonates have a trial with pyridoxine, pyridoxal phosphate (or only pyridoxal phosphate) and folinic acid. Pyridoxal phosphate can be used as fi rst-line treatment as it stops both pyridoxine- and pyridoxal-phosphate- dependent seizures. The recommended dose is 30–60 mg/ kg/day in at least three doses. To evaluate effi cacy, ther- C5.7.3 Infancy apy should be maintained for 5 days. CSF (and urine) studies should be performed before treatment in order to In infancy, several disorders present with epileptic have a biochemical marker, but should not delay treat- encephalopathy, among them untreated PKU has virtu- ment. If therapy is successful, the appropriate genetic ally disappeared in many countries with neonatal screen- studies should be performed as well. ing, biotinidase defi ciency, GLUT1 defi ciency, infantile If pyridoxal phosphate is not available, pyridoxine neuronal ceroid lipofuscinosis (CLN1), GAMT defi -

(B6) should be used ﬁ rst: 100 mg in a neonate (or ciency (also CRTR deﬁ ciency) and Menkes disease. 30 mg/kg) either intravenously or orally in a single Late-onset forms of pyridoxine dependency may start dose, preferably with EEG monitoring. High doses during infancy and early childhood. In these disorders, may be necessary to control seizures, at least initially. seizures are (partially) resistant to treatment with con- In classical cases, we suggest a starting dose of 100 mg ventional antiepileptic drugs. 140 A. García-Cazorla et al.

GLUT1 Defi ciency and a diet enriched in ornithine and low in arginine, the latter in order to lower the guanidino compounds Glucose enters the brain by crossing the blood-brain appear to be toxic for the CNS. Diet is effi cient for barrier. Its uptake into the brain is mediated by the seizure control even if started late in the course of glucose transporter type 1 (GLUT1). GLUT1 defi - the disease. In AGAT defi ciency, creatine must be ciency is characterised by persistent hypoglycorrha- supplemented. There is no treatment for CRTR chia (glucose CSF/serum quotient < 0.45) in the defi ciency. absence of hypoglycemia and with normal or low lactate in the CSF. Although the neurologic phenotype may vary from case to case, epilepsy starting in infancy is the predominant feature. Clinical manifestations vary from mild to severe and include Biotinidase Defi ciency acquired microcephaly, developmental delay, pyra- Epilepsy often starts at 3 or 4 months of life. West midal signs, a complex movement disorder with syndrome is the most frequent epileptic syndrome, dystonia, ataxia and spasticity, and different forms and conventional antiepileptic drugs are ineffective. of seizures, including myoclonic seizures resistant Alopecia and (perioral) dermatitis are useful clinical to anticonvulsant drugs. GLUT1 defi ciency can be clues. Psychomotor development is severely delayed, treated effectively with the ketogenic diet. Mutation and, if untreated, optic atrophy and deafness develop. analysis of SLC2A1 encoding GLUT1 and measure- Lactate is mostly elevated as are 3-methylcrotonylg- ment of erythrocyte uptake of 3-O-methyl-D- lycine, 3-hydroxyisovaleric, 3-hydroxypropionic and glucose confi rm the diagnosis. Mutations are usually 2-methylcitric acid in urine and other body fl uids. de novo, but familial cases have been described. Low doses of biotin (5–10 mg/day) stop the seizures. Diagnosis is easily made by measuring biotinidase activity,which is possible in dried blood spots. GAMT Defi ciency and Other Creatine Defi ciency Syndromes Creatine is crucial for energy metabolism and is synthesised in a two-step process: the fi rst is medi- Menkes Disease ated by AGAT (arginine–glycine amidinotransferase) and the second by GAMT (guanidinoacetate This is an X-linked condition which starts in early methyltransferase). Furthermore, a CRTR encoded infancy. Neonatal period is usually normal. First by a X-chromosomal gene is required for creatine symptom is often epilepsy with refractory focal sei- uptake into brain and muscle. Disorders of creatine zures and even focal status epilepticus or West syn- synthesis or transport produce psychomotor delay drome. Development is severely delayed, muscle and epilepsy, which is frequently refractory to con- hypotonia profound. Spasticity develops later. Hairs ventional antiepileptic drugs. In all the disorders, are abnormally brittle and steely; the microscopical low creatine concentration in the brain is demon- aspect is that of pili torti. Copper and ceruloplasmin strated by a severe decrease or absence of the nor- are low. The genetic defect involves a copper-trans- mal creatine peak in brain MRS (spectroscopy). All porting ATPase (ATP7A). Copper defi ciency also disorders of creatine metabolism can be reliably involves other structures, especially bones and con- diagnosed by analysis of guanidino compounds in nective tissue. Bone fractures and subdural haema- urine or plasma. In GAMT defi ciency, guanidinoac- tomas sometimes evoke the diagnosis of etate and other guanidino compounds are elevated. non-accidental injuries. In the cranial MRI, cerebral Epilepsy is especially prominent in this defect and vessels show increased tortuosity (Fig. C5.3). includes manifestation as West syndrome. It is resis- Treatment consists of subcutaneous copper-histi- tant to conventional anticonvulsant therapy. GAMT dine, but its effects are only marginal if started after defi ciency can be treated by creatine supplementation manifestation of the disease. C5 Neurological Disease 141

a bc

Fig. C5.3 Menkes disease. In this infant with Menkes disease, frequent ﬁ nding. Hairs are steely and fragile and were never cut wormian bones in the lambda and sagittal sutures are seen (a), (c). The MRI at the age of 4 shows tortuous and enlarged vessels (b) depicts a serial rib fracture of ribs 4–8 on the right, another in these axial T2w images (d) and (e)

C5.7.4 Childhood and Adolescence Late Infantile Neuronal Ceroidlipofuscinosis (CLN2, Jansky–Bielschowsky) In this age group, late infantile (CLN2) and juvenile This autosomal recessive disorder is caused by (CLN3) forms of neuronal ceroid lipofuscinoses mutations in the gene coding for TPP1, a lysosomal (NCL) and mitochondrial disorders are the most com- enzyme important for protein degradation. The dis- mon neurometabolic conditions with prominent sei- ease starts between age 2 and 5 years, usually with zures. If MRI shows stroke-like lesions in the occipital progressive ataxia and epilepsy. Generalised tonic– or temporal regions, a mitochondrial disease is the clonic seizures are frequent in the beginning; later on most probable diagnosis. Alpers disease and also myoclonic and focal clonic seizures are seen. Epilepsy MELAS may manifest with refractory status epilep- is difﬁ cult to treat, especially myoclonus. Dementia ticus in a previously normal child, a difﬁ cult clini- proceeds quickly. The retina is also involved, and cal situation. Regarding antiepileptic therapy, drugs due to pigmentary retinopathy vision deteriorates. should be chosen depending on the epileptic syn- EEG shows background changes and spikes with drome with the exception of valproic acid, which slow photic stimulation, which can trigger seizures must not be used in mitochondrial disease, espe- (Fig. C5.4). MRI displays cerebellar and less supra- cially Alpers disease, as the risk of fatal liver failure tentorial atrophy and mild white matter changes. is high. 142 A. García-Cazorla et al.

Fig. C5.4 Slow photic stimulation triggers posterior spikes in this child with late-infantile neuronal ceroid lipofuscinosis

Alpers Disease Alpers syndrome are recessive mutations in polymerase g 1 (POLG1), an enzyme that is involved in This autosomal recessive disorder starts at any age mtDNA maintenance. mtDNA is depleted in liver. from infancy to young adulthood, most frequently In some patients, lactate and protein are elevated in in childhood. First symptom is often refractory sta- CSF; in others, all investigations including activity tus epilepticus in a previously healthy child with of respiratory chain enzymes in muscle remain normal or almost normal development. EEG in this repeatedly negative. phase shows rhythmic high-amplitude delta with superimposed (poly) spikes (RHADS) involving either left or right posterior region (see Fig. C5.5). The progressive myoclonic epilepsies are a group of Liver function at that stage is usually normal. If sta- genetic disorders where epilepsy with myoclonic and tus epilepticus is survived, most children develop a generalised seizures appears in combination with cog- relentlessly downhill course with refractory sei- nitive deterioration and usually also ataxia. Myoclonus zures, especially epilepsia partialis continua, optic is often exaggerated by external stimuli, such as light, atrophy and dementia. Some children have a more sound, or touch, and they are very difﬁ cult to treat. This stable course with almost complete recovery after group of disorders comprises Unverricht–Lundborg dis- status. Administration of valproic acid almost ease, which has a more benign long-term course (muta- invariably triggers liver failure. If liver transplanta- tions in Cystatin B, CSTB, formerly known as EPM1), tion is done, neurologic outcome is extremely poor Lafora disease (80% of patients carry mutations in and survival short. Liver failure can also develop EPM2A, Laforin; another gene involved is EPM2B or without valproic acid. The most frequent cause of NHLRC1), myoclonic epilepsy with ragged red ﬁ bres (MERRF; mutations in MTTK which is located in the C5 Neurological Disease 143

Fig. C5.5 RHADS over the left posterior region at manifestation of Alpers disease; the child was compound heterozygous for two mutations in POLG1 mtDNA), some forms of the NCL, and the most fre- development of structured diagnostic and therapeutic quent being late infantile NCL, sialidosis (neuramini- approaches in the light of age-dependant physiological dase defi ciency; a cherry red-spot is common) and development and pathophysiological responses of dentatorubral-pallidoluysian atrophy (DRPLA). gross and fi ne motor functions. Again a logical series of investigations should be followed with the primary aim of early diagnosis of treatable conditions. Remember Only a few metabolic epilepsies beyond the neonatal period are amenable to causal treatment of a metabolic cause. In the majority of patients, conven- C5.9 Ataxia tional antiepileptic drugs must be used according to the seizure type. Valproic acid must be avoided in Ataxia is defi ned as an inability to maintain normal children with Alpers disease and other mitochon- posture and smoothness of movement while force and drial disorders as it can trigger fatal liver failure sensation are intact. There are many variations in the (Table C5.6). clinical phenotype, ranging from fi ndings of pure cerebellar dysfunction to mixed patterns of CNS (extrapyramidal pathways, brainstem and/or cerebral cortical participation) or peripheral nervous system involve- C5.8 Movement Disorders ment. A wide range of molecular defects has been identifi ed, among them classical metabolic disorders (Parker Our understanding of pediatric movement disorders is et al. 2003), as well as also other neurogenetic defects. rapidly evolving, stimulated by progress in the discov- It is important to fi rst consider the (partially) treatable ery of monogenic neurogenetic disorders and the disorders that give rise to ataxia, including vitamin E 144 A. García-Cazorla et al.

Table C5.6 Checklist of laboratory tests in epilepsy focusing C5.9.1 Intermittent/Episodic Ataxias on monogenic disorders Laboratory tests in epilepsy with neonatal onset These are mainly defects of intermediary metabolism. Basic laboratory tests (blood glucose, lactate, ammonia, Ataxia is a frequent sign during acute or subacute acid–base status, calcium, magnesium) decompensation of amino acid (especially MSUD), Pipecolic acid and 5-aminoadipic semialdehyde (pyridoxin- dependent seizures) organic acid and urea cycle disorders. Defects of CNS and plasma amino acids (non-ketotic hyperglycinemia, energy metabolism presenting with intermittent ataxia serine biosynthesis defects, congenital glutamine may involve exclusively the nervous system and are defi ciency) often more diffi cult to diagnose. Lactate may be ele- Neurotransmitters (PNPO defi ciency) vated only intermittently with clinical abnormalities. CSF GABA (GABA transaminase defi ciency) Sulphite test (molybdenum cofactor defi ciency and sulphite Intermittent ataxia can be seen in Hartnup disease; oxidase defi ciency) additional symptoms are pellagra-like skin changes, VLCFA (peroxisomal disorders) photic dermatitis and psychiatric symptoms. Laboratory tests in epilepsy with infantile onset (<1 year) Carbohydrate-sensitive ataxia is a feature of mild pyruvate dehydrogenase defi ciency and occurs only Basic laboratory tests (blood glucose, lactate, ammonia, acid–base status, calcium, magnesium) in boys. GLUT1 defi ciency may present with inter- CSF and plasma glucose (GLUT1 defi ciency) mittent ataxia as the only fi nding, mimicking other Amino acids (PKU) episodic ataxias. Ataxia is usually worse before meals. CSF lactate (mitochondrial disorders) Some children improve greatly with frequent carbo- Organic acids (biotinidase defi ciency, MHBD defi ciency, hydrate-rich snacks and do not need ketogenic diet. organic acidurias) Mild forms of biotinidase activity may also give rise Biotinidase activity Creatine, creatinine and guanidinoacetate in urine (creatine to intermittent ataxia. The so-called “episodic atax- synthesis defects and CRTR defect) ias” may be mistaken for metabolic disorders, Copper and ceruloplasmin (Menkes disease) although they belong to the channelopathies. Correct VLCFA (peroxisomal disorders) diagnosis is important, as effective treatment exists Purines and pyrimidines in urine also for these disorders. As a rule, metabolic param- Consider lysosomal studies (e.g. CLN1, Tay–Sachs disease) eters may be normal between bouts of ataxia; testing Pipecolic acid and 5-AASA (pyridoxine-dependent seizures) is therefore mandatory when patients are symptom- Laboratory tests in epilepsy with onset in late infancy and atic and usually involves CSF investigations also. An childhood (and usually additional signs and symptoms as ataxia or regression) important non-metabolic differential diagnosis for intermittent ataxia is that of intoxication, especially Basic laboratory tests (lactate, ammonia, acid–base status) with benzodiazepines and other centrally acting CSF lactate (mitochondrial disorders) CSF and plasma glucose (GLUT1 defi ciency) drugs. Creatine, creatinine and guanidinoacetate in urine (creatine synthesis defects and CRTR defect) Lysosomal studies (e.g. CLN2) Pipecolic acid and 5-AASA (pyridoxine-dependent seizures) for atypical late-onset cases, also consider therapeutic C5.9.2 Non-Progressive Ataxia trial Purines and pyrimidines in urine Non-progressive ataxias are mostly secondary to cerebellar malformations, e.g. in Joubert syndrome. True metabolic causes of non-progressive ataxias include CDG type Ia and other types of congenital glycosyla- defi ciency, biotinidase defi ciency, abetalipoproteine- tion defects, Marinescu–Sjögren syndrome, 4-hydroxy- mia, thiamine-responsive pyruvate dehydrogenase butyric aciduria defi ciency and L-2-hydroxyglutaric defi ciency, cerebrotendinous xanthomatosis and pri- aciduria. In CDG Ia, MRI displays cerebellar atrophy mary coenzyme Q10 defi ciency. A pragmatic approach (Poretti et al. 2008) or hypoplasia is usually accompa- classifi es ataxias according to their clinical evolution: nied by brainstem hypoplasia. Mitochondrial disorders intermittent or episodic, stable or progressive. rarely show non-progressive ataxia. C5 Neurological Disease 145

C5.9.3 Progressive Ataxia lesion) and cardiomyopathy. MRI is usually normal, but may show spinal atrophy later in the disease. Many neurometabolic and neurodegenerative disor- Metabolic investigations are normal. The effect of ders involve the cerebellum and are accompanied by a antioxidant therapy with idebenone, a coenzyme more or less prominent ataxia, which is most often Q10 homologue, is still under debate; cardiac hyper- progressive. In some disorders, ataxia is the most trophy may improve, but neurologic symptoms prominent sign and these are usually called the heredo- appear to remain constant, although there might be ataxias. Friedreich’s ataxia is part of this group, albeit an effect with higher doses. not a classical metabolic disorder, also the vitamin E-responsive ataxias. Ataxia is a prominent symptom in Refsum disease, cerebrotendinous xanthomoatosis and mevalonic aciduria. The relatively recent group of C5.9.4 Vitamin E-Responsive Ataxias ataxias with coenzyme Q defi ciency consists of sev- 10 (AVED, Ataxias with Isolated eral disorders without clear molecular defect. Infantile Vitamin E Defi ciency) neuroaxonal dystrophy (INAD) is a rare neurodegenerative disorder starting in the second year of life with ataxia, hypotonia and nystagmus. Vitamin E defi ciency results from recessive mutations in the gene for a-tocopherol transfer protein (TTP1). Laboratory fi ndings include low-to-absent serum vita- Remember min E and high serum cholesterol, triglycerides, and The rare treatable causes for progressive ataxia should b-lipoprotein. High doses of vitamin E (400–1200 IU/ be checked in all the patients by determining – day) improve neurologic function. This disorder can start from childhood to adulthood. MRI is usually nor- vitamin E levels, phytanic acid, possibly Q10 levels in mononuclear cells or muscle, and if clinical man- mal. Retinitis pigmentosa, peripheral neuropathy and ifestations are suggestive, also sterols. pyramidal signs may be present; cardiomyopathy is rare. Abetalipoproteinemia (Bassen–Kornzweig disease) is a rare disorder resulting from a dysfunction in Non-metabolic disorders are another frequent cause of the microsomal triglyceride transfer protein (MTP) hereditary progressive ataxia. An important group of gene. Acanthocytosis in peripheral blood smears is a the DNA repair disorder – ataxia telangiectasia (AT) is constant fi nding. Decreased serum LDL and VLDL the most frequent example. Ataxia with oculomotor and increased HDL cholesterol levels together with apraxia (AOA) 1 and 2 also belong to this group; a-feto- low triglyceride levels are also present. protein is elevated in AT and AOA2, making for a simple and cheap screening test in progressive ataxias; it belongs to the initial evaluation of an ataxic child. Refsum Disease Friedreich Ataxia Accumulation of phytanic acid is the laboratory Friedreich ataxia (FRDA) is the most common spi- hallmark of this disorder, which is due to mutations nocerebellar degeneration. Onset is before the age in the genes coding for phytanoyl-CoA-hydroxylase of 25, usually before the age of 16. It is an auto- (PAHX or PHYH) or peroxin 7 (PEX7). Affected somal recessive disorder, and virtually all the patients have peripheral neuropathy and retinits affected individuals carry expanded GAA repeats in pigmentosa, sometimes cardiac involvement and the gene coding for frataxin, which appears to be impaired hearing. Ichthyosis and multiple epiphy- involved in mitochondrial iron homoeostasis and seal dysplasia are possible associated symptoms. iron sulphur centre regulation; oxidative stress is The disease starts usually in adolescence or late probably increased. The main clinical signs associ- childhood. Plasmapheresis may be required to effec- ated with ataxia are pes cavus, spinocerebellar tively lower phytanic acid levels, and a diet without degeneration (polyneuropathy and pyramidal tract phytanic acid or chlorophyll should be followed. 146 A. García-Cazorla et al.

Table C5.7 Checklist of Laboratory Investigations in Ataxia Cerebrotendinous Xanthomatosis (CTX) Basal laboratory investigations (including blood gases, This autosomal recessive disorder is caused by lactate, ammonia and albumin) mutations in CYP27A, which is coding for sterol Plasma amino acids 27-hydroxylase. Progressive ataxia and involvement Urinary amino acids of pyramidal tracts start in childhood or adoles- Urinary organic acids CSF and plasma glucose cence, sometimes later. Characteristic features are CSF lactate (and pyruvate) bilateral cataracts. Neonatal liver disease and chronic Vitamine E diarrhoea are other early symptoms. Xanthomata Cholesterol

may appear only late. Cholestanol is elevated in this Coenzyme Q10 disorder. Therapy is with chenodeoxycholic acid Sterols and HMG-CoA-reductase inhibitors in order to Phytanic acid lower cholestanol levels. Transferrin electrophoresis or MS/MS a-Fetoprotein Consider lysosomal studies if regression Consider genetic testing for inherited ataxias

Coenzyme Q -Responsive Ataxias and 10 C5.10 Dystonia, Parkinsonism, Chorea Defects of Coenzyme Q10 Biosynthesis

Coenzyme Q10 defi ciency may produce ataxia as the Movement disorders in inborn errors of metabolism only neurologic manifestation. Q10 levels should be include dystonia, secondary parkinsonism, chorea, measured in muscle or mononuclear cells. High- tremor, myoclonus and tics (Fernández-Alvarez et al. dose coenzyme Q10 supplementation is effective in 2001). Different movement disorders may co-exist in some. The genetic background of ataxias with Q10 the same patient with a metabolic disease. However, defi ciency remains obscure in many cases. It may dystonia is the predominating type of movement disor- be secondary to other genetic defects as aprataxin der in patients with inborn errors of metabolism. In defi ciency in AOA1. Recently, mutations in CABC1 general, diseases causing parkinsonism in adults may (also called ADCK3) could be identifi ed in patients produce dystonia in children. with autosomal recessive ataxia and epilepsy. The When the movement disorder is associated with protein encoded by this gene is involved in biosyn- intercurrent illnesses, onset is usually abrupt and gen- thesis of Q10. The other known defects in Q10 bio- eralised (i.e. in glutaric aciduria type I). In other cases, synthesis – affected genes are PDSS1, PDSS2 and movement disorders develop late in the course of the COQ2 – do not give rise to isolated or predominant disease, with focal onset and progressive generalisa- ataxia, but to multi-system involvement including tion, causing major disability and wheelchair depen- renal disease. dency (i.e. homocystinuria, Niemann–Pick C disease and GAMT defi ciency). Although other signs of neurological dysfunction usually co-exist, some inborn errors of metabolism can initially present as isolated dystonia (Segawa syndrome, panthothenate-kinase-associated Remember neurodegeneration (PKAN), Leigh syndrome, pyruvate Treatable causes of ataxia (vitamin E defi ciency, dehydrogenase defi ciency, and juvenile form of metach-

abetalipoproteinemia, coenzyme Q10 deﬁ ciency and romatic leukodystrophy (MLD). possibly also cerebrotendinous xanthomatosis) should be routinely checked in patients with (progressive) ataxia. Intermittent ataxias are often due C5.10.1 Dystonia to metabolic disorders. Important differential diagnoses for the latter are channelopathies and intoxi- Clinically, the term dystonia is used for both a symptom cations (Table C5.7). (sustained, abnormal muscular contraction causing C5 Neurological Disease 147

Table C5.8 Dystonias Primary dystonia Lesch–Nyhan disease MELAS Pure 3-Methylglutaconic aciduria Idiopathic generalised torsion dystonia (DYT1) Methylmalonic acidemia Transient idiopathic infantile dystonia Mucopolysaccharidoses Paroxysmal kinesiogenic choreoathetosis Niemann–Pick disease Paroxysmal non-kinesiogenic dystonia 2-Oxyglutaric aciduria Paroxysmal exercise-induced dystonia Pterin defects Dystonia “Plus” GTP cyclohydrolase I defi ciency Infantile parkinsonism-dystonia 6-pyruvoyltetrahydropterin synthase defi ciency Myoclonic dystonia (DYT11) Sepiapterin reductase defi ciency Dopa-responsive dystonia (DYT5) Dihydropteridine reductase defi ciency Tyrosine hydroxylase defi ciency Propionic acidemia Aromatic L-amino acid decarboxylase defi ciency Pyruvate dehydrogenase defi ciency Hereditary myoclonic dystonia (DYT11) Sulphite-oxidase defi ciency Rapid-onset dystonia-parkinsonism Tyrosinemia type I Juvenile parkinsonism Triose phosphate isomerase defi ciency Dystonia secondary to metabolic disease Vitamin E defi ciency Biotinidase defi ciency Wilson disease Cerebral folate defi ciency syndrome Other genetic causes of dystonia Creatine defi ciency syndromes (guanidinoacetate Ataxia-oculomotor apraxia methyltransferase defi ciency) Ataxia-telangiectasia Ethylmalonic aciduria Benign familial chorea 4-Hydroxybutyric aciduria Infantile and juvenile Batten disease D-2-Hydroxyglutaric aciduria Dentato-pallidoluysian atrophy L-2-Hydroxyglutaric aciduria Dystonia and sensorineural deafness Niemann–Pick C Familial dystonic-amyotrophic paraplegia Fucosidosis PKAN (Hallervorden–Spatz) disease Galactosemia Hereditary non-progressive athetoid hemiplegia Glutaric aciduria type I Huntington disease GLUT1 defi ciency Intranuclear neuronal inclusion disease Hartnup disease Machado–Joseph disease (striatonigral autosomal Homocystinuria dominant degeneration) Infantile Gaucher disease Myocionic hereditary dystonia with nasal malformations Juvenile GM2 and GMI gangliosidosis Neuroaxonal dystrophy Juvenile metachromatic leukodystrophy Olivocerebellar atrophy Keratan sulphaturia Pelizaeus–Merzbacher disease Krabbe leukodystrophy Progressive calcifi cation of the basal ganglia Leber hereditary optic neuropathy plus dystonia Progressive pallidal degeneration Leigh syndrome Rett syndrome

fl uctuating tone and abnormal posturing) and a group of stage, but glutaric aciduria type I, Leigh syndrome and disorders, which hereafter will be distinguished from other mitochondrial cytopathies are among the most the symptom by the term primary dystonia. Whereas the frequent. group of primary dystonias consists only of fi ve entities If dystonia is focal, has progressed slowly, or with an additional seven dystonias “plus”, a wide range remained isolated over time, it is unlikely to be due to of neurogenetic and neurometabolic disorders can cause an inborn errors of metabolism. In contrast, if dystonia secondary dystonias (Tables C5.8 and C5.9). appears abruptly, settles rapidly, is generalised and In many different metabolic diseases dystonia is a postural from the very fi rst stages of the disease, then a major feature (Assmann et al. 2003). In fact, almost all metabolic cause should be strongly considered. GLUT1 neurometabolic disorders can cause dystonia at some defi ciency can cause paroxysmal exercise-induced 148 A. García-Cazorla et al.

Table C5.9 Checklist of laboratory tests in dystonia and other neurotransmitter defi ciencies), creatine defi - 1) Treatable causes of dystonia: ciency syndromes, cerebral folate defi ciency syndrome, Biogenic amines, pterines, glucose and folate in the CSF Wilson disease, homocystinuria, biotinidase, vitamin E Plasma amino acids and total homocysteine in blood and GLUT1 defi ciencies. Creatine and guanidinoacetate compounds in urine Copper (plasma and urine), ceruloplasmin (plasma) Plasma biotinidase activity Inherited Disorders of Biogenic Amines 2) Pure dystonia: The disorders of dopamine and serotonin synthesis Biogenic amine metabolites in the CSF are aromatic l-amino acid decarboxylase defi ciency, DYT-1 gene tyrosine hydroxylase defi ciency and disorders of tet- 3) Dystonia associated with other neurologic signs: Parkinsonism: Biogenic amines and pterines in the CSF, rahydrobiopterin (BH4) synthesis. In contrast to clas-

copper and ceruloplasmin, lactate, pyruvate, plasma sical forms of BH4 defi ciency Segawa disease (GTP amino acids (consider respiratory chain activity and cyclohydrolase I defi ciency) and sepiapterin reductase mitochondrial DNA study), consider PANK2 gene defi ciency present without hyperphenylalaninemia depending on MRI and other clinical fi ndings, juvenile parkinsonism genes and are not detected by neonatal screening for phe- Myoclonus: urine organic acids, DYT5 gene, consider nylketonuria. Childhood-onset dystonia or parkin- mitochondrial and lysosomal investigations sonism-dystonia is the hallmark of functional Developmental delay and spasticity: uric acid plasma and dopamine defi ciency and the most suggestive clinical urine, purines in urine, lactate, pyruvate, plasma symptom of pediatric neurotransmitter diseases. In amino acids, organic acids, depending on other data consider mitochondrial and lysosomal investigations very young infants, the symptoms are less specifi c, Cerebellar ataxia: organic acids in urine, plasma and patients often presenting with truncal hypotonia, urine amino acids, plasma vitamin E with cholesterol restlessness, feeding diffi culties or motor delay. and triglyceride studies, consider mitochondrial Hypokinesia, increased limb tone, oculogyric crises, investigations, aprataxin gene (if oculomotor apraxia) and immunological studies (ataxia-telangiectasia), ptosis and faulty temperature regulation are also some depending on other data consider Niemann–Pick C of the common signs. Segawa disease, also called studies and spinocerebellar inherited diseases dopa-responsive dystonia, is an autosomal dominant 4) Abrupt generalised dystonia (especially in a catabolic disease characterised by progressive dystonia that state): normally appears during the fi rst decade of life, is Organic acids (including glutaric and 3-hydroxyglutaric acids), lactate, pyruvate, plasma amino acids not associated with cognitive impairment and has a (consider other mitochondrial and PDH studies) dramatic and lifelong responsiveness to levodopa. 5) Dystonia associated with other signs: Tyrosine hydroxylase, amino acid decarboxylase and Visceral signs: lysosomal investigations, urine organic sepiapterin defi ciencies may also respond in different acids degrees to levodopa. The most important biochemi- Ocular signs: if optic atrophy gen LHON, if oculomotor apraxia gen aprataxin, if ocular telangiectasia, cal investigations for the diagnosis of these neuro- immunologic studies and gen ataxia-telangiectasia, if logical diseases are cerebrospinal fl uid investigations retinitis pigmentosa, plasma vitamin E, mitochon- for neurotransmitter metabolites and pterins (see drial, lysosomal investigations later). Timely diagnosis is especially important for Deafness: plasma biotinidase activity, glycosaminogly- those conditions with specifi c treatments. cans and oligosaccharides in urine, consider mitochondrial investigations and dystonia/deafness gene Glutaric Aciduria Type I Glutaric aciduria type I is an autosomal recessive dyskinesia and also complex movement disorders with disease caused by the defi ciency of glutaryl-CoA elements of dystonia, ataxia and spasticity, even with- dehydrogenase. Classically, patients have an abrupt out epilepsy (García-Cazorla et al. 2008). presentation, usually between 6 and 18 months of Obviously, in all the cases it is important to consider age, with encephalitis-like symptoms following an fi rst those inborn errors of metabolism for which some acute illness. Later psychomotor regression and effective therapeutic intervention is possible, such as abnormal movement disorders appear; dystonic or dopa-responsive dystonia syndromes (Segawa disease C5 Neurological Disease 149

choreo-athetotic movements, focal, segmental or also be apparent. A small number of patients with generalised, present as twisting and torsion postures alternating variants of HPRT did not display abnor- of hands and feet in a child otherwise alert, with pro- mal behaviour; so enzyme assay is indicated in any found hypotonia of the neck and trunk and stiff arms patient with the neurologic features of this disease. and legs. Typically T2-weighted MRI shows fron- Allopurinol is indicated for renal manifestations but totemporal atrophy and high signal in the striatum. does not improve neurological symptoms. Levodopa, Macrocephaly is present in about 70% of the cases. dopaminergic agonists, dopamine-depleting agents In a minority of cases, the onset is insidious with have been used to treat movement disorders with- slowly developing psychomotor delay, hypotonia out convincing results. Pimozide, haloperidol, fl u- and dystonic posturing. Urine organic acid analysis phenazine and risperidone have been used to control reveals elevations of glutaric and 3-hydroxyglutaric self-injurious behaviour; baclofen and benzodiaz- acids. Isolated slight increases of 3-hydroxyglutaric epines may be useful to control spasticity. acid only or normal values of both the acids have been described. Increased ratios of acylcarnitines to free carnitine in plasma and urine as well as eleva- Wilson Disease tions of glutaryl-carnitine in body fl uids can also be detected. Treatment consists of a diet combined with This is an autosomal recessive disorder due to a oral supplementation of l-carnitine and an intensifi ed defect in the copper adenosine triphosphatase trans- emergency treatment during acute episodes of inter- porter (ATP7B) resulting in abnormal deposition of current illness. This strategy has signifi cantly reduced copper in liver, brain and other tissues. Hepatic the frequency of acute encephalopathic crises in early abnormalities are the fi rst manifestations of the dis- diagnosed patients. Anticholinergic drugs and botuli- ease in 50% and can co-exist with neurological num toxin type A have proved benefi cial as symp- forms. Neurological manifestations usually appear tomatic treatment for severe movement disorders. after the age of 10 years, although onset of neurological symptoms in patients as young as 4 years has been reported. In children, neurological symptoms may begin insidiously. Dysarthria, incoordination of Lesch–Nyhan Disease voluntary movements and tremor are common Lesch–Nyhan disease is caused by the X-linked symptoms, as are dystonia or a rigid-akinetic syn- recessive defi ciency of the purine salvage enzyme drome. There may be involuntary choreiform move- hypoxanthine-guanine phosphoribosyltransferase ments, and the gait may be affected. Psychiatric (HPRT). Affected patients exhibit over-production of manifestations and behaviour disorders are also uric acid, a characteristic neurobehavioural syn- common. Ocular abnormalities are usually silent but drome that includes mental retardation, recurrent have a great diagnostic value. The Kayser–Fleischer self-injurious behaviour and a complex spectrum of ring (see Fig. C2.2 Chap. C2) is practically pathog- motor disturbances. Pathogenesis of the neurologic nomonic for the disease and precedes the appear- and behavioural features remains incompletely ance of neurological abnormalities. MRI usually understood but has been related to dopaminergic shows hypointensity on T1-weighted and hyperin- function in the basal ganglia. All patients exhibit pro- tensity on T2-weighted sequences in the lenticular found motor disabilities. Extrapyramidal and pyra- nuclei, thalami, brainstem, claustrum and white midal signs typically begin to develop between 6 and matter (Fig. C5.6). The contrast between the normal 18 months of age. The most prominent feature of the low intensity of the red nuclei and substantia nigra motor syndrome in all patients is dystonia affecting and the abnormal high-intensity signal in the mid- all parts of the body. Less than half of the patients brain tegmentum results in the “face of the giant also had chorea less severe than dystonia, and it typi- panda sign”. Plasma ceruloplasmin and copper lev- cally emerged only with stress or excitement (Jinnah els are decreased, whereas cupruria is augmented. et al. 2006). Other movement disorders as tremor, Because of frequent side effects and initial neuro- opisthotonus and extensor spasms of the trunk may logic deterioration with penicillamine therapy, the 150 A. García-Cazorla et al.

Fig. C5.6 Wilson disease Axial T2w images of an adolescent demonstrating (a) symmetric hyperintense signal changes and atrophy of caudate nucleus and putamen and (b) hyperintense signal changes in the midbrain. The “sign of the giant panda” is marginally visible in this patient

less toxic substances trientine or zinc have gradually muscles is the main sign in juvenile onset cases fol- replaced penicillamine over the past few years as the lowed by intellectual deterioration, seizures and fi rst-line treatment for Wilson disease. Initial zinc ataxia. Parkinsonism is the predominant clinical therapy for asymptomatic/presymptomatic patients manifestation in late-onset (adult) cases. MRI shows and maintaining zinc therapy in patients after long- bilateral T2 high signal in the medial part of the term chelation seem to be safe and effective. pallidum surrounded by a larger zone of markedly low signal, the so-called eye-of-the-tiger image (Fig. C5.7). In some cases, only a markedly decreased Panthothenate-Kinase-Associated signal of the pallidum without central hyperintensity Neurodegeneration is found. If MRI is done early in the course of the The disease is caused by mutation in the gene for disease, the typical hypointense signal may not be pantothenate kinase 2 (PANK2) located on chromo- present on MRI. The pallidum may be hyperintense some 20. It is characterised by a progressive move- instead, mimicking a mitochondrial disorder. Only ment disorder, mainly consisting of dystonia or symptomatic treatment is available. parkinsonism, usually associated with dementia and pyramidal tract signs. The most specifi c pathological fi ndings include dysmyelination and deposition Remember of iron-staining pigments in the pallidum and the pars reticulata of the substantia nigra, axonal swell- ¼ Dystonia is a major symptom in many neuro- ing in the cerebral cortex and basal ganglia, increased metabolic diseases and is usually associated lipofuscin deposition and, in a few cases, Lewy bod- with other neurological signs. ies. In the early onset form, there is an initial silent ¼ If dystonia is associated with intercurrent ill- period, then unstable gait appears and regression nesses, appears abruptly and is suddenly gen- begins between 5 and 10 years. Dystonia (often in eralised or if it is focal but has a progressive the upper limbs, trunk and oromandibular muscles) generalisation, it is very probably caused by a may be an early manifestation but may not be appar- metabolic disorder. ent until 1 year after onset of regression. Retinopathy ¼ CSF studies are necessary if fi rst-line examina- and acanthocytosis are common. Progressive deteri- tions have not detected the aetiology. Also, l-dopa oration leads to death between 11 and 15 years of should be tried, especially when CSF analysis life. Dystonia in the lower limbs and oromandibular reveals low values of dopamine metabolites. C5 Neurological Disease 151

C5.10.2 Parkinsonism or Rigid Akinetic C5.10.4 Spasticity Syndrome Spasticity is a very common clinical situation in pediatric and adult neurology. It refers to dysfunction of Metabolic disorders causing the rigid-akinetic syn- the motor system in which certain muscles are contin- drome as the main clinical symptom include: (1) neu- uously contracted. This contraction gives rise to mus- rotransmitter defi ciencies (especially early onset cle stiffness interfering with voluntary movements. forms) such as tyrosine hydroxylase, aromatic l-amino Control of voluntary movements is achieved through acid decarboxylase and pterin defects; (2) early onset the upper motor neurons in the brain motor cortex, disorders of energy metabolism such as pyruvate car- which send their axons via the corticospinal tract to boxylase defi ciency or some mitochondrial DNA connect to lower motor neurons in the spinal cord. defects and (3) panthothenate-associated neurodegen- Spasticity is the result of damage to upper motor neu- eration (PKAN). In Wilson disease onset of neurologi- rons or to the corticospinal tract. Symptoms may vary cal symptoms in childhood is exceptional. However, as from mild stiffness to severe muscle spasms and Wilson disease is a treatable disorder, copper studies include hypertonia, brisk deep tendon refl exes, patho- are still strongly recommended from early school age. logical refl exes, clonus and weakness. Depending on the affected anatomical region, spasticity may be more evident or restricted to the lower extremities (spastic C5.10.3 Chorea diplegia or paraplegia) to one side of the body (spastic hemiplegia) or affecting all four limbs (spastic quad- The term chorea is a derived from the Greek word riplegia or tetraparesis). “choreia” for dancing and refers to involuntary rapid In children, most events related to motor damage spasmodic movements of the face, neck and proximal occur during late pregnancy and delivery such as pre- limb muscles. It may extend to the oropharyngeal mus- maturity, neonatal asphyxia, birth trauma or infections. cles generating swallowing diffi culties. The consequent clinical manifestations are usually Choreic movements appear in glutaric aciduria type grouped under the general term cerebral palsy. In con- I (in the context of severe acute dyskinetic syndrome), trast, traumatic injury to and infections of the CNS that Lesch–Nyhan disease, PKAN and homocystinuria and take place later on as well as genetic conditions (includ- Niemann–Pick C among others (Table C5.10). ing inborn errors of metabolism), may also result in spasticity. Since some of these metabolic disorders are Table C5.10 Differential diagnosis of chorea treatable and offer family genetic counselling it is Benign familial chorea important to include them in the diagnostic approach Canavan disease of spasticity. Dentatorubropallidoluyisian atrophy Spasticity in metabolic disorders is in general asso- Familial inverted choreoathetosis ciated with involvement of additional neurological Friedreich ataxia functions or organs. It may appear acutely and related Galactosemia Glutaric aciduria I to signs of metabolic intoxication such as loss of con- Guanidinoacetate methyltransferase defi ciency sciousness or vomiting. However, some disorders can Homocystinuria start with isolated spastic paraparesis such as X-linked Huntington chorea adrenoleukodystrophy(X-ALD), homocysteine rem- Infantile NCL ethylation defects, HHH syndrome (hyperammone- Lesch–Nyhan syndrome mia, hyperornithinemia and homocitrullinuria), Neuroacanthocytosis arginase defi ciency and Segawa disease. Niemann–Pick C Mutations in NKX2.1 The main components of the motor system (motor PKAN neurons and myelin) are extremely vulnerable to inborn Pontocerebellar hypoplasia type 2 errors of metabolism. This is the reason why disorders of Propionic acidemia both intermediary metabolism and complex molecules Pterin defects may exhibit spasticity. Disorders that interfere with Wilson disease myelin metabolism, defects in energy production or small 152 A. García-Cazorla et al. toxic molecules often cause pyramidal tract lesions. In folate concentration in every patient with isolated spas- fact, almost all progressive neurometabolic diseases end ticity, as it may be the only clinical sign over a long up manifesting different degrees of spasticity. This sec- period. The combination of betaine (up to 10 g/day in 3 tion will deal with those inborn errors of metabolism in doses), folic acid (5–10 mg/day) and hydroxycobalamin which spasticity is the dominant or one of the most prom- (0.5–1 mg/day orally or 1 mg i.m. monthly) is not as inent signs in the clinical picture (Sedel et al. 2007). effective as it is in resolving other neurological or psychiatric symptoms related to this same defect. Remember Cerebral folate defi ciency is characterised by low CSF 5-methyltetrahydrofolate concentration and early psy- ¼ The possibility of an inborn error of metabolism chomotor delay, microcephaly, movement disorders, should be considered in every child with the diag- ataxia and spastic paraplegia; clinical improvement fol- nosis of cerebral palsy but without a history of lows high doses of folinic acid (3–5 mg/kg/day). It is not perinatal or postnatal brain injury (prematurity, clear if this syndrome is an inborn error of metabolism, hypoxia, infections and traumatic brain injury). because these patients were shown to have autoantibod- ¼ In inborn errors of metabolism, spasticity tends ies against folate receptors suggesting an acquired auto- to be syndromic or complicated (other neuro- immune mechanism. Furthermore, there are secondary logical signs and/or other organs are involved). causes of cerebral folate defi ciency such as Aicardi– However, in some cases spasticity may remain Goutières, Rett syndrome and mitochondrial disorders. isolated for a long time. Cerebrotendinous xanthomatosis is a sterol disorder ¼ Spasticity is probably the most common neuro- that may present with progressive spasticity from the logic sign in neurometabolic disorders. It is second decade of life. Early diagnosis and appropriate therefore important to search carefully for other treatment may obviate this complication. Other clini- associated clinical signs. cal signs such as neonatal liver disease, cataracts or peripheral neuropathy are often present. Elevated levels of plasma cholestanol and bile acid precursors (in C5.10.5 Treatable Inborn Errors of plasma or urine) are diagnostic. Treatment with chen- Metabolism with Prominent odeoxycholic acid (750 mg/day) and statins improve and even reversal the neurological disability. Di/Tetraparesis: Dopamine synthesis defects especially GTPCH I defi - ciency may exhibit pyramidal signs, and, in some Urea cycle disorders such as arginase defi ciency, result cases, lower limb dystonia can mimic spastic parapare- in progressive spasticity, seizures and mental retarda- sis. Therefore, CSF study and a trial of levodopa should tion with relatively mild hyperammonemia. HHH syn- be advisable in all patients with unexplained spastic drome (hyperammonemia, hyperornithinemia and paraparesis or dystonic cerebral palsy. homocitrullinuria) is a disorder of ornithine transport Vitamin E defi ciency (see also Ataxia). Spasticity may between cytoplasm and mitochondria that causes pro- appear together with peripheral neuropathy and retini- gressive spastic diplegia in addition to other signs of tis pigmentosa. Laboratory fi ndings include low-to- neurologic dysfunction. Routine investigation should absent serum vitamin E and high serum cholesterol, include plasma ammonia and amino acids (both in triglycerides, and b-lipoprotein. High doses of vitamin plasma and urine) (Scaglia et al. 2006). Therapeutic E (400–1200 IU/day) improve neurologic function. strategies include diet and ammonia-lowering agents. Biotinidase defi ciency may also manifest with progres- Remember sive spastic paraparesis. It may improve with biotin ¼ (5–20 mg/day). Measurement of plasma biotinidase Measure ammonia, amino acids, biotinidase activity reveals the diagnosis. activity, total homocysteine, folate, cholestanol Homocysteine remethylation defects can lead to demy- and vitamin E, in all patients with spasticity of elination of the pyramidal tracts and produce a subacute unknown origin ¼ combined degeneration of the spinal cord. It is important Consider a trial of l-dopa and CSF studies to to measure plasma amino acids, total homocysteine and measure neurotransmitters and folate C5 Neurological Disease 153

C5.10.6 Progressive Spasticity Associated ¼ Consider the possibility of a brain iron disorder with Multiple Neurologic Signs, in a patient with progressive spasticity, especially Irritability and Global if episodes of regression are triggered by infec- Deterioration tious events, and even in the absence of specifi c brain MRI fi ndings (basal ganglia involvement). This is a group of heterogeneous disorders that usually manifest a complex neurological picture in which progressive spasticity is one of the main features. C5.10.7 Spastic Tetraparesis Associated Peripheral neuropathy, visual, auditory and visceral with Ichthyosis involvement are frequently present. Brain MRI may disclose specifi c white matter patterns that can be very This association is typical of SjögrenÐLarsson syn- helpful in the diagnostic approach. drome (fatty alcohol NAD oxidoreductase defi ciency). In lysosomal disorders, some of the most represen- The skin alteration consists of yellowish-brown tative diseases giving rise to spasticity are Krabbe dis- hyperkeratosis (ichthyosis). Glistening dots are pres- ease, MLD, MPS III, fucosidosis and mannosidoses. ent in the macular fundus. Mental retardation is also X-linked adrenomyeloneuropathy can present with present. Cutaneous signs may remain isolated for a spasticity as an isolated sign for a long time, whereas long time and spastic tetraparesis can appear in adult- in adrenoleukodystrophy of childhood, cognitive and hood. High leukotriene B in urine, enzymatic activity behavioural problems may appear before the motor 4 in fi broblasts or leukocytes and mutation analysis disturbances. Brain MRI is usually very helpful in are the diagnostic tools. Zileuton improves the cuta- diagnosis approach. neous symptoms and may ameliorate the neurological In mitochondrial disorders, pyramidal tract involve- disease (Gordon 2007). ment is also frequent, however, other signs and symp- Multiple sulphatase defi ciency combines features toms often guide the diagnostic approach. of mucopolysaccharidosis, MLD and ichthyosis, starts Neuroaxonal dystrophy and related high brain iron in infancy and is usually fatal in early childhood. disorders are due to mutated PLA2G6, encoding a Glycosaminoglycans are increased in urine, and phospholipase A2. They manifest progressive neuro- enzyme studies show defi ciencies of many sulphatases logical regression and distended axons (spheroid bod- (Tables C5.11 and C5.12). ies) in the nervous system as well as high basal ganglia iron. Progressive spastic paraparesis, extrapyramidal signs and usually dementia, retinitis pigmentosa and optic atrophy are present. C5.11 Neuroradiology Some cerebral organic acidurias such as Canavan disease (high urine and brain MRS N-acetylaspartate) Imaging of the brain is one of the most important diag- or L-2-hydroxyglutaric aciduria are examples of child- nostic tools in this fi eld. Morphological evaluation can hood leukodystrophies exhibiting different degrees of be done best with MRI where brain structures can be progressive spasticity and well-defi ned brain MRI adequately viewed. Cranial computer tomography patterns. (CCT) is still important when looking for calcifi cations or in the emergency situation when MRI takes Remember too long in an instable patient. Proton MR spectros- 1 ¼ Check nerve conduction, visual, auditory func- copy ( H-MRS) is a powerful tool to assess the main tion, skeleton examination (X-ray), urine gly- cerebral metabolites and is diagnostic for some cosaminoglycans/ oligosaccharides, in patients disorders. with multiple neurological signs and progres- Interpretation of cerebral imaging studies is not sive spasticity. easy, and care should be taken that in patients with the ¼ Some brain MRI specifi c patterns can give the diagnosis or suspicion of an inborn error of metabo- diagnostic clue. lism, images should be reviewed personally together with a neuroradiologist experienced in this fi eld. 154 A. García-Cazorla et al.

Table C5.11 Differential diagnosis of spasticity Clinical signs associated to spasticity Disorders Additional neurological signs Peripheral neuropathy CTX, mitochondrial disorders (axonal neuropathy), biotinidase defi ciency, b-mannosidosis, sialidosis type I, Krabbe disease, MLD, homocysteine remethylation defects, vitamin E defi ciency Leukoencephalopathy Krabbe disease, MLD, Cannavan disease, L-2-hydroxyglutaric aciduria, X-ALD, homocysteine remethylation defects, some mitochondrial disorders, multiple sulphatase defi ciency, Schindler disease Ataxia CTX, cerebral folate defi ciency, biotinidase defi ciency, HHH syndrome, some mitochondrial disorders, L-2-hydroxyglutaric aciduria Movement disorders Dopamine synthesis defects, cerebral folate defi ciency, mitochondrial disorders, brain iron disorders Epilepsy Homocysteine remethylation defects, cerebral folate defi ciencies, mitochondrial disorders, sialidosis, Schindler disease, multiple sulphatase defi ciency, Canavan disease, L-2-hydroxyglutaric aciduria Microcephaly Cerebral folate defi ciency, homocysteine remethylation defects, arginase defi - ciency, mitochondrial disorders Macrocephaly Cerebral organic acidurias, MPS Developmental delay/mental retardation In all of them except GTPCH I (dominant) and sialidosis type I Additional non-neurological signs Visceral Urea cycle disorders (liver involvement, cyclic vomiting), CTX (diarrhoea), X-linked adrenoleukodystrophy (adrenal insuffi ciency), most lysosomal disorders (organomegaly) Cutaneous signs CTX (xanthomas), multiple sulphatase defi ciency and Sjoegren–Larsson (ichthyosis), biotinidase defi ciency (alopecia, dermatitis), X-linked ALD (melano- derma), sialidosis II (angiokeratoma) Ocular signs Homocysteine remethylation defects, (retinitis pigmentosa, optic nerve atrophy), CTX (cataracts), biotinidase defi ciency, (optic neuropathy), Sjoegren–Larsson (retinopathy), cerebral folate defi ciency (optic atrophy), different lysosomal disorders (cherry red spot), brain iron disease, mitochondrial disorders, vitamin E defi ciency (retinitis pigmentosa) CTX Cerebrotendinous xantomatosis; MLD metachromatic leukodystrophy; X-ADL X-linked adrenoleukodystrophy

“Normal” MRIs should be re-evaluated as some Remember abnormalities escape detection by neuroradiologists unfamiliar with metabolic disorders. In infancy, nor- Neuroimaging should be repeated if new neurologic mal brain maturation – the process of myelination symptoms appear, if there is clincal regression and which is more or less completed at the age of 24 also if the ﬁ rst study has been during the ﬁ rst 2 months at least in the MRI – has to be taken into years of life to assess whether myelination has been account; there may be subtle abnormalities. The signal completed. of mature, myelinated white matter is dark in T2w images and bright in T1w images. In infants (and patients with hypomyelination), myelination is A pattern recognition approach greatly facilitates dif- assessed best using T1w sequences; it is complete in ferential diagnosis. In this approach, affected struc- Tw1 images at the age of 9 months. After that age, tures are analysed and compared with known disorders. T2w images are most informative regarding progress It is a powerful method not only for established disor- of myelination and also involvement of white matter ders, but also for the differentiation of new disorders, in metabolic and non-metabolic disorders. especially leukoencephalopathies (Lyon et al. 2006, C5 Neurological Disease 155

Table C5.12 Checklist of Laboratory Tests in Spasticity Treatable causes of spasticity Plasma ammonia, plasma and urine amino acids. Biotinidase activity (plasma) Folate and total homocysteine (blood) Cholestanol (plasma), bile acid precursors (urine and plasma) Vitamin E, triglycerides, cholesterol and fractions, erythrocyte morphology (plasma) Folate and biogenic amine metabolites in the CSF Progressive spasticity with signs of neurologic deterioration First consider treatable disorders Glycosaminoglycans, oligosaccharides, sialic acid (urine) Lysosomal enzymes (blood) Very long-chain fatty acids (plasma) Lactate, pyruvate (plasma) Organic acids (urine) Consider PLA2G6 mutation depending on clinical and MRI fi ndings Spasticity with icthyosis Glycosaminoglycans in urine Enzymatic activity of fatty alcohol NAD oxidoreductase in fi broblasts Other non-metabolic causes of spasticity Hereditary diseases: spinocerebellar atrophy, familiar spastic paraparesis Infections: AIDS, HTLV-1 Immunologic disorders: multiple sclerosis Fig. C5.7 PKAN. This axial T2w image displays the pathogno- Malformations: Arnold–Chiari, cervical/lumbar monic eye-of-the-tiger sign, a hypointense pallidum with central spondylosis hyperintense gliosis Cerebral palsy (prematurity, hypoxia, infections) Neoplasm white matter) and other general characteristics of white matter involvement (contrast enhancement, vacuolisa- Schiffmann R et al. 2004). Typical and diagnostic tion or cysts, calcifi cations, swelling and small vs. large patterns include the classical form of X-ALD, Cana- and isolated vs. confl uent lesions). van disease, INAD (Fig. C5.1), PKAN (Fig. C5.7) or In cerebral 1H-MRS, four main physiological L-2-hydroxyglutaric aciduria. Table C5.13 gives an metabolites can be assessed easily: choline, creatine overview of cerebral structures affected in important and phosphocreatine, N-acetylaspartate (NAA) and inborn errors of metabolism. The most obvious differen- myo-inositol. Lactate is not normally visible in CNS tiation is between affected grey or white matter. If grey spectra. Concentration of these metabolites has tradi- matter is involved, it must be discriminated between cor- tionally been expressed as relation to creatine, but it tex, deep grey matter structures (basal ganglia and thala- should be properly quantifi ed. Metabolite levels are mus) and cerebellar grey matter, also between prolonged age related; adequate control groups are therefore (hyperintense signal) or decreased (hypointense signal) important. 1H-MRS is diagnostic for creatine defi - T2 abnormalities. If primarily white matter is involved, ciency syndromes, Canavan disease, ribose-5-phos- differentiation must be made between the location of phate isomerase defi ciency (accumulation of polyols affected white matter (subcortical/arcuate fi bres, central, in the CNS) and probably complex II defi ciency where periventricular, corpus callosum and brainstem), the gra- CNS succinate is elevated (Table C5.14). All these dis- dient (symmetry, anterior vs. posterior white matter, orders are rare and can also be diagnosed by biochemi- central vs. peripheral white matter and rostral vs. caudal cal analysis of body fl uids. Complete absence of 156 A. García-Cazorla et al.

Table C5.13 MRI ﬁ ndings in inherited metabolic disorders Grey matter Cerebellar atrophy Unspeciﬁ c; present in many metabolic and degenerative conditions

High T2w signal of cerebellar cortex INAD, Marinescu–Sjögren syndrome, CoQ10 defi ciency, mitochondrial disorders Basal ganglia and thalamic involvement (symmetrical) long T2 Mitochondrial disorders, Wilson’s disease, organic acidurias, Alpers disease (may be unilateral) short T2 (pallidum) PKAN, INAD short T2 (thalamus) INCL, Tay–Sachs disease, Krabbe disease Dentate involvement L-2-hydroxyglutaric aciduria, cerebrotendinous xanthomatosis Cerebral atrophy Unspecifi c, present in many neurodegenerative disorders Stroke-like lesions MELAS, Alpers disease, urea cycle disorders Polymicrogyria Zellweger disease Cobblestone lissencephaly Walker–Warburg syndrome and other O-glycosylation defects White matter Delayed myelination Unspecifi c, present in many inherited and acquired conditions Hypomyelination Tay–Sachs disease, INCL, Salla disease, non-metabolic disorders (e.g. Pelizaeus–Merzbacher disease) Early involvement of subcortical white Galactosemia, L-2-hydroxyglutaric aciduria matter Central white matter/centrum semiovale Krabbe disease, metachromatic leukodystrophy, X-ALD, phenylketonuria, Lowe syndrome, disorders of cytosolic methyl group transfer, various other disorders Miscellaneous Subdural effusions Glutaric aciduria type I, Menkes disease Elongated and tortuous arteries Menkes disease Reduced opercularisation Glutaric aciduria type I Agenesis of the corpus callosum PDHc defi ciency, mitochondrial disorders Caudothalamic cysts Zellweger syndrome, glutaric aciduria type I Calcifi cations Mitochondrial disorders (especially Kearns–Sayre syndrome), folate defi ciency, other disorders (e.g. Aicardi-Goutières syndrome)

Table C5.14 Typical 1H-MRS abnormalities in inherited cerebral NAA which is a neuronal and axonal marker metabolic disorders has been described in one single child; the metabolic 1 H-MRS fi nding Disease defect has not yet been elucidated. This disease is not Absent creatine peak Creatine synthesis (GAMT, amenable to biochemical diagnosis. One of the ques- AGAT) and transport 1 (CRTR) defi ciencies tions H-MRS can answer is whether lactate is elevated Elevated lactate Mitochondrial disorders, in the CNS, which is possible even with normal CSF pyruvate dehydrogenase lactate levels. This may be the only metabolic indicator defi ciency, other IEMs, of a mitochondrial disorder, hence the importance of non-metabolic conditions 1H-MRS for neurometabolic disorders. (ischemia, infection, neoplasia) As a minimal protocol, MRI should include axial T1w, Elevated polyols Ribose-5-phosphate isomerase T2w and FLAIR images and sagittal Tw1 images. If there defi ciency are cerebellar abnormalities, coronar T2w or FLAIR Strongly increased NAA Canavan disease images should be acquired. Diffusion-weighted imaging Absent NAA Putative NAA synthesis defect is part of many routine studies nowadays and should Increased choline Demyelination (e.g. metachromatic leukodystrophy, always be done if stroke-like episodes or “metabolic X-ALD) stroke” is suspected (Roach et al. 2008). Fibre-tracking is Increased succinate Complex II defi ciency a novel method to depict connections within the CNS. If Increased lipids Sjögren–Larsson syndrome possible, 1H-MRS should be done together with C5 Neurological Disease 157 conventional MR imaging, at least one single voxel. evoked potentials help to detect early involvement of the Optimally, voxels of interest in 1H-MRS should be located optic nerve, e.g. in mitochondrial disorders, INAD or in the basal ganglia, in the centrum semiovale, in the cor- Alpers disease. ERG is important in detecting retinal tex (usually in the occipital cortex) and also in the cerebel- involvement,which is of use in the differential diagnosis lum (Barkovich et al. 2005; van der Knaap et al. 1999). of neurodegenerative disorders. Retinal involvement is part of the NCL, but also of panthotenate-associated neurodegeneration (PKAN), mitochondrial disorders and many others (see also Chap. C8). C5.12 Neurophysiology

Neurophysiologic studies are important for diagnosis and follow-up in neurometabolic disorders, especially if epi- C5.13. Diagnostic Lumbar Puncture lepsy or neuropathy are part of the clinical picture. There are few pathognomonic or typical fi ndings. Of major In an increasing number of encephalopathies of unknown interest for neurometabolic disorders are EEG, elec- origin metabolic investigations of CSF can be instrumen- troretinography (ERG), measurement of nerve conduc- tal in identifying the underlying neurometabolic disorder. tion velocities and evoked potentials. Electromyography As there is still widespread uncertainty about when to (EMG) is done if myopathy or motor neuropathy are sus- perform specialised CSF investigations and what to inves- pected. It can reveal myotonia-like discharges as typical tigate in many patients remain undiagnosed, although fi nding in juvenile type II glycogenosis. they often have recognisable phenotypes. As a conse- EEG may be of value in the diagnosis of several quence futile diagnostic searches continue, and the often neurometabolic disorders. In INAD, it shows a pro- available rational therapy cannot be instituted. On the nounced, diffuse fast b–activity in the absence of medi- other hand, a lumbar puncture should only be performed cation, especially in stage I and II sleep which helps in in the diagnostic work-up of a suspected neurometabolic the diagnosis of this rare disorder (Fig. C5.2). In late- disorder after basic analyses have been carried out in infantile neuronal ceroid lipofuscinosis, slow photic blood and urine (see Table C5.15) and after the results of stimulation leads to occipital spikes (Fig. C5.4) and neuroimaging studies have been carefully evaluated. may even trigger focal occipital seizures. In the infan- There is no place for a selective screening in CSF. Reliable tile form, EEG shows early a slowing of the back- results of specialised CSF investigations can only be ground, later in the course an isoelectric tracing. In the obtained if the appropriate protocol is strictly adhered to. neonatal manifestation of maple syrup urine disease, This should be discussed beforehand with the neurometa- EEG displays comb-like rhythms. A burst-suppression bolic laboratory (see (D2) Biochemical Studies). pattern is not specifi c and may be found in different White cell hexosaminidase, sphingomyelinase, palmi- epileptic encephalopathies with neonatal onset the most toylprotein thioesterase activity, aryl sulphatase, frequent being non-ketotic hyperglycinemia. In patients with homocystinuria, centrotemporal spikes are often Table C5.15 Investigations for neurometabolic diseases in present resembling the epileptiform potentials in benign blood and urine epilepsy with centrotemporal spikes. If cortical abnor- Blood/plasma/serum: Full blood count and reticulocytes, malities are present, EEG refl ects their localisation and usual chemistry profi le including Ca, P, alkaline quality – in cobblestone lissencephaly, it shows genera- phosphate, creatinine, uric acid, copper, ceruloplasmin, T3, T4, TSH, thyroid binding globulin, prolactin b lised -activity, usually of high amplitude. In Alpers Amino acids, total homocysteine, lactate and pyruvate, disease, EEG is very valuable in the early stage of the ammonia, biotinidase, very long-chain fatty acids, disease and diplays, albeit not in all patients, RHADS, pristanic acid, pipecolic acid, transferrin isoelectric usually over the posterior regions (Fig. C5.5). focussing Somatosensory evoked potentials (SSEP) are helpful Urine: Organic acids, lactic acid, uric acid (urate/creatinine ratio, guanidino compounds, sulphite, purines and in delineating posterior column involvement, e.g. in pyrimidines, bile acid intermediates, mucopolysaccaccha- FRDA or cobalamin defi ciency. Giant SSEP are found rides, oligosaccharides, sialic acid), creatinine (preferably in some of the progressive myoclonic epilepsies includ- 24-h urine) ing late infantile neuronal ceroid lipofuscinosis. Visual Blood or urine: Acyl-carnitine esters 158 A. García-Cazorla et al. glucocerebrosidase, galactocerebrosidase, fucosidase and tripeptidyl peptidase activity Lactate CSF Glucose The diagnosis of monogenic defects of neurotrans- glucose mission is almost exclusively based on the quantitative lactate or normal lactate determination of the neurotransmitters and/or their or normal glucose metabolites in CSF, i.e. the amino acids glutamate, CSF to plasma glycine and g-aminobutyric acid (GABA), the acidic glucose ratio < 0.45 metabolites of the biogenic monoamines, dopamine, Defects in CNS energy metabolism serotonin, epinephrine and norepinephrine, and indi- GLUT-1 defect vidual pterin species. In other neurometabolic disorders, results of CSF investigations are important, although not exclusive part of the diagnostic work-up, Fig. C5.8 Evaluation of CSF/blood results of glucose and lactate e.g. GLUT1 defi ciency, mitochondriopathies and serine synthesis disorders. Blood should be taken fi rst as glucose may rise stress related during the lumbar puncture. Remember Figure C5.8 depicts an algorithm for the interpreta- Whenever CSF investigations are performed, the tion of pathological CSF/plasma results of glucose and analysis should include quantitative determination lactate. Glucose and lactate must be looked together. of lactate, pyruvate and amino acids, the latter by Pathologically altered ratios may indicate a disease methods especially suited for CSF, in addition to intrinsic to the CNS. Elevations of CSF lactate together cells, glucose, protein, immunoglobulin classes, with a reduction in glucose results from inadequate specifi c immunoglobulins and an evaluation of the aerobic energy production, e.g. in mitochondriopathies blood-brain barrier (Table C5.16). but more commonly in the course of CNS infections. In contrast, reduced levels of CSF glucose in GLUT1 defi ciency is accompanied by low normal or even Preprandial plasma amino acids, serum glucose and reduced levels of lactate. Post-prandial or stress-related blood lactate should always be determined at the time elevated blood glucose is the most common cause of a of the lumbar puncture, as ratios are highly informa- reduced CSF/blood ratio for an erroneous suspicion of tive for a large number of disorders and almost indis- GLUT1 defi ciency. pensable for the diagnosis of some disorders, e.g. non-ketotic hyperglycinemia (CSF glycine almost Take Home Messages always > 30 mM and glycine CSF/plasma ratio > 0.04), glucose transport protein defi ciency (glucose CSF/ › Neurological disease can be considered as the plasma ratio < 0.45) and defects of serine synthesis most common consequence of inborn errors of (CSF serine < 14 mM and serine CSF/plasma ratio metabolism. < 0.2). Unreported blood contamination of CSF is the › A structured diagnostic approach should be most common cause for an erroneous diagnosis of based on: non-ketotic hyperglycinemia. Because amino acids -Age-dependant physiological developments and glucose change dramatically after a meal, timing -Predominant neurological manifestation of the lumbar puncture and blood taking should not be (mental retardation, epilepsy, motor distur- post- but pre-prandial, i.e. at least 4–6 h after a meal. bances) -Therapeutic possibilities. Table C5.16 Investigations for neurometabolic diseases in CSF › A high level of suspicion and close interaction Cells, protein, immunoglobulin classes and glucose (plus between paediatric neurologist and specialist plasma glucose and evaluation of blood-brain barrier) for inborn errors of metabolism are indispens- Lactate (plus preprandial blood lactate) able to effectively and timely identify patients Amino acids (plus preprandial plasma amino acids) in whom neurological disorders are the present- Biogenic amine metabolites ing and/or main symptoms of an inborn error. Individual pterin species 5-Methyltetrahydrofolate C5 Neurological Disease 159

Key References Moser H (1997) Adrenoleukodystrophy: phenotype, genetics, pathogenesis and therapy. Brain 120:1485–1508 Nance WE (2003) The genetics of deafness. Ment Retard Dev American Academy of Child and Adolescent Psychiatry Working Disabil Res Rev 9:109–119 Group on Quality Issues (1999) Practice parameters for the Parker CC, Evans OB (2003) Metabolic disorders causing child- assessment and treatment of children, adolescents, and adults hood ataxia. Semin Pediatr Neurol 10:193–199 with mental retardation and comorbid mental disorders. J Am Poretti A, Wolf NI, Boltshauser E (2008) Differential diagnosis Acad Child Adolesc Psychiatry 38(12 Suppl):5S–31S of cerebellar atrophy in childhood. Eur J Pediatr Neurol Assmann B, Surtees R, Hoffmann GF (2003) Approach to the 123:155–167 diagnosis of neurotransmitter diseases exemplifi ed by the Roach ES, Golomb MR, Adams R et al (2008) Management of differential diagnosis of childhood-onset dystonia. Ann stroke in infants and children. Stroke 39:1–48 Neurol 54(Suppl 6):S18–24 Scaglia F, Lee B (2006) Clinical, biochemical, and molecular Barkovich AJ (2005) Magnetic resonance techniques in the spectrum of hyperargininemia due to arginase I defi ciency. assessment of myelin and myelination. J Inherit Metab Dis Am J Med Genet C Semin Med Genet 142:113–120 28:311–343 Schiffmann R, van der Knaap MS (2004) The latest on leu- Fernández-Álvarez E, Aicardi J (2001) Movement disorders in kodystrophies. Curr Opin Neurol 17:187–192 children. Mac Keith Press, London Sedel F, Fontaine B, Saudubray JM (2007) Hereditary spastic García-Cazorla A (2008) Neurometabolic diseases: guidance for paraparesis in adults associated with inborn errors of metab- neuropaediatricians. Rev Neurol 47(Suppl 1):S55–63 olism: a diagnostic approach. J Inherit Metab Dis 30: Germain DP, Avan P, Chassaing A, Bonfi ls P (2002) Patients affected 855–864 with Fabry disease have an increased incidence of progressive Shevell M, Ashwal S, Donley D et al (2003) Practice parameter: hearing loss and sudden deafness: an investigation of twenty- evaluation of the child with global developmental delay. two hemizygous male patients. BMC Med Genet 11(3):10 Neurology 60:367–380 Gordon N (2007) Sjögren-Larsson syndrome. Dev Med Child Surtees R, Wolf N (2007) Treatable neonatal epilepsy. Arch Dis Neurol 49:152–154 Child 92:659–661 Jinnah HA, Visser JE, Harris JC et al (2006) Lesch-Nyhan van der Knaap MS, Breiter SN, Naidu S et al (1999) Defi ning Disease International Study Group. Delineation of the motor and categorizing leukoencephalopathies of unknown origin: disorder of Lesch-Nyhan disease. Brain 129:1201–1217 MR imaging approach. Radiology 213:121–133 Kokotas H, Petersen MB, Willems PJ (2007) Mitochondrial Van Karnebeek CDM, Jansweijer MCE, Leenders AGE et al deafness. Clin Genet 71:379–391 (2005) Diagnostic investigations in individuals with mental Lyon G, Fattal-Valevski A, Kolodny EH (2006) Leukodystrophies: retardation. Eur J Hum Genet 13:6–25 clinical and genetic aspects. Top Magn Reson Imaging Wolf NI, Bast T, Surtees R (2005) Epilepsy in inborn errors of 17:219–242 metabolism. Epileptic Disord 7:67–81 Morgan NV, Westaway SK, Morton JEV et al (2006) PLA2G6, Zschocke J, Hoffmann GF (2004) Vademecum Metabolicum. encoding a phospholipase A2, is mutated in neurodegenera- Manual of metabolic paediatrics, 2nd edn. Schattauer, tive disorders with high brain iron. Nat Genet 38:752–754 Stuttgart Metabolic Myopathies C6 Stephen G. Kahler

Key Facts › Diagnosis requires careful attention to dietary and › The metabolic disorders which affect muscle exercise history, and appropriate laboratory inves- can cause chronic weakness and hypotonia, or tigations. Exercise testing, electromyogram, and episodic exercise intolerance cumulating in muscle biopsy can provide essential information. rhabdomyolysis, or both. Rhabdomyolysis › Treatment depends on avoiding precipitating disorders can be conveniently separated factors and optimizing muscle energetics. according to tolerance of short, intense exercise compared to longer, milder efforts. › Most metabolic disorders which affect skeletal muscle do so by altering energy metabolism. Muscle at rest uses fatty acids as the main C6.1 General Remarks energy source. › During intense exercise, there will be anaero- Many metabolic disorders cause muscle dysfunction bic glycolysis and utilization of muscle glyco- or damage (Table C6.1). The pathophysiological basis gen. During sustained exercise, fatty acids in most is impairment of energy production when become the source of fuel. Exercise, fasting, stressed, particularly by exercise, cold, fasting, or cold, infections, and medications may elicit infection (especially of viral origin). In some situa- symptoms. tions, the problem is confi ned to skeletal muscle, but in Important causes of metabolic myopathy › many there is cardiac involvement as well. Liver, brain, include adenosine monophosphate (myoade- retina, and kidney, all of which have signifi cant energy nylate) deaminase defi ciency, and disorders of requirements, may also be involved. Even more exten- glycolysis, glycogenolysis, fatty acid oxida- sive or patchy involvement, e.g., pancreas and bone tion, and oxidative phosphorylation. In many marrow, is a characteristic of the mitochondrial disor- cases other organs are involved. ders where heteroplasmy may occur (see also Chaps. B2.3 and D6).

C6.1.1 Special Aspects of Skeletal Muscle Metabolism

S. G. Kahler Skeletal muscle relies on different fuel sources at differ- Department of Pediatrics, Division of Clinical Genetics, ent times and circumstances. Fatty acids are the primary University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205, USA fuel at rest. Glucose from the blood and derived from e-mail: [email protected] muscle glycogen is used during short-term intensive

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 161 DOI: 10.1007/978-3-540-74723-9_C6, © Springer-Verlag Berlin Heidelberg 2010 162 S. G. Kahler

Table C6.1 Major presentations of metabolic myopathies? Enzyme (position- in Rhabdomy- Hypotonia, Cramps Worsened Worsened Worsened Worsened Incr. pathway – Fig. C1) olysis weakness by fasting? by exercise? by cold? by Baseline CK infection?

RHABDOMYOLYSIS AND MYOGLOBINURIA PRESENTATION Intense exercise not tolerated; ++ + ++ ++ + second wind phenomenon GSD type V Muscle phosphorylase (II) ++ + ++ ++ + GSD type IX Muscle phosphorylase kinase (II) + + + + + GSD VII Muscle phosphofructokinase (8) + + + + ++ ++

PGK defi ciency Muscle phosphoglycerate ++ ++ ++ ++ kinase (III) PGAM defi ciency Phosphoglycerate mutase ++ ++ ++ ++ (III) b-Enolase defi ciency b-Enolase (III) ++ ++ ++ + LDH defi ciency Lactic dehydrogenase M ++ ++ subunit (12)

Myoadenylate deaminase Muscle AMP deaminase + + + + - defi ciency + + ++ ++ ++ ++ ++ + Short, intense exercise tolerated; prolonged exercise not tolerated Translocase defi ciency Carnitine-acylcarnitine translocase (22) CPT II defi ciency Carnitine palmitoyltrans- ++ ++ ++ ++ ++ ferase II (23)

VLCAD defi ciency Very-long chain acyl-CoA ++ + ++ + + + dehydrogenase (24) LCAD defi ciency Long-chain acyl-coA dehydrogenase (24) LCHAD/trifunctional enzyme Long-chain 3-hydroxyacyl- ++ defi ciency CoA dehydrogenase (26) MCAD defi ciency Medium-chain acyl-CoA − (+) ++ + - ++ dehydrogenase (24)

SCAD deﬁ ciency Short-chain acyl-CoA ++ dehydrogenase (24) Hydroxyacyl-CoA ++ + dehydrogenase (short-chain hydroxyacyl- CoA dehydrogenase) deﬁ ciency

HYPOTONIA AND WEAKNESS +++++ +

Carnitine transporter defi ciency Plasma membrane carnitine ++ ++ + transporter (20) Secondary carnitine depletion ++ + + + Mild multiple acyl-CoA Electron-transfer fl avoprotein + dehydrogenase defi ciency (ETF) or ETF (MADD) defi ciency dehydrogenase ETF-QO Lysosomal glycogen storage Lysosomal a-glucosidase ++ ++ disease (Pompe) GSD type III Glycogen debrancher (II) + ++ + ++

GSD type IV Glycogen brancher enzyme + (I) Neutral lipid storage disease I Comparative gene + + identiﬁ cation-58 Neutral lipid storage disease II ATGL + +

MITOCHONDRIAL MYOPATHIES Many disorders and ++++ ++++ mechanisms. (See Chap. B2.3 and Fig. B2.3.2 Coenzyme Q defi ciency Several known gene defects +++ and mechanisms See text for details about specifi c disorders D DNA (predominant or common mutation); E erythrocytes; F fi broblasts; H heart; K kidney; L liver; M muscle; W leukocytes. This list is only a guide. Which tissue is required and which test is to be done depends on the laboratory performing the test, and in some cases on which organs are affected ++ Often present or abnormal; + occasionally present or abnormal aHepatic dysfunction with encephalopathy is a “Reye-like syndrome”W C6 Metabolic Myopathies 163

Abnormal Muscle Organic Carnitine Acylcarnitine Cardiomy- Hepatic Encephal- Diagnostic Comments EMG biopsy acids level proﬁ le opathy dysfunc- opathya tissues tiona

++ Disorders of glycogenolysis and glycolysis; AMP deﬁ ciency

+ M Occ. severe infantile form. Proximal > distal + One form M Four syndromes, two involving skeletal muscle + ++ + M and E Hemolytic anemia, rare, hyperuricemia. Muscle cannot utilize glucose – worse after glucose, high-CHO meal. Severe infantile form with cardiomyopathy ++ - ++ M Hemolytic anemia. Neurologic abnormalities. X-linked -++- M Very rare

-++- M Very rare -- M Very rare. Lactate does not rise after ischemic exercise when pyruvate is elevated. Uterine stiffness +++ L Impaired ammonia production with ischemic exercise + ++ ++ + ++ ++ ++ ++ Disorders of fatty acid oxidation and carnitine-assisted transport into mitochondria

+ + ++ ++ ++ + F

++ + ? ++ ++ ++ + F, M, L, and W Heterozygote may have symptoms, be vulnerable to malignant hyperthermia. Lactic acidosis. Sensori-motor neuropathy. ++ ++ ++ ++ ++ ++ ++ F, W, and L

F, W, and L Uncommon; see text

++ ++ ++ ++ ++ ++ F, M, L, and D HELLP syndrome in pregnant heterozygote + ++ ++ + ++ ++ F, L, W, and D Myopathy is minimal; this is the commonest disorder of fatty acid oxidation. Most early cases called systemic carnitine deﬁ ciency + ++ ++ ++ F, M, and L Rare. Extremely variable

++ ++ F, M, and L Very rare

+ + + + + Carnitine deﬁ ciency or depletion; Pompe disease; glycogen brancher and debrancher deﬁ ciencies + ++ + ?? ++ ++ + F, M, L, and K

+ + + ?? + ++ + Occurs in many settings + + ++ + ++ ++ ++ ++ F, M, and L Mild forms exist – may respond to riboﬂ avin

++ ++ ++ W, M, and F Variable. Macroglossia, severe cardiomyopathy in infantile Pompe form ++ Most, but ++ E, F, H, L, M, Liver ± muscle. Distal > proximal. sx rare and W ++ + ++ L, M, W, E, F, Severe liver disease. Neuropathy, dementia in adult and D form +++ M Ichtyosiform nonbullous erythroderma

+ ++ + M Hepatomegaly + ++ + + + ++ + ++ M, W, L, H, Several syndromes. See Table D6.3. and F Lactate /pyruvate ratio usually increased

+ + + + M Renal tubular dysfunction, ataxia. Myopathic forms allelic with MADD 164 S. G. Kahler

PLASMA MC Fatty AcidsLC Fatty Acids Carnitine

3 LC Triglycerides 20 UDP-Gal UDP-Glu ATGL Fatty Acids 12 CGI-58 21 I Galactose Galactose-1-P Glucose-1-P LC-Acyl-CoA Carnitine 564 Glycogen Glucose Glucose-6-P II 7 LC-Acylcarnitine CoASH Fructose-6-P CYTOPLASM 22 8 10 9 Fructose Fructose-1-P 11 Fructose-1,6-bis-P Alanine + + III LC-Acylcarnitine NAD NADH + H 12 Lactate Pyruvate MITOCHONDRION 23 14 19 13 Acyl-CoA Acetyl-CoA Oxaloacetate 24 27 15

Malate Citrate 3-Oxo- Dehydro- Acyl-CoA BETA Acyl-CoA 18 OXIDATION Fumarate KREBS Isocitrate CYCLE 26 25 Succinate Acetoacetyl-CoA 3-Hydroxy- 17 Cis-Aconitate Acyl-CoA Acetone Succinyl-CoA 28 16 HMG-CoA Acetoacetate 30 29 3-OH-Butyrate + + 2-Oxoglutarate NADH + H NAD

Fig.C6.1 Pathway of carbohydrate metabolism illustrating the different steps of glycogenolysis and glycolysis exercise. Fatty acids predominate again during pro- defi ciency, and muscle phosphorylase kinase defi ciency) longed exercise and during fasting. Impairment of mus- results in signifi cant limitation when the patient attempts cle energy metabolism will lead to clinical symptoms. short, intense exercise. There will be diminished pro- The history of events that elicit the symptoms is a guide duction of pyruvate and hence lactate. The situation is to the likely area of the biochemical defect. magnifi ed, if the muscle being tested is deprived of oxy- Triglycerides are stored in the cytoplasm as drop- gen and continuous fuel by a tourniquet. This is the lets close to the mitochondria. The mobilization of free principle of the ischemic exercise test (see Chap. D8). fatty acids from the lipid droplets depends on hormone Defects of glycolysis in muscle, e.g., defi ciencies of sensitive lipases and involves the hydrolase ATGL and muscle phosphofructokinase (PFK), phosphoglycerate its activator protein comparative gene identifi cation-58 kinase (PGK), phosphoglycerate mutase (PGAM), (CGI-58), two recently identifi ed causes of lipid stor- b-enolase, and lactate dehydrogenase (LDH), can age myopathies (Fig. C6.1). cause symptoms similar to muscle phosphorylase defi - Resting muscle in the fed state uses fatty acids as the ciency. The pathways of glycogenolysis and glycolysis primary fuel; glucose is stored as muscle glycogen (in are shown in Fig.C6.1. the cytoplasm). Preformed high-energy phosphate com- When sustained muscle activity is initiated there is an pounds and muscle glycogen, in addition to glucose and initial reliance on glucose and glycogen as a fuel source. fatty acids in the blood stream, are a source of energy Metabolic disorders affecting these pathways typically for short-term intense activity. Lactate is the end prod- cause symptoms at this time. After a few minutes glyco- uct of anaerobic glycolysis. Impaired ability to utilize gen stores will be depleted, and fatty acids become more muscle glycogen (e.g., glycogen storage diseases (GSD) important as a fuel. The carnitine cycle and the b-oxidation III – debrancher defi ciency, V – muscle phosphorylase spiral of fatty acid oxidation are essential at this point, so C6 Metabolic Myopathies 165 defects in these pathways can result in easy fatigability (SCHAD) (see also Chap. B2.5), and mitochondrial and impaired tolerance of sustained exercise. oxidative phosphorylation disorders. Rhabdomyolysis may also be a chronic feature of the various muscular dystrophies, which are usually disorders of the struc- C6.1.2 Basic Patterns tural proteins of muscle (dystrophin, actin, tropomyo- sin, the dystroglycan complex, etc). of Metabolic Myopathies Remember The two major distinct syndromes of muscle metabolic Short, intense exercise stresses glycolysis and gly- disorders are exercise intolerance and rhabdomyolysis cogen utilization. (with or without myoglobinuria), and weakness (with Prolonged exercise requires adequate utilization or without hypotonia). Rhabdomyolysis can be further of fatty acids. divided into syndromes where it occurs during strenu- Fasting, cold, infection, and medications can ous exercise and those where it occurs afterwards. worsen many myopathies. Remember Myoglobinuria is an extreme result of rhabdomyoly- Major symptoms of myopathies are weakness, hypo- sis. When muscle cells lyse myoglobin is released. tonia, exercise intolerance, and rhabdomyolysis. Visible myoglobin in the urine indicates extensive damage. Typically, there is no myoglobin in the urine Rhabdomyolysis, the destruction of skeletal muscle if the CK is <10,000 IU, so the absence of myoglobin- cells, often results from failure of energy production, uria provides no reassurance regarding absence of leading to an inability to maintain muscle membranes. rhabdomyolysis. The hallmark of rhabdomyolysis is elevation of muscle Myoglobinuria is an emergency situation, as the enzymes in the blood, particularly creatine kinase (CK pigment may precipitate in the renal tubules, leading to or CPK). This elevation can persist for several days renal failure, which may become irreversible. Severe after an acute event. Chronic elevation of CK indicates rhabdomyolysis can raise the serum potassium level continuous damage. dangerously high, leading to cardiac rhythm distur- Rhabdomyolysis during short-term intensive exer- bances. Accordingly, dark urine in a patient suffering cise is a primary feature of the disorders of carbohy- from muscle symptoms (pain, weakness, cramping, drate metabolism, especially muscle phosphorylase etc) must be tested for myoglobin using a specifi c test (McArdle disease). After a period of intense pain with (to distinguish the pigment from hemoglobin). or without cramping, however, there may be consider- Hemoglobinuria most often accompanies hematuria, able relief and ability to continue exercise, called the readily detectable by fi nding erythrocytes on micro- “second wind” phenomenon, as the muscle switches to scopic analysis of the urine. Intravascular hemolysis increased use of fatty acids for fuel. Patients with will occasionally result in hemoglobinuria, without McArdle disease will also benefi t from glucose admin- hematuria. istration before exercise, because they are able to utilize Myoglobinuria is treated with diuresis, and careful glucose. In contrast, patients with a metabolic block in monitoring of electrolyte, fl uid status, and urine out- glycolysis, e.g., in PGK, cannot utilize neither glucose put, until the myoglobinuria resolves. Investigation of nor glycogen. Glucose administration even diminishes the underlying cause of myoglobinuria begins at the the concentrations of the alternative fuels triglycerides same time as its treatment. and ketone bodies (“out of wind” phenomenon). Chronic weakness and hypotonia are typical features Postexercise cramps and rhabdomyolysis are the of disorders of endogenous triglyceride catabolism, gly- more common pattern in fatty acid disorders, especially cogen breakdown, carnitine availability, fatty acid oxi- defi ciencies of carnitine palmitoyltransferase (CPT) II, dation, and oxidative phosphorylation. Important causes very long-chain acyl-CoA dehydrogenase (VLCAD), include lysosomal GSD (acid maltase defi ciency – Pompe long-chain hydroxyacyl-CoA dehydrogenase (LCHAD), disease), glycogen debrancher defi ciency, carnitine and short-chain hydroxyacyl-CoA dehydrogenase transporter defect and secondary carnitine defi ciencies, 166 S. G. Kahler

VLCAD, LCHAD, SCHAD defi ciencies, and mito- or mitochondrial dysfunction, need to be monitored chondrial myopathies. Chronic weakness may certainly carefully during infections. result from rhabdomyolysis and conse quent muscle The history of exercise can provide preliminary destruction from any cause, especially if recurrent. guidance in determining the most likely causes of a myopathy. An inability to perform sudden intense exercise suggests a problem with glycogenolysis or glycolysis, while inability to perform at a sustained C6.2 Approach to Metabolic Myopathies level suggests a problem with fatty acid oxidation. Many mitochondrial disorders of oxidative phos- The most urgent issues in the assessment of myopathy phorylation fi rst become apparent because of skeletal are to determine if there is weakness so severe to muscle weakness. Even isolated myopathies can impair respiration, if there is suffi cient damage to lead occur. Rhabdomyolysis is uncommon. Mitochondrial to myoglobinuria, and if there is cardiac involvement. disorders may have prominent muscle involvement. Hepatic involvement, often manifest as hypoglycemia Mitochondrial disorders can involve any organ at any and fasting intolerance, occurs in many metabolic dis- age. Other organs commonly affected include the orders, particularly those involving glycogen or fatty brain, retina, extraocular muscles, heart, liver, kidney, acid metabolism. A toxic encephalopathy, including pancreas, gut, and bone marrow. Systemic growth may cerebral edema, may also develop. be impaired. Mild hypertrichosis often accompanies The history of muscle dysfunction may be easy to systemic lactic acidosis. Despite the diversity of mito- elicit from an adult or a child (or the parents), but may chondrial dysfunction there are several common syn- be diffi cult with infants. Hypotonia and weakness may dromes in which many patients can conveniently be fi rst become evident as developmental delay. Careful grouped. They include MERRF, MELAS, and infantile assessment may then reveal that social, fi ne motor, and myopathy (Chap. D6). language skills are appropriate for age, and the only Two very rare neutral lipid storage diseases could be area of delay is in gross motor skills. recently identifi ed. Neutral lipid storage disease type I As a young child grows older, problems with exer- or Chanarin–Dorfman syndrome manifests as a slowly cise intolerance and easy fatigability become easier to progressive proximal myopathy sparing the axial mus- detect, particularly if there is an unaffected older sib- cles. The hallmark of the disorder is an ichthyosiform ling to serve as a reference point for the parents. nonbullous erythroderma. Neutral lipid storage disease Occasionally, a child with a muscle disorder is thought type II is due to mutations in the activator gene ATGL, to be “seeking attention” or malingering, but careful again leading to a lipid myopathy associated with car- history and observation can usually eliminate this pos- diac dysfunction and hepatomegaly. sibility quickly. Laboratory tests that convincingly demonstrate ongoing muscle injury (e.g., elevated CK) are most persuasive. Disorders made worse by fasting may not be evi- C6.2.1 Genetics dent in infancy, as most infants are fed frequently. An inability to tolerate intense or prolonged exercise will Most metabolic myopathies, like most other metabolic not be evident in infancy, and perhaps not until adult- disorders, are inherited in an autosomal recessive man- hood. Rhabdomyolysis in response to cold also may ner. All disorders of fatty acid oxidation and most dis- not become evident until adolescence or adulthood. orders of glycogen and glucose metabolism are inherited Rhabdomyolysis triggered by infection (usually viral), this way. However, other mechanisms including one or fasting, may present in infancy as sudden weakness, form of phosphorylase b kinase defi ciency, and PGK accompanied by dark urine. Rhabdomyolysis is often defi ciency, autosomal dominant (heterozygous CPT II quite painful, but may be painless. defi ciency), mitochondrial maternal transmission, and Some conditions that are not yet completely char- sporadic mitochondrial disorders occur. Specifi cs of acterized can cause severe and potentially fatal rhab- inheritance are mentioned when appropriate in the domyolysis in children, particularly in the setting of discussion of the various disorders. viral infection. Children with such conditions, like Because of the highly variable nature of most meta- children with named disorders of fatty acid oxidation bolic disorders of muscle, all siblings of patients should C6 Metabolic Myopathies 167 be checked for the condition which is in the family. If Third-order tests include electromyogram (often the disorder may be dominant, X-linked, or mitochon- coupled with nerve conduction studies), chest X-ray, drially inherited other appropriate relatives should also electrocardiogram, echocardiogram, and muscle biopsy be examined carefully. (perhaps together with nerve biopsy). Light and electron microscopic examination, and special stains for glycogen, lipid, and various enzymes may all be essen- C6.2.2 Physical Examination tial. “Classic” lipid storage is found in four conditions: primary carnitine defi ciency due to a defi ciency of the carnitine transporter, mild multiple acyl-CoA dehy- The general physical examination of a patient sus- drogenase defi ciency (MADD) with secondary coen- pected of myopathy includes assessment of growth zyme Q defi ciency, and the neutral lipid storage and development, and particular attention to other 10 diseases types I and II. organs. The muscles should be examined for bulk and Many enzymes can be studied in fi broblasts or lym- regional (proximal and distal) or local evidence of phocytes, and DNA can be obtained from blood or a buc- wasting, texture and consistency, and tenderness. Deep cal brush instead of a tissue biopsy. Mitochondria can be tendon refl exes, which are generally preserved in myo- prepared for functional and molecular studies from mus- pathies, but lost in peripheral neuropathies, should be cle (the preferred source) but also liver, leukocytes, and tested carefully. Attention should be especially directed other samples. Coenzyme Q is best measured in muscle. to extraocular movements and the retina, the tongue, Details of these tests are given in (Chap. D5). If an open the heart, and the size and character of the liver. muscle biopsy is done, a skin biopsy for fi broblast culture and DNA analysis can be taken from the edge of the incision. Some pathologists and mitochondrial laborato- C6.2.3 Laboratory Investigations ries are now able to analyze muscle tissue obtained by needle biopsy. Table C6.1 provides a guide to the princi- Laboratory investigation of suspected myopathies pal features of the major metabolic myopathies and the should be undertaken during the acute episode if pos- usefulness of the various diagnostic materials. sible, and later repeated as indicated. Routine serum electrolytes, measurement of glucose, urea and creatinine, and “muscle enzymes” including CK, LDH including isoforms, aldolase, SGOT (ALT), SGPT (AST), C6.3 Specifi c Disorders total and free carnitine, plasma or blood spot acylcar- of Muscle Metabolism nitines profi le, plasma lactate and pyruvate, phosphate, calcium, thyroid hormone, plasma and urine amino C6.3.1 Exercise Intolerance/ acids, and urine organic acids, may all provide useful Rhabdomyolysis and information. Following the assessment of the fi rst-order laboratory Myoglobinuria Presentation tests, further tests may be warranted. Functional testing using ischemic exercise (for suspected glycogen storage C6.3.1.1 Intense Exercise not Tolerated. Mild, and glycolytic disorders, and adenosine monophosphate Prolonged Exercise Tolerated. Fasting deaminase defi ciency) can be most helpful (see Chap. D8). Tolerated. Dietary Modifi cations Helpful Graded exercise or bicycle ergometry may help pinpoint the metabolic error, or defi ne the general area of impair- Muscle Glycogen Phosphorylase ment, if history and blood tests have not done so. A (Myophosphorylase) Defi ciency – McArdle Disease “diagnostic fast” to evoke abnormal metabolites or pro- (GSD Type V) voke symptoms should only be done if information cannot be obtained by another method – challenges are This dramatic disorder of muscle glycogen metabolism better put to fi broblasts or tissue samples. However, a is a relatively common cause of rhabdomyolysis and fast under controlled circumstances can provide valuable myoglobinuria. Although it is inherited in an autosomal information regarding how long it is safe for a particular recessive manner, most symptomatic patients are men. child to fast when healthy (see Chap. D8). Symptoms usually begin between late childhood and 168 S. G. Kahler late middle age. Strenuous exercise leads rapidly to recessive. Patients may have neurologic problems (men- cramping and fatigue, but, after a period of rest (adapta- tal retardation, behavioral abnormalities, seizures, and tion), exercise is tolerated. Some patients with McArdle strokes). The other disorders are autosomal recessive. disease have chronic progressive weakness and wasting, without pain or cramping. Exceptional cases include a rapidly fatal form in Lactate Dehydrogenase (LDH) Defi ciency infants or young children with hypotonia and generalized weakness, a late onset form with chronic weakness, and Lactate dehydrogenase catalyzes the conversion of a late onset form with severe symptoms (pain, cramping, pyruvate and NADH to lactate and NAD+. The enzyme weakness, and muscle swelling), after decades of normal is a tetramer of H and M peptides, produced from activity. Diagnosis after inadvertent discovery of elevated LDHB and LDHA genes. Homozygous defi ciency of CK in a child without symptoms has been reported. the M protein results in impaired muscle LDH activity. Diagnosis is suspected from the symptoms, and The result is impaired regeneration of NAD+ for anaer- response to ischemic exercise. The enzyme is expressed obic glycolysis, and impaired production of lactate mainly in muscle. Muscle biopsy may show myopathic (and resulting high levels of pyruvate) with exercise, changes and increased glycogen content. detectable by the ischemic exercise test (see Chap. D8). Specifi c treatment is generally not needed, as avoid- Cramps, weakness, and myoglobinuria can occur with ing strenuous exercise prevents symptoms in most strenuous exercise. Defi ciency of LDH can result in a patients. A high-protein, low carbohydrate diet has “false negative” result if LDH is being measured to been suggested to improve endurance. Others have assess tissue damage in other situations. No syndrome found increased carbohydrate intake (glucose and is attributable to LDHB defi ciency. fructose) immediately before exercise to be helpful.

Muscle Glycogen Phosphorylase Kinase Adenosine Monophosphate (Myoadenylate) Defi ciency (Formerly Phosphorylase Deaminase Defi ciency b Kinase Defi ciency, GSD IX) This autosomal recessive disorder of purine metabo- There have been a few men with defi ciency of muscle lism is probably the commonest metabolic myopathy, phosphorylase kinase. Weakness without cramps and with impaired exercise tolerance, postexercise cramps, cramps without weakness have both been reported. and myalgias. Myoglobinuria is uncommon, but CK is Increased muscle glycogen content, elevation of CK, and often elevated after exercise. Onset of symptoms (usu- rhabdomyolysis have been reported. Enzyme defi ciency ally pain after exercise) ranges from childhood to later was demonstrated in muscle. The gene encoding the adult life. In the US population perhaps 2% are alpha subunit of phosphorylase (PHKA1) in muscle is on homozygous for defi ciency, but most have no symp- the X chromosome. It is distinct from the liver isoform toms. Because AMP deaminase defi ciency is so com- (also on X). There are also three autosomal components mon, it has sometimes been found coincidentally with of the glycogen phosphorylase kinase system. Autosomal a less common muscle disorder that by itself would recessive defects have been found in the beta and gamma account for the symptoms, e.g., muscle phosphorylase peptides. No defects in the three delta subunit isoforms or PFK defi ciency. As the enzyme defi ciency will only (which are calmodulins) have been reported. be discovered by ischemic exercise testing, or specifi c assay or molecular test, there is a selection bias toward muscle problems. Muscle Glycolytic Disorders Many patients discovered to have AMP deaminase defi ciency have other symptoms as well, especially Defi ciencies of fi ve glycolytic enzymes in muscle are neuromuscular disease. Diminished synthesis of AMP rare causes of myopathy similar to muscle phosphory- deaminase occurs in a variety of situations. This is lase defi ciency. They are PFK, PGK, PGAM, b-enolase, termed acquired defi ciency and does not seem to have and triosephosphate isomerase (Fig.C6.1). Hemolytic a direct genetic basis, and the common mutation is not anemia can occur in all. PGK defi ciency is X-linked present at a frequency above the background rate. C6 Metabolic Myopathies 169

AMP deaminase catalyzes the deamination of AMP is defi cient, there will be diminished ammonia produc- to IMP (inosine monophosphate) in the purine nucle- tion. Muscle biopsy may be normal or show some otide cycle (see Fig. C6.2). During exercise there will myopathic changes. Specifi c staining for AMP deami- be increased production of IMP and ammonia, and nase is a generally reliable diagnostic test. Enzyme maintenance of the adenylate energy charge by pre- activity in defi cient muscle ranges up to 15% of nor- venting AMP accumulation. A decrease in ATP and mal; some authorities regard activity >2% as adequate increase in ammonia will also stimulate glycolysis by to prevent symptoms. A common mutation accounts increasing the activity of PFK. The increase in IMP for most cases of inherited AMP deaminase defi ciency. may also enhance glycogen phosphorylase. Finally, This common mutation is actually a pair of mutations during intense exercise AMP deaminase moves from in linkage disequilibrium, GLN12TER (p.Q12X) and the cytosol and becomes bound to myosin, which sug- PRO48LEU. The nonsense mutation in exon 2, gests that it is important in muscle metabolism during GLN12TER can result in a severely truncated protein. such times. However, an alternative splicing mechanism allows for The diagnosis of AMP deaminase defi ciency is phenotypic rescue by production of a shortened but approached by ischemic exercise ordinarily provoking functional protein, with PRO48LEU in exon 3 retained. a rise in blood ammonia level (see Chap. D8). If AMP This may account for the great variability in symptoms

Rlbose-5-p+ATP PRPP Synthesetase

5-Phosphorlbosyl-I-Phyrophosphate (PRPP)

PRPP Amidotransferance

5-Phosphoribosyl-I-Amine

5-Phosphoribosyl-5-Amino-4-Imidazole-Succinocarboxamide (SAICAMP) ASL

5-phosphoribosy1-5-Amino-4-Imidazole-Carboxamide (AICAMP) Nucloic acids Nuclelc acids AMPDA OH OH

N N N N Adenyllc acid N Fig. C6.2 Pathways of synthesis, salvage, and H N N N N Adenylosuccinlc 2 R æ P R æ P degradation of purines. The enzymatic steps in Guanylic Inosinic acid acid acid boxes indicate the sites of the commonly APRT PPI PPI encountered disorders of purine metabolism ADA Guanosine Inosine AMPDA muscle adenosine monophosphate Adenoslne PNP HRPT PNP Adenlne deaminase defi ciency; APRT adenine phosphori- PRPP PRPP bosyltransferase defi ciency; ASL adenylosuccinate OH OH N N lyase (adenylosuccinase) defi ciency; HPRT N N hypoxanthine–guanine phosphoribosyltransferase N N H N N N 2 defi ciency; PNP purine nucleoside phosphorylase Guanine Hypoxanthine defi ciency; SO sulfi te oxidase defi ciency; XO XO xanthine oxidase (xanthine dehydrogenase) Xanthine defi ciency XO Uric acid 170 S. G. Kahler in homozygotes for this mutation. The allele frequency and fatty acid metabolism) may precipitate episodes, was 0.13 (Caucasians) and 0.19 (African-Americans) which usually do not occur in children. Cardiac in one study, which accounts for the observed homozy- involvement is uncommon in this form of CPT II gote frequency of about 0.02. Polymorphisms of defi ciency. angiotensin I-converting enzyme may contribute to the Plasma or blood spot acylcarnitine analysis shows variability of symptoms seen with AMP deaminase prominent long-chain species, especially saturated and defi ciency. unsaturated C16 and C18 forms, and there may be AMP deaminase defi ciency has been found in dicarboxylic aciduria, similar to translocase defi ciency. patients with aldolase defi ciency, lactate dehydroge- A severe form of infantile CPT II defi ciency also nase M (LDHA) defi ciency, and b-enolase defi ciency. exists. Hepatic encephalopathy with hypoketotic hypoglycemia, severe cardiac involvement, renal malformations, and low plasma and tissue carnitine levels C6.3.1.2 Short, Intense Exercise Tolerated, may be present. This form is usually fatal, from car- Prolonged Exercise not Tolerated, diac complications. It is discussed in more detail in Fasting Detrimental, Diet Effects section (Chap. D1, Cardiac). Less Pronounced. Symptoms may Treatment includes avoiding of fasting and provid- be Triggered by Infection. Restriction ing adequate fuel for the muscles. Medium-chain fatty of Long-Chain Fats, with acids do not need the carnitine system in order to enter Supplementation of Medium-Chain the mitochondria, so they can be used in place of long- Lipids, may be Helpful, Especially chain fats as a source of energy. for Cardiomyopathy Although this is an autosomal recessive disorder, heterozygosity for a mutation may be associated with Carnitine-Acylcarnitine Translocase Defi ciency subtle myopathy and risk of malignant hyperthermia (MH) in response to anesthetics or muscle relaxants. An inability to import long-chain acylcarnitines into There is a form of CPT I that is solely expressed the mitochondrial matrix would be expected to cause in muscle. No clinical defi ciency has been recognized serious diffi culties, especially in cardiac and skeletal so far. muscle, and in the liver. The severe infantile form of translocase defi ciency typically does this, starting a day or two after birth. Cardiac (cardiomyopathy, usu- VLCAD Defi ciency ally hypertrophic) and hepatic dysfunction (hypoglycemia, vomiting, and hyperammonemia) are more VLCAD is the fi rst enzyme of the b-oxidation spiral. prominent than hypotonia and weakness. Urinary It is bound to the inner mitochondrial membrane. organic acids show dicarboxylic aciduria, and the Defi ciency of VLCAD is a common cause of meta- plasma/blood spot acylcarnitine profi le is dominated bolic myopathy and cardiomyopathy. For several by long-chain species (C16:1, C18:1, and C18:2), years, the enzyme now known as VLCAD was called which can be formed but not used, and dicarboxylic LCAD (long-chain acyl-CoA dehydrogenase); reports acylcarnitines. from before about 1993 regarding LCAD defi ciency almost always involve what is now called VLCAD. VLCAD utilizes fatty acids of 14–20 carbons. A CPT II Defi ciency major source of confusion is that fatty acids called very long chain fatty acids, of chain length >20, are CPT II defi ciency is the commonest disorder of fatty metabolized by a different system altogether, in the acid oxidation to cause episodic rhabdomyolysis. CPT peroxisomes. II is needed to synthesize long-chain acylcarnitines Impairment of VLCAD will lead to variable skeletal, once they have been translocated into the mitochon- cardiac, liver, and brain symptoms, including recurrent dria, so the clinical features are similar to translocase Reye syndrome with coma, and hypoketotic hypoglyce- defi ciency. Prolonged exercise, cold, infection, and mia. Muscle soreness and episodic rhabdomyolysis may emotional stress (which will increase catecholamines be provoked by infection, cold, fasting, or emotional C6 Metabolic Myopathies 171 stress (perhaps mediated by catecholamines). There is neuropathy and retinopathy. The basis for these com- usually dicarboxylic aciduria, although during severe plications is not yet known. metabolic derangement it may be overlooked because of The enzyme activity called LCHAD is found in an excessive lactic aciduria indistinguishable from a pri- octameric protein (a4b4) called the trifunctional protein, mary defect of the respiratory chain. Carnitine deple- for its ability to catalyze the 2-enoyl-CoA hydration, tion, with low plasma and tissue levels, can occur and 3-hydroxyacyl-CoA dehydrogenation, and 3-oxoacyl- acylcarnitine analysis shows prominence of C14:1 (tet- CoA thiolysis of long-chain acyl-CoAs. The fi rst two radecenoyl) species, derived from oleic acid (C18:1). activities reside in the a subunit, and thiolase activity in Hepatic dysfunction may result in hyperammonemia the b subunit. The two subunits depend on each other for and lipid accumulation. Muscle biopsy may show lipid stability. The trifunctional protein is in the mitochondrial storage, and the EMG is often myopathic. inner membrane. VLCAD defi ciency, like other disorders of fatty There is some relationship between mutation and acid oxidation, must be promptly treated during the symptoms. The commonest mutation (87% in one acute episode with glucose suffi cient to maintain the study), c.1528 G > C (p.E510Q) in the a subunit, usu- blood glucose level at 6–8 mM or even higher (see ally causes liver dysfunction with hypoketotic hypo- also Chap. B2. 8). The use of carnitine supplementa- glycemia in infancy. tion has been controversial on theoretical and experi- LCHAD defi ciency is an autosomal recessive disor- mental grounds, particularly because of fear that der, and carriers are generally symptom free. A par- long-chain acylcarnitines would accumulate and ticular complication of heterozygous (carrier) status provoke arrhythmias. However, there are few con- for LCHAD defi ciency, especially the p.E510Q muta- vincing reports of this actually happening. Long- tion, is serious liver disease during pregnancy when term management emphasizes adequate calories carrying an affected infant. The mother may suffer from carbohydrate, restricting long-chain dietary from acute fatty liver of pregnancy (AFLP, with nausea fats, avoiding fasting and other stressors, and supple- and anorexia, vomiting, and jaundice) or the HELLP mentation with medium-chain triglycerides, which syndrome of hypertension or hemolysis, elevated liver will provide a source of fuel that can be metabolized enzymes, and low platelets. It may be that the produc- without requiring VLCAD. Triheptanoin, a novel tion of abnormal fatty acid metabolites by the fetus treatment to provide a constant source of ketone overloads the mother’s ability to deal with them, on bodies for patients with defects of long-chain fatty top of the increased fatty acid mobilization that occurs acid oxidation is showing great promise in prelimi- during pregnancy. Prospective studies of women with nary studies. AFLP or HELLP syndrome, however, have not found The enzyme now known as the LCAD is in the an increased number of carriers of LCHAD defi ciency, mitochondrial matrix. Its major substrates are unsatu- indicating there are other causes for these conditions. rated long-chain fatty acids, 12–18 carbons in length. Women heterozygous for hepatic CPT I defi ciency, Defi ciency is very uncommon; manifestations are sim- which may cause a Reye-like syndrome in homozy- ilar to other disorders of fatty acid oxidation. gotes, may also suffer from AFLP when carrying an affected fetus. In untreated patients, the urine organic acids reveal LCHAD (Including Trifunctional Protein) Defi ciency increased saturated and unsaturated dicarboxylic and hydroxy species. The plasma acylcarnitine profi le typi- LCHAD defi ciency often results in chronic myopathy, cally shows elevation of hydroxy-C18:1 species, with rhabdomyolysis, which may be extensive, partic- which, in combination with an elevation of two of the ularly during viral infections. Like other disorders of three long-chain species C14, C14:1, and hydroxy- long-chain fatty acids there is often cardiomyopathy C16, identifi es over 85% of patients with high specifi c- and signifi cant liver dysfunction, both of which may be ity (<0.1% false positive rate). Blood spot acylcarnitine fulminant. The extent of chronic liver dysfunction can analysis is not quite as sensitive, because of higher lev- be greater than in other disorders, and fi brosis often els of long-chain species in blood samples. Dietary occurs. There may be Reye-like episodes of hepatic treatment (restriction of long-chain fats and supple- encephalopathy. In addition there may be peripheral mentation with medium-chain triglycerides) will lower 172 S. G. Kahler the long-chain acylcarnitine species, often to normal. results (plasma levels can be <5 mmol/L, normal » Plasma carnitine levels are usually low, especially dur- 45 mmol/L) will lead to tissue carnitine depletion as ing acute illness. Carnitine is sometimes given at such well. Hepatic carnitine depletion results in hypoketotic times. Its usefulness as a medication for this myopathy hypoglycemia. Onset of myopathy and cardiomyopathy (and whether it might provoke arrhythmias in certain can be in the fi rst few months, or not for several years. situations – see Chap. C1) is a subject of current Urine organic acids are typically normal. Muscle biopsy investigation. reveals lipid accumulation, and the muscle carnitine level is extremely low. Response to carnitine supplementation is dramatic, but because of the ongoing renal Short-Chain Acyl-CoA Dehydrogenase Defi ciency leak of carnitine, it is extremely diffi cult to maintain normal plasma carnitine levels or tissue levels, and exer- This enzyme defi ciency is extremely variable, having cise tolerance may be limited. Oral carnitine supple- been reported in infants and adults. Muscle symp- mentation to an amount just short of provoking a fi sh toms, when present, have ranged from a mild lipid odor (trimethylamine) by exceeding the oxidizing myopathy with low muscle carnitine, to a more severe capacity is the usual approach. Inheritance is autosomal condition with weakness, poor exercise tolerance, and recessive. myopathic EMG. Systemic symptoms of hepatic encephalopathy have occurred. The impaired metabolism of short-chain acyl-CoAs leads to short-chain C6.3.2.2 Secondary Carnitine Depletion dicarboxylic aciduria [ethylmalonic and adipic, similar to electron-transfer fl avoprotein (ETF) defi ciency] Adults are able to synthesize all the carnitine they and excess butyrate. Acylcarnitine analysis may show need. The major dietary source of carnitine is meat. increased C4 species. The carnitine content of human breast milk is similar to that of plasma. Carnitine defi ciency has occurred in several infants on prolonged parenteral nutrition (with- Hydroxyacyl-CoA Dehydrogenase out added carnitine), with resulting myopathy and car- (HADH) Defi ciency diomyopathy, impaired ketogenesis, and hepatic steatosis. Carnitine supplementation (intravenous or HADH defi ciency is a very rare condition, with recur- oral) was rapidly benefi cial. This experience suggests rent rhabdomyolysis, hypertrophic cardiomyopathy, that carnitine may be an essential nutrient in the very and hypoketotic/hyperinsulinemic hypoglycemia. Mild young, and that routine carnitine supplementation of dicarboxylic and hydroxydicarboxylic aciduria was TPN solutions for infants should be considered. reported. The enzyme can be assayed in muscle or in Severe carnitine depletion can result from a gener- mitochondria from skin fi broblasts. This enzyme was alized Fanconi syndrome, characteristic of cystinosis, previously called SCHAD. Lowe syndrome, mitochondrial disorders (especially, cytochrome C oxidase defi ciency), etc (see also Chap. C4). Recognition of the carnitine depletion usually C6.3.2 Weakness and Hypotonia occurs after the discovery of Fanconi syndrome. Plasma carnitine measurement and determination of Presentation the fractional excretion should be part of the investigation of all patients with Fanconi syndrome. Restoration C6.3.2.1 Carnitine Transporter Defi ciency of tissue carnitine levels may take a very long time even after correction of the renal leak, as after trans- The carnitine transport defect is the result of defi cient plantation for cystinosis. activity of the high-affi nity carnitine transporter Some medications, such as valproic acid and pivam- (OCTN2, encoded by the gene SLC22A5), active in picillin are essentially organic acids and there may be kidney, muscle, heart, fi broblasts, and lymphocytes. excretion of metabolites as carnitine esters causing Renal fractional excretion of carnitine, calculated signifi cant urinary losses of carnitine as valproylcarni- in relation to creatinine clearance, approaches 100% tine or pivaloylcarnitine, and consequently secondary (normal <5%). The severe carnitine depletion that systemic carnitine defi ciency. Use of pivampicillin C6 Metabolic Myopathies 173 has been linked to life-threatening crisis, especially (50–100 mg/day), the precursor of the cofactor fl avin in patients with an underlying metabolic disorder adenine dinucleotide, is often dramatic in the milder [e.g., medium-chain acyl-CoA dehydrogenase (MCAD) forms. A postulated disorder of ribofl avin transport defi ciency]. may be the cause of a similar disorder. MCAD defi ciency is unusual among fatty acid oxidation disorders for having only minimal skeletal muscle symptoms. Hepatic and cerebral symptoms (Reye-like C6.3.2.5 Lysosomal GSD Type II syndrome, hypoketotic hypoglycemia, and sudden unex- (Pompe Disease) plained death) are the usual features. However, carnitine depletion can occur. MCAD defi ciency accounts for Pompe disease is the severe infantile form of lyso- nearly all the patients described with “systemic carnitine somal a-glucosidase (acid maltase) defi ciency. It is defi ciency,” before the enzyme defi ciency was discov- one of the most common storage diseases presenting in ered. This may be the mechanism for chronic weakness infancy, and the fi rst one to be identifi ed. The infant and impaired exercise tolerance in some older patients typically presents in the fi rst few months with weak- with MCAD defi ciency. Long-term carnitine supplemen- ness and profound hypotonia. Feeding and respiratory tation, whose use in MCAD defi ciency is not universally diffi culties are common. There is macroglossia, but accepted, might be expected to ameliorate this situation. minimal hepatomegaly. At least 80% have signifi cant Longitudinal studies have not yet been reported. cardiac involvement. Progressive weakness and cardiomyopathy usually lead to death within a year. Despite the hypotonia the muscles feel fi rm or even woody. C6.3.2.3 2,4-Dienoyl-CoA Reductase Defi ciency There is a spectrum of defi ciency of a-glucosidase, with onset of symptoms reported as late as the eighth This extremely rare disorder has been described in one decade. Various terms, including juvenile and adult onset, infant with hypotonia, normal deep tendon refl exes, are used to describe late-onset patients. The age of onset poor feeding, and failure to thrive. There was plasma bears no relation to the rapidity of the course. The older carnitine defi ciency, and a unique unsaturated acylcar- the patient the more likely that symptomatic muscle nitine (C10:2) in plasma, shown to be derived from involvement will be patchy clinically and morphologi- long-chain unsaturated fatty acids. cally. However, enzyme activity will be defi cient, regardless of the appearance of the cells. The function of lysosomal glycogen is not known. a-Glucosidase activity C6.3.2.4 MADD–ETF OR ETF-Dehydrogenase can be measured in leukocytes, as well as in muscle or in Defi ciency (Synonymously Glutaric liver biopsy, or in cultured skin fi broblasts. Muscle biopsy, Aciduria Type II) which is not necessary for diagnosis if enzyme assay can be performed, shows enlarged lysosomes engorged with The severe form of this disorder causes overwhelming glycogen, altering the ultrastructure of the cell. acidosis shortly after birth. Impairment of the ETF sys- There was no satisfactory treatment of Pompe dis- tem blocks many different dehydrogenation systems ease until the development of enzyme replacement for fatty acids and amino acids degradation. Milder therapy. Recombinant a-glucosidase can arrest and defi ciency of the same system can cause a lipid storage even reverse the disease if begun early enough, so early myopathy that may not become apparent for years or diagnosis is now an urgent matter. decades. Gradual onset of weakness and easy fatigability may be overlooked initially, and the history may fi rst suggest an infl ammatory myopathy. There may be C6.3.2.6 Glycogen Debrancher liver dysfunction. Urine organic acids can show dicar- Defi ciency – GSD Type III boxylic aciduria, including ethylmalonic, adipic, and (Cori or Forbes Disease) glutaric acids. Plasma acylcarnitine analysis shows elevation of short- and medium-chain species. A sec- This relatively common disorder of glycogen metabo- ondary defi ciency of coenzyme Q10 (CoQ) may occur. lism results from varying defi ciencies of amylo-1,6- Mitochondrial studies may show defi cient activity glucosidase, 4-a-glucoanotransferase, the debrancher complex I and II. Response to supplemental ribofl avin enzyme, a remarkable peptide which has two separate 174 S. G. Kahler catalytic activities (transferase and glucosidase). There reported with a muscle form of the disease, which is always liver involvement, but muscle involvement is caused muscle weakness, reduced exercise capacity, variable, and does not occur at all in about 15% (GSD and hypertrophic cardiomyopathy in the fi rst decade. IIIb). In infancy and childhood the liver symptoms dominate, with hepatomegaly, hyperlipidemia, and fasting hypoglycemia similar to GSD I. Muscle weak- C6.3.2.9 Mitochondrial Myopathies ness may not be apparent. After puberty the liver and Coenzyme Q Defi ciency symptoms subside, but the myopathy may persist as weakness, and may worsen with time. CK may be ele- The mitochondrial myopathies are an extremely hetero- vated, but may be normal even if there is muscle geneous group of disorders. Symptoms may be confi ned involvement. There can be distal wasting, myopathic to muscle, or may involve other organs, particularly the EMG, and peripheral neuropathy. There is usually car- brain, heart, liver, and kidneys. Symptoms may already diac involvement (mild) as well. be present at birth or not appear for decades. Myopathy Diagnosis is suspected from the history. Enzyme is particularly evident in the syndromes of chronic pro- assay and gene analysis can be done using fi broblasts, gressive external ophthalmoplegia (CPEO) including lymphocytes, muscle, or liver. Liver and muscle biop- the Kearns–Sayre syndrome (KSS) or ophthalmoplegia- sies are often done to obtain direct information about plus; mitochondrial encephalomyopathy, lactic acidosis, these organs. Analysis of glycogen structure in muscle and stroke-like episodes (MELAS); mitochondrial or liver can demonstrate abnormalities due to the lack of encephalomyopathy with ragged-red fi bers (MERFF); normal glycogen breakdown. The differences in tissue fatal infantile mitochondrial myopathy; depletion of the expression are traceable to alternate splicing of exon 1 mitochondrial DNA; and autosomal dominant and of the gene. Differences in phenotype correlate with dif- recessive mitochondrial myopathies. Molecular defects ferent mutations. Two mutations in exon 3 account for can be in the mitochondrially encoded tRNAs, mito- 90% of patients with GSD IIIb (i.e., no muscle involve- chondrial- and nuclear-encoded subunits of the oxida- ment). If these mutations are not present, muscle biopsy tive phosphorylation complexes, and many other must be done to ascertain if there is muscle involve- proteins. Some of the most relevant disorders and etiol- ment. Support of the glucose availability is achieved ogies are shown in Table 3, Chap. D6. with corn starch, similar to GSD I. Treatment of the Muscle symptoms are generally those of chronic myopathy using high-protein meals and high-protein weakness and impaired exercise tolerance. Cramps are enteral feeds overnight has been attempted, but it does unusual. Rhabdomyolysis can occur, particularly, in not seem to improve the long-term outlook. the setting of sustained exercise or febrile illness. Malignant hyperthermia may occur with anesthesia or muscle relaxants. C6.3.2.7 Glycogen Brancher Systemic lactic acidosis may be present at rest or Defi ciency – GSD Type IV elicited with exercise. Some infants will have acidosis (Anderson Disease) from the work of breathing, but be chemically normal with ventilator support. CK may be elevated. Plasma Type IV GSD, glycogen brancher defi ciency, can cause amino acids may show increased alanine. Urinary hypotonia and weakness, but the clinical picture is domi- organic acids may show increased of lactate, citric acid nated by hepatic fi brosis and dysfunction. Cardiomyopathy cycle intermediates, and dicarboxylic fatty acids. The may be signifi cant in the severe infantile form. plasma carnitine level may be normal or low. Plasma acylcarnitine analysis may show a generalized increase in short- and medium-chain species, especially acetyl- C6.3.2.8 Glycogen Synthase carnitine, or be unrevealing. Defi ciency – GSD type 0 EMG may be normal or suggest myopathy; nerve conduction studies may reveal a peripheral neuropa- This disorder results in insuffi cient glycogen reserves thy, usually axonal. in the liver, so that fasting hypoglycemia occurs rela- Muscle tissue can be analyzed for carnitine content tively soon after a meal, and excessive lactate is pro- and acylcarnitine species and coenzyme Q level. duced from dietary glucose. One family has been Muscle biopsy may show dense clusters of abnormal C6 Metabolic Myopathies 175 mitochondria, especially near the surface of the cell C6.3.4 Malignant Hyperthermia membrane (“ragged red fi bers”), as well as increased lipid, but may be normal (see also Chap. D5). Cells Malignant hyperthermia (MH) in response to anesthet- may stain strongly for succinate dehydrogenase (com- ics occurs in many different situations and myopathies. plex II) yet not stain for cytochrome oxidase (COX), MH is most commonly due to the failure of regulation of particularly in CPEO, KSS, and MERFF. Maternally calcium concentration in the sarcoplasmic reticulum. inherited Leigh syndrome patients may have defi cient Excess calcium permits continuous muscle contraction, COX staining, but no ragged-red fi bers. Electron leading to heat generation, and a rise in body tempera- microscopy may show abnormal mitochondrial mor- ture. Severe myoglobinuria, irreversible kidney damage, phology, including paracrystalline inclusions. and death from hyperthermia or arrhythmia due to Studies of oxidative phosphorylation are best car- hyperkalemia may occur. For these reasons, all patients ried out in fresh muscle biopsy tissue. Some laborato- with a myopathy must be especially carefully monitored ries will work with frozen muscle tissue or freshly during surgery or any other procedure where anesthesia isolated platelets. Mitochondrial DNA studies are opti- is used, and the most risky anesthetics (e.g., halothane) mally performed from muscle biopsy as well. If there and muscle relaxants (e.g., suxamethonium) should be is a heteroplasmic disorder in the mtDNA, other tis- avoided. Premedication with dantrolene can lessen the sues (leukocytes and fi broblasts) can sometimes give a risk of untoward reactions. Several genes are now known misleading normal result. Mitochondrial myopathies for MH syndromes. MHS1 is due to mutations in the are a subset of the overall group of mitochondrial ryanodine receptor RYR1 (with or without central core cytopathies, which are discussed in Chap. D6. disease). MHS2 is due to mutations in the alpha subunit Mitochondrial disorders of oxidative phosphorylation of the gated sodium channel IV, SCN4A, also altered in are generally treated with a high-fat, low carbohydrate hypokalemic periodic paralysis, paramyotonia congen- diet, and supplemental vitamins and antioxidants, espe- ita, and related disorders. MHS3 is due to mutations in cially coenzyme Q (ubiquinone) and ribofl avin (which the calcium channel CACNL2A. MHS5 is due to may be quite helpful for complex I defi ciency myopa- changes in another calcium channel, CACNA1S. All of thy). Vitamin C, thiamin, vitamin E, vitamin K3 (as an these conditions can be dominantly transmitted. Patients artifi cial electron acceptor-donor), dichloroacetate, car- with muscular dystrophies or the recessive Native nitine, and succinate have been used in various situa- American myopathy with cleft palate and congenital tions. Responses are generally subtle, but occasionally a contractures are also vulnerable. Even for these high- patient responds dramatically to CoQ or other therapies. risk conditions MH does not occur with each exposure The synthesis of coenzyme Q involves nine steps; to a triggering agent. Of the disorders of intermediary recessive defects have been discovered in SPSS1, metabolism, which are the primary topic of this book, SPSS2, CABC1, COQ2, and APTX. Besides myopa- the greatest risk is to patients with CPT II defi ciency thy there may be ataxia, deafness, encephalopathy, (perhaps even in the heterozygous state), and with mito- liver disease, renal tubular dysfunction, and cardiac chondrial disorders, but all patients with metabolic myo- valvulopathy, depending on the defect. In many patients pathy should be regarded as at potential risk for MH. suffering from the myopathic form of CoQ defi ciency MADD defi ciency is the primary defect.

Key References C6.3.3 Myopathies with Major Cardiac Involvement Bonnefont J-P, Taroni F, Cavadini P, et al (1996) Molecular analysis of carnitine palmitoyltransferase II deﬁ ciency with hepa- tocardiomuscular expression. Am J Hum Genet 58: 971–978 The cardiac manifestations of several disorders Bruno C, DiMauro S (2008) Lipid storage myopathies. Curr discussed in this chapter, including GSD types II and IV, Opin Neurol 21:601–606 fatty acid oxidation disorders including carnitine transport DiMauro S (2007) Muscle glycogenoses: an overview. Acta Myol 26:35–41 defect, deﬁ ciencies of carnitine-acylcarnitine translocase, DiMauro S, Gurgel-Giannetti J (2005) The expanding pheno- CPT II, VLCAD, and LCHAD/tri-functional enzyme, type of mitochondrial myopathy. Curr Opin Neurol and mitochondrial disorders are discussed in Chap. C1. 18:538–542 176 S. G. Kahler

Leonard JV, Schapira AH (2000) Mitochondrial respiratory Pestronk A. Washington University Neuromuscular Disease chain disorders I: mitochondrial DNA defects. Lancet Center website. http://neuromuscular.wustl.edu 355:299–304 Vladutiu GD, Bennett MJ, Smail D et al (2000) A variable myo- Leonard JV, Schapira AH (2000) Mitochondrial respiratory pathy associated with heterozygosity for the R503C muta- chain disorders II: neurodegenerative disorders and nuclear tion in the carnitine palmitoyltransferase II gene. Mol Genet gene defects. Lancet 355:389–394 Metab 70:134–141 Psychiatric Disease C7 Ertan Mayatepek

Key Facts C7.1 General Remarks › Psychiatric manifestations may be the only symptom of inherited metabolic diseases Inherited metabolic diseases can manifest as acute before additional neurological or other clinical attacks of delirium or psychosis as well as intellectual signs are recognized. disintegration, mental regression, or chronic psychosis. › Inherited metabolic diseases can manifest Being aware of these manifestations allows to initiate acutely as attacks of delirium (visual), hallucina- appropriate diagnostic investigations and, if available, tions, mental confusion, hysteria, schizophrenia to institute rationale effective therapy (Table C7.1 and or psychosis, e.g., in urea cycle disorders, organic C7.2). acidurias, maple syrup urine disease, porphyrias, In the course of many neurometabolic diseases, psy- methylene tetrahydrofolate reductase defi ciency, chiatric manifestations often become of critical impor- cobalamin metabolism defects, Morbus Fabry, tance, and it is most relevant to inform families and to and metachromatic leukodystrophy. evaluate and treat them appropriately. Typical constellations in early childhood range from severe distur- › In infancy, autistic features may be a leading bances of mood and vegetative symptoms in patients clinical feature of metabolic diseases, e.g., in with neurotransmitter defects, and Smith–Lemli–Opitz urea cycle disorders, inborn errors of biop- syndrome to hyperactivity in patients with Sanfi llipo terin, or purine metabolism. disease and pterin defects to (auto-) aggressive behav- Psychiatric manifestations are mostly com- › ior in Lesh–Nyhan disease. These aspects are beyond bined with regression of cognitive functions, the scope of this book, except to stress the necessity of e.g., in X-linked adrenoleukodystrophy or close collaboration with colleagues from child psychi- mucopolysaccharidosis type III (Sanfi lippo). atry in these instances. › Patients with late-onset lysosomal storage disorders may initially present with psychiatric diagnoses such as dementia, psychosis, or emotional illness. C7.2 Acute Psychiatric Manifestations › Psychiatric manifestations can become most important in the long-term management of many patients with metabolic disorders. Acute psychiatric attacks may be the fi rst clinical correlate of an underlying metabolic defect. Presentations include mental confusion, hysteria, delirium, dizziness, aggressiveness, anxiety, bizarre behavior, agitation, agony, hallucinations or schizophrenic-like behavior, frank psychosis, and fi nally coma (see also Chap. B3.7). E. Mayatepek Especially dramatic are acute attacks of hyperammone- Department of General Pediatrics, University Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany mia, i.e., urea cycle disorders [e.g., ornithine transcar- e-mai: [email protected] bamylase (OTC) defi ciency and organic acidurias or

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 177 DOI: 10.1007/978-3-540-74723-9_C7, © Springer-Verlag Berlin Heidelberg 2010 178 E. Mayatepek

Table C7.1 Psychiatric symptoms suggestive of inherited Psychiatric manifestations are classically found in metabolic diseases acute intermittent porphyria (AIP) or hereditary Association with organic symptoms (organomegaly, coproporphyria. In AIP, a disorder of heme bio neurological features, etc.) synthesis leading to intermittent elevations in porpho- Cognitive decline Confusion bilinogen and related porphyrins, psychiatric mani- Visual hallucinations festations of acute attacks comprise anxiety, de Catatonia pression, psychosis, or altered mental status. AIP is Aggravation with therapy often mistaken for schizophrenia or hysteria. Before an initial attack there is also a high frequency of his- trionic personality traits in many patients with AIP. Table C7.2 Investigations for the differential diagnosis of An even higher incidence of anxiety disorder is psychiatric manifestations of inherited metabolic diseases observed in otherwise asymptomatic carriers. For Plasma/serum patients with AIP, it is essential to get the correct Ammonia diagnosis early because many psychotropic drugs Lactate may induce or exacerbate an acute attack of porphy- Amino acids ria. The metabolic crisis may induce or aggravate Homocysteine psychiatric symptoms, leading to a long-term psychi- Very long-chain fatty acids Ceruloplasmin atric career because of misinterpretation as treatment Copper resistance. Diagnosis of porphyria requires the dem- Sterols onstration of pathological metabolites in urine (e.g., Urine porphobilinogen). Amino acids Patients suffering from methylene tetrahydrofolate Organic acids reductase deﬁ ciency are sometimes initially (mis-) Mucopolysaccharides (electrophoresis) diagnosed as suffering from schizophrenia or psycho- Porphyrins (especially porphobilinogen) sis. In these patients, further symptoms often include Purines and pyrimidines Oligosaccharides strokes, peripheral neuropathy, and a progressive myel- N-Acetylneuraminic acid opathy. Similar psychiatric signs are also found in Copper (24-h collection) cobalamin metabolism defects (Cbl C and Cbl G). Histology including electron microscopy (skin biopsy, bone Besides symptoms like paraesthesias stroke events, marrow, lymphocytes) patients with Fabry disease are at higher risk of depres- Biogenic amines and pterins in cerebrospinal ﬂ uid sion and even suicide. Enzyme studies (lysosomal enzymes, respiratory chain enzymes) Mitochondrial DNA Brain MR spectroscopy C7.3 Chronic Psychiatric Manifestations

Psychiatric symptoms are predominantly recognized in maple syrup urine disease]. All these are in principle older children, adolescents, or adults suffering from neu- treatable disorders, especially the late-onset variants. rometabolic diseases. The decisive manifestations in these However, if the appropriate metabolic investigations disorders are mental deterioration and/or progressive neu- are not initiated, disease courses will become chronic rological manifestations (see Chap. C5). However, these leading to permanent handicap and early death. Topping may be less obvious than psychiatric symptoms for some the list of investigations in any patient presenting with time. Psychiatric features can be the only presenting acute encephalopathy including psychiatric presenta- symptom before any signifi cant neurological or extraneu- tion has to be ammonia. All too often acute psychosis rological signs are recognized. These manifestations after pregnancy or drunkenness were fatal misdiagnoses include behavior disturbances, changes of personality and in patients suffering from urea cycle disorders, espe- character, mental regression, dementia, psychosis, depres- cially hemizygous females with OTC defi ciency. sion, or schizophrenia. Such presentations are especially C7 Psychiatric Disease 179 common in X-linked adrenoleukodystrophy (cerebral In mucopolysaccharidosis type III (Sanfi lippo), form), Hallervorden–Spatz disease, Huntington chorea, major clinical manifestations include regression of urea cycle disorders, especially hemizygous OTC defi - high-level achievements as well as loss of speech. ciency, and Wilson disease. Affected children often show agitation, autism, and It is helpful to consider the relationship of psychiat- disintegrative behavior. Mental retardation with epi- ric manifestations to the age of the patient. sodes of psychosis, hyperactivity, or confusion may be seen in a- or b-mannosidosis.

C7.3.1 Infancy (1–12 months) C7.3.3 Childhood and Early Adolescence (5–15 years) In infancy, autistic traits may be a leading feature of metabolic disease. They have been observed in Progressive neurological and mental deterioration untreated phenylketonuria (PKU) and inborn errors of along with psychiatric manifestations can be found in biopterin metabolism. Autistic features may be also a number of neurometabolic diseases such as juvenile present in infants affected with late-onset subacute neuronal ceroid lipofuscinosis (SpielmeyerÐVogt). forms of disorders associated with hyperammonemia, Intellectual deterioration and depression develops in especially in urea cycle disorders, as well as in infants addition to the loss of sight and retinitis. In patients with succinic semialdehyde dehydrogenase defi - with this disease alteration in behavior may be the pre- ciency, SmithÐLemliÐOpitz syndrome, adenylosucci- senting complaint and may be seen years before other nase defi ciency, dihydropyrimidine dehydrogenase manifestations are evident. defi ciency, homocystinuria, nonketotic hyperglycine- Dementia and deterioration are found alongside mia, or sulfi te oxidase defi ciency. Children with the psychiatric manifestations in metabolic diseases with treatable pyrimidine nucleotide depletion disease due predominant cerebellar ataxia such as mitochondrial to cytosolic 5'-nucleotidase superactivity develop a disorders, e.g., MELAS, and in cerebrotendinous xan- pervasive developmental disorder. Patients with thomatosis, GM1 gangliosidosis, Gaucher disease, SmithÐLemliÐOpitz syndrome often present severe NiemannÐPick disease type C, the juvenile form of sleeping problems as well as excessive screaming in Krabbe, Lafora disease, and metachromatic leu- early childhood. kodystrophy (MLD). Psychiatric manifestations are most often present in the late juvenile and adult age group and are characterized by psychosis with disorga- nized thoughts, delusions, and auditory hallucinations. C7.3.2 Early Childhood (1–5 years) The subsequent rapid intellectual deterioration to dementia should be the clue to the suspicion of an underlying metabolic disease. Episodes of psychotic behavior, depression, and mania are typical psychiatric signs of the late infantile form Remember of GM gangliosidosis (TayÐSachs and Sandhoff). The 2 ¼ disease is characterized mainly by developmental In childhood psychotic behavior, depression, regression, spinocerebellar degeneration, ataxia, and and mania are typically found in GM2 ganglio- spastic fright reaction. In this age group, it is important sidosis. to be aware of other diseases with arrest or regression of cognitive function, most importantly Rett syndrome. In Rett syndrome, girls are affected and they present Severe behavioral problems in combination with mental with characteristic behavior, regression of develop- retardation may be seen in creatine transporter defi - mental achievements, and sterotyped movements of ciency, in monoamine oxidase A defi ciency, as well as fi ngers and hands. in maternal PKU. 180 E. Mayatepek

C7.3.4 Late Adolescence and Take Home Messages Adulthood (>15 years) › Many neurometabolic diseases manifest symptoms compatible with various psychiatric diag- In late adolescence and adulthood, progressive neurologic noses. Psychiatric features may be a leading and mental deterioration along with preponderant psy- clinical correlate of certain metabolic diseases as chosis and dementia may be found in a variety of meta- well as an important sequel in long-term care. bolic diseases discussed in the preceding section including With improvements in treatment and prognosis, urea cycle disorders, MLD, NiemannÐPick disease type C, manifestations that affect long-term quality of ceroid lipofuscinosis, cerebrotendinous xanthomatosis, life such as psychiatric problems become increas- Huntington chorea, and Lafora disease. In adult, onset ingly important in an older patient population. MLD psychotic changes may be those of schizophrenia. › Recognition of metabolic diseases in the dif- This is also true of Nieman–Pick type C disease. Paralysis ferential diagnosis of psychiatric manifesta- of upward gaze is the characteristic give away sign, but it tions is important since some metabolic has been a missed in psychiatric evaluations. diseases may be mistaken for diagnoses such In Wilson disease, toxic tissue levels of copper cause as schizophrenia. In AIP, psychiatric symp- damage primarily the liver and basal ganglia (see also toms may be at the forefront of clinical mani- Chap. C2). Psychiatric manifestations may vary and festations. Careful examination of mental and include personality changes, depressive episodes, cog- neurological status, psychiatric history, and nitive dysfunction, and psychosis. The overall preva- recognition of psychotropic drugs or treatment lence of psychiatric symptoms in Wilson disease is regimens potentially exacerbating metabolic >20%. In total, 10% of the patients present with psy- crises, e.g., in porphyrias or in Wilson disease, chiatric symptoms alone. Effective chelation treatment are mandatory. does improve the majority of psychiatric symptoms. However, initiation of chelation treatment in Wilson disease may precipitate an acute psychiatric crisis. Psychiatric abnormalities have been reported in patients with homocystinuria due to cystathionine b-synthase (CBS) defi ciency. Major diagnostic categories in patients with CBS defi ciency include, in order Key References of, decreasing incidence, personality disorders, chronic disorders of behavior, episodic depression, and chronic Abbott MH, Folstein SE, Abbey H et al (1987) Psychiatric obsessive-compulsive disorder. Schizophrenia or psy- manifestations of homocystinuria due to cystathionine chotic episodes are uncommon in CBS-defi cient beta-synthase defi ciency: prevalence, natural history, and patients. relationship to neurologic impairment and vitamin B6-responsiveness. Am J Med Genet 26:959–969 PKU in late treated or untreated patients results Argov Z, Navon R (1984) Clinical and genetic variations in the not only in mental retardation but also varying syndrome of adult GM2 gangliosidosis resulting from hexo- degrees of psychiatric pathology especially (auto-) saminidase A defi ciency. Ann Neurol 16:14–20 aggression. Some adult patients with early treated Crimlisk HL (1997) The little imitator-porphyria: a neuropsychi- atric disorder. J Neurol Neurosurg Psychiatry 62:319–328 PKU have exhibited an atypical pattern of psychopa- Dening TR, Berrios GE (1989) Wilson’s disease. Psychiatric thology with a variety of symptoms of anxiety and symptoms in 195 cases. Arch Gen Psychiatry 46:1126–1134 depression. PKU patients no longer observing dietary Estrov Y, Scaglia F, Bodamer OAF (2000) Psychiatric symptoms restriction have an increased risk of psychosocial of inherited metabolic diseases. J Inherit Metab Dis 23:2–6 Grewal RP (1993) Psychiatric disorders in patients with Fabry’s diffi culties; agoraphobia has been recognized as a disease. Int J Psychiatry Med 23:307–312 common symptom. It is also possible that psychiatric Rauschka H, Colsch B, Baumann N et al (2006) Late-onset disease, being common, simply coexists in some of metachromatic leukodystrophy: genotype strongly infl u- these patients. Interestingly, psychiatric symptoms ences phenotype. Neurology 67:859–863 Sedel F, Lyon-Caen O, Saudubray JM (2007) Inborn errors of observed in PKU are not clearly related to phenylala- metabolism in adult neurology – a clinical approach focused nine levels. on treatable diseases. Nat Clin Pract Neurol 3:279–290 Eye Disorders C8 Alberto Burlina and Alessandro P. Burlina

Key Facts C8.1 General Remarks › Eye involvement represents important and specifi c processes in the development of inher- The eye is the most highly specialized of the sensor ited metabolic disease. organs and is often implicated in metabolic disorders › Ocular manifestations are common in inher- that affect the senses. Moreover, the eye has important ited metabolic diseases. They result in charac- physiological links to the central nervous system and, teristic signs, which are very useful in the thus, tends to be involved in diseases affecting the cen- diagnosis of the metabolic defect. tral nervous system. Because the eye is such a complex organ, one or more of its structural or functional com- › Ophthalmological manifestations can occur ponents may be affected by a single disease. early in life with bilateral symmetrical involve- Ophthalmological manifestations occur in various ment. Monitoring of eye disease progression is metabolic disorders. In some instances, the occurrence necessary in many inherited metabolic dis- of eye abnormalities suggests that it might be induced eases (i.e., for cataracts in galac tosemia). by direct toxic mechanisms of abnormal metabolic Specifi c localized therapies are available (mainly › products or accumulation of normal metabolites; by surgical approaches) and, as for other organs, defects of synthetic pathways; or by defi cient energy involved in inherited metabolic diseases, the metabolism. In other diseases, it remains to be eluci- main treatment consists of the primary therapy dated how the systemic metabolic abnormalities con- of the metabolic defect. tribute to ocular defects. Two situations may arise in clinical practice. Either the patient presents with a known metabolic disorder, and the ocular defect appears to be as expected manifestation of the disease, or the patient presents primarily with eye abnormalities and a metabolic disorder can be suspected. For many metabolic diseases, symmetrical bilateral involvement is the rule. Severe visual impairment from birth is often not recognized until around 2 months of age, when normal sighted children have developed eye contact. Severe poor vision needs to be detected within the fi rst weeks of life. In some conditions, anomalies of the eye can be more easily detected, such as cataracts in galactosemia. A. Burlina ( ) In other conditions, such as in some peroxisomal Department of Pediatrics, Division of Metabolic Disorders, University Hospital, Via Giustiniani 3, 35128 Padova, Italy diseases, fundoscopic examination may still be nor- e-mail: [email protected] mal in the neonatal period, whereas recordings of

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 181 DOI: 10.1007/978-3-540-74723-9_C8, © Springer-Verlag Berlin Heidelberg 2010 182 A. Burlina and A. P. Burlina electroretinogram and visual evoked responses are are relatively easy to detect using simple instruments already abnormal. such as a hand light or the ophthalmoscope. More sub- In inborn errors of metabolism, we can detect the tle changes can be seen by slit-lamp examination. following eye pathology: ¥ Corneal clouding ¥ Lens defects and dislocations ¥ Retinal degeneration Remember ¥ Optic atrophy Corneal clouding is relatively easy to detect using instruments such as a hand light or the ophthalmoscope. C8.2 Corneal Clouding

The three components of corneal tissue are epithelium, Inherited metabolic diseases that affect the cornea are stroma and Descemet’s membrane. Corneal transpar- numerous and severe (Table C8.1). ency depends, in part, on stromal constituents, i.e., collagen fi brils and proteoglycans. The composition of proteoglycans in the cornea is involved in the organization of the collagen fi brils including fi brillar ultra- Remember structure, fi bril packing, organization, stability of the corneal lamellae, fi bril size, as well as corneal hydra- The most frequently inherited metabolic diseases tion. About 80% of the stromal dry weight is collagen, which present corneal clouding are lysosomal dis- primarily type I, together with others such as type V orders (including AndersonÐFabry disease), lipid and VI collagens. The collagen fi brils are arranged in disorders, and tyrosinemia type II. lamellae with adjacent lamellae arranged at right angles, forming an orthogonal grid. Corneal clarity is maintained by a crystalline array of stromal fi bers and Table C8.1 Corneal clouding multiple translucent endothelial layers. The three com- Lysosomal disorders ponents of corneal tissue (epithelium, stroma, and Mucopolysaccharidoses: types I, II, IV, VI, VII (severe Descemet’s membrane) may be involved separately or form) simultaneously, depending on the disease. Mannosidosis Sialidosis (severe infantile) Galactosialidosis Fabry’s disease Mucolipidosis I, II, IV Fucosidosis type III Remember Multiple sulfatase defi ciency Farber’s disease The cornea is optically sensitive to abnormal stor- Cystinosis age products, which may accumulate as a result of a Lipid disorders systemic disorder. Homozygous familial hypercholesterolemia Lecithin:cholesterol acyltransferase defi ciency (Tangier disease) Fish-eye disease Amino acid disorders If the abnormal substrate is produced by the corneal Tyrosinemia type II tissue, it may be found throughout the cornea. If it is Alkaptonuria found in elevated amounts in the blood, it is more com- Metal disorder monly accumulated in the corneal periphery. Lesions Wilson’s disease C8 Eye Disorders 183

C8.2.1 Lysosomal Disorders and subsequently optic atrophy, pigmentary retinopathy have been reported in patients, often masked by corneal problems. Disease Info: Mucopolysaccharidoses In the past, the ocular management of many In the mucopolysaccharidoses (MPS), some clinical patients with MPS has been conservative due to manifestations such as coarse facial features, thick- their short life span and intellectual impairment. ened skin, organomegaly, and eye lesions result Now, new treatments such as bone marrow trans- from the reduction in activity of specifi c lysosomal plantation and enzyme replacement therapy are enzymes involved in the breakdown of glycosamin- leading to a longer and better-quality life for many oglycans (GAG). MPS patients, and these treatments appear to be Ocular pathology is common in all types of MPS benefi cial in reducing, but not eliminating, the ocu- and results in frequent impairment of vision. The lar manifestations. ocular complications include corneal opacifi cation, retinopathy and raised intraocular pressure. Ocular hypertension and glaucoma are often diffi cult to Disease Info: Anderson–Fabry disease diagnose and monitor in patients with MPS because AndersonÐFabry disease, commonly named Fabry’s of coexistent corneal opacifi cation and thickening, disease, is an X-linked lysosomal disease that results and the physical and intellectual problems of some from a defi cient activity of the hydrolase a-galactosi- individuals. dase. It is associated with severe multiorgan dysfunc- Progressive corneal opacifi cation (characteristi- tion. The subtle eye manifestations of Fabry’s disease cally described as having the appearance of ground are visually insignifi cant to the patient. However, they glass) affects all patients with MPS types I, IV, and may prove to be useful in the diagnosis, in under- VI. Corneal opacifi cation is mild in MPS IS (Scheie standing the natural history of the disease and in disease) and MPS II (Hunter syndrome), and rarely assessing the response to enzyme replacement ther- requires corneal transplantation, and is not a promi- apy. In Fabry’s disease, ophthalmological abnormali- nent feature of MPS III (Sanfi lipo syndrome). ties occur mostly at the level of the conjunctival and Progressive corneal opacifi cation is also seen in retinal vessels, the cornea and the lens (Fig. C8.1). MPS VII (Sly syndrome). The lesion is due to der- They result from a progressive deposition of gly- matan sulfate deposition in the cornea that is not cosphingolipids in ocular structures. present in normal cornea. Corneal opacifi cation in A vortex keratopathy (“cornea verticillata”) repre- MPS IV (Morquio disease) is due to keratin sulfate sents the ocular manifestation most commonly deposition. reported in Fabry’s disease. They can be the only ocu- Glycosaminoglycan deposition in the corneal lar sign present in Fabry patients. The prevalence of stroma has been suggested by some authors to cause cornea verticillata is similar in different age groups. It progressive increase in corneal thickness. can be detected in 70% of Fabry patients. Cornea ver- In animals with systemic MPS, corneal clouding ticillata is well recognized only by slit-lamp micros- results from storage of GAG in stromal keratocytes. copy. The earliest lesion is a diffuse haziness in the The corneal epithelium is normal (MPS VI and VII) subepithelial layer that later appears as fi ne, straight or minimally affected (MPS I), and stromal edema or curved lines radiating from the periphery toward is not a feature even though the corneal endothe- the center of the cornea in a characteristic pattern. lium demonstrated variable pathology. The corneal They usually do not affect vision. Indistinguishable clouding is the result of storage in stromal kerato- drug-induced phenocopies of Fabry cornea verticil- cytes rather than corneal edema from endothelial lata have been reported in patients on long-term chlo- dysfunction. roquine or amiadarone therapy. Other ocular manifestations are also common in A small number of patients exhibit a characteristic MPS. Glaucoma, cataracts, optic nerve swelling lens opacity with a “spoke-like” pattern usually referred 184 A. Burlina and A. P. Burlina

Other lysosomal defects may lead to corneal manifes- to as “Fabry cataract.” It consists of two types, anterior tations as the earliest indication of a metabolic disease. and posterior subcapsular cataracts. The former, which In mucolipidosis type IV, corneal clouding with major only occurs in hemizygous males, appears as granular, visual impairment is a prominent feature. Corneal radially arranged wedges. The latter has the appear- clouding is one of the early symptoms; later, retinal ance of nearly translucent spoke-like or dendritic pro- degeneration and blindness may develop. Cytoplasmic jections; they may also occur in heterozygous female membranous bodies are found in diverse tissues, carriers. These signs can be detected by basic slit-lamp including the conjunctiva, fi broblasts, liver, and spleen. examination, a procedure that is noninvasive, inexpen- Affected patients are usually mentally retarded. The sive, and not time consuming. late-onset form of a-mannosidosis also involves cor- Conjunctival and retinal vascular lesions are also neal clouding, in addition to cataracts, changes in bone common and represent part of the diffuse systemic and hearing loss. In Farber’s disease, the severe form, vascular involvement. Conjunctival and retinal ves- presents with eye changes including a cherry-red spot, sels are tortuous and may exhibit aneurysmal dila- a paint gray ring around the cornea, modular corneal tations. Vessel tortuosity shows an association with opacity and a pingueculum-like conjunctival lesion. both cornea verticillata and Fabry cataract; hence, In the juvenile/adult type of galactosialidosis, cor- the isolated presence of tortuous vessels (especially neal clouding with loss of visual acuity develops in the in the fundus), without any corneal or lens involve- second decade of life. Additional ophtalmologic abnor- ment, is not diagnostic of Fabry’s disease. Till now, malities include bilateral cherry-red spots, punctate there are no studies in the literature that report any lens opacities and color blindness. Other clinical symp- changes in cornea verticillata or conjuctival and toms include coarse face, growth disturbance, cardiac retinal vessels appearance after enzymatic replace- involvement, hernias, angiokeratomata, hearing loss, ment therapy. joint stiffness, vertebral changes and a progressive neurologic course with mental retardation, seizures, myoclonus and ataxia. In steroid sulfatase defi ciency, a corneal opacities are small punctate or fi liform lesions located in the deep corneal stroma.

Disease Info: Cystinosis The disease is a systemic metabolic disorder affecting the conjunctiva, cornea, iris, choroid and retinal pigment epithelium, as well as the kidney and other organs. It results from the accumulation of cystine within lysosomes. The disease is caused by mutations b in the CTNS gene coding for cystonin, a lysosomal carrier protein. In the cornea, the crystals are located in the anterior stroma; they are iridescent and poly- chromatic, presenting fi rst in the periphery and extend- ing centrally. The corneal changes and associated photophobia are due to the anterior location of the crystal deposition. They may be present before nephropathy is severe; thus, they can be the fi rst sign of the disease. The anterior location of the crystals can predispose the patient to recurrent erosions. Photophobia, watering and blepharospasm may become disabling; Fig. C8.1 Eye abnormalities associated with Fabry’s disease: these symptoms are often related to erosions of the (a) cornea verticillata (vortex opacities located in the superfi cial corneal epithelium, leading eventually to keratopathy. corneal layers); (b) conjunctival vessel tortuosity C8 Eye Disorders 185

Sight may be progressively reduced, leading to blind- diagnosed and treated, ocular damage including ness in a severe form of the disease. Cataract and pig- corneal opacities, visual impairment, corneal plana, mentary retinopathy also develop. nystagmus, amblyopia and glaucoma are frequent Cysteamine eye drops are effective in reducing complications. the deposits of crystals in the cornea and in improv- Treatment consists of a phenylalanine and tyrosine ing the extreme photophobia. Corneal transplanta- restricted diet. There is no consensus as to the opti- tion may be indicated for visual rehabilitation, as mal blood level of tyrosine, but a level <500 mM is a well as for recurrent erosions. reasonable goal. The eye symptoms resolve within a short period of treatment (few weeks). Treatment with systemic steroids should be avoided because C8.2.2 Lipid Metabolism Disorders the disease can worsen with such therapy.

Some disorders of high-density lipoproteins (HDL) metabolism, including Tangier disease, fi sh-eye dis- Alkaptonuria is a rare autosomal recessive condition ease, lecithin cholesterol acyltransferase defi ciency and caused by defi cient homogentisic acid oxidase. apoprotein A-1 (Apo A-I) defi ciency must be included Homogentisic acid is excreted in excess in the urine, in the differential diagnosis of corneal opacity. The but its oxidized pigment derivatives (alkapton) also central corneal changes are gray dots occupying the bind collagen, leading to pigment accumulation in full thickness of the stroma, mainly centrally, with connective tissue of the nose, sclera and ear lobes. peripheral condensation to form the arcus-like changes. Affected patients develop a degenerative arthropathy. Occasionally an arcus-like structure develops due to Ocular changes occur in 70% of patients. Just inside the deposition of a variety of phospholipids, low-den- the limbus, the cornea develops a black “oil-droplet” sity lipoproteins and triglycerides in the stroma of the pigmentation that appears similar to spheroidal degen- peripheral cornea. Patients with these diseases have eration. Pigmentation gradually increases throughout hypoalphalipoproteinemia with low HDL, low Apo A-I adulthood. and elevated triglycerides. Those with Tangier disease Wilson’s disease may be diagnosed by character- have striking large yellow tonsils or pharyngeal plaques. istic ocular fi ndings. It is an autosomal recessive dis- They also have peripheral neuropathy manifested by order that causes deposition of copper in the liver, weakness, paresthesias, autonomic dysregulation and corneas, kidneys and nervous system. The KayserÐ ptosis. In addition, abnormal rectal mucosa, anemia, Fleischer ring is the single most important diagnos- renal failure, and hepatosplenomegaly may occur. tic fi nding of the disease. When fully developed, it is Corneal clouding is the only clinical manifestation in seen with the naked eye, but subtle changes can be patients with fi sh-eye disease. seen by slit-lamp examination (see Fig. 2 in Chap. C2). It consists of brownishÐgreenish deposit of copper in the Descemet’s membrane just within the limbus of the cornea; it can be especially prominent at C8.2.3 Amino Acid Disorders the upper pole, therefore it requires lifting of the eyelid for recognition. It is present in 60% of children at the stage of acute or subacute liver disease. It Disease Info: Tyrosinemia Type II may be present in asymptomatic affected individual as well. Tyrosinemia type II (oculocutaneous tyrosinemia, The ring is not absolutely pathognomonic of RichnerÐHanhart syndrome) is due to a defect in Wilson disease, because other causes of liver failure cytosolic tyrosine aminotransferase. Photophobia, such as carotenemia and multiple myeloma may lead redness, watering eyes, and pain are often the pre- to similar rings. In Wilson’s disease, another rare but senting symptoms. At slit-lamp examination, cen- characteristic abnormality is the “sunfl ower” subcap- tral dendritic corneal erosions that stain poorly with sular cataract. The rings improve with effective fl uorescein are present. The lesions are bilateral in decoppering therapy or after liver transplantation, as contrast to herpetic ulcers that are unilateral. If not does cataract. 186 A. Burlina and A. P. Burlina

Table C8.2 Cataracts according to age of presentation Remember Newborn period The cornea verticillata and the KayserÐFleischer Galactosemia (all defects) ring are important diagnostic ocular fi ndings for Sorbitol dehydrogenase defi ciency Fabry’s disease and Wilson’s disease, respectively. Zellweger syndrome Rhizomelic chondrodysplasia puntata Lowe’s syndrome Childhood C8.3 Lens Defects and Dislocations Carbohydrate disorders Galactosemias Sorbitol dehydrogenase defi ciency C8.3.1 Cataracts Aldose reductase defi ciency Lysosomal disorders Oligosaccharidoses: a-mannosidosis; sialidosis; Cataract is defi ned as any opacity within the lens. galactosialidosis Cataracts and dislocation of the lens are anomalies Fabry’s disease; neuronal ceroid lipofuscinosis (juvenile of the lens detectable in many inherited metabolic form) defects. At birth lens opacities, if not diagnosed Amino acid disorders or removed, are a major cause of blindness or ambly- Delta1-Pyrroline-5-carboxylate synthase defi ciency opia. Hyperornithinemia (ornithine aminotransferase defi ciency) Lysinuric protein intolerance Lowe’s syndrome Lipid disorders Remember Sjögren–Larsson syndrome Neutral lipid storage disorder Early cataract investigation: look for the orange or Cholesterol metabolism defects red retinal refl ex after a dilute dilating eye drop at Cerebrotendinous xanthomatosis (cholestanol lipidosis) the fi rst pediatric examination after birth with an Mevalonate kinase defi ciency (severe form) ophtalmoscope. ConradiÐHunermann syndrome SmithÐLemliÐOpitz syndrome Peroxisomal disorders Peroxisome biogenesis defects Often, the retinal refl ex can be visualized with an oph- Rhizomelic chondrodysplasia punctata talmoscope alone. When cataracts are bilateral, they Mitochondrial oxidative phosphorylation disorders lead to irreversible nystagmus and amblyopia by 3 Senger disease Senger-like disease months of age. Thus, these cataracts must be quickly b-Methylglutaconic aciduria removed by surgery, preferably within the fi rst few mitochondrial DNA mutations days or weeks of life. Copper disordsers Wilson’s disease Menkes disease

Remember (galactose and polyol pathways), peroxisomal disor- The etiologic classifi cation of congenital cataract is ders (peroxisomal biogenesis) and Lowe syndrome. diverse and more then 90% remain unexplained. Cataracts develop later in life in lysosomal disorders The most common etiologies include infections, (sialidosis, a-mannosidosis and Fabry’s disease), metabolic disorders, and genetic syndromes. Wilson’s disease, Menkes disease, lipid disorders, some amino acid defects, and some mitochondrial disorders. Rarely, cataracts have been reported in metach- A number of inherited metabolic diseases presents with romatic leukodystrophy, hypobetalipoproteinemia, cataracts (Table C8.2). It is a constant and early fi nding vitamin E or D defi ciencies and lactose intolerance. in neonates in defects of carbohydrate metabolism Hypoglycemic episodes of various origins during the C8 Eye Disorders 187 perinatal period or in early infancy may result in lens opacities.

Remember Cataracts are an early ﬁ nding in defects of carbohydrate metabolism (galactose and polyol pathways), peroxisomal biogenesis, cholesterol biosynthesis and amino acid transport.

C8.3.2 Carbohydrate Metabolism

Disease Info: Galactosemia The lens is avascular by nature, receiving its nutrients from the aqueous humor. Most of the glucose metabolized in the lens is handled by anaerobic glycolysis. The citric acid pathway produces about 20Ð30% of the total ATP in the lens, even though only about 3% of the glucose passes through the citric acid cycle. Three defects in galactose metabolism are known: galactose-1-phosphate uridyltransferase (GALT), galactose1-phosphate epimerase and galactokinase. At an early stage, “oil droplet” cataracts which are not true cataracts but refracture changes in the lens nucleus are present (Fig. C8.2). The lesion appears in retroillumination as a drop in the center of Fig. C8.2 Galactosemia: oil droplet cataract in untreated patient the lens like an oil droplet fl oating in the water. The visible by slit-lamp examination accumulation in the lens of galactitol, a metabolite of galactose, creates a shift of water into the lens, due to the lack of permeability of galactitol, with ultimate Remember disruption of the lenticular structure. In GALT defi - In all type of galactosemia, cataracts are usually ciency, major manifestations include liver failure, reversible after the start of a galactose-free diet. jaundice, and tubulopathy. Demonstration of GALT defi ciency establishes the diagnosis. This can be achieved using the fl uorescent spot test used in neo- The polyol pathway consists of two enzymes: aldose natal screening (Beutler test) followed by quantita- reductase and sorbitol dehydrogenase. Aldose reductase tive tests for confi rmation (enzyme activity in reduces hexose sugars such as glucose and galactose to erythrocytes) and by mutation analysis. In epimerase their respective polyols, sorbitol and galactitol. Polyol defi ciency, patients may have a symptomatology accumulation has been demonstrated to cause cataracts resembling transferase defi ciency; enzyme activity is owing to increases in intracellular fl uid resulting in lens measured in erythrocytes and leukocytes. In galac- swelling, increased membrane permeability, and elec- tokinase defi ciency, cataracts may not be recognized trolyte abnormalities. In sorbitol dehydrogenase defi - until later because no other symptoms develop. The ciency, cataracts present at birth as the only sign. Patients enzyme is measured in red blood cells. In all disor- with glucose-6-phosphate dehydrogenase defi ciency, ders, cataracts are usually reversible after the intro- who usually come to the clinician’s attention because of duction of a galactose-free diet. hemolytic anemia, may also develop cataracts. 188 A. Burlina and A. P. Burlina

C8.3.3 Lipid Metabolism Disorders Cataracts are also present in the following forms of spastic paraplegias: SPG9 (autosomal dominant form), SPG21 and SPG25, both autosomal recessive The lens membrane contains the highest cholesterol disorders. content of any known membrane, indicating the importance of normal cholesterol metabolism in lens maintenance. Disorders of cholesterol biosynthesis (mevalonate kinase defi ciency, Conradi–Hunermann syndrome and C8.3.5 Amino Acid Disorders Smith–Lemli–Opitz syndrome) present with a large and variable spectrum of morphogenic and congenital In Lowe’s oculo-cerebro-renal syndrome, cataracts anomalies. Cataracts are present early in the very severe are a constant and hallmark fi nding. The lesion is forms but due to the clinical and biochemical continuum already present prenatally as early as in the 24th week of these defects may not develop in milder forms. In of gestation. Additional features are kidney pathology cerebrotendinous xanthomatosis bilateral, irregular, (Fanconi syndrome) and severe neurological impair- corticonuclear and anterior polar or posterior capsular ments including muscular hypotonia, arefl exia and cataracts can occur in the fi rst decade. It may be associ- mental retardation. ated with opacities of the crystalline lens. The lesion is In other aminoacidopathies, including ornithine associated with xanthomata and a progressive neuro- aminotransferase (OAT) defi ciency (gyrate atrophy of logical syndrome of ataxia, pyramidal signs, cognitive the choroid and retina), delta1-pyrroline-5-synthase impairment, epilepsy and peripheral neuropathy. defi ciency and lysinuric protein intolerance, cataracts Patients with the Sjögren–Larsson syndrome have can be a presenting sign. cataracts and it may also be present in another syndrome characterized by ichthyosis, the so-called neutral lipid storage disorder. This syndrome, whose etiology is still unknown, includes ataxia, myopathy, and hepatomegaly C8.4 Lens Dislocations (Ectopia Lentis) as further clinical features. Vacuolated lymphocytes are a frequent fi nding in the peripheral blood.

Remember In the diagnostic work-up for patients with sublux- C8.3.4 Peroxisomal Disorders ation of the ocular lens, total plasma homocysteine must always be measured. Congenital cataracts in association with craniofacial dysmorphic features, hepatomegaly and renal cysts are frequently present in disorders of peroxisome biogen- Dislocations of the ocular lens (ectopia lentis) are esis. To this group belong Zellweger syndrome and two frequent, severe and characteristic sequels of both allied conditions: neonatal adrenoleukodystrophy and homocystinuria and Marfan syndrome. The probable infantile Refsum disease. Other ocular abnormalities mechanism for lenticular dislocation in Marfan syn- include pigmentary degeneration of the retina, corneal drome is microfi bril abnormalities of the lens cap- opacities and glaucoma. Differences between these sule. The lens subluxation in homocystinuria most syndromes relate to the severity of the neurological commonly occurs downwards, whereas in Marfan abnormalities and modifi cations of clinical and patho- syndrome the lens usually subluxes upwards, logical features. Measurement of plasma very long- although, it can occur in any direction in both the chain fatty acids allows the diagnosis. Chondrodys- diseases. Marfan syndrome is due to alteration in plasia punctata and rhizomelic dwarfi sm combined microfi brils caused most commonly by mutations of with congenital cataracts lead to the diagnosis of chon- the fi brillin-1 gene. The involvement of the ocular drodysplasia punctata. Normal very long-chain fatty system includes a fl at cornea, an increased axial acids and low plasmalogens levels in tissues and red length of the globe with hypoplastic iris, or hyp- blood cells are the diagnostic abnormalities. oplastic ciliary muscle causing decreased miosis. C8 Eye Disorders 189

Table C8.3 Ectopia lentis (dislocation of the lens) Disease Info: Homocystinuria Homocystinuria In patients with homocystinuria, subluxation of Marfan syndrome ocular lens is very characteristic and frequent ocu- Molybdenum cofactor defi ciency lar feature, estimated to occur in over 90% of Sulfi te oxidase defi ciency WeillÐMarchesani syndrome patients (Fig. C8.3). The presence of the detached lens is often heralded by the clinical recognition of iridodonesis. It seldom occurs before 3 years of age and is usually present by 10 years of age. Prior Table C8.3 lists metabolic disorders in which ectopia patients usually develop rapidly worsening myopia, lentis occurs. as well as astigmatism and sometimes glaucoma. Staphyloma may result from increased ocular pressure. Cataracts which can be congenital may occur in the lens. All these manifestations may be secondary to the ectopia lentis. The patient may also have C8.5 Degeneration of the Retina retinal detachment and optic atrophy may follow central retinal artery occlusion. Among the disorders that might be expected to affect Although the body habitus of patients with the pigmented epithelium of the retina are diseases homocystinuria resembles that of Marfan syndrome, caused by storage of specifi c components or diseases the etiology is different. Increased levels of plasma in which the normal ability to synthesize pigment is and urine homocysteine are found in all patients. absent. Included are disorders characterized by the Hypermethioninemia is an important fi nding. The presence of retinal pigmentation and a progressive red- diagnosis is confi rmed by assays of the enzyme cys- corneal dystrophy as a prominent feature of retinal tathionine b-synthetase in cultured fi broblasts, lyn- degeneration. phoblasts, or mutation analysis. Prevention of the lens dislocation is possible in patients detected through neonatal screening and early treated with low methionine formulas or pyridoxine in the responsive disease variants. C8.5.1 Retinitis Pigmentosa (RP) In homocystinuria, due to 5,10-methylentetrahy- drofolate reductase defi ciency, ectopia lentis has RP is a clinically and genetically heterogeneous group never been reported. of hereditary disorders in which a progressive loss of photoreceptor and pigment epithelial function occurs. Diagnostic criteria include bilateral involvement, loss of peripheral vision, rod dysfunction and progressive loss of photoreception function. The history should include information regarding the nature of the earliest symptoms, the age at onset, and progression. The age at onset of RP varies but often begins in early childhood or infancy.

Remember RP is characterized by progressive loss of photoreceptor function causing visual problems such as defective adaptation to the dark, or night blindness, Fig. C8.3 The lens dislocation in homocystinuria is usually visual ﬁ eld decrease, and reduction of visual activity. downward, while in Marfan syndrome it is upward 190 A. Burlina and A. P. Burlina

In adults, careful questioning often elicits a history atrophy of the choroid and retina caused by OAT starting in childhood or adolescence when patients are defi ciency. asked to recall diffi culties with outdoor activities at dusk or with indoor activities at night in minimal light- Disease Info: OAT Defi ciency ing. Patients rarely note a loss in peripheral vision as an early symptom, although they may be considered Patients come to the attention of the ophtalmologist clumsy before constricted visual fi elds are detected. in late childhood or puberty for evaluation of increas- Other symptoms include pendular eye movements and ing myopia or decreased night vision. At this age, nystagmus. Patients who present with initial symptoms sharply demarcated, circular areas of chorioretinal of photophobia, sensations of fl ashing lights, abnormal degeneration are present in midperiphery of the central vision, abnormal color vision or marked asym- ocular fundus (Fig. C8.4). In a few years, the retinal metry in ocular involvement may not have RP, but degeneration accelerates, the lesions enlarge, rather, another retinal disease. coalesce and extend toward the posterior pole of the fundus. Frequently, a posterior subcapsular cataract develops in the second decade. By the third decade, Remember much of the fundus is involved and increased pig- The diagnosis of RP is typically made by seeing a mentation is common in the macula area, while the pigmentary retinopathy on fundus examination. An optic disc remains pink and does not become atro- electroretinogram is an important confi rmatory test phic. Visual acuity decreases gradually and visual fi elds are progressively and concentrically reduced. OAT defi ciency results in a 10Ð20-fold increase in ornithine in body fl uids including aqueous humor The ocular examination should include measurements and a lesion in photoreceptors rather than the retinal of the patient’s best-corrected visual acuity, refraction, pigment epithelium. Therapeutic approaches include examination of the anterior segment, and measurement stimulation of residual OAT activity with pharmaco- of intraocular pressure. Attention should be given to the logical doses of pyridoxine, dietary reduction of lens, vitreous, optic disc, retinal vessels, macula and ornithine through arginine reduction, increasing renal retinal periphery. The earliest ophtalmoscopic fi ndings losses by administration of pharmacological doses of are a dull retinal refl ex and a thread-like aspect of the lysine and/or administration of creatine and proline. retinal arteries. The electroretinogram is abnormal Because of slow disease progression, the evaluation before gross fundoscopic evidence of RP is found. of therapeutical approaches are diffi cult. In hyperornithinemiaÐhyperammonemiaÐhomo- citrullinuria (HHH syndrome), the retina is not Remember affected. The most frequent RP associated with metabolic defects are Sjögren–Larsson syndrome, neuronal Secondary RP is most often associated with neurologi- ceroid lipofuscinoses, abetalipoproteinemia, cal disease, dysmorphic features, myopathy, nephropa- 3-hydroxyacyl-CoA-dehydrogenase defi ciency, thy, deafness and skin abnormalities. Some of these Refsum disease, KearnsÐSayre syndrome and conditions are well-defi ned inherited metabolic dis- OAT defi ciency. eases (Table C8.4). The neuronal ceroid lipofuscinoses (CLNs) are a group of progressive encephalopathies, which are char- The pigmented retinopathies can be divided into two acterized by neural and extraneural accumulation of groups: primary RP, in which the disease process is ceroid and lipofuscin storage material (see also Chap. confi ned to the eyes and secondary RP, in which reti- C5). Ceroid lipofuscinoses are among the most com- nal degeneration is associated with involvement of mon neurodegenerative disorders. Three main types additional organs. The fi rst group includes gyrate that cause RP have been distinguished on clinical, C8 Eye Disorders 191

Different disorders of lipid metabolism present with RP. Abetalipoproteinemia is caused by the absence of apoprotein B and the malabsorption of fat and fat- soluble vitamins, especially vitamins A and E. The most common clinical manifestations are diarrhea and failure to thrive from early infancy. Other manifestations involve the nervous system, with signs of peripheral neuropathy, spinocerebellar ataxia and muscle weakness. Although the retinal dystrophy may occur at early age, it most commonly presents in late childhood. Fundus examination may be normal in the early stages, later there may be peripheral pigmentary retinopathy or a picture similar to retinitis punctata albescens with scattered white dots Fig. C8.4 Gyrate atrophy of choroid and retina in ornithine ami- at the level of the retinal pigment epithelium. The elec- notransferase defi ciency: the mid- and far-perphery show the typical atrophic areas in peripheral retina troretinogram may be normal initially but later becomes abnormal with scotopic responses fi rst to be lost. The retinal and neurologic complications may be prevented neurophysiologic and genetic criteria: the infantile or stabilized by early supplementation with vitamin E. SantavuoriÐHaltia disease (CLN1), with onset between Disorder of fatty acid oxidation, mostly 3-hydroxyacyl- 6 and 18 months of age, regression of psychomotor C-A-dehydrogenase defi ciency, present with a general- development, myoclonic epilepsy, visual failure, micro- ized red-cone dystrophy. cephaly and “vanishing electroencephalogram (EEG)”; the late infantile JanskyÐBielschowski disease (CLN2), with onset between 2 and 4 years of age, epilepsy, Disease Info: Sjögren–Larsson Syndrome regression of mental skills, ataxia, myoclonic jerks, pathologic EEG, electroretinogram and visual evoked Sjögren–Larsson syndrome, caused by a defi - potentials (VEP); and the juvenile SpielmeyerÐVogtÐ ciency of fatty aldehyde dehydrogenase, is charac- Batten disease (CLN3), with onset of visual impairment terized by a triad of intellectual disability, spastic between the ages of 4 and 10 years, psychological dis- diplegia or tetraplegia, and congenital ichthyosis turbances, motor dysfunction and epilepsy, retinal with associated ocular features, which include degeneration and vacuolated lymphocytes. pigmentary changes in the retina. Ocular features are characterized by a crystalline maculopathy and bilateral, glistening yellowÐwhite dots involving Table C8.4 Secondary retinitis pigmentosa (RP) the foveal and parafoveal area from the age of 1Ð2 Lysosomal disorders years. The number of dots may increase with age, Neuronal ceroid lipofuscinoses although the extent of macular involvement does Mucopolysaccharidoses: all except Morquio disease Mucolipidosis IV, Krabbe disease (late onset) not correlate with the severity of systemic features. Lipid disorders Color vision, electroretinography and electroocu- Abetalipoproteinemia lography have been shown to be normal. Most Peroxisomal disorders: peroxisome biogenesis disorders; patients exhibit photophobia, subnormal visual Refsum disease acuity, myopia and astigmatism. It is unknown if b-Oxidation defects: long-chain hydroxyacyl-CoA reduced visual acuity is due to retinal deposits or dehydrogenase defi ciency, mitochondrial trifunctional protein defi ciency due to demyelination of the optic pathways, the Sjögren-Larsson syndrome latter having been demonstrated by MRI in the Mitochondrial Disorders affected patients. KearnsÐSayre syndrome NARP (neuropathy, ataxia, and RP) 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase Peroxisomal disorders are best diagnosed by determin- defi ciency ing plasma concentrations of very-long-chain fatty 192 A. Burlina and A. P. Burlina acids. Disorders with pigmentary retinopathy include neurological regression, dystonia and acanthocytosis), Zellweger syndrome, neonatal adrenoleukodystrophy Laurence–Moon–Biedl (obesity, polydactyly and men- and isolated enzyme defects, such as acyl-CoA oxi- tal retardation), Usher type II (deafness and severe dase defi ciency and peroxisomal thiolase defi ciency. mental retardation), Joubert (mental retardation, cere- Nystagmus is common and ocular fi ndings include bellar vermis atrophy and attacks of hyperventilation), retinal dystrophy, corneal clouding and cataract. and Cockayne (dysmorphia, hypotonia, intacranial cal- Extensive photoreceptor degeneration and loss of gan- cifi cations and deafness) syndromes. glion cells with gliosis of nerve fi bre layers have been reported. Patients with Refsum disease, an inborn error caused by phytanic acid oxidase defi ciency, present C8.5.2 Pigment Retinopathies with RP (Fig. C8.5), peripheral polyneuropathy and elevated cerebrospinal fl uid protein. Other symp- Pigment retinopathies due to the storage of specifi c toms include cerebellar ataxia, deafness, anemia, compounds include many lysosomal storage diseases. ichthyosis and skeletal and cardiac manifestations. The cherry-red spot is due to ganglioside accumulation Night blindness may be the fi rst clinical symptom at in the retinal ganglion cells. The absence of ganglion school age. cells at the fovea gives rise to the red spot surrounded Defects in the mitochondrial electron transport chain by white cells fi lled with storage material. As the gan- cause a variety of oculars manifestations. Chronic exter- glion cells die, the cherry-red spot fades, and optic nal ophtalmoplagia and retinal dystrophy similar to RP atrophy becomes apparent. are often found. It is seen in several mitochondrial disor- The differential diagnosis mainly includes lyso- ders. Electroretinogram shows evidence of rod and cone somal disorders (Table C8.5). It is present at an early dysfunction with the rods being more severely affected. stage in most patients affected with GM2 gangliosido-

Retinal degeneration has been consistently reported only sis (GM2 type I, II, and III) and GM1 gangliosidosis in the Kearns–Sayre syndrome, a progressive multisys- (type I). The electroretinogram is consistently normal, tem disorder, with onset usually before the age of 20 but the VEP are abnormal from the early stages of the years. Clinical features include chronic progressive disease, and the cortical response is generally abol- external ophtalmoplegia, ptosis, retinopathy, cardiac ished from the ﬁ rst months of age. conduction defects and deafness. It is caused by deletion Niemann–Pick disease type A also leads to corneal ± duplications of mitochondrial DNA (mtDNA). opaciﬁ cation and dislocation of the anterior lens Finally, several well-known autosomal recessive capsule. disorders comprise RP; these include pantothenate In sialidosis (mucolipicosis type I), formerly known kimase - assosiated neurodegeneration, (PKAN), (severe as cherry-red spot myoclonus syndrome, the lesion presents in adulthood with a progressive myoclonus and normal intelligence. In Farber’s disease, metachromatic leucodystrophy and Gaucher disease type II, a faint or irregular cherry-red spot can be present.

Table C8.5 Metabolic diseases with cherry-red spots Sialidosis type I and II Galactosialidosis (early-infantile)

TayÐSachs disease [gangliosidosis type 2 (GM2), variant B, infantile]

Sandhoff disease (GM2, variant O, infantile) Gangliosidosis type 1-gangliosidosis (infantile) NiemannÐPick disease type A Gaucher disease type II Farber disease Fig. C8.5 Refsum’s disease showing retinal pigment abnormality Metachromatic leukodystrophy C8 Eye Disorders 193

Retinal degeneration may occur in other defects of Disease Info: LHON intermediary metabolism including defects of intracellular cobalamin metabolism (Cbl C/D defects) LHON is an inherited form of acute or subacute loss and congenital disorders of glycosylation syn- of central vision affecting predominantly young dromes (especially type Ia, phosphomannomutase males. The disease is the paradigm of mitochondrial defi ciency). optic neuropathies where a primary role for mitochondrial dysfunction is certifi ed by maternal inheritance and caused by missense mutations in different mtDNA genes, especially complex I subunits. The C8.6 Optic Atrophy typical presentation is a rapid painless loss of central vision in one eye. Usually, fading of colors (dys- chromatopsia) in one eye is followed by a similar Optic atrophy results from loss of ganglion cell involvement of the other eye, within days, months or axons that form the optic nerve, and/or a loss of the rarely years. Visual acuity stabilizes at or below supporting microvascular tissue surrounding the 20/200 within a few months. The accompanying optic nerve. Symptoms of optic atrophy include visual fi eld defect usually involves the central vision decreased visual acuity (ranging from no light per- in the form of a large centrocecal absolute scotoma. ception to mild decreases in visual acuity), visual Fundus examination during the acute/subacute fi eld defects and/or abnormalities in color vision and stage mostly reveals characteristic changes as contrast sensitivity. follows. (1) circumpapillary telangiectatic microangiopathy, (2) swelling of the nerve fi ber layer around the disc Remember (pseudoedema), and (3) absence of leakage on fl uorescein angiography The hallmark clinical sign of optic atrophy is optic (in contrast to true edema). nerve pallor at fundoscopic examination. Thus, the optic disc appears hyperemic, occasionally with peripapillar hemorrhages and the axonal loss rapidly leads to temporal atrophy of the optic disc. Optic neuropathy is a more general term for optic With time, the optic disc turns pale. The microan- nerve dysfunction and includes the early phase of the giopathy may be present in a number of asymptom- disease, before clinical signs of optic atrophy may be atic at-risk family members along the maternal line, present. in whom it may remain stable over the years. Mostly no effective treatment exists, although cor- Optic atrophy with permanent severe loss of cen- rection of an underlying cause or elimination of an tral vision but with relative preservation of pupillary offending medication may halt progression. light responses is the usual endpoint of the disease. Genetic defects are responsible for a substantial However, spontaneous recovery of visual acuity has portion of optic atrophy. The lesion can be the only occasionally been reported even years after onset. clinical feature (primary) or associated to various neu- Visual function may improve, sometimes suddenly, rological and systemic symptoms (secondary). with contraction of the scotoma or reappearance of small islands of vision within it (fenestration). In long-lasting LHON, cupping of the optic disc has frequently been reported as a sign of the chronic C8.6.1 Primary Optic Atrophy stage of the pathological process. LHON is due to homoplasmic mtDNA mutations, typically with wide variability in phenotypic pene- In primary optic atrophies, optic atrophy is often the trance. To date, LHON is the only human disease for only clinical feature of the disease. An example is which the infl uence of mtDNA background (haplo- Leber’s hereditary optic nauropathy (LHON) or Costeff groups) has been solidly documented, particularly optic atrophy syndrome (OPA 3). 194 A. Burlina and A. P. Burlina

Table C8.6 Secondary optic atrophy on the T14484C/ND6 and G11778A/ND4 LHON Mitochondrial disorders mutations. Recently, the association of LHON with a MERRF (mitochondrial encephalopathy with ragged-red region of chromosome X suggests that one or more fi bers and stroke-like episodes) nuclear genes may act as modifi ers, possibly explain- MELAS (mitochondrial encephalomyopathy, lactic ing the male prevalence. acidosis, stroke-like episodes) The recent identifi cation of mutations in the KearnsÐSayre syndrome nuclear gene OPA1 as the causative factor in domi- NARP(neuropathy, ataxia, retinitis pigmentosa) Leigh syndrome nant optic atrophy (DOA, Kjer’s type) brought the Peroxisomal disorders unexpected fi nding that this gene encodes for a Zellweger spectrum disorders mitochondrial protein, suggesting that DOA and Adrenoleukodystrophy LHON may be linked by a similar pathogenesis. Primary hyperoxaluria type I Polymorphisms in this very same gene may be Lysosomal disorders associated with normal tension glaucoma (NTG), Mucopolysaccharidoses which might be considered a genetically determined Oligosaccharidoses optic neuropathy that again shows similarities with NiemannÐPick type C NiemannÐPick type A or B both LHON and DOA. GM1 gangliosidosis Sandhoff disease Costeff optic atrophy syndrome or Type III 3-methyl- Multiple sulfatase defi ciency glutaconic aciduria (OPA 3) is a neuroophthalmologic Krabbe disease disease that consists of early onset bilateral optic atrophy Metachromatic leukodystrophy Neuronal ceroid lipofucinoses and late-onset spasticity, extrapyramidal dysfunction Cystinosis and cognitive defi cits. Urinary excretion of 3-methyl- Other metabolic disorders glutaconic acid and of 3-methylglutaric acid is increased. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase The disorder is mapping to chromosome 19q13.2Ðq13.3, defi ciency and the causative gene has been identifi ed in the Cobalamin C/D disorders FLJ22187-cDNA clone, which consists of two exons Propionic acidemia Homocystinuria and encodes a peptide of 179 amino acid residues. Milder SmithÐLemliÐOpitz syndrome mutations in OPA3 should be sought in patients with Mevalonic aciduria optic atrophy with later onset, even in the absence of Canavan disease additional neurological abnormalities. Behr syndrome is Alexander disease clinically similar to Costeff syndrome, but it is distin- PelizaeusÐMerzbacher disease guished by the absence of 3-methylglutaconic aciduria. Menkes disease

dysfunction, optic atrophy may occur in mitochondrial C8.6.2 Secondary Optic Atrophy encephalomyopathies due to mtDNA point mutations in tRNA genes, such as myoclonic epilepsy, ragged- Secondary optic atrophy occurs often in inherited met- red-fi bres (MERRF) and mitochondrial encephalomy- abolic diseases and is due to accumulation or shortage opathy, lactic acidosis, and stroke-like syndrome of substrates or formation of harmful metabolites such (MELAS), NARP (neuropathy, ataxia and pigmentary as in mitochondrial defects, peroxisomal defects and retinopathy) and Leigh syndrome (see also Chap. D1). lysosomal defects and occasionally in some other met- Although the visual system is not usually empha- abolic defects (Table C8.6). sized, optic atrophy is frequently reported in Leigh syndrome in addition to the bilateral necrotic lesions affecting the periventricular white matter, basal gan- C8.6.2.1 Mitochondrial Disorders glia and brainstem. Given the early onset of Leigh syndrome, it is diffi cult to document visual loss, but In addition to the relative selective involvement of the according to the few histopathological reports of the optic nerve in some disorders with mitochondrial visual system there is a typical loss of retinal C8 Eye Disorders 195 ganglion cells and nerve fi ber layer dropout in the ganglion cells. Single peroxisomal enzyme defects papillomacular bundle. There is progressive deterio- that lead to optic atrophy are X-linked adrenoleu- ration of brainstem functions, ataxia, seizures, periph- kodystrophy and primary hyperoxaluria type I. Patients eral neuropathy, intellectual deterioration, impaired with X-linked adrenoleukodystrophy show loss of hearing and poor vision. Visual loss may be second- retinal ganglion cells and sporadic macrophages sur- ary to optic atrophy or retinal degeneration. A variety rounding the optic nerve fi bres. Patients with primary of molecular defects in both nuclear DNA (nDNA) hyperoxaluria type I harbor oxalate crystals in various and mtDNA have been identifi ed. Leigh syndrome tissues, including the eye, due to a defi ciency of the can be inherited, depending on the particular defect, peroxisomal enzyme alanine:glyoxylate aminotrans- maternally (mtDNA defects), as an X-linked reces- ferase. Optic atrophies can occur secondary to sive trait (pyruvate dehydrogenase complex, PDHC increased intracranial pressure caused by impeded defect) or as an autosomal recessive disease (defects cerebrospinal fl uid drainage due to oxalate crystals. in complexes I and II nuclear genes; defects in SURF1 Crystals within the retinal ganglion cells can also gene for complex IV assembly). directly cause apoptosis. Optic atrophy has also been reported in mitochondrial diseases caused by defects in nuclear genes such as hereditary spastic paraplegia due to mutations in the C8.6.2.3 Lysosomal Disorders paraplegin gene, and in the deafness-dystonia-optic atrophy syndrome (Mohr–Tranebjaerg syndrome) due Lysosomal storage diseases can cause optic atrophy. In to mutations in the X-linked DDP1 gene. In this dis- the MPS, pathology is optic disc swelling. Secondary ease patients manifest optic atrophy with severely con- raised intracranial pressure must be excluded. In the stricted visual fi elds, a pattern opposite to LHON and absence of raised intracranial pressure, optic nerve the other optic neuropathies that affect primarily the swelling and consequently atrophy arises from com- central visual fi eld due to predominant loss of the pap- pression of the nerve by a thickened dura and sclera, illomacular bundle. resulting in compression at the level of the lamina cri- brosa. Also, accumulation of glucosaminoglycans within ganglion cells is thought to eventually lead to Remember degeneration and optic atrophy. The most characteristic and primary manifestation Optic atrophies may also occur secondary to retin- of LHON is visual loss due to optic nerve dys- opathy or can be caused by glaucoma. function. Among the oligosaccharidoses, the gangliosidoses

(GM1 gangliosidosis, TayÐSachs, and Sandhoff disease) and, occasionally, NiemannÐPick A or B disease, present with distinct ocular fi ndings and optic C8.6.2.2 Peroxisomal Disorders neuropathies. Causes of optic neuropathies in sphingolipidoses could be loss of myelinated nerve fi bres or thickening of the pial septum of the optic nerve. Remember Mostly, ocular pathology in sphingolipidoses relates Ocular fi ndings in peroxisomal biogenesis disor- to the damage of the retinal ganglion cells that form ders include profound demyelination of the optic the optic nerve. Abnormal accumulation of lipid nerve. material and loss of retinal ganglion cells results in optic atrophy. Another lysosomal storage disease with optic atro- Ocular fi ndings in all peroxisomal biogenesis disor- phy is Krabbe disease (globoid cell leukodystrophy). ders, such as Zellweger syndrome, neonatal adreno- In this disease, the lesion is a consequence of the leukodystroph and infantile Refsum disease, include severe loss of myelin and oligodendroglia that is char- profound demyelination of the optic nerve, reduced acteristic of this disorder. Although the optic atrophy numbers of optic nerve fi bres, and inclusion-bearing occurs early, it is usually overshadowed by the neuro- macrophages surrounding the optic nerve retinal logical deterioration. Optic atrophy is also a frequent 196 A. Burlina and A. P. Burlina cause of severely impaired vision in metachromatic Take Home Messages leukodystrophy. In addition, some disorders of lysosomal mem- › Ocular manifestations occur frequently in var- brane transport such as NiemannÐPick C disease and ious inherited metabolic disorders. Accurate infantile sialic acid storage disease can present with examination of the eye by clinical means and marked ocular abnormalities and sporadic optic neu- with the help of the ophthalmoscope and slit ropathies. In cystinosis, benign intracranial hyperten- lamps may detect the following pathogno- sion has led to optic atrophy. In neuronal ceroid monic abnormalities: corneal clouding, lens lipofuscinoses, neuro ophthalmological abnormalities defects and dislocation, retinal degeneration like optic atrophy, maculopathy and RP have been and optic atrophy. Ophthalmology evalua- reported. It is likely that lysosomal storage of metabo- tion is a key twist in the diagnostic focus of lites indirectly affect the optic nerve and its supporting many inherited metabolic diseases, e.g. cata- system. racts in galactosemia, ectopia lentis in homocystinuria, cornea verticillata in Fabry’s disease, macular cherry-red spot in a number of sphingolipidoses. C8.6.3 Other Secondary Metabolic › For some inherited metabolic diseases, spe- Optic Atrophies cifi c treatment can restore or prevent the ocular manifestation in a short period of time, e.g. tyrosinemia type II, galactosemia and Optic atrophies are described in a variety of inherited homocystinurina. metabolic disorders. It should be emphasized that, while optic atrophy has been noted in these disorders, it is not a constant fi nding. In some cases, it may be a secondary effect of a metabolic crisis. The fi rst group includes disorders where critical metabolites are either Acknowledgements We thank Dr. L. Pinello and Dr. E. Zanin defi cient or accumulate within the retinal ganglion (Department of Paediatrics, University of Padova) for providing ophthalogical pictures reported in the chapter. cells or the supporting cells. Disorders in this group include biotinidase defi ciency, Smith–Lemli–Opitz syndrome, Menkes disease and a subset of disorders of amino acid metabolism such as homocystinuria, coba- Key References lamin C disease and propionic acidemia. A second group involves myelination defects of the Fernandes J, Saudubray JM, van den Berghe G, Walter JH (eds) optic nerve. Disorders in this group include some (2006) Inborn metabolic diseases Ð diagnosis and treatment, 4th edn. Springer, New York hereditary ataxias such as Friedreich ataxia, certain Huizing M, Brooks BP, Anikster Y (2005) Optic atrophies in forms of CharcotÐMarieÐTooth disease, Canavan dis- metabolic disorders. Mol Genet Metab 86:51Ð60 ease, abetalipoproteinemia, Pelizaeus–Merzbacher Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic disease, Alexander disease and the spastic paraplegia diseases, 2nd edn. Hodder Arnold, London Poll-The BT, Maillette de Buy Wenniger-Prick LJ, Barth PG, form with peripheral polyneuropathy (spastic paraple- Duran M (2003) The eye as a window to inborn errors of gia, optic atrophy and neuropathy). metabolism. J Inherit Metab Dis 26:229Ð244 Skin and Hair Disorders C9 Carlo Dionisi-Vici, May El Hachem, and Enrico Bertini

Key Facts C9.1 General Remarks › A correct classifi cation of skin and hair signs as well as the knowledge of characteristic A correct classifi cation of skin and hair signs as well as cutaneous symptoms is important to under- the knowledge of cutaneous symptoms of inherited stand and diagnose inherited metabolic metabolic diseases is an important prerequisite to help diseases. understand and diagnose these complex and heteroge- › The profi le of cutaneous signs, the age of the neous diseases. The profi le of cutaneous signs, the age patient when manifestations initially occurred of the patient when manifestations initially occurred and the presence of associated symptoms, is of and the presence of associated symptoms is of particu- particular importance. lar importance. Sometimes cutaneous signs may represent a hallmark for a specifi c metabolic disorder, while › Dermatological evaluation can help identify in other instances a careful dermatological evaluation complications of the disease or side effects of can help identify complications of the disease or treatments. adverse side effects of (over-)treatment. › Cutaneous signs of several types often occur in a single given disorder. › The principal lesions can be grouped into the following main groups: vascular lesions, skin Remember eruptions, ichthyosis, papular and nodular skin Skin lesions can be grouped in the following main lesions, abnormal pigmentation, photosensi- categories: vascular lesions, skin eruptions, ich- tivity, hair disorders, and skin laxity. thyosis, papular and nodular skin lesions, abnormal pigmentation, photosensitivity, hair disorders, and skin laxity.

Cutaneous signs of several types often occur in a single given disorder, and the principal lesions can be grouped in the following main categories: vascular lesions, skin eruptions, ichthyosis, papular and nodular skin lesions, abnormal pigmentation, photosensitivity, hair disorders, and skin laxity. We have summarized in the tables, for simplicity, containing the list of disorders that frequently C. Dionisi-Vici ( ) cluster for each skin or hair lesion. For most disorders, Division of Metabolism, Bambino Gesu Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy skin and hair abnormalities have been obtained from the e-mail: [email protected] clinical synopsis of Online Mendelian Inheritance in

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 197 DOI: 10.1007/978-3-540-74723-9_C9, © Springer-Verlag Berlin Heidelberg 2010 198 C. Dionisi-Vici et al.

Man (OMIM), available at http://www.ncbi.nlm.nih. C9.2 Vascular Lesions gov/sites/entrez. As shown in Table C9.1, vascular lesion can be sub- classiﬁ ed in different forms. C9.2.1 Angiokeratoma

The angiokeratomata are ﬂ at or raised vascular skin lesions, and they develop classically as clusters, punc- Remember tuate, reddish or blueÐblack angiectases. They do not blanch with pressure and the largest lesions may appear The main vascular abnormalities include angioker- hyperkeratotic. atoma, hemangiomas, petechiae, acrocyanosis, and In Fabry disease, a metabolic disorder in which telangiectasia. the skin lesions are a dominant feature of the clinical

Table C9.1 Vascular skin lesions Disease MIM number Type of vascular skin lesions Additional dermatological Hair manifestations Fabry disease 301500 Angiokeratoma Hypohidrosis Gangliosidosis GM1 230500 Angiokeratoma Dermal Hypertrichosis corporis diffusum melanocytosis Fucosidosis 230000 Angiokeratoma Thin, dry skin Anhidrosis Heavy eyebrows Aspartylglucosaminuria 208400 Angiokeratoma Acne corporis diffusum Galactosialidosis 256540 Angiokeratoma Widespread hemangiomas Mannososidosis 248510 Angiokeratoma Schindler disease 609242 Angiokeratoma Hyperkeratosis type II corporis diffusum Dry skin Telangiectasia on lips Maculopapular and oral mucosa eruption Mucolipidosis II 252500 Cavernous hemangioma Tight skin Ethylmalonic 602473 Petechiae encephalopathy Orthostatic acrocyanosis Hyperoxaluria type I 259900 Livedo reticularis Acrocyanosis Neuraminidase 256540 Widespread hemangiomas defi ciency SmithÐLemliÐOpitz 270400 Facial capillary hemangioma Severe Blonde hair syndrome photosensitivity Eczema Transaldolase 606003 Telangiectases Cutis laxa Hypertrichosis defi ciency of the skin CDG type Ie 608799 Telangiectasia Dysplastic nails Hemangiomas CDG type IIa 212066 Midfrontal capillary hemangioma CDG type IIf 603585 Petechiae Ecchymoses Prolidase defi ciency 170100 Diffuse telangiectases Crusting erythematous dermatitis Severe progressive ulceration of lower extremities C9 Skin and Hair Disorders 199 picture, angiokeratomata also known as angiokerato- angiokeratomata have been reported to occur along mas corporis diffusum, may be the fi rst manifestations with mental retardation, coarse facial features, dysos- of the disease, and in some heterozygous females, they tosis multiplex, and hepatosplenomegaly. Schindler may be the only sign of disease, but males with this disease type II, also known as Kanzaki disease, is an X-linked disorder have usually a number of years of fre- adult-onset disorder characterized by angiokeratoma quent excruciating and unexplained pains. Pain appears corporis diffusum, hyperkeratosis, dry skin, maculo- most commonly in the extremities, particularly in the papular eruption, telangiectasia on lips, and oral mucosa lower extremities, but patients may present with unex- and mild intellectual impairment. plained pain in the abdomen or elsewhere. Affected boys may have been referred to a psychiatrist because it is so diffi cult to fi nd why they are complaining so vehe- mently. The appearance of the skin lesions creates an C9.2.2 Acrocyanosis, Angiomas, entirely different scenario. They are dark red, and they and Telangiectasia do not blanch with pressure. They seek out pressure points such as buttocks or knees, but they are often Acrocyanosis appears as a bilateral mottled discolor- profuse in distribution over the scrotum and penis. ation of the entire feet or hands. In relation to a vasos- They increase in number with time. Older lesions also pasm of small arterioles and venules with secondary increase in size. They are usually fl at at fi rst, but may dilatation of capillaries, the skin’s color becomes become slightly palpable. Lesions may be seen on the bright red, with no trophic changes and pain. Orthostatic oral mucosa or on the conjunctiva. Skin signs may also acrocyanosis is one of the principal signs of ethylma- include hypohidrosis. lonic encephalopathy, a devastating mitochondrial disorder affecting the brain, gastrointestinal tract, and peripheral vessels (Fig. C9.1). Remember Angiokeratomas corporis may be the fi rst manifes- Remember tations of Fabry disease. Orthostatic acrocyanosis is a key sing of ethylmalonic encephalopathy. Angiokeratoma may also occur in some other lysosomal storage disorders. One or two angiokeratomata and dermal melanocytosis may be seen in GM1 gangli- Acrocyanosis may also be observed in other mitochon- osidosis, but usually these patients will already have drial disorders with onset in early infancy and multisys- been diagnosed because of hepatosplenomegaly, devel- tem involvement (Bodemer et al. 1999). Acrocyanosis opmental delay, coarse features, and dysostosis multi- and livedo reticularis may be observed in hyperoxaluria plex that occur very early in infancy, whereas the skin type I along with urolithiasis, nephrocalcinosis, renal lesions are rare before the fi rst year of age. Similarly, a failure, peripheral vascular insuffi ciency, arterial occlu- patch of angiokeratomata with thin and dry skin may sion, and Raynaud phenomenon. be seen in fucosidosis, but again not among the earliest Capillary malformations appearing as light pink features, although they may appear from 6 months to 4 to deep-red angiomas or as telangectasias may occur years; organomegaly, bone changes and developmen- in several metabolic diseases. Widespread heman- tal delay are evident earlier. Similar lesions may be giomas, coarse facies, conjunctival telangiectases, seen in childhood in aspartylglucosaminuria, but long severe developmental delay seizures, and skeletal after the patient is known to have coarse features, acne, abnormalities can be observed in neuraminidase defi - cataracts, joint laxity, and neurodegeneration. They ciency. Facial capillary hemangioma, severe photo- may be seen in the juvenile form of galactosialidosis sensitivity, eczema, and blond hair are typical features together with corneal opacities, bone fi ndings, neuro- of the SmithÐLemliÐOpitz syndrome, a disorder of logical degeneration, and widespread hemangiomas cholesterol biosynthesis presenting with characteristic that bring the patient to attention again usually before facial appearance, ambiguos genitalia, failure to thrive, the skin lesions appear. Also in b-mannosidosis, syndactily, microcephaly, and intellectual impairment 200 C. Dionisi-Vici et al.

scalp, the skin folds, and the sebaceous follicle-rich areas of face and trunk. The lesions appear pink to erythematous, with or without edema, covered with greasy yellowÐbrown scales. The scaling on the scalp can vary from fi ne to thick and could be mild to moderate on the folds. The facial involvement is most prominent on the forehead, the eyebrows and around the nose. Eczema is an infl ammatory dermatosis characterized by the appearance of erythema, edema, vesicles, scaling, and crusts. The lesions can be single, multiple or confl uent and pruritus is generally present and intense. Psoriasiform lesions consist of erythemato-squamous papules, sharply demarcated with clear-cut borders. Hyperkeratosis is a horny thickening that fi rmly adheres to the skin that may be epidermal or follicular. Vesico-bullous lesions are circumscribed, translucent elevated lesions containing fl uid arising from cleavage at various level of the skin. Ulcer is the result of loss of the entire epidermis and at least the upper dermis (pap- Fig. C9.1 Orthostatic acrocyanosis inethylmalonic enceph- illary), which heals with scarring. alopathy

Remember

(see also paragraph on photosensitivity). In transaldo- Skin eruptions include seborrheic dermatitis, lase defi ciency, a newly recognized metabolic disease eczema, skin rashes, psoriasiform lesions (erythema- of pentose phosphate pathway reported in patients tous squamous lesions), lupus-like lesions, hyperk- with liver failure and cirrhosis, dysmorphic facial fea- eratosis, vesciculo-bollus lesions, and ulcers. tures, renal, cardiac and hematological involvement, cutaneous signs include teleangectases, cutis laxa, and hypertrichosis (Valayannopoulos et al. 2006). More In multiple carboxylase defi ciency, either due to holo- rarely, vascular signs have been observed in patients carboxylase synthetase or to biotinidase defi ciency, with congenital disorder of glycosylation type Ie, type skin and hair abnormalities are prominent fi ndings. The IIa, and type IIf along with neurologic, facial and usual initial presentation is the classic organic aciduria other multisystem abnormalities (Dyer et al. 2005). emergency with massive ketosis and acidosis progres- Prolidase defi ciency, which also presents with diffuse sive to coma. Patients surviving the initial episode of teleangiectases, will be discussed in the skin eruption metabolic decompensation often develop the dermato- paragraph. sis. Cutaneous signs consist in a bright red patchy or generalized body eruption, associated with alopecia. Lesions are often desquamative and typically periorifi - cial. Complication by monilial infection is very com- C9.3 Skin Eruptions (Table C9.2) mon, especially around the mouth, the eyes and in the diaper area. Skin lesions become vesicular when they The main lesions discussed in this paragraph include are complicated by mucocutaneous fungal infection. seborrheic dermatitis, eczema, skin rashes, psorias- The clinical manifestations in holocarboxylase syn- iform lesions (erythematous squamous lesions), lupus- thetase defi ciency are more severe than in biotinidase like lesions, hyperkeratosis, vesciculo-bollus lesions, defi ciency, with an earlier age at onset and some patients and ulcers of the skin. Seborrheic dermatitis is associ- unresponsive to biotine therapy. In biotinidase defi ciency ated to increased sebum production and involves the patients are less likely to develop life threatening C9 Skin and Hair Disorders 201

Table C9.2 Skin eruptions Disease MIM number Type of skin eruptions Additional Hair dermatological manifestations Biotinidase 253260 Skin rash Cat odor Alopecia defi ciency Holocarboxylase 253270 Seborrheic dermatitis Loss of eyelashes synthetase defi ciency 3-Methylcrotonyl-CoA 210210 Skin infections Loss of eyebrows carboxylase defi ciency Periorifi cial dermatitis Methylmalonic aciduria 251000 Scalded skin, superfi cial Fine hair desquamation Propionic aciduria 606954 Periorifi cial dermatitis Alopecia Psoriasis-like lesions Mevalonic aciduria 610377 Skin Rash Edema and arthralgia during crisis Hyper-IgD syndrome 260920 Morbilliform rash Erythematous macules or papules Acrodermatitis 201100 Bullous, pustular dermatitis Impaired would Alopecia of scalp enteropatica of extremities, oral, anal, healing and genital areas Paronychia Alopecia of eyebrows Dermatitis, symmetric pattern Alopecia of eyelashes Hyperzynchemia with 601979 Skin rash functional zinc depletion Sulfi te oxidase defi ciency 272300 Mild eczema Fine hair SmithÐLemliÐOpitz 270400 See Table C9.1 syndrome Prolidase defi ciency 170100 Diffuse telangiectases Severe progressive ulceration of lower extremities Crusting erythematous dermatitis Lysinuric protein 222700 Lupus-like erythematous Hyperelastic skin Fine sparse hair intolerance squamous lesions Hyperzincaemia and 194470 Infl ammatory skin lesions Pyoderma hypercalprotectinaemia gangrenosum Tyrosinemia type II 276600 Painful punctate keratoses of digits, palms, and soles

ketoacidosis. They may present with laryngeal stridor, carboxylase defi ciency. The differential diagnosis convulsions and, if undiagnosed and untreated, all are includes acrodermatitis enteropathica. In fact the fi rst hypotonic and most continue to become mentally patients described with biotinidase defi ciency carried retarded. In biotinidase defi ciency, the effect of biotin the diagnosis of acrodermatitis enteropathica. Patients treatment is spectacular with rapid and complete remis- with acrodermatitis enteropathica have diarrhea, are sion of skin, neurological and metabolic abnormalities. zinc defi cient, and respond to treatment with zinc, but Visual and auditory impairment are often irreversible the diagnosis may be a wastebasket term, and any infant despite biotin therapy. More rarely, patients with the with this diagnosis should at the least have organic acid isolated defect of 3-methylcrotonyl-CoA carboxylase analysis of the urine and biotinidase of the plasma, as may present with skin signs similar to multiple well as a work-up for immunodefi ciency. 202 C. Dionisi-Vici et al.

Remember supplemented. As a presenting clinical manifestation of untreated disease, cheilitis and diffuse erythema with Generalized body eruption with desquamative and erosions and desquamation has been reported in two periorifi cial skin lesions associated with alopecia patients with methylmalonic aciduria with homocystinu- are the characteristic signs of multiple carboxylase ria, cobalamin C type. In both the cases, skin lesions defi ciency, either due to holocarboxylase synthetase were already present prior to diagnosis, in the absence of or to biotinidase defi ciency. iatrogenic nutritional restrictions or defi ciency (Howard et al. 1997).

The typical skin lesions of methylmalonic aciduria and Remember propionic aciduria usually occur in patients with the severe forms of these diseases, with no residual enzyme Acrodermatitis enteropathica like lesions may com- activity and subjected to a very severe natural protein- plicate any of the organic acidurias or disorders of restricted diet (Fig. C9.2). Cutaneous manifestations amino acid metabolism that are overtreated with an could be divided into fi ve categories: superfi cial scalded excessive dietary restriction of natural protein and skin, superifi cial desquamation, bilateral and periorifi cial other essential nutrients. dermatitis, psoriasiform lesions, and alopecia (Bodemer et al. 1999). Different skin lesions can coexist in a given case and may be due to the enzyme defi ciency itself or Recurrent morbilliform rashes and erythematous macules may be part of a multidefi ciency syndrome. Similar pic- and papules can be frequently observed in mevalonic aci- tures, that strongly resemble to acrodermatitis entero- duria a disorder of cholesterol biosynthesis (Fig. C9.3). pathica, may complicate any of the organic acidurias or Patients have also facial dysmorphic features, global disorders of amino acid metabolism that are overtreated developmental delay, cataract, arthralgias with periarticu- with an excessive dietary restriction of natural protein lar edema, recurrent febrile crises with lymphadenopathy, and other essential nutrients. In patients with disorders hepatosplenomegaly, vomiting, and diarrhea. Rash, related to branched-chain amino acid metabolism, skin edema, and arthralgia may occur during crisis. Similar lesions are often due to a selective defi ciency of isoleu- skin signs can also be observed in hyper-IgD syndrome, cine and rapidly resolve when this essential amino acid is the less severe allelic variant of mevalonic aciduria.

Fig. C9.2 Propionic aciduria with extensive skin lesions characterized by superﬁ cial scalded skin, desquamation and perioriﬁ cial dermatitis C9 Skin and Hair Disorders 203

Ulcers in adolescence and adulthood may also develop in classical homocystinuria caused by cystathionine b-synthase deﬁ ciency; these ulcers result from the grounds of thromboembolic disease usually in the lower extremities.6pInﬂ ammatory skin lesions of various degree along with recurrent infections, growth failure, hepatosplenomegaly, arthritis, anemia, and persistently raised concentrations of C-reactive protein are the clinical features of hyperzincemia and hypercalprotectinemia, a newly described disorder of zinc metabolism, probably caused by dysregulation of calprotectin metabolism. Hyperkeratotic lesions are a characteristic sign of oculocutaneous tyrosinemia or tyrosinemia type II. The combination of ocular and these lesions has also been called the RichnerÐHanhart syndrome. The major manifestation of this disease is keratitis and corneal ulcers associated with skin lesions occurring on the palms and soles. The skin lesions that usually begin in

Fig. C9.3 Skin rash in mevalonic aciduria

Cutaneous ulcers, mainly severe progressive ulceration of lower extremities, can represent the hallmark fi nding of prolidase defi ciency. The ulcers may be further complicated by secondary infection. Skin changes also include teleangiectases, purpuric ecchymoses, rashes, or crusting erythematous dermatitis. In addition, patients may have mental retardation, ophthalmoplegia, splenomegaly, dysmorphic features, and susceptibility to infections. Severe immunological abnormalities, fulfi lling the criteria for diagnosis of systemic lupus erythematous, have been reported in prolidase defi ciency (Shrinath et al. 1997). Interestingly, skin lupus-like lesions (Fig. C9.4) and immunological abnormalities have also been observed in lysinuric protein intolerance, a transport defect of dibasic aminoacid that usually presents with postprandial hyperammonemia, failure to thrive, severe renal, and pulmonary involvement (Dionisi-Viciet al. 1998).

Remember Skin lesions and severe immunological abnormalities fulfi lling the criteria for diagnosis of systemic lupus erythematous can be observed in prolidase defi ciency and in lysinuric protein intolerance. Fig. C9.4 Lupus-like skin lesions in lysinuric protein intolerance 204 C. Dionisi-Vici et al. early infancy, are painful, nonpruritic, and frequently Most ichthyoses are not part of an inborn error of metab- associated with hyperhidrosis. Bullous lesions may olism, but in many metabolic diseases ichthyosis may occur and progress rapidly to erosions that become be a very prominent presentation. As shown in the table, crusted and hyperkeratotic. Rarely, lesions have a sub- a large number of metabolic diseases Ð belonging to the ungueal localization. group of syndromic ichthyoses Ð are listed. The purest form is X-linked ichthyosis resulting from defective activity of steroid sulfatase, which leads to deposition of cholesterol sulfate. These patients may have no other C9.4 Ichthyosis (Table C9.3) manifestations of the disease than dark, scaly skin. Mild corneal opacities, which do not interfere with vision, are Ichthyosis is a very striking scaling dermatosis that found in about 25% of patients. Mental retardation, results from overactive proliferation of skin cells. Extra hypogonadism, shortness of stature and chondodyspla- skin piles up in scales, reminiscent of the skin of fi sh, sia punctata have been observed. Patients with multiple and falls off. Dependent on the rate of the process, the sulfatase defi ciency are most debilitated by manifesta- scales may appear old and black or there may be the tions of metachromatic leukodystrophy and those of pronounced erythroderma of new skin. Ichthyosis is mucopolysaccharidosis. A patient with features of both characterized by abnormal differentiation (cornifi ca- and who also has ichthyosis doubtless has multiple sul- tion) of epidermis. Several features are useful to distin- fatase defi ciency, also known as Austin disease. guish different forms of ichthyosis. They generally In patients with Refsum disease, the skin may be the may appear with fi ne or thick scales, covering the skin key to the diagnosis of this multisystem disease. Ataxia red or normally colored, sometimes sparing the folds, is an early manifestation. Deafness, peripheral neurop- palms and plantar-areas. In some forms, it can be athy, and retinitis pigmentosa complete the syndrome. observed superfi cial fi ssuring or blistering especially Patients with SjogrenÐLarsson syndrome in which on the folds. The scales could be generalized or local- the fatty alcohol oxidoreductase is defective have cata- ized. In lamellar ichthyosis, they are accentuated on racts and ichthyosis (Fig. C9.6). In addition, they are the extremities, large plate-like brown covering most mentally retarded and have spastic paraplegia. Retinitis of the body. Collodion baby is the most severe presen- pigmentosa may be evident on ophthalmoscopy; there tation of congenital ichthyosis (Fig. C9.5). The child is may be glistening dots in the area of the macula. born encased in a translucent membrane which is taut Cataracts and ichthyosis are also seen in neutral and may impair ectropion, eclabion, ears deformities, lipid storage disorder, also known as ChanarinÐDorfman and respiration and sucking. During the fi rst 2 weeks syndrome. There are vacuolated lymphocytes and, to of life, the membrane breaks up and peels off, often distinguish it from SjogrenÐLarrson disease, hepato- leaving fi ssures, with exposure to infection and water megaly. Patients also have muscle weakness ataxia and loss. This can lead to diffi culties in thermal regulation myopathy. and increased risk of infection and dehydratation. On Ichthyosis is seen as an early, sometimes prenatal, the basis of a clinical classifi cation, two major group congenital manifestation in neuronopathic Gaucher of ichthyosis can be identifi ed: (1) primary ichthyoses disease. There may even be a collodion baby appear- limited to the skin and (2) syndromic ichthyoses, in ance (Fig. C9.6). With time the skin may appear nor- which associated features are present. mal, but neurodegeneration is progressive. Ichthyotic lesions are also prominant in infants with CHILD syndrome, the acronym for congenital hemidysplasia with ichthyosiform. Remember Erythroderma and limb defects. Unilateral hypome- lia (digital hypoplasia to complete limb absence), elbow Ichthyoses can be classifi ed in primary ichthyoses and knee webbing, joint contractures, and ipsilateral and syndromic ichthyoses, in which associated fea- epiphyseal stippling complete the clinical fi ndings. The tures are present. Ichthyoses, in inborn error of disorder is characterised by unilateral ichthyotic skin metabolism, usually belong to the group of syndro- lesions with a sharp demarcation at the midline of the mic ichthyoses. trunk with the facial area typically spared, although the C9 Skin and Hair Disorders 205

Table C9.3 Ichthyosis Disease MIM number Skin Hair Steroid sulfatase defi ciency 308100 Hypertrophic ichthyosis Multiple sulfatase 272200 Dark adherent skin scales defi ciency Ð Austin disease Ichthyosis Refsum disease 266500 Ichthyosis SjogrenÐLarsson syndrome 270200 Ichthyosis ChanarinÐDorfman 275630 Nonbullous congenital Diffuse alopecia syndrome/neutral lipid ichthyosiform erythroderma storage disorder Collodion baby Gaucher disease type II 608013 Erythematous skin ichthyosis, Collodion skin Desquamation of skin soon after birth Petechiae Purpura ConradiÐHuenermann 302960 Congenital ichthyosiform erythroderma Coarse, sparse hair syndrome Follicular atrophoderma Patchy areas of alopecia “Orange peel” skin Ichthyosis Sparse eyebrows Sparse eyelashes CHILD syndrome 308050 Unilateral erythema and scaling Unilateral alopecia Sharp midline demarcation Hyperkeratosis Onychorrhexis Destruction of nails Rhizomelic chondrodysplasia 215100 Ichthyosis Alopecia punctata type I Chondrodysplasia 302960 Congenital ichthyosiform Coarse, sparse hair punctata type 2 erythroderma Follicular atrophoderma Patchy areas of alopecia “Orange peel” skin Ichthyosis Sparse eyebrows Sparse eyelashes CDG type If 609180 Ichthyosis CDG type Im 610768 Ichthyosis Sparse eyebrows Sparse eyelashes Minimal hair growth Serine defi ciency Ichthyosis Ichthyosis, split hairs, 242550 Lamellar ichthyosis Split hair and aminoaciduria Collodion skin

scalp may be involved. Studies of skin cell kinetics have Besides ichthyosis, ConradiÐHuenermann syndrome is indicated the lesions in this disease to be psoriasis. characterized by the typical calciﬁ cations and asymmet- Punctate calciﬁ cations are present in the epiphyses and ric rhizomelic limb shortness. Cataract and mental retar- other cartilaginous structures of the affected side, which dation have been found in a few affected females. The is usually the right side. The disease is inherited as an skin lesions may have a whorled pattern of hyperkera- X-linked disorder and is thought to be lethal in males. totic, white adherent scales with underlying red skin and Ichthyosis may also be seen in the ConradiÐHuenermann palmar and plantar hyperkeratosis and disappear by 3Ð6 or X-linked dominant chondrodysplasia punctata. months. Biochemically, ConradiÐHuenermann syndrome 206 C. Dionisi-Vici et al.

patients have normal or decreased cholesterol levels and elevated concentrations of 8-dehydrocholesterol and 8(9)-cholestenol in plasma and tissues due to a defect of 3-b-hydroxysteroid- D8, D7-isomerase. The sterol-4-demethylase, the enzymatic step just prior to sterol-8-isomerase is the underlying defect in CHILD syndrome. Ichthyosis has recently been described along with psychomotor retardation, night blindness, and dwarfi sm in siblings with a distinct form (type 1f) carbohydrate defi cient glycoprotein (CDG) syndrome. Sialotransferrin pattern in the serum was that of type 1. Activities of phosphomannose isomerase, phosphomannose mutase, and GDP-mannose synthase were normal. Another Fig. C9.5 Collodion skin in a neonate with Gaucher disease type II form of CDG syndrome (type 1m) causing hypoketotic hypoglycemia and death in early infancy can present with ichthyosis and hair abnormalities. Ichthyosis has been observed in a girl with serine defi ciency of unknown cause, presenting a progressive polyneuropathy, growth retardation, and delayed puberty. Despite no defi ciency of any of the serine biosynthetic enzymes was detected, treatment with serine resulted in a clear improvement of neuromuscular and cutaneous signs (de Koning and Klomp 2004). Differential diagnosis of syndromic ichthyosis include other rare conditions such as KID syndrome (keratitis, hepatitis, ichthyosis and deafness, MIM 148210), NISH syndrome (neonatal ichthyosis-scleros- ing cholangitis syndrome MIM 697626), Netherton syndrome (MIM 256500), Rud syndrome (MIM 308200) and other related disorders, and ichthyosis with hepatomegaly and cerebellar ataxia (MIM 242520).

C9.5 Papular and Nodular Skin Lesions (Table C9.4)

Papules are small elevated lesions that may have a variety of shapes. Papules can be differentiated according to the color, margin, number, surface, and distribution. A nodule is a circumscribed, palpable, solid, round, or ellipsoidal lesion up to 1 cm in size. Depth of involvement distinguishes papule from nodule. Nodules can be located deep dermal, dermal-subdermal, or in the subcutaneous fat. Fig. C9.6 Ichtyosis and spastic diplegia in a patient with The clinical expression of Farber disease, result- Sjögren–Larrson syndrome ing from defi ciency of ceramidase, is striking and the C9 Skin and Hair Disorders 207 diagnosis can be easily suspected with a careful clini- In Hunter disease, uniquely among the mucopolysac- cal evaluation. The characteristic features are nodular charidoses, there are localized nodular accumulations of lesions in the subcutaneous tissues around joints and mucopolysaccharide in the skin of the scapular area. pressure point areas and painful swelling of joints Mitochondrial disorders have long been regarded as appearing in early infancy. Lesions are located in the diseases of the neuromuscular system and internal interphalangeal and metacarpal regions as well as in organs. However, since almost every tissue, including the ankle, wrist, knee, and elbow. The patient com- the skin, is dependent on mitochondrial energy supply, plains of pain and stiffness of the joints and may be it is not surprising to observe dermatological signs in carrying a diagnosis of arthritis. Hoarseness is another this extremely heterogeneous group of disorders. feature. Development may be severely and progres- Reviewing the literature of skin disorders associated sively delayed. Deep tendon refl exes may be dimin- with mitochondrial encephalomyopathies the most ished or absent. Interstitial pneumonia is an infi ltrative recurring signs are lipomas (Birch-Machin 2000). component of the disease. Infantile systemic hyalino- Lipomas usually occurs in the adult forms of mitochon- sis (MIM 236490) presents with similar features and drial disorders in patients bearing mutations of mito- should be considered in the differential diagnosis. chondrial DNA and appear as multiple and symmetrical

Table C9.4 Papular and nodular skin lesions Disease MIM number Papular and nodular Additional dermato- Hair skin lesions logical manifestations Farber lipogranulomatosis 228000 Lipogranulomatosis Periarticular subcutaneous nodules MPS type II 309900 Pebbly skin lesions on back, Tight blue cutaneous Hypertrichosis upper arms pigmentation Mitochondrial diseases Lipomas Hyperpigmentation See Table C9.7 Hypopigmentation Acrocyanosis Skin rashes Erythema Keratoderma Anhidrosis Purpuric lesions Familial 143890 Xanthomatosis hypercholesterolemia Sitosterolemia 210250 Tendinous and tuberous xanthoma Lipoprotein lipase 238600 Xanthomatosis defi ciency 107741 Apolipoprotein E defi ciency Cerebrotendinous 213700 Tuberous xanthoma xanthomatosis Xantelasma Glycogenosis type I a/ b 232200 Xanthoma 232220 NiemannÐPick 257200 Xanthoma disease type A CDG type Ia 212065 Abnormal subcutaneous fat Sparse hair tissue distribution Fat pads Trichorrhexis “Orange peel” skin nodosa Pili torti Inverted nipples CDG type IIc 266265 Localized cellulitis 208 C. Dionisi-Vici et al. lesions. The mechanisms underlying the appearance of patients with lipoprotein lipase defi ciency and with lipomas have not yet been fully understood. A recent other forms of chylomicronemia are very different. study demonstrated that lipomatosis in a patient with Severe elevation of circulating triglyceride concentra- tRNA(Lys) mutations was associated with a pattern of tions is associated with eruptive skin xantomata that altered expression of master regulators of adipogenesis appear as yellow papules on a slightly erythematous and with a distorted pattern of brown vs. white adipo- base. Typical locations are over the buttocks, shoul- cyte differentiation (Guallar et al. 2006). ders, and extensor surfaces of the extremities. They In lipoprotein disorders, lipoproteins can enter the occur when triglyceride levels are very high, and they skin, subcutaneous tissues, and tendons producing disappear promptly on dietary reduction of triglycer- xantomata through lipid accumulation and infi ltration. ides. These patients have chylomicronemia and do not Different species of lipoproteins produce different develop vascular disease, whereas they are subject to types of xantomata and characteristic phenotypes asso- recurrent attacks of pancreatitis. Diagnosis may be ciated with specifi c metabolic defects. According to made by the appearance of the blood, which looks like the morphological characteristics, different types of milky tomato soup. In cerebrotendinous xantomatosis, lipid infi ltrates can be distinguished: eruptive-, tubero- accumulation of abnormal sterol derivates is responsi- eruptive-, tuberous-, tendineous-, planar-, and subcuta- ble for the development of xantomata in the brain and neous-xantomata, xantelasma, corneal arcus, and tendons. Patients are at risk for myocardial infarction tonsillar infi ltration (Goldsmith 2003). Diffuse cutane- and also present with several neurologic signs and ous xanthomata of tuberous and subcutaneous types juvenile cataracts. Xantomata may be observed as a are seen along with tendinous xanthomata, xantelasma, late event in glycogen storage disease type 1 with poor and corneal arcus in familial hypercholesterolemia metabolic control in relation to increased serum levels homozygotes (Fig. C9.7). This disorder of low-density of triglycerides and cholesterol. In NiemannÐPick dis- lipoprotein metabolism leads to early coronary artery ease type A, xantomata may also occur along with disease and myocardial infarction; early adult myocar- extreme hepatosplenomegaly, severe developmental dial infarction is seen in heterozygotes. In homozygotes delay and dystonia. the xanthomata are large, fl at, and sometimes distinctly The enlarged fat pads of the patient with CDG syn- yellowish. A similar picture can occur in sitosterolemia, drome type 1a are unique and absolutely diagnostic which, if untreated, also results in premature atheroscle- (Fig. C9.8A). The enlargement of fat pads is usually rosis and occasionally hemolysis. The xanthomata of evident toward the end of the fi rst year. They are

Fig. C9.7 Diffuse cutaneous xanthomata in homozygous hypercholesterolemia C9 Skin and Hair Disorders 209 typically located over the upper and outer areas of the Besides oculocutaneous albinism, differential diagnosis buttocks or lower back, but they may be seen elsewhere of skin hypopigmentation includes several genetically including the lateral thighs and upper arms. The skin determined disorders often associated with immune dys- may feel thickened and later there may be lipoatrophy functions [e.g., HermanskyÐPudlak syndrome (MIM leaving streaks on the lower extremities. Hair changes, 203300), ChediakÐHigashi syndrome (MIM 214500), consisting of sparse appearance, slow growing, coarse Griscelli syndrome type 1 (MIM 214450) type 2 texture and trichorrexis nodosa have also been observed (607624) and type 3 (609227), and the so called (Dionisi-) (Silengo et al. 2003). These infants present with pre- Vici syndrome (MIM 242840 immunodeﬁ ciency with dominant neurological signs, dysmorphic facial fea- cleft lip/palate, cataract, hypopigmentation and absent tures, failure to thrive, inverted nipples (Fig. C9.8B) corpus callosum)] as well as some metabolic diseases. associated with systemic manifestations affecting heart, kidneys, and gastrointestinal system. Remember Differential diagnosis of skin hypopigmentation Remember includes some metabolic diseases and several The enlarged fat pads of the patient with CDG syn- genetically determined disorders often associated drome type 1a are unique and diagnostic. with immune dysfunctions.

Patients with phenylketonuria prior to the development C9.6 Abnormal Pigmentation of screening diagnosis and early treatment were fair of (Table C9.5) hair and skin and blue-eyed in the majority of cases. Early treatment has made it clear that it is not the gene, but the abnormal chemical environment in the untreated C9.6.1 Hypopigmentation state that interferes with normal pigmentation. Also patients with cystinosis and some with homocystinuria Skin hypopigmentation is in most cases caused by an have thin hypopigmented skin, and ﬁ ne brittle hair. enzyme deﬁ ciency involving the production, metabo- Moreover, skin hypopigmentation, along with early lism, or distribution of melanin. Oculocutaneous albi- onset severe developmental delay, coarse facial fea- nism, not discussed in this chapter, is a group of tures, hepatosplenomegaly, failure to thrive, and dys- inherited disorders characterized by a generalized ostosis multiplex is one of the characteristic signs of reduction in pigmentation of hair, skin, and/or eyes. infantile sialic acid storage disease.

Fig. C9.8 Enlarged fat pads (a) and inverted nipples (b) in carbohydrate deﬁ cient glycoprotein syndrome type Ia 210 C. Dionisi-Vici et al.

Table C9.5 Abnormal pigmentation Disease MIM number Abnormal pigmentation Additional dermato- Hair logical manifestations Phenylketonuria 261600 Pale pigmentation Dry skin Blond hair Eczema Scleroderma Mousy odor Cystinosis 219800 Light skin pigmentation Recurrent corneal Light hair erosions pigmentation Homocystinuria 236200 Hypopigmentation Livedo Fine, brittle hair Ulceration Infantile sialic acid 269920 Hypopigmented skin Fair hair storage disease Methionine 250900 White hair malabsorption syndrome Alkaptonuria 203500 Ochronosis Adrenoleukodystrophy/ 300100 Hyperpigmentation Scarce and thin hair adrenomyeloneuropathy Alopecia Glycerol kinase deﬁ ciency 307030 Hyperpigmentation with adrenal insufﬁ ciency KearnsÐSayre syndrome 530000 Hyperpigmentation Hemochromatosis 235200 Hyperpigmentation Alopecia Telangiectases Wilson disease 277900 Hyperpigmentation Gaucher disease type I 230800 Hyperpigmentation

C9.6.2 Hyperpigmentation Remember Skin hyperpigmentation is characteristic of adrenal In alkaptonuria, defective activity of homogentisic insuffi ciency and may be the fi rst substantial clue acid oxidase leads to the accumulation of homogenti- in late childhood to the presence of adrenoleu- sic acid, which is then oxidized to form black insoluble kodystrophy. pigment. Pigment deposition is greatest in cartilage; so it is prominent in the ears. It may also be seen early in the sclerae and in the nose, initially as salt and pepper Adrenal insuffi ciency is also seen in those patients spots, but later these may become confl uent. In an with glycokinase defi ciency who have a contiguous older patient it may be widely distributed, especially gene deletion syndrome. Dependent on the size of the distally on the fi ngers. Deposition in joint cartilage deletion, some also have Duchenne muscular dystrophy leads to debilitating early osteoarthritis. The skin may and/or ornithine transcarbamylase defi ciency. Likewise, serve as an alerting clue to the diagnosis. adrenal insuffi ciency can cause skin hyperpigmenta- Skin hyperpigmentation is characteristic of adrenal tion in KearnsÐSayre syndrome. insuffi ciency and this may be the fi rst substantial clue In hereditary hemochromatosis, bluishÐgray hyperin late childhood to the presence of adrenoleukodystro- pigmentations are seen on the light exposed sebostatic phy or, later in life, of adrenomyeloneuropathy, an scaling skin areas. Bronze diabetes with liver cirrhosis, X-linked recessive trait characterized by progressive hypogonadism, and loss of libido are found in more demyelination of the nervous system, resulting in the advanced cases. Hyperpigmentation of mucous mem- accumulation of very-long chain fatty acids in the ner- branes and conjunctival membranes occur in 15Ð20% of vous system, adrenal gland, and testes, which disrupts patients. There can be a loss of axillary and pubic hair normal activity. because of hepatotesticular insuffi ciency. Besides skin C9 Skin and Hair Disorders 211 signs, fully developed clinical manifestations of heredi- prevent complications associated with prolonged tary hemochromatosis include a multiorgan involve- unprotected exposure to sunlight allowing recognition ment affecting liver, heart, pancreas, endocrine organs, of families at risk for rare heritable disorders associ- and joints. Patients with Wilson disease suffer from pro- ated with photosensitivity. Drugs and chemicals may gressive severe dystonia, and skin manifestations are char- interact with UV to induce photosensitivity, and recog- acterized by reticulated brownish hyperpigmentation of nition of the associated reaction patterns greatly assists the lower legs, blue lunulae, and corneal pigmentation clinical classifi cation of these disorders, which include known as KayserÐFleischer ring. There is additionally a some metabolic diseases, the DNA repair defi cient dis- premature graying of the hair and development of liver orders, and other genodermatoses. cirrhosis. Cutaneous hyperpigmentation can be observed Photosensitive skin lesions characterize many of in Gaucher type 1. the porphyries, a clinically and genetically heterogeneous group of diseases arising from enzymatic defects along the pathway of porphyrin-heme biosynthesis. In erythropoietic protoporphyria, photosensitivity begins C9.7 Photosensitivity (Table C9.6) early in life. Skin signs can occur acutely with burning, stinging, and pruritus in sun-exposed areas accompa- The phenotypic expression of diseases with increased nied by pain in the extremities. These are followed by photosensitivity mainly depends on UV or light expo- erythema, edema, erosions, and scarring. The vesicular sure. Early recognition and prompt diagnosis may lesions in response to sun tend to be smaller and more

Table C9.6 Photosensitivity Disease MIM number Photosensitivity Additional dermatological Hair manifestations Erythropoietic 177000 Light-sensitive Edema Mild scarring protoporphyria dermatitis Itching Burning Erythema Congenital erythropoietic 263700 Photosensitivity Conjunctivitis Hypertrichosis porphyria Blistering Corneal scarring Scarring Red stained teeth Alopecia Mutilating skin deformity Loss of eyelashes Pseudoscleroderma Loss of eyebrows Hyperpigmentation Hypopigmentation Porphyria cutanea 176090 Photosensitivity Mechanically fragile skin Facial tarda type I hypertrichosis Porphyria cutanea 176100 Blisters in sun-exposed tarda type II areas Pseudoscleroderma Hyperpigmentation in Fingernail onycholysis Alopecia sun-exposed areas Porphyria variegata 176200 Photosensitivity Coproporphyria 121300 Photosensitivity Hartnup disorder 234500 Light-sensitive Atrophic glossitis dermatitis Tryptophanuria with 276100 Cutaneous dwarfi sm photosensitivity SmithÐLemliÐOpitz 270400 See Table C9.1 syndrome 212 C. Dionisi-Vici et al. patchy than in the other porphyrias of childhood. They conversion of tryptophane to kynurenine.6pThe differ- leave tiny areas of fl at depressed atrophy of skin that ential diagnosis of photosensitive dermatoses includes may be very subtle but are telltale markers of the dis- the different forms of xeroderma pigmentosum and of ease. Unusual late complications include biliary tract Fanconi anemia, Cockayne syndrome type A (MIM stones and hepatic failure. The most dramatic and ear- 216400) and B (MIM 133540), Bloom syndrome liest in onset is congenital erythropoietic porphyria, (MIM 210900), and trichothiodystrophy (601675). All the disease in which there is pink urine and red teeth. of these disorders of DNA repair are important to rec- These patients may also have hemolytic anemia and ognize because of their frequent complication by splenomegaly. The skin lesions are vesicular and neoplasia. bullous. The skin appears quite fragile, and ulcers or erosions appear. There may be residual scarring and alternating hyperpigmentation and depigmentation. Remember Mutilation of fi ngers, nasal tips, and ears may eventually occur. Patients with porphyria cutanea tarda type The differential diagnosis of photosensitive derma- I, the heterozygous, autosomal dominant form of toses includes the different forms of DNA repair uroporphyrinogen decarboxylase-III defi ciency, pres- disorders. ent fi rst symptoms in adulthood. The skin is fragile and minor trauma leads to erosions. Patients also develop hepatic siderosis. In patients with hepatoerythropoietic C9.8 Hair Disorders porphyria (also called porphyria cutanea tarda type II), the homozygous form of uroporphyrinogen decarboxylase-III defi ciency, onset is neonatal or shortly there- C9.8.1 Hair Shaft Abnormalities after. Patients may also have hemolytic anemia and splenomegaly. The urine may be black or pink. The Hair shaft abnormalities are highly heterogeneous and skin is fragile and develops vesicles and blisters in range from changes in color, density, length, and struc- response to sun exposure. Hypertrichosis about the ture of the hair to absence of the hair (alopecia). The face is common. Liver disease is a complication. hair of patients with hair shaft diseases that may occur Patients with variegate porphyria have abdominal and as localized or generalized disorders feels dry and neurological crises. Onset is from puberty to adult- looks lusterless. Alopecia may be universal (loss of hood. Late changes in the skin may be pseudosclero- scalp hair, eyebrows and eyelashes), total (loss of scalp dermatous. Patients with hereditary coproporphyria hair), and partial. have hemolytic anemia and abdominal and neurologi- Trichorrhexis nodosa is a characteristic sign in cal crises. Onset is in adulthood. Abdominal or psychi- some patients with argininosuccinic aciduria. The atric symptoms may be precipitated by drugs that patient is recognized for an appearance at a distance of increase hepatic cytochrome P450. alopecia, but on close examination it is clear that very Hartnup disease is characterized by a pellagra-like short hairs are abundant. The hair shafts are fragile and light-sensitive rash, cerebellar ataxia, emotional insta- break easily. A longer hair under the microscope dis- bility, and aminoaciduria. Patients have a transport displays the characteristic nodules. Abnormal appearance order involving the intestine and the renal tubule, and of the hair is also seen in Menkes disease. The typical failure to absorb tryptophan leads to the dermatologi- appearance is that of pili torti, in which the hair shaft is cal picture of pellagra. Cutaneous manifestations of twisted. These patients may also have trichorrhexis this disease have been rare in the U.S., presumably nodosa or monilethrix in which there is segmental nar- because most eat such a high-protein diet that defi - rowing of the hair shaft. These hairs tend to break ciency is avoided. Tryptophanuria with dwarfi sm readily too, but the patient never appears to have alope- was described in three siblings with mental defect, cia. As a consequence of reduced tyrosinase activity, cutaneous photosensitivity, and gait disturbance resem- hair and skin appear hypopigmented. In addition, bling cerebellar ataxia. The clinical features resembled patients show skin laxity. Menkes disease, also called Hartnup disease but the chemical fi ndings were kinky hair disease, is a devastating cerebral degenera- different and the defect was thought to concern the tive disease, with refractory seizures, bone lesions, C9 Skin and Hair Disorders 213 skin and joint laxity, and tortuous cerebral arteries. In the occipital horn syndrome, the milder allelic variant of Menkes disease, the hair appears coarse (Fig. C9.9) and the skin is soft, mildly extensible and redundant with easy bruisability. In some cases of congenital disorder of glycosylation type 1 hair aspects are unusual, they appear sparse, slow growing, lacking luster and coarse in texture. Enhanced fragility in the form of trichorrexis nodosa, torsion of the shaft along the longitudinal axis, and pili torti has also been observed (Silengo et al. 2003). The classic presentation of sulfi te oxidase defi - ciency involves neonatal seizures, progressive encephalopathy, dislocation of lenses, and death at an early age. Dermatological signs include mild eczema and fi ne hair.6pAlopecia totalis is the characteristic appearance of holocarboxylase synthetase (Fig. C9.10). Fig. C9.10 Alopecia totalis with absence of eyebrows and eye- Patients have no hair on the head, no eyebrows, eyelashes in a child with multiple carboxylase defect due to holocarboxylase synthetase defi ciency lashes, or lanugo hair. Patients with biotinidase defi - ciency have patchy alopecia in the pattern of the patient with acrodermatitis enteropathica. Patients with organic acidurias, such as methylma- As shown in Table C9.7, in many other metabolic lonic aciduria, propionic aciduria, or maple syrup urine diseases already discussed in previous paragraphs of disease, all of whom must be treated with very strict this chapter, patients may have hair shaft abnormalities restriction of the intake of protein, may have fi ne hair or alopecia. or develop alopecia when the protein restriction is too stringent or when intercurrent infection increases demand (see Sect. C9.4). C9.8.2 Hypertrichosis (Table C9.8)

Hirsutism or hypetricosis is a common ﬁ nding in several lysosomal storage diseases. In most cases, patients have a characteristic coarse facial features, neurological abnormalities, intellectual delay, dysostosis multiplex, and hepatosplenomegaly.

Remember Hirsutism, hypetricosis, coarse facial features, neurological abnormalities, intellectual delay, dysostosis multiplex, and hepatosplenomegaly are common ﬁ ndings in several lysosomal storage diseases.

In the wide spectrum of clinical abnormalities of mitochondrial diseases, diffuse hypertichosis is a typical Fig. C9.9 Coarse, sparse, and fragile hair in Menkes disease sign of the Leigh syndrome encephalomayopathy with 214 C. Dionisi-Vici et al.

Table C9.7 Hair shaft abnormalities Disease MIM number Skin Hair Argininosuccinic aciduria 207900 Trichorrhexis nodosa Dry brittle hair Menkes disease 309400 Hypopigmentation, skin laxity Steely, kinky, sparse hair Occipital horn syndrome 304150 Soft, extensible and Coarse hair redundant skin Easy bruisability CDG type Ia 212065 See Table C9.4 Sparse hair Trichorrhexis nodosa Pili torti Sulfi te oxidase defi ciency 272300 Mild eczema Fine hair Mitochondrial diseases See Table C9.4 Hypertrichosis Alopecia Thin, dry, brittle, sparse hair Trichothiodystrophy Trichorrexis nodosa Pili torti Biotinidase defi ciency 253260 See Table C9.2 Alopecia Holocarboxylase 253270 Loss of eyelashes synthetase defi ciency 210210 Loss of eyebrows 3-Methylcrotonyl-CoA carboxylase defi ciency Congenital erythropoietic 263700 See Table C9.6 Hypertrichosis porphyria Alopecia Loss of eyelashes Loss of eyebrows Porphyria cutanea 176090 See Table C9.6 Facial hypertrichosis tarda type I Porphyria cutanea 176100 Alopecia tarda type II Hemochromatosis 235200 See Table C9.5 Alopecia Adrenoleukodystrophy/ 300100 See Table C9.5 Scarce and thin hair adrenomyeloneuropaty Alopecia ChanarinÐDorfman syndrome/ 275630 See Table C9.3 Diffuse alopecia neutral lipid storage disorder Rhizomelic chondrodysplasia 215100 See Table C9.3 Alopecia punctata type I CHILD syndrome 308050 See Table C9.3 Unilateral alopecia ConradiÐHuenermann 302960 See Table C9.3 Coarse, sparse hair syndrome Patchy areas of alopecia Sparse eyebrows Sparse eyelashes Acrodermatitis 201100 See Table C9.2 Alopecia of scalp enteropatica Alopecia of eyebrows Alopecia of eyelashes C9 Skin and Hair Disorders 215

Table C9.8 Hypertrichosis Disease MIM number Skin Hair MPS type I 607014 Skin thickening Hypertrichosis MPS type II 309900 Coarse facies Synophrys MPS type IIIA 252900 Pebbly skin lesions on back, Hirsutism upper arms MPS type IIIB 252920 Coarse hair MPS type IIIC 252930 Thigh blue cutaneous pigmentation MPS type IIID 252940 MPS type VI 253200 MPS type VII 253220 MPS type VIII 253230 Mucolipidosis type IIIA 252600 Gangliosidosis GM1 230500 Mannosidosis 248500 Sialuria 269921 Transaldolase defi ciency 606003 See Table C9.1 Hypertrichosis Leigh syndrome Ð SURF1 256000 Hypertrichosis mutations Infantile lactic acidosis/ 611224 Hypertrichosis SUCLG1 mutations Mitochondrial diseases See Table C9.4 See Table C9.4 cytochome-c-oxidase defi ciency and mutations in the provided by E. Morava). In addition to skin signs, SURF1 gene (Fig. C9.11). patients present with widely persistent fontanelles, slight oxycephaly, dental caries, frontal bossing, downward slanted palpebral fi ssures, hip dislocation, scoliosis, and inguinal hernia. Isoelectrofocusing of C9.9 Skin Laxity (Table C9.9) serum transferring shows a type II CDG pattern and the disorder is caused by loss-of-function mutations Type II autosomal recessive cutis laxa (MIM 219200), in the ATP6V0A2 gene, which encodes the alpha-2 characterized by loose skin with redundant folds, subunit of the V-type H + ATPase. The occurrence of slow return on stretching and unaffected facial skin, mutations in the same gene in wrinkly skin syndrome is a recently discovered defect of N-glycosylation at (MIM 278250) indicates that autosomal recessive the level of Golgi apparatus (Fig. C9.12, kindly cutis laxa type II and some cases of wrinkly skin

Table C9.9 Skin laxity Disease MIM number Skin Hair Type II autosomal recessive cutis 219200 Loose skin with redundant folds laxa Delta-1-pyrroline-5-carboxylate 138250 Skin hyperelasticity synthetase deﬁ ciency Menkes disease 309400 See Table C9.7 See Table C9.7 Occipital horn syndrome 304150 See Table C9.7 See Table C9.7 Transaldolase deﬁ ciency 606003 See Table C9.1 See Table C9.8 Lysinuric protein intolerance 222700 See Table C9.2 216 C. Dionisi-Vici et al.

Fig. C9.11 Diffuse hypertichosis in a child with Leigh syn- Fig. C9.12 Characteristic skin features in a child with type II drome, cytochome-c-oxidase defi ciency, and mutations in the autosomal recessive cutis laxa (kindly provided by Morava, SURF1 gene Nimegen, The Netherlands) syndrome may represent variable manifestations of Key References the same genetic defect. Skin hyperelasticity, lax joints, cataract, and mental Birch-Machin MA (2000) Mitochondria and skin disease. Clin retardation along with fasting hyperammonemia, hypo- Exp Dermatol 25:141Ð146 prolinemia, hypocitrullinemia, and hypoornithinemia Bodemer C, Rötig A, Rustin P et al (1999) Hair and skin disorders as signs of mitochondrial disease. Pediatrics 103: are the characteristic fi ndings of delta-1-pyrroline-5- 428Ð433 carboxylate synthetase defi ciency (MIM 138250) de Koning TJ, Klomp LW (2004) Serine-defi ciency syndromes. In Menkes disease, the skin appears loose and Curr Opin Neurol 17:197Ð204 redundant, particularly at the nape of the neck and on Dionisi-Vici C, De Felice L, el Hachem M et al (1998) Intravenous immune globulin in lysinuric protein intoler- the trunk. In occipital horn syndrome, the less severe ance. J Inherit Metab Dis 21:95Ð102 variant of Menkes disease, clinical fi ndings include Dyer JA, Winters CJ, Chamlin SL (2005) Cutaneous fi ndings in cutis laxa and joint hypermobility.. Skin laxity can also congenital disorders of glycosylation: the hanging fat sign. be observed in transaldolase defi ciency and in lysinu- Pediatr Dermatol 22:457Ð460 Goldsmith LA (2003) Xanthomatoses and lipoprotein disorders. ric protein intolerance. In: Eisen AZ, Wolf K et al (eds) Fitzpartick’s dermatology in Differential diagnosis of skin laxity includes the general medicine, McGraw-Hill, New York subtypes of EhlerÐDanlos syndrome, an umbrella term Guallar JP, Vilà MR, López-Gallardo E et al (2006) Altered which encompasses a heterogeneous group of connec- expression of master regulatory genes of adipogenesis in lipomas from patients bearing tRNA(Lys) point muta- tive tissue disorders with distinct inheritance patterns, tions in mitochondrial DNA. Mol Genet Metab 89: genetic defects, and prognostic implications. 283Ð285 C9 Skin and Hair Disorders 217

Howard R, Frieden IJ, Crawford D et al (1997) Methylmalonic Silengo M, Valenzise M, Pagliardini S. et al (2003) Hair changes acidemia, cobalamin C type, presenting with cutaneous in congenital disorders of glycosylation (CDG type 1). Eur J manifestations. Arch Dermatol 133:1563Ð1566 Pediatr 162:114Ð115 Online Mendelian Inheritance in Man. http://www.ncbi.nlm.nih. Valayannopoulos V, Verhoeven NM, Mention K et al gov/sites/entrez (2006) Transaldolase deﬁ ciency: a new cause of hydrops Shrinath M, Walter JH, Haeney M et al (1997) Prolidase deﬁ - fetalis and neonatal multi-organ disease. J Pediatr 149: ciency and systemic lupus erythematosus. Arch Dis Child 713Ð717 76:441Ð444 Physical Abnormalities in Metabolic Diseases C10

Ute Moog, Johannes Zschocke, and Stephanie Grünewald

Key Facts › Many disorders from the rapidly growing group of inherited glycosylation defects › Inborn errors of metabolism have been delin- (CDG) present with multi-organ involvement, eated as important causes of syndromes with mostly accompanied by neurologic symptoms, multiple congenital anomalies and/or dysmor- while defects of cholesterol biosynthesis result phism, often in association with mental in congenital malformation syndromes. retardation. › Finally, physical abnormalities can also be char- › The most frequent are lysosomal storage dis- acteristic features of mitochondrial disorders orders. Connective tissue, nervous tissue and, and homocystinuria. parenchymatous organs are primarily affected. It is, however, a misconception that all lysosomal storage disorders generally cause visceromegaly and characteristic skeletal changes. › A substantial group affects predominantly or C10.1 General Remarks exclusively the central nervous system, causing chronic progressive neurologic and psy- Over the last decades, the connection between physical chiatric dysfunction. (morphological) anomalies and inborn errors of metab- › Similarly, peroxisomal disorders present in olism has seen considerable re-evaluation. Traditionally, many different ways, including dysmorphisms inherited metabolic disorders were presumed to be and relentless neural degeneration in infancy characterised primarily by abnormal function leading or early childhood, small stature, adrenal fail- to abnormal laboratory parameters, an episodic or pro- ure, spino-cerebellar degeneration or renal gressive disease course and sometimes multi-organ stones. involvement. In contrast, primary genetic syndromes with multiple congenital anomalies (MCA) and/or dysmorphism, often in association with mental retardation (MR), were regarded to be caused by different groups of disorders. This apparently easy distinction changed most clearly in 1993 when Smith–Lemli–Opitz syndrome (SLOS), a classical MCA/MR syndrome without symptoms indicating a metabolic disease, was shown to be a disorder of cholesterol biosynthesis, thus bridging dysmorphology and metabolism. Subsequently, a variety of conditions with physical U. Moog ( ) abnormalities such as dysmorphic signs, brain and Institute of Human Genetics, Ruprecht-Karls-University, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany other congenital malformations, as well as eye and e-mail: [email protected] skin abnormalities, were shown to be due to primary

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 219 DOI: 10.1007/978-3-540-74723-9_C10, © Springer-Verlag Berlin Heidelberg 2010 220 U. Moog et al. enzyme deﬁ ciencies. In this chapter, we discuss which typically causes organomegaly and other morphologi- inborn errors of metabolism might present with physical features. There are no acute metabolic crises, cal abnormalities and which speciﬁ c anomalies can be although some conditions, notably Fabry disease, may expected, relating them to the underlying pathogenetic have acute presentations. mechanisms.

Remember C10.2 Defi nitions Cardinal clinical features of lysosomal disorders comprise: For this chapter, physical abnormalities are confi ned to ¼ Hydrops fetalis those detectable by physical examination and exclude ¼ Organomegaly and visceral disease both structural brain and eye anomalies. They com- ¼ Dysostosis multiplex and dysmorphism prise congenital malformations which are disorders of ¼ CNS disease – regression blasto- and organo-genesis, and minor anomalies which result from disturbed phenogenesis. Metabolic diseases sensu stricto are defi ned as inborn distur- In most cases, the typical clinical course is character- bances of biosynthesis or degradation of substances ised by normal appearance at birth and normal devel- within metabolic pathways; classical examples are opment for a variable time span. However, the age of genetic enzymopathies. Metabolic diseases in this onset varies greatly depending on the underlying dis- sense are essentially different from primary distur- order and the amount of residual enzyme activity; non- bances of structural proteins and their modifi cation, immunologic hydrops fetalis is the most severe form and from channelopathies and membrane diseases; and has the earliest (prenatal) onset. Severely affected consequently, these will not be considered. In addition, children may show facial dysmorphism or cardiomyo- primary endocrinopathies or disorders of hormone pathy at birth. More frequently, patients present with syntheses are not brought into focus; they may be muscular hypotonia and developmental delay. Coarse mediated by enzyme defi ciencies, but the main patho- facial features, typical skeletal changes (radiologically genetic mechanism is alteration of other effector mol- classifi ed as dysostosis multiplex) or thickening of the ecules often involved in intercellular signalling or skin in addition to hepatosplenomegaly and cardio- cellular regulation. Although arbitrary to some extent, megaly are important physical signs. Lysosomal stor- these defi nitions help to narrow down the vast fi elds of age disorders with predominant central nervous system physical anomalies and of diseases with biochemical involvement frequently present with ataxia, hyperex- abnormalities. In consequence, six subgroups and citability and spasticity; opthalmological examination mechanisms can be identifi ed which are presented in may reveal a characteristic cherry-red spot in the mac- this chapter illustrated by prototypic examples. ula region in some disorders. A differential diagnosis of typical fi ndings in lysosomal storage diseases is given in Table C10.1; however, clinical presentation is generally variable. C10.3 Lysosomal Storage Disorders: Diagnostic work-up in children with suspected lys- Disturbed Degradation of osomal storage disorders includes: Macromolecules in Lysosomes • Physical examination • Ultrasound of parenchymatous organs Genetic defects of lysosomal enzymes cause the accu- • Neurological examination, hearing tests, cranial mulation of incompletely degraded macromolecules in MRI scan to consider lysosomes resulting in progressive impairment of the • X-ray examinations for dysostosis multiplex function of affected cell systems such as connective • Cardiological examination (ECG, echocardiography) tissue, solid organs, cartilage, bone and, above all, • Opthalmological examinations (retina, macula, lens the nervous system. The storage of macromolecules and cornea) C10 Physical Abnormalities in Metabolic Diseases 221

attenuated, juvenile and adult forms of the disease. Remember The liver usually is not enlarged except in cardiac fail- Characteristic features of dysostosis multiplex are ure; CK is usually highly elevated. Enzyme replace- as follows: ment therapy is available. ¼ Lateral skull – enlarged sella turcica; thickened Mucopolysaccharidoses (MPS) arise from the lack diploic space of specifi c lysosomal enzymes involved in the degra- ¼ Hands – proximally pointed, distally broadened dation of GAGs, long chains of sulphated or acetylated metacarpals amino sugars attached to a protein skeleton. GAGs are ¼ Lateral spine – hypoplastic L1 major components of the viscous extracellular matrix. There are three major types of GAGs, and the clinical presentation of the different MPS types are linked to the tissue distribution of these subtypes. Dermatan sul- There are few general laboratory tests for lysosomal phate is found in connective tissue and skin as well as disorders. Several conditions are associated with bone, cartilage and tendons, internal organs, blood increased concentrations of glycosaminoglycans vessels, cornea and other tissues. Disorders affecting (GAGs) or oligosaccharides in the urine. Microscopic dermatan sulphate breakdown show typical progres- examination of fresh leukocytes (bed-side blood smear, sive morphological changes as seen, for example, in not from an EDTA tube), bone marrow cells or biopsies Hurler’s disease but are not necessarily associated with may show vacuoles in some conditions. Chiotriosidase, mental decline (intelligence is usually normal, e.g. in a chitinolytic enzyme and marker of monocyte/mac- MPS type VI, Maroteaux–Lamy). In contrast, disor- rophage activation, is highly elevated in several lyso- ders affecting heparan sulphate (a ubiquitous constitu- somal storage disorders including Gaucher and ent of glycoproteins and the basal membrane) are Niemann–Pick C diseases. It may be used for screening usually associated with mental retardation but, as in as well as monitoring of treatment; however, results Sanfi lippo’s disease (MPS type III), do not always may be false negative due to a common null allele of show the characteristic morphological changes. the CHIT1 gene, and chitotriosidase activity is also Similarly, in MPS type IV (Morquio), the defi cient increased in a large number of non-metabolic condi- breakdown of keratan sulphate, a constituent of bone, tions including atherosclerosis, sarcoidosis, b-thalas- cartilage and the cornea, causes a severe skeletal dys- semia or malaria. Reliable confi rmation or exclusion of plasia but usually is not associated with mental retar- lysosomal storage disorders involves the measurement dation. Most patients are recognised by GAG analysis of specifi c enzyme activities in leukocytes or fi broblasts in the urine but results may be false negative particu- and/or molecular genetic analysis. larly in MPS types III and IV requiring electrophoretic There has been considerable progress in the treat- analysis; the different MPS types may be distinguished ment of lysosomal storage disorders in the last years. through the electrophoretic separation of the different Bone marrow transplantation has proven benefi t in pre- GAGs. All MPS except the X-linked type II (Hunter) symptomatic patients in some disorders (e.g. MPS I, are inherited as autosomal recessive traits. late-onset Krabbe and metachromatic leukodystrophy), but not in others (MPS III, MPS IV). Enzyme replacement therapy is available for Gaucher diesease, Fabry disease and MPS I. For other disorders, treat- Disease Info: Hurler Disease, Scheie Disease ment is largely symptomatic. In Hurler disease (MPS IH,OMIM 607 014), the One of the most common lysosomal disorders, degradation of mucopolysaccharides, in particular Pompe disease, a glycogen storage disease caused by of dermatan sulphate, is disturbed due to a defi - the defi ciency of acid maltase, usually shows no obvi- ciency of a-l-iduronidase. It is the classical form ous morphological anomalies apart from sometimes an of MPS. Substrate accumulation takes place in both enlarged tongue. It is characterised by progressive lysosomes and the extracellular matrix, in particu- muscle disease which in the severe infantile form pres- lar those of chondrocytes (causing disturbance of ents in the neonatal period and leads to cardiac failure endochondral ossifi cation), hepatocytes, dermis and death usually in the fi rst year of life. There are also 222 U. Moog et al.

Table C10.1 Typical clinical ﬁ ndings and relevant biochemical tests in lysosomal storage diseases Neuro Coarse facial features Dysostosis multiplex Organo megaly Makrog lossia Cardiac involvement Hydrops fetalis Angio keratoma Mental retardation

Mucopoly saccharidoses Hurler (MPS I) ++ ++ + ++ + ++ Scheie (MPS I, atten.) + + + + (+) Hunter (MPS II) ++ (+) + + + ++ Sanﬁ lippo (MPS III) (+) (+) (+) (+) ++

Morquio (MPS IV) + (+) + + Maroteaux–Lamy ++ + (MPS VI) Oligosaccharidoses Fucosidosis ++ (+) (+) + (+) ++ a-Mannosidosis ++ + + (+) ++ b-Mannosidosis + (+) + Aspartyl + (+) (+) (+) (+) + glucosaminuria Schindler + Sialidose type I + Sialidose type II ++ (+) + + (+) + ++ Sphingolipidoses

GM1 gangliosidosis ++ + + + (+) + + ++ Tay-Sachs, Sandhoff (+) ++ Metachromatic ++ leukodystrophy Krabbe ++ Niemann–Pick ++ + + Gaucher type I ++ Gaucher type II ++ + ++ Fabry ++ Angiokeratoma = red to dark blue lesions ( (< 1 mm, slightly hyperkeratotic, do not blanche on pressure) mostly on buttocks, genitalia, lower trunk, thighs); Cherry-red spot = in the macula region; Cardiac involvement = cardiomyopathy, valve lesions, coronary artery disease; Vacuolated lymphocytes = typical vacuoles or evidence of storage in lymphocytes. F ﬁ broblasts; L leukocytes; S serum; M muscle ++ prominent feature, + often present, (+) sometimes present

and subcutis and synovia. Infl ammation and apop- the nose, full cheek and lips, a large tongue, hyper- tosis in cartilage and synovial tissue is caused by trophy of the gums and a mouth held open. The stimulation of lipopolysaccharide signalling path- patients are generally hirsute and have thick and ways. Somatic features affecting the skeleton, abundant hair. A protuberant abdomen, recurrent facies and internal organs, as well as neurologic (umbilical) hernias, corneal clouding, optic nerve features, evolve within the fi rst year of life. Death head swelling and retinal degeneration, frequent occurs by age 1–10 years, with a mean age at death ear, nose and throat infections, decreasing growth of 6 years. The craniofacial phenotype is character- velocity by the age of 2 years with resulting short ised by progressive coarsening apparent at 3–6 stature, short neck, and short, broad hands and months, a large scaphocephalic head with bulging feet, joint stiffness and cardiac disease are all fre- frontal bones, depressed nasal bridge, broad tip of quent physical fi ndings. Radiologically, a pattern of C10 Physical Abnormalities in Metabolic Diseases 223

Eyes Diagnosis Spasticity Peripheral neuropathy Myoclonic seizures Corneal clouding Cherry-red macuar spot Vacuolated lymphocytes (urine) GAG elevated Pathol. Oligosac- charides Enzyme studies in: OMIM

++ + L/F 607014 + + L/F 607016 + S/L/F 309900 + + (+) L/F 252920

(+) + L/F 253000 + + L/F 253200

+ + + + L/F 230000 (+) ++ + + L/F 248500 ++ + L/F 248510 (+) (+) + L/F 208400

+ + + L/F 104170 + + ++ ++ + + F 256550 (+) ++ + + F 256550

(+) + ++ + L/F 230500 + + ++ + L/F 272750 + ++ (+) L/F 250200

+ ++ (+) L/F 245200 (+) (+) (+) + F 257200 + L/F 230800 + + + L/F 230900 S/L/F 301500

skeletal changes called “dysostosis multiplex” may form of a-l-iduronidase defi ciency is denoted be seen. As in other lysosomal storage diseases, Scheie disease (MPS IS, OMIM 607 016); it has a progressive dermal melanocytosis, e.g. extensive much later onset, a milder course and symptoms Mongolian spots, may be a clue to the diagnosis, restricted to milder somatic features; stature is nor- particularly in infants with darker skin types. mal. Hurler–Scheie syndrome (MPS IH/S, OMIM Dermal melanocytosis results from arrest of trans- 607 015) has an intermediate phenotypic expres- dermal migration of melanocytes from the neural sion. Bone marrow transplantation has proven ben- crest to the epidermis. Pathogenetically, abnormal efi t in pre-symptomatic patients with MPS I. increases in nerve growth factor (NGF) activity Enzyme replacement therapy is licensed and avail- caused by the binding of accumulating metabolites able but has no benefi cial effect on brain manifesta- to the tyrosine kinase-type receptors for NGF has tion as the intravenously applied enzyme does not been discussed as a primary cause. The attenuated cross the blood-brain barrier. 224 U. Moog et al.

Dysostosis Multiplex severe MPS I, these interventions may stabilise but not improve joint function and stiffness. The benefi t Dysostosis multiplex is the typical skeletal abnor- of physical therapy in patients with severe orthope- mality found in MPS I and other lysosomal disor- dic compromise is controversial. Occupational ders. In severe MPS I, defective ossifi cation may therapists can improve the quality of life and func- not be evident until the characteristic gibbus defor- tional independence; the most effective occupa- mity of the lumbar spine is apparent at 6–14 months tional therapies are those that patients can perform of age. As vertebrae become progressively fl attened on their own. and beaked, spinal deformities, including kyphosis, (Bernhard Zabel, University Children’s Hospital, scoliosis and kyphoscoliosis, may develop. Hips Freiburg i. Brsg., Germany) may be affected, resulting in dysplasia or subluxation. Long-bone irregularities produce valgus and varus deformities, and genu valgum may occur in Oligosaccharidoses are disorders in the breakdown of the knees. Phalangeal dysostosis and synovial thick- complex carbohydrate side-chains of glycosylated ening produce the characteristic claw deformity and proteins (glycoproteins), leading to increased concen- trigger digits. Carpal tunnel syndrome and phalan- tration of oligosaccharides in the urine. They are less geal involvement diminish hand function. By the common than MPS which they resemble clinically time severely affected children are 2 years of age, with variable coarsening of the face, skeletal deformi- joint stiffening and progressive arthropathy affect ties, hepatomegaly and sometimes corneal clouding. all joints. Radiographs obtained at birth can detect Psychomotor development is usually delayed; progres- dysostosis in some patients with MPS I. sive neurological symptoms and seizures are common. Patients with attenuated MPS I have progressive Early presentation with hydrops fetalis or neonatal car- arthropathy, which ultimately leads to loss of joint diomegaly is more frequent than in patients with MPS. range of motion. Patients present with mild to severe Elevation of urinary oligosaccharides is also found in skeletal involvement and tend to have short stature. several sphingolipidoses such as GM1 and GM2 gan- Kyphosis and/or scoliosis are frequent presenting gliosidoses and galactosialidosis. symptoms, with attendant hip and back pain. Patients Sphingolipidoses are disorders in the breakdown of also may experience generalised pain and malaise, membrane lipids that are found throughout the body but which may be attributable to osteopenia and micro- are of special importance in the nervous tissue. Some fractures. Patients with moderate to severe skeletal sphingolipids are essential components of myelin sheaths; disease should be monitored by an orthopedic sur- others are prevalent particularly in the grey matter of the geon, preferably one who is familiar with MPS dis- brain. Sphingolipidoses thus usually present with pri- orders. Early detection of skeletal abnormalities, mary disturbances of the central or peripheral nervous such as kyphoscoliosis, before irreversible changes system; in addition, sphingolipids frequently accumulate occur may provide more surgical options. Spine in the reticuloendothelial system or other cells. Typical deformities may require fusion; acetabular hip dys- clinical features include progressive psychomotor retar- plasia can be addressed with osteotomy and genu dation and neurological problems such as epilepsy, ataxia valgum with epiphyseal stapling. Flexor tendon or and/or spasticity. Hepatosplenomegaly is not uncommon. carpal tunnel release can provide relief and the Progressive facial coarsening and dysostosis multiplex return of some hand function. Premature cessation are found in GM1 gangliosidosis; liver disease is a char- of skeletal growth may occur in MPS I and should acteristic feature of Niemann–Pick disease; a lysosomal be taken into account when surgical procedures are leukodystrophy is found in Krabbe disease and metach- being planned. Physical therapists can assess the romatic leukodystrophy. Several sphingolipidoses show degree of joint restriction and develop interventions a cherry-red macula spot, there may be foam cells in the to maintain joint function and muscle strength. In bone marrow or vacuolated lymphocytes. Neurological patients with attenuated MPS I, joint stiffness and and neuroradiological fi ndings are not always specifi c. pain may be lessened through passive and active The diagnosis is made by enzyme or molecular analyses. range-of-motion exercises and hydrotherapy. In All sphingolipidoses except Fabry disease are inherited as autosomal recessive traits. C10 Physical Abnormalities in Metabolic Diseases 225

of very long-chain fatty acids (VLCFA) and related Disease Info: Gaucher Disease, Fabry Disease substances, a-oxidation of 3-methyl fatty acids, and The most common form of Gaucher disease (OMIM biosynthesis of etherlipids, and bile acids. Peroxisomal 230800), the non-neuronopathic type I, is character- disorders are classifi ed into generalised defects of per- ised by accumulation of sphingolipds in cells of the oxisome biogenesis and single peroxisomal enzyme reticu loendothelial system leading to massive (hepato-) defi ciencies, but clinical phenotypes are variable and splenomegaly and anaemia, thrombocytopenia and overlapping. Generalised peroxisomal disorders are hematomas. Progressive bone changes may be associ- multi-system diseases that cause dysmorphic features ated with acute “bone crises” (pain, fever) as well as and skeletal changes (specifi cally proximal shortening with osteonecrosis, arthrosis and osteopenia. Long- of the limbs) as well as neurological and opthalmologi- term complications include growth retardation and cal abnormalities and hepatointestinal dysfunction. lung fi brosis. The diagnosis of Gaucher disease is sup- Morphological abnormalities such as rhizomelic chon- ported by highly elevated chitotriosidase activity in drodysplasia punctata are generally related to a defi - serum and the presence of characteristic Gaucher cells, ciency in biosynthetic functions and may be caused both for example, in bone marrow, and is confi rmed by by single enzyme defi ciencies in this pathway as well as enzyme analysis. Enzyme replacement therapy is generalised peroxisomal disorders. Variant, milder phe- available but again has no effect on brain manifesta- notypes are dominated by neurological manifestations. tion in the neuronopathic type II of Gaucher disease Patients may follow a neurodegenerative course after a (10% of cases). period of normal development. An important clue may Fabry disease (OMIM 301 500) typically pres- be white matter abnormalities consistent with the ents in the fi rst decade of life with episodes of acute pathologic appearance of sudanophilic leukodystrophy. painful acroparesthesias in arms or legs triggered, Peroxisomal disorders should therefore be included in for example, by physical exertion or temperature the differential diagnosis of infants and young children changes and lasting for hours or days. There may be with severe hypotonia and seizure disorders, but not in hypohidrosis and episodic fever; 80% of patients patients with mental retardation alone. show typical angiokeratoma skin lesions. Corneal Children with peroxisomal disorders may show and lenticular opacities as well as proteinuria are elevated transaminases and a tendency to hypoglyce- common. In middle age, deterioration of renal func- mia, as well as hypocholesterolemia and increased tion usually leads to renal failure and cardiovascular serum concentrations of iron and transferrin. Organic disease may occur. Intelligence is normal; there is acid analysis may reveal dicarboxylic aciduria, in par- no hepatosplenomegaly or facial dysmorphy. Fabry ticular elevated 2-hydroxysebacic acid refl ecting disease is inherited as an X-linked trait but carrier impaired peroxisomal b-oxidation. The specifi c labo- females frequently show complications in adulthood ratory fi nding in peroxisomal disorders (with the including progressive renal failure, hypertrophic exception of rhizomelic chondrodysplasia punctata) is cardiomyopathy and stroke. The diagnosis is con- an elevation of VLCFA in serum or cultured fi bro- fi rmed by a-galactosidase analysis or mutation test- blasts, usually determined by GC-MS. VLCFAs are ing. Enzyme replacement therapy is available. found consistently and reliably elevated in all disorders of peroxisome biogenesis as well as in single defects of VLCFA transport or b-oxidation. The ratio C10.4 Peroxisomal Disorders: of C26/C22 is the most sensitive index. The analysis of Disturbance of Generalised plasmalogens in erythrocytes is indicated in suspected rhizomelic chondrodysplasia punctata. If levels are Biogenesis or of Modifi cation decreased, the analysis of phytanic acid in plasma will of Substrates distinguish classical rhizomelic chondrodysplasia punctata, in which phytanic acid is increased (at least Many oxygen-dependent reactions take place in the after the fi rst months of life), from single enzyme peroxisomes to protect the cell against oxygen radicals; defects that may produce the same clinical and radio- logic features. If plasmalogens are normal, other disor- the produced H2O2 is metabolised by a catalase. Important peroxisomal functions include b-oxidation ders with punctate calcifications such as Warfarin 226 U. Moog et al. embryopathy (should be evident from the prenatal his- C10.5 Disorders of Catabolism tory) or X-linked chondrodysplasia punctata (due to a of Small Molecules: Substrate defect in of sterol biosynthesis) must be considered. Accumulation Can Affect Macromolecular Functions

Disorders of biosynthesis and catabolism of small Remember molecules, e.g. amino acids, organic acids, lipids, vita- The decisive marker for peroxisomal diseases, with mins and cofactors usually are not associated with the exception of rhizomelic chondrodysplasia punc- physical abnormalities. There are, however, excep- tata, is the elevation of VLCFA. Additional bio- tions, in particular when accumulating substrates or chemical investigations for peroxisomal disorders their metabolites affect macromolecules. A well- are not usually warranted. known example is homocystinuria (OMIM 236 200). The principal cause of homocystinuria is cystathionine b-synthase defi ciency leading to accumulation of homocystine which is the dimeric form of the amino acid homocysteine. The major clinical features of Disease Info: Zellweger Syndrome homocystinuria consist of MR, a characteristic mar- fanoid habitus but with joint limitations and genera- The prototypic disorder of disturbed peroxisomal lised osteoporosis, eye features such as ectopia lentis biogenesis with multiple enzyme abnormalities is and myopia, and vascular problems complicated by Zellweger syndrome (ZS, OMIM 214 100). It is thromboembolic events. However, clinical variability PEX caused by defects in a number of genes which is high. Homocystinuria may present as unspecifi c encode peroxins, proteins necessary for peroxi- MR, but about one-third of the patients have normal somal biogenesis and for the import of proteins. intelligence. Psychiatric problems are seen in about Children with ZS typically present in the neonatal 50% of patients. Physical fi ndings in homocystinuria period with severe hypotonia, no or little psycho- are partly due to the effects of the accumulating homo- motor development, seizures and other neurologic cystine on collagen, fi brillin and other elements of the disturbances. MRI of the brain often shows gross connective tissue. Lens dislocation results from dis- abnormalities refl ecting defects of early brain devel- ruption of disulphide bonds of fi brous proteins. The opment. The craniofacial features in ZS are charac- diagnosis is made through the identifi cation of highly teristic with macrocephaly, large fontanels and wide elevated concentrations of total homocysteine in sutures, a high forehead, fl at and square face, shal- plasma, or even a pathological sulphite test in urine. low supraorbital ridges, telecanthus, epicanthic About half of the patients respond well to substitution folds, broad and depressed nasal bridge, anteverted of the cofactor pyridoxine (vitamin B6), sometimes in nares, dysplastic ears and redundant skin in the combination with folic acid and betaine. In all other neck. The limbs show variable contractures; calci- patients including partial responders, a diet restricted fi ed stippling of the patella and other bones is seen in methionine, and intake of betaine must be initiated. in about half of the patients. Hepatomegaly, hepatic fi brosis and renal cysts are additional characteristic fi ndings. Eye anomalies include cataracts, nystag- C10.6 Disorders of Cholesterol mus, optic atrophy and retinal changes. Most patients with severe ZS die within the fi rst year of Biosynthesis: Disorders Affecting life; survivors often show severely impaired devel- Developmental Signalling opment, post-natal growth failure, blindness or Pathways severe visual handicap and signifi cant sensorineural hearing impairment, Facial dysmorphy in these Sterol synthesis defects present clinically as multi- cases may be less marked with a high forehead and, system disorders with dysmorphic features and vari- typically, attached ear lobules. able skeletal dysplasias; they should be considered in C10 Physical Abnormalities in Metabolic Diseases 227 cases of otherwise unexplained recurrent abortions and caused by the defi ciency of POR, a fl avoprotein that fetal dysmorphy. The biochemical diagnosis is not donates electrons to all P450 oxidoreductases including always straightforward: serum cholesterol is usually 3b-hydroxysterol 14a-demethylase. normal in all disorders except (sometimes) Smith– Mevalonic aciduria is caused by a defi ciency of Lemli–Opitz syndrome (SLOS, OMIM 270 400), and mevalonate kinase, which catalyses an early (pre- even specifi c sterol analysis may yield normal results. squalen) step in sterol biosynthesis. Affected children In these instances, the diagnosis can be reached by show facial dysmorphism and severe neurological mutation analysis or functional studies (fi broblasts abnormalities including progressive ataxia due to cer- cultured in sterol-free media). ebellar atrophy, psychomotor retardation and retinitis The identifi cation of SLOS as a disorder of choles- pigmentosa. In addition, there may be dystrophy and terol biosynthesis proofed that enzymopathies within recurrent crises with fever, skin rash, lymphadenopa- metabolic pathways can have a major impact on devel- thy and hepatosplenomegaly. Hyper-IgD syndrome opmental signalling pathways and lead to MCA/MR with recurrent febrile attacks represents an attenuated syndromes. Other disorders of post-sequalene choles- form of the disease, with residual enzyme function. terol biosynthesis include desmosterolosis (OMIM 602 398), X-linked dominant chondrodysplasia punctata (CDPX2; OMIM 302 960), CHILD syndrome Disease Info: Smith–Lemli–Opitz Syndrome (OMIM 308 050), lathosterolosis (607 330) and (SLOS) hydrops-ectopic calcifi cation-moth-eaten skeletal SLOS is the most common defect of the cholesterol dysplasia (HEM; 215 140). They are all associated pathway. It is caused by the defi ciency of 7-dehy- with congenital malformations. A summary of clini- drocholesterol reductase, the last step of cholesterol cal features in primary defi ciencies of sterol-biosyn- synthesis, leading to elevated levels of 7- and thetic enzymes is given in Table C10.2. Antley–Bixler 8-dehydrocholesterol and (often) reduced levels of syndrome or lanosterolosis, a multiple malformation cholesterol. As diagnostic markers, 7-and 8-dehy- syndrome with craniofacial dysmorphy, limb anoma- drocholesterol are measured by GC-MS in serum or lies and, in some patients, ambiguous genitalia, is

Table C10.2 Clincial features in disorders of distal (post-sequalene) sterol biosynthesis Clinical features Enzyme OMIM SLOS Psychomotor retardation, growth retardation, 7-dehydrocholesterol 270 400 microcephaly, facial dysmorphism, cleft palate, reductase genital anomalies in males, postaxial polydactyly, syndactyly of toes 2/3, photosensitivity Desmosterolosis Psychomotor retardation, facial dysmorphy, cleft 3b-Hydroxysterol 602 398 palate, multiple malformations, ambiguous D24-reductase genitalia, short limbs, osteosclerosis CHILD Unilateral ichthyotic skin lesions with a sharp Sterol-4-demethylase 308 050 syndrome demarcation at the midline of the trunk, stippled epiphyses on the affected side, limb defects; X-linked inheritance, lethal in males Greenberg Non-immune hydrops fetalis, severe 3b-Hydroxysterol 215140 dysplasia chondrodysplasia punctata, prenatally lethal D14-reductase (lamin B receptor; LBR gene) Pelger–Huët Psychomotor retardation, epilepsy (heterozygous 3b-Hydroxysterol 169400 anomaly mutations; additional skeletal abnormalities D14-reductase when homozygous for speciﬁ c mutations) (lamin B receptor; LBR gene) Chondrodysplasia Psychomotor retardation, rhizomelic short stature 3b-Hydroxysterol 302 960 punctata with asymmetric shortening of proximal limbs, D8,D7-isomerase Conradi–Hünermann stippled epiphyses, cataracts, ichthyosis; X-linked inheritance, lethal in males Lathosterolosis Severe malformations, overlapping the spectrum 3b-Hydroxysterol 607330 of Smith–Lemli–Opitz syndrome; lipid storage D5-desaturase 228 U. Moog et al.

phosphorylation, discussed in detail in Chap. D6. tissues. SLOS is characterised clinically by recogn- They are often multi-system disorders affecting neu- isable craniofacial features in childhood with micro- romuscular and other systems and as such may also cephaly and bitemporal narrowing, ptosis, broad affect morphogenesis. Whilst mitochondrial dys- nasal tip with anteverted nostrils, broad upper alve- function caused by mtDNA mutations is not usually olar ridges (and later, broad upper secondary inci- associated with physical abnormalities, mitochon- sors) and micrognathia. In addition, children with drial energy defi ciency resulting from mutations in SLOS show syndactyly of the second and third toes, nuclear encoded genes may occasionally result in genital anomalies in males, short stature, photosen- congenital malformations and (various) dysmor- sitivity and mostly severe mental retardation. The phism. An important disorder to consider is glutaric phenotypic spectrum, however, is very broad. Facial aciduria type II, also known as multiple acyl CoA appearances change with age and become less char- dehydrogenase defi ciency. acteristic in adulthood. Also, genital anomalies may Multiple acyl CoA dehydrogenase defi ciency is a become less prominent or even disappear. Structural defect in the mitochondrial transfer of electrons brain anomalies occur in 37%; not infrequently they from fatty acids to the electron transport chain, by belong to the holoprosencephaly spectrum, a failure genetic defects of the electron transfer fl avoprotein of normal bilobar development of the forebrain. (ETF) or ETF:ubiquinone oxidoreductase (ETFQO). Malformations of the heart, lungs and the gastroin- Attenuated forms of the disease are characterised by testinal system, may be associated. Dietary choles- predominant muscle and hepatic disease, but severe terol supplementation may have benefi ts especially multiple acyl CoA dehydrogenase defi ciency results on the behavioural problems in some SLOS patients. in the virtual absence of fatty acid oxidation with Trials combining high cholesterol diets with statin signifi cant physical abnormalities, overwhelming treatment so far have given inconclusive results. metabolic decompensation hence poor prognosis. Given the multiple biological functions of cho- Malformations are predominantly found in the brain, lesterol, the link between abnormal cholesterol kidneys, e.g. renal cysts and genitals. Craniofacial metabolism and abnormal morphogenesis in SLOS dysmorphism may be reminiscent of Zellweger syn- is complex and only partly unravelled. Some of the drome, as it is characterised by macrocephaly, large malformations relate to impairment of Sonic hedge- anterior fontanel, high forehead, fl at nasal bridge, hog (SHH) functioning, one of the major embry- increased inner canthal distance and malformed onic signalling pathways. Genetic disorders in this ears. An unpleasant acrid body odour may be noted. pathway cause holoprosencephaly, and mutations Acidosis, cardiomyopathy, liver dysfunction and in GLI3, one of the downstream effectors of SHH, epileptic encephalopathy all contribute to an early cause Pallister–Hall syndrome (PHS, 146 510) with fatal course. The diagnosis is made through the clinical features overlapping those of SLOS. The identifi cation of a wide variety of abnormal metabo- different conditions may thus be grouped to one lites in urinary organic acid analysis. syndrome family that share the same pathway and part of their phenotypic characteristics. Other SLOS features may relate to defi cient total sterols and alteration of cellular membrane properties, and to possible toxic effects of cholesterol precursors. C10.8 Glycosylation Defects: Disorders of Protein Modifi cation C10.7 Energy Defects: Mitochondrial Defects Potentially Affecting Glycosylation, i.e. the transfer of glycans to proteins or Morphogenesis lipids, is the most complex post-translational modifi cation of molecules in humans. Human disorders in gly- Mitochondrial disorders in a strict sense are disor- cosylation pathways are mainly known for N-linked ders of enzymes or enzyme complexes directly (attached to the amide group of an asparagine of the involved in the generation of ATP by oxidative protein) or O-linked glycans (to the hydroxyl group of C10 Physical Abnormalities in Metabolic Diseases 229 serine or threonine). N-glycosylation requires assembly Disease Info: Phosphomannomutase (PMM) of glycans in the cytosol and ER, transfer to the protein Defi ciency and a subsequent processing pathway mainly located in the Golgi apparatus. Multiple enzymes, transporters By far the most common and prototypic CDG is PMM and transferases are involved in that pathway. Type I defi ciency (OMIM 212065), previously denoted CDG disorders of N-glycosylation affect the early assembly 1a, caused by mutations in the PMM2 gene. The phe- pathway; in type II disorders, the processing pathway is notypic spectrum is very broad. Commonly, the cen- disturbed. O-glycosylation, mainly confi ned to the tral nervous system is involved and children show Golgi apparatus is a much shorter – however, more psychomotor retardation, hypotonia, hyporefl exia, variable – pathway, consisting of assembly and transfer ataxia and seizures. Dysmorphic features include a of the glycans to a protein. The diversity of prominent forehead, large ears and a thin upper lip. Fat O-glycosylation is due to variable sugars in the fi rst distribution is abnormal with fat pads at the buttocks position of the glycans, such like mannose in the or other sites and lipoatrophic changes. The nipples O-mannosylation pathway. However, in view of the are typically inverted. Retinitis pigmentosa, strabis- multitude of enzymes involved it has recently been sug- mus and myopia are the most common ophthalmo- gested to discontinue this system of classifi cation and logic signs. Skeletal manifestations consist of growth describe the individual disorders by their enzymatic retardation, osteopenia, contractures, spine anomalies, defi ciency. “bone-in-bone” appearance and other changes. In the The clinical features of congenital disorders of gly- multivisceral form of PMM defi ciency, multiple other cosylation (CDG) are as diverse as the numerous func- manifestations may exist, and include liver, cardiac, tions of glycoproteins which can be enzymes, renal and gastrointestinal involvements. transporter proteins, membrane components or hormones. CDGs may present as single organ diseases, but are often multi-system disorders, comprising structural malformations and abnormal function, occurring biopsy will show abnormal O-mannosylation. For in various combinations and covering a spectrum of the majority of CDG subtypes identifi ed so far, muta- severity. Symptoms comprise psychomotor retardation tional analysis can be performed to confi rm the and neurological abnormalities, multiple function defi - diagnosis. cits like bleeding diathesis, endocrine or gastrointesti- The group of CDGs is rapidly growing. At the time of nal disturbances, organ manifestations (e.g. heart or writing, nearly 40 CDGs have been characterised, 14 of kidney), skeletal manifestations (e.g. short stature, which affect N-glycosylation of proteins, 11 are linked to contractures and osteopenia), as well as other physical O-glycosylation pathways and in 15 others both or other abnormalities. Many CDG patients already display pathways are affected. Recently, not only dystroglycano- symptoms at birth; this is also the case in the most pathies but also several malformation syndromes have common primary glycosylation disorder, phospho- been recognised as glycosylation defects, e.g. the auto- mannomutase defi ciency (CDG-Ia). somal recessive Peters’ Plus syndrome which is caused Most N-glycosylation disorders are readily by mutations in B3GALTL resulting in an O-fucosylation detected by isoelectric focusing (IEF) of serum trans- defect. ferrin, although results may be false negative in some As so far only very few patients have been identi- disorders, and abnormal results may be caused by a fi ed for many of the CDGs, it is impossible to describe variety of other conditions. Abnormal O-glycosylation characteristic dysmorphic features. Major clinical involving the so-called core 1 O-glycans with the signs and symptoms of different CDGs as reported in fi rst sugar being N-acetylgalactosamine (the most the literature have been listed in Table C10.3. commonly observed O-glycosylation type) can be As there are many more glycosylation pathways, it detected by IEF of apolipoprotein CIII in serum. is to be expected that far more glycosylation disorders For other O-glycosylation defects, such like will be identifi ed in the middle-term future and one O-mannosylation defects (Walker–Warburg syn- needs to be open for any possible clinical presentation. drome or muscle–eye–brain disease), particular Certainly, the book of CDGs is not closed, and will staining of the a-dystroglycan complex in a muscle have to be updated rapidly. 230 U. Moog et al. ed bones ed brosis, coagulopathy, hyperinsulinemic brosis, coagulopathy, sm, macrocephaly, abnormal bones, precocious ossifi sm, macrocephaly, malformations, severe muscular hypotonia, severe MR, death in infancy muscular hypotonia, severe malformations, severe hypoglycemia multi-organ involvement, stroke-like episodes, seizures, cardiomyopathy, episodes, seizures, cardiomyopathy, stroke-like involvement, multi-organ nephrotic syndrome, hyperinsulinemic hypoglycemia, coagulopathy, hypogonadism hypergonadotropic epilepsy multiplex, drug resistant epilepsy, abnormal coagulation drug resistant epilepsy, MR, multiple exostosis, hypertrichiosis MR, multiple exostosis, 608210 601756 calcium deposits in skin and tissue Massive 608093 MR, seizures, esotropia, microcephaly 608177/ - N -mannosyltransferase 1 and 2 607423 anterior chamber cerebellar malformation, ventriculomegaly, II lissencephaly, Type O -acetylgalactosaminyltransferase 3 -acetylgalactosaminyltransferase cient protein OMIM Clinical features N transferase acetylglucosaminyltransferase -acetylglucosaminyltransferase 2-acetylglucosaminyltransferase 602616 disease liver seizures, dysmorphy, dysmorphism, stereotypic behaviour, MR, facial -1,4-galactosyltransferase 7 604327 abnormal bones, loose skin MR, short stature, macrocephaly, N Glucuronyltransferase/ b ) ) Mannosyltransferase 6 601110 arthrogryposis iris coloboma, secondary microcephaly, MR, optic atrophy, ) UDP-GlcNAc:Dol-P-GlcNAc-P ) Mannosyltransferase 8 607143 undetectable IgF1 and IgFB3, cardiomegaly MR, genital hypoplasia, microcephaly, ) Glucosyltransferase 2 608104 cataracts, osteopenia, MR hepatic failure, renal failure, Protein-loosing enteropathy, ) Glucosidase 1 606056 disease, early death Seizures, liver ) Mannosyltransferase 7–9 608776 early death renal cysts, seizures, hepatomegaly, microcephaly, Severe ) Mannosyltransferase 1 608540 nephrotic syndrome, early death MR, secondary microcephaly, ) Glucosyltransferase 1 603147 strabismus coagulopathy, MR, hypotonia, epilepsy, ) Flippase of Man5GlcNAc2-PP-Dol 611633 impaired vision, to thrive, retardation, arthrogryposis, failure Intrauterine growth ) Mannosyltransferase 2 607906 poor vision Intractable seizures, iris coloboma, hepatomegaly, ) Phosphomannose isomerase 602579 hepatic fi Protein-loosing enteropathy, Human genetic disorders of glycosylation -acetylgalactosaminylglycan synthesis -acetylgalactosaminylglycan N CDG-Ij CDG-Id CDG-IIa CDG-Ig CDG-Il CDG-Ih CDG-Ik CDG-Ic CDG-Ii CDG-IIb CDG-In CDG-Ib -acetylgalactosaminylglycan synthesis -acetylgalactosaminylglycan exotoses) syndrome) -xylosyl/ -mannosylglycan synthesis -xylosylglycan synthesis O-N O O SLC35D1 (Schneckenbecken dysplasia)SLC35D1 (Schneckenbecken 35 Solute carrier family 610804 Micromelic dwarfi Relevant geneRelevant of protein Disorders N-glycosylation PMM2 (CDG-Ia)MPI ( Defi Phosphomannomutase 2 601785 to thrive, pads, hypotonia, failure MR, cerebellar hypoplasia, abnormal fat * tumoral calcinosis)GALNT3 (familial * Polypeptide * POMT1/POMT2 Protein- ALG6 ( NOT56L ( NOT56L ALG 12 ( ALG 8 ( ALG2 ( ( DPAGT1 HMT1 ( DIBD1 ( RFT1 ( MGAT2 ( MGAT2 GLS1 ( TUSC3 of protein Disorders O-glycosylation O EXT1/EXT2 (multiple cartilaginous Oligosaccharyltransferase subunit 601385 Non-dysmorphic, mental retardation B4GALT7 (progeroid type of Ehlers-Danlos B4GALT7 Table C10.3 C10 Physical Abnormalities in Metabolic Diseases 231 res, late closure of the fontanelle adducted thumbs, cardiac defects, wrinkled skin, hyperthermia, early death infections 606822 ocular abnormalities, lissencephaly Congenital muscular dystrophy, 603590 brain abnormalities Congenital muscular dystrophy, 602576 Spondylocostal dysostosis 606978606973 retardation, hypotonia, growth dysmorphy, microcephaly, MR, progressive 606979 hypotonia microcephaly, retardation, progressive MR, growth to thrive psychomotor retardation, seizures, failure Severe 609056 epilepsy Amish infantile 603503 blindness epilepsy, MR, microcephaly, - N - N -1,2- b -2,3 sialyltransferase -1,3- -1,3-glucosyltransferase 610308 chamber defects, disproportionate short stature, cleft palate MR, anterior eye a b b c c -mannose O acetylglucosaminyltransferase protein acetylglucosaminyltransferase 1 complex 8 complex complex 7 complex (GM3 synthase) (Dol-P-Man synthase 1) -fucose-specifi -fucose-specifi -acetylglucosaminyltransferase-like -acetylglucosaminyltransferase-like -1,4-galactosyltransferase 1 607091 macrocephaly Dandy–Walker-malformation, MR, myopathy, N O O GDP-fucose transporter 605881 persistent neutrophilia, recurrent fever retardation, microcephaly, MR, growth Phosphatidylinositolglycan, class M 610273b thrombosis, absence seizures Portal venous CMP-sialic acid transporter 605634 haemorrhages, respiratory distress syndrome, opportunistic Thrombocytopenia, ) oligomeric Golgi Component of conserved ) oligomeric Golgi Component of conserved ) ) (GDP-fucose ) (CMP-sialic acid ciency) ciency) ) Lec35 (Man-P-Dol utilisation 1) 608799 retardation, ichthyosis, ataxia, pigmentary retinopathy MR, growth ) GDP-Man:Dol-P-mannosyltransferase ) Dolichol kinase 610768 early death Seizures, ichtyosis, dilated cardiomyopathy, CDG-IIe CDG-IIg CDG-IId CDG-IIf CDG-IIc CDG-If CDG-Ie ciency) CDG-Im -fucosylglycan synthesis defi transporter defi transporter defi O * Dolichol pathway COG complex V-ATPase FKTNFKRPLARGE Fukutin Fukutin-related protein 606596 Limb-girdle muscular dystrophy type 607440 Fukuyama congenital muscular dystrophy POMGNT1 Protein- * * * SCDO3 (spondylocostal dysostosis type 3) (Peters plus syndrome) B3GALTL COG1 defect ( COG8 defect oligomeric Golgi Component of conserved DK1 ( COG7 defect ( defect (cutis laxa type II)ATP6VOA2 H(+)-ATPase A2 of vesicular V0 subunit 611716 MR, wrinkled skin/cutis laxa, pachygyria, seizu Disorders of glycosphingolipid and glycosylphosphatidylinositol anchor of glycosphingolipid and glycosylphosphatidylinositol anchor Disorders glycosylation epilepsy) (Amish infantile SIAT9 Lactosylceramide PIGM (glycosylphosphatidylinositol SLC35C1 ( Disorders of multiple glycosylation functions and other pathways Disorders DPM1 ( GNE (hereditary inclusion body myopathy)SLC35A1 ( epimerase/kinase UDP-GlcNAc 600737 on biopsy Ascending muscle weakness, “Rimmed vacuoles” MPDU1 ( ( B4GALT1 232 U. Moog et al.

Key References Grünewald S (2007) Congenital disorders of glycosylation: rapidly enlarging group of (neuro)metabolic disorders. Early Hum Dev 83:825–830 Brown GK (2005) Congenital brain malformations in mitochon- Hennekam RC (2005) Congenital brain anomalies in distal drial disease. J Inherit Metab Dis 28:393–401 cholesterol biosynthesis defects. J Inherit Metab Dis 28: Clayton PT, Thompson E (1988) Dysmorphic syndromes with 385–392 demonstrable biochemical abnormalities. J Med Genet Herman GE (2003) Disorders of cholesterol biosynthesis: proto- 25:463–472 typic metabolic malformation syndromes. Hum Mol Genet de Lonlay P, Seta N, Barrot S et al (2001) A broad spectrum of 12 R75–88 clinical presentations in congenital disorders of glycosyla- Jaeken J, Matthijs G (2001) Congenital disorders of glycosylation I: a series of 26 cases. J Med Genet 38:14–19 tion. Annu Rev Genomics Hum Genet 2:129–151 Epstein CJ, Erickson RP, Wynshaw-Boris A (eds) (2008) Inborn Mudd SH, Skovby F, Levy HL et al (1985) The natural history errors of development, 2nd edn. Oxford University Press, of homocystinuria due to cystathionine beta-synthase deﬁ - Oxford, New York ciency. Am J Hum Genet 37:1–31 Faust PL, Banka D, Siriratsivawong R et al (2005) Peroxisome bio- Porter FD (2008) Smith-Lemli-Opitz syndrome: pathogene- genesis disorders: the role of peroxisomes and metabolic dys- sis, diagnosis and management. Eur J Hum Genet 16: function in developing brain. J Inherit Metab Dis 28:369–383 535–541 Gorlin RJ, Cohen MM, Hennekam RCM (2001) Syndromes of Wanders RJ (2004) Metabolic and molecular basis of peroxi- the head and neck, 4th edn. Oxford University Press, Oxford, somal disorders: a review. Am J Med Genet 126A: New York 355–375 Hematological Disorders C11 Ellen Crushell and Joe T R Clarke

Key Facts HPRT Hypoxanthine guanine phosphoribosyltransferase › Complete blood count and blood ﬁ lm are indi- IRT Immunoreactive trypsin cated in all metabolic diagnostic work-ups. IVA Isovaleric aciduria › Hematological disorders may be the lead LCAT Lecithin:cholesterol acyltransferase symptom or a secondary ﬁ nding in many met- MCV Mean corpuscular volume abolic disorders. ML Mucolipidosis › Hemolytic anemia may be the only sign of MLASA Mitochondrial myopathy an inherited disorder of red cell energy and sideroblastic anemia metabolism. MMA Methylmalonic aciduria › Macrocytic anemia is an important pointer to MPS Mucopolysaccharides/oses many metabolic disorders. NCL Neuronal ceroid-lipofuscinosis › Hematological abnormalities due to metabolic NP NiemannÐPick disease are rarely seen in isolation. OA Organic acid analysis PA Propionic acidemia RBC Red blood cell TG Triglycerides TPI Triose phosphate isomerase Abbreviations UMP Uridine monophosphate XLR X-linked recessive

ACE Angiotensin converting enzyme AD Autosomal dominant ALAS 5-Aminolevulinic acid synthase AR Autosomal recessive C11.1 General Remarks ASAT Anemia, sideroblastic, and spinocerebellar ataxia Hematologic abnormalities are common features of CBC Complete blood count inherited metabolic diseases. Many of these have other CDG Congenital disoders of glycosylation major manifestations, including neurological features. GSD Glycogen storage disease On the other hand, in some inherited metabolic dis- HDL High-density lipoprotein eases, particularly those involving red cell energy metabolism, the hematological abnormalities may be the only manifestation of disease. The identifi cation of a hematological abnormality, along with documentation of any nonhematological problems, often leads to E. Crushell () 26 Wainsfort Grove, Terenure, Dublin 6 W, Ireland the diagnosis of a specifi c inherited metabolic disease. e-mail: [email protected] A complete blood count and blood fi lm may provide

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 233 DOI: 10.1007/978-3-540-74723-9_C11, © Springer-Verlag Berlin Heidelberg 2010 234 E. Crushell and J. T. R. Clarke diagnostic clues and should be included in the work-up dehydrogenase defi ciency. It is an X-linked recessive of all patients with a suspected metabolic disorder. (XLR) condition that occurs with very high frequency While many metabolic disorders are associated in Mediterranean, African, and Asian populations. In with secondary hematological fi ndings [e.g., hyper- some regions of Greece, one in three males is affected; splenism in Gaucher disease, neutropenia in propionic in West Africa and some parts of India, the prevalence academia (PA) ], sometimes the hematological disor- is up to 20Ð25%; in China, including Hong Kong, der is the lead feature, as in Pearson marrow pancreas 3Ð5% of males are affected. Acute hemolysis is usu- syndrome or the macrocytic anemia of inherited disor- ally associated with exposure to oxidizing chemicals ders of cobalamin metabolism. In many instances, the or drugs or ingestion of certain foods, such as fava hematological abnormalities involve all the formed beans, hence the historical name for the disease: favism elements of the blood. In this chapter, these are dis- (Table C11.3). Some parts of the world have intro- cussed in the context of the most common or the most duced mass programs of newborn screening for the prominent abnormality. For example, type 1 Gaucher condition in an attempt to prevent the development disease often presents as thrombocytopenia; however, of acute hemolysis, severe hyperbilirubinemia, and neutropenia and anemia are almost always present, kernicterus. though rarely as prominent as the decrease in platelet Chronic isolated hemolytic anemia, characterized counts. by jaundice, gallstones, indirect hyperbilirubinemia, and splenomegaly, is also seen in patients with inherited defi ciencies of pyruvate kinase, 2,3 diphosphoglyceromutase, glucose phosphate isomerase, or C11.2 Abnormal Cell Morphology hexokinase. The diagnosis may be suspected from the red cell morphology; however, confi rmation gener- Abnormal cell morphology on blood fi lm or bone mar- ally requires analysis of the specifi c red cell enzymes; row aspirate may be the fi rst pointer toward a meta- pyruvate kinase defi ciency is the most common of bolic diagnosis. See Table C11.1 for various abnormal this group of nonspherocytic hemolytic anemias cell morphologies found in metabolic disease. It is (Table C11.2). important to be aware that various primary hemato- Defi ciencies of other enzymes involved in anaero- logical disorders (including malignancies) may pro- bic glycolysis also lead to hemolytic anemia with sig- duce similar morphological fi ndings. Also, a “normal” nifi cant non-hematological problems, such as mental blood fi lm does not rule out a metabolic diagnosis such retardation, myopathy, ataxia, chronic metabolic aci- as a mucopolysaccharidosis where vacuolated lym- dosis, or stroke (Table C11.4). phocytes may be few and far between. The extrinsic hemolytic anemias may be caused by any inborn error that produces severe liver disease in infancy, such as galactosemia and neonatal hemochromatosis. Erythropoietic protoporyphyria and Wilson C11.3 Hemolytic Anemia disease also lead to hemolytic anemia. Skin changes such as photosensitivity, ulcers, or abnormal pigmen- Hemolytic anemia may be caused by factors within the tation, should prompt a porphyria screen in neonates red cell (intrinsic) or in the environment of the cell and young children. Hemolytic anemia is often not the (extrinsic) (Table C11.2). Mature red cells contain no lead sign, but an associated fi nding, along with poor mitochondria and are totally dependent upon anaero- growth, poor feeding, vomiting, and hypotonia in bic glycolysis and the hexose monophosphate shunt to methylmalonic aciduria, PA, and isovaleric aciduria, meet their energy needs and requirements for NADPH. abetalipoproteinemia, and Wolman disease in the fi rst Defi ciencies of any of the enzymes involved may be year of life. associated with hemolytic anemia as a result of a defi - Early onset xanthomas and hypercholesterolemia ciency of ATP or of reducing equivalents. (and elevated phytosterols) are seen in phytoster- By far, the most common of the hereditary intrinsic olemia. Lecithin:cholesterol acyltransferase defi ciency defects causing hemolytic anemia is glucose-6-phosphate is associated with corneal opacities and proteinuria. C11 Hematological Disorders 235

Table C11.1 Abnormal blood cell morphology Cell type Morphology Disorder Red cells Target cells Liver disease Lecithin:cholesterol acyltransferase defi ciency Abetalipoproteinaemia Spherocytes Hypersplenism Liver disease Spiculated red cells Renal failure Ð echinocytes Vitamin E defi ciency (“burr”) or Abetalipoproteinaemia acanthocytes (“spur”) Wolman disease Cbl C defects HallervordenÐSpatz (panthothenate kinase) syndrome Pyruvate kinase defi ciency White cells Basophilic stippling Lead poisoning Iron defi ciency Hemolytic anemias Pyrimidine-5' nucleotidase defi ciency Vacuolated lymphocytes Aspartylglucosaminuria Multiple sulfatase defi ciency Ceroid lipofuscinoses Mucolipidosis II

GM1 gangliosidosis Mucopolysaccharidoses NiemannÐPick disease types A, C Pompe Sialidosis Wolman (Alder) Reilly bodies Mucopolysaccharidoses “Sea blue” histiocytes NiemannÐPick disease Ceroid lipofuscinoses Adult cholesterol ester storage disease

GM1 gangliosidosis Bone Marrow Foam cells NiemannÐPick disease types A, B, C, D Gaucher disease types 1, 2, 3

Gangliosidosis Ð GM1 and GM2 Sialidosis I, II (late infantile) Mucolipidosis II, III, IV Fucosidosis Mannosidosis Ceroid lipofuscinoses Farber disease Wolman disease Cholesterol ester storage disease Cerebrotendinous xanthomatosis Chronic hyperlipidemia Adapted from Lanzkowsky (2005) 236 E. Crushell and J. T. R. Clarke

Table C11.2 Hemolytic anemias Intrinsic defects Disorder/enzyme defi ciency Associated abnormalities and diagnosis Anaerobic glycolytic enzyme defi ciencies Isolated hemolytic anemia Pyruvate kinase Nonspherocytic hemolytic anemia, red cell enzyme assay Glucose phosphate isomerase Nonspherocytic hemolytic anemia, red cell enzyme assay Hexokinase Nonspherocytic hemolytic anemia, red cell enzyme assay 2,3-diphosphoglyceromutase Nonspherocytic hemolytic anemia, red cell enzyme assay + Neurological Phosphofructokinase Prominent myopathy, cardiomyopathy in infants with abnormalities severe disease. Diagnosis by enzyme assay or PFKM mutation analysis Triosephosphate isomerase Autosomal dominant, early onset, neurodegenerative disease. Diagnosis by red cell enzyme assay or TPI mutation analysis Phosphoglycerate kinase X-linked recessive (XLR) myopathy with chronic progressive encephalopathy. Diagnosis by red cell enzyme assay or PGK1 mutation analysis Hexose monophosphate shunt defects Glucose-6-phosphate XLR hemolytic anemia. Diagnosis by red cell enzyme dehydrogenase assay Glutathione synthetase Autosomal recessive, metabolic acidosis, pyroglutamic aciduria (5-oxoprolinuria). Diagnosis by GSS mutation analysis Glutathione reductase Nonspherocytic hemolytic anemia. Progressive neurological abnormalities in severe form. Diagnosis by red cell enzyme assay Abnormal erythrocyte nucleotide metabolism Adenosine triphosphatase Red cell enzyme assay Adenylate kinase AK1 mutation analysis Pyrimidine 5-nucleotidase Accumulation of pyrimidine nucleotides in red cells, most common disorder of nucleotide metabolism causing hemolytic anemia. Red cell assay Extrinsic causes of hemolysis Hypersplenism Gaucher disease Leukocyte or fi broblast b-glucosidase, GBA mutation analysis NiemannÐPick disease Leukocyte or fi broblast acid sphingomyelinase, SMPD1 mutation analysis Defects of porphyrin metabolism Congenital erythropoietic Autosomal recessive photosensitivity of the skin with porphyria blistering and scarring, red coloring of teeth and urine. Diagnosis by urinary porphyrin analysis, measurement of enzyme (uroporphyrinogen III cosynthetase), UROS mutation analysis Disorders of lipid metabolism Lecithin:cholesterol Corneal opacities, “target cell” anemia, proteinuria, acyltransferase (LCAT) progressive renal impairment, low plasma esterifi ed cholesterol. Diagnosis by plasma LCAT assay and by LCAT mutation analysis Abetalipoproteinemia Steatorrhea, ataxia, ‘burr’ cells (acanthocytes), hypocholesterolemia. Diagnosis by plasma apolipoprotein analysis, MTP mutation analysis Defects of sterol metabolism Phytosterolemia Premature coronary artery disease. Diagnosis by plasma (sitosterolemia) sterol analysis Intoxications Wilson disease Hepatocellular dysfunction, neurological abnormalities, KayserÐFleischer rings. Diagnosis by plasma ceruloplasmin analysis, urinary copper excretion, ATP7B mutation analysis C11 Hematological Disorders 237

Table C11.3 Partial list of agents inducing Antimalarials Antipyretics/analgesics Others hemolytic anemia in glucose-6-phosphate Primaquine Acetylsalicylic acid Chloramphenical dehydrogenase deﬁ ciency Pamaquine Acetanilide Isoniazid Sulfonamides Infections p-Aminosalicylic acid Sulfadiazine Viral respiratory infections Phenytoin Sulﬁ soxazole Viral hepatitis Vitamin K (water soluble) Nitrofurans Typhoid Naphthalene (moth balls) Nitrofurantoin Nalidixic acid

Table C11.4 Hemolytic anemia + neurological abnormalities Disorder/enzyme deﬁ ciency Gene Inheritance Diagnostic tests Triosephosphate isomerase TPI1 Autosomal dominant (AD) Red cell enzyme assay, TPI mutation analysis Phosphoglycerate kinase PGK1 XLR Red cell enzyme assay, PGK mutation analysis Phosphofructokinasea PFKM AR Ischemic exercise test, enzyme assay, in muscle, PFK mutation analysis Abetalipoproteinemia MTP AR Plasma apolipoprotein analysis, MTP mutation analysis Hypobetalipoproteinemia APOB AD Plasma apolipoprotein analysis, APOB mutation analysis Glutathione synthetase GSS AR Red cell GSS assay, urinary organic acids showing pyroglutamic aciduria, mutation analysis Wilson disease ATP7B AR Plasma ceruloplasmin, urinary copper excretion, mutation analysis aMyopathy

C11.4 Macrocytic Anemia Congenital (or acquired) disorders of nucleotide metabolism may also present with macrocytic Inherited disorders of absorption, transport, or metab- anemia. In orotic aciduria caused by uridine mono- olism of vitamin B12 (cobalamin) and folate may pres- phosphate synthase deficiency, pyrimidine biosyn- ent with macrocytic anemia as the leading sign (Table thesis is interrupted, while in LeschÐNyhan C11.5). Elevated homocysteine with low methionine (hypoxanthine guanine phosphoribosyltransferase points toward a remethylation defect of homocysteine deficiency) disease, purine nucleotide regeneration to methionine (Table C11.6). is impaired (the resulting macrocytic anemia is usually a late feature). Thiamine-responsive megalo- Remember blastic anemia is associated with diabetes and sensorineural deafness (in some, the full DIDMOAD ¼ Over 95% of patients with macrocytic anemia spectrum Ð diabetes insipidus, diabetes mellitus, in childhood are secondary to acquired defi - optic atrophy and deafness Ð is present) only the ciencies of folate or cobalamin. anemia, however, will respond to high-dose thia- ¼ Macrocytosis may be masked by iron defi ciency mine treatment; the underlying defect is in thiamine and thalassemia. transport. ¼ High homocysteine and low methionine sug- The sideroblastic anemia in Pearson marrow pan- gests inborn errors of cobalamin metabolism. creas syndrome associated with mitochondrial DNA ¼ Intermittent orotic aciduria may be seen in con- deletions is macrocytic and associated with exocrine genital folate defi ciency. pancreatic dysfunction, growth failure, and lactic aci- ¼ Absence of methlymalonic aciduria does not dosis. The clinical phenotype may be or evolve into rule out a cobalamin defect. that of KearnsÐSayre syndrome. 238 E. Crushell and J. T. R. Clarke

Table C11.5 Macrocytosisa Disorder Disorder/enzyme defi ciency Associated abnormalities and diagnosis Disorders of nucleotide metabolism Orotic aciduria Uridine monophosphate (UMP) Autosomal recessive megaloblastic anemia and synthase defi ciency marked orotic aciduria responsive to treatment with uridine. Confi rmation of diagnosis by UMPS mutation analysis Thiamine-responsive Thiamine transporter protein Autosomal recessive anemia responsive to megaloblastic anemia high-dose thiamine treatment. Diagnosis confi rmed by SLC19A2 mutation analysis LeschÐNyhan disease Hypoxanthine guanine X-linked recessive spasticity, variable psychomo- phosphoribosyltransferase defi ciency tor retardation, self-mutilation, with high plasma, and urinary urate. Diagnosis confi rmed by enzyme assay and mutation analysis Coblamin disorders Malabsorption ImerslundÐGrasbeck syndrome Autosomal recessive megaloblastic anemia, with

low plasma vitamin B12 levels

Intrinsic factor deﬁ ciency Low plasma vitamin B12 analysis; Schilling test Ð corrected by intrinsic factor Transport Transcobalamin II deﬁ ciency with normal Variable psychomotor retardation, hypotonia, serum cobalamin levels methylmalonic aciduria (MMA), hyperhomocysteinemia, low methionine, normal plasma

vitamin B12 levels. Diagnosis conﬁ rmed by measurement of plasma TC II Metabolism Defective synthesis of methylcobalamin Variable psychomotor retardation, associated with Ð cblE, cblG hyperhomocysteinemia with low methionine and without MMA, normal plasma folate and

vitamin B12. Diagnosis by complementation studies in ﬁ broblasts Defective synthesis of both Psychomotor retardation, variable retinal adenosylcobalamin and dystrophy, associated with MMA and methylcobalamin Ð cblC, cblD, cblF hyperhomocysteinemia with low methionine,

normal plasma folate and vitamin B12. Diagnosis by complementation studies in fi broblasts Folate disorders Malabsorption Congenital folate malabsorption Chronic encaphalopathy, movement disorder, psychomotor retardation, pancytopenia, hypomethioninemia, low plasma folate Metabolism Methylene-tetrahydrofolate reductase Psychiatric disturbances, psychomotor (MTHFR) defi ciency retardation, movement disorder, with hyperhomocysteinemia, and low methionine. Diagnosis confi rmed by enzyme assay or MTHFR mutation analysis Glutamate formiminotransferase defi ciency Variable psychomotor retardation, marked increase in urinary FIGLU excretion Dihydrofolate reductase defi ciency Phenotype unclear, mild psychomotor retardation? Features of folic acid defi ciency that respond to treatment with N(5)-formyltetrahydrofolate but not to folic acid. Confi rmation by DHFR mutation analysis Others Mevalonic aciduria Mevalonic acid kinase defi ciency Developmental delay, failure to thrive, hepatosplenomegaly, dysmorphic facies, recurrent fever with lymphadenopathy, with urinary excretion of mevalonic acid. Diagnosis confi rmed by enzyme assay or MVK mutation analysis aMCV > 85 fL and megaloblasts in bone marrow C11 Hematological Disorders 239

Table C11.6 Key metabolic investigations of macrocytic The acquired sideroblastic anemias are caused by anemia inhibitors of 5-aminolevulinate synthase, such as iso- Plasma cobalamin, plasma and red cell folate levels niazid, cylcoserine, and lead. Chloramphenicol and Plasma total homocysteine, amino acids, lactate, urate ethanol also inhibit mitochondrial function resulting in Urinary organic acids sideroblastic anemia. Urinary orotic acid Urine amino acids Partial response to pyridoxine treatment has been Results from these initial investigations will determine further seen in some of the congenital and acquired sidero- testing Ð e.g., Schilling test, folate challenge, mutation analy- blastic anemias. sis, speciﬁ c enzyme, or complementation studies

C11.5 Sideroblastic Anemia

Sideroblasts are red cell precursors with iron-loaded C11.6 Cytopenias (Prussian blue staining) mitochondria clustered in a ring around the nucleus. Sideroblastic anemia is Decreased numbers of the formed elements of blood caused by mitochondrial dysfunction that may be pri- may occur as a result of defects in production or mary or acquired. The acquired forms far outnumber increased destruction. Pancytopenia is virtually a the congenital forms; however, a hereditary form constant fi nding with propionate intoxication, which is should be particularly sought in the young and in a feature of some of the organic acidurias during acute those without a history to suggest acquired forms decompensation. The mechanism is not understood, (e.g., alcohol ingestion) (Table C11.7). Four of the and there is no specifi c treatment other than restoration eight enzymes necessary for the biosynthesis of heme of metabolic control in these disorders. During periods are located in mitochondria (the other four are cyto- of good metabolic control, the hematological picture plasmic and defi ciencies do not result in sideroblasts, generally normalizes. e.g., the porphyrias). Defective mitochondrial heme Neutropenia is a chronic fi nding seen in glycogen synthesis causes iron accumulation within the mito- storage disease 1b/c; patients often develop infl am- chondria resulting in oxidative mitochondrial dam- matory bowel disease later in the course of the dis- age and the formation of sideroblasts. Sideroblastic ease. The neutropenia responds to granulocyte-colony anemia is usually microcytic or normocytic with stimulating factor treatment. The neutropenia in hypochromia; megaloblastic sideroblastic anemia Barth syndrome (XLR inheritance) may have a cycli- suggests Pearson syndrome or thiamine-responsive cal pattern associated with intermittent ulcers of the megaloblastic anemia. mouth.

Table C11.7 Sideroblastic anemias Disorder Gene Inheritance Diagnostic tests Pearson marrow-pancrease mtDNA Maternal/sporadic Plasma immunoreactive trypsin (IRT), stool syndromea trypsin, analysis of mtDNA for deletions Mitochondrial myopathy and PUS1 Autosomal recessive Mutation analysis sideroblastic anemia b Anemia, sideroblastic, and ABC7 X-linked recessive Mutation analysis spinocerebellar ataxia (XLR) Pyridoxine-responsive ALAS2 XLR Response to high-dose pyridoxine treatment, megaloblastic anemia mutation analysis aPancreatic insufﬁ ciency often precedes development of anemia bWith lactic acidosis 240 E. Crushell and J. T. R. Clarke

C11.6.1 Pancytopenia (Table C11.8)

Table C11.8 Pancytopenia Associated fi nding Disorder Diagostic tests Splenomegaly Gaucher disease types 1 and 3 Leukocyte or fi broblast b-glucosidase, GBA mutation analysis NiemannÐPick disease type B Leukocyte or fi broblast acid sphingomyelinase, SMPD1 mutation analysis + Neurological abnormalities Gaucher disease type 3 Leukocyte or fi broblast b-glucosidase, GBA mutation analysis NiemannÐPick disease type A Leukocyte or fi broblast acid sphingomyelinase, ASM mutation analysis Ketoacidosis, hyperammone- Propionic acidemia, methylmalonic Urinary organic acids, plasma acylcarnitine profi le mia, hypotonia aciduria, and isovaleric aciduria

Failure to thrive, diarrhea Folate malabsorption Plasma and red cell folate, plasma vitamin B12, folate challenge test

Transcobalamin II deﬁ ciency Plasma transcobalamin II level, plasma vitamin B12 (normal)

C11.6.2 Neutropenia (Table C11.9)

Table C11.9 Neutropenia Associated fi ndings Disorder Diagnostic tests Hepatomegaly, hypoglycemia, Glycogen storage disease 1b Glucose challenge. Analysis of glucose-6- short stature, infl ammatory phosphatase in fresh (unfrozen) liver and bowel disease (late) liver that has been frozen and thawed for demonstration of latency. G6PT mutation analysis. Hyperammonemia, failure to thrive, Lysinuric protein intolerance Plasma and urine amino acids interstitial pneumonia, diarrhea Ketoacidosis, hypotonia, failure to Propionic acidemia, methylmalonic Urinary organic acids, blood acylcarnitine thrive, developmental delay aciduria, isovaleric aciduria profi le Cardiomyopathy and skeletal Barth syndrome Urinary organic acids, cardiolipin electropho- myopathy, short stature in a male resis, TAZ mutation analysis Macrocytic anemia, developmental Orotic aciduria Urinary orotic acid, uridine monophosphates delay, and failure to thrive synthase (UMPS) enzyme assay or UMPS mutation analysis Coarse facies, developmental delay, Aspartylglucosaminuria Urine oligosaccharides, enzyme studies, AGA hepatomegaly, skeletal abnormalities mutation analysis

C11.6.3 Thrombocytopenia (Table C11.10) Table C11.10 Thrombocytopenia Associated fi nding Disorder Diagnostic tests Splenomegaly Gaucher disease type 1 Leukocyte or fi broblast b-glucosidase, GBA mutation analysis NiemannÐPick disease type B Leukocyte or fi broblast acid sphingomyelinase, SMPD1 mutation analysis Ketoacidosis, hypotonia, failure Methylmalonic aciduria, Propionic Urinary organic acids, plasma acylcarnitine profi le to thrive, developmental acidemia, Isovaleric aciduria delay Cardiomyopathy, ketoacidosis, Cobalamin defects Plasma homocysteine, amino acids, urinary organic macrocytic anemia acids, plasma cobalamin, complementation studies on cultured skin fi broblasts, specifi c mutation analysis Lactic acidosis, hypotonia, Holocarboxylase synthetase Plasma lactate, ammonia, urinary organic acids, dermatosis, alopecia defi ciency enzyme assay on fi broblasts C11 Hematological Disorders 241

C11.7 Others C11.7.5 Methemoglobinemia

C11.7.1 Bleeding Tendency Methemoglobinemia is the result of the failure of reduction of oxidized circulating hemoglobin, either as the result of exposure to strong oxidizing agents, such Coagulopathy may be seen in any metabolic disorder as nitrites or failure of the normal enzymatic reduction associated with severe liver disease, such as galac- of heme. Congenital methemoglobinemia may occur tosemia, fructosemia, tyrosinemia type I, and neonatal as a result of defi ciency of NADH-cytochrome b5 hemochromatosis. Because of defi cient glycosylation, reductase that is limited to red cells (type I) or a gener- coagulation factors (especially XI) may be defi cient in alized defi ciency of the enzyme (type II), both are congenital disoders of glycosylation (CDG). Bleeding caused by mutations of the CYB5R3 (formerly DIA1) tendency associated with (hepato)splenomegaly, but gene. In type I, the only clinical abnormality is chronic without signifi cant hepatic dysfunction, is found in persistent cyanosis resulting from methemoglobin Gaucher disease type 1 and glycogen storage disease accumulation and in type II, the methemoglobiemia is type Ia and Ib/c. associated with a chronic progressive encephalopathy.

C11.7.2 Hypercoagulability Key References

The classic disorder associated with hypercoagulable Cappellini MD, Fiorelli G (2008) Glucose-6-phosphate dehy- states is homocystinuria. It has been shown that good drogenase defi ciency. Lancet 371:64Ð74 metabolic control through dietary and medical inter- Chiarelli LR, Fermo E, Zanella A et al (2006) Hereditary erythrocyte pyrimidine 5'-nucleotidase defi ciency: a biochemical, vention reduces the number of vascular events in clas- genetic and clinical overview. Hematology 11:67Ð72 sical homocystinuria. CDG is also associated with Finsterer J (2007) Hematological manifestations of primary hypercoagulable states. mitochondrial disorders. Acta Haematol 118:88Ð98 Lanzkowsky, P (2005) Manual of pediatric hematology and oncology, 4th edn. Burlington, MA, Elsevier Academic Press. Merle U, Schaefer M, Ferenci P et al (2007) Clinical presenta- C11.7.3 Hyperleukocytosis tion, diagnosis and long-term outcome of Wilson’s disease: a cohort study. Gut 56:154 Ogier de Baulny H, Saudubray JM (2002) Branched-chain Hyperleukocytosis >100,000/mm3 has been described organic acidurias. Semin Neonatol 7:65Ð74 in CDG IIc (GDP fucose transporter 1) or leukocyte Rampoldi L, Danek A, Monaco AP (2002) Clinical features and adhesion defi ciency S. molecular bases of neuroacanthocytosis. J Mol Med 80:475Ð491 Rosenblatt DS, Whitehead VM (1999) Cobalamin and folate defi ciency: acquired and hereditary disorders in children. Semin Hematol 36:19Ð34 C11.7.4 Hemophagocytosis Sassa S (2000) Hematologic aspects of the porphyrias. Int J Hematol 71:1Ð17 Zanella A, Fermo E, Bianchi P et al (2005) Red cell pyruvate Hemophagocytosis is encountered occasionally in kinase defi ciency: molecular and clinical aspects. Br J Haematol patients with carnitine palmitoyltransferase I defi ciency, 130:11Ð25 Zimran A, Altarescu G, Rudensky B et al (2005) Survey of the propionic aciduria, hemochromatosis, lysinuric protein hematological aspects of Gaucher disease. Hematology 10: intolerance, Gaucher disease, and NiemannÐPick disease. 151Ð156 Immunological Problems C12 Ertan Mayatepek

Key Facts enteropathica, biotinidase and holocarboxylase synthetase defi ciencies, hereditary orotic › Immunological problems can develop as per- aciduria, defi ciency of intestinal folic acid manent key manifestations of a metabolic dis- absorption, a-mannosidosis, and in some ease (e.g., adenosine deaminase defi ciency) or patients with methylmalonic aciduria. can occur inconstantly in individual patients. › Phagocytic dysfunction and neutropenia are › Inherited metabolic diseases associated with characteristic fi ndings in glycogen storage dis- T cell immunodefi ciency mainly feature purine ease type I non-a. Dysfunction of phagocytes nucleoside phosphorylase defi ciency. This dis- or neutropenia is also frequently observed in ease is characterized by severe viral, bacterial, classical galactosemia, X-linked cardioskele- or fungal infections. Milder impaired T cell tal myopathy (Barth syndrome), glutathione function is sometimes seen in lysinuric protein synthetase defi ciency, or Pearson syndrome. intolerance, Menkes disease or Zellweger › NK cell immunodefi ciency is very rarely asso- syndrome. ciated with inborn errors of metabolism. In sin- › B cell immunodefi ciency has been observed in gle cases, this immunological dysfunction has patients with transcobalamin II defi ciency or been reported in lysinuric protein intolerance. propionic acidemia. › Adenosine deaminase defi ciency represents the best characterized inherited metabolic disease with combined T and B cell immunodefi - ciencies. Recurrent infections caused by a broad variety of microorganisms often become C12.1 General Remarks life-threatening. Bone marrow transplantation has been successful in some patients. A wide range of immunological problems has been iden- › Besides adenosine deaminase defi ciency, immu- tifi ed in association with inherited metabolic diseases, nological dysfunction affecting both the B and T sometimes in single case reports, and in others as a con- cell lines has been reported in acrodermatitis stant clinical manifestation. In clinical practice, two situations arise: (1) the patient presents with a known metabolic disorder and immunological problems appear as recognized manifestations of the disease or (2) the patient presents with immunological problems and an underlying metabolic disorder is sought. Some metabolic defects lead to symptoms such as chronic or recurrent infection, or infection with unusual agents. An example of an inher- E. Mayatepek ited metabolic disease linked to an infl ammatory periodic Department of General Pediatrics, University Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany fever syndrome is mevalonic aciduria. Mutations and e-mai: [email protected] consecutively reduced activity of mevalonate kinase have

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 243 DOI: 10.1007/978-3-540-74723-9_C12, © Springer-Verlag Berlin Heidelberg 2010 244 E. Mayatepek been found in mevalonic aciduria as well as in hyperim- Table C12.2 Investigations for differential diagnosis of munoglobinemia D syndrome. Besides congenital inherited metabolic diseases in patients with immunological problems malformations, hepatosplenomegaly, failure to thrive, developmental retardation and others, the clinical course Urine Purines and pyrimidines of mevalonic aciduria is characterized by recurrent crises Organic acids of fever, vomiting, and diarrhea, suggesting an infectious Amino acids or autoimmune disease. Orotic acid Clinically signifi cant immunodefi ciency presents Oligosaccharides with both, an unusual history of infection and corre- Plasma/Serum sponding confi rmatory laboratory tests. Other patients Lactate may have milder manifestations or sometimes only Copper Zinc laboratory evidence of immunological abnormalities. Very long-chain fatty acids One or several major components of the immune sys- Folic acid tem can be affected in distinctive metabolic diseases, Transcobalmin II single or combined T or B cell immunodefi ciencies, Enzyme studies phagocyte immunodefi ciency, or natural killer (NK) Galactose-1-phosphate uridyl transferase cell immunodefi ciency (Table C12.1 and C12.2). Biotinidase Lysosomal enzymes Mutational analysis (e.g., GSD type I non-a or Pearson syndrome) C12.2 Inherited Metabolic Diseases Associated with T Cell most important metabolic disease associated with clin- Immunodefi ciency ically relevant isolated T cell immunodefi ciency resulting in severe immunodefi ciency. The number of T cells Purine nucleoside phosphorylase is required for nor- is greatly reduced leading to lymphopenia and cutane- mal catabolism of purines. In purine nucleoside phos- ous anergy. Recurrent infections are usually obvious at phorylase defi ciency, substrates accumulate that affect least by the end of the fi rst year. There is an enhanced the immune and nervous systems. This disease is the susceptibility to viral diseases, such as varicella, mea- sles, cytomegalovirus, and vaccina. Severe pyogenic or fungal infections also occur. T cell dysfunction may Table C12.1 Diagnostic tests in patients with a known worsen in the course of the disease. Affected patients metabolic disorder and frequent infections may have autoantibodies and autoimmune hemolytic History anemia. Neurological symptoms include abnormal Physical examination motor development, ataxia, and spasticity. Bone mar- Differential blood count row transplantation can be curative. Sedimentation rate Lysinuric protein intolerance is characterized by Urine analysis Chest X-ray defective transport of the dibasic amino acids lysine, Immune work-up arginine, and ornithine in the intestine and renal B cell disorders tubules. Biochemically, this defect leads to decreased Quantitative immunoglobulins levels of these amino acids in plasma and increased Isohemagglutinin levels in urine interfering with the urea cycle and Specifi c antibody titers consecutively hyperammonemia. Intestinal protein T cell disorders intolerance, failure to thrive, hepatosplenomegaly, Skin tests for tetanus, mumps and monilia (>3 years of age) Lymphocyte count/differentiation and osteoporosis as well as progressive encephal- Lymphocyte stimulation opathy develop. Decrease in CD4+ T cell number, Thymus on X-ray studies lymphopenia, and leukopenia have been reported, Phagocyte disorders as well as decreased leukocyte phagocytic activity. Granulocyte count As in purine nucleoside phosphorylase deficiency, Nitro blue tetrazolium test varicella infection may follow an especially severe Neutrophil function tests course. C12 Immunological Problems 245

Impaired T cell function has been also described in C12.4 Inherited Metabolic Diseases Menkes disease. This disorder is caused by a defect in Associated with Combined T a membrane copper transport channel which interferes and B Cell Immunodefi ciencies with the absorption of copper and its distribution to the cells, resulting in generalized copper defi ciency. Clinical symptoms of classical Menkes disease include Adenosine deaminase defi ciency represents the best neonatal hypothermia, unconjugated hyperbilirubine- characterized metabolic disease leading to combined mia, mental retardation, seizures, typical facies, immunodefi cieny. Adenosine deaminase defi ciency “kinky” hair, and abnormalities of connective tissue accounts for up to 50% of the patients with autosomal and bone. Immunological problems or infections are recessive severe combined immunodefi ciency (SCID) rare, but pulmonary infection secondary to inhalation disease. Adenosine deaminase converts adenosine may prove lethal. and deoxyadenosine to inosine and deoxyinosine. The Thymic hypoplasia and defective T cell function resulting accumulation of deoxyadenosine and ade- has been noted in some patients with Zellweger syn- nosine exerts toxicity to lymphocytes. Obviously, the drome, the most severe of the disorders of peroxisome severity of the disease correlates with accumulation of biogenesis. Affected infants exhibit extreme muscular toxic metabolites and inversely with residual adenos- hypotonia, seizures, liver dysfunction, dysmorphic ine deaminase expression. Multiple, recurrent infec- skeletal, and eye abnormalities, failure to thrive, tions are usually more severe than in purine nucleoside and mostly early death due to the progressive enceph- phosphorylase defi ciency and become rapidly life alopathy. Immunological problems are not usually of threatening. Typical laboratory fi ndings include lym- high clinical relevance. phopenia (usually <500 total lymphocytes per cubic millimeter), involving both the B and T cells as well as hypogammaglobulinemia. While IgM defi ciency C12.3 Inherited Metabolic Diseases may be detected early, IgG defi ciency manifests only Associated with B Cell after the age of 3 months, when the maternal supply becomes exhausted. Further immunologic abnormali- Immunodefi ciency ties include a defi ciency of antibody formation following specifi c immunization, and absence or severe Transcobalamin II is necessary for intestinal absorp- diminution of lymphocyte proliferation induced by tion of cobalamin and transport to tissues. Defi ciency mitogens. This condition is progressive, since residual of transcobalamin II leads to severe megaloblastic B and T cell function that may be found at birth disap- anemia with hypocellular bone marrow, leukopenia, pears later on. Only a few patients have been reported thrombocytopenia, vomiting, failure to thrive, with delayed (up to 3 years of age) or late (up to diarrhea, and lethargy. A frequent fi nding in transcoba- 8 years of age) onset. Infections can be caused by a lamin II defi ciency is the presence of hypogammaglob- broad variety of microorganisms. Localization of ulinemia, particularly IgG. Less often, levels of IgA infections are predominantly the skin, and the respira- and IgM are found decreased. Failure to produce spe- tory as well as the gastrointestinal tract, where they cifi c antibodies against diphtheria or poliomyelitis has often lead to intractable diarrhea and malnutrition. In been demonstrated in several patients. Although phago- patients older than 6 months of age, hypoplasia or cytic killing is usually normal, a specifi c impairment of apparent absence of lymphoid tissue may constitute a neutrophils against Staphylococcus aureus has been diagnostic sign. In about half of the patients bony reported in a single patient. Immunological abnormali- abnormalities include prominence of the costochon- ties usually resolve after cobalamin supplementation. dral junctions. In some patients neurologic abnormal- Propionic acidemia is caused by defi ciency of ities are found, including spasticity, head lag, propionyl-CoA carboxylase, a biotin-dependent en movement disorders, and nystagmus. Injections of zyme. In the long-term mental retardation, an extrapy- bovine adenosine deaminase conjugated to polyethyl- ramidal movement disorder and osteoporosis develop ene glycol (PEG-ADA) results in normalization of in most patients. Decreased levels of IgG and IgM as T cell number and cellular and humoral responses. well as B cell lymphopenia have been observed during Bone marrow transplantation has been very success- periods of metabolic decompensation. ful in some patients. 246 E. Mayatepek

The disturbance of zinc homeostasis in acroderma- and ataxia. Recurrent infections are an occasional clini- titits enteropathica results from a partial block in intes- cal feature. Inconstantly found immunologic abnormali- tinal absorption. Reduced zinc absorption leads to ties include decreased levels of IgM, IgG, and IgA as impairment of the function of many zinc metalloen- well as decreased proliferation to phytohemagglutinin zymes, which are involved in major metabolic path- and pokeweed mitogen (PWM). ways. Symptoms usually start in infancy. The most a-Mannosidosis caused by defi ciency of a-mannosi- dramatic clinical feature is a characteristic skin rash. In dase leads to the accumulation of mannose-rich oligosac- patients with zinc defi ciency states, impaired humoral charides in neural and visceral tissues. This lysosomal and cell-mediated immune responses can usually be storage disease is characterized by progressive mental demonstrated. Secondary infections are common, retardation, deafness, cataracts, corneal clouding, dysosto- mostly with Candida or Staphylococci. Mucosal sis multiplex, progressive ataxia, hernias and hepatomegaly. lesions include gingivitis, stomatitis, and glossitis. Many patients with a-mannosidosis have recurrent infec- Further symptoms include diarrhea, failure to thrive, tions. Immunologic abnormalities may include decreased alopecia, irritability, and mood changes. Zinc therapy IgG levels, impaired lymphoproliferation to phytohe- leads to clinical remission. magglutinin, defective chemotaxis, phagocytosis, and Biotin is a cofactor for carboxylation of 3-methylcrot- bactericidal killing. In one patient, pancytopenia result- onyl-CoA, propionyl-CoA, acetyl-CoA, and pyruvate. ing from antineutrophil antibodies has been reported. Biotinidase defi ciency and holocarboxylase synthetase Leukopenia occurs in about 50% of patients with defi ciency result in multiple carboxylase defi ciency. methylmalonic aciduria, a classical organic aciduria Symptoms include lactic acidosis, muscular hypotonia, affecting branched-chain amino acid metabolism. seizures, ataxia, psychomotor retardation, skin rashes, Immunologic abnormalities associated with methylma- hair loss, and immune defects. Immunologic dysfunction lonic aciduria may include neutropenia, pancytopenia, affecting both the B and T cell lines has been reported in decreased B and T cell numbers, low IgG levels, and several children with biotinidase defi ciency. Symptoms impaired phagocyte chemotaxis. Specifi c lack of respon- include mucocutaneous candidiasis, absence of delayed siveness to Candida antigen has been observed resulting hypersensitivity as assessed by skin testing and by in in extensive dermatosis. In addition, methylmalonic acid vitro lymphocyte responses to Candida challenge, inhibits bone marrow stem-cell growth in vitro. decreased IgA levels, poor antibody formation to pneu- mococcal immunization, subnormal amounts of T lymphocytes, reduced leukocyte killing against Candida, lack C12.5 Inherited Metabolic Diseases of myeloperoxidase acitivity in neutrophils, impaired Associated with Phagocyte lymphocyte suppressor activity, and decreased prosta- Immunodefi ciency glandin E2 production in vitro. One child with biotinidase defi ciency was diagnosed initially as having SCID and was treated with bone marrow transplantation, but the Galactosemia results from galactose-1-phosphate uri- symptoms were not ameliorated until biotin was given. In dyl transferase defi ciency and is characterized by jaun- general, immunological fi ndings in biotinidase defi ciency dice, hepatomegaly, nuclear cataracts, mental disability, are inconsistent. However, biotin treatment corrects the and feeding diffi culties. Neonates with galactosemia immunologic as well as the metabolic abnormalities. are at increased risk for life-threatening sepsis from Hereditary orotic aciduria is an inborn error of Escherichia coli. Granulocyte chemotaxis is impaired, pyrimidine metabolism characterized by growth retar- whereas bactericidal activity is usually not affected. In dation, developmental delay, and megaloblastic anemia vitro exposure of neutrophils from affected neonates to unresponsive to cobalamin and folic acid. Lymphopenia galactose results in impaired function. and increased susceptibility to infections, including Glycogen storage disease (GSD) type I non-a pres- candidiasis, bacterial meningitis, and fatal varicella ents with hepatomegaly, hypoglycemia acidosis, and have been observed. Immunologic abnormalities are growth failure. The condition is clearly distinguished variable and include low T cell number, impaired from GSD type Ia by the occurrence of recurrent severe delayed-type hypersensitivity response, reduced T cell- bacterial infections and immunologic abnormalities, mediated killing, and decreased levels of IgG and IgA. resulting from neutropenia as well as defective function Defi ciency of intestinal folic acid absorption leads to of leukocytes. Neutrophil function is variable, in most megaloblastic anemia, pychomotor retardation, seizures, patients random movement, chemotaxis, microbial C12 Immunological Problems 247 killing, and respiratory burst are diminished. In contrast, clear but probably related to the accumulation of CoA monocytes have decreased respiratory burst, but usually esters of organic acids. have normal random and directed motility. T cell, B cell, Other inborn errors of metabolism including acro- and NK-cell functions are normal. In most patients, dermatitis enteropathica, methylmalonic aciduria, recurrent or chronic bacterial infections become a major propionic acidemia, Gaucher disease, lysinuric protein clinical problem. These infections are underlined by intolerance, Niemann-Pick disease, or a-mannosidosis a decreased number of neutrophils (usually below are at least in some patients associated with phagocyte 1500/mL) combined with defective neutrophil and dysfunction or macrophage activating syndrome. monocyte functions. Bone marrow examination shows hypercellularity. GSD type I non-a is caused by mutations in the glucose-6-phosphate translocase gene. This C 12.6. Inherited Metabolic Diseases gene encodes a microsomal transmembrane protein that Associated with NK Cell is expressed in numerous tissues, including monocytes Immunodefi ciency and neutrophils. Frequently observed symptoms, such as infl ammatory bowel disease similar to Crohn disease, Lysinuric protein intolerance is one of the very few oral lesions, and perianal abscesses are presumably inborn errors of metabolism in whom NK cell immuno- related to defective neutrophil function. Neutrophil cell defi ciency has been reported at least in single instances. counts and some but not all neutrophil functions improve Other syndromes such as the ChediakÐHigashi syn- after subcutaneous treatment with granulocyte colony- drome, Sutor syndrome, Griscelli syndrome, or xero- stimulating factor. derma pigmentosum form this specifi c subgroup of X-linked cardioskeletal myopathy (Barth syndrome) immunodefi ciency disorders. is characterized by a congenital dilated cardiomyopathy and mitochondrial myopathy with growth failure. In most patients, moderate to severe neutropenia is a Take Home Messages persistent feature leading to recurrent serious bacterial › Patients with inherited metabolic diseases may infections. have immunological problems resulting in Glutathione synthetase defi ciency causes severe increased susceptibility to infections. In addi- metabolic acidosis and hemolytic anemia. In the course tion, infections may be secondary to chronic of the disease progressive neurological symptoms may disease, malnutrition, movement disorders, or develop, including psychomotor retardation, seizures, poor control of swallowing and resulting aspi- ataxia, and spasticity. In about 10% of affected patients, ration. In most metabolic defects, immunologi- recurrent bacterial infections have been reported cal abnormalities are secondary to the metabolic resulting from impaired bacterial killing. The patient’s derangement. Immune dysfunction can affect neutrophils fail to assemble microtubules during any of the major components of the immune phagocytosis leading to damage to membranous system: T cells, B cells (including immuno- structures. However, the susceptibility to infections is globulins), phagocytes, e.g., neutrophils, mono- relatively mild. Treatment with vitamins E and C can cytes, macrophages, natural killer (NK) cells, restore abnormal immunologic functions. and complement. Pearson syndrome is caused by large deletions and › Primary metabolic immunodefi ciency diseases duplications in the mitochondrial DNA and character- include adenosine deaminase defi ciency or ized by exocrine pancreatic and liver dysfunction, fail- purine nucleoside phosphorylase defi ciency. ure to thrive as well as neuromyopathy. In addition to Other inherited diseases with often compro- anemia and thrombocytopenia, neutropenia is a fre- mised immunity include, beside others, inborn quent fi nding. errors of cobalamin and folate metabolism, In isovaleric aciduria, as in propionic acidemia or organic acidurias, and disorders of carbohy- methylmalonic aciduria, neutropenia, and pancytopenia drate metabolism. In most of them, correction can develop during periods of acidosis. Neonatal sepsis of the metabolic defect restores normal can lead to early death. The bone marrow contains large immune function. numbers of immature cells, suggesting an arrest of maturation. The underlying mechanism, however, is not 248 E. Mayatepek

Key References Hirschhorn R (1993) Overview of biochemical abnormalities and molecular genetics of adenosine deaminase defi ciency. Pediatr Res 33:35SÐ41S Carapella-de Lacua E, Aiuti F, Lucarelli P et al (1975) A patient Houten SM, Kuis W, Duran M et al (1999) Muations in MVK, with nucleoside phosporylaswe defi ciency, selective T-cell encoding mevalonate kinase, cause hyperimmunoglobulinae- defi ciency and autoimmune hemolytic anemia. J Pediatr mia D and periodic fever syndrome. Nature Genet 22:175Ð177 93:1000Ð1003 Kobayashi R, Blum P, Gard S et al (1980) Granulocyte function Cowan MJ, Wara DW, Packman S et al (1979) Multiple biotin- in patients with galactose-1-phosphate uridyl transferase dependent carboxylase defi ciencies associated with defects defi ciency (galactosemia). Clin Res 28:109A in T-cell and B-cell immunity. Lancet 2:115Ð118 Ming JE, Stiehm ER, Graham JM Jr (2003) Syndromic immu- Desnick RJ, Sharp HL, Grabowski GA et al (1976) Mannosidosis: nodefi cienies: genetic syndromes associated with immune clinical, morphologic, immunologic, and biochemical stud- abnormalities. Crit Rev Clin Lab Sci 40:587Ð642 ies. Pediatr Res 10:985Ð996 Spielberg SP, Boxer LA, Oliver JM et al (1979) Oxidative dam- Dionisi-Vici C, de Felice L, el Hachem M et al (1998) Intravenous age to neutrophils in glutathione synthetase defi ciency. immunoglobulin in lysinuric protein intolerance. J Inherit Br J Haematol 42:215Ð223 Metab Dis 21:95Ð102 Gitzelmann R, Bosshard WU (1993) Defective neutrophil and monocyte function in glycogen storage disease type Ib: a literature review. Eur J Pediatr 152:33SÐ38S Newborn Screening for Inherited Metabolic Disease D1

Piero Rinaldo and Dietrich Matern

Key Facts D1.1 General Remarks › The classic presentation of inborn errors of metabolism is with a free period of apparent Routine screening of all newborns for inherited dis- health that may last days or even years, but it orders began in the 1960s after Horst Bickel had is followed by overwhelming life threatening established an effective dietary therapy for phenylke- disease. tonuria (PKU), and Robert Guthrie introduced a sim- › The episode usually follows catabolism intro- ple bacterial inhibition assay to detect elevated duced usually by acute infection; sometimes concentrations of phenylalanine in dried blood spots, after surgery. a type of specimen universally now known as the “Guthrie card.” Over time, newborn screening was › Initial laboratory evaluation needs only the extended to several other treatable metabolic and routine clinical laboratory to establish acidosis endocrine disorders including galactosemia, maple or alkaoisis, hyperammonemia, ketosis, hypo- syrup urine disease, biotinidase deﬁ ciency, hypothy- clycimia, or latic acidemia. roidism, congenital adrenal hyperplasia (CAH), and hemoglobinopathies. In recent years, the scope of newborn screening has leaped forward exponentially by the introduction of acylcarnitine and amino acid analysis in Guthrie cards by tandem mass spectrometry (MS/MS). This multiplex platform allows the concurrent detection of disorders of amino acid, organic acid, and fatty acid intermediary metabolism and thus several of the most prevalent treatable inborn errors of metabolism. Although the implementation of MS/MS is not pursued consistently in different countries, there is a notable trend toward overall screening expansion. For example, by July 2009, all babies born in the United States were screened for the metabolic conditions included in the uniform panel recommended by the American College of Medical Genetics (ACMG). This chapter contains P. Rinaldo ( ) information on how to proceed when the results of Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, 200 First Street S. W., Rochester, MN 55905, USA ﬁ rst tier newborn screening for certain disorders are e-mail: [email protected] reported to be abnormal.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 251 DOI: 10.1007/978-3-540-74723-9_D1, © Springer-Verlag Berlin Heidelberg 2010 252 P. Rinaldo and D. Matern

D1.2 Hyperphenylalaninemias plasma concentrations of phenylalanine must be kept below 360 mmol/L throughout gestation in order to minimize the potential teratogenic effects of phe- Newborn hyperphenylalanemia can be caused by a nylalanine on a developing fetus. variety of conditions. The primary genetic defi ciency is that of phenylalanine hydroxylase, the enzyme that catalyzes the conversion of phenylalanine to tyrosine. Missing phenylalanine hydroxylase activity is the cause of PKU. In addition, abnormalities in the biosynthesis D1.3 Galactosemia or regeneration of tetrahydrobiopterin (BH4), the cofactor of this enzyme, are also detected. Secondary causes The activated 1-phosphate metabolites of both galac- of an elevated concentration of phenylalanine include tose and fructose are highly toxic, particularly for parenteral nutrition, drugs (trimethoprim, chemothera- liver, kidneys, and brain. In classical galactosemia, peutic agents), and liver disease. Persistent hyperphe- galactose-1-phosphate (Gal-1-P) accumulates because nylalaninemia above 360–600 mmol/L is harmful to the of a defective synthesis of UDP-galactose catalyzed developing brain, leading to progressive mental retar- by galactose-1-phosphate uridyltransferase (GALT). dation, epilepsy, spasticity, and psychiatric problems. It Affected children show symptoms such as vomiting, can be prevented by early diagnosis and dietary restric- diarrhea, and jaundice progressing after the start of tion of phenylalanine intake, ideally beginning as soon milk (lactose) feedings, usually from the 3rd or 4th as possible after birth but not later than 14 days of age. day of life onwards. Untreated, the disease usually Most neonates with signifi cant hyperphenyla- progresses to hepatic and renal failure, and death; there laninemia suffer from phenylalanine hydroxylase may be progressive bilateral cataracts. The severe defi ciency; the incidence in most Caucasian popula- acute manifestations of galactosemia can be prevented tions is between 1:4,000 and 1:12,000. For practical by exclusion of galactose from the diet. Late compli- purposes, two forms of phenylalanine hydroxylase cations such as ovarian failure and impaired speech defi ciency are distinguished: PKU, which requires and language development may occur despite good treatment, and mild hyperphenylalaninemia (pheny- compliance with treatment. The preventive potential lalanine levels <600 mmol/L), which does not. Flow- of newborn screening may not be fully realized if chart 1 depicts the work-up of a neonate with infants become symptomatic before the newborn hyperphenylalaninemia recognized through newborn screening results are available. The incidence of clas- screening, according to the ACT sheet established sic galactosemia in newborn screening programs in by ACMG (available online at http://www.acmg. the United States has ranged from 1:55,000 to 1:80,000. net/). High phenylalanine values should be con- False negative fi ndings in newborn screening may be fi rmed through quantitative analysis of plasma amino observed after blood transfusion. acids so that concentrations of tyrosine as well as Newborn screening is carried out either by mea- phenylalanine are known. Analysis of urinary pter- surement of the activity of GALT alone or (less fre- ins and the activity of dihydropteridin reductase in quently in the US) in combination with determination blood should always be carried out to exclude BH4 of the total galactose concentration. The combined cofactor defi ciency. A positive response of the BH4 approach allows identifi cation not only of GALT loading test is of immediate therapeutic relevance. defi ciency, but also of galactokinase defi ciency and Treatment of PKU should be started immediately epimerase defi ciency. A positive screening result is with a phenylalanine restricted diet and supplemen- followed up by measurement of GALT activity in red tation of essential amino acids. Recommended ther- blood cells. When GALT activity is reduced, further apeutic phenylalanine values differ between diagnostic characterization is possible by isoelectric countries but should not be above 240–360 mmol/L focusing of the GALT protein on a gel, which helps in the fi rst 6–10 years of life. Most diets are more to determine either the classic GG variant, the other liberal for teenagers. There is no consensus with common variant Duarte (DD), compound heterozy- regard to whether or not treatment is necessary in gotes (DG), or heterozygotes. A large number of dif- adulthood, with the exception of pregnancy, when ferent mutations have been identifi ed in the GALT D1 Newborn Screening for Inherited Metabolic Disease 253 gene. The Duarte variant has an allele frequency of D1.4 Biotinidase Defi ciency ca. 6% and is associated with a 50% reduction in enzyme activity, which does not usually require The carboxylation of 3-methylcrotonyl-CoA, propio- dietary intervention. Even in compound heterozy- nyl-CoA, acetyl-CoA, and pyruvate is biotin depen- gotes with a severe mutation on the other allele, diet dent. The individual apoenzymes need to be covalently may not be necessary. Therefore, a majority of chil- bound to biotin in order to generate the active holoen- dren with elevated galactose concentrations found in zymes. This reaction is catalyzed by the enzyme holo- newborn screening may have a mild form of GALT carboxylase synthetase. A defi ciency of this enzyme defi ciency that does not require treatment. In these causes severe, multiple carboxylase defi ciency, which children, galactose and Gal-1-P concentrations on an usually presents in the newborn period. Affected chil- ordinary diet often normalize within a few weeks or dren show severe metabolic decompensation typical months. for an organic aciduria and progressive neurological When galactose is <20 mg/dL (1.1 mmol/L) it is problems; in addition, there are usually skin rashes suffi cient to check the general condition (feeding, and alopecia. Another, milder but therefore often vomiting, weight gain, and liver size) and to send insidious form of multiple carboxylase defi ciency is another blood spot sample for galactose measurements caused by an impaired release of covalently bound (preferentially taken 60 min after a milk feed) to the biotin from dietary and endogenous proteins, a reac- newborn screening laboratory. tion that is catalyzed by the enzyme biotinidase. The When galactose is between 20–50 mg/dL (1.1– defi ciency of biotinidase causes a depletion of free 2.8 mmol/L) or when it has been determined that the biotin and progressive neurological and dermatolog- infant has a GG or DG phenotype, determination of ical symptoms usually starting in infancy. plasma galactose and erythrocyte Gal-1-P are carried Symptomatology includes seizures, ataxia, hearing out in 1–3 mL of EDTA blood shipped at ambient tem- loss, optic atrophy, spastic diplegia, and developmen- perature. Molecular genetic testing of the GALT gene tal delay as well as skin rash and alopecia. These char- is also available to confi rm the diagnosis. EDTA blood acteristic symptoms may occur in half of the affected may be stored at room temperature for a couple of patients, but less specifi c symptoms such as hypoto- days, if necessary. Lactose-free milk feedings are rec- nia, developmental delay, and seizures may occur in ommended until fi nal results are available. some cases. Because of the insidious onset of symp- Immediate hospital admission is necessary in the toms, the diagnosis is often delayed or even missed. presence of galactose concentrations above 50 mg/dL This is a major concern since this form of multiple (>2.8 mmol/L), laboratory signs of liver disease, or carboxylase defi ciency can be effectively treated with clinical distress. Lactose-free feedings should be com- biotin. A semiquantitative colorimetric or fl uorometric menced as soon as the appropriate blood and urine measurement of biotinidase activity in dried blood samples have been taken (amino acids and reducing spots has therefore been included in newborn screen- substances in the urine, determination of plasma galac- ing programs in many countries. tose and erythrocyte Gal-1-P, complete blood cell False positive results may occur with improper han- count). Coagulation studies and blood cultures dling of samples (e.g., exposure to excessive heat). On (Escherichia coli sepsis is common) should be consid- the other hand, false negative fi ndings in newborn ered in all patients not clinically normal. screening may be observed after blood transfusion or The indication for long-term therapy ultimately when the neonate received a catecholamine infusion rests on degree of GALT defi ciency and the concen- during blood sampling. Residual biotinidase activity tration of Gal-1-P in erythrocytes, which is normally may vary, depending on the underlying mutations in below 0.3 mg/dL (11 mmol/L) but may rise up to the biotinidase gene. Mild forms of biotinidase defi - 100 mg/dL (~4 mmol/L) in classical galactosemia. It ciency with relatively high enzyme activity levels is impossible to obtain completely normal Gal-1-P occur that do not cause disease and do not require levels in most GG patients because of a substantial treatment. Nevertheless, it is advisable to start biotin endogenous production of galactose. Therapeutic supplementation immediately after an abnormal new- target concentrations of 2–4 (at most fi ve) mg/dL are born screening result has been reported, before confi r- realistic. matory tests are completed. It is also advisable to 254 P. Rinaldo and D. Matern collect samples for blood ammonia, plasma lactate, The initial screening test is the assay of thyroid and urinary organic acids before commencing biotin stimulating hormone (TSH) to detect an elevated con- supplementation to evaluate the baseline metabolic centration (usually >20 mU/mL). However, in some status. Treatment is simple and does not involve com- laboratories the assay of thyroxine (T4) is preferred, plicated dietary regimens as in some other disorders others measure both. Newborns with TSH >50 mU/mL that are included in newborn screening programs. are considered very likely to have congenital hypothy- Temporary initiation of biotin supplementation does roidism, and, therefore, require immediate follow-up not interfere with breastfeeding and parent–child inter- testing of plasma and treatment. Those having a less action. The recommended starting dose is 10 mg/day. prominent elevation of TSH are evaluated by confi r-

If a reduced biotinidase activity is conﬁ rmed in the matory serum tests (free T4, T3 resin uptake, and repeat blood spot screening test, biotinidase activity TSH). should be determined in serum. A residual activity of Treatment includes oral l-T4 at a dosage to main-

0–10% indicates profound biotinidase defi ciency. tain blood TSH concentration <4 mU/mL and T3/T4 Long-term treatment with 5–20 mg biotin/day is usu- in the age-related range. Dosage and follow-up should ally adequate. Treatment can be reevaluated by deter- be coordinated in consultation with a pediatric endocri- mining the activity of carboxylases in lymphocytes. nologist. Biotinidase activity between 10 and 25% indicate partial defi ciency which may not require long-term treatment. Many centers recommend treatment of these D1.6 Congenital Adrenal children with 10 mg/day for the fi rst year of life. It is not yet clear what regimens are optimal for control of Hypoplasia (CAH) late complications, particularly to the optic and auditory nerves. Infants with CAH have a defi ciency of one of several adrenal enzymes involved in steroid biosynthesis resulting in limited cortisol production and, in some cases, limited aldosterone production. The pituitary gland D1.5 Congenital Hypothyroidism senses the cortisol defi ciency and produces increased amounts of ACTH. The adrenal glands enlarge but con- Congenital hypothyroidism occurs in infants who are tinue to produce inadequate amounts of cortisol. Some born without the ability to produce adequate amounts of of the precursors of cortisol are virilizing hormones. As thyroid hormone. Thyroid hormone is essential for nor- a result of cortisol defi ciency, affected infants are mal growth and brain development. If untreated, con- unable to respond adequately to the stress of injury or genital defi ciency of thyroid hormone results in mental illness. Because of aldosterone defi ciency, sodium and retardation and stunted growth. Infants with untreated water are lost in the urine resulting in dehydration. congenital hypothyroidism may appear clinically nor- Potassium accumulates in the blood, causing irritability mal up to 3 months of age, by which time some brain or lethargy, vomiting, and muscle weakness, including damage will already have occurred. When symptoms or cardiac muscle irritability and weakness, leading to clinical signs are present, they may include prolonged shock and death in a salt-wasting crisis. newborn jaundice, constipation, lethargy, poor muscle Male infants with CAH usually appear normal at tone, feeding problems, macroglossia, mottled and dry birth. Female infants usually show the effects of ele- skin, distended abdomen, and umbilical hernia. vated virilizing hormones: an enlarged clitoris and The most common causes are total or partial failure fusion of the labia majora over the vaginal opening. of the thyroid gland to develop (aplasia or hypoplasia) Occasionally, the female infant may be virilized so as or its development in an abnormal location (an ectopic to appear to have a male penile structure with hypospa- gland). These types of hypothyroidism rarely recur in dia. Such newborns should not have a palpable gonad siblings. Less commonly, the hypothyroidism results in the labial/scrotal sac. Their ovaries, uterus, and from a hereditary inability to manufacture thyroid hor- Fallopian tubes are normal. mones, maternal medications during gestation (iodine Several types of genetic defects cause the enzymatic and antithyroid drugs), or maternal antibodies. defi ciencies of CAH. All are autosomal recessive. The D1 Newborn Screening for Inherited Metabolic Disease 255 traditional newborn screening test is designed to detect techniques of sample preparation and injection into the the accumulation of 17-hydroxy progesterone (17-OHP), instrument. In most laboratories, acylcarnitines and the result in most cases (>90%) of underlying 21-hydrox- amino acids are derivatized as butyl esters. ylase defi ciency. In clinical practice, however, one should Amino acids are of course the building block of pro- remember that an abnormal newborn screening test can teins. Acylcarnitines are formed from free carnitine and also indicate rarer enzyme defi ciencies which cause acyl-CoA moieties derived from fatty acids and organic CAH, for example, 11-hydroxylase defi ciency. acids (which may have been derived from amino acids) The screening test for CAH is an immunoassay for through the action of one of several carnitine-acyl-CoA 17-OHP, a precursor of cortisol. Cross reactivity with other transferases. The concentration of acylcarnitine species steroids is a frequent interference, in particular among pre- increases in response to many possible metabolic defects, mature newborns. If screening indicates the possibility of leading to complex profi les with as many as 18 informa- CAH, an emerging, cost-effective approach is to perform tive markers, each of them requiring various degrees of on the same blood spot a second tier test by LC-MS/MS differential diagnosis. In other words, the many pro- for the determination of 17-OHP, androstenedione, and gresses achieved at the analytical level have no remedy, most importantly cortisol, the end product of the pathway or substitute, for the inevitable complexity of post-ana- (Lacey et al. 2004). This approach can eliminate more lytical interpretation (Rinaldo, Cowan, Matern 2008). than 90% of potential false positive results of the primary More than 60 metabolic conditions could be detected screening (frequently >1% of all newborns, and a much by multiplex analysis of acylcarnitine and amino acid. greater proportion of premature babies), and could include Forty-two of them have been included in the screening additional informative markers such as 11-deoxy cortisol panel recommended by the American College of and 21-deoxy cortisol (Minutti et al. 2004). Overall, ste- Medical Genetics ACMG in 2006, which includes a roid profi ling by LC-MS/MS achieves improved specifi c- total of 29 primary targets (collectively described as the ity, and could potentially evolve into a fi rst tier screening uniform panel) and 25 secondary targets (Watson 2006a with clinically validated cutoffs based on birth weight, & b). The metabolic conditions screened for by MS/ gender, and comparison with disease ranges. MS are divided in 20 primary and 22 secondary targets, Effective treatment for CAH is hormone replace- based on our understanding of the natural history and ment. Decisions about hormonal treatment should be severity of the disorders and available evidence of effec- made in consultation with a pediatric endocrinologist tive modalities of treatment. However, it must be under- and may include hydrocortisone and mineralocorti- scored that sorting, and “elimination,” of conditions that coids. Medications need to be adjusted as the child are detected based on exactly the same markers is unre- grows. Serum adrenal hormone levels and renin are alistic and potentially dangerous. In reality, all but one monitored. Female infants who have virilization of the of the secondary targets, 2–4 dienoyl-CoA reductase genitalia may need surgical correction. This is usually defi ciency, are involved to some degree in the differen- done in stages, with the fi rst surgery before the age of tial diagnosis of one or more primary targets. 2 years. Infants with CAH, if detected early and treated Table D1.1 summarizes the main characteristics of with appropriate doses of medication, can have normal the 42 metabolic conditions included in the ACMG growth, development, and intellectual potential. In recommended panel. In addition to basic disease infor- addition, fertility is usually normal. mation (MIM number, common name if applicable, and name of the defi ciency enzyme or function), the table lists the analytes and major ratios relevant to the detection of each condition. This information is com- D1.7 Expanded Newborn Screening bined with an estimate of the likelihood of correct identifi cation (sensitivity) and of detection of carriers. of Metabolic Conditions by MS/MS The latter situation is usually encountered when a newborn investigated for an abnormal result is found to be Tandem MS/MS has been used for the detection and an obligate heterozygote born to an affected, possibly analysis of acylcarnitines since the 1980s. This tech- asymptomatic and therefore undiagnosed mother. nique has been extended to newborn screening because Table D1.2 focuses primarily on the analytes rather of the development of automated, high-throughput than the conditions. It also highlights the critical need 256 P. Rinaldo and D. Matern (maternal cases) (maternal cases) – – – – – Possible Possible – – – – – – – – – – Possible Detection of carriers by NBS m mol/L to be high negatives and succinylacetone is not measured et al. (Turgeon 2008) test) measured test) >150 to be low at age to be low <7 days Unknown, but likely likely but Unknown, High Potential false Potential false Low if Asa not Low High (with 2nd tier Low if based on Tyr if based on Tyr Low Unknown, but likely likely but Unknown, Likelihood of Likelihood cation by NBS identifi based on using cutoffs disease ranges ratios Phe/Tyr High – Cit/Arg High Phe/Tyr High – AC/Cit High C14:1/C16 Met/Phe High Cit/Arg High C16-OH/C16 High Met/Phe High (with 2nd tier ASA/Arg ASA/Arg Cit/Arg Xle/Ala Val/Phe Phe/Tyr if cutoff Low Phe/Tyr High C16-OH/C16 High C18:1-OH C18-OH C18:1-OH C18-OH Phe Tyr Arg Phe Tyr C0 C16:1-OH C16-OH C14:2 C14:1 C14 C14:1/C2 Succinylacetone – Met Cit C16:1-OH C16-OH Met Cit, Arg, Met, Thr Cit, Arg, ASA, Cit Ile + Leu (Xle), Val Xle/Phe Phe Phe Informative analytesInformative Informative Biochemical markers cient ciency reductase, pterin -4 a -carbinolamine dehydratase 2 pyruvate acid pyruvate oxidase (dioxygenase) tetrahydropterin synthase transporter ( a , b subunit) acyl-CoA dehydrogenase acyl-CoA adenosyltransferase I/III, hydrolase, S-adenosylhomocysteine Glycine N-methyltransferase defi dehydrogenase b -synthase carrier (citrin) lyase -keto acid dehydrogenase a -keto hydroxylase enzyme or function Arginase Plasma membrane carnitine Trifunctional protein Trifunctional Methionine Argininosuccinate synthetase Argininosuccinate Cystathionine Aspartate glutamate Argininosuccinate Argininosuccinate Phenylalanine Phenylalanine hydroxylase Name of defi Fumarylacetoacetate hydrolase BIOPT (REG) Dihydropteridine TYR III 4-Hydroxyphenyl ARG BIOPT (BS) 6-pyruvoil GTP cyclohydrolase, CUD TYR II transaminase Tyrosine TFP VLCAD long-chain Very MET CIT LCHAD Long-chain l -3-hydroxy HCY CIT ASA MSUD Branched-chain H-PHE Phenylalanine PKU Code TYR I ciency ciency ciency regeneration biosynthesis defect protein defi dehydrogenase defi dehydrogenase defi (newborn/adult) acidemia disease (PKU) (classic) Disorders of biopterin Disorders of biopterin Tyrosinemia type III Tyrosinemia Argininemia Carnitine uptake Carnitine uptake Trifunctional Trifunctional Very long-chain acyl-CoA long-chain acyl-CoA Very Tyrosinemia type II Tyrosinemia Long-chain 3-OH acyl-CoA Long-chain 3-OH acyl-CoA Hypermethioninemias Citrullinemia type II Citrullinemia type I Argininosuccinic Argininosuccinic Maple syrup urine Phenylketonuria Phenylketonuria Tyrosinemia type I Tyrosinemia Homocystinuria Hyper-phenylalaninemia 261630 261640 250850 606664 126090 600225 276710 207800 212140 609015 201475 276600 609016 180960 605814 603472 215700 207900 248600 261600 276700 236200 261600 MIM number Common name of Medical Genetics recommended panel Inborn errors included in the American College D1.1 AA AA AA AA FAO FAO FAO AA FAO AA AA AA AA AA AA AA AA AA Conditions (IEMs detected by mass spectrometry only) Group Table D1 Newborn Screening for Inherited Metabolic Disease 257 a (maternal cases) (maternal cases) (maternal cases) (maternal cases)a Possible – – – – negatives test) test) test) test) Potential false Potential false High – High Possible Uncertain Possible High Possible High – High – High – High – High – HighHigh – – High (with 2nd tier High (with 2nd tier High (with 2nd tier High (with 2nd tier C5DC/C8 C5DC/ C16 C5-OH/ C0 C5/C3 C8/C10 C5-OH/ C0 ratios C18:1)/ C2 C5/C3 C4/C8 ratios C18) C4/C8 C3/C16 C3/C16 C3/C16 C3/C16 All related C16 C5-OH (C0) C5-OH/C8 C6 C8 C10:1 C10 C8/C2 C14 C16 C18 High – C5-OH C5:1 C5-OH/C8 Uncertain C0 C16 C18 C0/(C16 + C8 C8-OHC4-OH Unknown Unknown – – C10:2 Unknown – C3 C3/C2 C3 C3/C2 C3 C3/C2 ) 18:1 + C 18 + C 16 + C 3 + C subunit) 2 -ETF ETF-QO C4 C5 C8 C5DC C14 b b , a ( dehydrogenase translocase dehydrogenase palmitoyltransferase Ia palmitoyltransferase thiolase ketoacyl-CoA dehydrogenase reductase mutase synthesis transferase, MMADHC protein (C2ORF25) + C 0 -ETF -Ketothiolase C5-OH C5:1 C4-OH C5-OH/C8 High – b a 3MCC carboxylase 3-Methylcrotonyl-CoA CACT Carnitine:acylcarnitine MCAD Medium-chain acyl-CoA HMG 3-Hydroxy-3-methylglutaryl-CoA lyase C5-OH C6DC C5-OH/C8 MCD Holocarboxylase synthetase C3 C5-OH All related CPT II II Carnitine palmitoyltransferase C14 C16 C18 (C16 + 2MBG dehydrogenase 2-Methylbutyryl-CoA C5 C5/C0 C5/C2 GA II 2M3HBA 2-Methyl 3-hydroxybutyryl-CoA IBG dehydrogenase Isobutyryl-CoA C4 C4/C2 C4/C3 CPT I Carnitine M/SCHAD Short-chain L-3-hydroxy acyl-CoA SCADMCKAT dehydrogenase Short-chain acyl-CoA Medium-chain C4 C4/C2 C4/C3 DE-RED 2,4-Dienoyl-CoA MUT Methylmalonyl-CoA Cbl A B Adenosylcobalamin Cbl C D methyl MMA mutase, Hcy:MTHFR sum of selected species (C of the newborn ciency ciency ciency ciency ciency AC ciency ciency ciency BKT ciency ciency (L) ciency ciency ciency ciency ciency ciency acyl-CoA dehydrogenase acyl-CoA defi carboxylase defi acidemia defi translocase defi II defi nase defi type II defi dehydrogenase defi palmitoyl-transferase palmitoyl-transferase Ia defi 3-OH acyl-CoA 3-OH acyl-CoA dehydrogenase defi defi dehydrogenase defi dehydrogenase defi acidemia (Mut) acidemia (Cbl A, B) acidemia (Cbl C, D) -Ketothiolase defi -Ketothiolase b Glutaric acidemia Methylmalonic Methylmalonic 231675 608053 251110 611935 OA 231670 Glutaric acidemia type I GA I Glutaryl-CoA deydrogenase C5DC (C0) C5DC/C5-OH FAO 607008 Medium-chain OA 210200 3-Methyl crotonyl-CoA OAOA 203750 246450 3-Hydroxy 3-methyl glutaric OA 243500 acidemia Isovaleric IVA dehydrogenase Isovaleryl-CoA C5 C5/C0 C5/C2 OA 253270 Multiple carboxylase FAO 255110 Carnitine:acylcarnitine FAO 255110 Carnitine palmitoyl-transferase OA 600301 dehydroge- 2-Methyl butyryl-CoA FAO 130410 OAOA 250950 611283 3-Methyl glutaconic acidemia dehydrogenase Isobutyryl-CoA 3MGA hydratase 3-Methylglutaconyl-CoA C5-OH C5-OH/C8 High – OA 248360 Malonic acidemia MAL decarboxylase Malonyl-CoA C3DC C3DC/C10 High – OA 300256 2-Methyl 3-hydroxybutyryl-CoA FAO 255120 Carnitine FAOFAO 601609 222745 Medium/short-chain reductase 2,4 Dienoyl FAOFAO 201470 602199 Short-chain acyl-CoA Medium-chain ketoacyl-CoA OA 251000 Methylmalonic OA 251100 OA 277400 OA 606054 Propionic acidemia PA carboxylase Propionyl-CoA C3 C3/C2 Detection is possible due to secondary carnitine defi Table D1.2. of metabolites see Table abbreviations For a 258 P. Rinaldo and D. Matern C8 see High Low Urgency of Urgency clinical action rma- UAC C8 PC, PAC, UOAPC, PAC, High see C5-OH tory testing HCY – PC, PAC Moderate – PAAAllo-Ile UOA PAA, Low High C18) C3/C2 MMA MCA Critical ratio 2nd tier test C0/(C16 + Gln/Cit Orotic acid UOA PAA, Met/Phe Uncertain HCY tHCY PAA, High def (mat) 3MCC) LA CPS OTC OH-PRO Cbl G, Cbl D v1 – – PAC UOA, Uncertain OTHER OTHER conditions – Phe/Tyr(CUD, GA I, – UPTR PAA, High S/MCHAD (BS), BIOPT (REG) Cb C, D B12 SUCLA2, Vit SECONDARY SECONDARY conditions CBL A,B PRIMARY PRIMARY conditions Differential diagnosisDifferential Initial confi 302 IVA 2MBG, GA II EE – PAC UOA, UAG, High 287288 – – SCAD, IBG SCAD, IBG, GA II EE, FIGLU FIGLU EMA PAC, UOA, UAG, UOA PAC, Low weight ester) (butyl 186 – — OXO-PRO by mass spectrometry screening Metabolites investigated (C5) acid (FIGLU) (C4) acid (Gln + Pyrog) Hexanoylcarnitine (C6)Hexanoylcarnitine 316 MCAD GA II – – see Isovaleryl-/2-methylbutyrylcarnitine Isovaleryl-/2-methylbutyrylcarnitine OH Butyrylcarnitine (C4-OH) 304 BKT 2M3HBA, Formiminoglutamic Formiminoglutamic Butyryl-/isobutyrylcarnitine (C5:1)Tiglylcarnitine 300 BKT 2M3HBA – – Arginine (Arg)Arginine Citrulline (Cit) (Tyr)Tyrosine Glutamic acid (Glu) acid (ASA)Argininosuccinic Carnitine (C0) (C3)Propionylcarnitine 459 231 (215) 232 260 238 CIT I, ASA ASA ARG 218 274 TYR I CIT II – MUT, CIT II CUD PA, TYR II, III CPS PC OTC – – CPT I – – CPS OTC Maternal cases ASA/Arg Glu/Cit UOA PAA, – SUAC – Orotic acid High UOA PAA, UOA PAA, UOA PAA, Moderate Uncertain PAA High Moderate Informative markerInformative Glycine (Gly)Alanine (Ala) Molecular Proline (Pro) (Val)Valine Threonine (Thr)Glutamine + Pyroglutamic Leucine/Isoleucine/OH Proline (Xle) 132 188 146Ornithine + Asparagine (Orn Asn) (Lys)Lysine 172 189Methionine (Met) 176 174 – MSUD (Phe)Phenylalanine – – – MSUD – 206 – 203 – 222 – – – HCY CIT II – PDH (E3), NKHG Phenylketonuria LA H-PHE, BIOPT II, I, PRO PRO PDH (E3), VAL – H-ORN, HHH MET RED MTHFR, Cbl E, Allo-Ile – H-LYS – UOA PAA, CSF GLY PAA, – High UAA PAA, Low L and P PAA, Uncertain PAA Low – Low PAC PAA, Low Table D1.2 D1 Newborn Screening for Inherited Metabolic Disease 259 high for other conditions see C16-OH see C16 see C16 see C16 see C16-OH see C16-OH see C8see C8 see C8 see C8 see C14:1 UOA, PACUOA, Low see C14:1 see C14:2 see C5-OH see C14:1 HCY – UAC PAC, UOA, for 3MCC, Low – DNA UOA, PAC, High C5-OH/ C8 C14:1/ C16 urinary organic acids urinary organic –– –– – – UOA PC, PAC, High UOA CACT, CPT II, CACT, GA II CACT, CPT II, CACT, GA II CACT, CPT II CACT, CPT I (low), CPT I (low), 2M3HBA, 3MGA2M3HBA, (partial) BIOT C5-OH/C0, CACT, CPT IICACT, – – GA IIGA II – – C4:1/C2, – total homocysteine; total homocysteine; tHCY TFP BKT, MCD BKT, TFP TFP TFP plasma carnitine; PC 360 –374 Cbl A,B MUT, MAL, MCKAT – – SUCLA2 C3DC/C10 – PAC UOA, MMA MCA 388 GA I GA II – C5DC/C5OH – UAC PAC, UOA, High plasma acylcarnitines; plasma acylcarnitines; PAC (C3DC) (C4DC) Decanoylcarnitine (C5DC) Decanoylcarnitine plasma amino acids; OH Hexadecenoylcarnitine (C16:1-OH)OH Hexadecenoylcarnitine 470 (C16-OH)OH Palmitoylcarnitine (C18:2)Linoleylcarnitine (C18:1)Oleylcarnitine 472 LCHAD/TFPStearylcarnitine (C18) 480 – LCHAD/TFP (C18:1-OH)OH Oleylcarnitine 482OH Stearylcarnitine (C18-OH) – LCHAD/TFP 484 498 500 VLCAD, LCHAD/ – CPT II VLCAD LCHAD/TFP – LCHAD/TFP – – – CPT I (low), C16-OH/C16 – – – – PC,PAC,UOA – High – – Octanoylcarnitine (C8)Octanoylcarnitine Octanoylcarnitine Malonyl-/OH (C10:2)Decadienoylcarnitine (C10:1)Decenoylcarnitine (C10)Decanoylcarnitine 344Succinyl-/methylmalonylcarnitine 368 370 372 MCAD – MCAD MCAD GA II, MCKAT – DE-RED GA II GA II – – C8/C2 – – PAC UOA, UAG, High – – – PAA PAC, Uncertain OH Isovalerylcarnitine (C5-OH)OH Isovalerylcarnitine 318 3MCC, HMG, Palmitoylcarnitine (C16)Palmitoylcarnitine 456 VLCAD CPT I (low), Tetradecanoylcarnitine (C14)Tetradecanoylcarnitine 428 VLCAD, LCHAD/ Glutaryl-/OH (C12:1)Dodecenoylcarnitine (C12)Dodecanoylcarnitine Methylglutarylcarnitine (C6DC) (C14:2)Tetradecanedioylcarnitine 398 (C14:1)Tetradecenoylcarnitine 402 424 400 426 VLCAD HMG VLCAD, LCHAD/ VLCAD – VLCAD, LCHAD/ – – – – – – – – PAAs 260 P. Rinaldo and D. Matern

Fig D1.1 Short-term Neonatal Screening (19.08.09) follow-up algorithm for elevated phenylalanine detected by newborn Elevated Phenylalanine screening. Reproduced from http://www.acmg.net, with permission Assay Plasma amino acids

Elevated phenylalanine Normal/Reduced tyrosine Normal

Begin low Phenylalanine diet; Assay urine pterins; RBC DHPR assay

Urine Urine pterins-abnormal Urine pterins-abnormal pterins-normal DHPR activity-normal DHPR activity-reduced

PKU BH4 deficiency DHPR deficiency Newborn screening Continue diet Evaluate for specific Initiate treatment with BH4 result was false Consider PAH pterin defect; initiate treatment and neurotransmitter positive. No further genotyping with BH4 and neurotransmitter precursors. action required. precursors. for differential diagnosis, the availability of particu- defi ne evidence-based, clinically driven cutoff target larly informative ratios, and the existence of second ranges for all analytes detected by MS/MS, and calcu- tier tests which, like in the case of CAH, could lated ratios. The cutoff target range could be either dramatically improve the overall performance of a above (high) or below (low) the range of the normal screening program (Matern et al. 2007). Finally, the population. Briefl y, the high target range is defi ned as table mentions the confi rmatory tests to be performed the interval between the cumulative 99%ile of the nor- as fi rst line of confi rmatory evaluation, and a subjec- mal population and the lowest 5%ile of disease ranges, tive assessment of the urgency of clinical action. if the analyte is informative for multiple conditions. On Obviously, many factors may have a role in the timing the other hand, the low target range is defi ned as the of clinical intervention, and such estimates should be interval between the highest 99%ile of disease ranges, considered carefully on a case by case basis. if the analyte is informative for multiple conditions, The inherent complexity of these tables should be and the 1%ile of the normal population. When the suffi cient to call attention to the need to carefully mon- degree of overlap between normal population and dis- itor the performance of a screening laboratory, and to ease range makes it inapplicable to use the criteria assess it based on objective metrics (Rinaldo, Zafari stated above, one or both limits are modifi ed to give 2006). A collaborative effort that involves more than priority to the disease range, and may require the use of 100 laboratories worldwide has set the following tar- a second tier test to maintain adequate specifi city (low gets of adequate performance for newborn screening false positive rate) (Matern et al. 2007 and Oglesbee by MS/MS: (1) detection rate of at least 1:3,000 births et al. 2008). This effort is based on data from more than (assuming testing for most of the 20 conditions included 7,500 true positive cases, 3–5 new cases are added in the uniform panel); (2) False positive rate <0.3%; daily. More information about this initiative can be and (3) Positive predictive value >20%. To achieve found at the project website at: http://www.region4ge- these targets, the collaborative project collects data to netics.org. D1 Newborn Screening for Inherited Metabolic Disease 261

implementation of MS/MS-based second tier tests: the Mayo Take Home Messages Clinic experience (2004–2007). J Inherit Metab Dis 30:585–592 › NBS is a universal public health program Minutti CZ, Lacey JM, Magera MJ, Hahn SH, McCann N, Schulge A, Cheillan D, Dorche C, Chace DH, Lymp JF, aimed to avoid mortality, morbidity and dis- Limmerman D, Rinaldo P, Matern D (2004) Steroid profi l- abilities by early identifi cation of treatable ing by tandem mass spectrometry improves the positive pre- conditions and pre-symptomatic intervention dictive value of newborn screening for congenital adrenal Many biochemical markers detected by MS/ hyperplasia. J Clin Endocrinol Metab 89:3687–3693 › Oglesbee D, Sanders KA, Lacey JM, Magera MJ, Casetta B, Strauss MS require a careful differential diagnosis KA, Tertorelli S, Rinaldo P, Matern D (2008) 2nd-Tier test for between multiple conditions quantifi cation of alloisoleucine and branched-chain amino acids › Guidelines for clinical and laboratory follow in dried blood spots to improve newborn screening for maple up of an abnormal NBS result are available on syrup urine disease (MSUD). Clin Chem 54:542–549 Rinaldo P, Zafari S, Tortorelli S, Matern D (2006) Making the line, see for example www.acmg.net. case for objective performance metrics in newborn screening by tandem mass spectrometry. MRDD Res Rev 12:255–261 Rinaldo P, Cowan TM, Matern D (2008) Acylcarnitine profi le analysis. Genet Med 10:151–156 Turgeon C, Magera MJ, Allard P, Torterelli S, Gavrilov D, O gles- bee D, Raymond K, Rinaldo P, Matern D (2008) Combined Key References newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots. Clin Chem 54: 657–664 Watson MS, Lloyd-Puryear MA, Mann MY, Rinaldo P, Howell RR Lacey JM, Minutti CZ, Magera MJ, Tausher AL, Casetta B, (eds) (2006a) Newborn screening: toward a uniform screening McCann M, Lymp J, Hahn SH, Rinaldo P, Matern D (2004) panel and system (main report). Genet Med 8(Supplement):12S- Improved specifi city of newborn screening for congenital 252S adrenal hyperplasia by second tier steroid profi ling using tan- Watson MS, Mann MY, Lloyd-Puryear MA, Rinaldo P, Howell dem mass spectrometry. Clin Chem 50:621–625 RR (eds) (2006b) Newborn screening: toward a uniform Matern D, Tortorelli S, Oglesbee D, Gavrilov D, Rinaldo P (2007) screening panel and system (executive summary). Genet Reduction of the false positive rate in newborn screening by Med 8(Supplement):1S-11S Biochemical Studies D2 K. M. Gibson and C. Jakobs

Key Facts This chapter draws extensively on previously published work: Hoffmann et al. (2002). The authors › Evidence for the presence of an inherited meta- acknowledge the use of that material. bolic disease may often be derived from detailed clinical evaluation of the patient and examination of the family history (Nyhan et al. 2005). › Important stumbling blocks in identifying an D2.1 General Remarks inherited metabolic disease include the fact that signs and symptoms are often nonspecifi c, Most known inherited metabolic diseases are identifi ed leading to initial testing to exclude routine via biochemical analyses of various body fl uids, pre- childhood illnesses and delaying consideration dominantly blood and urine, but also cerebrospinal, of metabolic disorders. vitreous, and even bile fl uids. Concentrations of physi- › Even when appropriately suspected, ordering ologically relevant metabolites* in plasma or serum are physicians may be unfamiliar with important generally tightly controlled, and thus increases/ biochemical interrelationships and the appro- decreases of specifi c intermediates may have diagnostic priate diagnostic tests to order, occasionally relevance. Normative data for many compounds of leading to inappropriate sample collection and intermediary metabolism are highly dependent upon the storage. metabolic state at sampling, and appropriate interpreta- › Absence of acute metabolic decompensation (e.g., tion of assay results requires knowledge of intake and hyperammonemia, hypoglycemia, overwhelming other physiological data, including fasting or postpran- metabolic acidosis, and anion gap) does not nec- dial status or postexercise status. Some disorders may essarily rule out an inherited metabolic disease. only be identifi ed through specifi c function tests (e.g., › Consultation and coordination with a licensed loading) that stress metabolic conditions or result in sup- clinical biochemical genetics laboratory helps raphysiological increases in metabolite load (see Chap. insure that appropriate tests are ordered, the D8). Such tests have inherent risks and escalate the correct samples are obtained, and the limita- potential for metabolic overload and decompensation; tions of the testing scheme are clearly defi ned accordingly, such tests should only be instituted by expe- prior to metabolic work-up. rienced clinicians in the appropriate hospital setting, and only when other diagnostic options that carry less patient risk have been exhausted (Table D2.1). Many inherited metabolic disorders induce the accumulation of substrates, which are metabolized via alternative processes (alternate pathways, liver K. M. Gibson () biotransformation) and/or removed via excretion in the Department of Biological Sciences, Michigan Technological urine. Studies carried out in urine for many such dis- University, Dow 740, ESE 1400, Townsend Drive, Houghton, MI 49931, USA eases may be more straightforward and sensitive than e-mail: [email protected] plasma/serum analyses. Differences in fl uid intake and

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 263 DOI: 10.1007/978-3-540-74723-9_D2, © Springer-Verlag Berlin Heidelberg 2010 264 K. M. Gibson and C. Jakobs

Table D2.1 An overview of metabolic investigations described laboratories must adhere to the accepted practices of in this chapter internal and external QC schemes, which insure ongo- Simple colorimetric evaluations in urine ing education of laboratory staff and competence in Amino and organic acids, carnitine, and acylcarnitines analytical performance. External schema are particu- Lactate, pyruvate, nonesterifi ed fatty acids, and ketones larly important, providing data on accuracy and bias Congenital disorders of glycosylation Purines and pyrimidines, orotic acid of results. Participation in external QC schemes (and Sugars and polyols acceptable performance) is a requirement for external Glutathione accreditation of the laboratory. In the USA, the Mucopolysaccharides and oligosaccharides College of American Pathologists (CAP) offers profi - Very long-chain fatty and pristanic acids (peroxisomal ciency testing for urine and blood amino acids, urine function) organic acids, plasma acylcarnitines, and qualitative Creatine and folate mucopolysaccharide analyses. European Research Sterols, bile acids, and porphyrins Biogenic amines and pterins Network for Inherited Disorders of Metabolism offers these and a more extensive menu of special assays in *see also Blau et al. 2003, 2005 urine and blood, profi ciency testing for purine and pyrimidine analysis, and has recently instituted a pilot urinary dilution, and their effect on metabolite concen- program for lysosomal enzyme analysis. Depending trations, are usually accounted for by correcting urine on the country or region, laboratory accreditation may metabolite levels with creatinine output. Urine analy- be optional or mandatory, the latter being the case in ses are generally less infl uenced by metabolic and the USA (CAP). nutritional changes, since the specimen is collected over a time period and often (but not always) there are signifi cant differences between normal and pathological values that are readily recognized. As a general D2.2 Simple Colorimetric rule, a spot urine sample (morning void to enhance Evaluations of Urine metabolite concentrations) is suffi cient for most studies. Exceptions may occur, however, as in the case of A number of simple tests are available that may be car- some fatty acid oxidation disorders that frequently ried out in nonspecialized hospitals or at the bedside, show urinary abnormalities only under fasting or load- and provide important fi rst clues for the diagnosis of ing conditions, or in the case of certain disorders metabolic disorders. (e.g., cystinuria, porphyrias, etc.) where a 24-h urine collection may be required. Laboratory investigations for inherited metabolic diseases are complex and susceptible to technical D2.2.1 Dinitrophenylhydrazine problems. To maintain acceptable standards, laborato- (DNPH) Test ries performing biochemical investigations for inherited metabolic diseases should process a suffi ciently Method high number of samples to maintain diagnostic acu- Mix 1.0 mL urine with 1.0 mL 0.2% DNPH men and should participate in quality assurance/qual- solution. ity control (QA/QC) processes. Referring physicians should bear in mind that many analyses are often qualitative and not quantita- The DNPH assay detects urine a-keto acids via forma- tive (although the expanding implementation of tan- tion of hydrazones that precipitate out of solution. dem mass spectrometry (MS–MS) is changing this Several metabolic disorders may be detected, including paradigm), thereby leading to a certain level of sub- phenylketonuria (PKU), maple syrup urine disease jective interpretation. Furthermore, the conditions (MSUD), tyrosinemia type I, tyrosyluria, histidinemia, examined are biochemically heterogeneous in their and methionine malabsorption syndrome. Acetone also expression, which can complicate identifi cation of yields a positive result and may suggest ketonuria asso- subtle abnormalities. For these reasons, diagnostic ciated with hyperglycinemia, branched-chain organic D2 Biochemical Studies 265 acidurias, and glycogen storage disorders. A 2 N HCl D2.2.1.2 Rapid Urinalysis test is performed on all DNPH positives to determine the presence of false positive results. Substances with low acid solubility may cause interference. Mandelamine Method (methenamine mandelate), an antibacterial medication, Commercially available reagent test strips (Multistix and radiopaque contrast material will form a precipitate 10 SG/Multistix PRO 11®, Bayer Corporation). immediately upon the addition of DNPH. The color and immediacy of precipitate formation distinguishes it Multistix 10 SG and Multistix PRO 11 provide qualita- from the yellowish precipitate of a-keto acid hydra- tive colorimetric analysis of protein, blood, leukocytes, zones. Information on medications is critical prior to nitrite, glucose, ketones (acetoacetic acid), pH, spe- use of the DNPH test. The DNPH test may be carried cifi c gravity, creatinine, bilirubin, and urobilinogen in out by caregivers and may be useful in the management urine. Methodology, interpretation, and characteristics of patients with MSUD living at some distance from of the approximate linearity of these measurements are the Metabolic Center. provided in the package insert. In addition, for each test expected values, limitations, and interfering substances are described. This screening procedure can D2.2.1.1 Reducing Substances in Urine provide insight into likelihood of bacterial infection/ contamination, kidney and liver function, acid–base balance, and/or carbohydrate metabolism. Method A number of interferences can occur for each Commercially available test tablets (e.g., Clinitest®, metabolite estimated. Certain medications (e.g., ribo- Bayer Corporation). fl avin or drugs containing azo dyes, or nitrofurantoin) may lead to urine discoloration and test interference. False negatives for glucose may be encountered with Numerous disorders lead to the urinary excretion of ascorbate levels >50 mg/dL or in the presence of sugars and other reducing substances, some of which ketones. Ascorbate interferes with bilirubin estima- are described in Table D2.2. Urine reducing substances tion, as does indican (indoxyl sulfate). Estimation of may be detected with commercially available test tab- ketones is hampered by the presence of highly pig- lets which provide a color change in the presence of mented samples, levodopa metabolites, and sulfhydr- reducing substances. This test, in conjunction with the yls. False positives for heme (blood) may arise from Multistix analysis described later, can assist in differ- oxidizing contaminants (hypochlorite) or microbial entiating between glucose-related and nonglucose- peroxidase (urinary tract infection). Finally, protein related reducing substances. in the urine may be overestimated in highly buffered or alkaline samples or in urine contaminated with quaternary ammonium compounds (antiseptics and Table D2.2 Urine reducing substances (with associated detergents). disorders or conditions) Galactose (classical galactosemia, galactokinase defi ciency, liver disease, secondary galactose intolerance) Fructose (fructose intolerance, essential fructosuria) D2.2.2 Cyanide Nitroprusside Test 4-Hydroxyphenylpyruvate (tyrosinemia types I/II, bacterial (Brand Reaction) contamination of urine) Homogentisic acid (alkaptonuria) Xylose (pentosuria) Glucose (diabetes mellitus, Fanconi syndrome) Method Oxalate (hyperoxaluria) Add 0.4 mL of 5% NaCN to 1.0 mL urine. After Salicylates, ascorbic acid (drugs) 10 min add 0.2 mL of 0.5% Na-nitroprusside Uric acid (hyperuricosuria) (Na-nitroferricyanide). Mix and immediately assess Hippurate (sodium benzoate treatment of hyperglycinemia and hyperammonemia) the color. 266 K. M. Gibson and C. Jakobs

Table D2.3 Intermediates generating a positive nitroprusside Urobilin is responsible for the characteristic color of reaction (and associated disorder or condition) urine, while sterobilin is a major pigment of feces. Cystine (cystinuria, hyperaminoaciduria) Indoles in the urine may also give a positive Ehrlich Homocysteine (homocystinuria, cobalamin disorders, test. cystathioninuria, urinary tract infection) Individuals with porphyria may present with acute Glutathione (disorders of the g-glutamyl cycle) Drugs (see text) attacks, skin lesions, or both, but rarely prior to the onset of puberty. An attack usually consists of severe abdominal pain and may be associated with neuro- The Brand reaction identifi es free sulfhydryl or disul- logical fi ndings. Such attacks are associated with fi de compounds in urine. Cyanide reduces any disul- excessive amounts of d-aminolevulinic acid (ALA) fi des to free sulfhydryls. In the subsequent reaction, a and PBG in the urine. PBG deaminase defi ciency reddish color results when free sulfhydryl groups com- (acute intermittent porphyria) is the most common plex with nitroprusside. A positive result is usually due form of porphyria. A positive Ehrlich test, coupled to cystine in the urine. Familial cystinuria is among the with hepatosplenomegaly, indicates that evaluation of most common aminoacidurias. Disulfi des are also tyrosine metabolites in urine (nitrosonaphthol test) excreted in other metabolic disorders such as homo- should be pursued. cystinuria and b-mercaptolactate-cysteine disulfi duria. Both will produce a positive result. The urine specimen should be at approximately neutral pH. If the sample has been preserved with acid, a false positive D2.2.4 Nitrosonaphthol Test reaction may occur. Drugs such as N-acetylcysteine, (Tyrosine Metabolites) 2-mercaptoethanesulfonate, 2-mercaptopropionylgly- cine, captopril, penicillamine, and large amounts of Method synthetic penicillin metabolites and acetoacetate will yield false positives; accordingly, a listing of medica- Three drops of clear urine are sequentially treated tions must be obtained. Bacterial contamination may with 1.0 mL 2.6 N nitric acid, 1 drop 2.5% sodium also generate a false positive. Cystathionine, methion- nitrite, and 0.2 mL 1 mg/mL 1-nitroso-2-naphthol. ine, and taurine are not detected (Table D2.3). After 15 min, the color formation is recorded. As blank, water is substituted for urine; N-acetyl-l- tyrosine (0.04 mg/mL 1 M HCl) is employed to develop a qualitative standard curve. D2.2.3 Ehrlichs Aldehyde Reagent

4-Hydroxylated phenolic acids (and to a limited extent Method hydroxylated indoles derived from tryptophan) conju- Add 1.0 mL of urine to 0.1 mL 2% p-dimethylamin- gate with 1-nitroso-2-naphthol in the presence of nitric obenzaldehyde in 2 N HCl. Mix and assess color acid to yield orange/red chromophores. The correspond- formation after 10 min. ing tyrosine analogs include 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate and 4-hydroxyphenylacetate, The Ehrlichs test detects porphobilinogen (PBG) or and tyrosine itself. A limitation of this method is tran- urobilinogen in urine. PBG yields a red color in urine sient newborn tyrosinemia, a common ﬁ nding as the for the patient with acute porphyria. Urobilinogen, a hepatic enzymes involved in tyrosine metabolism may component of heme degradation, results when biliru- develop slowly. 4-Hydroxyphenylacetate may be ele- bin analogs are secreted into the bile and further vated as a result of intestinal bacterial metabolism, mal- degraded by intestinal bacteria. In the normal patient, absorption and other disorders, and may lead to false some urobilinogen is reabsorbed and transported to the positives. Some disorders of carbohydrate metabolism, kidneys where it is converted to urobilin (yellow) and and liver dysfunction, may also alter tyrosine metabo- excreted. However, the majority of urobilinogen is lism and produce positives. Patients undergoing paren- converted microbially to sterobilin (deep red-brown). teral nutrition are often supplemented with tyrosine D2 Biochemical Studies 267 analogs, which can lead to difﬁ culties in interpretation. D2.3.1 Plasma Patients with adrenal tumors may excrete increased homovanillic acid (HVA) and/or 5-hydroxyindoleacetic acids (HIAA) (end products of dopamine and serotonin Sample metabolism, respectively), which yield pink and purple chromophores with nitrosonaphthol. Any positive One to two milliliter heparinized blood, with plasma should be correlated with clinical history and/or fol- immediately separated. If shipped, the sample lowed-up with more specialized testing (blood amino should be sent on dry ice. acids/urine organic acids).

Quantitative amino acid analysis in plasma (or serum) D2.2.5 Sulfi te Test by ion-exchange chromatography or HPLC (ninhydrin or phenylisothiocyanate derivitization) provides pertinent information on alterations in amino acid Method homeostasis and is one of the fi rst-line investigations Commercially available dipstick (Merckoquant performed in a patient with suspected inherited met- 10013, Merck Darmstadt, Germany); fresh urine. abolic disease. It is also the confi rmatory adjuvant for follow-up of positive newborn screening results (see Chap. E1). Analysis should be performed The sulfi te test recognizes increased concentrations of promptly in patients with hyperammonemia or with sulfi te in the urine that may be a marker of primary an acute presentation of a suspected amino acid dis- defi ciencies of the enzyme sulfi te oxidase or molybde- order; the results should be available STAT. Routine num cofactor defi ciency. Children affected with one of (fasting) amino acid analysis is also necessary in these disorders typically suffer from severe epileptic patients that are protein restricted or receiving spe- encephalopathy, which usually starts in the neonatal cifi c metabolic dietary therapy, in order to adjust period or infancy. Testing for urinary sulfi te should be amino acid intake and to identify any defi ciencies of part of baseline investigations in any child with unex- essential amino acids. Many amino acids (but not all plained psychomotor delay particularly in combina- as yet, especially the dibasic amino acids) can also tion with severe epilepsy. The test may be falsely be quantifi ed reliably in dried blood spots (Guthrie negative if old urine samples are used for testing. False cards) by electrospray tandem MS–MS or fast atom positive results may be caused by various drugs and bombardment (FAB) MS (see later). For some disor- other conditions that cause increased urinary sulfi te ders such as PKU this is a superior technology concentrations. (throughput, time savings) to classical chromatographic methods (analysis time from 2 to 3 h) with less invasive approaches for the patient. The newest technological advance for amino acid analysis is the D2.3 Amino Acids UPLC–MS/MS methodology. This system employs high fl ow rate (to 20,000 psi) and small resin particle Physiological amino acids occupy essential positions size to enable extremely rapid analysis and resolu- in intermediary metabolism as the building blocks of tion. Analysis of a sample from a suspected MSUD proteins, but also are involved in numerous other meta- patient can be achieved in minutes with absolute bolic processes including methylation reactions, neu- quantifi cation (stable isotope internal standards). An rotransmission, energy production, and others. A long added advantage over current electrospray MS–MS list of inherited metabolic diseases either directly or is that stereoisomers (e.g., leucine/isoleucine) are indirectly affect amino acid metabolism (both cata- separated on the UPLC system. This technology is bolic and anabolic processes), and lend themselves to rapidly developing, and may eventually replace older detection through careful analysis of the appropriate (yet still robust) ninhydrin-based amino acid analyz- physiological fl uid. ers widely in use. 268 K. M. Gibson and C. Jakobs

results in essentially useless values for some amino Remember acids (e.g., arginine or taurine), either through the action Although amino acid analysis can be quantitatively of erythrocyte enzymes such as arginase (which con- performed, slight deviations in one (or even a few) verts arginine to ornithine) or through concentration intermediate(s) do not necessarily indicate a defect gradients between cells and fl uid (e.g., liberation of tau- of amino acid metabolism. The entire pattern of rine from platelets). Exact arginine concentrations are physiologically relevant amino acids should be essential for diagnosis and treatment monitoring of urea reviewed by an experienced biochemical geneticist cycle defects. Even after prompt separation, shipment at whose training has included evaluation of numer- ambient temperature results in questionable values for ous chromatograms over a number of years. several amino acids, e.g., glutamate, aspartate (both artifi cially elevated), glutamine, asparagine, cysteine, When looking at 20–30 physiologically relevant amino and homocyst(e)ine (reduced). The concentrations of acids in plasma, artifacts are a common occurrence some amino acids (phenylalanine, tyrosine, valine, iso- (Table D2.4), linked to other pathological states, infec- leucine, and leucine) are less affected by overnight ship- tion, nutritional status, and a host of other variables. ment of room temperature plasma or serum, but frozen Plasma amino acid levels are dependent upon meta- samples are optimal to avoid artifactual results. bolic status, and routine sampling should occur 4–6 h Certain amino acids, notably homocysteine and tryp- after the last meal. Furthermore, it may be useful to tophan, require specifi c methods for exact quantifi ca- quantify amino acids in postprandial (and fasting) tion. Determination of total homocysteine is critical for samples, particularly when disordered energy metabo- the evaluation of hyperhomocysteinemias, due to both lism is suspected. For postprandial analyses it may be cystathionine-b-synthase defi ciency and cobalamin dis- advisable to provide a defi ned meal to achieve stan- orders. For optimal results, immediately centrifuge the dardized substrate intake (see Chap. D8 for more infor- blood sample, order total homocysteine, and transport mation). Postprandial samples may reveal signifi cant plasma to the laboratory (if short transit time, room elevations of essential amino acids. For example, an temperature is acceptable; for longer transport times, excessive alanine increase indicates impaired pyruvate freeze plasma and ship on dry ice). Analytically, homo- handling and may suggest a mitochondrial disorder. cysteine is released from disulfi de conjugates by treat- Conversely, fasting results in a marked elevation of the ment with reducing agents, and the total homocysteine branched-chain amino acids (leucine, isoleucine, and concentration is determined. Normative data for total valine), while most other amino acids are low. homocysteine (fasting) in children <10 year approxi- For optimal results in amino acid analysis, it is mates 3.5–9 mmol/L; >10 year, 4.5–11 mmol/L; women important to separate plasma or serum from cells as (premenopausal) 6–15 mmol/L; and postmenopausal soon as possible. Hemolysis or transport of whole blood 6–19 mmol/L; men 8–18 mmol/L. Reference ranges, however, must be established in the individual laborato- Table D2.4 Facts and artifacts for selected plasma amino acid ries and include normal and pathological samples. elevations (and associated conditions) Glycine (organic acidurias, nonketotic hyperglycinemia, valproate treatment) D2.3.2 Urine Serine (tracks with glycine as a result of metabolic interconversion; decreased in serine biosynthetic defects) Phenylalanine (phenylketonuria and biopterin disorders, tyrosinemias, liver disease) Sample Tyrosine (tyrosinemia I, II, III; liver disease) Minimum 10 mL urine, preferably a morning void Arginine (argininemia; decreased in trauma, shock, hemolysis). but a random sample is acceptable, without preser- Methylhistidines, anserine, carnosine (consumption of fowl) vatives. If shipped, transport on dry ice. Methionine (homocystinuria, cobalamin disorders, pernicious anemia, liver disease) Citrulline (argininosuccinic aciduria and citrullinemias, Quantitation of urine amino acids is usually less reveal- decreased in certain forms of pyruvate carboxylase ing than comparable studies in plasma since amino defi ciency and urea cycle disorders (ornithine transcar- acids are normally well reabsorbed in the renal tubules; bamylase, carbamylphosphate synthetase, NAGS) ) accordingly, subtle to moderate changes in amino acid D2 Biochemical Studies 269 homeostasis cannot be recognized using urine analy- Table D2.5 Inherited metabolic diseases requiring CSF a ses. Various methodologies are available, including investigations for diagnosis standard quantitative analysis (above) and qualitative Disorder Relevant parameter assessment by thin-layer or paper chromatography. Glucose transporter Glucose ratio (cerebrospinal defi ciency fl uid (CSF)/blood) The qualitative analysis is relatively inexpensive, pro- Selected mitochondrial CSF lactate, pyruvate, vides insight into renal tubular function and has the encephalomyopathies alanine, threonine capacity to identify several amino acidopathies/amino Nonketotic hyperglycinaemia Glycine ratio (CSF/plasma) acidurias; it is therefore a key element of routine Serine biosynthesis CSF amino acids (low serine; selective screening for inherited metabolic diseases. defects serine CSF/plasma ratio) Quantitative urine amino acid analysis is appropriate Cohen syndrome b-Alanine in CSF Isolated pipecolic Pipecolic acid in instances in which a renal tubular reabsorption oxidase defi ciency defect such as cystinuria is suspected, and (in conjunc- Defects of monoamine CSF monoamines tion with plasma analysis) in hyperammonemia when metabolism and metabolites increased urinary excretion of specifi c amino acids Autosomal dominant GTP CSF biogenic amines (e.g., argininosuccinate and citrulline) may be diag- cyclohydrolase I and pterins defi ciency nostic for certain urea cycle defects and in lysinuric Sepiapterine reductase CSF biogenic amines protein intolerance (Hommes 1991). defi ciency and pterins GABA transaminase CSF GABA, b-alanine, defi ciency homocarnosine Folate-binding protein CSF D2.3.3 Cerebrospinal Fluid defi ciency 5-methyltetrahydrofolate Cerebral folate defi ciency CSF 5-methyltetrahydrofolate Selected cases of glutaric CSF glutarate Sample aciduria I One milliliter cerebrospinal fl uid (CSF), freeze Selected cases of leucine CSF 3-hydroxyisovaleric catabolic defects acid immediately, in conjunction with a plasma sample aModifi ed from Hoffmann et al (1998) obtained at the time of lumbar puncture. If shipped, always transport on dry ice. CSF samples are unusable; slightly blood contaminated specimens should be rapidly centrifuged and the Quantitation of amino acids in CSF may be pursued in supernatant frozen (notify the laboratory). Immediate patients with suspected neurometabolic disorders, in deep-freezing and special analytical methods are particular severe (neonatal) epileptic encephalopathy required for the determination of numerous CSF (see also Table D2.5). It is highly desirable to obtain a metabolites (free and total GABA, homocarnosine, concurrent plasma sample for amino acid analysis as carnosine, biogenic amines, and pterins). calculation of the plasma/CSF ratio of specifi c amino acids may be required for the diagnosis of nonketotic hyperglycinemia (glycine CSF/plasma ratio >0.06) and serine biosynthesis defi ciency (3-phosphoglycer- D2.4 Organic Acids ate dehydrogenase defi ciency; serine CSF:plasma ratio <0.2). An increased glycine CSF/plasma ratio is only meaningful, however, in the presence of elevated Samples plasma glycine. If plasma glycine is abnormally low, Urine elevation of the CSF/plasma ratio in the face of a nor- Minimum 10 mL random (morning) urine, with- mal CSF glycine level is not consistent with nonketotic out preservatives. If transported, send on dry ice. hyperglycinemia. Raised levels of alanine and threo- Plasma, CSF, and vitreous fl uid (only limited, nine in CSF are indicators of mitochondriopathies. specifi c indications) Again, interpretation of CSF amino acid values beyond Minimum 1 mL, freeze immediately and send on normal limits is more reliable if concurrent plasma dry ice. concentrations are available. Heavily bloodstained 270 K. M. Gibson and C. Jakobs

The major source of organic acids in mammals include Canavan disease (N-acetylaspartic acid). Stable iso- the amino acids, lipids and carbohydrates, and to a lim- tope dilution assays (either by routine GC-MS or via ited extent nucleic acids and steroids. Urine organic liquid chromatography-tandem MS) are the methods acid analysis is most readily performed by gas chro- of choice for prenatal diagnoses, when metabolite matography-mass spectroscopy (GC-MS), which pro- levels may be low and accurate quantifi cation is vides insight into a wide range of metabolic pathways required. For postmortem studies, urine should be and is accordingly a mainstay of selective metabolic obtained via bladder puncture; organic acid analysis screening. Urine organic acid by GC alone is not rec- in postmortem plasma, CSF or vitreous fl uid is much ommended, as slight elevations of metabolites are less informative. missed and confi rmatory identifi cation via fragmenta- tion patterns is not possible. In addition to the classical organic acidurias, urine organic acid analysis is a key Remember diagnostic component in the evaluation of patients with suspected amino acid disorders, fatty acid oxida- Organic acid analyses in urine are, for the most part, tion defects, or disorders of mitochondrial energy run in a qualitative or only semiquantitative fash- metabolism. ion. Exact quantitation of the hundreds of normal Organic acids may be extracted from urine follow- (and abnormal) intermediates detected in a human ing acidifi cation with mineral acids and are then con- urine sample is laborious, technically challenging, verted to derivatives (e.g., trimethylsilyl- or methyl and time-consuming, but is offered in some labora- esters) for analysis by GC-MS. One or more non- tories. A key to diagnosis for qualitative analysis is physiological internal standards are included in the pattern recognition, especially in those instances in analysis for internal QC and for retention time stan- which elevations may be only very slight. An expe- dardization (e.g., 2-phenylbutyrate, undecanoic acid). rienced diagnostic laboratory, with ample involve- Organic acid analysis in urine is suggested in the ment in internal/external QC/QA, is optimal for the patient with systemic intoxication, unexplained met- best diagnostic outcome. abolic crisis, or unexplained laboratory fi ndings of disturbed intermediary metabolism such as metabolic acidosis, elevated lactate, elevated anion gap, hypoglycemia, ketonemia, neonatal ketonuria, or hyper- Remember ammonemia. Furthermore, organic acid analysis is indicated in children with unclear hepatopathy, neu- Organic acid analysis in plasma is almost never of rological/neuromuscular symptoms including epilep- diagnostic value. Urine is the fl uid of choice for tic encephalopathy, and in children with multisystem analysis. However, in selected instances of fatty disorders (particularly when symptoms fl uctuate or acid oxidation defects, increases of C14:0 and progress). C14:1 fatty acids can provide evidence for long- Organic acids are optimally analyzed in urine as chain hydroxyacyl-CoA dehydrogenase defi ciency. they are usually effi ciently excreted via the kidneys This disorder is readily detected by plasma acylcar- (high water solubility) and show higher concentra- nitine analysis (see later). tions in urine than other body fl uids. Nonetheless, exact quantifi cation of specifi c organic acids in plasma or CSF with isotope dilution analysis (only in D2.5 Acylcarnitine Analysis selected laboratories) may be useful in selected cases for exclusionary purposes or for therapeutic assessment. Examples (Table D2.5) include glutaric acidu- Sample ria type I (glutaric acid and 3-hydroxyglutaric acid), Three to six dried blood spots on fi lter paper cobalamin defects (methylmalonic acid), tyrosinemia (Guthrie card) or 1–2 cc EDTA blood (plasma rap- type I (succinylacetone) and various cerebral organic idly separated). Plasma sent on dry ice, blood spots acidurias such as succinic semialdehyde dehydroge- at room temperature. nase defi ciency (4-hydroxybutyric aciduria) and D2 Biochemical Studies 271

Acylcarnitine analysis in dried blood spots by electro- tivity makes it the method of choice in emergency spray tandem MS–MS or FAB MS–MS facilitates situations. diagnosis of most organic acidurias and fatty acid oxidation defects. This method has enabled the massive growth of expanded newborn screening around the world, not only in North America and Europe but also D2.6 Carnitine Status in Australia, Saudi Arabia, Qatar, and many other countries. The rapidity of analysis (<2 min/sample for newborn screening) lends itself to high throughput Sample analysis. For acylcarnitine analysis, the characteristic One milliliter serum/plasma, ± 5 mL urine, shipped daughter ion of all acylcarnitines is m/z 85 (a frag- to the laboratory frozen. ment of the parent carnitine moiety), an ion selectively monitored for quantitation. Tandem MS–MS analysis of acylcarnitines in plasma is also a primary evalua- Long-chain fatty acids are transported into the mito- tion for selective screening of inherited metabolic dis- chondrion as their respective carnitine esters, a pro- eases, with diagnostic relevance to organic acidurias, cess that requires transesterifi cation of acyl-CoA fatty acid oxidation defects, and amino acid disorders species at the inner and outer sides of the inner mito- (when the appropriate amino acid ion fragment is chondrial membrane. Similarly, acyl-CoA compounds quantifi ed). that accumulate within the mitochondrial matrix may In selected laboratories, acylcarnitine analysis in transesterify to l-carnitine esters, with transport of the cultured fi broblasts can provide important diagnostic mitochondrion and urinary excretion. This process of information on the primary disorder. For example, detoxifi cation induces secondary carnitine depletion fi broblasts may be cultured in the presence of [U–13C] in disorders that alter the metabolism of mitochondrial leucine, isoleucine, or valine with added l-carnitine. CoA-activated carboxylic acids (e.g., organic acidu- Isolation of cell culture medium and fi broblasts, fol- rias, fatty acid oxidation defects, and respiratory chain lowed by lysis, enables analysis for 13C-carnitine esters abnormalities). In the work-up of a patient with a sus- of the corresponding acyl-CoA intermediates, which pected inherited metabolic disease, reduced serum occur in amino acid degradation (l-carnitine transes- carnitine may be regarded as one potential indicator of terifi es the acyl-CoA species). Accumulation of any these disorders. Quantifi cation of total, free, and ester- one Acylcarnitine provides evidence for the site of the ifi ed carnitine in serum or plasma (carnitine status) specifi c defect in the pathway. Acylcarnitine analysis using MS–MS is mandatory for recognizing carnitine by tandem MS–MS may also be employed in an emer- defi ciency and monitoring carnitine supplementation gency in children with acute metabolic crises or hypo- in these disorders, as well as identifying primary car- glycemia; it is faster than the standard organic acid nitine transporter defi ciency. Many laboratories still analysis for the diagnosis of organic acidurias, and pro- successfully utilize spectrophotometric methods for vides more rapid and reliable identifi cation of the fatty carnitine analysis on the autoanalyzer. acid oxidation defects. The analysis of many (but not Free carnitine is effi ciently reabsorbed in the renal all) relevant amino acids, as well as orotic acid, which tubule, while fi ltered acylcarnitine species accumulate are required in the emergency of metabolic decompen- in the urine. Urine carnitine analyses should be per- sation can be quantifi ed simultaneously by MS–MS. formed in conjunction with plasma analyses, and may Quantifi cation of total and free carnitine by MS–MS be indicated if plasma values are abnormal or for moni- using Guthrie cards is inaccurate and requires specifi c toring treatment. Increased urinary acylcarnitines are plasma/sera analysis (see later). often detected in organic acidurias and fatty acid oxidation defects, and in the context of normal plasma carni- Remember tine values suggest good detoxifi cation capacity. High urine concentrations of free carnitine and a reduced Acylcarnitine analysis in plasma has become a pri- renal tubular reabsorption rate (<90%) may reveal renal mary adjuvant for the routine analysis of inherited tubular dysfunction as a cause of systemic carnitine metabolic diseases. Its rapid throughput and sensi- depletion or primary carnitine transporter defi ciency. 272 K. M. Gibson and C. Jakobs

D2.7 Lactate, Pyruvate, Ketone Bodies, To accurately quantify the keto species (acetoacetate and Nonesteriﬁ ed Fatty Acids and pyruvate), blood should be immediately deproteinized with perchloric acid at the bedside.

Sample The analysis of NEFA in plasma or serum, in addition One milliliter serum/plasma, ship on dry ice (to to the studies outlined above, provides important infor- avoid lipolysis). If determinations of pyruvate and mation on lipid catabolism and ketogenesis and is acetoacetate are requested, rapid deproteinization at essential for any patient with acute metabolic coma or the bedside (using perchloric acid) is fundamental hypoglycemia. NEFA quantifi cation is a critical inves- for an accurate value. tigation at the end of a fasting test. Normal values vary greatly according to the metabolic state. High insulin Lactate, pyruvate, and the ketone bodies (primarily levels in the fed state inhibit lipolysis, resulting in low 3-hydroxybutyrate and acetoacetate, but also acetone) concentrations of both NEFA (<300 mmol/L) and are intermediates in blood which provide key infor- ketone bodies (<100 mmol/L). Conversely, increased mation on a number of metabolic processes, including lipolysis during fasting leads to a continuous rise of pyruvate metabolism, the citric acid cycle, gluconeo- NEFA levels up to 2–3 mmol/L within 24 h and a cor- genesis, hepatic glycogenolysis, oxidation of fatty responding increase of ketone bodies to concentrations acids, ketogenesis, and the respiratory chain. When that generally exceed those of NEFA. Interpretation of quantifi ed in blood in conjunction with glucose and the results at an early stage of the fast may be inconclu- nonesterifi ed fatty acids (NEFA), a wealth of infor- sive, since NEFA may exceed 1 mmol/L prior to eleva- mation may be gleaned concerning the function of tions of ketone bodies. A molar concentration of ketone intermediary metabolism in the patient. All of these bodies is less than NEFA after a 24 h fast suggests a intermediates may be determined employing spectro- disorder of fatty acid oxidation or ketogenesis; a molar photometric assays (commercial kits available), gen- concentration of ketone bodies <50% of NEFA is erally relying on NAD+/NADH coupled systems. strongly suggestive of such disorders. The determina- During fasting, lactate is employed in the liver for tion of NEFA without simultaneous determination of glucose production; in the fed state, lactate is a source ketone bodies has little diagnostic utility. of energy for muscle and heart. Pyruvate is a key product of carbohydrate, fat, and protein breakdown that enters the citric acid cycle as acetyl-CoA (cata- D2.8 Investigations for Congenital lyzed by pyruvate dehydrogenase). The concentration Disorders of Glycosylation (CDG) of ketone bodies in the circulation is regulated by the interplay between hepatic ketogenesis and peripheral consumption as fuel source. Especially during the Sample fasting state, ketone bodies are critical for brain One milliliter serum or plasma, shipped to the labo- energy needs. Also in the fasting state, free fatty acids ratory on dry ice. are oxidized in the mitochondrion to acetyl-CoA species, which are interconverted in the liver (hepatic 3-hydroxy-3-methylglutaryl-CoA lyase activity) to In the discipline of inherited metabolic diseases, no other ketone bodies (acetoacetate primarily). area has grown at such a staggering rate as that of the CDG disorders. To date, at least 12 different forms of Remember CDG I and six forms of CDG II diseases have been described. The two main subcategories are differentiated Lactate, pyruvate, and the ketone bodies may be by the pattern of serum transferrins following isoelectric quantifi ed in plasma, urine, and cerebrospinal fl uid. focusing, the most common approach for the diagnosis Nutritional status must be known (fasting, postpran- of the CDG disorders. The phenotypic spectrum is broad dial, or post-loading with triglyceride or other diet). and covers every system; many of the disorders have a neurological component, but some may have only D2 Biochemical Studies 273 visceral (gastrointestinal) involvement, while others may broad, encompassing immunodefi ciency, seizures, feature mainly immune dysfunction. One CDG disorder nephrolithiasis, developmental delay, autistic fea- features accumulation of a specifi c tetrasaccharide in the tures, and growth abnormalities. Purine/pyrimidine urine of affected patients. Thus, evaluation of serum nucleotides are involved in nucleic acid synthesis, transferrins has become a common analysis in selective formation of phospholipids and glycogen, and are screening for inherited metabolic diseases. critical for sialylation reactions involved in protein glycosylation. The HPLC analysis of purines and pyrimidines in urine may be particularly indicated in Remember patients with renal calculi and related problems, neu- Isoelectric focusing of serum transferrins, while an rological problems, or both. Other symptoms may excellent screening tool to detect CDG, does not include arthritis, muscle cramps, and muscle wasting. provide 100% detection. Purine and pyrimidine excretion is signifi cantly infl u- enced by diet and may vary considerably during the day; thus, a 24-h urine collection may be optimal for In humans, the glycosylation process may involve more analysis, but purines are a favorite food for microor- than 300 glycosyltransferases, glycosidases and sugar ganisms, and thus 24 h collections are accurate only transporters, all residing in cytosolic as well as endo- when each voided sample is added to a container in a plasmic reticulum and Golgi compartments. More than freezer. For diagnostic work, a spot sample assayed 30 different enzymes are involved in the production of promptly is preferable. Urinary tract infections result oligosaccharide side chains that may be either lipid- or in a markedly reduced concentration of purines or protein-linked. Diagnosis and differentiation of several pyrimidines in the urine. Foodstuffs such as coffee, types of CDG involves the demonstration of pathologi- black tea, cocoa, or licorice contain methylxanthines cal glycosylation patterns in the isoelectric focusing and should be avoided during urine collection and pattern of serum transferrins. This test does not, how- 24-h prior. Succinylaminoimidazole carboxamide ever, detect all forms of CDG, and notably in two forms ribonucleoside, the marker metabolite of of CDG II the serum isoelectric focusing pattern is normal. Once an abnormal transferrin pattern is detected, additional studies may include HPLC of the oligosac- Remember charides, enzymology, immunocytochemistry, and mutational studies. An emerging approach for CDG Patients with dihydropyrimidine dehydrogenase identifi cation is electrospray MS–MS for analysis of (DHPD) defi ciency and other pyrimidine degreda- glycosylation patterns of serum transferrins. Secondary tion defects can manifest a life-threatening response disturbances of glycosylation may be linked to chronic to treatment with the antitumor agent 5-fl uoroura- alcoholism, classic galactosemia, or fructose intoler- cil, making DHPD defi ciency one of the classical ance (defi cient mannose-6-phosphate synthesis). pharmacogenetic disorders.

D2.9 Purines and Pyrimidines adenylosuccinase defi ciency, is very unstable at room temperature; thus, if this disorder is suspected, the urine specimen must be maintained frozen and shipped Sample on dry ice. For the diagnosis of disorders of pyrimidine Minimum 10 mL urine, preferably morning void or breakdown, urine needs to be examined for dihydropy- 24 h urine collection (refrigerate and avoid light). rimidines via GC-MS (as in organic acid analysis), Sample must be sent refrigerated or on dry ice (espe- since these substances are not recognized by HPLC cially for diagnosis of adenylosuccinase defi ciency). alone. Some groups have begun to extend screening for inherited metabolic diseases in bloodspots to the use of fi lter papers soaked with urine for analysis of The spectrum of clinical features in the disorders of purines and pyrimidines using HPLC coupled to elec- purine and pyrimidine metabolism is exceptionally trospray MS–MS methodology. 274 K. M. Gibson and C. Jakobs

D2.9.1 Orotic Acid Specifi c analyses (galactose, galactose-1-pho- shate, galactitol, enzyme studies, and mutation analysis Sample ) EDTA whole blood (2 cc) preferably 30 min post Minimum 10 mL urine, sent on dry ice. milk feed, send at ambient temperature. Red blood cells (RBCs) are used for enzyme studies, plasma Pyrimidine biosynthesis, in which orotic acid is a major for metabolite determination; leukocytes may be intermediate, is regulated at the level of carbamylphos- employed for isolation of genomic DNA for muta- phate synthetase II (CPS II), the enzyme catalyzing the tion screening. Store only at room temperature (no fi rst reaction in this pathway. The isoenzyme CPS I (a refrigeration or freezing), since hemolysis leads to mitochondrial enzyme) catalyzes the production of car- signifi cant artifact and galactokinase is a membrane bamylphosphate during the process of urea synthesis. bound protein lost with RBC hemolysis). Under conditions in which mitochondrial carbamylphosphate accumulates (e.g., urea cycle disorders), cytosolic carbamylphosphate levels rise, and urine oro- Inherited metabolic defects of galactose metabolism tic acid excretion increases. This may be recognized in are recognized through determination of galactose organic acid analysis, but orotic acid is a charged nitro- concentration and GALT activity in dried blood spots gen species which has very limited solubility in organic (Guthrie card), a core part of newborn screening in solvents, and the amount detected by routine GC-MS is most countries. More specifi c analyses are needed for a fraction of the actual concentration. Thus, accurate those newborns with pathological screening tests quantifi cation of orotic acid requires HPLC or electro- (Chap. B2). Several laboratories quantify galactose in spray tandem MS–MS methodology. Urine orotic acid plasma (normal <3 mg/dL). During intervention in analysis is one of the key investigations in signifi cant patients with galactosemia (GALT defi ciency), galac- hyperammonemia for the diagnosis of ornithine tran- tose-1-phosphate is measured in erythrocytes (normal scarbamylase (OTC) defi ciency. In the past, heterozyo- <0.3 mg/dl). Furthermore, many laboratories quantify gous female carriers for OTC defi ciency have been urine galactitol (a further metabolite of galactose) detected via urine orotic acid quantifi cation following using HPLC or stable isotope dilution methodology ingestion of allopurinol, a compound that blocks the with GC-MS (normal <100 mmol/mol creatinine, age conversion of orotic acid to uridine monophosphate. 0–1 year; <25 mmol/mol creatinine, age >1 year). However, the allopurinol loading test is no longer widely employed, because mutational analysis provides more accurate information. Too, the allopurinol D2.11 Sugars test may be normal in the presence of some OTC variants. Increased urine orotic acid may be found in other disorders as well, including mitochondrial disease, Rett syndrome, and Lesch–Nyhan syndrome. Sample Minimum 10 mL urine, which may be transported refrigerated or on dry ice. D2.10 Disorders of Galactose Metabolism The detection of pathological urinary excretion of sugars is routinely assessed by thin-layer chromatography, Samples which although not technologically advanced is still Screening (galactose, GALT (galactose-1-phosphate suffi cient. This methodology has utility for the charac- uridyltransferase) activity terization of renal tubular defects and to provide Three to six dried blood spots on fi lter paper evidence for selected disorders of carbohydrate metab- (Guthrie card), routine mail. olism. These include (but are not limited to) galactokinase defi ciency, GALT defi ciency (see above), UDP D2 Biochemical Studies 275 galactose-4-epimerase defi ciency (rare), fructoaldo- have driven the expansion of this area of screening. The lase (aldolase A), fructose-1,6-bisphosphatase (aldo- fi rst is transaldolase defi ciency, which features primar- lase B) defi ciency, and essential fructosuria. The sugars ily hepatic dysfunction and the elevated excretion of routinely tested erythritol, arabitol, ribitol, and the C7 compounds sedoheptitol and sedoheptulose. The other defect, ribose-5- phosphate isomerase defi ciency, is rare (a single case). Remember In that patient, a progressive leukoencephalopathy was Urinary accumulation of glycerol may suggest associated with considerably increased urine concen- X-linked heritable glycerol kinase defi ciency, fruc- trations of arabitol and ribitol, with considerably higher tose-1,6-bisphosphatase defi ciency, and may also be concentrations of these species in CSF and brain. an artifact from the use of glycerin gels/ointments.

D2.12 Glutathione (and Analogs) using thin-layer methodology include glucose, galactose, fructose, and lactose (laboratory specifi c). The determination of liver glycogen (the key storage form of Sample liver glucose), when a disorder of glycogen storage is Three milliliter EDTA whole blood. Centrifuge, suspected, is more complex and requires liver biopsy. remove, and freeze plasma immediately. Deproteinize Glycogen is quantifi ed only in a limited number of spe- erythrocyte fraction with 5% sulphosalicylic acid cialized laboratories. GC (with and withoutMS) method (ratio approx. 1:1), shake/vortex thoroughly (until by which a number of sugar and polyols have been mea- homogenous brown color), centrifuge twice at 5000 sured (e.g., C4-polyols threitol and erythritol; C5-sugars/ × g, remove, and freeze clear supernatant. Send polyols arabinose, xylulose, ribose, xylose, ribulose, plasma and erythrocyte extract on dry ice. xylitol, arabitol, and ribitol; C6-sugars/polyols fucose, fructose, glucose, mannitol, sorbitol, and galactitol; and some C7-sugars/polyols including seduheptulose and Defects of the g-glutamyl cycle lead to a range of clini- sediheptitol) was crucial in the discovery of two new cal problems including neonatal metabolic acidosis, defects in the pentose phosphate pathway (see D2.11.1). hemolytic anemia, electrolyte disturbances, and a progressive neurological syndrome. All four enzyme defects are inherited in autosomal-recessive fashion; glutathione synthetase defi ciency is the most prevalent D2.11.1 Polyols disorder of this group. Initial investigations include the analysis of organic acids (elevated 5-oxoproline, i.e., pyroglutamic acid), in conjunction with quantifi cation Sample of glutathione and its metabolites in urine, erythrocytes, leukocytes, and/or fi broblasts. Enzyme studies are per- Minimum 10 mL urine, shipped on dry ice or refrig- formed in erythrocytes or other nucleated cells (leuko- erated if transit time is not extensive. cytes, fi broblasts), but erythrocytes lack g-glutamyl transpeptidase and 5-oxoprolinase activities. As in the case of the CDG, defects in polyol (sugar alcohols) metabolism represent an expanding area of interest for screening of a patient with suspected inher- D2.13 Glycosaminoglycans ited metabolic disease. The polyols include C4 (erythri- (Mucopolysaccharides) tol and threitol), C5 (ribitol, arabitol, and xylitol), C6 (galactitol, sorbitol, and mannitol), and the C7-polyols (e.g., sedoheptitol and perseitol). These intermediates Sample may be quantifi ed utilizing GC, GC-MS, or electro- Ten milliliter urine, sent on dry ice or refrigerated if spray MS–MS with internal standards. Two recently transit time is not extensive. discovered disorders in the pentose phosphate pathway 276 K. M. Gibson and C. Jakobs

Glycosaminoglycans (GAGs; mucopolysaccharides) are D2.14 Oligosaccharides protein-bound oligosaccharides. Mucopolysaccharidoses (MPS) are the result of defective metabolism of certain GAG which accumulate in the lysosomes and are Sample excreted in the urine. All MPS represent progressive diseases with considerable variability in phenotypic Ten milliliter urine shipped refrigerated or on dry presentation. ice. When possible, 24 h urine collections are optimal, but not necessary. Remember

Only MPS II is an X-linked disorder (the remaining Many lysosomal storage disorders result in accumula- MPS disorders are transmitted as autosomal-reces- tion of specifi c oligosaccharides that accumulate and are sive traits), which may often (but not always) be excreted in the urine. This is most readily recognized identifi ed in relation to the creamy-colored skin through separation of individual oligosaccharides by lesions that are unique to this disease. thin-layer chromatography. The chromatographic separation is generally on silica gel, and the plates are devel- These disorders may be detected using quantitative oped with solvent systems in a single dimension. For analysis of total urinary GAG concentration (in rela- detection, oligosaccharides are reacted with the orcinol tion to creatinine), usually through determination of reagent followed by heating at 100°C. Quantitative anal- total uronic acid via Alcian blue or the carbazole reac- ysis is available only for free neuraminic acid (sialic tion. MPS disorders are more reliably detected through acid), which accumulates in sialic acid storage disease electrophoretic separation of different GAG species, (Salla disease). Oligosaccharide analysis provides evi- usually on cellulose acetate plates. For example, dence for the following disorders: fucosidosis, a- and increased dermatan sulfate (associated with skeletal b-mannosidoses, aspartylglycosaminuria, Schindler dis- and organ changes) is found in MPS types I, II, VI, and ease, sialidosis, GM1- and GM2-gangliosidoses, galacto- VII; elevated heparan sulfate (associated with mental sialidosis, Gaucher disease, Pompe disease, and Salla retardation and, especially behavioral disturbances in disease. However, pathological oligosaccharide patterns MPS III (Sanfi lippo syndromes) is detected in MPS are often variable and may be a challenge to recognize. types I, II, III, and VII. The increased presence of A laboratory with extensive experience in this analysis is preferable. A high degree of clinical suspicion may be Remember developed on the basis of coarse facial features, dysostosis multiplex, and/or ocular involvement (also fi ndings in Morquio syndrome cannot be identifi ed by quanti- the MPS disorders). If the phenotype is strongly sugges- tation of urine GAGs, as the characteristic metabo- tive of one of these disorders, it is preferable to perform lite (keratan sulfate) does not react with reagents enzyme studies even in the face of normal oligosaccha- typically employed to quantify uronic acids. ride fi ndings. Other lysosomal storage disorders generally require enzyme analysis for defi nitive diagnosis. keratan sulfate (primarily associated with skeletal changes, including scoliosis and/or kyphoscoliosis) is pathognomonic for MPS type IV (the Morquio syn- D2.15 Peroxisomal Function – Very drome). Increased chondroin sulfate is characteristi- Long-Chain Fatty, Phytanic, cally found in MPS type VII and less consistently in and Pristanic Acids MPS type IV, but chondroitin sulfate is a normal constituent of urine. Quantifi cation of total urinary GAGs may produce borderline or false normal results, particularly in MPS types III and IV. In those instances, Samples electrophoretic GAG separation is the method of choice One milliliter serum/plasma for very long-chain fatty for identifi cation. The determination of quantitative acids (VLCFAs), shipped on dry ice to avoid lipoly- GAG levels is amenable to MS–MS methodology, and sis (hemolysis is to be avoided as RBCs contribute some laboratories are employing this platform. D2 Biochemical Studies 277

extensively to VLCFA levels, and the patient should D2.16 Creatine Disorders be fasted at draw); 1 mL EDTA whole blood for the analysis of plasmalogens in erythrocytes, express delivery, room temperature. Samples AGAT (arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase) As cellular organelles, the peroxisomes catalyze a defi ciency. number of important processes. These include b-oxi- 10 cc urine (preferably morning void) and 1–2 cc dation of VLCFAs, bile acids, biosynthesis of ether heparinized plasma, for analysis of creatine and phospholipids, and a-oxidation of branched fatty acids guanidinoacetate in both fl uids. such as phytanic acid. The methodological procedure SLC6A8 (creatine transporter) defect. of choice is quantifi cation of VLCFA, phytanic, and 10 cc urine (preferably morning void), for cre- pristanic acids using isotope dilution atine determination expressed in relation to creatinine. Remember Combined analysis of VLCFA, pristanic, and phytanic acids has great utility in diagnosing peroxi- Much like the CDG and polyol disorders, the defects somal disorders. However, diseases such as in creatine biosynthesis and transport have become rhizomelic chondrodysplasia types II and III and an additional emerging fi eld in the selective screening hyperoxaluria type I require additional studies (e.g., for inherited metabolic disorders. The biosynthetic erythrocyte plasmalogens, urine glyoxylate and disorders include l- AGAT defi ciency (featuring low oxalate, etc.). guanidinoacetate in urine and plasma), guanidinoacetate methyltransferase (GAMT) defi ciency (elevated guanidinoacetate in urine and plasma), and the GS-MS, with deuterium labeled internal standards. X-linked creatine transporter (SLC6A8) defect Quantifi cation of VLCFA via GC alone relies on peak (increased creatine in urine, expressed in relation to area and retention time, and is not highly recom- creatinine). The latter represents an emerging focus mended. Peroxisome biogenesis disorders manifest of research into X-linked mental retardation. The multisystem pathology in affected patients. Most dis- nonspecifi c clinical features in all disorders may eases of peroxisomal biogenesis result in defective include developmental delay, language and speech peroxisomal b-oxidation, and result in accumulation disorders, autistic behavior, and seizures, occasion- of VLCFA species in serum or plasma. Additional ally associated with an extrapyramidal movement studies to pinpoint the exact biochemical defect may disorder. A sensitive investigation for all disorders is in vivo proton MRS of the brain, which reveals a signifi cant reduction of creatine. Remember Circulating VLCFA, phytanic acid, and pristanic Remember acid are essentially present in esterifi ed form. Accordingly, they are not fi ltered by the kidney and X-linked mental retardation syndromes should give cannot be detected in urine. rise to a high clinical suspicion for fragile X syndrome as well as the creatine transporter (SLC6A8) defect. include bile acid analysis, plasmalogens, and other species. An isolated elevation of serum phytanic acid is detected in adult Refsum disease. Disorders of etherphos- The method of choice for determining guanidinoacetate pholipid biosynthesis, such as rhizomelic chondrodyspla- and creatine in body fl uids is isotope dilution GC-MS, sia punctata or its variants, entails erythrocyte plasmalogen although liquid chromatography (LC)/MS–MS meth- determination in more specialized laboratories. ods are becoming more widely available. 278 K. M. Gibson and C. Jakobs

D2.17 Folate Metabolites most circumstances. However, mevalonate excretion in the hyper IgD syndrome can be low, and a high degree of clinical suspicion (periodic fever, rash) can Samples lead to specifi c quantitation of mevalonic acid by stable isotope dilution assay or directly to enzyme analy- 1–2 cc EDTA plasma (hemolysis must be avoided, sis for mevalonate kinase activity (leukocytes and as it artifi cially increases 5-methyltetrahydrofolate fi broblasts). (MTHF) levels). Plasma must be deep frozen immediately at −80°C. CSF (any blood must immediately be removed by centrifugation), 1 cc, also deep fro- Remember zen immediately at −80°C. Immune dysfunction and skin abnormalities (rash, psoriasis) may, in conjunction with other clinical features, suggest a disorder of cholesterol synthesis. The active and principle form of circulating folate is 5-MTHF, one-carbon donor in a number of reactions. Biochemical processes including serine and glycine With the exception of the Smith–Lemli–Opitz syn- interconversion, homocysteine, and methionine inter- drome (SLOS), the remaining post-squalene disorders conversion, as well as purine biosynthesis, depend are exceedingly rare. Plasma analysis of 7-dehydro- heavily on 5-MTHF as co-substrate. Serum folate defi - cholesterol and (in some laboratories) 8-dehydrocho- ciency leads to megaloblastic anemia, hyperhomo- lesterol, employing stable isotope dilution GC-MS, is cysteinemia, and neural tube defects in the newborn. the fi rst-line investigation for identifi cation of SLOS. The recently recognized cerebral folate defi ciency is a Other suspected cholesterol defects (e.g., lathoster- neurological disorder featuring psychomotor retarda- olemia, 4-methylsterol oxidase defi ciency, Antley– tion, paraplegia, cerebellar ataxia, and dyskinesia. In Bixler syndrome, Greenberg dysplasia, desmosterolosis, the latter, 5-MTHF in CSF is low. Quantitation of and Conradi–Hünerman–Happle syndrome) require 5-MTHF is readily achieved in a number of laborato- detailed sterol profi ling in specialized laboratories, ries employing HPLC separation with electrochemical generally employing GC with the appropriate internal detection (see Table D2.5). standards. In all of these disorders, clinical suspicion is raised by patients with abnormalities in morphogenesis, such as hand/toe polydactyly, internal organ defects, D2.18 Cholesterol Biosynthetic Defects skeletal and/or skin abnormalities.

Sample D2.19 Bile Acids 1–2 cc EDTA/heparin serum/plasma, sent on dry ice.

Sample Cholesterol is a key membrane component, and the Ten milliliter urine, 2 mL EDTA/heparin plasma, or precursor for numerous intermediates such as oxys- 2 mL bile fl uid, shipped express delivery on dry ice. terols, hormones, vitamins, and bile acids. Cholesterol Stool may also be evaluated by specialist laboratories. is fundamental for membrane fl uidity, and with sphingomyelin forms the lipid rafts/caveolae, sites at which intracellular signaling molecules aggregate. The pre- The primary bile acids, cholic acid (CA) and chenode- dominance of disorders of cholesterol formation occurs oxycholic acid (CDCA), are the end products of cho- at the postsqualene step, a step at which isoprenes lesterol metabolism that conjugate with glycine or formed in the biosynthetic pathway diverge to form taurine to form the excreted bile salts. A large group ubiquinone, dolichol, or cholesterol. Two disorders of enzymes, located in the endoplasmic reticulum, (severe mevalonic aciduria and hyper IgD syndrome) mitochondria, and peroxisomes of hepatocytes is represent defects of pre-squalene synthesis and can be involved in the pathway of bile acid synthesis. identifi ed via routine urine organic acid analysis under Therefore, alterations in concentrations of specifi c bile D2 Biochemical Studies 279 acids may be observed in various peroxisomal disor- dermatological fi ndings primarily in sun-exposed body ders, and also as specifi c defects of bile acid biosynthe- regions. Congenital erythropoetic porphyria (Günther sis. Defective side chain oxidation of cholesterol is disease) features discoloration of the urine (brown, detected in patients with cerebrotendinous xanthoma- red-fl uorescent spots in the diapers). Generally, ALA tosis (CTX), characterized by mitochondrial 27-hydrox- and PBG are readily detected in urine. They may be ylase defi ciency. CTX results in accumulation of separated from other contaminants by ion-exchange cholestanol and chromatography. ALA in urine may be derivatized to a pyrrole analogue, and both ALA and PBG may be Remember derivatized as dimethylaminobenzaldehyde derivatives (seeD2.2.3), which, in the presence of mineral Serum bile acid levels in healthy (fasting) individu- acids, yields strongly fl uorescent analogs for both als are generally <2 mg/mL, but may climb artifac- compounds (Ex = 405 nm; Em = 615 nm). Less polar tually in liver cirrhosis, obstructive jaundice, and porphyrins (e.g., coproporphyrin and protoporphyrin) viral hepatitis. are often only detected in bile secretions. Commercially available kits can be utilized for determination of ALA and PBG. cholesterol in most tissues, whereas serum CA and CDCA acids are signifi cantly decreased. Although a number of methodological approaches are utilized to D2.21 Biogenic Amines and Pterins quantify bile acids in different fl uids, the most reliable include GC-MS, LC-MS–MS, and FAB-MS. Older (Specialized CSF/Urine Analyses) methodologies, including HPLC, radioimmunoassay, enzymatic analysis, and/or GC alone are more time CSF consuming, laborious, and less sensitive than MS or Collect several samples (age <1 year: 0.5 mL frac- MS–MS methodologies. tions, use fractions 2–4 for metabolic investigations; age >1 year: 1 mL fractions, use fractions 3–5). Freeze samples immediately at the bedside D2.20 Porphyrins (dry ice or liquid nitrogen), store at −70°C. The analysis of pterins requires the addition of specifi c antioxidants (contact the referring laboratory for Samples explicit instructions). If bloodstained: centrifuge Twenty milliliter urine (random sample), 5 mL feces, before freezing. It is strongly recommended that 5–10 mL heparinized whole blood; no additives; two CSF samples (of approx. 1 mL each) remain in store cool and dark, may be sent overnight at ambi- storage at −70°C for follow-up studies that may be ent temperature. In many instances, a 24 h urine col- indicated from preliminary studies. lection is desirable. Urinary pterins Ten milliliter random urine sample, keep cool and dark (dark urine collection bag), ship on dry Porphyrias are inherited abnormalities in heme bio- ice. Alternatively: 5 mL urine, add 6 M HCl to pH synthesis. Heme formation is highest in the erythro- 1.0–1.5, add 100 mg MnO2, shake for 5 min (ambi- cyte progenitors of the marrow but is also high in ent temperature), centrifuge for 5 min (4,000 rpm), hepatocytes. Accordingly, the heritable porphyrias send supernatant protected against light (aluminum manifest signifi cant hepatic involvement. The clinical foil) by express mail. presentation of the porphyrias facilitates their subdivi- sion into two broad categories, including the acute and nonacute porphyric syndromes. The acute disorders For a suspected neurometabolic disorder, a lumbar punc- feature debilitating abdominal pain, nausea, vomiting, ture should only be pursued after basic analyses have constipation, and psychiatric fi ndings. Urine levels of been performed in blood and urine, and following neu- the heme precursor d- ALA and PBG are signifi cantly roimaging studies (see Chap. 5). The use of CSF is not elevated. Conversely, the nonacute disorders feature warranted in selective screening, and there must be a 280 K. M. Gibson and C. Jakobs high degree of clinical suspicion to suggest a lumbar with age and sex, and thus it is fundamentally important puncture. Nonetheless, several neurometabolic disorders to only send samples to a highly experienced, special- can only be diagnosed by specifi c CSF studies (Table ized laboratory. D2.5), especially the disorders of monoamine metabo- Quantifying pterins in all body fl uids (urine, CSF, lism. As a rule with any CSF investigation, the analysis plasma, etc.) is the most common and reliable method should include quantitative determination of lactate, for diagnosing the inborn errors of BH4 metabolism. pyruvate, amino acids, cell count, glucose, protein, BH4 is a mandatory cofactor in the phenylalanine immunoglobulin classes, specifi c immunoglobulins, and hydroxylase reaction, and its intracellular level regu- an evaluation of the integrity of the blood–brain barrier. lates tyrosine and tryptophan hydroxylases, and nitric oxide synthase. Pterin analysis should be included in any study quantifying monoamines in CSF as well. A Remember number of defects have been demonstrated in the bio- The analysis of monoamines in CSF requires analy- synthetic pathway of BH4 synthesis, including GTP sis in only well-skilled, specialized laboratories cyclohydrolase defi ciency, 6-pyruvoyl-tetrahydropterin with the appropriately derived collection protocols synthase defi ciency, and sepiapterin reductase defi - and age-related control ranges necessary for correct ciency. Quantitation of pterin levels is a valuable tool evaluation of the fi ndings. for identifi cation of these disorders. The analysis of neopterin, biopterin, 5-MTHF, 5-HIAA, and HVA serves to differentiate between mild and severe forms of

Remember BH4 defi ciencies. Because of its key role in phenylalanine metabolism, selected screening for BH defi ciency CSF monoamine concentrations show diurnal vari- 4 should be pursued in newborns whose plasma phenyla- ation and a rostrocaudal gradient of concentration. lanine exceeds 120 mmol/L. Notably, neopterin is also Exact collection protocols must be followed for col- employed as a marker of T helper cell-derived cellular lection of CSF to insure meaningful data are immune activation. Pterin analogs are generally present obtained. in physiological fl uids in reduced forms, but may be artifi cially oxidized to highly fl uorescent species prior to HPLC analysis with fl uorescent detection. Neopterin, Remember biopterin, and primapterin are blue-fl uorescing species, Many specialized laboratories provide collection whereas sepiapterin is a yellow-fl uorescing compound. tubes for CSF samples, as well as detailed instructions. Selected tubes in the collection set will con- Take Home Pearls tain pertinent antioxidant compounds (e.g., for › Screening for an inherited metabolic disease tetrahydrobiopterin (BH4) ) that will insure accurate measurements. requires interaction of both physician and laboratory personnel. Testing must be ordered in the context of the clinical symptoms and fi ndings. › Physicians who suspect a metabolic disease Generally speaking, the term biogenic amine is equiva- are best served to discuss the clinical features lent to monamines, but in the case of inherited meta- and general laboratory fi ndings with a special- bolic disorders encompasses catecholamines (dopamine, ist who is trained and/or Board-certifi ed in noradrenaline) and serotonin. These derive from the Clinical Biochemical Genetics. amino acid precursors, tyrosine and tryptophan. The › Key stumbling blocks in diagnostic testing are end products of dopamine and serotonin metabolism not knowing which tests to order, the limitations include HVA and 5- HIAA. Defects in these pathways of the test, and the care and handling of the have a signifi cant component of neurological dysfunc- appropriate body fl uids. This further underscores tion. Species including HVA, 5-HIAA, dopamine, the value of discussion and interaction with the 3,4-dihydroxyphenylacetate, 5-hydroxytryptophan, and Biochemical Genetics laboratory director. 3-O-methyldopa are quantifi ed by HPLC with electrochemical detection. Normative data vary extensively D2 Biochemical Studies 281

Key References Hoffmann GF, Nyhan WL, Zschocke J, Kahler SG, Mayatepek E (2002) Inherited metabolic diseases – biochemical studies. Core handbooks in pediatrics, Lippincott Williams & Blau N, Duran M, Blaskovics ME, Gibson KM (eds) (2003) Wilkins, Philadelphia, pp 95–109 Physician’s guide to the laboratory diagnosis of metabolic Hommes FA (ed) (1991) Techniques in diagnostic human bio- diseases, 2nd edn. Springer, Berlin chemical laboratories: a laboratory manual. Wiley-Liss, New Blau N, Hoffmann GF, Leonard J, Clarke JTR (eds) (2005) York Physician’s guide to the treatment and follow-up of meta- Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of metabolic bolic diseases. Springer, Berlin diseases, 2nd edn. Hodder Arnold, London Hoffmann GF, Surtees RAH, Wevers RA (1998) Investigations of cerebrospinal ﬂ uid for neurometabolic disorders. Neuropediatrics 29:59–71 Enzymes, Metabolic Pathways, Flux Control Analysis, and the Enzymology D3 of Speciﬁ c Groups of Inherited Metabolic Diseases

Ronald J. A. Wanders, Ben J. H. M. Poorthuis, and Richard J. T. Rodenburg

Key Facts enzyme studies should be performed to resolve › Enzyme functions in a particular metabolic the underlying enzymatic defect. Biochemical diagnostics of mitochondrial dis- pathway are best described by Km and Vmax › values. orders usually involves a broad screening of › The central parameter in metabolic fl ux analy- mitochondrial enzyme activities and analysis sis is that of control strength, alternatively of fl uxes through the citric acid cycle and oxi- called fl ux control coeffi cient. It is described dative phosphorylation system. The analysis of by the fl ux control coeffi cient which is set fl uxes can only be performed in freshly obtained between 0 and 1. A fl ux control coeffi cient of tissue samples (usually muscle). 1 means that the particular enzyme has full › Determination of the activity of lysosomal control of fl ux through the pathway, whereas a enzymes in leukocytes, plasma or dried blood fl ux control coeffi cient of 0 implies that the spots, or in fi broblasts is the core in the laboratory particular enzyme exerts virtually no control diagnosis of lysosomal storage disorders. of fl ux. › In any patient with a Zellweger spectrum-like phenotype in which very long-chain fatty acids have been found abnormal, a full enzymatic D3.1 General Remarks study in fi broblasts is warranted in order to resolve whether the patient has a defect in the D3.1.1 Enzymes biogenesis of peroxisomes or is affected by a single peroxisomal enzyme defi ciency. › The introduction of tandem mass spectrometry The key players of metabolism are the enzymes, which has revolutionized the diagnosis of fatty acid are the true catalysts of biological systems. These oxidation (FAO) disorders. Acylcarnitine anal- remarkable molecular devices convert a particular sub- ysis is the fi rst-line test in patients suspected to strate into a specifi c product, a process called cataly- suffer from an FAO disorder. If abnormal, sis. Nearly all the known enzymes are proteins, although proteins do not have an absolute monopoly on catalysis, since the discovery of catalytically active RNA molecules (ribozymes). R. J. A. Wanders () A remarkable feature of enzymes is their high level Laboratory of Genetic Metabolic Diseases, Academic Medical of specifi city. Indeed, an enzyme usually converts only Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands one or at most a few substrates into the corresponding e-mail: [email protected] products. The underlying basis for this specifi city is

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 283 DOI: 10.1007/978-3-540-74723-9_D3, © Springer-Verlag Berlin Heidelberg 2010 284 R. J. A. Wanders et al.

2 5 Slope=Km/Vmax 1/vi Vmax 4 1/Vmax 3 1 Vi 2 1a 1b 1

0 0 0 5 10 15 20 25 30 35 -0.5 0 0.5 1 1.5

Km [S] 1/ [S] −1/Km

Fig. D3.1 Effect of the concentration of substrate S on the initial velocity (vi) of an enzyme-catalyzed reaction represented in a V vs. [S] plot (a) or a double-reciprocal, Lineweaver–Burke plot of 1/vi vs. 1/[S] (b) that the active site at which catalysis takes place, gener- as the concentration of substrate at which the enzyme ally takes the form of a three-dimensional cleft or crev- operates at 50% of its maximal velocity, whereas Vmax ice formed by steric groups that usually come from is deﬁ ned as the turnover number of an enzyme, which different parts of the amino acid sequence. Indeed, is the number of substrate molecules converted into amino acid residues far apart in the primary amino acid product by an enzyme molecule in a unit time under sequence may interact more strongly than adjacent resi- conditions such that the enzyme is fully saturated with dues in the sequence, thus constituting the active site. substrate. Figure D3.1a shows a plot of the reaction

For instance, in lysozyme, which is an enzyme that velocity (vi) as a function of the substrate concentra- degrades the cell walls of some bacteria, the active site tion [S] for an enzyme that obeys Michaelis–Menten is formed by amino acids 35, 52, 62, 63, 101, and 108 in kinetics, which clearly illustrates that the maximal the sequence of 129 amino acids. The three-dimensional velocity (Vmax) is approached asymptotically. The active site shields the substrate(s) from the solvent and Michaelis constant (Km) can best be determined with creates a unique microenvironment, which allows catal- the aid of a double-reciprocal or Lineweaver–Burke ysis to take place. What actually happens is that the plot (Fig. D3.1b). A simple, direct measure describing active site of enzymes facilitates formation of the tran- the effi ciency of a particular enzyme, is the catalytic sition state, thereby providing the rate enhancement, effi ciency of an enzyme defi ned as kcat/Km, or, alterna- which is so characteristic of enzymes. tively, as Vmax/Km. Enzymes may accelerate reactions by factors of as When localized within the cell, the activity of an much as a million or more. Indeed, most reactions in enzyme may be changed by several distinct regulatory biological system do not take place at any perceptible mechanisms. One key mechanism is allosteric control, rate in the absence of enzymes. Even a reaction as sim- in which the activity of an enzyme is stimulated or ple as the hydration of CO2 is catalyzed by an enzyme, decreased by effectors of the enzyme, which bind at an i.e., carbonic anhydrase. The transfer of CO2 into the allosteric site. Examples of allosteric enzymes are as blood and then to the alveolar air would be less effi - follows: (1) carbamylphosphate synthase (allosteric cient in the absence of carbonic anhydrase. effector (positive): N-acetyl glutamate), (2) pyruvate In order to understand how a certain enzyme func- carboxylase (allosteric effector (positive): acetyl-CoA), tions in a particular metabolic pathway it is important (3) aspartate transcarbamylase (allosteric effector (neg- to know the properties of the enzyme in terms of its ative): CTP), and (4) carnitine palmitoyltransferase 1 ability to convert a certain substrate [S] into a particu- (CPT1) (allosteric effector (negative): malonyl-CoA). lar product [P]. These properties are best described in A second level of control is exerted by the reversible the Km and Vmax values of that particular enzyme for a covalent modifi cation of enzymes. Many modifi cations certain substrate. The Michaelis constant (Km) is defi ned are known at present, ranging from glycosylation, D3 Enzymes and Inherited Metabolic Diseases 285 hydroxylation, acetylation, acylation, farnesylation, extended by the groups of Kacser and Burns (1979) phosphorylation, ubiquitination, etc. Phosphorylation and Heinrich et al. (1977). The central parameter in of proteins is again achieved by enzymes, so-called metabolic fl ux analysis is that of control strength, protein kinases, which catalyze the transfer of the alternatively called fl ux control coeffi cient, as defi ned terminal phosphoryl group of ATP to the hydroxyl by the fractional change in fl ux through a pathway, groups of seryl, threonyl, or tyrosyl residues, thus induced by a fractional change in activity of the enzyme forming O-phosphoseryl, O-phosphothreonyl, and/or catalyzing a particular step in the pathway involved. O-phosphotyrosyl residues. Typical examples of mam- The value of a fl ux control coeffi cient is usually malian enzymes undergoing phosphorylation/dephos- between 0 and 1. A fl ux control coeffi cient of 1 means phorylation are as follows: acetyl-CoA carboxylase, that the particular enzyme has full control of fl ux glycogen synthase, pyruvate dehydrogenase, and through the pathway. In the old nomenclature, such an HMG-CoA reductase. enzyme would be classifi ed as “rate-controlling,” The activity of enzymes can also be regulated by “rate-limiting,” or “pacemaker enzyme.” On the other changing its quantity. Indeed, some enzymes are constitu- hand, a fl ux control coeffi cient of 0 means that the par- tive in nature, which implies that the concentration remains ticular enzyme exerts virtually no control of fl ux. essentially constant over time, whereas other enzymes are Several methods are available for the determination inducible. Examples of the latter category are as follows: of fl ux control coeffi cients. The fi r s t method makes (1) the enzymes of the urea cycle, as induced by a high- use of specifi c inhibitors of particular enzymes. In protein diet; (2) the enzymes of the mitochondrial fatty practice, this means that the effect of adding increas- acid oxidation (FAO) system, as induced by a high-fat diet ing concentrations of a certain inhibitor on pathway and prolonged fasting; and (3) HMG-CoA reductase and fl ux is measured. From such plots the fl ux control the other enzymes of the cholesterol biosynthesis path- coeffi cient of that enzyme can be calculated, espe- way, as induced by low cholesterol. cially if irreversible or noncompetitive inhibitors are available. This approach has been used successfully for instance by Groen et al. (1982a) who used a vari- D3.1.2 Control of Flux Through Metabolic ety of different inhibitors to resolve the importance of the ATP/ADP-carrier as well as other steps in the con- Pathways trol of the rate of mitochondrial oxidative phosphorylation (OXPHOS). These studies revealed that the Many of the enzymes involved in the major metabolic control of fl ux of mitochondrial OXPHOS is distrib- pathways have been purifi ed and their kinetic proper- uted among different steps and furthermore that the ties determined. Despite all this detailed knowledge, it value of the different fl ux control coeffi cients is very has remained diffi cult to resolve the extent to which a much dependent on the relative rate at which mito- particular enzyme contributes to the control of fl ux chondria respire between state 4 (resting state) and through the metabolic pathway in which the enzyme is state 3 (maximal rate of respiration) (Groen et al. involved. Quantitative studies of control mechanisms 1982a). The same inhibitor-based approach, which in metabolic pathways are hampered by the fact that makes use of inhibitors, was also used to determine the fl ux through a pathway cannot be described ade- the fl ux control of other metabolic pathways including quately by a rate equation analogous to that for a reac- citrulline synthesis in mitochondria (Wanders et al. tion catalyzed by a single enzyme. In order to 1984), fatty acid b-oxidation in mitochondria (Eaton circumvent this diffi culty, computer simulations of 2002), and gluconeogenesis in hepatocytes (Groen metabolic pathways have been devised, although this et al. 1982b, 1986). Another means of determining approach has major drawbacks. fl ux control coeffi cients is to measure the so-called A breakthrough came in 1965 when Higgins (1965) elasticity coeffi cients described later. introduced the concept of control strength as a quanti- Measurement of the fl ux control coeffi cient of a tative measure to describe the contribution of a partic- particular enzyme in a certain pathway provides con- ular enzyme to the control of fl ux through a metabolic siderable information and quantitatively describes how pathway. This concept was later reformulated and much control is exerted by that particular enzyme. It 286 R. J. A. Wanders et al. does not, however, reveal how this “control” is actually brought about. This issue has been resolved by Kacser Enzyme 1 Enzyme 2 and Burns (1979, 1981) when they formulated their S I P connectivity theorem, which links the kinetic properties of individual enzymes in the pathway to the control exerted by these enzymes. The theorem states that Fig. D3.2 Simplifi ed scheme of a metabolic pathway consisting there is a reciprocal relationship between the fl ux con- of two enzymes which convert the substrate S into product P trol coeffi cients of adjacent enzymes in a pathway and with I as intermediate the so-called elasticity coeffi cients of the two enzymes toward their common intermediate whereby the elas- inhibition by carbamoyl phosphate (high Ki-value for ticity coeffi cient is defi ned as the fractional change in carbamoyl phosphate), whereas the next enzyme in the rate through an enzyme (dvi/vi) induced by a frac- line, which is ornithine transcarbamylase, has a high tional change in substrate or product concentration affi nity for carbamoyl phosphate (low Ki-value). This (dSi/Si) while keeping all other factors able to infl uence implies that fl ux through the urea cycle is predomi- the rate of the enzyme reaction constant. The elasticity nantly controlled by carbamoyl phosphate synthase coeffi cient is thus a quantitative expression for the with little control exerted by any of the subsequent responsiveness or sensitivity of an enzyme toward a steps, including ornithine transcarbamylase, arginino- change in substrate of product concentration. succinate synthetase, argininosuccinate lyase, and A simple example may be helpful here. Let us take arginase. Another example is HMG-CoA reductase, a simple two-enzyme pathway(S→I→P) in which the which converts HMG-CoA into mevalonate with initial substrate [S] is converted into the intermediate mevalonate being a poor product inhibitor of HMG- product [I], which is then converted to the fi nal prod- CoA reductase. uct [P] by enzyme 2 (Fig. D3.2). Let us further assume Important to know is that the elasticity coeffi cients that enzyme 2 has a high affi nity for its substrate [I] of enzymes in metabolic pathway can be determined

(Km = 1.0 mM) and enzyme 1 a low affi nity for [I] as experimentally and can be used to subsequently calcu- refl ected in an inhibitor constant (Ki) of 100 mM for late the fl ux control coeffi cients (see Groen et al. enzyme 1. Let us also assume that the concentration (1982b) for detailed information). of [I] during the experiment would be 1.0 mM. What The importance of certain enzymes for the overall will happen now if we take away a little bit of enzyme fl ux through a metabolic pathway as refl ected in their 1, say 1%? At a constant concentration of [S], this fl ux control coeffi cients is also important for inherited will immediately lead to a decrease in the concentra- metabolic diseases, although this concept has not been tion of [I]. Since the concentration of [I] was in the widely introduced in the fi eld. Indeed, the concept of fl ux same range as the Km of enzyme 2 toward [I], the drop control analysis gives a logical explanation for generally in [I] will lead to an immediate drop in the formation known facts, which are often intuitive. In the fi eld of of [P]. Since enzyme 1 is practically insensitive to [I] inherited metabolic diseases, it is, for instance, generally due to the Ki of 100 mM, the drop in [I] will not affect accepted that the effect of a particular residual activity of enzyme 1 so that in the new steady state, pathway fl ux an enzyme in a patient is very much dependent on the will be decreased by about 1%. This means that in this enzyme involved. It is clear that a similar residual activ- example, enzyme 1 exerts almost full control on fl ux ity of for instance 5% has a markedly different effect on through the pathway and this is explained by the low pathway fl ux if the enzyme has a fl ux control coeffi cient sensitivity of the enzyme toward its product [I] in of 1.0 as compared to a fl ux control coeffi cient of 0.01. combination with the high sensitivity of enzyme 2 Flux control analysis also provides a logical basis for toward [I]. the known fact that heterozygosity for some enzyme defi - In general, enzymes in metabolic pathways that ciencies is associated with clinical signs and symptoms, show little product inhibition like enzyme 1 in the whereas in most other cases heterozygotes show no example of Fig. D3.2 exert major control on pathway abnormalities whatsoever. In addition, fl ux control analy- fl ux. A good example of such an enzyme is carbamoyl- sis provides a logical framework for the concept of syner- phosphate synthase, which shows minimal product gistic heterozygosity as advanced by Vockley (2008). D3 Enzymes and Inherited Metabolic Diseases 287

D3.2 Peroxisomal Disorders etherphos pholipid biosynthesis, (3) fatty acid a -oxidation, and (4) glyoxylate metabolism as most important peroxisomal functions, at least from the perspective of human D3.2.1 Background disease (Wanders and Waterham 2006). Accordingly, the group of single peroxisomal enzyme defi ciencies can be The peroxisomal disorders (PDs) represent a group of subdivided into subgroups based on whether the enzyme inherited diseases in man in which there is an impair- defect affects b-oxidation, a-oxidation, etherphos pho- ment in either the biogenesis of peroxisomes or one of lipid biosynthesis, or glyoxylate metabolism. Table D3.1 the metabolic functions of peroxisomes (Wanders lists the PDs as identifi ed until now with details on the 2004; Weller et al. 2003). Accordingly, the PDs are mutated gene and dysfunctional enzyme/transporter pro- usually subdivided into two subgroups: (1) the peroxi- tein in each of the diseases. some biogenesis disorders (PBDs) and (2) the single peroxisomal enzyme defi ciencies. The group of PBDs is again subdivided into two categories, including the following: (1) the Zellweger spectrum disorders (ZSDs) D3.2.2 Laboratory Diagnosis with Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease as representatives Once a patient is suspected to suffer from one of the and (2) rhizomelic chondrodysplasia punctata type 1. PDs listed in Table D3.1 on clinical grounds, metabolite The principle features of the biogenesis of peroxi- analysis of specifi c (sets of) metabolites should be initi- somes have been elucidated in recent years. In many ated, which differs for each of the PDs (see Table D3.1). respects, the biogenesis of peroxisomes resembles that In general, most of the PDs known can be screened for of mitochondria, although peroxisomes do not possess reliably by analysis in plasma and/or erythrocytes. This their own DNA, like mitochondria do, which implies is true for the ZSDs plus acyl-CoA oxidase defi ciency that the genetic information for all peroxisomal pro- and D-bifunctional protein defi ciency by performing teins, irrespective of their localization in the peroxi- plasma very long-chain fatty acid (VLCFA) analysis. somal membrane or the peroxisomal matrix, is nuclear This is also true for the different forms of rhizomelic DNA encoded (Platta and Erdmann 2007). chondrodysplasia punctata if plasmalogen analysis is Peroxisomal proteins are targeted to peroxisomes done in erythrocytes and also for Refsum disease if by virtue of distinct targeting signals (PTS), which are plasma phytanic acid is measured. The same applies to recognized by the peroxisomal protein uptake machin- 2-methylacylCoA racemase (AMACR) and SCPx defi - ery, followed by their incorporation into either the ciency if plasma phytanic acid and/or the bile acid inter- peroxisomal membrane or the peroxisomal matrix. mediates are measured. Alternatively, the latter can also Multiple so-called peroxins encoded by PEX genes are be determined in urine. Finally, plasma VLCFA analy- involved in this process. This immediately explains sis is also very reliable for the identifi cation of X-linked why the group of PBDs, notably the ZSDs, is geneti- adrenoleukodystrophy, especially for hemizygote detec- cally heterogeneous. Indeed, the molecular basis of the tion. Identifi cation of the true defect in each of these ZSDs is remarkably diverse and involves mutations in cases warrants detailed enzymatic studies in fi broblasts 12 different PEX genes, including PEX 1, 2, 3, 5, 6, as described later. 10, 12, 13, 14, 16, 19, and 26 (Ebberink et al. 2009). Interestingly, mutations in PEX7, which codes for a specifi c peroxin, involved in the import of a distinct set of peroxisomal proteins, carrying the so-called PTS2 Remember signal, are only found in patients with rhizomelic chondrodysplasia punctata, but not in patients having a Most PDs known can be screened for reliably by ZSD-like phenotype. analysis in plasma and/or erythrocytes (analysis of With respect to the metabolic role of peroxisomes, VLCFA, phytanic acid, and the bile acid intermedi- peroxisomes are known to be involved in various meta- ates in plasma, plasmalogen analysis in eryth- bolic processes, including (1) fatty acid b-oxidation, (2) rocytes). 288 R. J. A. Wanders et al.

Table D3.1 Peroxisomal disorders plus information on the enzyme and gene defects, biochemical abnormalities and fi rst-line tests Enzyme defect Gene defect Plasma Erythrocytes VLCFA BA Ph Pr Pi Plasmalogens Peroxisome Zellweger Generalized PEX1,2,3,5, Ý ↑↑↑↑↓ biogenesis spectrum 6,10,12,13, disorders disorders 14,16,19, 26 + DLP1 Rhizomelic PTS2-proteins PEX7 NNÝ NNß chondrodys- plasis punctata type 1 Single Peroxisomal peroxi- b-oxidation somal defects enzyme defi ciencies X-linked ALDP ABCD1 Ý NNNNN adrenoleukodystrophy Acyl-CoA oxidase ACOX1 ACOX1 Ý NNNNN defi ciency D-bifunctional DBP HSD17B4 Ý ↑↑↑NN protein defi ciency SCPx-defi ciency SCPx SCP2 N ÝÝÝNN Methylacyl-CoA AMACR AMACR N ÝÝÝNN racemase (AMACR) defi ciency Plasmalogen biosynthesis defects RCDP type 2 Dihydroxyacetone GNPAT NNNNNß phosphate acyltransferase RCDP type 3 ADHAPS AGPS NNNNNß Phytanic acid a-oxidation defects Refsum disease PhyH/Phax PHYH NNÝ NNN Glyoxylate metabolism defects Hyperoxaluria Alanine glyoxylate AGXT NNNNNN type 1 aminotransferase BA bile acid intermediates; Ph phytanic acid; Pi pipecolic acid; Pr pristanic acid; VLCFA very-long-chain fatty acids The fi rst-line test for each individual disorder is a double arrow (Ý/ ß) Explanation of symbols: Ý = increased; N = normal; ß = decreased.

D3.2.3 Enzymology of the PDs or minus abnormalities in any of the other peroxisomal parameters measured, including the bile acid intermediates, phytanic acid, pipecolic acid, and Following the analysis of metabolites as described ear- erythrocyte plasmalogens, a full enzymatic study in lier, enzymatic studies are required for most of the PDs ﬁ broblasts is warranted in order to resolve whether with some exceptions as described later in order to the patient has a defect in the biogenesis of peroxi- identify the ultimate genetic defect. The latter is espe- somes or is affected by a single peroxisomal enzyme cially important if prenatal diagnosis is required in the deﬁ ciency, notably at the level of acyl-CoA oxidase future. We describe the enzymology of the different (Ferdinandusse et al. 2007) or D-bifunctional pro- groups of PDs: tein (Ferdinandusse et al. 2006a). Such a study 1. In any patient with a ZSD-like phenotype in which includes: (1) measurement of C26:0 and pristanic VLCFAs have been found abnormal in plasma plus acid b-oxidation, (2) plasmalogen biosynthesis, (3) D3 Enzymes and Inherited Metabolic Diseases 289

phytanic acid a-oxidation, and (4) immunofl uores- den Brink et al. 2003). Apparently, mutations in the cence microscopy analysis of peroxisomes using PEX7 gene can give rise to two different pheno- antibodies raised against catalase. If all parameters types including a Refsum-like phenotype in case of including immunofl uorescence microscopy analy- mild mutations (van den Brink et al. 2003), and rhi- sis are abnormal, a PBD is very likely and subse- zomelic chondrodysplasia punctata in case of severe quent complementation analysis, followed by mutations (Motley et al. 2002). molecular analysis of the relevant PEX gene, is nec- 4. In a few other patients with some signs and symp- essary to identify the gene that is defective (see toms reminiscent of Refsum disease, but, in addi- Wanders (2004) ). If the abnormalities as observed tion, showing signs and symptoms not found in in fi broblasts are restricted to the peroxisomal Refsum disease, the defect has turned out to be at b-oxidation system only, one is probably dealing some other level including SCPx (Ferdinandusse with either acyl-CoA oxidase (Ferdinandusse et al. et al. 2006b) and AMACR (Ferdinandusse et al. 2007) or D-bifunctional protein defi ciency, which 2006a). can be resolved by direct measurement of the activ- 5. Analysis of plasma VLCFA is also the fi rst line of ity of these enzymes in fi broblasts, followed by testing for the identifi cation of X-ALD. Especially analysis of either ACOX1 or HSD17B4 (see Wanders for hemizygote detection, plasma VLCFA analysis (2004) ). has turned out to be fully conclusive (Bezman et al. 2. In any patient with a rhizomelic chondrodysplasia 2001). For X-linked ALD, detailed enzymatic stud- punctata-like phenotype, plasmalogens should be ies in fi broblasts may not be warranted and instead analyzed in erythrocytes and, if abnormal, it is fully molecular analysis of the X-ALD gene, i.e., ABCD1, sure that one is dealing with one of the peroxisomal might be initiated right away. At present, >700 dif- forms of rhizomelic chondrodysplasia punctata, be it ferent mutations have been identifi ed (see www. type 1, 2, or 3, as caused by mutations in either PEX7, XALD.nl). GNPAT or AGPS. The latter two genes code for the 6. Heterozygote detection is much less straightforward, two peroxisomal enzymes involved in etherphos pho- especially if the patient is from a family having no lipid biosynthesis, i.e., dihydroxyacetone phosphate family history of X-linked adrenoleukodystrophy. In acyltransferase (DHAPAT) and alkyl-DHAP syn- such cases, a full study should be done: (1) in plasma thase. Resolution between these three forms of rhi- including VLCFA analysis (which is only abnormal zomelic chondrodysplasia can be done via enzymatic in 80% of obligate heterozygotes) and (2) fi bro- studies in fi broblasts, which includes direct measure- blasts, which should include immunofl uorescence ment of DHAPAT and alkyl-DHAP synthase, fol- microscopy analysis of ALD protein. If the pre- lowed by molecular analysis of the different genes. sumed mutation affects, the stability of ALD protein Importantly, erythrocyte plasmalogen analysis has as is the case in about 65% of the mutations found in turned out to be an extremely powerful initial screen- the ABCD1 gene until now, one will see a mosaic ing method since all the patients with RCDP, who pattern upon immunofl uorescence microscopy anal- have been analyzed by us, have shown clearly defi - ysis, using an antibody raised against ALD protein cient plasmalogen levels in erythrocytes independent with some cells showing a punctuate fl uorescence whether it was RCDP type 1, 2, or 3, with only one pattern, whereas other cells are lacking this. exception, which concerns a patient with a very mild 7. Hyperoxaluria type 1 is also a peroxisomal disorder Refsum-like phenotype (Horn et al. 2007). as it is caused by a functional defi ciency of the per- 3. In patients with a Refsum-like phenotype in whom oxisomal enzyme alanine glyoxylate aminotrans- plasma phytanic acid levels are elevated, fi broblast ferase (AGT) encoded by the AGXT gene. Interestingly, studies should be done with prime emphasis on a functional defi ciency of AGT can be caused either phytanic acid a-oxidation, which, if abnormal, by a defi cient activity of the enzyme or by the mistar- should be followed up by measurement of phytanoyl- geting of the enzyme to the wrong organelle, i.e., the CoA hydroxylase activity and molecular analysis of mitochondrion (Danpure et al. 1993). This leads to the PHYH genes. In some atypical patients, these the bizarre situation that the detoxifi cation of glyox- studies have failed to show mutations in the PHYH ylate in the peroxisome is impaired thus leading to gene. Subsequent studies have shown that in these hyperoxaluria, not because the AGT enzyme is cata- patients it is the PEX7 gene that is defective (van lytically inactive, but because of a functionally active 290 R. J. A. Wanders et al.

AGT enzyme, which is not able to detoxify glyoxy- b-oxidation and the acetyl-CoA units generated in each late in the peroxisome, due to its aberrant localization cycle of b-oxidation can subsequently be degraded to

in the mitochondrion. This immediately suggests CO2 and H2O in the Krebs cycle or used for the synthe- that there is no place for enzymatic analysis of AGT sis of the ketone bodies acetoacetate and 3-hydroxybu- in liver biopsies in the absence of parallel studies to tyrate, which occurs primarily in the liver (Rinaldo determine the subcellular localization of AGT. For et al. 2002). this reason, direct analysis of the AGXT gene by sequence analysis of all exons and fl anking introns is the method of choice now for the identifi cation of hyperoxaluria type 1 in any patient suspected to suf- D3.3.2 Laboratory Diagnosis fer from hyperoxaluria type 1, based on clinical of FAO Disorders grounds and/or laboratory fi ndings including elevated oxalic acid, glycolic acid, and/or glyoxylic The introduction of tandem mass spectrometry (MS) in acid in urine (van Woerden et al. 2004). laboratories dealing with the diagnosis of inherited metabolic diseases in general and FAO disorders in particular, has revolutionized the diagnosis of FAO dis- D3.3 Mitochondrial FAO Disorders orders. This is due to the fact that acylcarnitine species, which previously were notoriously diffi cult to measure, are relatively easy to quantify by means of tan- D3.3.1 Background dem MS techniques. For this reason, acylcarnitine analysis is a fi rst-line test in patients suspected to suffer from an FAO disorder. In fact, the same technique is Fatty acids (FAs) are an important source of energy for also used in neonatal screening programs designed to human beings. Most organs are able to oxidize FAs identify neonates affected by a certain FAO disorder. If with some exceptions, notably erythrocytes and the abnormal, enzyme studies should be done to resolve brain. On the other hand, the heart is very much depen- the underlying enzymatic defect as specifi ed later. dent on mitochondrial FAO with 60–90% of the energy equivalents being generated by means of mitochondrial fatty acid b-oxidation. Although both mitochondria and peroxisomes are able to oxidize fatty acids, mito- D3.3.3 Enzymatic Analysis chondria are responsible for the oxidation of the bulk of fatty acids, derived from our daily diet. FAs destined of FAO Disorders for mitochondria enter the cell via a mechanism which has remained ill-defi ned until now. Inside the cell, the In principle, there are two scenarios for the enzymatic FAs are rapidly esterifi ed with coenzyme A (CoA) by work-up of patients suspected to suffer from an FAO one of several acyl-CoA synthetases to generate the disorder. The fi rst scenario is that plasma or blood spot corresponding acyl-CoA esters. Acyl-CoA esters can- acylcarnitine analysis has been done and has shown a not traverse the mitochondrial membrane and fi rst need characteristic abnormal profi le, which immediately to be converted into the corresponding acylcarnitine suggests a specifi c defect in the FAO system. Figure esters by the enzyme CPT1, whereby the carnitine D3.3 reveals that many of the currently known FAO required for this reaction enters the cell via a coupled disorders may show characteristic acylcarnitine pro- carnitine/Na+ -importer called OCTN2. The acylcarni- fi les. This is true for (1) OCTN2 defi ciency (primary tine generated in the CPT1 reaction then enters the carnitine defi ciency), (2) CPT1 defi ciency, (3) CPT2/ mitochondria via the mitochondrial carnitine acylcar- CACT defi ciency, (4) VLCAD defi ciency, (5) MCAD nitine translocase (CACT), which exchanges acylcar- defi ciency, (6) SCAD defi ciency, (7) LCHAD/MTP nitines for free carnitine. Once inside the mitochondrion defi ciency, and (8) SCHAD defi ciency. In case of such the acylcarnitine ester is reconverted into the corre- a diagnostic acylcarnitine profi le, the enzyme that is sponding acyl-CoA ester via the enzyme carnitine supposed to be defi cient should be measured right palmitoyltransferase 2 (CPT2). The acyl-CoA ester away, preferably in lymphocytes, in order to identify can then be broken down via subsequent rounds of the enzyme defect as soon as possible. The activities of D3 Enzymes and Inherited Metabolic Diseases 291

FATTY ACID Free carnitine acylcarnitines

acyl-CoA synthetases

Extra- mitochondrial acylCoA space

CPT1 ↑/n reduced acylcarnitine levels

acylcarnitineout

CACT ↓/n 16:0 ↑, 18:1 ↑, 18:2 ↑, 18:0 ↑

acylcarnitinein

CPT2 ↓/n 16:0 ↑, 18:1 ↑, 18:2 ↑, 18:0 ↑

↓/n 14:0 ↑, 14:1 ↑, 16:0 ↑, 16:1 ↑ acyl-CoA in VLCAD 16:2 ↑, 18:0 ↑, 18:1 ↑, 18:2 ↑

acyl-CoA MCAD ↓/n 6:0 ↑, 8:0 ↑, 10:0 ↑, 10:1 ↑ dehydrogenases enoyl-CoA SCAD ↓/n 4:0 ↑

intra- LCEH enoyl-CoA mitochondrial hydratases SCEH (crotonase) space 3OHacyl-CoA ↓/n 3OH16:0 ↑, 3OH16:1 ↑ LCHAD 3OH18:0 ↑, 3OH18:1 ↑ 3OHacyl-CoA dehydrogenases SCHAD 3OH4:0 ↑ 3ketoacyl-CoA

LCKT 3ketoacyl-CoA thiolases single case reported (Kamijo et al.) MCKT no information on acylcarnitines acylCoA acetyl-CoA

Fig. D3.3 Plasma acylcarnitine proﬁ ling and its importance for guiding the way toward the enzyme defect

Table D3.2 Mitochondrial fatty acid b-oxidation disorders plus information on the enzyme defect and enzyme tests Mitochondrial FAD Defi cient enzyme Mutated gene Enzyme assay Enzyme assay in disorders in fi broblasts lymphocytes 1. Primary carnitine defi ciency OCTN2 OCTN2 + +a 2. Carnitine palmitoyl CPT1 CPT1 + +a transferase 1 defi ciency 3. Carnitine acylcarnitine CACT CACT + +a translocase defi ciency 4. Carnitine palmitoyltrans- CPT2 CPT2 + + ferase 2 defi ciency 5. VLCAD defi ciency VLCAD ACADVL + + 6. MCAD defi ciency MCAD ACADM + + 7. SCAD defi ciency SCAD ACADS + + 8. LCHAD/MTP defi ciency LCHAD/MTP HADHA/HADHB + + 9. SCHAD defi ciency SCHAD HADH + + aIn principle feasible, but not yet validated in our laboratory

CPT2, VLCAD, MCAD, SCAD, LCHAD/MTP, and as compared to fi broblasts so that fi broblasts remain SCHAD can all be measured reliably and reproducibly the best material for enzymatic analysis of OCTN2 in peripheral blood mononuclear cells. Although, and CPT1 (see Table D3.2). OCTN2 and CPT1 are also expressed in lymphocytes, In contrast to the clear-cut situation described earlier the activity of these two gene products is relatively low in which patients exhibit a characteristically abnormal 292 R. J. A. Wanders et al. acylcarnitine profi les, patients may show up with a less which is transported into the mitochondria and subse- abnormal acylcarnitine profi le. In addition, there are quently converted into acetyl-CoA by the pyruvate patients with signs and symptoms suggestive for an dehydrogenase complex (PDHc), a key enzyme in FAO defect such as hypoketotic hypoglycemia, cardio- energy metabolism. The activity of PDHc is regulated myopathy, and/or muscle weakness, in the absence of allosterically by its products acetyl-CoA and NADH, any characteristic plasma acylcarnitine abnormalities. and by specifi c kinases and phosphatases, which are In such cases, a full enzymatic study should be done in activated depending on the energy demands of the cell. cultured skin fi broblasts, which fi rst involves whole Acetyl-CoA formed by PDHc and from other sources cell b-oxidation analysis, to establish whether fl ux (e.g., oxidation of fatty acids) is taken up by the tricar- through the mitochondrial b-oxidation machinery is boxylic acid (TCA) cycle. In the TCA cycle, the reduc- defective or not. Two different tests are advisable here. tion equivalents NADH and FADH2 are formed, which The fi rst test is the tritium release assay in which intact are the substrates for the OXPHOS system. The TCA fi broblasts are incubated with one of different tritium cycle is a dynamic cycle of enzyme reactions, and labeled fatty acids, notably myristic acid (C14:0), metabolites can enter or leave at various steps within palmitic acid (C16:0), and oleic acid (C18:0). Use of the cycle. Levels of intermediates of the TCA cycle can such FAs has proven very helpful in the identifi cation increase due to defects in subsequent steps of the mito- of disturbances in the mitochondrial FAO system chondrial energy generating system and can often be (Olpin et al. 1997). detected in urine of mitochondrial patients. The prod-

The second test, originally developed by Nada et al. ucts of the TCA cycle, NADH, and FADH2, are oxi- (1995) involves loading of intact fi broblasts with deu- dized by respiratory chain complexes I and II, terated fatty acids, notable deuterated palmitic acid, respectively, and electrons are transferred via coenzyme followed by acylcarnitine analysis of the suspending Q, complexes III, cytochrome c, and IV to molecular medium (Ventura et al. 1999). If one of the two differ- oxygen. The electron transfer is accompanied by proton ent tests is abnormal, additional enzymatic analysis translocation by respiratory chain complexes I, III, and must be done to identify the underlying defect. In the IV. In this way, a mitochondrial inner-membrane poten- Amsterdam laboratory, we have methods available for tial is maintained, which provides the energy for com- the measurement of all individual enzyme activities plex V to convert ADP into the fi nal product ATP. The and also molecular analysis of all the different genes fi ve enzyme complexes of the OXPHOS system are involved has been established in our laboratory. multi-subunit assemblies of proteins, iron–sulfur clusters, fl avins, cupper ions, and heme groups. The largest OXPHOS enzyme is complex I, consisting of 45 different subunits, of which 7 are encoded by the mtDNA and D3.4 Mitochondrial Disorders 38 by the nuclear genome. There is evidence for a higher degree of organization of respiratory chain enzymes into supercomplexes, containing different D3.4.1 Background combinations of respiratory chain enzyme complexes. Some of these can function as respirasomes with

Mitochondrial disorders can be defi ned as disorders NADH:O2 oxidoreductase activity (Acin-Perez et al. caused by a defect in the mitochondrial energy generat- 2008). It has been suggested that supercomplexes can ing system. In “classical” mitochondrial disorders, the form even larger structures, the so-called respiratory primary defect leading to a reduced mitochondrial strings (Wittig et al. 2006), although this remains to be energy generating capacity is located in the OXPHOS established. system, the citric acid cycle, or pyruvate dehydroge- The clinical spectrum of mitochondrial disorders is nase. In addition, there is a growing number of mito- very broad, and moreover, the genotype–phenotype chondrial disorders in which the reduced mitochondrial correlation of mitochondrial disorders can be very energy generating capacity is caused by other defects, poor. It seems likely that the complexity of the mito- for example, in mitochondrial metabolite transporters chondrial energy generating system, as described ear- (Mayr et al. 2007; Molinari et al. 2005). A major sub- lier, is one of the reasons for this. There are hundreds strate for mitochondrial energy production is pyruvate, of proteins involved, and it can be envisioned that the D3 Enzymes and Inherited Metabolic Diseases 293 effect of a mutation in one gene can be modulated, or presence of carnitine is indicative of an OXPHOS enhanced, by polymorphisms in genes encoding other defect (Janssen et al. 2006). Also defects in the citric proteins involved in the mitochondrial energy generat- acid cycle and in a number of mitochondrial carriers ing system. For mtDNA mutations, evidence has been can be detected in this way (Mayr et al. 2007). In addi- found for a role of the mtDNA haplotype in the patho- tion to the analysis of fl uxes, individual enzymes can genicity of mtDNA mutations (Pulkes et al. 2000). be examined. Often muscle is the tissue of choice, but Striking examples of clinical heterogeneity due to in certain cases a liver or heart biopsy should be con- mutations in a single gene are disorders caused by sidered. All of the enzymes mentioned earlier can be mutations in POLG1, a gene encoding a subunit of the determined by spectrophotometric assays. Although mtDNA polymerase. Originally described as a gene for pyruvate dehydrogenase spectrophotometric analy- involved in progressive external ophthalmoplegia, and sis is possible, a more sensitive radiochemical assay later in Alpers syndrome, which seem quite different with 14C-labeled pyruvate is the preferred method of clinical phenotypes, it is now recognized that the phe- enzymatic analysis. notype associated with POLG1 mutations is extremely diverse (Chinnery and Zeviani 2008). Remember Assays of individual enzyme activities are available Remember for pyruvate dehydrogenase, citric acid cycle It is worthwhile to include POLG1 mutation analy- enzymes, respiratory chain enzymes, and complex V. sis in an early stage of the diagnostic work-up of They can be performed both in freshly obtained tis- mitochondrial patients. sue samples but also in frozen tissue samples, cultured cells, and blood cells. Given the tissue specifi c character of part of the mitochondrial disorder spectrum, it is very important to examine enzyme activi- D3.4.2 Laboratory Diagnosis ties in clinically affected tissues. of Mitochondrial Disorders

Given the complexity of the system, reaching a diag- Once fl ux as well as individual enzyme assays have nosis is not always straightforward and often an in been performed in a patient’s muscle biopsy, there are depth evaluation of clinical, biochemical, and genetic a number of possible outcomes: (1) all parameters are information is necessary. Biochemical diagnostics normal, (2) mitochondrial energy generating system usually involves a broad screening of mitochondrial capacity is reduced and one or more enzymes are defi - enzyme activities and analysis of fl uxes through the cient, and (3) mitochondrial energy generating system citric acid cycle and OXPHOS system. The analysis of capacity is reduced, but no enzyme defi ciency is fl uxes can only be performed in freshly obtained tissue observed. In case all parameters are normal, a mito- samples (usually muscle), although assays for cultured chondrial disorder is unlikely, especially if both fi broblasts have also been described. There are three enzyme activities and fl uxes through the mitochondrial types of assays to determine fl uxes through the mito- energy generating system have been examined in the chondrial energy generating system, namely the analy- affected tissue. However, if this is not the case, e.g., in sis of the (1) ATP production rate, (2) substrate patients with central nervous system impairment, a oxidation rate, and (3) oxygen consumption rate. A mitochondrial disorder cannot be completely excluded. reduced rate of any of these parameters can be indica- In those cases, additional diagnostic tests may still be tive of a defect in mitochondrial energy generating worthwhile, such as examination of the mtDNA, as tis- system. Moreover, by using different combinations of sue specifi c expression of the disease may be due to substrates, indications for the localization of the defect differences in heteroplasmy levels in different tissues. within the system can be obtained. For example, a When only the mitochondrial energy generating sys- reduced oxidation rate of pyruvate in the presence of tem capacity is reduced, there are several possible malate, and a normal oxidation rate of pyruvate in the explanations. First, it could be a primary mitochondrial 294 R. J. A. Wanders et al. dysfunctioning due to a defect in a mitochondrial pro- Remember tein/enzyme that was not examined. Examples of previously unidentifi ed mitochondrial protein defects Once a mutation has been found, it is important to other than the classical mitochondrial defects are sub- verify the functional implications of the presumed strate carriers such as the glutamate, phosphate, or genetic defect in functional assays. pyruvate carrier (Brivet et al. 2003; Mayr et al. 2007; Molinari et al. 2005). In these cases, detailed analysis in muscle and in cultured cells provided important biochemical clues for the identifi cation of the primary D3.5 Lysosomal Storage Disorders defect. Second, as it has been shown that assays of the capacity of the mitochondrial energy generating system are more sensitive to detect certain mitochondrial D3.5.1 Background defects, e.g., mutations in the mtDNA (Janssen et al. 2008), in such a constellation sequence analysis of the Lysosomes play a very important role in the turnover mtDNA should always be considered. Third, it could of the cell. Turnover is the constant state of break- be a refl ection of secondary mitochondrial dysfunc- down and resynthesis of macromolecules leading to tioning, e.g., due to a muscle dystrophy, a very poor renewal of these molecules, while their concentration clinical status of the patient, or other reasons. remains constant. The main function of lysosomes is In case of a reduced capacity of the mitochondrial the breakdown of complex macromolecules by lyso- energy generating system and a defi ciency of one or somal enzymes to simple monomers and the transport more enzymes, several possibilities for subsequent of these monomers by specifi c membrane transporters diagnostic analysis are possible. In many cases, bio- out of the lysosomal compartment into the cytosol, chemical analysis of fi broblasts is useful. Both positive where they can be reutilized for biosynthesis. This and negative results are informative. In case of normal function is achieved by the presence of a large set of enzyme activities in fi broblasts, in particular in cases hydrolytic enzymes with an acidic pH optimum capa- with reduced activities of complex I, III, and/or IV in ble of hydrolyzing all cellular components including muscle, analysis of the mtDNA and of genes involved DNA, RNA, complex carbohydrates like glycolipids, in mtDNA maintenance are indicated. In case of clearly glycoproteins, and glycosaminoglycans as well as reduced activities in fi broblasts, mtDNA depletion is proteins, neutral lipids, and phospholipids. With few less likely, and defects in either mtDNA or in nuclear exceptions, the reactions catalyzed by lysosomal genes encoding either structural components of the hydrolases are irreversible. respiratory chain or assembly factors should be con- The pH within the lysosome is maintained around fi ve sidered. A BN-PAGE analysis can be informative as by an ATP-driven proton pump. Most lysosomal enzymes well, as certain defects give a characteristic pattern in are glycoproteins and are present in the matrix of the (2-dimensional) BN-PAGE. lysosome. They are synthesized as larger precursors (pre- As the number of genes in which defects have been proenzymes) and glycosylated during transport in the described is already approaching 100 and still increases endoplasmic reticulum and the Golgi apparatus. Some of each year, it is not possible to routinely screen all the the free mannose groups on the lysosomal precursor candidate genes with current technologies, although enzymes are phosphorylated to mannose-6-phosphate technical developments may allow for this in the near groups in the trans-Golgi, which are recognized by the future. Cell model systems, such as patient-derived mannose-6-phosphate receptors present in acidic endo- fi broblasts and myoblasts can be invaluable to estab- somes. These mannose-6-phosphate receptors are essen- lish the pathogenicity of newly identifi ed mutations, tial for the lysosomal targeting of matrix enzymes to the e.g., by viral complementation studies (Hoefs et al. lysosomes and are also present on the plasma membrane. 2008). Also at the fi nal stage of the diagnostic exami- After dissociation of the lysosomal precursor enzymes nation, functional assays, such as enzyme assays and from the mannose-6-phosphate receptor in the acidic assays for mitochondrial fl uxes, provide important environment, they fi nally reach the endosomal/lysosomal information to establish the diagnosis mitochondrial compartment (Kornfeld 1987). Once, they have reached disorder at the molecular genetic level. the endosomal/lysosomal compartments the precursor D3 Enzymes and Inherited Metabolic Diseases 295 enzymes may be further processed by proteolytic cleav- molecules, but are delivered to the lysosome by endo- age to smaller molecular weight mature enzymes. cytosis when their origin is outside the cell or phago- Lysosomal enzymes can be active as monomers, homodi- cytosis when they are of intracellular origin. They may mers (e.g., a-galactosidase and a-galactosaminidase and be taken up as single molecules or may be part of a b-hexosaminidase B), or heterodimers (b-hexosamini- larger molecular complex like a lipoprotein or even an dase A) and are mostly present as soluble enzymes. Like organelle or a microorganism. In some cases, the all other proteins lysosomal enzymes are also degraded uptake by endocytosis is a very effi cient process cata- in the lysosome and have a fi nite half-life. In most cell lyzed by the presence of a receptor such as the uptake types, the lysosomal enzymes appear to be constitutively of low-density lipoprotein (LDL), which is catalyzed expressed. by the presence of the LDL receptor. Also lysosomal Besides glycosylation, which is essential for the enzymes carrying a mannose-6-phosphate group can activity of all soluble lysosomal enzymes, another post- be very effi ciently taken up by most cell types because translational modifi cation is necessary for all sulfatases of the presence of a mannose-6-phosphate receptor on including the lysosomal sulfatases. This modifi cation the plasma membrane. involves the oxidation of an essential cysteine residue Lysosomal storage disorders are characterized by to a formylglycine group by the formylglycine generat- the presence of nondegraded material in endosomal/ ing enzyme, which is present in the endoplasmic reticu- lysosomal compartments. Any process that interferes lum (Dierks et al. 2005). There is no evidence that the with the lysosomal degradation or endosomal/lyso- activity of lysosomal enzymes is regulated by covalent somal transport of molecules can give rise to storage. modifi cations once they are in their mature form. The cause may be environmental – for example, in The enzymes b-galactosidase and a-neuraminidase drug-induced lipidoses or caused by the presence of are present in an enzyme complex that also contains a undegradable materials- or genetic in nature. Here, we third protein, the so-called “protective protein,” which will discuss the genetic lysosomal storage disorders. is actually an enzyme, cathepsin A, with proteolytic activity. The presence of this protein is essential for the stability of both b-galactosidase and neuraminidase. D3.5.2 Genetic Causes of Lysosomal In general, lysosomal enzymes have to work in con- cert in order to effi ciently catalyze the stepwise break- Storage Diseases down of macromolecules like glycosaminoglycans, glycoproteins, and glycolipids. However, with the D3.5.2.1 Single Enzyme Defi ciencies exception of the example discussed earlier there is no evidence that they are actually organized in enzyme The majority of the inherited lysosomal storage disor- complexes. ders are caused by mutations in the genes coding for a When the substrate is not water soluble as is the lysosomal enzyme (see Table D3.3). These mutations case with lipid molecules destined for degradation, may give rise to the complete absence or a severe which are present in internal membranes within the reduction in activity of the lysosomal enzyme. Since endosomal/lysosomal compartments, the lipid hydro- most lysosomal enzymes are exohydrolases the step- lyzing enzymes may need an activator protein for full wise breakdown of macromolecules stops at the step activity (Kolter and Sandhoff 2005). Examples are the which is normally catalyzed by the enzyme that is now

GM2-activator protein, which is specifi cally needed for defi cient. These defi ciencies give rise to the storage of the breakdown of GM2-ganglioside by b-hexosamini- specifi c macromolecules, which causes disease. The dase A and the saposins A–D. The latter activator pro- storage is a slow, but continuous process, which teins are proteolytic cleavage products of the precursor explains why lysosomal storage disorders are chronic, protein prosaposin and are needed for the hydrolysis of progressive diseases. Most lysosomal storage diseases complex glycolipids like sulfatide, globotriaosylcer- are autosomal recessive disorders with the exception amide, galactosylceramide, glucosylceramide, and of Fabry disease and mucopolysaccharidosis type II ceramide. (Hunter’s disease), which are X-linked. Since carriers Substrates for lysosomal hydrolysis usually do not are not affected, enzyme levels of 50% of normal are enter the lysosome by diffusion or transport of single suffi cient for normal lysosomal functioning. In fact, a 296 R. J. A. Wanders et al.

Table D3.3 Lysosomal storage disorders as classiﬁ ed in distinct subgroups Disease Eponyme OMIM Locus Gene Gene product Storage product Sphingolipidoses/lipidoses (single enzyme defects and activator protein deﬁ ciencies) Farber disease Lipogranulomatosis 228000 8p22 Acid ceramidase Cer ASAH Fabry disease Anderson–Fabry 301500 Xq22 a-Galactosidase A Gb3 GLA Gaucher disease Glucosylceramidosis 606463 1q21 b-Glucocerebrosidase GlcCer 230900 GBA 231000 230800

GM1-gangliosidosis 230500 3p21 b-Galactosidase GM1 230600 GLB1

GM2-gangliosidosis B Tay–Sachs 272800 15q23 b-Hexosaminidase a-subunit GM2 HEXA

GM2-gangliosidosis O Sandhoff 268800 5q13 b-Hexosaminidase b-subunit GM2 HEXB

GM2 gangliosidosis AB Tay–Sachs AB variant 272750 5q32 GM2A GM2 activator protein GM2 Krabbe disease Globoid cell 245200 14q31 b-Galactocerebrosidase GalCer leukodystrophy GALC Metachromatic Arylsulfatase A 250100 22q13 Arylsulfatase A Sulfatide leukodystrophy defi ciency ARSA Niemann–Pick types A and B 257200 11p15 Acid sphingomyelinase SM 607616 SPMPD1 Wolman disease Cholesteryl ester 278000 10q23.2 Acid lipase Cholesterolester, storage disease LIPA triglycerides Prosaposin defi ciency 176801 10q22 Prosaposin Multiple lipids PSAP Saposin B defi ciency Metachromatic 249900 10q22 Saposin B Sulfatide leukodystrophy PSAP variant Saposin C defi ciency Gaucher variant 610539 10q22 Saposin C GlcCer PSAP Glycogen storage disease (single enzyme defi ciency) Pompe disease Glycogen storage 232300 17q25 a-Glucosidase Glycogen disease type II GAA Mucopolysaccharidoses (MPS, single enzyme defi ciencies) MPS I Hurler 607015 4p16 a-Iduronidase DS, HS Hurler/Scheie 607015 IDUA Scheie 607016 MPS II Hunter 309900 Xq28 Iduronate sulfatase DS, HS IDS MPS IIIA Sanfi lippo A 252900 17q25 Heparan N-sulfatase HS SGS MPS IIIB Sanfi lippo B 252910 17q21 N-acetyl-a-glucosaminidase HS NAGLU MPS IIIC Sanfi lippo C 252930 8p11 a-Glucosaminide-acetyl-CoA HS TMEM76 transferase HGSNAT MPS IIID Sanfi lippo D 252940 12q14 N-acetylglucosamine-6- HS GNS sulfatase D3 Enzymes and Inherited Metabolic Diseases 297

Table D3.3 (continued) Disease Eponyme OMIM Locus Gene Gene product Storage product

MPS IVA Morquio A 253000 16q24 N-acetylgalactos-amine-6- KS, CS GALNS sulfatase

MPS IVB Morquio B 253010 3p21 Acid b-galactosidase KS GLB1 MPS VI Maroteaux–Lamy 253200 5q12 Arylsulfatase B DS ARSB MPS VII Sly 253220 7q21 b-Glucuronidase DS, HS, CS GUSB MPS IX Hyaluronidase 601492 3p21 Hyaluronidase 1 HA defi ciency HYAL1 Glycoproteinoses (single enzyme defi ciencies) Aspartylglucosaminuria 208400 4q32 Aspartylglucosaminidase Aspartylglucosamine AGA Oligosaccharides Fucosidosis 230000 1p34 a-Fucosidase Oligosaccharides FUCA a-Mannosidosis 248500 19q12 a-Mannosidase Oligosaccharides MAN2B1 b-Mannosidosis 248510 4q22 b-Mannosidase Oligosaccharides MANBA Sialidosis Sialidase defi ciency 256550 6p21 Sialidase, Oligosaccharides Mucolipidosis type I NEU1 a-neuraminidase-1 N-acetyl-a-glucosaminidase Schindler disease 609241 22q13 N-acetyl-a-glucosaminidase Oligosaccharides defi ciency Kanzaki disease 609242 NAGA Multiple enzyme defi ciencies Mucolipidosis type II I-cell disease 252500 12q23 UDP-GlcNac phosphotrans- Lipids and GNPTAB ferase, a/b subunits oligosaccharides Mucolipidosis type IIIA Pseudo-Hurler 252600 12q23 UDP-GlcNac phosphotrans- See above polydystrophy GNPTAB ferase, a/b subunits Mucolipidosis type IIIB Pseudo-Hurler 352605 16p UDP-GlcNac phosphotrans- See above polydystrophy GNPTG ferase, g-subunit Multiple sulfatase defi ciency Austin disease 272200 3p26 Formyl-glycine generating Sulfatide, SUMF1 enzyme mucopolysaccharides Galactosialidosis 256540 20q13 Protective protein, cathepsin Glycosphingolipids, PPCA A oligosaccharides Lysosomal transport defects Cystinosis 219800 17p13 Cystinosin Cystine 219900 CTNS 219750 Sialic acid storage disease Salla disease 604322 6q14 Sialin Sialic acid SLC17A5

Methylmalonic aciduria Vitamin B12 lysosomal 277380 Unknown Vitamin B12 carrier (CbIF) Vitamin B12 release defect Lysosomal/endosomal trafﬁ cking defects, defects in fusion/ﬁ ssion of vesicles Niemann–Pick type C1 257220 18q11 NPC1 Cholesterol, phospho- NPC1 lipids, glycosphingolipids (continued) 298 R. J. A. Wanders et al.

Table D3.3 (continued) Disease Eponyme OMIM Locus Gene Gene product Storage product

Niemann–Pick type C2 607625 14q24 NPC2 Cholesterol, phospholip- NPC2 ids, glycosphingolipids Mucolipidosis type IV 252650 19p13 Mucolipin-1 Phospholipids, MCOLN1 glycosphingolipids, mucopolysaccharides Danon disease Pseudoglycogenosis II 300257 Xq24 LAMP2 Glycogen LAMP2 Neuronal ceroid lipofuscinoses (NCL) (single enzyme deﬁ ciencies and other defects) CLN1 Haltia–Santavuori, 256730 1p32 PPT1, palmitoyl protein SAPs infantile NCL CLN1 thioesterase 1 CLN2 Janský–Bielschowsky, 204500 11p15 TPP1, tripeptidyl peptidase SCMAS late-infantile NCL CLN2 I CLN3 Spielmeyer–Sjögren 204200 16p12 CLN3 SCMAS Batten, juvenile NCL CLN3 CLN4A Kufs 204300 Unknown Unknown SCMAS CLN4B Parry disease 162350 Unknown Unknown SAPs CLN5 Variant Finnish 256731 13q22 CLN5 SCMAS late-infantile NCL CLN5 CLN6 Variant Indian or 601780 15q23 CLN6 SCMAS Czech late CLN6 infantile CLN7 Variant Turkish late 610951 Unknown Unknown SCMAS infantile CLN8 Northern epilepsy 600143 8p32 CLN8 SCMAS CLN8 CLN9 Variant Batten disease 609055 Unknown Regulator of dihydrocer- SCMAS amide synthase CLN10 Congenital NCL 610127 11p15 Cathepsin D SAPs CTSD Cer ceramide; CS chondroitin sulfate; DS dermatan sulfate; Gb3 globotriaosylceramide, gangliosides GM1 and GM2; GlcCer glucosylceramide; HA hyaluronan; HS heparansulfate; KS keratansulfate; SAPs sphingolipid activator proteins; SCMAS subunit c mitochondrial ATP synthase; SM sphingomyelin

reduction of enzyme activity to <10% of normal is enzyme activity and the steady-state concentration and usually necessary to cause disease. turnover rates of its substrate was derived and a good A wide spectrum of disease expression is usually found correlation was found in patients with severe and and it is customary to classify the individual lysosomal attenuated phenotypes of GM2-gangliosidosis and storage disorders according to age of onset in a (late) metachromatic leukodystrophy between the residual infantile, juvenile, and adult form. In practice, there is enzyme activities and the clinical phenotype often a continuum of clinical phenotypes varying from (Leinekugel et al. 1992). very severe to relatively mild. In several lysosomal dis- Although lysosomal enzymes are present in all cell orders, the residual activity expressed by the mutant types except red blood cells, cells and tissues may be enzyme correlates with the severity of the disease differentially affected depending on the enzyme defi - (phenotype). Examples are metachromatic leu- ciency involved. It is the fl ux of substrate through the kodystrophy caused by the defi ciency of the lysosomal endosomal/lysosomal system that determines which enzyme arylsulfatase A, and GM2-gangliosidosis type cells and tissues will be affected and the clinical phe- Tay–Sachs caused by a defi ciency of b-hexosamini- notype that emerges. Thus, it can be understood that dase A. A theoretical correlation between the residual the defi ciency of a-glucosidase in Pompe disease will D3 Enzymes and Inherited Metabolic Diseases 299 primarily affect the skeletal muscle, the heart, and the A defect in the formylglycine generating enzyme liver, which are most active in glycogen metabolism. leads to a disease called multiple sulfatase defi ciency in Similarly, the defect in the turnover of sulfatide – an which all sulfatases including at least seven lysosomal important component of myelin – due to the defi ciency sulfatases are defi cient. In its most severe form the dis- of arylsulfatase A will affect normal myelin function ease has Niemann–Pick features of a mucopolysaccha- and white matter disease in metachromatic leukodystro- ridosis, metachromatic leukodystrophy, and ichtyosis phy, and a defect in the turnover of gangliosides due to (due to steroid sulfatase defi ciency), but the clinical defi ciencies in b-hexosaminidase A and GM2- and presentation is very variable. A defect in the protective

GM1-gangliosidosis will lead to severe neuronal dys- protein/cathepsin A leads to a deﬁ ciency of both function. In reality, the pathophysiology is usually b-galactosidase and a-neuraminidase and the lyso- more complex, but these are important considerations. somal storage disorder galactosialidosis.

D3.5.2.2 Activator Protein Deﬁ ciencies D3.5.2.4 Lysosomal/Endosomal Trafﬁ cking (see Table D3.3) Defects (see Table D3.3)

Mutations in the genes encoding for the activator pro- Lysosomal storage diseases can also be caused by teins give rise to a functional defi ciency of glycolipid abnormalities in lysosomal/endosomal traffi cking or hydrolases, while the enzymes themselves are nor- vesicle fusion (Walkley 2001). mally present. The phenotypes are similar to the phe- Niemann–Pick type C is caused by two different notypes caused by the enzyme defi ciencies. Examples gene defects NPC1 and NPC2. NPC1 is a membrane are metachromatic leukodystrophy caused by saposin protein with 13 transmembrane domains and a choles- B defi ciency, Gaucher disease caused by saposin C terol sensing domain and is present in late endosomes, defi ciency, and GM2-gangliosidosis caused by GM2- whereas NPC2 is a soluble lysosomal protein that is tar- activator protein defi ciency. geted to lysosomes by the mannose-6-phosphate receptor pathway. Despite numerous studies, the function of NPC1 is still poorly understood. It may function as a D3.5.2.3 Multiple Enzyme Defi ciencies transmembrane effl ux pump. NPC2 serves probably as (see Table D3.3) a lysosomal lipid transfer protein with a high specifi city for cholesterol. Both defects give rise to a similar clini- A defi ciency of the enzyme UDP-N-acetylglucosamine: cal phenotype. Biochemically, both defects lead to a lysosomal enzyme N-Acetylglucosamine-1-phospho perturbed fl ow of substrate (primarily lipids) through transferase leads to mistargeting of lysosomal enzymes, the endosomal/lysosomal pathway and to a complex which cannot be delivered to lysosomes, but are instead pattern of lipid storage consisting of cholesterol, gly- excreted into the plasma because they lack the man- cosphingolipids, sphingoid bases, and phospholipids. nose-6-phosphate targeting signal. Interestingly, Mucolipidosis IV is a lysosomal storage disease with a although the phosphotransferase defi ciency is present heterogeneous storage pattern consisting of phospho- in all cells only cells of mesenchymal origin appear to lipids, glycosphingolipids, and mucopolysaccharides. be affected, suggesting that alternative routes of deliv- The disease is caused by a mutation in the MCOLN1 ering lysosomal enzymes to lysosomes must exist in gene encoding the mucolipin1 protein, a lysosomal other cell types. The phosphotransferase enzyme con- membrane protein with six transmembrane domains. sists of three subunits (a2b2g2) encoded by two genes, There is evidence that this protein functions as a cation one for the a and b subunits and the other one for the g channel. This defect may lead to a reduced capacity in subunit. the fusion and fi ssion of lysosomal membranes with Mutations in the gene for the a and b subunits give endosomal and plasma membranes. rise to mucolipidosis II (I-cell disease) and its attenu- Danon disease is an X-linked glycogen storage dis- ated form mucolipidosis III (pseudo-Hurler polydystro- order caused by a defect in the endosomal/lysosomal phy), while mutations in the g subunit only lead to membrane protein LAMP-2. There is a failure of mucolipidosis III. autophagic vacuoles to fuse with endosomes/lysosomes 300 R. J. A. Wanders et al. or of autophagolysosomes to mature into digestive true for the analysis of oligosaccharides, which is usu- organelles. ally only qualitative. Each lysosomal defect in the degradation of oligosaccharides shows a specifi c pattern of oligosaccharides in urine (see Table D3.3). D3.5.2.5 Transport Defects (see Table D3.3) Glycosphingolipid analyses in plasma and urine can be performed to obtain evidence for a sphingolipido- Lysosomal storage diseases due to a defect in the trans- sis. In the case of the sphingolipidoses, enzymatic tests port of small molecules out of the lysosome are cysti- are usually performed fi rst based on the clinical infor- nosis caused by a defect in the cystine transport protein mation. The analysis of the specifi c storage product in cystinosin, and sialic acid storage disease is due to a plasma or urine serves to confi rm the diagnosis and to defect in the lysosomal sialic acid transporter sialin. exclude pseudodefi ciencies, for example, in metachromatic leukodystrophy or in cases with normal results D3.5.2.6 Laboratory Diagnosis of the enzyme determination and strong clinical suspicion for Gaucher disease, metachromatic leukodysto- The technical aspects of the laboratory diagnosis of phy, or Krabbe disease to obtain evidence for an lysosomal storage disorders have been reviewed in activator protein defi ciency (see Table D3.3). detail recently and for the practical details the reader is referred to these reviews (Lukacs 2008; Poorthuis and Aerts 2008; Sewell 2008;Verheijen 2008). D3.5.3 Enzyme Analysis The laboratory diagnosis of lysosomal storage diseases consists of four different approaches, histology, metabolite analysis, enzymatic analysis, and mutation D3.5.3.1 Single Enzyme Defi ciencies analysis that are complementary to each other. (see Table D3.3)

Determination of the activity of lysosomal enzymes in D3.5.2.7 Histology leukocytes, plasma, or dried blood spots prepared from whole blood or in fi broblasts is the core in the labora- Lysosomal storage can be demonstrated by light or tory diagnosis of lysosomal storage disorders. Most electron microscopy in easily accessible cells like white lysosomal storage disorders due to single enzyme defi - blood cells or in skin biopsies (see Chap. D5). In some ciencies can be diagnosed reliably and defi nitively by diseases, biopsies are taken from affected organs such enzyme assays using artifi cial fl uorescent substrates. as bone marrow in Gaucher disease and a kidney biopsy Although the Km of the enzymes measured with these in Fabry disease to establish a diagnosis. However, it is substrates is usually much higher than with the natural recommended to perform enzyme analysis fi rst. substrate, the specifi city of the substrates is suffi ciently Abnormal fi ndings in the biopsy should always be con- high to allow reliable diagnostic performance. fi rmed by enzyme analysis. In some cases, histology is the fi rst choice to obtain evidence for a lysosomal storage disorder, for example, when mucolipidosis IV or a neuronal ceroid lipofuscinosis is suspected. D3.5.4 Multiple Enzyme Defi ciencies (see Table D3.3) D3.5.2.7 Metabolite Analysis Mucolipidoses II and III are diagnosed by the strongly Lysosomal storage can be demonstrated by analysis of increased levels of multiple lysosomal enzymes in plasma and urine for storage products such as glycosamin- plasma and their defi ciency in fi broblasts. Multiple sul- oglycans, oligosaccharides, and glycosphingolipids. The fatase defi ciency is diagnosed by the defi ciency of sev- combined quanlitative and quantitative analyses of gly- eral lysosomal sulfatases in leukocytes and/or fi broblasts. cosaminoglycans gives important information on the type Galactosialidosis is diagnosed by the defi ciency of both of mucopolysaccharidosis involved and limits the number b-galactosidase and a-neuraminidase in leukocytes of enzymes that need to be tested to defi nitively estab- and/or fi broblasts. Confi rmation can be obtained by lish the diagnosis (see Table D3.3). The same holds measuring cathepsin A in leukocytes or fi broblasts. D3 Enzymes and Inherited Metabolic Diseases 301

D3.5.5 Lysosomal Transport Defects discussed earlier), for carrier testing, which is not pos- (see Table D3.3) sible with certainty by enzyme analysis and – increasingly – in prenatal diagnosis, especially in cases where enzymatic analysis is diffi cult. Sometimes, there is a Cystinosis is diagnosed by measuring increased cys- relation between the genotype and the phenotype and tine levels in leukocytes or granulocytes. The sialic mutation analysis can help in making a prognosis about acid storage disorders are diagnosed by the increased the course of the disease. excretion of sialic acid in urine or increased levels of sialic acid in fi broblasts. A very rare defect in the release of Vitamin B from lysosomes gives rise to 12 Take Home Messages methylmalonic aciduria. The initial diagnosis can be established by organic acid analysis in urine. › Enzymes are the key players of metabolism, converting a specifi c substrate into a specifi c product, a process called catalysis. D3.5.3.2 Lysosomal/Endosomal Traffi cking › The fl ux through a metabolic pathway under Defects (see Table D3.3) certain conditions is determined by the con- certed action of all enzymes in the pathway, Niemann–Pick type C disease is diagnosed by show- although the extent to which each particular ing cholesterol storage in fi broblasts followed by muta- enzyme contributes to the control of fl ux may tion analysis to discriminate between NPC1 and NPC2. vary widely as defi ned by each enzyme’s fl ux Mutation analysis has superseded the laborious bio- control coeffi cient. chemical test used to show decreased cholesterol ester- › Most peroxisomal disorders can be screened ifi cation in fi broblasts. Mucolipidosis IV may be for by analysis in plasma (very long-chain diagnosed by a combination of electron microscopy of fatty acids) and erythrocytes (plasmalogens) the skin and mutation analysis. Danon disease may be but subsequent detailed enzymatic and molec- diagnosed by electron microscopy cardiac biopsies ular studies in fi broblasts are necessary to pin- and mutation analysis. point the true underlying enzymatic and subsequent genetic defect. D3.5.3.3 Neuronal Ceroid Lipofuscinosis › Disorders of mitochondria energy production represent a group of clinically and genetically Three of the neuronal ceroid lipofuscinosis (CLN1, diverse diseases caused by mutations in either CLN2, and CLN10, see Table D3.3) are single lyso- mitochondrial or nuclear DNA. Since metabo- somal enzyme defects and can be diagnosed by lyso- lite abnormalities, including lactate may vary somal enzyme assays with artifi cial fl uorescent from (grossly) elevated to normal, correct substrates. For the remaining NCLs, a combination of diagnosis of patients may be problematic electron microscopy of blood cells or skin biopsy and which requires a multiple approach, including mutation analysis is recommended. measurement of enzyme activities in clinically affected tissues. › Acylcarnitine analysis is a powerful fi rst-line test in patients suspected to suffer from a FAO D3.5.6 Mutation Analysis disorder, subsequently followed by enzymatic analysis, preferably lymphocytes, followed by The molecular basis of most of the lysosomal storage molecular analysis in case the enzyme defect diseases is known (see Table D3.3) and many muta- has been established. tions have been characterized. However, with few › The inherited lysosomal storage disorders are exceptions mutation analysis is rarely used as the initial a diverse group of diseases whose correct diag- step in the diagnostic work-up of patients suspected of nosis involves a multiple approach involving suffering from a lysosomal storage disorder. Rather histology, metabolite analysis, enzyme activity mutation analysis is complementary to enzymatic anal- measurements and molecular analysis. ysis. It is employed when enzyme determinations cannot be used to reach a diagnosis (see the examples 302 R. J. A. Wanders et al.

Key References Janssen AJ, Schuelke M, Smeitink JAM et al (2008) Muscle 3243A–>G mutation load and capacity of the mitochondrial energy-generating system. Ann Neurol 63:473–481 Acin-Perez R, Fernandez-Silva P, Peleato ML et al (2008) Janssen AJ, Trijbels FJ, Sengers RC et al (2006) Measurement Respiratory active mitochondrial supercomplexes. Mol Cell of the energy-generating capacity of human muscle mito- 32:529–539 chondria: diagnostic procedure and application to human Bezman L, Moser AB, Raymond GV et al (2001) Adre- pathology. Clin Chem 52:860–871 noleukodystrophy: incidence, new mutation rate, and results Kacser H, Burns JA (1979) Molecular democracy: who shares of extended family screening. Ann Neurol 49:512–517 the controls? Biochem Soc Trans 7:1149–1160 Brivet M, Garcia-Cazorla A, Lyonnet S et al (2003) Impaired Kacser H, Burns JA (1981) The molecular basis of dominance. mitochondrial pyruvate importation in a patient and a fetus Genetics 97:639–666 at risk. Mol Genet Metab 78:186–192 Kolter T, Sandhoff K (2005) Principles of lysosomal membrane Chinnery PF, Zeviani M (2008) Polymerase gamma and disor- digestion: stimulation of sphingolipid degradation by sphin- ders of mitochondrial DNA synthesis. Neuromuscul Disord golipid activator proteins and anionic lysosomal lipids. Annu 18:259–267 Rev Cell Dev Biol 21:81–103 Danpure CJ, Purdue PE, Fryer P et al (1993) Enzymological and Kornfeld S (1987) Traffi cking of lysosomal enzymes. FASEB J mutational analysis of a complex primary hyperoxaluria type 1:462–468 1 phenotype involving alanine:glyoxylate aminotransferase Leinekugel P, Michel S, Conzelmann E, Sandhoff K (1992) peroxisome-to-mitochondrion mistargeting and intraperoxi- Quantitative correlation between the residual activity of beta- somal aggregation. Am J Hum Genet 53:417–432 hexosaminidase A and arylsulfatase A and the severity of the Dierks T, Dickmanns A, Preusser-Kunze A et al (2005) Molecular resulting lysosomal storage disease. Hum Genet 88:513–523 basis for multiple sulfatase defi ciency and mechanism for Lukacs Z (2008) Mucopolysaccharides. In: Blau N, Duran M, formylglycine generation of the human formylglycine-gen- Gibson KM (eds) Laboratory guide to the methods in bio- erating enzyme. Cell 121:541–552 chemical genetics. Springer, Berlin Eaton S (2002) Control of mitochondrial beta-oxidation fl ux. Mayr JA, Merkel O, Kohlwein SD et al (2007) Mitochondrial Prog Lipid Res 41:197–239 phosphate-carrier defi ciency: a novel disorder of oxidative Ebberink MS, Mooyer PA, Koster J et al (2009) Genotype- phosphorylation. Am J Hum Genet 80:478–484 phenotype correlation in PEX5-defi cient peroxisome bio- Molinari F, Raas-Rothschild A, Rio M et al (2005) Impaired genesis defective cell lines. Hum Mutat 30:93–98 mitochondrial glutamate transport in autosomal recessive- Ferdinandusse S, Denis S, Hogenhout EM et al (2007) Clinical, neonatal myoclonic epilepsy. Am J Hum Genet 76:334–339 biochemical, and mutational spectrum of peroxisomal acyl- Motley AM, Brites P, Gerez L et al (2002) Mutational spectrum coenzyme A oxidase defi ciency. Hum Mutat 28:904–912 in the PEX7 gene and functional analysis of mutant alleles in Ferdinandusse S, Denis S, Mooyer PA et al (2006a) Clinical and 78 patients with rhizomelic chondrodysplasia punctata biochemical spectrum of D-bifunctional protein defi ciency. type 1. Am J Hum Genet 70:612–624 Ann Neurol 59:92–104 Nada MA, Rhead WJ, Sprecher H Clark S (1995) Evidence for Ferdinandusse S, Kostopoulos P, Denis S et al (2006b) Mutations intermediate channeling in mitochondrial beta-oxidation. in the gene encoding peroxisomal sterol carrier protein X J Biol Chem 270:530–535 (SCPx) cause leukencephalopathy with dystonia and motor Olpin SE, Manning NJ, Pollitt RJ, Clarke S (1997) Improved detec- neuropathy. Am J Hum Genet 78:1046–1052 tion of long-chain fatty acid oxidation defects in intact cells Groen AK, Van der Meer R, Westerhoff HV et al (1982b) using [9,10-3H]oleic acid. J Inherit Metab Dis 20:415–419 Control of metabolic fl uxes. In: Sies H (ed) Metabolic com- Platta HW, Erdmann R (2007) The peroxisomal protein import partmentation. Academic Press, New York, London machinery. FEBS Lett 581:2811–2819 Groen AK, van Roermund CWT, Vervoorn RC, Tager JM (1986) Poorthuis BJHM, Aerts JMFG (2008) Glycosphingolipids. In: Control of gluconeogenesis in rat liver cells. Flux control coef- Blau N, Duran M, Gibson MK (eds) Laboratory guide to the fi cients of the enzymes in the gluconeogenic pathway in the methods in biochemical genetics. Springer, Berlin absence and presence of glucagon. Biochem J 237:379–389 Pulkes T, Sweeney MG, Hanna MG (2000) Increased risk of Groen AK, Wanders RJA, Westerhoff HV et al (1982a) Quan- stroke in patients with the A12308G polymorphism in mito- tifi cation of the contribution of various steps to the control of chondria. Lancet 356:2068–2069 mitochondrial respiration. J Biol Chem 257:2754–2757 Rinaldo P, Matern D, Bennett MJ (2002) Fatty acid oxidation Heinrich R, Rapoport SM, Rapoport TA (1977) Metabolic regu- disorders. Annu Rev Physiol 64:477–502 lation and mathematical models. Prog Biophys Mol Biol Sewell A (2008) Oligosaccharides. In: Blau N (ed) Laboratory 32:1–82 guide to the methods in biochemical genetics. Springer, Higgins JJ (1965) Metabolic fl ux analysis. In: Chance B, Berlin Estabrook RW, Williamson JR (eds) Control of energy van den Brink DM, Brites P, Haasjes J et al (2003) Identifi cation metabolism. Academic Press, New York, London of PEX7 as the second gene involved in Refsum disease. Am Hoefs SJ, Dieteren CE, Distelmaier F et al (2008) NDUFA2 J Hum Genet 72:471–477 complex I mutation leads to Leigh disease. Am J Hum Genet van Woerden CS, Groothoff JW, Wijburg FA et al (2004) Clinical 82:1306–1315 implications of mutation analysis in primary hyperoxaluria Horn MA, van den Brink DM, Wanders RJA et al (2007) type 1. Kidney Int 66:746–752 Phenotype of adult Refsum disease due to a defect in per- Ventura FV, Costa CG, Struys EA et al (1999) Quantitative acyl- oxin 7. Neurology 68:698–700 carnitine profi ling in fi broblasts using [U-13C] palmitic D3 Enzymes and Inherited Metabolic Diseases 303

acid: an improved tool for the diagnosis of fatty acid oxida- Wanders RJA, van Roermund CWT, Meijer AJ (1984) Analysis tion defects. Clin Chim Acta 281:1–17 of the control of citrulline synthesis in isolated rat-liver Verheijen FW (2008) Sialic acid. In: Blau N, Duran M, Gibson mitochondria. Eur J Biochem 142:247–254 KM (eds) Laboratory guide to the methods in biochemical Wanders RJA, Waterham HR (2006) Biochemistry of mamma- genetics. Springer, Berlin lian peroxisomes revisited. Annu Rev Biochem 75: Vockley J (2008) Metabolism as a complex genetic trait, a sys- 295–332 tems biology approach: implications for inborn errors of Weller S, Gould SJ, Valle D (2003) Peroxisome biogenesis dis- metabolism and clinical diseases. J Inherit Metab Dis orders. Annu Rev Genomics Hum Genet 4:165–211 31:619–629 Wittig I, Carrozzo R, Santorelli FM, Schagger (2006) Supercomplexes Walkley SU (2001) New proteins from old diseases provide and subcomplexes of mitochondrial oxidative phosphorylation. novel insights in cell biology. Curr Opin Neurol 14:805–810 Biochim Biophys Acta 1757:1066–1072 Wanders RJA (2004) Metabolic and molecular basis of peroxisomal disorders: a review. Am J Med Genet 126A: 355–375 DNA Studies D4 Johannes Zschocke

Key Facts The last two decades have seen remarkable progress in the molecular understanding of inborn errors of › Mutation studies have become an important metabolism. The localization and structure of most component in the diagnostic work-up of genes involved in monogenic metabolic disorders have patients, but their use should be balanced with been characterized, and information gained through other (phenotypic) diagnostic methods. the human genome project and reverse genetics has › Molecular analyses have a limited sensitivity allowed the detection or confi rmation of many “new” as some disease-causing mutations are usually metabolic disorders. The identifi cation of various missed by standard methods, and failure to disease-causing mutations in the individual conditions identify a diagnostic genotype may not neces- has not only greatly enhanced the diagnostic options sarily exclude a diagnosis. Sensitivity depends but also led to an improved understanding of molecular on both genetic characteristics and the method disease mechanisms and sometimes new therapeutic employed. options. › Identifi cation of disease-causing mutations Mutation analyses are now frequently used for clin- confi rms the diagnosis, may provide prognostic ical purposes including confi rmation of diagnosis and information, and allows simple testing of other prediction of disease severity. Nevertheless, there is family members including prenatal diagnosis. still uncertainty about how molecular analyses can › The functional impact of novel genetic vari- complement or replace other diagnostic methods. Many ants identifi ed in a patient should be assessed metabolic disorders are reliably diagnosed and con- with great care. They should be denoted fi rmed through biochemical and enzymatic investiga- “unclassifi ed variants” unless they are likely to tions rather than through mutation analyses. Molecular cause disease. analyses have a limited sensitivity as some disease- › Mutation analyses (like all specialized tests) are causing mutations are usually missed by standard prone to errors. Confi rmatory repeat analyses methods, and failure to identify a diagnostic genotype (either on a new sample or by analysis at a sec- may not necessarily exclude a diagnosis. Also, techni- ond independent laboratory) may be considered cal and interpretative diffi culties may be underesti- when the results of molecular studies are impor- mated both by clinicians and laboratories, and quality tant for patient management but do not seem to assessment schemes even for a common condition fi t the clinician’s assessment of the case. such as phenylketonuria have given rather disconcert- ing results. Nevertheless, there is an increasing number of disorders in which molecular studies are indicated at an early stage in the diagnostic process, usually because the disease is caused by prevalent mutations in particular populations or because invasive J. Zschocke procedures are necessary to obtain samples for specifi c Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria enzyme studies, e.g., liver biopsies. A collation of e-mail: [email protected] useful Internet resources that provide molecular or

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 305 DOI: 10.1007/978-3-540-74723-9_D4, © Springer-Verlag Berlin Heidelberg 2010 306 J. Zschocke metabolic information is given in Chap. E3. Laboratories blood is not available, other materials including dried that offer diagnostic mutation analyses may be found blood spots on fi lter paper cards, coagulated blood, hair through the databases GeneTests (www.genetests.org, roots, buccal swabs, or even serum, urine, or feces may mostly North America), Orphanet (www.orpha.net), be used for extraction of genomic DNA or for poly- and EDDNAL (http://www.eddnal.com, both mostly merase chain reaction (PCR)-amplifi cation of specifi c European). sequences, but these methods are less reliable and may be considerably more expensive. An alternative template for genetic analysis is Remember mRNA obtained from cells in which the target gene Mutation analyses in children may only be per- is expressed. As a single-stranded molecule, mRNA is formed if there is an important medical consequence quite unstable and in the laboratory is converted into in childhood. Carrier analyses in healthy siblings of double-stranded complementary DNA (cDNA) which children with metabolic disorders are not indicated can be stored indefi nitely. cDNA analysis has certain and should not usually be performed even when advantages over genomic DNA. It does not have an requested by the parents. intronÐexon structure but contains the uninterrupted sequence that is translated into protein. The detection of unknown mutations may be more convenient from D4.1 Samples cDNA since fewer fragments are required for PCR- based analysis methods. Splicing mutations that cause the removal of whole exons from the translated sequence are easily recognized, thus confi rming the Remember pathological relevance of DNA variants in the introns. On the other hand, splicing variants that are observed Sample for most applications under physiological or cell culture conditions are some- 5Ð10 ml EDTA full blood, shipped at ambient tem- times not likely to cause disease and may be diffi cult to perature. For long distances it may be preferable to interpret; RNA fragments that contain premature stop extract the DNA and send that. codons may be eliminated by nonsense-mediated decay, and the preparation of cDNA is more tedious than that of genomic DNA. While cDNA analysis may Mutation analysis can be performed in any human sam- be the method of choice in conjunction with enzyme ple that contains cellular nuclei. The exact sample studies if organ tissue such as liver or skin fi broblasts is depends on the type of DNA that needs to be investi- available for investigation, genomic DNA analysis gated. Routine diagnostic mutation analyses are usually remains the method of choice for most applications. performed in genomic DNA which is most conveniently extracted from 5 to 10 ml of anticoagulated full blood D4.2 Indications (usually EDTA blood). Smaller amounts of blood down to a few 100 ml may be acceptable but less satisfactory as DNA extraction from small samples is less reliable, Remember less DNA is available, and the extraction method may It is a long way from the genotype to the phenotype be more expensive (discuss with laboratory). The sam- in metabolic disorders, and a diagnosis may be ple should not be centrifuged but shipped as native made on all levels full blood by normal (overnight) mail at ambient temperature. DNA is quite stable, and the sample may be ¼ Genotype = genetic information, may or may stored at room temperature or in the refrigerator for 1Ð2 not determine disease manifestation. days if necessary (e.g., on weekends). Alternatively, ¼ Enzymatic phenotype = measurable protein whole blood may be stored frozen for several weeks or function, mostly independent from external fac- may be sent on dry ice; enquire with the molecular lab- tors but often restricted to specifi c organs. oratory whether frozen blood is accepted. If EDTA D4 DNA Studies 307

¼ Metabolic phenotype = concentration of meta- Remember bolites, e.g., in body fl uids, may vary consider- It is often diffi cult to predict the functional effects ably depending on the external factors including of mutations, and the impact of novel genetic vari- treatment. ants identifi ed in a patient should be discussed with ¼ Clinical phenotype = result of genetic and great care. external factors, key to diagnosis.

Biochemical and enzymatic analyses, when available, D4.3 Methods are often preferable for the diagnosis of metabolic disorders as they refl ect phenotypical parameters (“enzy- A wide range of molecular methods is available for matic” or “metabolic phenotype”) and are therefore the identifi cation of mutations and other DNA vari- closer to the patient’s clinical phenotype and disease ants, most of which are based on the PCR. Different expression. Nevertheless, there are various circum- methods are used to screen for specifi c known vari- stances in which molecular studies are cheaper, faster, ants or to examine the gene for unknown mutations. more convenient, or the only reliable method for diag- There is no uniform strategy that is suitable for all nosis or confi rmation of diagnosis of an inherited dis- applications, and a combination of methods is often order. Many metabolic defects involve enzymes that used. The exact approach depends on gene character- are only expressed in specifi c organs such as the liver istics, type and frequency of mutations, and the sensi- or the brain, necessitating invasive procedures (if at all tivity required to answer the clinical question. For possible) for enzymatic confi rmation. Other disorders adequate requesting of tests and interpretation of involving structural, receptor, or membrane proteins results, it is important that the clinician is familiar that do not cause metabolic alterations are not open for with the sensitivity, specifi city, and indication of the enzyme testing and therefore may be diffi cult to con- most frequently used mutation detection strategies. In fi rm by traditional methods. Molecular studies may be this chapter, we concentrate on PCR-based methods fast and effi cient and therefore the method of choice for the detection of point mutations or small deletions/ for the confi rmation of disorders that are caused by insertions as these are by far the most frequent causes single prevalent mutations in the patient’s population, of inborn errors of metabolism. e.g., the prevalent Caucasian medium-chain acyl-CoA There are several widely used mutation screening dehydrogenase mutation c.985A>G (p.K329E). The methods for the specifi c identifi cation of selected DNA identifi cation of well-characterized mutations may variants. Most methods are relatively inexpensive; com- provide information on disease severity, prognosis, or mercial kits that test for several common mutations are other clinical parameters in disorders with good geno- available for some disorders such as cystic fi brosis. typeÐphenotype correlations. Common genetic vari- However, the sensitivity of these mutation-specifi c screen- ants that potentially infl uence disease course or ing methods for the detection of relevant DNA changes is treatment may be easily detected through specifi c limited and will remain so by defi nition since rare and analyses, e.g., the factor-V-Leiden mutation or the new mutations are not identifi ed. Homozy gosity or com- methyltetrahydrofolate reductase variant p.A222V. pound heterozygosity of disease-causing mutations is Identifi cation of disease-causing mutations in a par- diagnostic in the investigated patients, but failure to iden- ticular patient allows rapid, cost-effi cient and reliable tify a mutation does not exclude the disorder. Screened testing of other family members including prenatal mutations are usually well characterized and their clinical diagnosis. In any case, laboratories that offer molecu- relevance is well known. Mutation-specifi c screening lar analyses for clinical purposes need to have good methods are especially useful for the detection of com- knowledge of the tested disorder, genotypeÐphenotype mon DNA polymorphisms that may infl uence multifacto- correlations, alternative diagnostic approaches, as well rial disorders such as atherosclerosis as well as for the as the sensitivity and specifi city of mutation analysis confi rmation of inherited disorders that are caused by spe- in the individual patient. cifi c prevalent mutations in a particular population. 308 J. Zschocke

DNA sequencing of the whole coding region of rele- D4.4 Pitfalls vant genes is more demanding with regard to both the detection and the interpretation of DNA variants but is now regarded as method of choice for the majority of Remember conditions. Current technology involves semi-automated DNA sequencers that detect fl uorescently labeled DNA Mutation analysis, like any laboratory technique, fragments with specifi c laser systems. In the standard has a certain error rate, which is diffi cult to elimi- approach, the genomic target region is fi rst amplifi ed by nate completely. PCR. Fluorescently labeled base-specifi c fragments are then generated through cycle sequencing with unidirec- tional primers and ddNTP nucleotides, either of which is Incorrect results or interpretations may be generated labeled with a fl uorescent marker dye. These fragments either through analytical or sampling errors, method- are then separated on conventional acrylamide gels or ological limitations, insuffi cient knowledge of the through capillary electrophoresis. There is as yet no types of mutations causing a particular disease, or 100% reliable system for the automatic evaluation of the inadequate consideration of family or population infor- DNA sequences generated in this fashion, and skilled mation. The following factors should be considered in interpretation of the results is required particularly for the choice of mutation analysis methods and the inter- the detection or exclusion of heterozygous mutations. It pretation of the results: is also important to note that direct sequencing of coding exons detects neither large genomic rearrangements ¥ What type of mutation usually causes the disease? (deletions, duplications) nor intronic mutations outside Different approaches need to be chosen for differ- splicing regions. ent mutations such as point mutations, trinucleotide Some laboratories continue to use mutation scan- repeats, or large deletions. ning methods for rapid, cost-effi cient mutation analy- ¥ Are there certain prevalent disease-causing muta- sis particularly in large genes. These methods identify tions in particular populations? Screening for such regions in which a mutation or other genetic variant is mutations may be a cost-effi cient method for con- located and which are subsequently sequenced for fi rming the disease or reducing the likelihood of its characterization of the exact base change. Scanning presence in a patient. It is essential to take the eth- methods considerably reduce sequencing load but are nic origin of a patient into consideration when such sometimes diffi cult to interpret and differ in their sen- an approach is chosen. sitivity, i.e., the reliability with which mutations are ¥ What is the sensitivity of comprehensive mutation found. Failure to detect mutations in a patient by one analysis with PCR-based methods such as DNA of the less sensitive scanning methods such as single- sequencing? For some disorders, this may approach strand confi guration polymorphism analysis does not 100%, while for others, only a proportion of muta- exclude the disorder in question, albeit making it more tions is recognized. PCR-based methods are usually unlikely. Other methods such as denaturing gradient restricted to coding exons and adjacent intron gel electrophoresis or denaturing high-performance sequences of the particular gene and may fail to detect, liquid chromatography, when well designed, have a e.g., large gene deletions spanning several exons. sensitivity that equals direct sequencing. ¥ Could there be more than one mutation on the same The capacity for mutation detection for common chromosomal strand? Double mutants have been conditions will be greatly expanded through the intro- identifi ed in many genes. It is not justifi ed to conduction of DNA arrays probably within the next clude that there is compound heterozygosity when decade. DNA arrays are already used for the exclusion two mutations are identifi ed in a patient with a reces- of candidate gene loci for autosomal recessive disor- sive disease. This constellation is only one of the ders in consanguineous families or families with sev- reasons why inheritance of mutations on separate eral affected siblings. In these circumstances, DNA chromosomes should be confi rmed in samples from arrays may also allow the identifi cation of novel dis- parents or other relatives. In addition, identifi cation ease genes on a research basis. of common missense mutations through selective D4 DNA Studies 309

screening may not be suffi cient for predicting the and accreditation for diagnostic molecular genetic ser- phenotype in a particular patient as additional muta- vices. Confi rmatory repeat analyses (either on a new tions affecting protein structure may be missed. sample or by analysis at a second independent labo- ¥ What is the diagnostic specifi city of mutation identi- ratory) may be considered when the results of molec- fi cation? Only a proportion of variants in a gene affect ular studies are important for patient management but protein function and cause disease. Criteria that may do not seem to fi t the clinician’s assessment of the case. be used to estimate pathogenetic relevance include Adequate interpretation of the results requires good type of mutation (missense or nonsense, predicted knowledge of the respective disease and the under- impact on amino acid sequence), extent of DNA ana- lying mutations that determine its pathogenicity. It is lyzed, segregation with the disease in a family, preva- prudent to request molecular diagnostic services only lence of the mutation in the general population, and from laboratories that are familiar with the respec- functional assessment through expression analysis. tive disorders and the underlying genotypeÐphenotype ¥ How important are nongenetic factors of pathogen- correlations. esis? Disease penetrance may vary considerably even within single families. Strict genotypeÐpheno- type correlations are observed only in a proportion of metabolic disorders, and the clinical picture in a Key References patient may be insuffi ciently explained by the mutations in a single gene. Zschocke J, Janssen B (2008) Molecular genetics: mutation analysis in the diagnosis of metabolic disorders. In: Blau Clinicians who request mutation analyses should treat N, Duran M, Gibson KM (eds) Laboratory guide to the the results with care, just like all other laboratory tests. methods in biochemical genetics. Springer, Heidelberg, Quality assessment schemes, available only for very pp 805Ð829 Zschocke J, Aulehla-Scholz C, Patton S (2008) Quality of diag- few conditions anyway, show that signifi cant errors nostic mutation analyses for phenylketonuria. J Inherit occur even in laboratories with good technical facilities Metab Dis 31:697Ð702 Pathology − Biopsy D5 Hans H. Goebel

Key Facts D5.1 General Remarks › Neurometabolic single organelle-multiorgan disorders, i.e., lysosomal, peroxisomal, polyg- Pathology, i.e., pathomorphology concerning individ- lucosan, and mitochondrial disorders warrant ual groups of metabolic diseases, as summarized in biopsies for morphological (and often bio- Chaps. A1–A3, may be divided into disease-specifi c or chemical by direct tissue and/or indirect fi bro- pathognomonic pathology. Recognition allows precise blast culture) studies. nosological diagnosis. Examples are lysosomal inclu- › Tissues suitable for biopsies are lymphocytes, sions in certain lysosomal diseases or group-specifi c skin, conjunctiva, skeletal muscle, rectum, and pathological features, e.g., lysosomal vacuoles in cer- liver. tain lysosomal diseases, or abnormally structured mitochondria in mitochondrial diseases. Lesions non- › Different neurometabolic disorders show dif- specifi c for any disease may be characteristic of a par- ferent patterns of morphological expression in ticular class of metabolic diseases, e.g., sponginess of different tissues. cerebral tissue in amino acid disorders. Secondary Brain and peripheral nerves need not be biopsied › pathological phenomena may also be encountered, for because any neurometabolic disease morpho- instance, reactive cellular and fi brillar astrocytosis or logically expressed in the nervous system may demyelination in the brain following loss of nerve and also be encountered in any of the other tissues. glial cells, and fi brosis in liver or heart following atro- Only rectum, among noncerebral tissues, may phy or loss of hepatocytes or cardiomyocytes. show exclusive lysosomal neuronal storage. Inherited metabolic diseases are defi ned by bio- Accessibility and morphological manifesta- › chemical and molecular criteria, while morphological tion determine the target of biopsy in an indi- investigations of tissues express pathomorphology vidually suspected neurometabolic disorder. (Table D5.1). While not always disease-specifi c, the Preferential sites of biopsy are lymphocytes in › pathological picture may pave the way for relevant vacuolar and certain nonvacuolar lysosomal biochemical and molecular studies. Postbiochemical diseases, skin in many lysosomal and Lafora and postmolecular morphological investigations may diseases; skeletal muscle in many lysosomal, confi rm fi ndings. polyglucosan, and mitochondrial diseases; and The methodological spectrum of the anatomic liver in peroxisomal and lysosomal diseases. pathologist, the neuropathologist, and the paidop- athologist may encompass histological studies with a wide range of structural and histochemical stains, enzyme histochemical preparations requiring unfi xed frozen tissue, of lysosomal, mitochondrial and peroxi- H. H. Goebel somal enzymes, and electron microscopy. When anti- Department of Neuropathology, Mainz University Medical Center, Langenbeckstrabe 1, 55131 Mainz, Germany bodies against enzyme substrates or enzyme proteins e-mail: [email protected] are available, immunohistochemistry is employed.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 311 DOI: 10.1007/978-3-540-74723-9_D5, © Springer-Verlag Berlin Heidelberg 2010 312 H. H. Goebel

Table D5.1 Neurometabolic diseases, morphologically expressed Table D5.2 Metabolic diseases with morphological pathology in various tissues in various tissues suitable for biopsy (single organelle-multi Frequently biopsied organ disorders) Lymphocytes Lysosomal diseases Skeletal muscle Lymphocytes Skin Bone marrow Infrequently biopsied Brain Bone marrow Liver Brain Rectum Conjunctiva Skeletal muscle Intestine Skin Kidney Mitochondrial diseases Liver Brain Lymph nodes Liver Peripheral nerves Skeletal muscle Tonsils Peroxisomal diseases Kidney Liver Adrenal glands These techniques require different approaches, imme- Peripheral nerves diately after having obtained the biopsied tissue, e.g., Polyglucosan diseases Brain fi xation in formalin for regular histological studies and Liver frozen unfi xed tissue for certain histochemical and Skeletal muscle enzyme histochemical methods. Immunohistochemical Skin preparations depend on the suitability of antibodies for fi xed, paraffi n-embedded or frozen tissues. Immediate proper fi xation, i.e., of small tissue fragments in the metabolic diseases are multi-organ diseases, and specifi c fi xative glutaraldehyde, is required for elec- respective pathology may be recognized in more than tron microscopy. one tissue or organ, allowing biospy of variously Biopsy obtains tissues of living patients, while available tissue, such as lymphocytes, skin, or con- autopsies may confi rm a diagnosis and document the junctiva (Table D5.2). distribution of the disease and the intensity of With the enormous expansion of biochemical and the pathology. Clinico-pathological and clinico- molecular assays in metabolic diseases, signifi cance of radiological correlations allow for tissue-specifi c bio- a diagnostic biopsy or morphological study has con- chemical studies, e.g., muscle tissue in postinfantile siderably decreased. The number of biopsies is shrink- type-II glycogenosis or in mitochondrial diseases. In ing as is knowledge of postsurgical morphological many fatal inherited metabolic diseases, the diagnosis fi ndings. Hence, earlier comprehensive reviews are has been established by the time of autopsy. important (Goebel 1997, 1999; Goebel and Jänisch Biopsies in patients with metabolic diseases are 1995; Goebel and Warlo 1990a, b). invasive, diagnostic surgical procedures. Skin, con- Biopsies of extracerebral tissues in patients with junctiva, rectum, and skeletal muscle may be investi- metabolic diseases while often pathognomonic may gated by open biopsy, allowing proper orientation and be considered optional, whereas an earlier category removal of tissue as well as tissue for different mor- of “essential” biopsies (Goebel 1999) has lost its phological preparations. Tissues of visceral organs value. Considering the diversity of tissues involved in and sometimes skeletal muscle may also be obtained metabolic diseases, each condition or group of condi- by needle biopsies. A limited number of lysosomal tions may require different approaches to different disorders may morphologically be recognized in tissues or possibly a “sequential” order, such as in the blood lymphocytes, requiring only venipuncture. It is neuronal ceroid-lipofuscinoses (NCL) commencing essential to know that the biopsied organ will contain with lymphocytes and, if unyielding or equivocal, pathology of the disease, which is suspected or proceeding to skin and other tissues such as muscle already ascertained by the clinician. Many inherited and rectum. D5 Pathology − Biopsy 313

Disease-specifi c morphological evaluation and lysosomal nature of vacuoles may be further confi rmed advice as to respective biopsy procedures will thereby enzyme histochemical demonstration of increased fore be provided according to tissues and organs. activity of acid phosphatase, the marker enzyme for lysosomes. In most lysosomal diseases vacuoles only appear in lymphocytes, but in certain mucopolysac- Remember charidoses there are granules in polymorphonuclear Biopsies are rewarding for morphological diagno- leukocytes known as Alder bodies. sis when disease-typical lesions are present in the At the electron microscopic level, non-vacuolar tissue. Metabolic single organelle-multi-organ dis- lysosomal residual bodies may be ascertained. orders are such conditions. Lesions may be identi- Although lymphocytes are present in the buffy coat, fi ed by electron microscopy. The tissue target of numerical predominance of granulocytes may impede biopsy is dictated by the morphological manifesta- careful electron microscopic studies of such a speci- tion of the disease and the accessibility of the men. Therefore, isolation of circulating lymphocytes, tissues. using the Ficoll technique, greatly facilitates ultrastructural investigation. For this reason, heparinised blood requires immediate isolation of the lymphocytes before fi xation as a pellet in buffered glutaral- D5.2 Circulating Blood Cells dehyde and, then, routine embedding in resin for regular electron microscopic workup. Only at the In certain metabolic disorders, particularly lysosomal electron microscopic level, may swollen mitochon- diseases (Table D5.3), the easiest cells to obtain are dria be distinguished from membrane-bound lyso- circulating white blood cells. A blood smear may show somal vacuoles; the mitochondrial double membrane vacuolated lymphocytes in certain lysosomal disorders and marginal remnants of ruptured cristae provide marked by lysosomal vacuoles, e.g., mucopolysacc- clear distinction. When disease-specifi c lysosomal inclusions of vac- haridoses, oligosaccharidoses, GM1-gangliosidosis. However, the procedure may only be considered sup- uolar or non-vacuolar nature are present, they should portive rather than proving since swollen mitochondria be photographed for permanent documentation. When may give the spurious light microscopic impression absent, one may safely count in the electron micro- of lysosomal vacuoles. A PAS stain may show scope 200 consecutive properly identifi ed lymphocytes carbohydrate-containing intralysosomal contents. The to be sure that lysosomal storage is not present in the preparation. Among metabolic diseases, lysosomal storage or residual bodies are vacuolar in mucopolysacchari- Table D5.3 Lysosomal pathology in lymphocytes doses, oligosaccharidoses, mucolipidoses, GM1- Vacuoles gangliosidosis (Fig. D5.1) and in juvenile or CLN3 Aspartylglucosaminuria NCL. In the latter vacuoles may occasionally con- GM1-gangliosidosis Mannosidosis tain fingerprint profiles, but their number is consid- Fucosidosis erably lower than the number of vacuoles. Thus, Juvenile neuronal ceroid-lipofuscinosis (NCL) empty lysosomal vacuoles usually predominate in Mucolipidosis I and II juvenile NCL. On the other hand, non-vacuolar, i.e., Mucopolysaccharidoses I–VII granular or lamellar inclusions, often of disease- Salla disease specific nature, are seen in type-II glycogenosis, Type-II glycogenosis Niemann-Pick disease, and Fabry disease. They are Solid non-vacuolar Infantile NCL granular in infantile NCL/CLN1, curvilinear bodies Late-infantile NCL in classical late-infantile NCL/CLN2 and ususally Late-infantile variant of NCL few but regularly shaped solid non-vacuolar lipop- Mucolipidosis IV igments with a granular component and fingerprint Niemann-Pick disease profiles in late-infantile variants of NCL, i.e., CLN5, Type-II glycogenosis (when glycogen is well preserved) CLN6, CLN7, and CLN8. 314 H. H. Goebel

Fig. D5.1 Electron microscopic abundant lysosomal membrane-bound vacuoles in a blood lymphocyte, GM1 gangliosidosis

complexes are often located in the cutis–subcutis Remember region, while epidermis is of limited reward (Kaesgen Lymphocytes are only suitable for morphological and Goebel 1989). investigations in lysosomal diseases, not in mito- The catalogue of morphologically diagnosable meta- chondrial or peroxisomal disorders. Electron micros- bolic diseases affecting the skin in children includes lyso- copy not light microscopy − may show lysosomal somal and peroxisomal diseases as well as Lafora disease vacuoles or compact lysosomal inclusions which (Table D5.4). Mitochondrial disorders − even those asso- vary in ultratructure, according to the type of lyso- ciated with abnormally structured mitochondria are seen somal disease. predominantly in skeletal muscle ﬁ bers − and are not

Table D5.4 Pathology of metabolic diseases in skin D5.3 Skin Vacuolar lysosomal diseases MPS, ML, oligosaccharidosis Avacuolar lysosomal disorders Skin is widely used for morphological investigations Globoid cell leukodystrophy

(Table D5.4). It offers easy accessibility by punch GM1 gangliosidosis biopsy; a large diversity of cell types, i.e., epithelial, GM2 gangliosidosis (Sandhoff type) mesenchymal, and neuroectodermal, allowing meta- Farber disease bolic conditions to be differently expressed in these Fabry disease Metachromatic leukodystrophy (when nerves are present) cell types. In addition, fi broblast cultures may be Neuronal ceroid-lipofuscinosis obtained from separate biopsies of skin. Niemann-Pick disease A prerequisite for gainful skin biopsy is that the Type-II glyocogenosis clinically suspected disease manifests itself morpho- Peroxisomal diseases logically in the skin or in cultured fi broblasts. A Adrenoleukodystrophy rewarding biopsy requires a full-thickness skin biopsy, Polyglucosan diseases as diagnostically crucial eccrine sweat gland epithelial Lafora disease D5 Pathology − Biopsy 315 convincingly expressed in skin. Amino acid and other Of the lysosomal diseases, type-II glycogenosis, small-molecule disorders do not display meaningful and the NCL show ubiquitous lysosomal pathology pathomorphological data in skin. affecting epithelial, mesenchymal, and neuroectoder- In skin obtained by diagnostic biopsy, morphologi- mal cells. In type-II glycogenosis, there is intralyso- cal investigations are largely to be performed with the somal glycogen accretion and a lysosomal vacuolar electron microscope because it is only at the ultrastruc- appearance when glycogen is badly preserved in tissue tural level that lesions appear distinct and sometimes handling after biopsy. In NCL, there are ultrastructur- disease-specifi c. Light microscopic studies may give ally distinct lipopigments. Granular lipopigments spurious results even when employing special tech- without any lipid droplets, the latter a conspicuous niques, such as enzyme histochemistry, immunohis- component of regular lipofuscin, may be encountered tochemistry, or fl uorescence microscopy. However, in genetic CLN1 or its infantile, late-infantile, juve- Lafora disease may be recognized at the light micro- nile, and adult clinical forms; curvilinear bodies are scopic level by strongly PAS-positive diastase-resistent found in genetic CLN2 or classical late-infantile form, polyglucosan bodies in ductal cells of eccrine sweat while other late infantile variant forms, genetic CLN5, glands. Lysosomal vacuolation may be excessive in CLN6, CLN7, and CLN8 types show a mixture of cur- respective lysosomal disorders, such as mucopolysac- vilinear profi les and fi ngerprint patterns. In genetic charidoses, but confi rmatory electron microscopy CLN3 or classic juvenile NCL, fi ngerprint profi les or remains desirable. Another hint at electron microfi ngerprint bodies prevail while, occasionally, vacuola- scopic lysosomal lesions may be increased enzyme tion of lysosomes is also encountered − as in CLN3 in histochemical activity of acid phosphatase at unusual lymphocytes. sites, e.g., smooth muscle cells of arrectores pilorum Dermal nerves contain myelinated and unmyeli- muscle or mural cells of vessels. The secretory gran- nated nerve fi bers, covered by Schwann cells and, ules in secretory cells of eccrine sweat glands are PAS- when still situated in fascicles, surrounded by perineu- positive, a nonspecifi c physiological feature, which is rium or perineurial cells. Schwann cells of both myeli- not to be taken as evidence of pathological lysosomal nated and unmyelinated axons and perineurial cells storage of glycolipids, glycoproteins, or glycosamino- may contain pathological lysosomes of different glycans. Even semi-thin, 1 mm-thick, plastic sections lamellar types, such as in mucolipidosis IV, sialidosis, stained with toluidine or methylene blue serve to iden- Niemann-Pick disease, Sandhoff disease, and fore- tify various cytological components in the skin speci- most the lysosomal leukodystrophies with involve- men for subsequent electron microscopic examination ment of the peripheral nervous system, such as rather than to ascertain unequivocal pathology. metachromatic (MLD) and globoid cell (GCL) leu- Among the biochemically heterogeneous lysosomal kodystrophies. When myelinated nerves are encoun- disorders, different entities are morphologically tered in the skin specimen, metachromasia of stored expressed in different cytological components of the sulfatides in MLD may be demonstrated as brownish skin. Some, like purely in particular neuronal forms, material in acid cresyl violet-stained fi xed frozen sec- Tay-Sachs disease, or Gaucher disease do not express tions and brownish material in toluidine blue-stained any pathology in the skin. semi-thin sections. Disease-specifi c lysosomal resid- As in lymphocytes, dermal cells in lysosomal disor- ual bodies accrue in Schwann cells, particularly of ders may be characterized by lysosomal vacuolation or myelinated nerve fi bers, including prismatic or tufa- by the formation of non-vacuolar compact lysosomal ceous bodies in MLD and needle-like inclusions in residual bodies. Hence, known lysosomal disorders GCL. In addition, similar compact lysosomal residual marked by lysosomal vacuolation, such as mucopoly- bodies may be encountered in macrophages within the saccharidoses, mucolipidoses, and oligosaccharidoses, endoneurium, derived from damage to and breakdown show lysosomal vacuoles in mesenchymal cells, such of myelinated nerve fi bers. The ultrastructure of lyso- as fi broblasts and mural cells of vessels. Certain lysosomal inclusions in GCL differs in the infantile and somal disorders, such as sialidosis and mucolipidosis non-infantile forms in that needle-like structures are IV, may show both lysosomal vacuolation and com- largely seen in infantile GCL and rather vacuolar lyso- pact lysosomal residual bodies of lamellar type in dif- somes fi lled with indistinct lamellae are seen in non- ferent cell types of the skin. infantile GCL (Goebel et al. 1990). A remarkable 316 H. H. Goebel distinction in dermal morphological manifestation Remember between MLD and GCL is the involvement of secretory cells of eccrine sweat glands with similar needle- Skin biopsy in the axillar region is not advisable like inclusions in GCL, but no abnormal lysosomal when suspecting a lysosomal disorder because there storage in MLD. are physiological inclusions. While mural cells of vessels are affected by lysosomal storage in a large number of lysosomal diseases, they are particularly involved in Fabry disease. They Both, epithelial cells of apocrine sweat glands and duc- are even demonstrable in manifesting carriers of this tal cells of eccrine sweat glands may contain nonspe- X-linked inherited disorder. cifi c normal inclusions, the former rather uniform and Important tissue components of skin are the eccrine compact, the latter of a lamellar ultrastructure. These sweat glands, the secretory cells of which display a cytoplasmic inclusions should not be confused with wide variety of lysosomal residual bodies both vacuo- pathological storage bodies. Melanin- and melano- lar and non-vacuolar in different disease. These lysosome-containing cells as well as mast cells also harbor somal residual bodies can often be distinguished from cell type-specifi c inclusions, which should not be con- secretory granules, both compact or electron-lucent. fused with true pathological lysosomal inclusions. Sometimes, there is a disturbingly large number of Not infrequently, axons, usually unmyelinated and membrane-bound vacuoles in secretory eccrine sweat in their terminal course, are nonspecifi cally enlarged gland epithelial cells, not associated with any lyso- by mitochondria and dense bodies, the latter possibly somal disorder, a nonspecifi c morphological feature of degenerating mitochondria (Dolman et al. 1977; Walter unknown connotation. While the ductal cells of eccrine and Goebel 1988), but hardly ever by disease-specifi c sweat glands are not affected by lysosomal storage in lysosomal residual bodies. These have only been seen lysosomal disorders, they are the main cell type in the gangliosidoses and in mucolipidosis IV. When involved in formation of polyglucosan bodies, not lim- encountering such enlarged axons, a lysosomal disor- ited by a unit membrane, in Lafora disease (Fig. D5.2) der may be suspected. The enlargement of the axons (Goebel and Bönnemann 2004). Similarly, polygluco- may result from impaired axoplasmic transport as a san bodies may be encountered in apocrine sweat gland result of lysosomal storage in respective neuronal epithelial cells of the axilla. perikarya.

Fig. D5.2 Several electron microscopic non-membrane bound polyglucosan bodies in ductal sweat gland epithelial cells of the skin, Lafora disease D5 Pathology − Biopsy 317

Cultures of dermal fi broblasts provide valuable sources of biochemical and molecular investigations, while morphological studies of cultured fi broblasts are largely unrewarding, except in mucolipidosis II or I-cell (inclusion cell) disease. Nonspecifi c lysosomal residual bodies may accrue over time in cultured fi broblasts giving rise to erroneous interpretations. Hence, performing a skin biopsy solely to produce tissue cultures may be considered incomplete in disorders in which meaningful morphological investigations may be made.

Remember Skin is an important biopsy target in lysosomal diseases both of vacuolar and nonvacuolar forms because of the diverse cell types and accessibility. Electron microscopy is required for proper morphological diagnosis. When nerves are present, electron microscopy may permit the recognition of lysosomal leukodystrophies (metachromatic and globoid cell forms) and peroxisomal disorders (adrenoleukodystrophies and infantile Refsum disease). Mitochondrial diseases are not morphologically reliably expressed in skin.

D5.4 Conjunctiva

Fig. D5.3 Electron microscopic appearance of needle-like Conjunctiva has occasionally been biopsied in chil- structures in Schwann cells in adrenoleukodystrophy dren as a target alternative to skin. This may refl ect a preference of the clinician, e.g., an ophthalmologist. It is seldom preferred by the pathologist. Sweat glands Diagnostically informative cytological components are absent from conjunctiva; vessels and nerves are, in skin are often widely spaced and scarce, and there is however, more abundant than in skin and are informa- an abundance of noninformative collagen fi bril aggre- tive. Mural cells of vessels and Schwann cells of both gates. Disease-specifi c lesions in affected patients may myelinated and unmyelinated axons may harbor dis- not be present in the individual skin specimen biop- ease-specifi c lysosomal residual bodies, both vacuo- sied. A second skin biopsy or biopsy of another tissue lar and nonvacuolar. Axons usually do not show any may be required. This may occasionally happen in the pathology in metabolic disorders except for nonspe- NCL. cifi c loss or regeneration. When axons are myelinated, Among the peroxisomal disorders, those forms their Schwann cells may harbor very typical disease- marked by needle-like inclusions (Fig. D5.3) in specifi c lysosomal bodies in lysosomal leukodystro- Schwann cells, such as adrenoleukodystrophy, and phies, so-called prismatic or herring-bone inclusions infantile Refsum disease, may show group-specifi c in MLD. Examples are the needle-like inclusions in pathology while other cell types, such as epithelial and GCL or Krabbe leukodystrophy, and similar but mesenchymal cells do not seem to be involved in mor- not identical needle-like inclusions in peroxisomal phological pathology. adrenoleukodystrophies. 318 H. H. Goebel

Fig. D5.4 An electron microscopic aggregate of membrane-bound lysosomal lamellar bodies in the Schwann cell cytoplasm of a myelinated peripheral nerve ﬁ bre of a patient with metachromatic leukodystrophy

D5.5 Peripheral Nervous System Sandhoff disease, may also involve non-neuronal cell

types and tissues as well, while GM2 gangliosidosis of Tay-Sachs type, as a purely neuronal lysosomal Because peripheral nerves are often encountered in disease, can safely be ascertained by morphological dermal and conjunctival biopsy specimens, the biopsy studies on nerve cells only, i.e., neurons of the rectum of peripheral nerves as an invasive, purely diagnostic and other tissues. Other nerve cell-containing regions procedure will seldom provide more information on of the peripheral nervous system located in dorsal root metabolic diseases than skin and conjunctiva may and autonomic ganglia are hardly ever a target of yield. In mitochondrial disorders, peripheral nerves biopsy. In GM gangliosidosis of all biochemical and are equally unsuitable as biopsy targets since they 2 molecular forms and in GM gangliosidosis membra- may give ambiguous and, thus, unreliable results. 1 neous cytoplasmic bodies are the characteristic intran- However, in specifi c constellations biopsy of nerves euronal lysosomal inclusions. can yield useful information (Fig. D5.4) (Table D5.5). Polyglucosan bodies within axons may be an occasional nonspecifi c fi nding, but they may be increased Table D5.5 Morphological expression of neurometabolic in number in conditions associated with polygluco diseases in peripheral nerves san body formation, polyglucosan diseases, such as Lysosomal diseases type IV glycogenosis. Vacuolar forms MPS, ML, oligosaccharidoses Non-vacuolar forms Fabry disease Globoid cell leukodystrophy D5.6 Rectum Glycogenosis type-II

GM1 gangliosidosis Metachromatic leukodystrophy Neuronal perikarya or nerve cell bodies of the periph- Neuronal ceroid-lipofuscinosis eral nervous system, largely situated in the rectum Peroxisomal disorders (Table D5.6), may serve as biopsy substitutes for neu- Adrenoleukodystrophies rons of the brain, because both are affected by lyso- Infantile Refsum disease somal storage in many lysosomal diseases. Certain Polyglucosan diseases neuronal lysosomal disorders, e.g., mucolipidosis IV or Polyglucosan body myopathy D5 Pathology − Biopsy 319

Table D5.6 Morphological expression of neurometabolic encephalomyopathies which do not show mitochon- diseases in rectum drial pathology in biopsied muscle, such as Leigh Lysosomal diseases syndrome. Vacuolar forms In addition, the vast achievements in biochemical MPS, ML, oligosaccharidoses and molecular data affecting the brain have deplorably Non-vacuolar forms Fabry disease been insufﬁ ciently correlated with postmortem brain Farber disease pathology. Gaucher disease Globoid cell leukodystrophy Glycogenosis type-II

GM1 gangliosidosis Remember

GM2 gangliosidosis Mucolipidosis IV Brain biopsy can now be considered an obsolete Neuronal ceroid-lipofuscinoses (NCL) diagnostic procedure to procure pathomorphology Niemann-Pick disease of metabolic disorders, but autopsies of the brain Peroxisomal disorders are still important to conﬁ rm in vivo diagnosis and Adrenoleukodystrophies secure morphological information in newly deﬁ ned Infantile Refsum disease metabolic conditions. Polyglucosan diseases Lafora disease Type-IV glycogenosis D5.8 Liver

Remember For morphological studies (Table D5.7), a liver biopsy may be a very rare procedure because its pathology in As nerves are wide-spread in skin and conjunctiva, numerous neurometabolic diseases is equally well peripheral nerve biopsies in metabolic disorders, expressed in other tissues more easily accessible, such especially in lysosomal diseases, are unnecessary. as skin or skeletal muscle. Only in peroxisomal disor- Likewise, when peroxisomal disorders affect periph- ders (Powers 2004), which may be divided into those eral nerves in skin and conjunctiva as seen in the with abnormal or absent peroxisomes and those with adrenoleukodystrophies and infantile Refsum disease, nerve biopsies may be replaced by biopsies of skin and conjunctiva. Table D5.7 Morphological expression of neurometabolic diseases in liver D5.7 Brain Peroxisomal diseases Infantile Refsum disease Neonatal adrenoleukodystrophy In metabolic diseases, premortem morphological stud- Zellweger disease ies of brain tissue for diagnostic purposes have become Lysosomal diseases rather obsolete. Among the metabolic disorders, there Vacuolar forms are hardly any which display disease-specifi c morpho- MPS, ML, oligosaccharidoses logical fi ndings confi ned to the brain and not encoun- Non-vacuolar forms tered in extracerebral tissues. Conversely, pathology of Fabry disease those metabolic disorders clinically predominant in Gaucher disease Glycogenosis type-II the brain does not appear disease-specifi c, such as in GM1 gangliosidosis amino acid disorders or neurotransmitter defects. NCL Moreover, brain biopsy is largely confi ned to the cor- Niemann-Pick disease tex and subcortical white matter, and this would fail to Polyglucosan diseases identify subcortical pathology and respective diseases Lafora disease as seen in Wilson disease or those mitochondrial Type IV glycogenosis 320 H. H. Goebel

Fig. D5.5 Electron microscopic membrane- bound lysosomal vacuoles with some electron-dense dark amorphous material in hepatocytes of liver in Niemann-Pick disease type B

regular peroxisomes, liver as biopsy target may be the smallness of peroxisomes and those defi ned by best choice, before or after respective biochemical and individual enzyme defi ciencies when peroxisomes molecular investigations. Peroxisomes are absent in are present in hepatocytes. Although lysosomal dis- Zellweger syndrome, neonatal adrenoleukodystrophy eases widely affect the liver, there are more easily and infantile Refsum disease. At the light microscopic accessible tissues, such as lymphocytes and skin. In level, the absence of the marker enzyme for peroxi- non-neuronopathic Gaucher and Niemann-Pick dis- somes, catalase, may suggest absence of peroxisomes eases, the liver is a warranted biopsy target. which may then be confi rmed by electron microscopic examination. In other peroxisomal conditions, peroxisomes may be present, but enlarged or abnormally struc- affecting liver but not skeletal muscle, such as the tured, and “angulate lysosomes” may be encountered. mitochondrial DNA depletion syndromes. Among lysosomal disorders, Gaucher disease may be recognized morphologically in liver but not in skin or skeletal muscle. It is of course recognized in bone marrow. Many other lysosomal diseases are morpho- D5.9 Skeletal Muscle logically evident in liver (Fig. D5.5). Although the liver may be affected in mitochondrial diseases and The main cytological components of skeletal muscle are even contain abnormally structured mitochondria, an the multinucleated striated muscle fi bers (Table D5.8). advantage of liver biopsy over skeletal muscle biopsy They may show a remarkable gamut of abnormal mito- exists among the mitochondrial disorders only in those chondria, i.e., increase in number, increase in size, abnormal shape, and pathological solid inclusions. Remember Abnormal mitochondria are seen in a large number but Liver is the most important biopsy target in peroxi- not all mitochondrial diseases. However, individual somal disorders to distinguish between those mitochondrial myopathies do not show different ultra- with defective biogenesis resulting in absence or structural patterns of mitochondria. In light microscopic specimens, accumulation of abnormal mitochondria in D5 Pathology − Biopsy 321

Table D5.8 Morphological manifestation of neurometabolic Among the lysosomal diseases, type-II glycogeno- diseases in skeletal muscle sis, in both the infantile and non-infantile types, is best Mitochondrial diseases studied in skeletal muscle (Fig. D5.6). Lysosomal gly- Lysosomal diseases cogen storage is not confi ned to striated muscle fi bers Vacuolar forms but is apparent in other tissue components of skeletal MPS, ML, oligosaccharidoses Non-vacuolar forms muscle, such as satellite cells, mural cells of vessels, Fabry disease and fi broblasts. Mucolipidosis IV may show lysosomal Glycogenosis type-II lamellar bodies in muscle fi bers and lysosomal vacu-

GM1 gangliosidosis oles in interstitial cells. Niemann-Pick disease, espe- Mucolipidosis IV cially type-C, may affect compartments of both striated Neuronal ceroid-lipofuscinoses and non-striated cells with the formation of typical Niemann-Pick disease lysosomal inclusions. On the other hand, vacuolar lys- Polyglucosan diseases osomal disorders, such as mucopolysaccharidoses, Lafora disease Type IV glycogenosis may readily be recognized in interstitial cells within Polyglucosan myopathy skeletal muscle but not in striated muscle fi bers. The presence of nerve fascicles may provide evidence of lysosomal leukodystrophies, such as metachromatic muscle fi bers may give rise to “ragged red fi bers” when and globoid cell forms. In Lafora disease, membrane- employing the modifi ed Gomori trichrome stain, to bound glycogen and debris displaying increased activ- “ragged blue fi bers” in oxidative enzyme histochemical ity of acid phosphatase suggest lysosomal involvement, preparations, such as NADH tetrazolium reductase and together with fi brillar glycogen which may be more succinic dehydrogenase (SDH) or “ragged brown apparent as polyglucosan bodies in type IV glycogeno- fi bres” in cytochrome-C oxidase preparations. Many sis and its non-infantile variants. The formation of times, however, ragged red fi bers may lack enzyme his- lysosomal glycogen in type-II glycogenosis is ubiqui- tochemical activity of cytochrome-C oxidase, being so- tous. Type-specifi c lipopigments of different genetic called COX-negative muscle fi bers, while SDH in (CLN) forms of NCL are encountered in striated mus- ragged blue fi bers is well expressed, an observation cle fi bers and interstitial cells, largely granular lipopig- which is quite conspicuous in a combined COX-SDH ments (so-called granular osmiophilic deposits enzyme histochemical preparation (Vogel 2001). (GROD) ) in the genetic CLN1 form. Curvilinear/

Fig. D5.6 Light microscopic deposition of lysosomal glycogen in and destruction of muscle fi bres of skeletal muscle, type-II glycogenosis, modifi ed Gomori trichrome stain 322 H. H. Goebel rectilinear, but not fi ngerprint profi les are encountered perform a light and electron microscopic study but in the genetic forms CLN2, CLN3, CLN5, CLN6, also to provide tissue for important biochemical CLN7, and CLN8. Finely granular lipopigments also and mitochondrial genomic investigations (mito- accrue in skeletal muscle and peripheral nerve in vita- chondrial diseases, glycogenoses, and lipid disor- min-E defi ciency, both the hereditary and acquired ders). Preservation of unfi xed frozen muscle tissue forms. This group includes abeta-lipoproteinemia or and archival storage is a diagnostic prerequisite. Bassen–Kornzweig syndrome. Biopsied unfi xed frozen muscle may also be a suitable organ for biochemical studies, such as the muscle-specifi c biochemical abnormalities seen in D5.10 Other Tissues mitochondrial DNA-related, organ-defi ned mitochondrial disorders and in non-infantile type-II glycogenosis. The latter condition may show normal acid Morphologically non-systematic observations concern maltase levels in circulating blood cells, often the biopsies of rather unusual tissues such as kidney, source for biochemical investigation of type-II gly- lymph nodes, spleen, appendix, or even urinary sedi- cogenosis, but absent or reduced activities in skeletal ment. The latter contains lysosomal residual bodies muscle (Sharma et al. 2005). derived from degenerated sloughed-off renal tubular Other glycogenoses, such as types III, V, and VII, cells. Bone marrow is particularly useful in Gaucher displaying increased storage of sarcoplasmic non- and Niemann-Pick diseases. membrane-bound glycogen in skeletal muscle fi bres, are associated with enzyme histochemical defi ciency of the respective two enzymes myophosphorylase and phosphofructokinase in glycogenoses V and VII. The Take Home Messages sarcoplasmic glycogen is strongly PAS-positive but › Certain neurometabolic disorders are marked liable to digestion by diastase; it may fade during pro- by cerebral and extracerebral morphological longed postsurgical tissue-handling rendering the manifestations of individual, i.e. lysosomal, myopathology that of vacuolar myopathies. Increased peroxisomal, mitochondrial, and polyglucosan amounts of sarcoplasmic lipid droplets, often confl u- diseases, these being single-organelle/multi- ent and then appearing as larger droplets at the light organ disorders. The extracerebral biopsy microscopic level, suggest a lipid myopathy, so-called facilitates and corroborates the diagnostic neutral lipid storage disease with or without associated armamentarium in these conditions, according mitochondrial defects, while lysosomal lipid accumu- to selective tissue manifestation and accessi- lation is evidence of a rare lysosomal disorder, the bility by biopsy. The preferential sites for lipase-defi cient Wolman disease. biopsy are blood lymphocytes, skin, and rec- Since cardiomyocytes, though uninuclear cells, tum in lysosomal diseases, skeletal muscle in show a similar constitution as skeletal muscle fi bres, mitochondrial diseases, liver in peroxisomal they may also be affected in metabolic disorders; thus disorders, and skin and skeletal muscle in skeletal muscle biopsy may serve as a substitute for polyglucosan diseases. Electron microscopy is cardiac biopsy. the most rewarding diagnostic technique requiring immediate post-bioptic fi xation of Remember the tissues, especially in lysosomal and peroxi- Skeletal muscle is the primary and most important somal diseases. Many mitochondrial diseases target for biopsy in mitochondrial diseases, in are also recognised in skeletal muscle by light muscle-affecting glycogenoses, and in neutral microscopy including mitochondria-related lipid storage diseases. For proper diagnosis in enzyme histochemial preparations. skeletal muscle tissue, it is essential not only to D5 Pathology − Biopsy 323

Key References dren. In: Cruz-Sánchez FF et al (eds) Neuropathological diagnostic criteria for brain banking. IOS Press, Amsterdam, pp 136–147 Dolman CL, MacLeod PM, Chang E (1977) Fine structure of Goebel HH, Warlo I (1990a) Biopsy studies in neurodegenera- cutaneous nerves in ganglioside storage disease. J Neurol tive diseases of childhood: part I. BNI Q 6:19–27 Neurosurg Psychiatry 40:588–594 Goebel HH, Warlo I (1990b) Biospy studies in neurodegenera- Goebel HH (1997) Neurodegenerative diseases: biopsy diagno- tive diseases of childhood: part II. BNI Q 6:24–30 sis in children (Chapt. 14). In: Garcia JH, Budka H, Kaesgen U, Goebel HH (1989) Intraepidermal morphologic man- McKeever PE, Sarnat HB, Sima AAF (eds) Neuropathology. ifestations in lysosomal diseases. Brain Dev 11:338–341 The diagnostic approach. Mosby, St. Louis, pp 581–635 Powers JM (2004) Peroxisomal disorders (Chapter 31). In: Goebel HH (1999) Extracerebral biopsies in neurodegenerative Golden JA, Harding BN (eds) Pathology and genetics: diseases of childhood. Brain Dev 21:435–443 developmental neuropathology. International Society of Goebel HH, Bönnemann C (2004) Disorders of carbohydrate Neuropathology (ISN), Basel, pp 287–295 metabolism. Polyglucosan disorders (Chapter 27.2). In: Sharma MC, Schultze C, von Moers A et al (2005) Delayed or Golden JA, Harding BN (eds) Pathology and genetics: late-onset type II glycogenosis with globular inclusions. developmental neuropathology. International Society of Acta Neuropathol (Berl) 110:151–157 Neuropathology (ISN), Basel, pp 221–225 Vogel H (2001) Mitochondrial myopathies and the role of the Goebel HH, Harzer K, Ernst JP et al (1990) Late-onset globoid pathologist in the molecular era. J Neuropathol Exp Neurol cell leukodystrophy: Unusual ultrastructural pathology and 60:217–227 subtotal b-galactocerebrosidase deﬁ ciency. J Child Neurol Walter S, Goebel HH (1988) Ultrastructural pathology of dermal 5:299–307 axons and Schwann cells in lysosomal diseases. Acta Goebel HH, Jänisch W (1995) The neuropathology of metabolic Neuropathol (Berl) 76:489–495 encephalopathies and neurodegenerative diseases in chil- Suspected Mitochondrial Disorder D6 Nicole I. Wolf

Key Facts Mendelian inheritance is caused by mutations in nuclear DNA and follows dominant, recessive, or X-linked › Lactate elevations in blood, CSF, or urine or modes of inheritance. The mitochondrial DNA (mtDNA) frank lactic acidosis are a hallmark of disorders is a circular structure of 16,569 bp, which codes for two of energy metabolism, more speciﬁ cally respi- ribosomal RNAs and 22 tRNAs needed for protein syn- ratory chain disorders, disorders of oxidative thesis within the mitochondria, and 13 additional pep- phosphorylation, mitochondrial disorders, or tides as components of the respiratory chain. MtDNA is mitochondriopathies. These can involve any maternally inherited; the result of maternal inheritance is organ at any age. that familial mitochondrial disorders typically affect all › Organs commonly affected include muscle, brain, the children of the affected women, but not children of retina, extraocular muscles, heart, liver, kidney, the affected men. Paternal inheritance has shown to be pancreas, gut, and bone marrow (Table D6.1). possible in one case but is exceedingly rare and not rele- Systemic growth may be impaired. It is some- vant for genetic counseling. times said that the greater the number of organ Every cell in the human body, besides erythrocytes, systems involved, the greater the likelihood of contains thousands of mtDNA molecules. Usually, their a mitochondrial disorder. sequence is identical (homoplasmy). If there are mutations, usually not every mtDNA molecule in the body will be affected, and wild type and mutated mtDNA will be present together even within one cell or tissue Within a cell, mitochondria and the mitochondrial (heteroplasmy). Levels of mutant mtDNA may be very respiratory chain are at the center of all energy-related different from one oocyte to another and from one organ processes. Secondary respiratory chain dysfunction is to another, depending on random segregation of mutant common in many disorders – not only in disorders mtDNA during oogenesis, oocyte division, and blasto- affecting intermediate metabolism, e.g., organic acidu- cyst development. Only, the mildest mutations can be rias or fatty acid oxidation defects, but also in many tolerated in homoplasmic mode. Heteroplasmic muta- other inherited diseases not primarily affecting meta- tions may be devastating to the function of an individual bolic pathways. Primary respiratory chain defects are mitochondrion in high numbers, but if present in a small disorders that directly involve oxidative phosphoryla- percentage of mitochondria, or cells, they can be toler- tion and the electron transfer chain. Inheritance can be ated. The threshold proportion for symptoms differs maternal or Mendelian, and a myriad of genes is widely for different mutations and tissues. Whether lev- involved, making the speciﬁ c genetic diagnosis of a els of mutant mtDNA are important for disease severity mitochondrial disorder still a challenge. in mtDNA disorders remains still unclear for most mutations. Mitochondria proliferate throughout life, so there is an opportunity for the proportion of normal and N. I.Wolf mutant to change within the various tissues, often with Department of Child Neurology, VU Medisch Centrum, 1007 MB Amsterdam, The Netherlands an increase in abnormal forms, and deterioration of e-mail: [email protected] symptoms over time. Over the last years, an increasing

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 325 DOI: 10.1007/978-3-540-74723-9_D6, © Springer-Verlag Berlin Heidelberg 2010 326 N. I. Wolf

Table D6.1 Important symptoms and fi ndings in respiratory important symptoms associated with mitochondrial dys- chain defi ciency function. An otherwise unexplained combination of Organ or tissue Signs and symptoms symptoms in different organ systems is the strongest indi- Brain Seizures, stroke-like episodes, cator of a mitochondrial disease. Mitochondrial disorders myoclonus, ataxia, Parkinsonism, migraine, may have prominent muscle involvement, although this dementia, presentation is rare in infancy and childhood. Other organs leukoencephalopathy commonly affected include brain, retina, extraocular Eye Optic atrophy, pigmentary muscles, heart, liver, kidney, pancreas, gut, bone marrow, degeneration, cataract and the endocrine systems (Table D6.1). Physical growth Extraocular muscles Ophthalmoplegia, ptosis may be impaired. Mild hypertrichosis is an unspecifi c Ear Deafness Skeletal muscle Myopathy, exercise intolerance sign of mitochondrial disorders as is thrombocytosis. Bone marrow Pancytopenia or failure of The consideration of mitochondrial disease proceeds specifi c cell lines along three axes – clinical symptoms, metabolic investi- Heart Cardiomyopathy, conduction gations, and functional assays. Molecular defects can be defects present in the mitochondrially encoded tRNAs, mito- Liver Hepatic dysfunction, liver failure, cirrhosis, bile stasis chondrial, and nuclear-encoded subunits of the oxidative Intestine Pseudo-obstruction phosphorylation complexes, and many other proteins. Testes and ovaries Gonadal failure There are many areas of overlap – the same mutation can Kidney Tubulopathy, Fanconi give rise to different syndromes, and the same syndrome syndrome can be caused by different functional impairments or Pancreas Diabetes mellitus, exocrine mutations in different genes; so investigations are neces- failure, pancreatitis Endocrine organs Failure of hormone secretion sarily wide ranging, and it is diffi cult, even impossible, to (thyroid, parathyroid, provide guidelines which can by applied for all patients in adrenal, pituitary gland, all settings. Functional defi ciencies of a single respiratory and pancreatric b cells) chain complex may be due to a mutation involving one of Growth Failure to thrive, wasting the subunits or assembly factors involved; mutations in Blood, CSF, and urine Increased lactate or alanine genes involving translation usually give rise to multiple complex defi ciency. The latter can also be found in nuclear gene defects impairing mtDNA maintenance or number of mutations in nuclear genes that affect replication; however, respiratory chain activity can also mtDNA proliferation and stability and, if defective, that be normal in some of these defects (see Table D6.2). cause different diseases could be identifi ed. Clinically, there are some well-defi ned mitochon- Despite the importance of mtDNA, most subunits drial syndromes. Some of them can be heterogeneous, of the fi ve complexes of the mitochondrial respiratory involving either several mitochondrial genes or several chain are encoded by nuclear genes. The same is true nuclear genes or even mitochondrial or nuclear genes for all transport proteins, proteins for mtDNA synthe- such as Leigh syndrome. Table D6.3 gives an overview sis and maintenance, proteins required for transcrip- over the most important syndromes. Leigh syndrome is tion and translation, and assembly factors of the fi ve one of the most frequent manifestations of a mitochon- complexes (Fig. D6.1). There are at least 1,000 nuclear drial disorder in infancy and childhood and illustrates genes involved in mitochondrial biogenesis, mainte- the diffi culties in the diagnosis of mitochondrial disor- nance, and functioning. During the last years, muta- ders. Affected patients present with developmental tions in nuclear genes coding for subunits of respiratory delay or a neurodegenerative course including extrapy- chain complexes (e.g., complex I) or assembly factors ramidal movement disorder, ataxia, strabismus, and (e.g., complex IV) have been increasingly recognized, swallowing diffi culties. MRI reveals symmetric hyper- especially in mitochondrial disorders of infancy and intensities of basal ganglia, mesencephalon, and brain childhood (see Table D6.2). stem (Fig. D6.2). Additional symptoms as cardiomyo- Mitochondrial disorders are among the most common pathy or renal insuffi ciency are possible and may help to metabolic disorder affecting ~ 1:5,000 already in child- pinpoint the genetic defect. Leigh syndrome is extremely hood. They can involve any tissue at any age with any heterogeneous and can be caused by mutations in the degree of severity. Table D6.1 lists some of the most mtDNA, especially T8993G/C in MTATP6 coding for D6 Suspected Mitochondrial Disorder 327

Intermembrane space

+ + H+ H H

e- - C0 I Co Q e e- Cyt C Co IV Co V - e Co II Co III e- e-

Matrix + FADH2 FAD + 2H H+ + + + O2 + H H2O ADP + P ATP NADH NAD + H i

Complex I: Complex II: Complex III: Complex IV: Complex V:

7 subunits 1 subunits 3 subunits 2 subunits mtDNA mtDNA mtDNA mtDNA

36 subunits 4 subunits 10 subunits 10 subunits 14 subunits nDNA nDNA nDNA nDNA nDNA

Fig. D6.1 Mitochondrial respiratory chain. Complex I (NADH:ubiquinone oxidoreductase), complex II (succinate:ubiquinone oxidoreductase), complex III (ubiquinol: cytochrome C oxidoreductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase) one of the complex V subunits, mutations in different In daily clinical work, there are three different clini- nuclear genes, e.g., involving complex I subunits and cal scenarios where mitochondrial disorders are sus- assembly factors for complex IV and also mutations in pected (Fig. D6.3). First, clinical symptoms are so the gene coding for the alpha subunit of pyruvate decar- suggestive of a mitochondrial disease that subsequent boxylase, the fi rst of three enzymes in the pyruvate investigations are warranted. Second, in the diagnostic dehydrogenase complex (PDHA1). In the differential work-up of a patient with more or less nonspecifi c diagnosis of Leigh syndrome, muscle biopsy is neces- symptoms results of either laboratory or other, e.g., neu- sary in order to narrow down the genetic investigations. roradiological investigations, point into the direction of Over 100 pathogenic point mutations and 200 dele- a mitochondrial disorder. Third, in patients with unex- tions, insertions, and rearrangements of mtDNA have plained symptoms and signs a mitochondrial disorder is been identifi ed since the fi rst mutations were described considered as possible differential diagnosis, even within 1988. The nuclear-encoded oxidative phosphoryla- out typical clinical or laboratory hallmarks supporting tion disorders and other mitochondrial syndromes are this idea. In the fi rst scenario, it is possible to go directly still diffi cult to elucidate although there has been great to DNA analysis if the symptoms are typical of a certain progress over the last few years. In infants and chil- syndrome as mitochondrial encephalomyopathy, lactic dren, nuclear defects are much more common than acidosis and stroke-like episodes (MELAS) or Alpers mtDNA mutations that cause only 10–20% of respira- disease; if this turns out to be negative, muscle biopsy is tory chain disorders in this age group. As mostly the necessary. In the second case, muscle biopsy is the fi rst molecular basis of most nuclear defect is as yet step, and depending on the results, tailored genetic unsolved, extended biochemical analysis in fresh mus- investigations would follow (e.g., in complex I defi - cle remains the gold standard for the diagnosis of many ciency sequencing of genes coding for its subunits), defects of the respiratory chain. This permits the deter- perhaps also additional investigations as proteome anal- mination of defects of regulation, posttranslational ysis, candidate gene sequencing, etc. in a research set- modifi cation, signaling, import, quality control, fold- ting. The third scenario is the most diffi cult one, as it is ing, and assembly of the oxidative phosphorylation virtually impossible to exclude a mitochondrial disor- apparatus as a whole. der even with the most sophisticated work-up (save for 328 N. I. Wolf

Table D6.2 Nuclear genes involved in mitochondrial disorders Biochemical defect Nuclear genes involved Gene function Other laboratory abnormalities Defi ciency of single enzymes Complex I defi ciency NDUFA11, NDUFS1, Structural subunits of complex I NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2 B17.2L, C6ORF66, Assembly factors of complex I C20orf7, NDUFAF1 Complex II defi ciency SDHA, SDHC, SDHD Structural subunits of complex II Succinate elevated in cerebral 1H-MRS Complex III defi ciency BCS1L Assembly factor of complex III Complex IV defi ciency SURF1, COX15 Assembly factor of complex IV SCO1, SCO2 Copper incorporation COX10 Heme synthesis LRPPRC Complex IV mRNA stabilization Complex V defi ciency ATP12 Complex V assembly gene

Deﬁ ciency of multiple enzymes Impaired translation EFG1, TSFM, EFTu Mitochondrial translation elongation MRPS16, MRPS22 Mitochondrial ribosomal proteins Impairment of coenzyme Q synthesis PDSS1, PDSS2, COQ2, Coenzyme Q biosynthesis Decreased ubiquinone (decreased activity of complex I + CABC1 content in muscle III and II + III) Impaired intergenomic communica- POLG1 mtDNA replication (autosomal tion (activity of respiratory chain dominant and recessive enzymes may be normal; mutations in several depletion or multiple deletions of disorders) mitochondrial DNA (mtDNA) common) TK2 Nucleotide salvage (mutated in CK strongly elevated mitochondrial mypoathy) DGUOK Nucleotide salvage (mutated in hepatocerebral mtDNA depletion) MPV17 Mitochondrial inner membrane protein with unknown function RRM2B p53-Controlled ribonucleotide Severe mtDNA depletion reductase subunit in muscle (1–2%); ragged red ﬁ bres SUCLA2 ADP-forming succinyl-CoA Elevated excretion of synthetase methylmalonic acid SUCLG1 Succinate-coenzyme A ligase Elevated excretion of (fatal infantile lactic methylmalonate and acidosis) methylcitrate C10ORF2 Twinkle, mtDNA replication RARS2 Mitochondrial arginyl-tRNA Only complex I may be synthetase (mutated in a abnormal type of pontocerebellar hypoplasia) D6 Suspected Mitochondrial Disorder 329

ANT1/SLC25A4 ADP/ATP translocator ECGF1 Thymidine phosphorylase Elevation of thymidine (mutated in mitochondrial and deoxyuridine neurogastrointestinal encephalopathy) PUS1 Pseudouridine synthase 1 Sideroblastic anemia (mutated in MLASA) Normal activity of respiratory chain complexes FXN Frataxin, implied in iron homoestasis (mutated in Friedreich’s ataxia) OPA1 mtDNA maintenance, mutated in patients with optic atrophy DDP1 Mitochondrial transport protein, mutated in x-linked dystonia deafness syndrome SPG7 Paraplegin (mutated in hereditary spastic sparaple- gia type 7) ABCB7 Iron export (mutated in x-linked Sideroblastic anemia sideroblastic anemia with spinocerebellar ataxia) DARS2 Mitochondrial aspartyl-tRNA Lactate elevated in synthetase (mutated in 1H-MRS, typical LBSL) neuroimaging DLP1 Dynamin-like protein, defi cient Lactic acidosis and mild fi ssion of mitochondria and elevation of VLCFA peroxisomes TAZ Tafazzine (mutated in Barth Elevated excretion of syndrome) 3-methylglutaconic acid another fi rm diagnosis) and diffi cult to decide how far When muscle biopsy is performed, care must be one should go with invasive and expensive diagnostic taken that all necessary investigations are made. In procedures as muscle biopsy. adults, it is current practice to do only morphological studies, and the presence or absence of ragged Remember red fibres is for some, enough to make or discard the diagnosis of a mitochondrial disorder (see In every patient diagnosed or suspected with a mito- Chap. D5). However, morphological studies are not chondrial disorder, organs must be systematically enough! Every muscle biopsy in a patient with sus- screened for possible involvement. This includes cere- pected mitochondrial disease has to be examined 1 bral MRI (and, if possible, H-MRS to assess intrace- morphologically (if possible including electron rebral lactate), CSF studies for lactate, alanine and microscopy to look at mitochondrial ultrastructure) protein, ECG and echocardiography to detect rhythm as well as functionally. The latter investigation, the abnormalities and cardiomyopathy, urine studies to assessment of global respiratory chain function and look for tubulopathies and pathological elevations of activity of single complexes of the respiratory chain organic acids, liver function tests, assessment of ret- can be done wholly only in fresh muscle. Single ina, optic nerve, and hearing. Diabetes mellitus should complex activities can also be investigated in be excluded. If these investigations remain normal freshly frozen tissue, but its diagnostic yield is sig- and no other fi nal diagnosis has been reached, they nificantly inferior to the overall investigation of have to be repeated at regular intervals. fresh tissue. 330 N. I. Wolf

Table D6.3 Important mitochondrial syndromes Syndrome Genes involved Inheritance Important clinical features LHON (Leber hereditary Missense mutations in different Mt, often be Acute or subacute mid-life painless optic neuropathy) mitochondrial DNA genes, homoplasmic visual loss. Smoking as risk especially complex I subunits factor. Additional mild neurological symptoms possible, also preexcitation syndromes MELAS (mitochondrial Most common mutation A3243G Mt Stroke-like episodes, epilepsy, small encephalomyopathy, in MTTL1; other mtDNA stature, diabetes mellitus, lactic acidosis and mutations deafness, cardiac conduction stroke-like episodes) defects MERFF (Mitochondrial Most common mutation A8344G Mt Progressive myoclonic epilepsy, encephalopathy with in MTTK; other mtDNA ataxia, dementia ragged red fi bers and mutations stroke-like episodes) Kearns–Sayre syndrome Large mtDNA deletions (most Mt (usually sporadic) Progressive ataxia, dementia, cardiac (KSS) common 4977 bp) of mtDNA conduction defect, deafness, elevated CSF protein and lactate, typical neuroimaging Pearson marrow-pancreas Large mtDNA deletions Mt, sporadic Exocrine pancreas insuffi ciency and syndrome (identical to CPEO (chronic anemia, evolves into KSS progressive external ophthalmoplegia) and KSS) CPEO Large mtDNA deletions; Mt (also sporadic); Progressive external ophthalmople- POLG1, ANT1, POLG2, AD; AR gia, additional myopathy possible C10ORF2 NARP (neuropathy, ataxia T8993G/C in MTATP6 Mt Neuropathy, ataxia, retinitis and retinitis pigmentosa. If high rate of pigmentosa) heteroplasmy → Leigh syndrome Leigh syndrome Many different genes, especially Mt, AR, XL Neurodegenerative disease with SURF1 (nuclear DNA) and onset in infancy or early T8993G/C childhood: movement disorder, swallowing diffi culties, strabismus. Additional symptoms possible. MRI with symmetric abnormalities in basal ganglia and brainstem Alpers disease POLG1, various mutations in AR, mtDNA Neurodegenerative disease with mtDNA; other unknown onset in childhood: status nuclear genes epilepticus, epilepsia partialis continua, liver failure (may be triggered by valproic acid), gastrointestinal dysmotility Mitochondrial neurogastro- ECGF1 (coding for thymidine AR Ptosis, CPEO, gastrointestinal intestinal phosphorylase) dysmotility with pseudoobstruc- encephalopathy tion, peripheral neuropathy, myopathy. MRI: diffuse leukoencephalopathy AD Autosomal dominant; AR Autosomal recessive; Mt Mitochondrial; XL X-linked D6 Suspected Mitochondrial Disorder 331

Fig. D6.2 T2w axial images of a child with Leigh syndrome. Nucleus caudatus, pallidum, and the periaquae- ductal area in the mesencephalon show elevated signal. Additionally, there are atrophy and white matter changes. 1H-MRS of basal ganglia displays a strongly elevated lactate and decreased NAA. Courtesy of Dr. Inga Harting, Department of Neuroradiology, University Hospital Heidelberg

Prenatal diagnosis in mitochondrial disorders is Remember straightforward if inheritance is Mendelian and the Muscle biopsy is necessary to confi rm the diagno- genetic defect is known. If Mendelian inheritance is sis of a mitochondrial disorder and to help guiding suspected, e.g., autosomal–recessive inheritance in a further genetic investigations in most patients. consanguineous couple with an affected child, prenatal Functional studies of respiratory chain function in diagnosis is possible if a biochemical defect on the fresh muscle tissue remain the gold standard in level of the respiratory chain has been proven in muscle diagnosis. Morphological studies alone are not and fi broblasts. For mtDNA mutations, prenatal diag- suffi cient. nosis and assessment of disease severity in the offspring 332 N. I. Wolf

Symptoms and signs Ye s Positive characteristic of a Direct genetic analysis Diagnosis confirmed mitochondrial syndrome

Negative No

Further workup (including Normal CSF studies and search Reconsider diagnosis for organ involvement)

Abnormal

Normal Muscle biopsy Other diagnosis?

Abnormal No

Further genetic investigations, possibly Molecular diagnosis fibroblast studies reached?

Ye s

Diagnosis confirmed

Fig. D6.3 Flowchart for the diagnostic approach in a patient with a suspected mitochondrial disorder

is diffi cult, even impossible, due to the random segre- inherited metabolic disorders, among others the group gation of mtDNA, although there are efforts to assess of congenital disorders of glycosylation, the glycog- levels of mutant DNA in oocytes, blastocysts, and cho- enoses or fatty acid oxidation defects, can mimic mito- rionic villi. These investigations are performed only in chondrial disorders very closely. Biotinidase defi ciency a few highly specialized laboratories. causes lactic acidosis, deafness, and optic atrophy and has to be sought for in every child with elevated lactic Remember acid, as it is treatable. Defi ciency of the PNPO gene encoding pyridox(am)ine 5-phosphate oxidase may Prenatal diagnosis of mitochondrial disorders is lead to greatly increased CSF lactate. Thiamine defi - straightforward if nuclear genes are affected and the ciency also results in severe lactic acidosis. Hypoxia, mutation is known. In selected cases, biochemical sepsis, and low cardiac output are systemic causes of assessment of respiratory chain function in chorionic lactate elevation. In several neurological disorders of villi is an option. With mtDNA mutations, prenatal childhood, lactate has been found to be intermittently diagnosis and risk assessment remain diffi cult. increased in some patients, among them patients with Rett and Angelman syndromes. Even single enzyme There are many possible differential diagnoses for pri- defi ciencies or decreased ATP production have been mary respiratory chain disorders. Metabolites in propi- demonstrated in patients with variable other diagnoses. onic and methylmalonic acidurias directly affect As correct diagnosis in these cases is important for respiratory chain function and can thereby give rise to prognosis and correct genetic counselling, clinical sus- “mitochondrial” symptoms. The presentation of other picion of other disorders must be high. D6 Suspected Mitochondrial Disorder 333

Remember Key References

Besides respiratory chain disorders, there are many Haas RH, Parikh S, Falk MJ et al (2007) Guidelines for the gen- differential diagnoses for lactate elevation/lactic aci- eralist on the diagnosis of mitochondrial disease. Pediatrics dosis including nonmetabolic inherited and acquired 120:1326–1333 disorders. The most common situation in which the The Mitochondrial Medicine Society’s Committee on Diagnosis, Haas RH, Parikh S, Falk MJ et al (2008) The in-depth evalu- concentration of lactic acid in blood is elevated is ation of suspected mitochondrial disease. Mol Gen Metab factitious, the result of improper technique, the use of 94:16–37 a tourniquet, or diffi culty in drawing the blood. Depending on the clinical setting, treatable disorders as biotinidase or PNPO defi ciency or thiamine defi - ciency must be considered fi rst.

Take Home Messages › Respiratory chain disorders can present with a myriad of symptoms, at any age. › Lactate (and alanine) elevation are common in respiratory chain disorders, but it can occur also in a variety of other inborn errors of metabolism, infection or circulatory failure or can be factitious. › The gold standard for the diagnosis of a respiratory chain disorder is measurement of oxidation rates and single enzyme activities in fresh muscle tissue. › Genetic investigations usually take the result of the muscle biopsy into account; if the clinical picture is very suggestive, the appropriate genetic test can be performed without a muscle biopsy. › Secondary abnormalities of oxidation rates and single enzyme activities in muscle can be encountered in other genetic and acquired disorders. Postmortem Investigations D7 Piero Rinaldo

Key Facts (Bennett and Rinaldo 2001) and, particularly, because of the broad implementation of expanded newborn › All cases of sudden and unexpected death in screening for these disorders (Watson et al. 2006). childhood should be evaluated for a possible Based on numerous observations, it has been postu- underlying metabolic disorder lated that without preventive intervention (i.e., new- › A history of "normal" newborn screening for born screening), FAO disorders might be responsible metabolic disorders is not a suffi cient reason for up to 5% of children who die suddenly and unex- to decline post-mortem evaluation pectedly from birth to 5 years of age, particularly › Most important specimens to collect at autopsy among those with evidence of acute infection that rou- are blood and bile, spotted on fi lter paper tinely would not represent a life-threatening event (Boles et al. 1998). Figure D7.1 shows a fl owchart for the evaluation of sudden and unexpected death that is centered on the analysis of acylcarnitines in blood and bile spots The high mortality rate that is associated with acute (Rinaldo et al. 2002). Postmortem blood (Chace et al. episodes of metabolic decompensation has lead to a 2001) and bile (Rashed et al. 1995) could be conve- perceived association between sudden unexpected niently collected on a single fi lter paper card (Fig. D7.2), death in early life and several inborn errors of amino very similar to those used for newborn screening, which acid, organic acid, and energy metabolism (Dott et al. can be shipped at room temperature once properly dried. 2006). However, in most cases, these conditions do It is important to underscore the fact that both speci- cause acute illness with obvious clinical symptoms that mens should be collected in order to detect patients who precede death by hours or days. The situation is signifi - may show mild or no apparent abnormalities in blood cantly different when considering the large number of alone (Rinaldo et al. 2005). In cases with a higher level cases affected with a fatty acid oxidation (FAO) disor- of suspicion, an effort should be made to collect a fro- der who were diagnosed either postmortem or, retro- zen specimen of liver (Boles et al. 1994) and a skin spectively, after the identifi cation of an affected sibling. biopsy, which could be kept frozen to reduce cell cul- The latter situation has become a relatively common ture workload (Gray et al. 1995). It has been possible to event since the achievement of greater awareness grow a viable line of cultured fi broblasts from a biopsy of the Achille’s tendon collected as long as 72 h after death. Although fatty infi ltration of the liver and/or other organs (heart and kidneys) is a common observation in FAO disorders, the fi nding of macroscopic steatosis cannot be used as the only criterion in deciding P. Rinaldo whether to investigate a possible underlying FAO Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, 200 First Street S. W., Rochester, MN 55905, USA disorder during the postmortem evaluation of a case e-mail: [email protected] of sudden death (Boles et al. 1998; Dott et al. 2006).

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 335 DOI: 10.1007/978-3-540-74723-9_D7, © Springer-Verlag Berlin Heidelberg 2010 336 P. Rinaldo

Fig. D7.1 Protocol for Sudden and unexpected death postmortem screening of fatty acid oxidation disorders with permission from Rinaldo et al (2002) • Retrieve newborn screening card for blood N Autopsy spot acylcarnitine analysis • Evaluation of siblings and parents

Blood and bile spots Risk N Liver N collected on filter paper Factors Steatosis (screening card) for acylcarnitine analysis

• Blood and bile spots on filter paper for acylcarnitine analysis • Frozen liver for Oil Red O stain, metabolic profile and carnitine • Fibroblast culture for in vitro enzymatic and molecular studies

Name of Deceased − Last Name, First Name, Middle Name Autopsy Number

Date of Birth Date of Death Time of Death Date of Autopsy Time of Autopsy Race or Ethnicity Blood Month Day YearMonth Day Year AM Month Day Year AM White Native American Asian PM PM Black Hispanic Other COMPLETELY FILL ALL CIRCLES.

ALLOW TO AIR DRY (3 HRS) Pathologist’s Name − Last Name, First Name Pathologist’s Phone Number Sudden, unexpected death Ye s No DO NOT HEAT Preliminary Diagnosis on Death Certificate

MML Client Number (if known) Client Name

Risk Factors Addltlonal Speclmens Available? Bile Ye s No Family history of sudden infant death syndrome (SIDS) or sudden unexplained death at any age Ye s No Liver Ye s No Family history of Reye Syndrome Ye s No Skin Ye s No Maternal complications of pregnancy (acute fatty liver of preganancy, HELLP syndrome, others) Ye s No Urine Bar Code Label Ye s No Lethargy, vomiting, fasting in the 48 hours prior to death Ye s No Plasma Ye s No Macroscopic findings at autopsy • Fatty infiltration of the liver and/or other organs Ye s No Other • Dilated or hypertrophic cardiomyopathy Ye s No Allegation of child abuse State of Birth Newborn Screening Card requested Ye s No Autopsy evidence of an infection not considered life-threatening Ye s No

Fig. D7.2 Example of ﬁ lter paper card for collection of postmortem blood and bile specimen

Special attention should be paid to the risk factors resuscitation efforts, which may still be available in listed in Table D7.1, and allegations of child abuse the clinical laboratory. In cases when no autopsy is should also be fully investigated, with the exception performed, retrieval of any unused portion of the of obvious cases of trauma/physical harm. The fro- blood spots collected for newborn screening could zen liver and skin biopsy could be discarded at a be arranged via a request submitted in writing to the later time without further testing when a credible local laboratory (for a template, see http://mayo- cause of death has been established, but could other- medicallaboratories.com/articles/communique/ wise be crucial to reach a proper diagnosis and con- archive.html), as long as the period of storage, rang- clusive conﬁ rmation in vitro. If parental permission ing from only a few weeks to indeﬁ nitely, had not to perform an autopsy is not granted, it might be pos- expired already in the state where the patient was sible to retrieve leftover specimens collected during born (http://www2.uthscsa.edu/nnsis/) D7 Postmortem Investigations 337

Table D7.1 Factors which increase the risk of an undiagnosed D7.1.3 Skin/Tendon for Fibroblast fatty acid oxidation disorder Culturing Birth at a location not yet providing expanded newborn screening by MS/MS Family history of sudden infant death syndrome or other In cases with any risk factors, a 5 × 5-mm skin speci- sudden, unexplained deaths at any age men should be collected and placed in culture media. Family history of Reye syndrome If culture media is unavailable, the specimen can be Maternal complications of pregnancy (acute fatty liver of pregnancy, HELLP syndrome) placed in sterile saline. Although saline is not an opti- Lethargy, vomiting, fasting in the 48 h prior to death mal media for skin, it will be suffi cient in most cases if Macroscopic fi ndings at autopsy the specimen is forwarded immediately for cell cultur- Fatty infi ltration of the liver and/or other organs ing. The skin specimen should be shipped at room Dilated or hypertrophic cardiomyopathy temperature via overnight delivery. Allegation of child abuse (excluding obvious cases of trauma, physical harm) Autopsy evidence of infection that routinely would not represent a life-threatening event D7.1.4 Urine

If urine is present, it should be collected and stored for D7.1 Specimen Requirements all cases of sudden, unexpected death. Urine may be collected on a second fi lter paper card by swabbing the D7.1.1 Blood and Bile bladder. The specimen should be allowed to dry for 2–3 h. The urine specimen should be stored at room temperature until the results of the postmortem screen Blood specimen collection is usually drawn into hep- on blood and bile, and the results from the original arin-containing tubes from the proximal aorta or by newborn screening card are available. intracardiac puncture. Bile collection is obtained by direct puncture of the gallbladder. These specimens are well preserved when spotted on a fi lter paper card. Two circles of the card are used for blood, two Key References circles for bile (each 25 mL of volume). Blood and bile have to be dried before sending the fi lter paper card to the laboratory which performs the analysis. Bennett MJ, Rinaldo P (2001) The metabolic autopsy comes of age. Clin Chem 47:1145–1146 Relevant demographic patient information should Boles RG, Buck EA, Blitzer MG et al (1998) Retrospective bio- be provided, together with a summary of relevant chemical screening of fatty acid oxidation disorders in post- autopsy fi ndings. mortem liver of 418 cases of sudden unexpected death in the fi rst year of life. J Pediatr 132:924–933 Boles RG, Martin SK, Blitzer MG et al (1994) Biochemical diagnosis of fatty acid oxidation disorders by metabolite analysis of post-mortem liver. Hum Pathol 25:735–741 D7.1.2 Liver Chace DH, DiPerna JC, Mitchell BL et al (2001) Electrospray tandem mass spectrometry for analysis of acylcarnitines in dried postmortem blood specimens collected at autopsy A liver specimen of approximately 1–5 g should be from infants with unexplained cause of death. Clin Chem 47: collected and stored in all cases of sudden, unexpected 1166–1182 death. The specimen should be frozen as soon as pos- Dott M, Chace D, Fierro M et al (2006) Metabolic disorders detectable by tandem mass spectrometry and unexpected sible either at −70 °C, if feasible or at −20 °C. The liver early childhood mortality: a population-based study. Am should not be fi xed or otherwise treated with a preser- J Med Genet A 140:837–842 vative. This specimen should be stored until the results Gray RGF, Ryan D, Green A (1995) The cryopreservation of of the postmortem screen on blood and bile, and the skin biopsies – a technique for reducing workload in a cell culture laboratory. Ann Clin Biochem 32:190–192 results from the original newborn screening card, are National Newborn Screening Report – 1997, National Newborn available. Screening and Genetics Resource Center (NNSGRC), 338 P. Rinaldo

Austin, TX, pp. 31–32. http://www2.uthscsa.edu/nnsis/ Rinaldo P, Matern D, Bennett MJ (2002) Fatty acid oxidation Accessed May 2001 disorders. Ann Rev Physiol 64(16): 1–26 Rashed MS, Ozand PT, Bennett MJ et al (1995) Diagnosis of The metabolic autopsy: postmortem screening in cases of inborn errors of metabolism in sudden death cases by acyl- sudden, unexpected death. Mayo Reference Services Communiqué. carnitine analysis of postmortem bile. Clin Chem 41: http://mayomedicallaboratories.com/articles/communique/ 1109–1114 archive.html. Accessed Sep 2003 Rinaldo P, Hahn SH, Matern D (2005) Inborn errors of amino Watson MS, Mann MY, Lloyd-Puryear MA, Rinaldo P, Howell RR, acid, organic acid, and fatty acid metabolism. In: Burtis CA, (eds) (2006) Newborn screening: toward a uniform screen- Ashwood ER, Bruns DE (eds) Tietz textbook of clinical ing panel and system (Executive summary). Genet Med chemistry and molecular diagnostics, 4th edn. Elsevier 8(Supplement):1S–11S Saunders, St. Louis, MO, pp 2207–2247 Function Tests D8 Johannes Zschocke

Key Facts hypoglycemia or suspected hyperinsulinism– › The fasting test has been partly superseded by hyperammonemia syndrome. improved diagnostic methods such as acylcar- › The phenylalanine challenge test may be useful for nitine analysis. Remaining indications include the identiﬁ cation of disorders of pterin metabolism in the differentiation of disorders of gluconeo- patients with unclear dystonic movement disorders, genesis from those with defective oxidation of in particular when Segawa syndrome is suspected.

pyruvate in patients with lactic academia; diag- › The tetrahydrobiopterin (BH4) test is intended

nostic work-up in patients with recurrent epi- to identify individuals with primary BH4 deﬁ -

sodes of symptomatic ketonemia, recurrent ciency or BH4-sensitive phenylketonuria.

cyclic vomiting, recurrent intermittent meta- › The vitamin B12 test aims at identifying respon- bolic acidosis, or the suspicion of ﬂ uctuating siveness to pharmacological doses of vitamin

neurometabolic disease; controlled determina- B12, found in some defects of 5-deoxyadeno- tion of fasting tolerance to ﬁ ne-tune the therapy sylcobalamin metabolism. of metabolic disorders; and the assessment of › The allopurinol test may be used for the diagno- the duration of safe intervals between feedings, sis of heterozygous or mild ornithine transcar- e g., in patients with mitochondrial disorders. bamylase deﬁ ciency in cases of unclear transient › The standardized determination of metabolic or intermittent hyperammonemia, unclear coma- parameters before and after meals is particu- tose or encephalopathic episodes in both sexes, larly useful in the diagnosis of mitochondrial or stepwise regressing neurodegenerative disease disorders, which typically show pathological in girls or women, especially those who show postprandial increases of lactate, alanine, and epilepsy and ataxia as prominent symptoms. other small amino acids. › The glucagon stimulation test is used as a pro- › A controlled glucose challenge is useful to vocative test for the diagnosis of glycogenosis assess cellular respiration in patients with sus- type I (von Gierke disease). pected disorders of energy metabolism, in whom › The major indication for the fructose tolerance lactate values have been repeatedly normal. test is a suspicion of hereditary fructose intol- › The leucine challenge test examines the insulin erance in patients in whom molecular analysis reaction to food intake and the function of the of the aldolase B gene failed to identify dis- blood sugar/insulin feedback mechanism and ease-causing mutations. may be useful in patients with postprandial › The forearm ischemia test examines both anaerobic glycolysis and the purine nucleotide cycle through measurement of lactate and ammonia concentrations before and after anerobic exercise. It may be useful in patients with muscle cramps J. Zschocke Divisions of Human Genetics and Clinical Genetics, Medical or other muscular symptoms on exertion. University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 339 DOI: 10.1007/978-3-540-74723-9_D8, © Springer-Verlag Berlin Heidelberg 2010 340 J. Zschocke

D8.1 General Remarks therefore is almost contraindicated in all patients with cardiomyopathy. Following a careful selection of patients and under careful observation it can be never- Many metabolic disorders show biochemical abnor- theless carried out safely. malities only intermittently under metabolic stress, and normal fi ndings in the interval may not reliably exclude a diagnosis in a particular patient. It is often possible to pinpoint the site or at least the area of a Remember patient’s metabolic abnormality by the use of a prop- A fasting may cause life-threatening complications erly chosen challenge. A variety of in vivo function and should only be carried out with careful moni- tests are used to create conditions that allow the assess- toring in a specialized hospital setting, and only ment of metabolism in a controlled manner. Frequently, after other, less risky investigations have been com- this entails ingestion of specifi c substances that give pleted without a clear diagnosis. rise to diagnostic metabolites in certain disorders. Most tests are fairly safe; many are inconvenient, but some (including the frequently performed fasting test) can lead to potentially serious complications and Fasting results in a series of hormonal and metabolic should only be carried out by experienced pediatri- responses to assure an endogenous supply of energy cians after other diagnostic options including mutation after cessation of exogenous intake. After each feed- analysis of candidate genes have been exhausted. It is ing, nutrients are supplied via gastrointestinal absorp- essential to carefully plan the collection of samples tion. Depending on the amount and composition of the during the test and to prepare emergency measures in food, this absorption period can last for up to 6 h in case complications occur. adults. It is more often <4 h in infants and small chil- Rapid advances in enzymatic, molecular, and other dren. With diminishing exogenous supply of glucose, diagnostic techniques permit an increasing number of plasma glucose concentrations fall and insulin levels diagnoses to be obtained without tests of tolerance. decrease in parallel. The decrease of insulin and However, functional studies may provide phenotypic increase of counteracting hormones diminishes glu- information that more closely refl ects the metabolic cose consumption in muscle and peripheral tissues. risk situation in the individual patient and will con- The body begins to utilize glycogen reserves by tinue to have their place in the diagnostic work-up as glycogenolysis. well as in tailoring therapy. In principle, hypoglycemia may be the consequence of endocrine or metabolic disease. The whole picture can therefore only be obtained if insulin, cortisol, and growth hormone as well as metabolic parame- D8.2 Tests of Fatty Acid Oxidation ters are measured simultaneously in patients who develop hypoglycemia. Patients with hyperinsulinism often have the shortest and sometimes variable fasting D8.2.1 Monitored Prolonged Fast tolerance. After 8–10 h of fasting, free fatty acids begin to sub- Monitored fasting is a powerful tool in unraveling stitute glucose as the primary energy source in muscle, the nature of metabolic disorders of energy metabo- while it often takes 17–24 h to deplete ordinary stores lism. It must, however, be emphasized that fasting is of glycogen. Two central metabolic adaptations to pro- potentially dangerous, and should only be carried out longed fasting are initiated in the liver. Glucose is syn- after other, less risky investigations have been com- thesized via glyconeogenesis from alanine and from pleted without a clear diagnosis. Fasting can cause oxaloacetate derived from amino acids as well as from life-threatening cardiac complications particularly in glycerol resulting from fatty acid oxidation; and most patients with long-chain fatty acid oxidation defects. importantly, fatty acid oxidation funnels into ketone Sudden cardiac arrest may occur even under controlled body production providing an alternative source of conditions in an intensive care unit and a fasting test energy. D8 Function Tests 341

may completely disappear with restoration of glucose Remember homeostasis. Patients with defects of glycogenolysis (glycogen storage disorders) often become hypoglycemic directly after the absorption period. Patients with Remember defects of gluconeogenesis may become hypoglycemic after 10–20 h of fasting, while those with defects Fatty acid oxidation defects should be ruled out of ketogenesis or ketolysis develop hypoglycemia at before a diagnostic fast by urinary organic acids and 15–24 h. acylglycine excretion patterns as well as analysis of plasma free and total carnitine, and especially acylcarnitine profi les in dried blood spots. Infants and small children may have shorter tolerance of fasting. In prolonged fasting, the body fi nally draws selectively on its lipid resources to spare vitally needed In fatty acid oxidation disorders, acylcarnitine profi les proteins. Depending on the nutritional state, an adult usually remain abnormal in the nonfasting state. with a defect in fatty acid oxidation may not become Phenylpropionic acid (PPA) loading may confi rm symptomatic until fasting has been prolonged for 36 h. medium-chain acyl-CoA dehydrogenase (MCAD) defi - In general, hypoglycemia following a short period of ciency or show an increased excretion of phenylhy- fasting signifi es disordered carbohydrate metabolism; dracrylic acid as a marker of long-chain hydroxyacyl-CoA hypoglycemia that occurs only after prolonged fasting dehydrogenase (LCHAD) defi ciency, but again this test signifi es disordered fatty acid oxidation or ketolysis. has been largely superseded by acylcarnitine analysis. Some patients with electron transport defects follow Analysis of fatty acid oxidation enzymes can be carried the prolonged pattern, because fatty acid oxidation out in leukocytes or fi broblasts. The diagnosis of MCAD may be impaired secondarily. defi ciency may be confi rmed in most patients of Free fatty acids can be oxidized in most body tis- European descent through the analysis of the common sues as an energy source. As they do not cross the mutation K329E in the MCAD gene. Early analysis of blood-brain barrier; homeostasis of the brain energy the common LCHAD gene mutation E510Q is indi- supply depends on adequate production of ketone bod- cated if the clinical and biochemical symptoms are sug- ies. Defects of fatty acid oxidation, in which ketone gestive of LCHAD defi ciency – progressive or episodic production may be impaired and toxic intermediates myopathy or cardiomyopathy and specifi c acylcarnitine are produced, may cause acute encephalopathy accom- pattern with or without bouts of hypoketotic hyoglyce- panied by hepatocelluar dysfunction, producing a mia and hydroxydicarboxylic aciduria. Reye-like syndrome. Determination of fasting tolerance through a moni- The fasting test has lost some importance with the tored fast is indicated in patients with recurrent episodes advent of acylcarnitine analyses in dried blood spots of apparently fasting-related symptoms, such as epi- and is now largely irrelevant if not contraindicated for sodes of decreased consciousness or especially recur- the diagnosis of fatty acid oxidation defects. These rent documented hypoglycemia or Reye-like disease in disorders frequently show clinical symptoms only at whom other analyses (including acylcarnitines) were times of fasting when there may be marked hypoke- inconclusive. In particular, patients with a defi ciency totic hypoglycemia and massive excretion of dicar- of mitochondrial 3-hydroxy-3-methylglutaryl-(HMG-) boxylic acids without appropriate ketones in the urine. CoA synthase, an isolated disorder of ketogenesis, may The single most important investigation is the deter- show a normal acylcarnitine profi le even during hypo- mination of free fatty acids (elevated) and ketone bod- glycemic episodes. A fasting test may reveal the typi- ies (no suffi cient rise) in a serum or plasma sample cal, hypoketotic hypoglycemia and a unique spectrum at the time of symptomatic hypoglycemia. Diagnostic of urinary organic acids. Primary mutation analysis is problems arise when the acute hypoglycemic illness the method of choice to confi rm the diagnosis. is treated without prior collection of at least a serum A fasting test is also useful in patients with lactic sample for analyses, as biochemical abnormalities acidemia to distinguish those with disorders of 342 J. Zschocke gluconeogenesis from those with defective oxidation everybody involved in advance. Special care must be of pyruvate (see Chap. B2.3). Other indications for taken to ensure complete collection of samples. It is controlled fasting include the following: advisable to fi ll out the forms and assemble and label all the tubes including day and timing of the samples • Recurrent episodes of symptomatic ketonemia. prior to the start of the test. • Recurrent cyclic vomiting. The aim, nature, and possible adverse effects of the • Recurrent intermittent metabolic acidosis. fasting test must be explained in detail to the patient • Suspicion of fl uctuating neurometabolic disease. and family to ensure optimal cooperation as well as • Controlled determination of fasting tolerance not early notifi cation of symptoms. Informed consent must only to fi ne-tune the therapy of metabolic disorders, be obtained from the parents prior to the test. as in patients with glycogen storage diseases and An intravenous line must be established in order to nesidioblastosis, but also to judge the duration of provide immediate access for the treatment of hypo- safe intervals in between feedings, e.g., in patients glycemia. It may be maintained with 0.9% NaCl or a with mitochondrial disorders. heparin lock.

D8.2.1.1 Preparations D8.2.1.2 Procedure A fasting test should be performed only if the patient is After baseline studies of plasma free fatty acids and in a stable, steady-state condition. Caloric intake for ketone bodies (acetoacetate and 3-hydroxybutyrate) the last 3 days should have been adequate for age. The blood sugar and urinary organic acids, fasting is con- test must be postponed in case of even minor intercur- ducted with careful bedside monitoring of the concen- rent illness. What is especially important is a detailed trations of glucose in blood and ketones in urine. The history with respect to the individual fasting tolerance, patient is allowed to drink water and unsweetened tea, and events and time courses of adverse reactions to but no juices or soft drinks including “diet” beverages. previous fasting. With this information and that of the Heart rate may be monitored by ECG. presumptive diagnoses, the timing of the fast can be An example of a fasting test of 24 h is given in Table optimally planned. The duration of the fasting period D8.2. Under normal conditions, ketogenesis is brisk may be scheduled according to the age of the child and especially after 15–17 h concentrations of acetoac- (Table D8.1) unless a shorter period is indicated by the etate and 3-hydroxybutyrate rise sharply, and gluco- clinical history. In general, it is usually safe to allow neogenesis occurs preserving normoglycemia. The fasting at night as long as the child usually goes with- fast is stopped at any time for the development of out eating, but the period beyond should take place hypoglycemia, and with close monitoring it is usually during the daytime. If hypoglycemia occurs, it should possible to avoid symptoms of hypoglycemia. be at a time of optimal stafﬁ ng. Blood samples should be obtained at 15, 20, and 24 h A monitored fast should be undertaken only in set- and always at a time of hypoglycemia when the fast is tings in which the entire staff is experienced with the stopped. The following basic laboratory parameters procedure, with close clinical supervision and well set should be measured: blood sugar, free fatty acids and out guidelines as to response to hypoglycemia or other ketone bodies (acetoacetate, 3-hydroxybutyrate), lactate, adverse events. Necessary details must be explained to electrolytes, blood gases, transaminases, and creatine kinase. In addition, dried blood spots must be obtained for the analysis of the acylcarnitine proﬁ le. Serum carni- Table D8.1 Suggested time periods for fasting in different age groups tine status and amino acids may be considered, and a Age < 6 6–8 8–12 1–7 > 7 spare serum sample should be obtained in case addi- Mmonths Mmonths Months years years tional tests may be indicated. Hormone studies includ- Starting 4 a.m. Midnight 7 p.m. 5 p.m. 3 p.m. ing insulin, glucagon, and growth hormone must be time carried out during hypoglycemia. Urine is collected in Duration 8 h 15 h 20 h 24 h 24 h 8 h aliquots for the quantitative determination of organic D8 Function Tests 343

Table D8.2 Twenty-four hour fast in a 6-year-old childx

Begin fast at time T = 0 h at 4 p.m. T(ime) = 0 h (4 p.m.) End of last meal with intake documented T = 1 h (5 p.m.) Serum glucose, electrolytes, creatine kinase. Blood gases. Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate from perchloric acid tube. Plasma amino acids, free fatty acids, and carnitine. Acylcarnitines. Start collection of urine in 8 h aliquots. Monitor ketones in urine at the bedside T = 1 h until T = 12h Blood glucose concentration is monitored 3-hourly at the bedside. From T = 12h (4 a.m.) onwards Collect glucose levels hourly. Heart rate may be monitored by ECG. T = 15 h (7 a.m.) Serum glucose, electrolytes. Blood gases. Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate. Plasma amino acids, free fatty acids T = 20 h (noon) Serum glucose, electrolytes. Blood gases. Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate. Plasma amino acids, free fatty acids T = 24 h (4 p.m.) or at the time of hypoglycemia Serum glucose, electrolytes, creatine kinase. Insulin, cortisol, growth hormone and additional specimen for follow-up hormonal investigations. Blood gases. Lactate, pyruvate, 3-hydroxybutyrate, acetoacetate. Plasma amino acids, free fatty acids. Acylcarnitines Quantitative analysis of organic acids is performed on the ﬁ rst and last aliquots. The middle collection is to ensure a sample for analysis in a patient developing hypoglycemia during that period aIn this sample case, no adverse effect to overnight fasting was to be anticipated from the history Parameters that are indispensible for evaluation and interpretation are highlighted in italics (see also Table D8.3)

acids. Hourly glucose determinations are made after the D8.2.1.3 Treatment of Adverse Reactions fi rst missed feeding and are repeated more frequently if the blood sugar falls <50 mg/dL. All urine passed should The fasting test must be terminated if the intravenous line be checked for ketones. Comprehensive investigation of is lost and cannot be immediately replaced or if the patient intermediary metabolites and hormones in blood and develops symptoms due to hypoglycemia or ketoacido- urine are especially important at the scheduled end of sis, such as irritability, sweating, and drowsiness. It should the fast or at the time of developing hypoglycemia, when also be discontinued at any sign of cardiac arrhythmia. the fast is terminated. The test is also terminated if hypoglycemia (blood glu- In the investigation of lactic acidemia fasting is cose <40 mg/dL or 2.3 mmol/L) or signifi cant metabolic employed to distinguish disorders of gluconeogenesis acidosis (bicarbonate <15 mmol/L) is documented. from defects of oxidative phosphorylation (see Chap. For treatment of hypoglycemia, 2 mL of 20% or B2.3). For this purpose, it is useful to give glucagon at 4 mL of 10% glucose/kg b.w. is given intravenously as 6 h into the fast (not in the evening or the night e.g. dur- a bolus followed by 3–5 mL 10% glucose/kg b.w./h ing a 24 hour fast). This may provide a presumptive until normoglycemia is restored and the patient retains diagnosis in a patient with glycogen storage disease oral food and fl uid. type I. In addition, it depletes the liver of glycogen derived from exogenous glucose, and as the fast continues, gluconeogenesis is required to keep from hypogly- D8.2.1.4 Interpretation cemia. A schedule is shown in Table 1 B2.3. If the suspicion for a defect of oxidative phosphorylation is If the patient develops hypoglycemia, the fasting test is especially high, it is possible to combine the fasting test abnormal. Accurate interpretation requires knowledge with a glucose and/or protein load as the last feed. of age-dependant hormonal and metabolic responses 344 J. Zschocke

Table D8.3 Control ranges of metabolites of energy metabolism in response to controlled prolonged fasting Glucose Ketones 3-OH-BA 3-OH-BA/ FFA FFA/ FFA/3- Glucose × Lactate L/P mmol/L mmol/L mmol/L Acetoacetate mmol/L Ketones OH-BA Ketones mg % Infants 15-h fasting 3.8–5.3 0.1–1.6 0.1–1.0 1.4–2.7 0.4–1.6 0.6–5.4 0.9–4.4 0.5–6 0.9–2.3 11–21 68–95 20-h fasting 3.5–4.7 0.6–3.5 0.4–2.5 1.7–3.1 0.6–1.4 0.3–1.7 0.5–2.1 3–12 0.8–2.0 12–19 63–85 24-h fasting 2.6–4.6 1.4–4.1 1.0–2.9 2.2–2.9 1.1–1.7 0.3–0.7 0.5–0.9 7–12 0.8–2.1 11–20 47–83 1–7 years 15-h fasting 3.5–5.1 0.1–2.2 <0.1–1.0 1.2–3.2 0.6–1.8 0.6–4.0 0.9–11 0.7–8 0.6–1.7 12–18 63–92 20-h fasting 2.8–4.5 1.0–3.8 0.6–2.9 2.3–3.3 0.8–2.6 0.4–1.7 0.4–2.1 4–12 0.5–1.8 10–19 50–81 24-h fasting 2.8–3.9 2.1–6.1 1.5–3.4 2.5–3.5 1.0–2.9 0.4–0.9 0.4–1.3 8–13 0.6–1.8 10–18 50–70 7–18 years 15-h fasting 3.8–5.3 < 0.1–0.8 <0.1–0.5 0.5–2.6 0.2–1.3 1.7–8 3.3–22 0.2–2.1 0.6–1.0 11–15 68–95 20-h fasting 3.5–4.7 0.1–1.5 <0.1–1.2 1.3–3.0 0.6–1.4 0.7–4.1 1.5–7.8 0.4–5.1 0.6–1.1 10–17 63–85 24-h fasting 2.6–4.6 0.7–4.1 0.5–1.6 1.5–3.1 0.9–1.8 0.5–2.0 1.1–2.4 2.4–8.1 0.4–1.0 8–18 47–83 FFA free fatty acids; Ketones sum of 3-hydroxybutyrate and acetoacetate; L/P lactate/pyruvate; 3-OH-BA 3-hydroxybutyrate. Glucose concentration are converted from mg% into mmol/l by division by 18 and vice versa The table has been modifi ed from the work of Bonnefort et al. (1990) following the grouping of subjects and parameters considered and expanding the ranges from experiences with an additional 25 control subjects studied at the authors departments to fasting as well as reference values from control indi- Reliable quantifi cation of acetoacetate and pyru- viduals. Table D8.3 is based on experience with close vate is diffi cult. For the evaluation of the fasting to 100 control subjects from two published series and response, ketone bodies (in particular 3-hydroxybu- additional control subjects, which were selected after tyrate) may be measured in a plasma or serum sample. extensive metabolic investigations failed to give any However, the ratio of 3-hydroxybutyrate to acetoace- indication of an inherited metabolic disease. tate is important in the work-up of a suspected defect In older children and adults, ketone body produc- of pyruvate metabolism or oxidative phosphorylation, tion may not be maximal until the 2nd or 3rd day of and analysis should be carried out in deproteinized fasting because of greater stores of glycogen and effi - blood samples (perchloric acid extraction) if such a cient gluconeogenesis. Prolonged fasting extended up disorder is suspected. Additional clues to a defect of to 36 or even 48 h may be justifi ed in adults. Infants pyruvate carboxylase are bouts of hyperammonemia and small children have much lower glycogen stores and elevated levels of citrulline and lysine on amino and higher capacities to form and utilize ketone bod- acid analysis. ies. Signifi cant ketone body production occurs before A good indicator of blood pyruvate concentrations 24 h. Interestingly, infants show an intermediate is the simultaneously determined level of alanine in response to fasting as compared to toddlers and older plasma. An alanine level of 450 mmol/L corresponds to children. This can be explained by larger glycogen a blood pyruvate of 100 mmol/L, i.e., the upper limit of stores in infants because of high calorie meals at regu- the normal range. Other amino acids to be evaluated at lar intervals. Ketone body production for children the end of a fasting test are the branched-chain amino older than seven was variable and in some, only mod- acids isoleucine, leucine, and valine that are physio- erate even after 24 h (see Table D8.3). Metabolic logically elevated during acute starvation. However, if defects of ketogenesis or ketolysis would still be the increase becomes excessive and allo-isoleucine expected to be diagnosed after 24-h fast. detectable, a variant of maple syrup urine disease may D8 Function Tests 345

Table D8.4 Response to fasting in disorders of carbohydrate and energy metabolism Presumptive Glucose Blood ketones 3-OH-butyrate/ FFA FFA/ketones Lactate L/P diagnosis acetoacetate Glycogenosis I ØØ N − ¯ N N N − ≠≠ N Glycogenosis III, VI ¯ NNNNN and 0 Defects of ØØ N − ¯ N N N − ≠≠ N gluconeogenesis Defects of fatty acid ØØ ØØ N − ¯ N ≠≠ N oxidation Defects of ketolysis − N − ¯ ≠≠ NNØ NN Defects of pyruvate ¯ Ø NN ≠≠ Ý carboxylase Defects of pyruvate ¯ N − ¯ NNN − ≠≠ N dehydrogenase Defects of oxidative N − ¯ − N N N − ≠≠ ≠ − ≠≠ phosphylation FFA free fatty acids; L/P lactate/ pyruvate ratio; N normal values (see Table D8.3) pathologically elevated and ¯ decreased values Parameters of specifi c diagnostic value are highlighted in bold and larger font be the cause for recurrent episodes of hyperketotic hormones. Single growth hormone determinations in hypoglycemia. response to hypoglycemia are of little value for the A diagnostic algorithm of differential metabolic diagnosis of growth hormone defi ciency, and different responses to fasting in disorders of carbohydrate and provocative tests should be employed if the diagnosis is energy metabolism is elaborated in Table D8.4. In gen- clinically suspected. Catecholamine defi ciency on the eral, a high level of free fatty acids and low levels of basis of dopamine-b-hydroxylase defi ciency or tyrosine 3-hydroxybutyrate and acetoacetate indicate a disorder hydroxylase defi ciency is an exceedingly rare condition of fatty acid oxidation. In defects of ketolysis, an ele- with a tendency to hypoglycemia. It can be ascertained vated product of blood glucose times ketones during in patients primarily suffering from severe orthostatic fasting is the most suggestive parameter. A summary hypotension by analysis of catecholamines in urine and of metabolic and hormonal disorders in which patho- biogenic amines in CSF. logical responses are elucidated by fasting is given in A presumptive diagnosis of glycogen storage dis- Table D8.5. eases is usually made before the diagnostic fast on the Congenital hyperinsulinism (previously described basis of signifi cant hepatomegaly and repeated bouts as nesidioblastosis or persistent hyperinsulinemia of of hypoglycemia. Fatty acid oxidation requires the infancy), the most common cause of persistent symp- coordinated action of at least 17 different enzymes and tomatic hypoglycemia in neonates and small infants, one additional transport protein. In each metabolic leads to hypoketotic hypoglycemia with low levels of center, there are patients with defi nitive diagnoses of free fatty acids. This disorder can be due to defects of the defective fatty acid oxidation, in whom the exact enzy- sulfonylurea receptor. The same constellation of neona- matic defect could as yet not be determined. In addi- tal hypoglycemia and impaired lipolysis can be caused by tion, there are a number of enzymes involved in fatty hyperproinsulinemia. In such patients, insulin levels are acid oxidation and ketolysis for which no human low and proinsulin grossly elevated. defects have yet been discovered. Diagnosis of hormonal disorders depends on the There are otherwise completely healthy children who correct collection of specimens during fasting and inter- can develop severe metabolic decompensation with pretation in connection with the blood glucose concen- excessive ketosis with or without hypoglycemia during trations. Diagnosis needs to be ascertained by repeated intercurrent illnesses. In these children, similar reactions determinations of insulin or detailed studies of pituitary can be provoked by prolonged fasting. Although this function and additional investigations in patients with is not a homogenous group of patients, an exaggerated insuffi ciency of one or more of the counteracting uncoordinated production of ketone bodies and signifi cant 346 J. Zschocke

Table D8.5 Differential diagnosis of pathological responses to fasting Disorder Response to fasting Diagnostic markers (in addition to hypoglycemia) Fatty acid oxidation disorders Hypoketotic hypoglycemia Free fatty acids/ketones >2 Carnitine defi ciency, except for CPT I with elevated free carnitine. Dicarboxylic aciduria Variable elevations of lactate Acute illness – increased creatine kinase and uric acid Glycogenoses I, III and VI Hypoglycemia Massive hepatomegaly. Variable elevations of lactate, urate, cholesterol, triglycerides, creatine kinase, and transaminases. Hypophosphatemia Defects of gluconeogenesis Hypoglycemia Hepatomegaly. Elevations of lactate Defects of ketolysis Hyperketotic hypoglycemia Persistently elevated free fatty acids and ketonesa. Ketones × glucose >15 (fasting) Mitochondrial disease Hyperketotic hypoglycemia Multisystem disease. Elevations of lactate and ketones as well as of L/P and 3-OH-BA/acetoacetate ratios Maple syrup urine disease Hyperketotic hypoglycemia Maybe ataxia. Elevations of branched-chain amino (intermittent variant) acids including allo-isoleucine Congenital hyperinsulinism Hypoketotic hypoglycemia Increased insulin levels >5 mU/L at glucose <30 mg% (nesidioblastosis) or >8 mU/L at glucose <40mg%. Insulin (mU/L)/ glucose (mg%)/ >3. Low-free fatty acids and ketones Hypocortisolism, growth hormone Hypoketotic hypoglycemia, but Cortisol <400 nmol/L. Defi ciency of growth and/ or defi ciency, panhypopituitarism sometimes signifi cant ketosis thyroid hormone in patients with Addison disease Catecholamine defi ciency Hypoketotic hypoglycemia Decreased catecholamines Cyclic vomiting (diminished Hyperketotic hypoglycemia No specifi c abnormalities glycogen and protein stores) CPT I carnitine palmitoyl transferase I defi ciency; Ketones sum of 3-hydroxybutyrate and acetoacetate; L/P lactate/ pyruvate; 3-OH-BA 3-hydroxybutyrate aIn normal fed children, the concentration of ketone bodies in the steady state are always <0.2 mmol/L metabolic acidosis leading to nausea and protracted D8.2.2 Phenylproprionic Acid vomiting appears to be a common pathogenetic mechanism. The susceptibility to these reactions slowly dimin- Loading Test ishes with age but may persist into adolescence and young adulthood. True hypoglycemia is uncommon in PPA is a nontoxic substance that, through a single these children, who appear to have a defect in ketone round of b-oxidation, is normally converted to hippu- utilization. This condition is diffi cult to distinguish from ric acid, which is excreted in the urine. PPA oxidation abdominal migraine. Relatively low stores of fat and gly- is catabolized by enzymes that are specifi c for medium- cogen appear to be another common denominator for chain acyl-CoA compounds, and the PPA loading test such exaggerated ketogenesis. Affected children are is traditionally performed to confi rm MCAD defi - often slim and have relatively low muscle mass. Children ciency. Like the other tests of b-oxidation function, the with pathological muscular wasting, such as patients PPA loading test has now been largely superseded by with spinal muscular atrophy, who have severely dimin- acylcarnitine analysis in conjunction with enzyme ished glycogen and protein stores, are at especially high studies or mutation analyses. Indeed, it has been shown risk for metabolic decompensation during fasting. that the PPA loading test can be normal in attenuated Similar metabolic constellations occur in milder forms MCAD defi ciency, which is still picked up by acylcar- of succinyl-CoA:3-oxoacid-CoA transferase defi ciency. nitine analysis in dried blood spots. D8 Function Tests 347

D8.2.2.1 Procedure Many biochemical parameters show marked variation with food intake and should be routinely examined under The test is usually carried out in the morning (fasting) preprandial conditions in order to determine baseline values. Reduced concentrations of amino acids or other • Obtain urine sample before PPA administration for metabolites may be diagnostically relevant but are some- organic acid analysis. times only found in preprandial samples. On the other • PPA is given orally in a dose of 25 mg/kg. The sub- hand, some metabolic disorders that affect substrate uti- stance is not licensed as a medical drug but may be lization give rise to abnormal metabolite concentrations obtained from metabolic laboratories. It has an only in the postprandial state. In order to reliably detect unpleasant cinnamon taste and should be mixed, relevant abnormalities it is often necessary to examine e.g., with jam or administered in 50–100 mL tea metabolic parameters before and after defi ned food through a nasogastric tube. The child should be intake. In practice, this may be a normal meal enriched encouraged to drink plenty of water after ingestion with protein and carbohydrates, the exact composition of of PPA and is allowed normal meals. which is less important. This test is particularly useful in • Collect urine for 6 (–12) h after PPA administration the diagnosis of mitochondrial disorders which typically for organic acid analysis. show pathological postprandial increases of lactate, alanine, and other small amino acids. Alanine, in contrast to lactate, is not affected by cuffi ng or crying and when D8.2.2.2 Interpretation elevated is a more reliable indicator of disturbed energy metabolism. This test is also able to recognize amino Normal activity of medium-chain b-oxidations is indi- acidemias and urea cycle defects but may trigger or cated by excessive excretion of hippuric and benzoic aggravate acute neurological symptoms. acids in urine, while marked excretion of phenylpropi- onylglycine and insuffi cient hippuric acid is indicative of MCAD defi ciency. Increased excretion of phenylhy- D8.3.1.1 Procedure dracrylic acid is another pathological response characteristic of LCHAD defi ciency. However, the diagnostic • Preprandial samples should refl ect a neutral metabolic sensitivity is far lower than in MCAD defi ciency. state (not a fasting reaction) and are best obtained 5–6 h Insuffi cient rise of hippuric acid without other abnor- (up to 8 h in older children) after the last meal. Measure malities indicates insuffi cient PPA intake and thus blood gases, blood sugar, amino acids, lactate, and makes the test results invalid. ammonia; obtain a deproteinized blood sample (perchloric acid extraction) for pyruvate and ketone bodies in case that lactate is elevated. In urine, check for D8.3 Tests of Energy Metabolism ketones (ketostix) and measure lactate and/or organic acids and orotic acid if not previously normal. • Give normal meal enriched with protein and sugar D8.3.1 Preprandial/Postprandial to attain a total amount of »1 g/kg b.w. each of pro- Analyses (Protein/Glucose tein and carbohydrate/sugar. Challenge) • Postprandial blood sample should be obtained 90 min after the meal; urine should be collected for 2 h. Measure the same parameters as in the preprandial samples. Remember The standardized analysis of metabolic parameters D8.3.1.2 Interpretation in the preprandial and postprandial state may provide important functional clues for the diagnosis of Blood lactate should not rise by >20% over base- metabolic disorders. line values and should not reach pathological values 348 J. Zschocke

(>2.1 mmol/L). When lactate is elevated, measure metabolic decompensation. In such cases, appropri- pyruvate, acetoacetate, and 3-hydroxybutyrate in the ate enzyme studies (muscle biopsy) and possible perchloric acid extract to determine the redox ratio. molecular analyses should be undertaken immedi- Acid–base status should remain normal. Most amino ately upon completion of the basic biochemical ana- acids will be elevated in the postprandial sample, but lyses. Other indications include unclear hypoglycemic the plasma concentration of alanine should stay under episodes and suspected glycogen storage disease 600–700 mmol/L and the alanine/lysine ration should (see Chap. B2.3). stay below three.

D8.3.2.1 Preparations D8.3.2 Glucose Challenge • Basic investigations including amino acids and organic acids should have been completed; blood lactate should have been normal in repeated measurements Remember before and after meals at different times of the day. A controlled glucose challenge is useful to assess • Glucose challenge should be carried out after over- cellular respiration in patients with suspected disor- night fasting in the morning, in younger infants at ders of energy metabolism in whom lactate values least 4–5 h after the last meal. have been repeatedly normal. • Secure intravenous access.

For aerobic generation of energy, glucose is catabo- D8.3.2.2 Procedure lized to pyruvate, transferred into the mitochondrion and fully oxidized via the Krebs cycle and the respira- • Measure baseline blood lactate, blood sugar, and tory chain. High lactate (the reduced form of pyruvate) acid–base status; obtain 10 mL urine for lactate and/ is the most valuable diagnostic marker of disturbed or organic acids before test. mitochondrial energy metabolism in respiratory chain • Give glucose 2 g/kg (max. 50 g) as 10% oral solu- defects and other disorders that affect cellular respiration. The solution may be administered through a tion. However, frequently lactate is elevated only after nasogastric tube (fl ush with water) in small chil- intake of glucose or glucogenic amino acids; single dren; for administration in older children, the solu- normal lactate values do not exclude a primary mito- tion may be stored in the refrigerator as it is more chondriopathy (see Chap. B2.3). A controlled glucose pleasant to drink when cool. challenge is useful to assess cellular respiration in • Measure blood lactate, blood sugar, and acid–base patients with suspected disorders of energy metabo- status 15, 30, 45, 60, 90, 120, and 180 min after the lism in whom lactate values have been repeatedly nor- test; collect urine for 2 h for lactate and/or organic mal. It is relatively inexpensive as lactate can be acids. Obtain deproteinized blood samples (per- measured in all general and pediatric hospitals but it chloric acid extraction) for pyruvate and ketone requires frequent venipunctures and lactate concentra- bodies in case lactate is elevated. tions may be affected by cuffi ng or crying of the child. The measurement of pyruvate is not necessary when lactate is normal, but deproteinized blood samples D8.3.2.3 Interpretation (perchloric acid extraction) should be obtained to determine the redox ration (lactate/pyruvate) when Blood sugar should be elevated after the test, but lac- lactate is high. A glucose challenge should not be car- tate should not rise by >20% over baseline values and ried out when lactate has been consistently elevated or should not reach pathological values (>2.1 mmol/L). when a signifi cant postprandial increase of lactate Acid–base status and urine measurements should has already been demonstrated as it may cause acute remain normal. D8 Function Tests 349

D8.4 Tests of Protein Metabolism D8.4.2 Phenylalanine Loading Test

D8.4.1 Leucine Loading Test The hydroxylation reactions of phenylalanine, tyrosine,

and tryptophan require tetrahydrobiopterin (BH4) as a cofactor. A defi ciency in the biosynthesis or recycling Insulin secretion from the pancreas is controlled by a of BH may cause reduced synthesis of monoamine complex system involving several enzymes and trans- 4 neurotransmitters. Frequently, this is not noticeable in membrane carriers. In some persons, inappropriate plasma amino acid concentrations but can be demon- insulin release is triggered by food intake, and more strated in the kinetics of phenylalanine hydroxylation specifi cally by the ingestion of large amounts of leucine after oral challenge. The phenylalanine challenge test (leucine-sensitive hypoglycemia). The leucine chal- may be useful for the identifi cation of disorders of lenge test examines the insulin reaction to food intake pterin metabolism in patients with unclear dystonic and the function of the blood sugar/insulin feedback movement disorders, in particular when Segawa syn- mechanism and may be useful in patients with postpran- drome is suspected. dial hypoglycemia or suspected hyperinsulinism–hyperammonemia syndrome. It is important to appreciate, however, that some patients with hyperinsulinism syndrome may suffer severe, life-threatening hypoglycemia after quite small amounts of leucine, and appropriate Remember emergency measures must be prepared before the test is The phenylalanine challenge has little use in the started. A leucine challenge is not suitable for the diag- diagnostic work-up of patients with phenylketonuria nosis of maple syrup urine disease. In these circum- (PKU). stances allo-isoleucine would be used, which does not cause severe symptoms in affected patients.

D8.4.2.1 Procedure D8.4.1.1 Procedure The phenylalanine challenge should be carried out at The test is carried out in the morning after overnight least 1 h after a light breakfast (minimal protein). No fasting. Insert an intravenous cannula and prepare an food is permitted until the end of the test. Obtain two intravenous 20% glucose solution for emergency appli- separate samples of 1 mL EDTA blood for the deter- cation in case of severe hypoglycemia. mination of basal values of phenylalanine, tyrosine, • Obtain blood samples for baseline measurements of and plasma pterins. For pterin analysis, it is impor- blood sugar, insulin, and ammonia. tant to immediately centrifuge the samples and • Give l-leucine orally in a dose of 150 mg/kg or freeze the plasma in two portions. Plasma should be intravenously in a dose of 50 (75) mg/kg. stored at −70 °C or sent on dry ice to the metabolic • Determine blood sugar, insulin, and ammonia 15, laboratory. 30, 45, 60, and 90 min after leucine challenge. • Give 100 mg/kg l-phenylalanine in orange juice, if necessary through a nasogastric tube. Do not use D8.4.1.2 Interpretation drinks containing protein or aspartame to mix. Phenylalanine does not dissolve well; therefore, stir Patients with leucine-sensitive hyperinsulinism usu- the mix just before drinking, and rinse the residual ally develop hypoglycemia caused by excessive insu- phenylalanine with additional juice. lin secretion (>3 mU/L at blood sugar <2.0 mmol/L) • Obtain blood samples 1, 2, 4, and 6 h after phenyla- approximately half hour after leucine administration. lanine ingestion; again centrifuge and freeze plasma Concomitant hyperammonemia is indicative of a defect immediately in two portions for the analysis of phe- of glutamate dehydrogenase. nylalanine/tyrosine and pterins. 350 J. Zschocke

D8.4.2.2 Interpretation classical PKU but may rapidly normalize phenylalanine in patients with a primary defect of pterin synthe- Plasma phenylalanine levels should rise sharply after sis or recycling. Also in many patients with mild or ingestion with a maximum around 60 min and then moderate PKU, oral administration of BH4 enhances decline continuously through conversion of phenylala- PAH activity and thus reduces plasma Phe concentra- nine into tyrosine. Biopterin concentrations should rise tions. The BH4 test is intended to identify individuals several fold above baseline (to >18 nmol/L). Plasma with either of these conditions, i.e., primary BH4 defi - concentrations of phenylalanine should not exceed ciency or BH4-sensitive PKU. tyrosine more than fi vefold after loading. A protracted As responsiveness to BH4 may be of immediate rise and slow decrease of phenylalanine together with therapeutic relevance, a BH4 test should be carried out a delayed rise of tyrosine indicate a reduced hydroxy- prior to the introduction of a Phe-reduced diet in all the lation capacity, which may be caused either by pres- neonates with plasma phenylalanine concentration ence of a (heterozygous) mutation in the phenylalanine above 400 mmol/L (6.5 mg/dL). For the reliable exclu- hydroxylase (PAH) gene or by reduced availability of sion of cofactor defi ciency, it is also necessary to deter-

BH4. Further differentiation is possible by examination mine pterin concentrations in urine and to measure of pterins in blood, mutation analysis of the PAH gene, dihydropteridine reductase (DHPR) activity in a ﬁ lter or repeating a modiﬁ ed phenylalanine challenge. This paper card (Guthrie card), which is usually sent time BH4 (20 mg/kg b.w.) is administered 1 h before or together with the samples of the BH4 test to the meta- alternatively 3 h after phenylalanine (100 mg/kg b.w.). bolic laboratory. The BH4 test is not reliably performed

In case of reduced availability of BH4, administration when phenylalanine levels are <400 mmol/L as the of BH4 prior to phenylalanine results in a complete therapeutic effect in cofactor deﬁ ciency is more difﬁ - normalization of the test. If BH4 is given after 3 h into cult to recognize, and most MHP patients show BH4 the test, an immediate decrease of phenylalanine occurs sensitivity, which, anyway, is not of therapeutic rele- together with a rise of tyrosine leading to normaliza- vance due to the harmless nature of the condition. tion of metabolite levels (see later). In the latter setting, plasma amino acids are determined after another 1, 3, and 5 h, i.e., amino acids are determined prior to Remember and 1, 2, 4, 6, and 8 h after phenylalanine loading. A BH4 test in conjunction with Phe loading is not recommended since interpretation is unreliable unless results are compared with the results of Phe loading without BH administration. D8.4.3 BH4 Test 4

Neonatal screening in most Western countries includes the diagnosis of PKU, the severe defi ciency of the D8.4.3.1 Procedure hepatic enzyme PAH, which untreated causes highly elevated phenylalanine (Phe) concentrations in blood • Collect 5–10 mL urine for pterin analysis, protect (hyperphenylalaninaemia) and mental retardation. against light (urine may need to be collected in a dark More than 600 variants or mutations in the PAH gene bag) and freeze. Obtain 2 mL EDTA full blood, centri- with variable effect on enzyme function have been fuge, and freeze plasma for amino acid analysis. identifi ed, and depending on dietary Phe tolerance, Collect a few drops of blood on a fi lter paper card severe, moderate, and mild forms of PKU have been (Guthrie card) for the analysis of Phe and tyrosine distinguished. The attenuated form of PAH defi ciency (Tyr) as well as DHPR activity. Plasma and urine sam- that does not require treatment is denoted “mild hyper- ples should be sent on dry ice to the laboratory. phenylalaninemia” (MHP). PAH requires BH4 as a Alternatively, urine may be oxidized with MnO2 and cofactor, and hyperphenylalaninemia is occasionally sent at ambient temperature by express mail (for this due to a primary defi ciency of BH4 biosynthesis. Oral purpose acidify 5 mL urine with 6 M HCl up to a pH administration of BH4 does not signifi cantly reduce of 1.0–1.5, add 100 mg MnO2, shake for 5 min at plasma phenylalanine concentrations in newborns with ambient temperature, centrifuge for 5 min at 4,000 rpm, D8 Function Tests 351

and mail supernatant protected against light in alumi- mitochondrial enzyme methylmalonyl-CoA mutase num foil or in a dark container). (MCM, EC 5.4.99.2) or by one of the many defects in

• Give 20 mg/kg BH4 diluted in water 30 min before a the uptake, transport, or synthesis of 5-deoxyadenosyl-

normal meal. Beware of the photosensitivity of BH4. cobalamin, the cofactor of MCM, the active metabolite

• Obtain dried blood spots or plasma samples 1, 4, 8, of vitamin B12. Primary deﬁ ciencies of MCM are fur-

and 24 h after BH4 administration for Phe and Tyr ther subdivided into defects without residual activity analysis. Collect urine 4–8 h after administration of (mut0) and defects with residual activity (mut−), caused

BH4 for pterin analysis (samples should be prepared by mutations in the apomutase locus or in genes cod- in the same way as the baseline samples). ing for the biosynthesis of cobalamin: cblA, cblB, cblC, cblD, cblD (variant 1 and 2), cblE, cblF, and cblG. The infant or older patient may be fed or may eat regu- MCM situated in the mitochondrium is part of the cat- larly throughout the test. abolic pathways of the amino acids isoleucine, valine, methionine, and threonine as well as of odd chain fatty D8.4.3.2 Interpretation acids, propionic acid coming from gut bacteria and cholesterol linking the degradation of these metabolites to the Krebs’ cycle. The BH4 test is regarded as positive if Phe concentrations fall by at least 30%; this should usually correspond Some defects of 5-deoxyadenosylcobalamin metabolism respond to pharmacological doses of vitamin B . with a rise of Tyr concentrations. In primary BH4 defi - 12 ciency, Phe concentrations rapidly normalize within the While cblC and cblD defects are routinely treated by vitamin B , only some patients with cblA and cblB or fi rst few hours after BH4 administration. The differential 12 diagnosis should be clarifi ed through pterin analysis: even with mut− disease will respond. Although response

to vitamin B12 has been identifi ed in many studies as an • GTP cyclohydrolase I defi ciency: both neopterin important prognostic factor and hallmark of therapy and biopterin absent (or very low). (Hörster et al. 2007), there is a large variation of prac- • 6-Pyruvoyl-tetrahydropterin synthase defi ciency: tice at least in European centers. Therefore, Fowler elevated neopterin but absent (or very low) biopterin. et al. (2008) have proposed the following protocol to • Pterin-4a-carbinolamin dehydratase defi ciency: ele- test the response to vitamin B in a standardized way. vated concentrations of various pterins, specifi cally 12 primapterin. • DHPR defi ciency: markedly elevated biopterin, normal D8.4.4.1 Procedure or mild elevation of neopterin, low DHPR activity. Patients with (severe) PKU also show increased uri- • The patient should be clinically stable on the same nary excretion of biopterin and neopterin, with neop- treatment for at least 1 month. The intakes of protein terin usually more markedly elevated than biopterin. A and energy should be specifi ed and recorded. • If the patient is already receiving cobalamin, this positive BH4 test in PKU patients may indicate BH4 sensitivity, but the therapeutic relevance of this phe- should be stopped for at least 1 month before the nomenon in the individual case has not yet been fully test. If the patient appears to deteriorate, restart vita- clarifi ed. min B12 and defer the test. Note a general rule that patients with MMA excretion >10,000 mmol/mol creatinine and those who are clinically unstable rarely respond to vitamin B . D8.4.4 Vitamin B Test 12 12 • Baseline urine collections: at least three specimens should be collected on different days. Plasma con- Friederike Hörster centrations may only be used if a sensitive assay (stable-isotope dilution assay) is available. Methylmalonic acidurias comprise a heterogenous • Give hydroxocobalamin 1 mg intramuscularly on group of diseases with accumulation of methylmalonic three consecutive days. acid (MMA) in urine and other body fl uids as the com- • After the cobalamin injection, collect urine (or mon denominator. They are caused by a defect of the plasma) specimens on alternate days for 10 days. 352 J. Zschocke

• The urine or plasma samples should be analyzed in those who show epilepsy and ataxia as prominent the same run in a laboratory participating in a rec- symptoms. ognized quality control scheme for MMA using gas chromatography–mass spectrometry, in Europe ERNDIM EQA (http:// www.erndim.unibas.ch). D8.4.5.1 Procedure and Interpretation

Avoid caffeine (decaffeinated coffee is acceptable), tea, coffee, cocoa, chocolate, chocolate biscuits, any cola D8.4.4.2 Interpretation drink, or benzoate-containing beverages 24 h before the test. Otherwise, there is no need for a special diet prior A decrease of the mean urine and plasma MMA con- or through the test. Women should be 7–12 days after centrations of >50% should be regarded as indicative their last menstrual period if possible. The test is usu- of a benefi cial response. ally started in the morning. In parallel to this test, the enzymology including in vitro response to adenosylcobalamin and if possible • Collect 10 mL urine for baseline measurement of the complementation group should be determined. orotic acid and orotidine, which should be done by high-pressure liquid chromatography and not by a colorimetric method. D8.4.5 Allopurinol Test • Give allopurinol orally in a dose of 100 mg for preschool children, 200 mg for children between 6 and 10 years of age, or 300 mg for older children and Various genetic disorders especially urea cycle defects adults. show increased urinary excretion of the pyrimidine • Collect urine over 24 h in four 6-h fractions (0–6 h, uridine or its precursor orotic acid. In the case of 7–12 h, 13–18 h, and 19–24 h); send 10 mL of each urea cycle defects, this is thought to be caused by fraction. The samples should be sent together fro- mitochondrial accumulation of carbamoylphosphate, zen or, after conservation with three drops of chlo- which is transferred into the cytosol and channeled roform, at ambient temperature by express mail. It into pyrimidine biosynthesis, thus bypassing the rate- is important to label sample tubes accurately and to limiting first step in that pathway catalyzed by the inform the laboratory of all medication taken dur- cytosolic enzyme carbamoylphopsphate synthase II ing the test as well as on the preceding days. (CPS II) (see Chap. B2.6 and Fig. B2.6.1). CPS II is different from the mitochondrial isoenzyme CPS I, Remember which is required for the detoxification of ammonia. Orotic aciduria is a diagnostic feature particularly Both false positive and false negative results of the in ornithine transcarbamylase (OTC) deficiency allopurinol test have been reported. which otherwise lacks specific amino acid changes. Nevertheless, orotic acid may be normal in the inter- Excessive rise of orotic acid and/or orotidine indicates val in mild forms of OTC defi ciency and particularly increased throughput in pyrimidine synthesis as typi- in heterozygous carrier women. In these cases, it may cally caused by mild (heterozygous) OTC defi ciency. be possible to demonstrate an increased through- Positive tests can also be found in other genetic disor- put in pyrimidine biosynthesis through an excessive ders including Rett syndrome, amino acid transport rise of orotic acid and its metabolite orotidine after defects, creatine synthesis disorders, and mitochon- allopurinol-mediated blockage of uridine monophos- drial disorders. A negative allopurinol test (or normal phate synthase, the enzyme that converts orotic acid to orotic acid after protein challenge) does not fully uridine monophosphate. The allopurinol test may be exclude heterozygous OTC defi ciency as mosaicism in used for the diagnosis of heterozygous or mild OTC the liver (lyonization) may be skewed in favor of nor- defi ciency in cases of unclear transient or intermittent mal hepatocytes to a degree that renders the detection hyperammonemia, unclear comatose or encephalo- of metabolic effects impossible. OTC gene mutation pathic episodes in both sexes, or stepwise regressing analysis should be considered if OTC defi ciency neurodegenerative disease in girls or women, especially remains a possibility. D8 Function Tests 353

D8.5 Tests of Carbohydrate Metabolism In patients with glycogen synthase defi ciency, glucagon administration in the fasting state is followed by no elevation of glucose, lactate, or alanine. In the fed D8.5.1 Glucagon Stimulation state, glucagon is followed by a glycemic response. These patients can be distinguished from those with Glucagon is a counterregulatory hormone whose secre- glycogenosis III by the elevation of lactate that occurs tion is normally stimulated by hypoglycemia. It acts to with a glucose tolerance test. stimulate hepatic glycogenolysis. It does this by stimulating phosphorylase, but glucose-6-phosphatase activity is required for release of free glucose into the blood. Thus, the glucagon stimulation test is an excellent pro- D8.5.2 Fructose Loading Test vocative test for von Gierke disease, glycogenosis type I, in which the hepatic activity of this enzyme is in most patients absent or nearly absent (see also Chap. B3.3). Remember The test is usually done following a fast for at least Mutation analysis is the primary method of choice 6 h. An overnight fast is usually employed in normal for the diagnosis of hereditary fructose intolerance. individuals. The duration of fasting in an infant with glycogenosis I depends on tolerance and may have to be 4 h or less. The dose of glucagon we generally employ The major indication for the fructose tolerance test is a is 0.5 mg intramuscular. Doses of 0.03–0.1 mg/kg have suspicion of hereditary fructose intolerance in patients in been used up to a maximum of 1 mg. We measure blood whom molecular analysis of the aldolase B gene failed concentrations of glucose at time 0 and 15 min intervals to identify disease-causing mutations. Evaluation of the after injection for 60 min, at 90 and 120 min. Lactate response to fructose loading can also provide valuable and alanine may also be measured in each sample in a diagnostic information for patients with suspected patient of suffi cient size. In a young infant, these deter- defects of gluconeogenesis, such as fructose-1,6-diphos- minations could be done on every other sample. phatase defi ciency. It provides no additional information In control individuals, glucagon administration is fol- in patients with glycogen storage disease. It may be use- lowed by a prompt glycemic response, and the concen- ful in guiding therapy in those patients with defects of trations of lactate and alanine do not increase. In a patient oxidative phosphorylation in whom fructose administra- with glycogenosis I, the curve for glucose may be fl at or tion leads to a major elevation of lactic acid. The test there may be a decline. In some patients, there may be a may cause potentially life-threatening hypoglycemia small elevation of glucose concentration in the blood, and should be performed with great care and only after because 6–8% of the glucose residues of glycogen are other diagnostic options (including mutation analysis of released as free glucose by the debranching enzyme. the aldolase B gene) have been exhausted. Close clinical However, the increase is usually not prompt and does observation is essential, and resuscitation facilities must not exceed 50% of the fasting level within 30 min. In be available. The test may cause severe nausea and/or glycogenosis type I, levels of lactate and alanine increase abdominal pain 15–90 min after oral fructose intake. after glucagon. The level of lactate rises rapidly and may Intravenous fructose challenge is preferable as nausea go very high, even over 15 mM. and vomiting are less common (15–50 min after intrave- In glycogenosis type III or debrancher defi ciency, a nous administration), but intravenous fructose prepara- presumptive diagnosis can be made by determining the tions are currently not available in most countries. response to glucagon in the fed and fasted state. Administration of glucagon after a 12–14 h fast is followed by little or no increase in blood glucose. The D8.5.2.1 Preparations patient is then fed and the glucagon test repeated 2–6 h later at which time the glycemic response is normal. • Normal results of aldolase B gene mutation analysis. These patients do not have lactic acidemia, and con- • The child should have been on a fructose-free diet centrations of lactate do not increase following gluca- for at least 2 weeks and liver function tests should gon. Their concentrations of alanine are low. have normalized. 354 J. Zschocke

• Explain the potential risks (hypoglycemia, nausea, D8.5.2.5 Interpretation and vomiting) to the parents and obtain informed consent. In hereditary fructose intolerance, fructose loading • Blood sugar before test should be in upper normal leads to profound metabolic disturbances. The picture range to reduce the risk of life-threatening hypo- is dominated by progressive hypoglycemia. An even glycemia. sharper fall of inorganic phosphate occurs within a few • An intravenous line should be maintained with minutes. A sharp rise in fructose and uric acid is fol- 0.9% NaCl to allow sampling of specimens and lowed by a slight rise of magnesium. Levels of rapid infusion of glucose if necessary. transaminases may increase, and there may be a slight • Prepare intravenous glucose solution (20%) for fall in serum potassium. Lactate, pyruvate, alanine, emergency administration in case of hypoglycemia. free fatty acids, glycerol, ketone bodies, and growth hormone may also rise. Fructose may be found in the urine in amounts depending on the level of hyperfruc- D8.5.2.2 Procedure – Oral Fructose Challenge tosemia. The renal threshold is 20 mg/dL. Transitory renal tubular dysfunction may lead to mild aminoaci- • Basal values: measure blood sugar, phosphate, mag- duria and proteinuria. The fall in glucose and phos- nesium, uric acid, and lactate; determine fructose if phate along with the rise in urate are suffi cient for an possible. interpretation of hereditary fructose intolerance. • Give oral fructose 1 g/kg as 20% solution over 5–7 min. A hypoglycemic response to fructose administration • Obtain blood samples after 15, 30, 45, 60, 90, and is also observed in fructose-1,6-diphosphate defi ciency. 120 min; measure the same parameters as in the This disorder may be differentiated from fructose intol- basal sample. erance by a history of fasting hypoglycemia and lactic acidemia and an absence of aversion to fruits and sweets. Enzyme assay of biopsied liver provides the defi nitive D8.5.2.3 Procedure – Intravenous Fructose diagnosis in both. Challenge

• Basal values: measure blood sugar, phosphate, D8.6 Tests of Exercise Tolerance magnesium, uric acid, and lactate; determine fructose if possible. D8.6.1 Forearm Ischemia Test • Give intravenous fructose 0.25 g/kg as 10% solution over 2–4 min. • Obtain blood samples after 5, 10, 15, 30, 45, 60, Adequate functioning of glycolysis is necessary for and 90 min; measure the same parameters as in the energy production during muscle exercise. With suf- basal sample. fi cient oxygen supply, glucose-6-phosphate is fully • Optionally, measure sugars (including fructose) and oxidized in the mitochondrion, while anerobic gly- amino acids in a baseline urine as well as urine col- colysis results in the production of lactate. Also lected over the 6–12 h after the load. required for adequate muscle function is the purine nucleotide cycle, which involves the deamination of adenosine monophosphate to inosine monophosphate D8.5.2.4 Treatment of Adverse Reactions and the release of ammonia. The forearm ischemia test examines both systems through measurement of In case of hypoglycemia (blood glucose <40 mg% or lactate and ammonia concentrations before and after 2.3 mmol/L) or if the patient becomes symptomatic anaerobic exercise. It may be useful in patients with with irritability, sweating, and drowsiness, give 2 mL muscle cramps or other muscular symptoms on exer- of 20% or 4 mL of 10% glucose/kg b.w. intravenously tion. Normally, there is a marked increase in the con- as a bolus, followed by 3–5 mL 10% glucose/kg b.w./h centration of both parameters after anaerobic exercise. until normoglycemia is restored and the patient is able Adequate rise of ammonia with absent rise of lactate to take and retain oral food and fl uid. indicates a defi ciency in the breakdown of glycogen D8 Function Tests 355 or in glycolysis as in glycogen storage disease types lactate, ammonia, and creatinine kinase. Myoglobin V (McArdle), III (Cori), or VII (Tauri). In contrast, is determined in the fi rst urine produced after mus- absent rise of ammonia with normal lactate production cle exercise. indicates a defi ciency in the purine nucleotide cycle such as muscle-AMP deaminase defi ciency. The test is invalid if neither lactate nor ammonia is elevated as this D8.6.1.2 Interpretation implies insuffi cient muscle work. Signifi cant increase of creatinine kinase after the test may indicate different Normally, lactate should rise by >2 mmol/L and ammo- metabolic disorders affecting the muscle, e.g., a long- nia by >50 mmol/L above basal values. Insuffi cient rise chain fatty acid oxidation defect. of both lactate and ammonia indicates insuffi cient muscle activity and makes the test invalid. Insuffi cient rise only of lactate indicates defi cient glyco(geno) lysis, while insuffi cient rise only of ammonia suggests D8.6.1.1 Procedure muscle-AMP-deaminase defi ciency. Elevations of creatinine kinase or myoglobin may refl ect muscle cell • Twenty to thirty minutes before the test, a urine damage which may be caused by metabolic disorders sample should be obtained to determine myoglobin, such as a long-chain fatty acid oxidation defect or elec- and the patient should rest in bed. tron chain disorders. • Just before the test, a large intravenous cannula is inserted into the cubital vein of the test arm, which should be the right arm in right-handed persons. A blood sample is taken for the measurement of lac- Key References tate, ammonia, and creatinine kinase. • An infl atable cuff for blood pressure measurements Bonham JR, Guthrie P, Downing M et al (1999) The allopurinal test is attached to the upper test arm and pumped to a lacks for primary urea cycle defects but may indicate unrecog- pressure of 250 or 20 mmHg above systolic blood nized mitochondrial disease. J Inherit Metab Dis 22:174–184 Bonnefont JP, Specola NB, Vassault A et al (1990) The fasting pressure of the patient. The palmar arc may be test in paediatrics: application to the diagnosis of pathologi- blocked with a small cuff across the wrist. The patient cal hypo- and hyperketotic states. Eur J Pediatr 150:80–85 is than asked to maximally exert the forearm and Cahill GF (1982) Starvation. Trans Am Clin Climatol Assoc hand muscles. This may require strong encourage- 94:1–21 Fowler B, Leonard JV, Baumgartner MR (2008) Causes of and ment and is diffi cult to achieve particularly in chil- diagnostic approach to methylmalonic acidurias. J Inherit dren, but insuffi cient exercise makes the test invalid. Metab Dis 31:350–360 A standardized approach may involve a dynamome- Hörster F, Baumgartner MR, Viardot C, Suormala T, Burgard P, ter which is best set at 80% maximum strength. Fowler B, Hoffmann GF, Garbade SF, Kölker S, Baumgartner ER (2007) Long-term outcome in methylmalinic acidurias is Alternatively, a hand-powered torch may be used, influenced by the underlying defect (muto, mut-, cbIA, which also gives feedback on muscle work (bright- cbIB). Pediatr Res 62:225–30 ness of the light). Squeezing a fi rm roll such as a Hyland K, Fryburg JS, Wilson WG et al (1997) Oral phenylala- towel as hard as possible every 2 s does not usually nine loading in dopa-responsive dystonia: a possible diagnostic test. Neurology 48:1290–1297 cause a suffi cient rise of lactate and ammonia and Morris AAM, Thekekara A, Wilks Z et al (1996) Evaluation of therefore is not a good alternative. After the end of fasts for investigating hypoglycaemia or suspected meta- muscle exercise the cuffs are released. bolic disease. Arch Dis Child 75:115–119 • Blood samples are obtained from the intravenous Touatti G, Huber J, Saudubray JM (2006) Diagnostic procedures: function tests and postmortem protocol. In: Fernandes line immediately after muscle exercise and the fol- J, Saudubray JM, Van den Berghe G, Walter, J (eds) Inborn lowing 1, 3, 5, and 7 min for the measurement of metabolic diseases, 4th edn. Springer, Berlin, pp 59–79 Family Issues, Carrier Tests, and Prenatal Diagnosis D9

Johannes Zschocke

Key Facts including an explanatory letter to the parents is usually beneﬁ cial even in disorders with an apparently simple › Carrier tests should not be performed in autosomal recessive inheritance. Genetic counselling also asymptomatic children unless there is a medi- involves taking a standardised family tree and may help cal consequence of the result for the tested to identify other relatives with a high risk for an affected individual in childhood. child, such as consanguineous partners in the same › Standard invasive procedures required for pre- extended family. Genetic counselling should be recom- natal analysis of inborn errors of metabolism mended to these couples as well. have risk of abortion of 0.5Ð1%. Chorionic villus biopsy is the method of choice › The human genome as a shelf of cook books for DNA-based prenatal tests. It can usually be A simple way to explain the human genome and carried out from the 11th to the 12th week of the molecular basis of inherited disorders to patients pregnancy onward is a comparison with a kitchen shelf of books. The › Preimplantation genetic diagnosis requires in genetic information is stored in each cell in a collec- vitro fertilisation and is illegal in many coun- tion of 46 books (chromosomes) that are comprised tries. An alternative is polar body analysis, of a total of more than 23,000 recipes (protein cod- which is more limited in its applications. ing genes). Each gene contains the information for a particular function that the cell may need to fulﬁ l. Quite importantly, there are not 46 different books but 23 pairs of the same books with the same recipes, and each person thus has two copies of most recipes. D9.1 General Remarks The only exceptions are the sex chromosomes: females have two X chromosomes, but males have only one and an additional Y chromosome (which Most inborn errors of metabolism are inherited as auto- used to be an X chromosome many million years ago somal recessive traits. Diagnosis in a child thus implies but over time has lost most of its genes, with the a high recurrence risk for subsequent pregnancies in the exception of a few genes that include some that trig- family. Although clinicians caring for the child usually ger male sex development). When people have chil- explain the genetic basis of the condition at an early dren, they pack one of the two copies of each book stage (text box), many parents do not really appreciate into the parcel for the child, which thus contains only all the implications, and formal genetic counselling 23 different exemplars. As a child gets a parcel from each parent, it again receives a total of 46 books. The text of the recipes consists of four letters ACGT, J. Zschocke and one set of books has more than three billion let- Divisions of Human Genetics and Clinical Genetics, Medical ters. Because the books are copied by hand from University Innsbruck, Schöpfstr. 41,6020 Innsbruck, Austria generation to generation, they contain the occasional e-mail: [email protected]

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 357 DOI: 10.1007/978-3-540-74723-9_D9, © Springer-Verlag Berlin Heidelberg 2010 358 J. Zschocke

Carrier analyses in siblings of affected children or spelling mistake or typing error, and there may be in more distant relatives may appear harmless on ﬁ rst different variants (alleles) of the same recipes. sight. Nevertheless, there have been cases where the Polymorphisms are variants that are quite common presence of mutations in completely healthy persons (>1% of gene copies in a population), while muta- was misunderstood by the affected individual or others tions are variants that change the information con- as a kind of disease and at worst had an adverse impact tent and in consequence change or remove the on insurance or employment. It is thus generally agreed normal function. It may be useful to point out that, that carrier tests should not be performed in underage for example, humans do not have two PAH genes siblings of patients with recessive disorders. This is (PAH codes for the enzyme phenylalanine hydroxy- true for all kinds of genetic tests. These should not be lase deﬁ cient in phenylketonuria) but have two cop- carried out in asymptomatic children unless there is a ies of the one PAH gene; just like having two copies medical consequence of the result for the tested indi- of the same recipe for chocolate cake does not mean vidual already in childhood. It is much wiser to let the that one has two chocolate cake recipes. Any genetic child grow up to an age where she or he understands abnormality may be explained with this example, the implications and may request carrier testing in the from X-chromosome inactivation (only one book course of genetic counselling. Growing up with the is used, the other exemplar is sealed) to genomic knowledge of the result may rob the child of the option imprinting (a recipe can only be read on either the to later actively work on this issue in the process of paternal or the maternal copy of the book, the other making a decision on testing. version is sealed) or balanced translocations (two books have fallen apart and have been wrongly glued together again). D9.3 Prenatal Diagnosis

Many parents who experienced a severely debilitating D9.2 Carrier Tests or fatal inherited disease in a child and who have a high risk for the same disease in future pregnancies Once disease-causing mutations have been identifi ed request prenatal diagnosis with the option of termina- in a patient, it is easily possible to test other family tion of pregnancy. The associated social and legal members for the familial mutation(s). In autosomal issues differ markedly between countries and are not recessive conditions, both the parents are normally discussed here. There is generally a window of oppor- carriers for the disease and mutation testing may not be tunity between the 11th and 12th week of pregnancy necessary for confi rmation. Sometimes, however, it is when the placenta and foetus are suffi ciently large for advisable to confi rm the results in the child through invasive testing, and the 22ndÐ23rd week when the carrier testing in both parents. In a child with homozy- foetus may survive outside the womb and termination gosity for a mutation, for example, it may be advisable of pregnancy is often regarded as much more problem- to exclude the presence of a large deletion on one allele atic for ethical reasons. Termination of pregnancy in that may lead to PCR non-amplifi cation on the other the second trimenon involves the induction of labour gene copy. When a child appears to be compound and delivery of a foetus which dies through the proce- heterozygous for two mutations, it may be important to dure, in contrast to the fi rst trimenon when curettage is confi rm that the two mutations are indeed in trans, i.e. usually suffi cient. After the 22ndÐ23rd week, the foe- on the two different chromosomal strands and not on tus is killed in the womb through injection of potas- the same chromosomal strand/in the same gene copy. In sium into the heart (foeticide) in order to avoid that the the latter case, the child is heterozygous for a double child is born alive, a procedure that is emotionally and mutation, and the genotype does not confi rm the dis- ethically very challenging or unacceptable for many ease. It should be kept in mind, however, that carrier parents and doctors. Parents should be offered genetic tests in the parents may also lead to the identifi cation of and/or psychosocial counselling when termination of non-paternity which may be embarrassing (or worse) pregnancy is considered. The decision for or against both for the couple involved and the doctor. termination of pregnancy rests solely with the couple D9 Family Issues, Carrier Tests, and Prenatal Diagnosis 359 or the mother. Non-directive counselling is mandatory allele are identifi ed in the foetal sample. CVS has a and is possible even when termination is requested by risk for miscarriage of approximately 1%, i.e. 1 in the parents but is rejected by the doctors or is deemed 100 women loses a healthy child after the procedure. incompatible with legal regulations in the specifi c Because of the early availability of foetal DNA, circumstances. CVS is the method of choice for prenatal diagnosis Prenatal diagnosis should be organised by a clini- through mutation analysis. cal geneticist in close contact with the gynaecologist 2. Amniocentesis is usually possible from the 14th to who performs both the invasive procedure and possi- 15th week of pregnancy onwards. Amniotic fl uid bly the termination. Parents should be advised to con- contains foetal cells derived from the urinary tract tact the geneticist as early as possible once a preg nan cy or other sources and is obtained transabdominally has been confi rmed. Traditionally, there are different with a long, relatively small needle. There is not approaches to prenatal diagnosis including metabolic usually suffi cient cellular material for DNA extrac- analyses in amniotic fl uid and enzyme tests in chorionic tion and amniocytes have to be cultured for 7Ð10 villus cells or amniocytes, but direct mutation analysis is days both for chromosomal and molecular analyses. now mostly regarded as the method of choice. Because of DNA is thus available for testing at least 4 weeks the enormous progress in the understanding of inherited later than after CVS. In contrast to CVS, there is disorders over the last years, it is now possible to test a smaller risk of maternal contamination unless there foetus for almost all inborn error of metabolism once a is a signifi cant amount of maternal blood in the disease has been recognised in a family. amniotic fl uid (which may be recognised, e.g. Metabolic conditions are not usually recognised through a reddish colour). Compared with CVS, through abnormal ultrasound scans and invasive sam- amniocentesis has a slightly lower risk for miscar- pling of foetal cells or fl uid is necessary for prenatal riage of approximately 0.5%, although these fi gures testing. There are three main approaches: also depend on the expertise of the gynaecologist who performs the procedure. 1. Chorionic villus sampling (CVS) is usually possible 3. Foetal blood sampling is generally possible after from the 11th week of pregnancy onwards. Small the 20th week of pregnancy and has a risk of mis- fragments of placental tissue are taken with a mod- carriage of approximately 2%. Blood is taken trans- erately large needle either through the abdomen or abdominally with a long needle from an umbilical the cervix. Chorionic villi have both foetal and vein at the insertion of the umbilicus into the pla- maternal components, and it is essential to carefully centa. As this foetal sampling approach becomes remove the latter after the sample has been taken. possible only relatively late in pregnancy, it is usu- Cells may be either cultured, e.g. for chromosome ally restricted to special indications. analysis or enzyme studies, or may be used for DNA extraction. Chorionic cells are derived from the tro- phoblast and the extraembryonic mesoderm, and D9.4 Preimplantation Genetic Diagnosis chromosomal abnormalities found in these cells (particularly when the abnormality is found only in and Polar Body Analysis a proportion of cells) do not necessarily refl ect the chromosomal status of the foetus. For karyotype As an alternative to prenatal diagnosis after several analysis, therefore, two different cell culture types months of pregnancy, disease status may also be deter- are used. This is not necessary for molecular analy- mined in the course of in vitro fertilisation (IVF) prior ses, but it is mandatory to exclude maternal contam- to implantation of the embryo into the womb. For pre- ination when chorionic villi are used for genetic implantation genetic diagnosis (PGD) one or two cells tests as presence of maternal cells may lead to false are taken from the embryo 2Ð3 days after fertilisation negative results. For this purpose, highly polymor- (in the four or eight cell stadium). PCR-based mutation phic microsatellite markers in DNA extracted from analysis is performed after DNA extraction and ampli- chorionic villi and maternal blood are compared; fi cation. This procedure requires a highly controlled maternal contamination is excluded when only one laboratory setting and may have to be specifi cally estab- of the two maternal alleles as well as one paternal lished for the individual case. PGD is prohibited in 360 J. Zschocke several countries as discarding an embryo is deemed to necessarily covered by health insurances. However, violate its right to life when there is no confl icting inter- they are options for parents who for religious or ethical est of the mother (there is as yet no pregnancy), and reasons object to termination of pregnancy. there is a perceived risk of a “slippery slope” of easy selection of preferable characteristics in a child con- ceived through IVF. As an alternative to PGD in these countries, mutation status may be determined in DNA Key References extracted from the polar bodies of an egg prior to fertilisation (polar body analysis). This method only allows Besley GTN (2005) Prenatal diagnosis of inborn errors of testing for mutations carried by the mother and not the metabolism. In: John M. Walker JM, Rapley R (eds) Medical father, again may need to be specially established for biomethods handbook. Humana Press, Totowa, New Jersey the individual case and is offered by few specialised Sasi K, Sanderson D, Eydoux Cartier h, Scriver CR, Treacy E (1997) Prenatal diagnosis for inborn errors of metabolism laboratories only. Both approaches require IVF, which and haemoglobinopathies: the Montreal Children’s Hospital may be stressful and carries additional risks, and is not experience. Pren Diagn 17:681Ð685 Part E Appendix Differential Diagnosis of Clinical and Biochemical Phenotypes E1

Acidosis Arthritis Hyperchloremic diarrhea Alkaptonuria Acrodermatitis, enteropathica Farber disease Infectious Gaucher type I Lactase defi ciency Gout-HPRT defi ciency; PRPP overactivity Sucrase defi ciency Homocystinuria Renal tubular acidosis (RTA) I cell disease Cystinosis Lesch–Nyhan disease Fanconi syndrome Mucolipidosis III Galactosemia Mucopolysaccharidosis IS; IIS Glucose, galactose malabsorption Bleeding Tendency Hepatorenal tyrosinemia Mitochondrial electron transport defect Abetalipoproteinemia Osteopetrosis and RTA a-1-Antitrypsin defi ciency Topamax Congenital disorders of glycosylation (CDG) Chediak–Higashi syndrome Alopecia Fructose intolerance An (hypo) hidrotic ectodermal dysplasia Gaucher disease Biotin defi ciency Glycogenoses types I and IV Cartilage hair hypoplasia Hermansky–Pudlak syndrome Conradi–Hünermann syndrome Peroxisomal disorder Multiple carboxylase defi ciency – holocarboxylase Tyrosinemia type 1 synthetase and biotinidase defi ciencies Calcifi cation of Basal Ganglia Trichorrhexis nodosa-argininosuccinic aciduria Vitamin D-dependent rickets-receptor abnormalities Albright syndrome Bilateral striato-pallido-dentate calcinosis Angiokeratomas Carbonic anhydrase II defi ciency Fabry disease Cockayne syndrome Fucosidosis Down syndrome Galactosialidosis Fahr disease Familial progressive encephalopathy with calcifi cation GM1 gangliosidosis Sialidosis of the basal ganglia (Aicardi–Goutieres syndrome) GM1-gangliosidosis Apparent Acute Encephalitis Hallervorden–Spatz disease Glutaric aciduria I Hypo-, hyper-parathyreoidism NARP Krabbe leukodystrophy Propionic acidemia Lipoid proteinosis

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 363 DOI: 10.1007/978-3-540-74723-9_E1, © Springer-Verlag Berlin Heidelberg 2010 364 Appendix

Microcephaly and intracranial calcifi cation Biotinidase defi ciency Mitochondrial cytopathies Carnitine palmitoyltransferase II defi ciency Multiple endocrine neoplasia I Cockayne syndrome Neurofi bromatosis GM2 gangliosidosis Pterin defects L-2-hydroxyglutaric aciduria Dihydropteridine reductase defi ciency Hypoparathyroidism GTP cyclohydrolase I defi ciency Kearns–Sayre syndrome 6-pyruvoyletetrahydropterin synthase defi ciency Krabbe disease Sepiapterin reductase defi ciency MELAS Spondyloepiphyseal dysplasia Osteopetrosis and renal tubular acidosis (carbonic anhydrase II defi ciency) Cardiomyopathy Mitochondrial disorders Congenital muscular dystrophy Danon disease Cerebral Vascular Disease Disorders of fatty acid oxidation Electron transport chain abnormalities Fabry disease Fabry disease Familial hypocholesterolemia Glycogenosis type III Homocystinuria Hemochromatosis Menkes disease D-2-Hydroxyglutaric aciduria 5,10-Methylenetetrahydrofolatereductase defi ciency 3-Methylglutaconic aciduria II (Barth disease) Myocardial infarction Mucopolysaccharidosis Pompe disease Cerebrospinal Fluid Lymphocytosis Cataracts Ð Lenticular Opacity Aicardi–Goutieres syndrome Delta1-pyrroline-5-synthase defi ciency Cerebrotendinous xanthomatosis Cerebrospinal Fluid Protein Elevation Electron transport chain disorders Congenital disorders of glycosylation CCDG Fabry disease L-2-Hydroxyglutaric aciduria Galactokinase defi ciency Kearns–Sayre syndrome Galactosemia Krabbe disease Homocystinuria MELAS – Mitochondrial encephalomyopathy, lactic Hyperferritinemia-cataract syndrome acidemia, and stroke-like episodes Hyperornithinemia (ornithine aminotransferase defi ciency) MERFF – Myoclonic epilepsy with ragged-red fi bers Lowe syndrome Metachromatic leukodystrophy Lysinuric protein intolerance Multiple sulfatase defi ciency Mannosidosis Neonatal adrenoleukodystrophy Mevalonic aciduria Refsum disease Multiple sulfatase defi ciency Neonatal carnitine palmitoyl transferase (CPT II Cherry Red Macular Spots defi ciency) a'-Pyrroline-5-carboxylate synthase defi ciency Galactosialidosis

Peroxisomal disorders GM1 gangliosidosis Mucolipidosis I Cerebral Calciﬁ cation Multiple sulfatase deﬁ ciency Abnormalities of folate metabolism Niemann–Pick disease Adrenoleukodystrophy Sandhoff disease Aicardi–Goutiere syndrome Sialidosis Biopterin abnormalities Tay–Sachs disease Appendix 365

Cholestatic Jaundice Peroxisomal disorder Hepatorenal tyrosinemia Alagille syndrome Niemann–Pick type C a-1-Antitrypsin defi ciency Peroxisomal disorders Byler disease Pyruvate kinase defi ciency (progressive familial intrahepatic cholestasis Transaldolase defi ciency (PF1C1, BRIC1)) Tyrosinemia type I PFIC2 (bile salt excretory pump (BSEP)) Wilson disease PFIC 3 (MDR3) Citrin defi ciency Corneal Opacity Cystic fi brosis Dubin–Johnson syndrome Cystinosis Fructose bisphosphate aldolase (B) defi ciency (HNF-1B) Fabry Hepatic nuclear factor 1b gene mutation Fish eye disease (LCAT defi ciency) Hepatorenal tyrosinemia Galactosialidosis

LCHAD deﬁ ciency GM1 gangliosidosis Mevalonic aciduria Hurler disease (MPS I) Niemann–Pick type C disease I-cell disease Peroxisomal biogenesis disorders Mannosidosis Rotor syndrome Mucolipidosis III Tyrosinemia, hepatorenal Multiple sulfatase deﬁ ciency

Chondrodysplasia Phenotypes Corpus Callosum Agenesis Conradi–Hünermann syndrome Adrenocorticotrophic hormone (ACTH) defi ciency Peroxisomal disorders Aicardi syndrome Warfarin embryopathy Mitochondrial disorders (especially pyruvate dehydrogenase defi ciency) Chronic Pancreatitis Nonketotic hyperglycinemia Hereditary (dominant) (with or without lysinuria Peroxisomal disorders (cystinuria)): with or without pancreatic lithiasis or Creatine Kinase Ð Elevated portal vein thrombosis With hyperparathyroidism in multiple endocrine ade- Aldolase A defi ciency nomatosus syndrome Carnitine palmitoyl transferase II defi ciency MELAS Disorders of fatty acid oxidation Organic acidemias Drugs – Toxins, alcohol, statins Pearson syndrome Glutaric acidemia (I) Regional enteritis (Crohn) Glycogenosis – III Trauma – pseudocyst Glycogenosis – V – McArdle Glycogenosis – posphofructokinase Cirrhosis of the Liver D-2-Hydroxyglutaric aciduria a-1-Antitrypsin defi ciency Infl ammatory myopathy – dermatomyositis, polymyositis Citrin defi ciency (citrullinemia) Infectious myositis Cystic fi brosis 3-Oxothiolase defi ciency Congenital disorders glycosylation Mevalonic aciduria Defects of bile acid synthesis Myoadenylate deaminase Electron transport chain disorders Muscular dystrophy – Duchenne, Becker Galactosemia 3-Oxothiolase defi ciency Glycogen storage disease type IV Oxphos abnormalities (Neonatal) Hemochromatosis Traumatic muscle injury 366 Appendix

Dermatosis B6/pyridoxal-phosphate dependencies Drug overdose (e.g., phenobarbital) Acrodermatitic enteropathica Molybdenum cofactor defi ciency Biotinidase defi ciency Nonketotic hyperglycinemia Holocarboxylase synthetase defi ciency Organic acidemias (neonatal encephalopathy-propionic Diabetes Mellitus Ð Erroneous Diagnosis academia) Purine metabolism defects Congenital disorders of glycosylation Isovaleric acidemia Methylmalonic acidemia Exercise Intolerance 3-Oxothiolase defi ciency Defects of glycogenolysis Propionic acidemia Disorders of fatty acid oxidation Diarrhea 3-Oxothiolase defi ciency Mitochondrial disorders Abetalipoproteinemia Myoadenylate deaminase defi ciency Congenital chloride diarrhea Electron transport disorders Fever Syndromes Enterokinase defi ciency Glucose galactose malabsorption Familial Mediterranean fever Johansson–Blizzard syndrome Hyperimmunoglobulin D syndrome (mevalonic Lactase defi ciency aciduria) Lysinuric protein intolerance Muckle–Wells syndrome (neonatal onset multisystem Pearson syndrome infl ammatory disease syndrome) Schwachman syndrome Tumor necrosis factor receptor-associated periodic Sucrase defi ciency syndrome Wolman disease Dysostosis Multiplex Glycosuria Galactosialidosis Cystinosis Generalized GM1 gangliosidosis Diabetes mellitus Hurler, Hurler–Scheie disease Hepatorenal tyrosinemia Hunter disease Fanconi–Bickel syndrome – GLUT-2 mutations Maroteaux–Lamy disease Glycogen synthase defi ciency Mucolipidosis II, I-cell disease Pearson syndrome Mucolipidosis III Renal Fanconi syndrome Multiple sulfatase defi ciency Wilson disease Sanfi lippo disease Sly disease Hair Abnormalities Ectopia Lentis (dislocation of the lens) Argininosuccinic aciduria Homocystinuria Kinky hair, photosensitivity, and mental Hyperlysinemia retardation Marfan syndrome Menkes disease (pili torti, trichorrhexis nodosa, Molybdenum cofactor defi ciency monilethrix) Sulfi te oxidase defi ciency Pili torti: isolated, MIM 261900 Weiss–Marchesani syndrome Pili torti with deafness or with dental enamel hypoplasia MIM 262000 (Björnstad syndrome (complex 3)) EEG Burst Suppression Pattern Trichothiodystrophy: trichorrhexis nodosa, ichthyosis, Anesthesia-deep stages and neurological abnormalities (Pollit syndrome) MIM Anoxia, cerebral hypoperfusion 27550 Appendix 367

HDL (lipoprotein) Low Phosphoenolpyruvate carboxykinase defi ciency Progressive intrahepatic cholestasis Lecithin cholesterol acyltransferase (LCAT) defi ciency Thalassemia (fi sh eye disease) Transaldolase defi ciency Tangier disease Urea cycle disorders Hypoalphalipoproteinemia Wilson disease Hemolytic Anemia Wolman disease

Defects of glycolysis Hepatic Failure Ð Acute 5-Oxoprolinuria Purine and pyrimidine disorders a-1-Antitrypsin defi ciency Wilson disease Fatty acid oxidation disorders Galactosemia Hemophagocytosis (Erythrophagocytosis) Hepatorenal tyrosinemia Carnitine palmitoyl transferase I Hereditary fructose intolerance Familial hemophagocytic lymphocytic histocytcosis Mitochondrial depletion syndromes, esp. POLG1 and (perforin defi ciency, PRF1) MPV17 mutations Gaucher disease Neonatal hemochromatosis Hemochromatosis Niemann–Pick types B and C Lysinuric protein intolerance Urea cycle disorders Niemann–Pick disease Wilson disease Propionic acidemia Wolcott–Rallison syndrome Wolman disease Hydrops Fetalis Hepatic Carcinoma Carnitine transporter defi ciency a-1-Antitrypsin defi ciency Congenital disorders of glycosylation Galactosemia Farber disease (disseminated lipogranulomatosis) Glycogen storage disease types I and IV Galactosialidosis Hemochromatosis Glycogenosis type IV Hepatorenal tyrosinemia GM1 gangliosidosis Progressive intrahepatic cholestasis Gaucher disease Thalassemia Infantile free sialic acid storage disease (ISSD) Wilson disease Mucolipidosis II – I-cell disease Neonatal hemochromatosis Hepatic Cirrhosis Niemann–Pick disease a-1-Antitrypsin defi ciency Niemann–Pick disease type C Cholesteryl ester storage disease Pearson syndrome (anemia) Congenital disorder of glycosylation (CDG-X) Siali inverted nipples dosis Cystic fi brosis Sly disease-b-glucuronidase defi ciency Coeliac disease Wolman disease Electron transport disorders 3-Hydroxyglutaric Aciduria Fructose intolerance Galactosemia Glutaryl-CoA dehydrogenase defi ciency Gaucher disease Short-chain hydroxyacyl-CoA dehydrogenase Glycogenosis types I and IV defi ciency Hematochromatosis Carnitine palmitoyltransferase I defi ciency Hepatorenal tyrosinemia Hyperammonemia Hypermethioninemia Mitochondrial DNA depletion N-acetylglutamate synthetase defi ciency Niemann–Pick disease a-1-Antitrypsin defi ciency 368 Appendix

Argininemia Pearson Argininosuccinic aciduria X-linked hypophosphatemic rickets Carbamoyl phosphate synthetase defi ciency Hypouricemia Chemotherapy-induced hyperammonemia Citrullinemia Fanconi syndrome, cystinosis, any proximal renal Fatty acid oxidation disorders tubular dysfunction HHH syndrome Isolated renal tubular defect (Dalmatian dog model) HMG-CoA lyase defi ciency Molybdenum cofactor defi ciency Isovaleric acidemia Phosphoribosyl pyrophosphate synthetase defi ciency Lysinuric protein intolerance Purine nucleoside phosphorylase defi ciency MCAD defi ciency Wilson disease Methylmalonic acidemia Xanthine oxidase defi ciency Multiple carboxylase defi ciency Ichthyosis Ornithine transcarbamylase defi ciency Pyruvate carboxylase defi ciency CHILD syndrome (congenital hemidysplasia ichthyosis Pyruvate dehydrogenase complex defi ciency and limb defects) Propionic acidemia Congenital disorders of glycosylation (CDG) type 1f Hyperthermia, malignant carnitine palmitoyl Gaucher disease transferase-II defi ciency Krabbe disease Transient hyperammonemia of the newborn Multiple sulfatase defi ciency Urinary tract infection – urea splitting bacteria Refsum disease Valproate Sjogren–Larsson syndrome Wilson disease X-linked ichthyosis – steroid sulfatase defi ciency Hypertyrosinemia Ichthyosis and Retinal Disease Defi ciency of 4-hydroxyphenylpyruvate dioxygenase Refsum syndrome Drug – toxin Sjogren–Larrson syndrome Hepatic infection Inverted Nipples Hepatorenal tyrosinemia Hyperthyroidism Biopterin synthesis disorders Oculocutaneous tyrosinemia Citrullinemia Postprandial Congenital disorders of glycosylation Scurvy Glycogenosis 1b Transient tyrosinemia of the newborn Hyperphenylalaninemia Treatment with NTBC Isolated – dominant (MIM163610) Tyrosinemia type III Isovaleric acidemia Menkes disease Hypoketotic Hypoglycemia Methylmalonic acidemia Carnitine transporter defi ciency Molybdenum cofactor defi ciency CPT I defi ciency Niemann–Pick type C HMG-CoA lyase defi ciency SCAD defi ciency LCAD Propionic acidemia LCHAD/MTP Pyruvate carboxylase defi ciency MCAD VLCAD defi ciency VLCAD Weaver syndrome Hypophosphatemia Isolated Defi ciency of Speech as Presentation in Metabolic Disease Fanconi syndrome Hyperparathyroidism Ethylmalonic aciduria MELAS D-glyceric aciduria Appendix 369

Histidinemia Pyruvate carboxylase defi ciency 3-Methylglutaconyl-CoA hydratase Tay–Sachs disease Lactic Acidemia Megaloblastic Anemia Electron transport chain Cobalamin metabolic errors-methylmalonic acidemia disorders and homocystinuria-Cbl C and D Fatty acid oxidation disorders Folate metabolism, abnormalities of Lues, congenital B12 defi ciency – vegan or breast-fed infant of vegan MELAS mother MERRF CblF cobalamin lysosomal transporter defi ciency Organic acidemia, e.g., propionic Dietary folate defi ciency acidemia Folate malabsorption – hereditary – protein coupled Pyruvate carboxylase defi ciency folate transport (PCFT) defi ciency Pyruvate dehydrogenase defi ciency Intestinal B12 transport defi ciency – Imerslund– Leigh Syndrome Grasbeck-cubilin defi ciency Methylmalonic acidemia-homocystinuria-Cbl C + D Biotinidase defi ciency Mevalonic aciduria Electron transport chain disorders Orotic aciduria Fumarase defi ciency Pearson syndrome 3-Methylglutaconic aciduria Pernicious anemia – intrinsic factor Pyruvate carboxylase defi ciency defi ciency Pyruvate dehydrogenase complex Transcobalamin II defi ciency defi ciency Sulfi te oxidase defi ciency Metabolic Acidosis and Ketosis Leukopenia with or without Thrombopenia and Fabry disease Anemia Familial hypocholesterolemia Homocystinuria Abnormalities of folate metabolism Isovaleric academia Isovaleric acidemia Menkes disease Johansson–Blizzard syndrome Methylcrotonyl-CoA carboxylase defi ciency Methylmalonic acidemia Methylmalonic/propionic acidemia 3-Oxothiolase defi ciency Multiple carboxylase defi ciency Pearson syndrome 3-Oxothiolase defi ciency Propionic acidemia Shwachman syndrome Methylmalonic aciduria Transcobalamin II defi ciency B12 defi ciency, pernicious anemia, including Macrocephaly autoimmune Bannayan–Ruvalcaba–Riley syndrome Cobalamin A Canavan disease Cobalamin C, D Glutaric aciduria type I Imerslund–Gräsbeck – cobalamin enterocyte Hurler disease malabsorption 4-Hydroxybutyric aciduria Muto, Mut− L-2-hydroxyglutaric aciduria Succinyl-CoA synthase defi ciency 3-Hydroxy-3-methylglutaric aciduria Transcobalamin II defi ciency Krabbe disease Mongolian Spot Ð Extensive Mannosidosis

Multiple acyl-CoA dehydrogenase deﬁ ciency GM1 gangliosidosis Multiple sulfatase deﬁ ciency Hurler syndrome Neonatal adrenoleukodystrophy Niemann–Pick disease 370 Appendix

Myocardial infarction-cerebral vascular disease Mevalonic aciduria Mitochondrial energy metabolism, defects in – including Fabry disease Leber hereditary optic neuropathy (LHON) Familial hypercholesterolemia Multiple sulfatase defi ciency Homocystinuria NARP Menkes disease Neonatal adrenoleukodystrophy Oxothiolase defi ciency Propionic acidemia Propionic acidemia Sandhoff disease SCHAD defi ciency Tay–Sachs disease Neonatal Hepatic Presentations in Metabolic Orotic Aciduria Diseases UMP synthase defi ciency (hereditary orotic aciduria) a-1-Antitrypsin defi ciency Urea cycle defects – ornithine transcarbamylase Cystic fi brosis defi ciency, citrullinemia, arginosuccinic aciduria, Fructose intolerance arginemia Galactosemia Purine nucleoside phosphorylase (PNP) defi ciency Glycosylation disorders, especially type Ib Phosphoribosylpyrophosphate (PRPP) synthetase Hemochromatosis defi ciency Hepatorenal tyrosinemia Long-chain hydroxy-acyl-CoA dehydrogenase defi ciency Osteoporosis and Fractures Mitochondrial DNA depletion syndromes Adenosine deaminase defi ciency Niemann–Pick type C disease Gaucher disease Wilson disease Glycogenesis I Wolman disease Homocystinuria Sly disease I-cell disease Odd or Unusual Odor Infantile Refsum disease Lysinuric protein intolerance Dimethylglycinuria Menkes disease Glutaric aciduria type II Methylmalonic acidemia Hepatorenal tyrosinemia Propionic acidemia Isovaleric acidemia Maple syrup urine disease Pain and Elevated Erythrocyte Sedimentation Phenylketonuria Rate Treatment of urea cycle disorder with phenylacetate Fabry disease Trimethylaminuria Familial hypercholesterolemia Optic Atrophy Gaucher disease ADP-ribosyl protein lyase defi ciency Pancreatitis Adrenoleukodystrophy (ALD) Carnitine palmitoyltransferase II defi ciency Biotinidase defi ciency Cytochrome c oxidase defi ciency Canavan disease Glycogenosis type I GM1 gangliosidosis Glycogenosis 1 plus apoE2 type III Homocystinuria hypertriglyceridemia Krabbe disease Hereditary dominant, with or without lysinuria; with or Menkes disease without pancreatic lithiasis or portal vein thrombosis Methylmalonic acidemia Homocystinuria MERRF Hydroxymethylglutaryl-CoA lyase defi ciency Metachromatic leukodystrophy Hyperlipoproteinemia type IV 3-Methylglutaconic aciduria, type III (Costeff) Isovaleric acidemia Appendix 371

Lipoprotein lipase defi ciency, also type IV Wilson disease Lysinuric protein intolerance Maple syrup urine disease Ptosis MELAS Dopamine defi ciency syndromes Methylmalonic acidemia Kearn–Sayre syndrome Ornithine transcarbamylase defi ciency MNGIE (mitochondrial neurogastrointestinal Pearson syndrome enceph alomyelopathy) Propionic acidemia Regional enteritis (Crohn) Ragged Red Fibers Trauma – pseudocyst Menkes disease With hyperparathyroidism in multiple endocrine Mitochondrial DNA mutations adenomatosus syndrome Red Urine Paralysis of Upward Gaze Beets Leigh, Kearns–Sayre syndromes Hematuria Niemann–Pick type C Hemoglobinuria Peripheral neuropathy Myoglobinuria Photophobia Drugs: rifampicin, phenolphthalein, nitrofurantoin, ibuprofen, pyridium Cystinosis Renal Calculi Oculocutaneous tyrosinemia APRT (adenosine phosphoribosyltransferase) Polycystic Kidneys defi ciency Carnitine palmitoyl transferase II (CPT-II) defi ciency Cystinuria Congenital disorders of glycosylation HPRT defi ciency-Lesch–Nyhan disease Glutaric aciduria type II, (multiple acyl-CoA dehydro- Oxaluria genase defi ciency) (GA II) PRPP synthetase abnormalities Zellweger syndrome Wilson disease Xanthine oxidase defi ciency Psychotic Behavior Renal Fanconi Syndrome Carbamoyl phosphate synthetase defi ciency Cbl disease Cystinosis Ceroid lipofuscinoses Electron transport defects Citrullinemia Galactosemia Hartnup disease Glycogenosis I and III Homocystinuria Hepatorenal tyrosinemia Hurler–Scheie, Scheie disease Idiopathic Krabbe disease Lowe syndrome Lysinuric protein Lysinuric protein intolerance b-mannosidosis Wilson disease Maple syrup urine disease Renal tubular acidosis (RTA) 5,10-Methylenetetrahydrofolatereductase defi ciency Metachromatic leukodystrophy Cystinosis Mitochondrial disease (MELAS) Fanconi syndrome Niemann–Pick type C disease Galactosemia Ornithine transcarbamylase defi ciency Hepatorenal tyrosinemia Porphyria Mitochondrial electron transport defect Sanfi lippo disease Osteopetrosis and RTA Tay–Sachs, Sandhoff-late onset Topamax 372 Appendix

Retinitis Pigmentosa Self Injurous Behavior Abetalipoproteinemia Lesch–Nyhan disease Congenital disorder of glycosylation Neuroacanthocytosis Ceroid lipofuscinosis Hunter disease Sensorineural Deafness Kearns–Sayre syndrome Biotinidase defi ciency LCHAD defi ciency Canavan disease Mevalonic aciduria Kearns–Sayre syndrome and other electron transport NARP chain disorders Peroxisomal biosynthesis disorders Peroxisomal disorders Primary retinitis pigmentosa PRPP synthetase abnormality Refsum disease Refsum disease Sjogren–Larsson syndrome (fatty alcohol oxidoreductase defi ciency) Spastic Paraparesis Reye Syndrome Presentation Argininemia Biotinidase defi ciency Gluconeogenesis, disorders of Fatty acid oxidation HHH syndrome disorders Metachromatic leukodystrophy Urea cycle disorders Pyroglutamic aciduria Electron transport chain disorders Sjögren–Larsson syndrome Fructose intolerance Stroke-like Episodes Organic acidemia Reynaud Syndrome Carbamyl phosphate synthetase defi ciency Chediak–Higashi syndrome Fabry disease Congenital disorders of glycosylation Ethylmalonic encephalopathy Rhabdomyolysis Fabry disease Aldolase A (fructose bisphosphate) defi ciency Familial hypercholesterolemia Disorders of fatty acid oxidation – LCHAD, Glutaric aciduria type I CPTII Homocystinuria Drugs – methylenedioxymethylamphetamine (MDMA) Hydroxymethylglutaryl-CoA lyase defi ciency Glutaric acidemia I Isovaleric acidemia Glycogenosis V-McArdle – myophosphorylase MELAS and other mitochondrial disorders VII – Tarui – phosphofructokinase Menkes disease Glycolysis – phosphoglycerate kinase Methylcrotonyl-CoA carboxylase defi ciency – phosphoglyceromutase Methylmalonic acidemia Infection – myositis 5,10-Methylenetetrahydrofolatereductase Ischemic injury defi ciency Mitochondrial DNA deletions (multiple) Multiple acyl-CoA dehydrogenase defi ciency Mitochondrial point mutations (MELAS) Ornithine transcarbamylase defi ciency Oxphos defects – complex I, complex II Propionic acidemia Quail ingestion – coturnism Phosphoglycerate kinase defi ciency Toxin-tetanus, snake venom, alcohol, cocaine, bee Progeria venom Pyruvate carboxylase defi ciency Pyruvate dehydrogenase defi ciency Scoliosis Respiratory chain disorders Congenital disorder of glycosylation Purine nucleoside phosphorylase defi ciency Homocystinuria Sulfi te oxidase defi ciency Appendix 373

Subdural effusions Isovaleric acidemia Methylmalonic acidemia Glutaric aciduria I 3-Oxothiolase defi ciency D-2-Hydroxyglutaric aciduria Phenylketonuria Menkes disease Propionic acidemia Pyruvate carboxylase defi ciency Vomiting and Erroneous Diagnosis of Pyloric Xanthomas Stenosis Cerebrotendinous xanthomatosis Ethylmalonic-adipic aciduria Familial hypercholesterolemia Galactosemia Lipoprotein lipase defi ciency HMG-CoA lyase defi ciency Niemann–Pick disease 4-Hydroxybutyric aciduria Sitosterolemia D-2-Hydroxyglutaric aciduria Reference Books E2

Blau N, Duran M, Blaskovics ME, Gibson KM (eds) Clarke JTR (2006) A clinical guide to inherited (2003) Physician’s guide to the laboratory diagnosis of metabolic diseases, 3rd edn. Cambridge University metabolic diseases, 2nd edn. Springer, Berlin. Press, Cambridge. Detailed collation of clinical and laboratory ﬁ nd- Hands-on description of ﬁ ve symptom-based presen- ings in individual disorders. Very helpful for meta- tations of metabolic diseases (CNS, heart or liver disease, bolic specialists and clinicians with experience of the acutely ill neonate, and storage disorders) as well as metabolic disorders but less well suited to clinicians other clinically relevant categories. who do not regularly see patients with inborn errors of metabolism. Fernandes J, Saudubray JM, van den Berghe G, Walter JH (eds) (2006) Inborn metabolic diseases – Blau N, Duran M, Gibson KM (eds) (2008) Laboratory diagnosis and treatment, 4th edn. Springer, Berlin. guide to the methods in biochemical genetics. Springer, Accessible pathway-based approach to inborn Berlin. errors of metabolism. Covers all the major groups of A prime resource for metabolic specialists working metabolic disorders and is particularly helpful for cli- in the laboratory that also provides excellent back- nicians who seek basic information on diagnosis and ground information for clinicians interested in meth- treatment of individual defects. Individual chapters odological background information. written by various authors and of variable quality.

Blau N, Hoffmann GF, Leonard J, Clarke JTR (eds) Firth HV, Hurst JA (2005) Oxford desk reference (2005) Physician’s guide to the treatment and follow- clinical genetics. Oxford University Press, Oxford. up of metabolic diseases. Springer, Berlin. A well-written, practical text. It is primarily aimed at The book gives detailed information with regard clinical geneticist and does not have a speciﬁ c focus on to the management of metabolic disorders. It is an metabolic disorders but is an extremely valuable intermediary between more general textbooks and resource for all clinicians who encounter patient’s dedicated original articles. A useful reference book genetic conditions. for clinicians who see metabolic patients. Nyhan WL, Barshop BA, Ozand PT (2005) Atlas of Bremer HJ, Duran M, Kamerling JP, Przyrembel H, metabolic diseases, 2nd edn. Hodder Arnold, London. Wadman SK (1981) Disturbances of amino acid metab- Detailed clinically oriented monographs of a wide olism: clinical chemistry and diagnosis. Baltimore- range of individual metabolic disorders with excellent Munich, Urban & Schwarzenberg. photographs. A unique, detailed source of information on amino acids and aminoacidopathies. Contains large tables of Rimoin DL, Connor JM, Pyeritz RE, Korf BR normal values in various body ﬂ uids. Somewhat out- (eds) (2006) Emery and Rimoin’s principles and prac- dated and unfortunately out of print, but worth an effort tice of medical genetics, 5th edn. Churchill Livingston, to get secondhand. New York.

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 375 DOI: 10.1007/978-3-540-74723-9_E2, © Springer-Verlag Berlin Heidelberg 2010 376 Reference Books

An excellent, comprehensive and mostly up-to-date The last printed version of the standard textbook textbook of clinical genetics. Chapters dealing with met- on inborn errors of metabolism, extended to four vol- abolic disorders are not always convincing. 3 Volumes, umes and nonmetabolic conditions. Special emphasis quite expensive. on molecular and pathophysiologal aspects of individual disorders. It has now turned into a huge online Sarafoglu K, Hoffmann GF, Roth K (eds) (2009) knowledgebase (“Online Metabolic and Molecular Pediatric endocrinology and inborn errors of metabolism, Bases of Inherited Disease”) covering the widest pos- McGraw Hill, New York. sible range of inherited disorders but unfortunately Concise, comprehensive, and reasonably prized requires an expensive annual subscription (www. clinical textbook covering the whole ﬁ eld of metabolic ommbid.com). as well as endocrine disorders. A must for every consultant pediatrician, not only endocrinologists and Zschocke J, Hoffmann GF (2004) Vademecum met- metabolic specialists. abolicum – manual of metabolic paediatrics, 2nd edn. Schattauer, Stuttgart. Scriver CR, Beaudet AL, Sly WS, Valle D (eds) A concise pocket book containing the essentials of (2001) The metabolic and molecular bases of inherited metabolic pediatrics, with short descriptions of most disease, 7th edn. McGraw-Hill, New York. inborn errors of metabolism. Internet Resources E3

Society for the Study of Inborn Errors of Metabolism, USA. Its “GeneReviews” are extremely useful mono- SSIEM (www.ssiem.org) graphs with detailed information on clinical presentation, diagnosis, differential diagnosis, management and Society for Inherited Metabolic Disorders (www. genetic counseling, molecular genetics, etc., available simd.org) now for a huge number of individual conditions. There The homepages of the SSIEM (international, focus are also directories of clinical centres and diagnostic in Europe) and the SIEM (USA) provide excellent laboratories particularly in the USA. GeneTests incorpo- links to various internet resources concerned with rates the previous GeneClinics Web site. inborn errors of metabolism, including learned societies, parent organisations, laboratory directories, etc. Orphanet (www.orpha.net) and EuroGentest (www. eurogentest.org) National Center for Biotechnology Information, Orphanet is an originally French database that devel- NCBI (www.ncbi.nlm.nih.gov) oped into a major European portal for rare genetic dis- The primary source for molecular biology informa- eases and orphan drugs. It was selected as the central tion on the Internet. Home to Pubmed, OMIM, database of EuroGentest, a more recent EU-funded GenBank, and a vast range of other databases. May be Network of Excellence looking at various aspects of diffi cult to navigate without some experience. genetic testing, including quality management, information databases, and education. Together the websites OMIM (http://www.ncbi.nlm.nih.gov/omim) provide a wealth of information on rare diseases and This is the online version of “Mendelian Inheritance various resources particularly in Europe, including a in Man,” the oldest collation of genetic disorders, origi- laboratory directory, clinical centres, patient support nally edited by Victor A. McKusick. It is freely available groups, etc. The information is available in different lan- from the NCBI. The database contains monographs of guages; moreover, access to the site is free of charge. individual disorders and genes together with references and extensive links to other online resources. OMIM has European Directory of DNA Laboratories, EDDNAL an emphasis on molecular information, but there are also (www.eddnal.com) plenty of clinical data that are useful in daily practice. EDDNAL provides information on genetic services The number allocated to entries in the OMIM catalog is and molecular genetic laboratories in Europe. It was used in scientifi c publications worldwide for the exact initiated by the European Molecular Genetics Quality identifi cation of individual genetic disorders. Network (EMQN) initiative that provides quality assessment schemes for molecular tests. Access to the GeneTests (www.genetests.org) site is free of charge. A freely available medical genetics information resource, specifi cally for physicians and other health care Human Genome Variation Society Website, HGVS providers, is funded by the National Institute of Health, (www.hgvs.org)

G. F. Hoffmann et al. (eds.), Inherited Metabolic Diseases, 377 DOI: 10.1007/978-3-540-74723-9_E3, © Springer-Verlag Berlin Heidelberg 2010 378 Internet Resources

The HGVS aims to “foster discovery and character- GeneCards (www.genecards.org) ization of genomic variations.” The website provides This is a concise, gene-oriented database that con- links to a vast number of locus-specifi c mutation data- tains information on various functions of human bases as well as standard recommendations for muta- genes including the respective proteins and associated tion nomenclature. An important resource for clinical disorders. There are copious links to other Internet molecular geneticists as well as clinicians and scientists databases. GeneCards are particularly useful for genet- trying to fi nd additional mutation-related information. icists and researchers in the areas of functional genomics and proteomics. Human Gene Mutation Database, HGMD (www. hgmd.cf.ac.uk) Online Metabolic and Molecular Bases of Inherited This database on mutations causing human disor- Disease (www.ommbid.com) ders, based in Cardiff, Wales, was originally established It is the online successor of the standard textbook for the study of mutational mechanisms in human genes. of inborn errors of metabolism, with detailed infor- Mutations were also identifi ed by scanning published mation on molecular and pathophysiological aspects articles, a major effort. However, content freely avail- of individual disorders. Though vastly expanded to able is not up-to-date, and the full “professional” ver- cover the widest possible range of inherited disor- sion provided by a commercial partner is extremely ders, it unfortunately requires an expensive annual expensive even for academic/nonprofi t users. subscription. Index

A cerebrotendinous xanthomatosis (CTX), 146

Abetalipoproteinaemia, 235 Coenzyme Q10, 146 Acetoacetate, 342-346, 348 Friedreich ataxia, 145 Acholic stool, 92 GLUT-1, 144 Acidosis, 363 INAD, 145 hyperchloremic, 118, 122 Refsum disease, 145 hypochloremic, 119 vitamin E defi ciency, 145 renal tubular, 118, 123–124 ATGL, hydrolase, 164 Acrocyanosis, 198–200, 207 Autism, 178 Acrodermatitis enterpathica, 201, 202, 213, 214 Acute immunization, 25 B Acylcarnitines, 29, 31–33, 37, 38, 43, 46, 47, 55, 255 Barth syndrome, 77 Acyl-CoA oxidase, defi ciency, 288 Bartter syndrome, 119, 124–125 Adenosine triphosphatase defi ciency, 234, 236 B cells, 244, 245, 247 Adenylate kinase defi ciency, 236 Behr syndrome, 194 Alagille syndrome, 92, 93 Benzoate/phenyl acetate

Alkalosis, hypokalemic, 124, 125 BH4 test, 350–351 Alkaptonuria, 20, 21, 185 Bile, 337 Allopurinol test, 352 Bile acid biosynthesis defects, 93 Alopecia, 200–202, 205, 210–214, 363 Bile acid metabolism, disorders of, 9 Alpers disease, 100, 157, 327 Bile acids, 278–279 Amino acid decarboxylase defi ciency, 148 Biliary atresia, 92, 93 Amino acids Bilirubin metabolism, disorders of, 9 cerebrospinal fl uid, 269 Biochemical studies/physiological fl uids, 263–280 metabolism, disorders, 3–4 Biogenic amines, 148, 264, 279, 280 plasma, 267 Biopsy transport disorders, 6 brain, 319 urine, 268–269 conjunctiva, 318 Ammonia, 26–28, 30, 33, 34 extracerebral, 322 Amniocentesis, 359 liver, 320 Anderson-Fabry disease, 183–184 lymphocytes, 320 Anesthesia, 63–66 lysosomal diseases, 320, 321 Angiokeratoma, 198–199, 363 mitochondrial diseases, 320 Angiokeratomata, 85 peripheral nerve, 318 Angiomas, 199–200 peroxisomal diseases, 314 Anion gap, 118, 123 polyglucosan diseases, 321 a1-Antitrypsin defi ciency, 92, 93, 96, 97, 101, 103, 104 skeletal muscle, 320 Anxiety, 177, 178, 180 skin, 317 Apparent acute encephalitis, 363 Biotinidase defi ciency, 152, 253–254 Argininemia, 26, 49 Bleeding tendency, 241, 363 Argininosuccinic aciduria, 26, 53 Blood, 337 Arthritis, 363 Blood cell morphology, 235 Ascites, 109, 112, 113 Blue diaper syndrome, 21 Ataxia, 32, 143–146 Bone marrow transplantation, 243–246 abetalipoproteinemia, 145 Butyrylcholinesterase, 63 CDG 1a, 144 Byler disease, 93–94

613 614 Index

C Coma, 26, 28–32 Calcifi cation of basal ganglia, 363 Comparative gene identifi cation-58 (CGI-58), 164 Canavan diseas, 153 Congenital adrenal hyperplasia (CAH), 254–255, 260 Candidiasis, 246 Congenital disorders of glycosylation (CDG), 9, 229–231, 241, Carbamyl phosphate synthetase, defi ciency of, 26, 31 272–273 Carbohydrate metabolism Congenital hypothyroidism, 254 fructose loading test, 353–354 Connectivity theorem, 286 glucagon stimulation, 353 Constipation, 109, 110, 112 Cardiomyopathy, 170–174, 364 Control strength, 283, 285 Carnitine, 27, 31, 33 Copper, 6 Carnitine-acylcarnitine translocase, defi ciency of, 170, 175, 291 Corneal clouding, 182–186 Carnitine defi ciency, 76, 172, 173 Corneal opacity, 365 Carnitine palmitoyltransferase - defi ciency, 290 Cornea verticillata, 184 Carnitine palmitoyl transferase (CPT) I, defi ciency of, 65 Cornstarch, 58 Carnitine palmitoyl transferase II (CPT II), defi ciency of, 65, Corpus callosum agenesis, 365 165, 170 Costeff optic atrophy syndrome (OPA 3), 193, 194 Carnitine status (total, free, esterifi ed), 271 Creatine Carnitine transporter, defi ciency of, 76, 172 defi ciency of, 12 Carrier testing, 358 disorders, 277 Cataracts, 182 kinase-elevated, 365 Cataracts-lenticular opacity, 364 Crigler-Najjar syndrome, 90 Cerebral calcifi cation, 364 Cystinosis, 122, 123, 184–185, 196 Cerebral vascular disease, 364 Cystinuria, 120 Cerebral edema, 31 Cerebrospinal fl uid (CSF), 157, 158 D lymphocytosis, 364 Danon disease, 298, 299, 301 protein elevation, 364 D-bifunctional protein 5-defi ciency, 287– 289 Cerebrotendinous xanthomatosis, 152 Deafness, 131–135 Chanarin-Dorfman syndrome, 166 chronic progressive external ophthalmoplegia (CPEO), 132 Cherry-red spots, 192, 364 Fabry disease, 133–134 Child abuse, 336 Kearns–Sayre (KSS), 132, 133 Chiotriosidase, 221 maternally inherited diabetes and deafness (MIDD), 132, Chloride diarrhea, 115 133 Cholestasis, 91–94, 103 mitochondrial encephalopathy lactic acidosis stroke-like Cholestatic jaundice, 365 episodes (MELAS), 132 Cholesterol biosynthesis defects myoclonic epilepsy with ragged red fi bres (MERFF), 133 Conradi-Hunermann syndrome, 188 Debrancher enzyme, defi ciency of, 173 mevalonate kinase defi ciency, 188 Dehydration, 118–119, 122, 124 Smith-Lemli-Opitz syndrome, 188 Delayed speech development, 131 Cholesterol biosynthesis, disorders of, 8, 9, 219, 226–228 Delta-l-pyrroline-5-synthase defi ciency, 188 Chondrodysplasia punctata, Dementia, 177–180 rizomelic, 225–227 Depression, 178–180 phenotypes, 365 Dermatan sulphate, 221 Chorea, 146–153 Dermatosis, 366 Chronic pancreatitis, 365 Deterioration, 178–180 Chorionic villus sampling, 359 Developmental delay, 135–137 Cirrhosis, 89, 92, 93, 96, 98, 100–104, 365 Diabetes insipidus, nephrogenic, 118 Citric acid cycle, 283, 292, 293 Diabetes mellitus-erroneous diagnosis, 366 Citrin defi ciency, 97 Diarrhea, 109, 110, 112–115, 366 Citrullinemia, 28 congenital chloride, 118, 119 Cobalamin disorders, 234, 237–240 2,4-Dienoyl-CoA reductase, defi ciency of, 173 Cobalamine metabolism, disorders of, 5–6 Dimethylglycinuria, 18, 20

Coenzyme Q10, 328 Dinitrophenylhydrazine (DNPH), 28, 29, 34 deﬁ ciency of, 167, 173–175 Disorders of fatty acid oxidation, 26, 29, 30, 39, 42, 44, Colorimetric analyses 46, 47, 63 brand test (sulfhydryls), 265–266 Disorders of ketolysis, 34 dinitrophenylhydrazine (DNPH), 264–265 D-Lactic acid, 36 Ehrlichs (aldehydes), 266 DNA nitrosonaphthol (tyrosine and metabolites), 266, 267 arrays, 308 reducing substances, 265 depletion syndromes, mitochondrial, 96, 99 sulﬁ te, 267 sequencing, 308 urinalysis, 265 studies, 305–309 Index 615

Dopamine, defi ciency of, 11, 12 Flux control coeffi cient, 286 Dubin-Johnson syndrome, 94 Foetal blood sampling, 359 Dysmorphic features, 16, 18 Foetal hydrops, 220, 222, 224, 227 Dysmorphies, 219, 220, 225–231 Folate, 278 Dysostosis multiplex, 220–224, 366 defi ciency, 152 Dysrhythmias, conduction disturbances, 83, 85 disorders, 237–240 Dystonia, 146–153 Folic acid metabolism, disorders of, 5–6 Forearm ischemia test, 354–355 E Free fatty acids, 39, 40, 42–44, 46, 48, 272, 340-346, 354 Ectopia lentis, 188–189, 366 Friedreich ataxia, 77–78 Eczema, 198–201, 210, 213, 214 Fructose intolerance, 29, 55, 58 Electroencephalography (EEG), 157 hereditary, 99 Electromyography (EMG), 157 Fructose loading test, 353–354 Electron microscopy, 311, 315, 322 Fructose metabolism, disorders of, 5 Electroretinography (ERG), 157 Emergency treatment, 55, 58 G Endocardial fi broelastosis, 73 Galactose (and metabolites), 274 Energy metabolism Galactose metabolism, disorders of, 5 glucose challenge, 348 Galactosemia, 26, 27, 55, 58, 90, 91, 94–96, 99, 102, 187, preprandial/postprandial analyses, 347–348 252–253 Enzymes, 283–301 Galactose-1-phosphate uridyltransferase (GALT), 252–253 Epilepsy, 137–143 Galactosialidosis, 184 Alpers Disease, 142 Gamma-aminobutiric acid (GABA), 158 Biotinidase defi ciency, 140 Gaucher disease, 225, 234–236, 240, 241, 296, 299, 300 creatine defi ciency, 140 Genotype, 305– 307, 309 GAMT defi ciency, 140 Gilbert syndrome, 90 GLUT1 defi ciency, 140 Gitelman syndrome, 119, 124, 125 Lafora disease, 142 Glucagon stimulation, 353 Menkes Disease, 140 Glucokinase gene, defi ciency of, 41, 252 neonatal seizures, 137–139 Gluconeogenesis neuronal ceroidlipofuscinosis, 141 defects, 37, 42, 55, 103, 104 non-ketotic hyperglycinemia (NKH), 139 disorders of, 5 pyridoxal phosphate-dependent seizures, 138–139 Glucose challenge, 348 pyridoxine-dependent seizures (PDS), 137–138 Glucose-galactose malabsorption, 114 Epimerase, defi ciency of, 252 Glucose 6 Phosphate dehydrogenase defi ciency, 234, 236, Erythropoietic porphyria, 234, 236 237 Etherphospholipid biosynthesis, 294, 298, 299 Glucose transporter 1 protein, defi ciency of, 30 Exercise test, ischemic, 164, 168 a-Glucosidase (acid maltase), defi ciency of, 173 Exercise test GLUT-1, 147, 158 tolerance, 354–355 Glutamate dehydrogenase, defi ciency of, 56–57 intolerance, 366 Glutaric aciduria, 148–149 type 1, 51 F type II, 173 Fabry disease, 134, 220, 221, 224, 225, 295, 296, 300 Glutathione, 275 Familial fever reductase defi ciency, 236 autosomal dominant/ Hibernian, 111–112 synthetase defi ciency, 236, 237 mediterranean, 111 Glycogenosis type I, 107 Family testing, 357 Glycogenosis type III, 107 Fanconi syndrome, 121–125 Glycogenosis type IV, 101–103 Farber’s disease, 184 Glycogenosis type IX, 107 Fasting test, 38 Glycogenosis type VI, 107 adverse reactions, 342, 343, 354 Glycogenosis type VIII, 107 differential diagnosis, 345-346 Glycogenosis type XI/ Fanconi-Bickel syndrome, 107 Fat pad, 207–209 Glycogen storage disease (GSD), 70, 78, 79, 87 Fatty acid oxidation type 0, 174 monitored prolonged fast, 340–346 type I, 37, 58, 166 phenylproprionic acid loading test, 346–347 type Ib, 239–241 Fatty acid oxidation defects type II, 166, 173 liver disease, 94, 96, 97 type III, 37, 58, 173–174 Fatty acid oxidation (FAO) disorder, 4, 5, 255, 283, 285, 287, type IV, 174 292, 335–336, 355 type V, 167–168 Fever syndromes, 366 Glycogen synthase, defi ciency of, 39, 40, 43 616 Index

Glycolysis, 164–166, 168, 169 mild, 252 disorders of, 5 screening for, 252 Glycosaminoglycans (GAGs), 221 Hyperpigmentation, 207, 210–212 Glycosaminoglycans (mucopolysaccharides), 275–276 Hypertrophy, 72, 73, 75 Glycosuria, 366 Hypertyrosinemia, 368 Glycosylation, disorders of, 9, 228–231 Hypobetalipoproteinaemia, 237 GM2-Gangliosidosis, 130 Hypogammaglobulinemia, 245 Guthrie card, 251 Hypoglycemia, 28, 30–34, 36–41, 64, 65 Gyrate atrophy of choroid and retina, 191 Hypoketotic hypoglycemia, 26, 30, 31, 39, 41, 43, 45, 368 Hypophosphatemia, 368 H Hypopigmentation, 207, 209–211, 214 Hair abnormalities, 366 Hypouricemia, 368 HDL (lipoprotein) low, 367 HELLP syndrome, 16 I Hematuria, 120 Ichthyosis, 197, 204–206, 368 Hemiplegia, 51, 52 retinal disease, 368 Hemochromatosis, 96–98, 104, 105 Immunodefi cieny, 244–247 Hemoglobinuria, 20, 21 Infantile neuroaxonal dystrophy (INAD), 131, 157 Hemolytic anemia, 367 Infantile Refsum disease, 287 extrinsic, 234, 236 Infection, 243–247 isolated, 234, 236 Inherited metabolic disease, 251–260 with neurological abnormalities, 236, 237, 240 Inhibitor constant (Ki), 286 Hemophagocytosis (Erythrophagocytosis), 367, 241 Intermediary metabolism, disorders, 3–6 Heparan sulphate, 221 Inverted nipples, 368 Hepatic carcinoma, 367 Iron, 6 Hepatic cirrhosis, 367 Isolated defi ciency of speech as presentation in metabolic Hepatic failure-acute, 367 disease, 368–369 Hepatomegaly, 89, 94, 97–99, 101, 103–107 Isoprenoid biosynthesis, disorders of, 8 Hereditary hemochromatosis (HH), 79–80 Isovaleric acidemia, 28, 33, 49 Hereditary spastic paraplegia, 195 Heteroplasmy, 325, 330 J Hirsutism, 213–215 Jaundice, 89–97, 99, 100, 102 HMG-Co A lyase, defi ciency, 45, 46 Juvenile hemochromatosis (JH), 80 HMG-Co A synthase, defi ciency, 21, 46 Holocarboxylase synthetase defi ciency, 240 K Homocystinuria, 66, 189, 190, 226 Kearns–Sayre syndrome, 76, 330 Hunter disease, 295 Keratan sulphate, 221 Hurler’s disease, 221 Ketogenesis, disorders of, 4–5 3-Hydoxy-3-methyglutarate (HMG), 45 Ketones, 272, 341-347 Hydrops fetalis, 220, 222, 224, 227, 367 Ketonuria, 29, 32–34, 41–43 non-immune, 113 b-Ketothiolase, 110, 112 Hydroxyacyl-CoA dehydrogenase (HADH), defi ciency of, Ketotic hypoglycemia, 39, 40, 42, 44–46, 64 172 Kidney stones, 119 3-Hydroxybutyrate, 26, 27, 30, 32–34, 342–346, 348 Kinky hair, 212 3-Hydroxyglutaric aciduria, 367 Krabbe disease, 131, 195 17-Hydroxyprogesterone (17-OHP), 255 Hyperammonemia, 27–29, 33, 34, 41, 44, 47–54, 57, 109, 112, 367–368 L disorders, 4, 6 Lactase defi ciency, intolerance, 114 Hyperbilirubinemia Lactate, 272, 325, 326, 329–333 conjugated/direct, 91 Lactate dehydrogenase, defi ciency of, 168, 170 conjugated/transient, 92 Lactic acidemia, 35–37, 47, 369 unconjugated/indirect, 90 Lactic acidosis, 325, 327–330, 332 Hypercholesterolaemia, 9 Laxity skin, 197, 212, 214–216 Hypercoagulability, 241 Leber’s hereditary optic atrophy (LHON), 193 Hyper-IgD syndrome, 112, 227 Lecithin:Cholesterol acyltransferase defi ciency (LCAT), Hyperinsulinemia, 39–41 234–236 Hyperkeratosis, 198–200, 205 Leigh syndrome, 194, 195, 331, 369 Hyperleukocytosis, 241 Lens defects and dislocation, 186–188 Hyperlipidaemia, 9 Lesch–Nyhan disease, 149, 237, 238 Hyperoxaluria Type, 288, 290 Leucine loading test, 349 Hyperphenylalanemia, 252 Leukopenia with or without thrombopenia and anemia, 369 Index 617

Lipid metabolism disorders, 185–188 inborn errors, 73–80 Lipid storage disorders, 8 investigations, 80–83 Lipoprotein disorders, 86–87 metabolic disorders, 71–72 Lipoprotein metabolism, disorders of, 9 patterns and pathophysiology, 70–73 Liver, 337 treatment principles, 83 failure, 89, 92–101 Metabolic emergencies, 25–59 transplantation, 90, 91, 93, 97, 98, 100, 102 Metabolic fl ux analysis, 285–286 L-Lactic acid, 36 Metachromatic leukodystrophy (MLD), 129 Long-chain acyl-CoA dehydrogenase, defi ciency of, 170, 171 Methemoglobinemia, 241 Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) 2-MethylacylCoA racemase, defi ciency, 287–288 defi ciency, 45, 74, 83, 171–172 Methylenetetrahydrofolate reductase, defi ciency of, 26, 54 Lowe syndrome, 186 Methylmalonic acidemia, 29, 31, 49 Lumbar puncture, 157 Methylmalonic aciduria, 369 Lupus, 200, 201, 203 Methylmalonyl-CoA mutase (MCM), 351 Lymphopenia, 244–246 Mevalonic aciduria, 227, 238 Lymphoproliferation, 246 Michaelis-Menten kinetics, 284 Lysinuric protein intolerance, 188, 240, 241 Microvillus inclusion disease, 115 Lysosomal diseases Mitochdondial encephalomyopathy, lactic acidosis and Fabry disease, 315 stroke-like episodes (MELAS), 327, 330 Farber disease, 315 Mitochondria, 287, 290–294, 298 Gaucher disease, 315 Mitochondrial disease, 5, 26, 31, 35, 59, 153, 228 globoid cell leukodystrophy, 319 Barth syndrome, 239, 240

GM1 gangliosidosis, 314, 318 Pearson syndrome, 234, 237, 239

GM2 gangliosidosis, 314, 318 Sideroblasts, 239 metachromatic leukodystrophy, 314, 318 Mitochondrial DNA (mtDNA), 293, 325, 328, 330 mucolipidoses, 313, 315 Mitochondrial encephalomyopathy with ragged red fi bres mucopolysaccharidoses, 313, 321 (MERRF), 330 neuronal ceroid-lipofuscinoses, 312, 319, 321 Mitochondrial myopathies Niemann-pick disease, 313– 315, 319– 322 CPEO, 174 oligosaccharidoses, 313, 315, 319, 321 Kearns–Sayre syndrome, 174 storage disorders, 7–8, 220–225, 283, 294–296, 298, 300, 301 MELAS, 174 type-II glycogenosis, 313, 315, 318, 319, 321 MERFF, 174, 175 Lysosomal disorders, 134, 153, 182–186, 191, 192, 194–196 Mitochondrial neurogastrointestinal encephalopathy (MNGIE), Lysosomal enzymes, 283, 294, 295, 298–300 113, 329 Lysosomal residual bodies Mohr-Tranebjaerg syndrome, 195 non-vacuolar, 313, 315, 316, 321 Molecular genetics, 309 ultrastructure, 315 Mongolian spot - extensive, 369 vacuolar, 311, 313– 315, 317– 319, 321 Monitored prolonged fast adverse reactions, treatment, 343 M interpretation, 343–346 Macrocephaly, 369 preparations, 342 Macrocytosis, 237, 238 procedure, 342–343 Malabsorption, 110, 114–116 Morquio disease, 64, 221, 222 Maldigestion, 109, 113–114 Movement disorders, 143 Malignant hyperthermia, 63, 66, 175 Mucolipidosis type IV, 184 Mania, 179 Mucopolysaccharidoses, 8, 134–135, 182, 183, 191, 194, Maple syrup urine disease, 25–29, 32, 34, 51, 53, 55–57, 65 221–224 Marfan syndrome, 188, 189 Mucopolysaccharidosis, 63, 64, 300 Maroteaux-Lamy disease, 221, 222 Multiple acyl-CoA dehydrogenase defi ciency (MADD), 45, 59, Massive ketosis, 28, 29, 31–34, 42, 48 74, 83, 173, 228 Maternal PKU, 16 Multiple sulphatase defi ciency, 153 McArdle disease, 165, 167–168 Muscle Medium-chain acyl-CoA dehydrogenase (MCAD), defi ciency biopsy, 332 of, 45, 49, 167, 291 energy metabolism, 164 Megaloblastic anemia, 369 glycogen phosphorylase (myophosphorylase), defi ciency of, Menkes disease, 113 167–168 Mental retardation, 135–137 glycogen phosphorylase kinase, defi ciency of, 168 Metabolic acidosis, 31, 36, 51 hypotonia, 4, 12 Metabolic acidosis and ketosis, 369 Mutation analysis, 308 Metabolic cardiomyopathy Myoadenylate deaminase, defi ciency of, 168–170 cardiac metabolism, 70 Myoglobinuria, 165–168, 175 diagnostic approach, algorithm, 81 Myocardial infarction-cerebral vascular disease, 370 618 Index

N Paralysis of upward gaze, 371 Natural killer (NK) cells, 244, 247 Parkinsonism, 146–153 Neonatal adrenoleukodystrophy, 290 Peptide metabolism, disorders of, 6 Neonatal hemochromatosis, 96–98 Periodic fever. See Familial fever Neonatal hepatic presentations in metabolic diseases, 370 Peroxins, 226, 287 Neonatal hepatitis syndrome, 92, 93 Peroxisomal disorders, 8, 9, 186, 188, 191–192, 194, 195, Nerometabolic disease, 127 225–226, 301 Nervous system, 127 chondrodysplasia punctata, 188 Neuroaxonal dystrophy, 153 neonatal adrenoleukodystrophy, 188 Neurodegeneration, 128, 129, 131 Refsun disease, 188 Neurological examination, 128 Zellweger syndrome, 188 Neurological symptoms, 127 Peroxisomal function Neuronal ceroid lipofuscinoses (NCL), 130–131, 157 phytanate, 276–277 Neuropathy, ataxia and retinitis pigmentosa (NARP), 330 pristanate, 276–277 Neurophysiology, 157 very long chain fatty acids, 225 Neuroradiology Peroxisome biogenesis, disorders, 288 brain MRI, 154–156 PEX genes, 287 INAD, 155 Phagocytosis, 246, 247 PKAN defi ciency, 155 Phenotype, 306, 307, 309 X-ALD, 155 Phenylalanine loading test, 349–350 Neurotransmitter defects, 11–12 Phenylbutyrate, 57 Neurotransmitters, 148 Phenylketonuria (PKU), 252 Neutropenia, 234, 239, 240 Phenylproprionic acid loading test, 346–347 Neutrophil function, 244, 246, 247 Phosphofructokinase defi ciency, 236, 237 Newborn screening, MS/MS, 251–260 Phosphoglycerate kinase defi ciency, 236, 237 Niemann–Pick disease, 92, 103, 235, 236, 240, 241, 301 Phosphomannomutase defi ciency, 229, 230 Niemann–Pick disease type C (NPC), 130 Phosphomannose isomerase, defi ciency of, 115 Nodules, 206, 207, 212 Phosphorylase b kinase, defi ciency of, 168 Nonketotic hyperglycinemia, 11 Photophobia, 371 Nutrient-defi ciency cardiomyopathies, 80 Photosensitivity, 197, 198, 200, 211–212 Physical abnormalities, 219–231 O Phytanic acid, 287–289 Oculogyric crises, 11 Phytosterolaemia, 234, 236 Odd or unusual odor, 370 Pigment retinophaties Oligosaccharidoses, 8, 186, 194, 195, 222, 224 Farber’s disease, 192 Optic atrophy, 370 Gaucher disease type II, 192 primary, 193–194 GM1 gangliosidosis, 192 secondary, 194–196 GM2 gangliosidosis, 192 Organic acidemia, 65 metachromatic leucodystrophy, 192 Organic acid oxidation disorders, screening for, 255 Niemann-Pick disease type A, 192 Organic acids (urine), 269–270 sialidosis, 192 Organic acidurias, 3–5, 153, 239 Plasmalogens, 225, 287 Organomegaly, 220 Polar body analysis, 359–360 Ornithine aminotransferase defi ciency, 186, 188, 191 Polycystic kidneys, 371 Ornithine transcarbamylase (OTC) defi ciency, 26, 30, 47, 51, Polymerase gamma (POLG1), 293 52, 58, 352 defi ciency of, 113 Orotate, 264 Polyols, 264, 275 Orotic aciduria, 47–49, 237–240, 370 defects, 7 Osteoporosis and fractures, 370 Pompe disease, 78, 165, 173, 221 Overwhelming neonatal illness, 28 Porphyrias, 9 Oxidative phosphorylation, 285, 325–327 acute intermittent, 111 Oxothiolase defi ciency, 29 hereditary coproporphyria, 110, 111 variegate, 110, 111 P Porphyrins, 279–280 Pain, abdominal, diffuse, 110 Postmortem investigations Pain and elevated erythrocyte sedimentation rate, 370 protocol, 335–336 Pancreatitis, 109, 110, 112, 370-371 specimen requirements, 337 Pancytopenia, 238–240 Preimplantation genetic diagnosis, 359–360 Panthothenate-kinase-associated neurodegeneration Prenatal diagnosis, 357–360 (PKAN), 157 Preprandial/postprandial analyses, 347–348 defi ciency, 150 Primary carnitine defi ciency, 290, 291 Papular lesions, 197, 199, 206–209 Progressive familial intrahepatic cholestasis (PFIC), 93–94 Index 619

Propionic acidemia, 26, 32, 33, 49, 52 Schizophrenia, 177, 178, 180 Protein-losing enteropathy, 115 Scoliosis, 372 Protein metabolism Seborrheic dermatitis, 200, 201 allopurinol test, 352 Segawa disease, 148

BH4 test, 350–351 Selenium, 80 leucine loading test, 349 Self injurous behavior, 372 phenylalanine loading test, 349–350 Sensorineural deafness, 372

vitamin B12 test, 351–352 Sepiapterin reductase defi ciency, 148 Psychosis, 177–180 Sequencing, 308 Psychotic behavior, 371 Serine biosynthesis, disorder of, 11 Pterins, 260, 264, 269 Serotonine, defi ciency of, 11 Ptosis, 371 Short-chain acyl-CoA dehydrogenase (SCAD), defi ciency of, Pulmonary arterial hypertension (PAH), 87–88 45, 172, 290, 291 Purine metabolism, disorders, 7 Short-chain hydroxyacyl-CoA dehydrogenase (SCHAD). See Purines and pyrimidines, 264, 273 also Hydroxyacyl-CoA dehydrogenase (HADH) Pyloric stenosis, 28, 33 defi ciency, 45 Pyridoxal phosphate, 12 Sideroblastic anemia, 237, 239 Pyrimidine metabolism, disorders of, 7 Sjögren–Larsson syndrome, 153 Pyrimidine 5-nucleotidase defi ciency, 235–237 Skin, 337 Pyruvate, 272 rash, 200, 201, 203, 207 carboxylase, defi ciency of, 37, 38, 53 Smith–Lemli–Opitz syndrome, 219, 227 dehydrogenase, defi ciency of, 32, 36–38, 53, 54, 58, 285, Spasticity, 152 292 Spastic paraparesis, 372 Sphingolipidoses, 8, 195, 196, 222, 224 Sterol biosynthesis, disorders of, 8 R Stroke-like episodes, 372 Ragged red fi bers, 371 Strokes, 31, 32, 36, 51–54, 85 Red urine, 371 Subdural effusions, 373 Refsum disease, 64–65, 287–289 Succinate semialdehyde dehydrogenase defi ciency, 11 retinal pigment abnormality, 192 Succinnylcholine, 63, 66 Renal calculi, 371 Succinyl CoA: oxoacid transferase defi ciency, 32, 34, Renal Fanconi syndrome, 371 38, 42 Renal tubular acidosis, 118, 123–124, 371 Sudden death, 335–337 Respiratory chain, 325–329, 331–333 Sugars (urine), 274–275 complexes, 292 Suicide, 178 Retinitis pigmentosa, 372 Sulfi te oxidase defi ciency, 12 abetalipoproteinemia, 191 Sulfonylurea receptor gene (SUR1), defi ciency of, 41 Cockayne syndrome, 192 HHH syndrome, 190 T 3-hydroxyacyl-CoA-dehydrogenase defi ciency, 190 Tafazzin (TAZ) gene, 77 Joubert syndrome, 192 Tandem mass spectrometry (MS/MS), 251, 255–260 Kearns-Sayre syndrome, 190, 192 T cells, 243–247 Laurence-Moon-Biedl syndrome, 192 Telangectasia, 199 neuronal ceroid lipofuscinois, 190–191 Tetrahydrobiopterin (BH4) test, 350-351 OAT defi ciency, 190 Tetrahydrobiopterin, defi ciency of, 11 peroxisomal disorders, 191–192 Thiamine, 80 PKAN, 192 Thiamine responsive megaloblastic anemia, 237–239 primary, 190 Thrombembolic disorders, 85–87 Refsum disease, 191, 192 Thrombocytopenia, 234, 240 secondary, 190, 191 Thymidine phosphorylase, defi ciency of, 113 Sjögren-Larsson syndrome, 191 Thyroid stimulating hormone (TSH), 254 Usher type II syndrome, 192 Transcobalamin II defi ciency, 238, 240 Reye syndrome, 30, 40, 43, 44, 48, 97, 372 Transient hyperammonemia of the newborn, 29, 49 Reynaud syndrome, 372 Trichorrexis nodosa, 209, 213, 214 Rhabdomyolysis, 63–66, 165–172, 174, 372 Trifunctional enzyme defi ciency, 74 Rhizomelic chondrodysplasia punctata, 287, 289 Trimethylaminuria, 18, 19 Rotor syndrome, 94 Triosephosphate isomerase defi ciency, 236, 237 RYR1 gene, 63, 66 Tubular dysfunction, renal, 117, 121–126 Tyrosine hydroxilase, 148 S Tyrosinemia Sanfi lippo disease, 131 transient neonatal, 98 Scheie disease, 221–223 type I, 58, 92, 96–99 620 Index

type II, 98, 182, 185, 196 Vitamin B12, deﬁ ciency of, 115

type III, 98, 99 Vitamin B12 test, 351–352 Vitamin E deﬁ ciency, 152 U Vomiting, 25, 26, 28, 32, 33, 40, 44, 52, 53, 56–58 Ulcer, 200, 201, 203, 210, 212 chronic or recurrent, 109 Unexpected death, 335–336 cyclic, 110 Urea cycle defects, 152 erroneous diagnosis of pyloric stenosis, 373 liver disease, 95–97 Urea cycle disorders, 3, 4, 52–55 W Uric acid Wilson disease, 97, 100–101, 104, 122, 149–151, 182, 185, clearance, 120 186, 234, 236, 237 excretion, 120, 121 Wolcott Rallison syndrome, 9 Urine, 337 color, 18, 20 X V Xantoma, 208, 373 Valvular dysfunction, 85 X-linked adrenoleukodystrophy (X-ALD), 130, 287, Ventricular hypertrophy, 75, 78, 79 289 Very long-chain acyl-CoA dehydrogenase (VLCAD) deﬁ ciency, 45, 74, 166, 170–171 Z Very long-chain fatty acids (VLCFA), 225, 226 Zellweger syndrome, 226, 228, 287, 288

Vitamin B6, 12 Zinc, 6