Argininosuccinic Aciduria: Clinical and Biochemical Phenotype Fi Ndings In

Malaysian J Pathol 2010; 32(2) : 87 – 95

ORIGINAL ARTICLE Argininosuccinic aciduria: Clinical and biochemical phenotype findings in Malaysian children

Msc, MRCP MPath CHEN Bee Chin NGU Lock Hock and *ZABEDAH Md Yunus

Department of Genetics, Kuala Lumpur Hospital and *Biochemistry Unit, Specialized Diagnostic Centre, Institute for Medical Research, Kuala Lumpur, Malaysia.

Abstract

Argininosuccinic aciduria is an inborn error of the urea cycle caused by defi ciency of argininosuccinate lyase (ASL). ASL-defi cient patients present with progressive intoxication due to accumulation of ammonia in the body. Early diagnosis and treatment of hyperammonemia are necessary to improve survival and prevent long-term handicap. Two clinical phenotypes have been recognized – neonatal acute and milder late-onset form. We investigated patients with hyperammonemia by a stepwise approach in which quantitative amino acids analysis was the core diagnostic procedure. Here, we describe the clinical phenotypes and biochemical characteristics in diagnosing this group of patients. We have identifi ed 13 patients with argininosuccinic aciduria from 2003 till 2009. Ten patients who presented with acute neonatal hyperammonemic encephalopathy had markedly elevated blood ammonia (>430 μmol/L) within the fi rst few days of life. Three patients with late- onset disease had more subtle clinical presentations and they developed hyperammonemia only during the acute catabolic state at two to twelve months of age. Their blood ammonia was mild to moderately elevated (>75–265 μmol/L). The diagnosis was confi rmed by detection of excessive levels of argininosuccinate in the urine and/or plasma. They also have moderately increased levels of citrulline and, low levels of arginine and ornithine in their plasma. Two patients succumbed to the disease. To date, eleven patients remained well on a dietary protein restriction, oral ammonia scavenging drugs and arginine supplementation. The majority of them have a reasonable good neurological outcome.

Keywords: Argininosuccinic aciduria, argininosuccinate lyase deﬁ ciency, hyperammonemia, urea cycle disorders, quantitative amino acid analysis

INTRODUCTION illness that rapidly progresses from poor feeding, vomiting, lethargy or irritability and tachypnea to Argininosuccinic aciduria (ASA, #MIM608310) seizure, coma and respiratory arrest. Early clinical is a rare autosomal recessive disorder caused by recognition and laboratory diagnosis, and urgent the defi ciency of argininosuccinate lyase. It is treatment to control hyperammonemia are crucial one of the six enzymes in the urea cycle pathway in order to prevent death and severe neurological that converts the toxic ammonium nitrogen into handicap.5 Patients with late-onset disease may urea before being excreted in the urine (Figure 1). present at any age outside of the newborn period. The gene for ASL defi ciency is located on Their clinical manifestations are generally less chromosome 7 and has been mapped to locus acute and more subtle than the neonatal-onset 7q11.2.1,2,3 The estimated worldwide incidence variant, and often are precipitated by stress such among general population is 1: 70,000.4 Two as infection and anesthesia.6,7 Their symptoms clinical phenotypes have been recognized may include anorexia, recurrent vomiting, – a neonatal acute form (the classical form) failure to thrive, epilepsy, developmental delay and a milder late-onset form. Patients with and behavioral problem.1,2,3 We report here our neonatal-onset disease present with severe experience in diagnosing and treating a cohort of hyperammonemic coma within the fi rst few 13 children with argininosuccinic aciduria. days of life. They usually have an overwhelming

Address for correspondence and reprint requests: Chen Bee Chin, Biochemical Genetics Unit, Department of Genetics, Kuala Lumpur Hospital, Jalan Pahang, 50586 Kuala Lumpur, Malaysia. Tel: 603-26155555 ext 6886, Fax: 603-26155705. Email: [email protected]

87 Malaysian J Pathol December 2010

Mitochondria NAGS N-acetylglutamate Glutamine Ammonia CPS I Carbamyl phosphate Orotic, – Orotidine HCO3 Citrulline & Uracil OTC Aspartate Urea Ornithine ASS

Arginase Argininosuccinate

ASL Arginine

Cytosol Fumarate

FIG. 1: Urea cycle pathway. The Urea cycle comprises of six enzymes: N-acetyl-glutamate synthase (NAGS), Carbamyl-Phosphate-Synthetase-I (CPS I), Ornithine Transcarbamylase (OTC), Argininosuccinate Synthetase (ASS), Argininosuccinate Lyase (ASL), and Arginase.

MATERIALS AND METHODS and the plasma was separated from the blood cells immediately by centrifugation. A minimum of We received samples and referral from 2 mL urine was collected in a sterile container. paediatricians nationwide for the diagnosis of Plasma and urine were frozen at -20°C if they urea cycle disorders (UCD) in children with could not be analyzed immediately. Samples were hyperammonemia. We followed a stepwise transported in an ice box and arrived frozen in diagnostic protocol as shown in Figure 2. the laboratory. Quantitative amino acid analysis in plasma and/ or urine (for patients suspected of having Chemicals argininosuccinic aciduria) is the most important Argininosuccinic acid, 5-sulphosalicylic acid diagnostic tool in the evaluation for UCD. (SSA), and physiological standard A and B were Presence of argininosuccinate in plasma or urine purchased from Sigma. The ultra physiological was mandatory in order to make a diagnosis of fl uid chemical kit was purchased from Biochrom ASL defi ciency/argininosuccinic aciduria. We Ltd., (Cambridge, UK). reviewed retrospectively the clinical records and laboratory data of more than 360 children Instrument from 8270 samples (4.35%) received who were Amino acids were quantifi ed by ion-exchange evaluated for hyperammonemia in our centre chromatography using a dedicated amino acids over a seven-year period (2003 – 2009). analyzer (Biochrom 30) and post column detection. In principle, the instrument system Samples works by pumping buffers of varying pH and Blood and urine samples were collected from ionic strength through a column of cation- acutely ill children when the basic metabolic exchange resin to separate the various amino screen showed signifi cant hyperammonemia. acids. The column eluent is mixed with the Blood (1-2 mL) was collected in a heparin tube ninhydrin reagent, and the mixture is then passed

88 ARGININOSUCCINIC ACIDURIA IN MALAYSIAN CHILDREN

Hyperammonemia

Plasma amino acid quantitative analysis

High plasma citrulline High plasma citrulline Low plasma citrulline

Arginase deficiency Argininosuccinate detection urine orotic acid in plasma and urine

Detected Not detected Elevated Low/normal

ASL deficiency ASS deficiency OTC deficiency CPS-I deficiency or NAGS deficiency

FIG. 2: Stepwise diagnostic protocol for the investigation of hyperammonemia. ASL: Argininosuccinate Lyase; ASS: Argininosuccinate Synthetase; CPS-I: Carbamyl-Phosphate-Synthetase-I; NAGS: N-Acetyl- Glutamate Synthase; OTC: Ornithine Transcarbamylase through a high temperature reaction coil. In Urine amino acids analysis the reaction coil, the ninhydrin reacts with the About 2mL of urine was required and the amino acid to form a coloured compound, and method for sample processing was similar to the amount of coloured compound produced is that of plasma. About 20 μL of fi ltrate was directly proportional to the quantity of amino injected into the amino acid analyzer. The acid present. The absorbance is measured by running time takes 2 hours for each sample. wavelengths at 570 nm and 440 nm. The whole Creatinine concentration was determined in the system is computer-controlled. urine sample by the modifi ed Jaffe (alkaline- picrate) using Modular Biochemistry Roche Plasma amino acids analysis Analyser and Roche reagents prior the analysis Plasma (100μL) was pipetted into an eppendorf of amino acids. tube and 100μL of 10% SSA solution was then added. The tube was capped, agitated for a few RESULTS seconds, and allowed to stand for 1 hour at We identifi ed 13 patients (from 12 families) with 40C. It was then centrifuged at 10,000 rpm for argininosuccinic aciduria (four boys and nine 5 minutes. The supernatant was fi ltered through girls). This is 0.16% of 8270 patients referred a 0.2 μm membrane to remove any remaining to our centre for investigation of possible particulate materials prior to analysis. The inborn errors of metabolism over seven year fi ltrate was transferred into a vial and loaded period. Table 1 and 2 summarize the clinical into an autosampler. The fi ltrate (20 μL) was presentations and laboratory data of our cohort then injected into the amino acid analyzer. A respectively. Eleven out of thirteen patients running time of 2 hours was required for each were Malay. Parental consanguinity was noted sample. in two families.

Malaysian J Pathol December 2010

Current status Current

Recurrent crises Recurrent Current age Current

ammonia scavenger drugs; c, mental retardation; 5, moder-

Acute treatment Acute

diagnosis Age at at Age

Clinical symptoms

Age at onset at Age consanguinity

: Patient 1 and 2 are siblings. *: until five years old.

# Parental Parental

Ethnic Gender B M no 2d coma, respiratory distress feeding refusal, vomiting, lethargy, B M no 7d a,b,c 8m 5y coma (following febrile illness) feeding refusal, vomiting, lethargy, yes 14m 1,2,5 c 4.5y no 1,2,4 B B M M no no 3d 3d seizure, coma feeding refusal, vomiting, lethargy, feeding refusal, vomiting, lethargy 15d a,c - no 4d Died a,b 3y no 1,2,3 G M G yes G C 3d G M no G coma feeding refusal, vomiting, lethargy, M no 10d G I no G 2d coma feeding refusal, vomiting, lethargy, M 3d no M coma feeding refusal, vomiting, lethargy, no G 13d seizure, coma feeding refusal, vomiting, lethargy, yes G 3d M coma feeding refusal, vomiting, lethargy, 5 d M coma feeding refusal, vomiting, hyperirritability, no seizure, coma feeding refusal, vomiting, lethargy, yes 1y 2m brittle hair epilepsy, developmental delay, recurrent vomiting, growth failure epilepsy, developmental delay, 10d c 5d 5y 21d b,c 3.5y b,c 23d - 5d 8 d 3m no c 2y a,b,c 11y a,c 20d 1,2,4 a,b,c no 2y 1.5y yes* no - 1,2,5 7y 1,2,3 no 1,2,4 no 6y 1,2,3 no no 1,2,4 Died 1,2,3 - 12y no 1,2,4 # # 5 12 4 6 7 8 9 10 11 13 m: month; y: year; acute treatment: a, dialysis b, intravenous B: boy; G: girl; M: Malay; C: Chinese; I: Indian; d: day, 2 3 ventilator support; current status: 1, protein restricted diet; 2, oral ammonia scavenger drugs; 3, normal development; 4, mild ate mental retardation. TABLE 1: Summary of the clinical phenotypes of 13 patients with argininosuccinic aciduria 1: Summary of the clinical phenotypes 13 patients with argininosuccinic TABLE 1

90 ARGININOSUCCINIC ACIDURIA IN MALAYSIAN CHILDREN mol/L μ mol/L mol/L μ mol/L mol/L μ mol/L mol/L μ mol/mmol μ mol/L mol/L creatinine of creatinine) μ 430 14.6 1,570 263 152 1,570 692 1,093 12 598 430 14.6 110 done 55 15 403 202 358 693 not 1,999 576 1,879 241 406 619 1,408 28 28 521 118 detected 147 229* 854 1,600* 3,913 39 572 19.2 not 5,448 576 780 elevated 160 184 262 36 20 1,716 88 1,101 1246 172 264 26.4 1,725 335 1,126 25 2,646 175 elevated 419 70 1,065 985 1,586 not done 1,084 570 done 259 1,600 640 done 1,586 not 2 324 1,993 3,397 1,172 not 17 406 160 4,970 1,183 1,035 13 22 593 711 530 2,357 379 2,862 265 187 done 1,205 117 1,878 247 29 1,719 1,848 not 56 800 600 mol/L, older mmol/mol ( Plasma Urine Urine Plasma Amino Acid μ 1 13 *Argininosuccinic acid was detected after protein challenge *Argininosuccinic child 50-80 child 3 6 7 9 11 12 <110 Reference range Neonate: 1.0-3.2 absent absent 3-36 17-119 <700 132-455 TABLE 2: Biochemical phenotypes of 13 patients with argininosuccinic aciduria 2: Biochemical phenotypes of 13 patients with argininosuccinic TABLE PATIENT 2 ammonia 4 5 orotic Arginino succinate succinate 8 Arginino Citrulline 10 Arginine Glutamine Alanine

91 Malaysian J Pathol December 2010

Ten patients (Patient 1 to 10) had the acute laboratory scientist that he/she may be dealing neonatal form of the disease, with symptoms of with argininosuccinic aciduria or a urea cycle hyperammonemia appearing between the second disorder in a sick child is raised blood ammonia. and thirteenth day after birth. The blood ammonia It is, therefore, essential to measure ammonia level ranged from 430μmol/L to 1,848μmol/L. early in every sick child without a clear diagnosis. Nine of the ten patients had argininosuccinic After excluding false hyperammonemia such as acid detected in blood (Fig. 3a) during the acute improper sample collection and transportation, episode. Argininosuccinate was detected in the struggling or a haemolysed blood sample, blood of Patient 8 only after a protein challenge. blood ammonia more than 200 μmol/L in a Argininosuccinate found in the urine was two previously healthy term newborn or more to ten times higher in the plasma levels. Plasma than 150 μmol/L in an older child is strongly glutamine and citrulline levels were elevated in suggestive of an underlying urea cycle disorders all patients, whereas arginine and ornithine levels such as argininosuccinic aciduria.2,9 This should were low. Urine orotic acid was measured in fi ve prompt the clinician to contact the diagnostic patients and; all of them had raised orotic acids laboratory for urgent plasma and urine amino levels, three to thirty times the normal limit. acids analysis . Three patients presented later (between the Plasma quantitative amino acid analysis age of two months and twelve months) with is necessary to confi rm a specifi c diagnosis milder clinical symptoms. Late-onset patients of urea cycle disorder. Argininosuccinic excreted signifi cantly less argininosuccinate aciduria is one of the 3 urea cycle disorders compared to the neonatal-onset group. In one of (the other two are citrullinemia and arginase the patient, argininosuccinic acid was detected defi ciency) in which changes in amino acids are only in urine. usually diagnostic without the need for further Two patients (Patient 1 and 10) with enzymatic or molecular testing.2,9,10 Presence of neonatal-onset disease died at the age of 12 argininosuccinate is the characteristic marker for days and 4 months when they had a recurrent diagnosis of argininosuccinic aciduria, which is hyperammonemic coma. However, nine usually not detected in a normal person.11 Other patients survived with a reasonably good signifi cant amino acids are citrulline and orotic neurological outcome; four patients have acid. In patients with argininosuccinic aciduria, normal developmental status and fi ve have mild the plasma citrulline is usually elevated to levels delayed development. Two patients (patient 4 of 150 to 250 μmol/L. Hyperglutaminemia and and 13) have severe neurological disabilities as hyperalaninemia are also often present. Elevated a consequence of recurrent hyperammonemic glutamine signifi es a hyperammonemic state episodes. as glutamine is an ammonia scavenger. Raised plasma alanine is a non specifi c fi nding. Under DISCUSSION normal circumstances, arginine is produced from argininosuccinate. Hypoargininemia will, Argininosuccinic aciduria is the second most therefore, be expected and is a common fi nding common disorder of inborn errors of the urea in argininosuccinic aciduria.11 cycle in European countries and the United States. Although plasma amino acid quantifi cation The reported incidence is about 1 in 70,000 live is diagnostic, potential pitfalls in amino births in the United States.8 Our study shows acid analysis need to be recognized. Firstly, a prevalent of 0.16% (13 positive) from 8270 argininosuccinic acid is not one of the usual patients referred to our centre for investigation of amino acids routinely detected in an amino various inborn errors of metabolism disorder. It is acids analysis and can easily be misidentifi ed, also considered to be the most common disorder because it may co-elute with other amino of urea cycle diagnosed in our country. acids especially leucine (Fig 3b).12 Secondly, The clinical presentation of argininosuccinic argininosuccinate acid is highly soluble and aciduria is rather non-specifi c, just like other rapidly cleared from blood. Therefore, the urea cycle disorders. Neonatal disease resembles amount present may be too little to be detected. a neonatal infection whereas late-onset disease As such urinary amino acid analysis is helpful can mimic many other neurological disorders.1,2 in confi rming argininosuccinic aciduria because As such the recognition of argininosuccinic of the marked excretion of argininosuccinate aciduria heavily relies on biochemical laboratory acid in urine.11 In addition, urine samples testing. The fi rst clue to alert the clinician and treated with heat or barium precipitation prior

92 ARGININOSUCCINIC ACIDURIA IN MALAYSIAN CHILDREN ent retention 10.Valine, 11. Methionine, 12. Leucine, 13. Tyrosine, Tyrosine, Methionine, 12. Leucine, 13. 11. 10.Valine, ciency ﬁ FIG. 3a: Plasma Amino Acid Chromatogram for Patient 1 with Argininosuccinate Lyase De Lyase Argininosuccinate Acid Chromatogram for Patient 1 with Amino FIG. 3a: Plasma pKa and hence eluted with differ The amino acids can be separated because of different This chromatogram was obtained at 570nm. was eluted immediately after leucine. times. In this chromatogram, argininosiccinate Alanine, 9. Citrulline Asparagine, 5. Glutamic acid, 6. Glutamine, 7. Glycine, 8. Threonine, 3. Serine, 4. Aspartic acid, 2. 1. Arginine. 18. Histidine, 19. Ammonium, 16. Ornithine, 17. Lysine, 14. Phenylalanine, 15.

93 Malaysian J Pathol December 2010 te are . mol/mmol μ 10. Valine, 11. Methionine, 12. Leucine, 13. 11. Valine, 10. excreted in large amount (1084 excreted in large ciency. ciency. ﬁ is the characteristic urinary marker, is is the characteristic urinary marker, argininosuccinate which argininosuccinate

ciency, ciency, ﬁ creatinine ), and some of these are converted into anhydride forms. In this chromatogram, the two anhydrides argininosuccina FIG. 3b: Urine Amino Acid Chromatogram for Patient 1 with Argininosuccinate Lyase De Lyase Argininosuccinate Acid Chromatogram for Patient 1 with Amino FIG. 3b: Urine In ASL de is eluted closely after the leucine peak eluted at the retention time of homocysteine and gaba peaks, whereas argininosuccinate Alanine, 9. Citrulline Asparagine, 5. Glutamic acid, 6. Glutamine, 7. Glycine, 8. Threonine, 3. Serine, 4. Aspartic acid, 2. 1. Tyrosine, 14. Phenylalanine, 15. Ammonium, 16. Ornithine, 17. Lysine, 18. Histidine, 19. Arginine. 18. Histidine, 19. Ammonium, 16. Ornithine, 17. Lysine, 14. Phenylalanine, 15. Tyrosine,

94 ARGININOSUCCINIC ACIDURIA IN MALAYSIAN CHILDREN to analysis will further improve the sensitivity metabolic diseases. Heidelberg, Germany: Springer; of detection by converting the argininosuccinic 2006. p.261–72. acid into anhydrides.12 Nevertheless, quantitative 4. Nagata N, Matsuda I, Oyanagi K. Estimated analysis of urine amino acids is generally not frequency of urea cycle enzymopathies in Japan, Am J Med Genet. 1991; 39: 228–9. useful for diagnosis of most amino acid disorders 5. Berry GT, Steiner RD. Long-term management of and other urea cycle disorders. This is because patients with urea cycle disorders. J Pediatr 2001;138 urine amino acids concentrations do not refl ect (Suppl 1): S56–S61. the true amino acid concentration in blood 6. Leonard JV. Inherited hyperammonemias. In: Blau due to the effect of renal reabsorption. Urine N, Hoffmann GF, Leonard VJ, Clarke JTR. editors. argininosuccinate quantitative analysis is one Physician’s guide to the treatment and follow-up of metabolic diseases. Springer-Verlag Berlin; of the few exceptions. Heidelberg, 2006.p.117-27. A favourable outcome can be achieved if 7. Bourrier P, Varache N, Alquier P, Rabier D, argininosuccinic aciduria is diagnosed early. Kamoun P, Lorre G, Alhayek G. Cerebral edema Immediate treatment may include acute dialysis with hyperammonemia in valpromide poisoning. to rapidly remove ammonia which is extremely Manifestation in an adult, of a partial defi cit in type toxic to the brain. Long term treatment will I carbamylphosphate synthetase. Presse Med.1988; normally include dietary protein restriction, 17: 2063-6. 8. Scaglia F, Brunetti-Pierr N, Kleppe S, et al. Clinical arginine supplementation, use of pharmacological consequences of urea cycle enzyme defi ciencies ammonia scavengers such as sodium benzoate and potential links to arginine and nitric oxide and sodium phenylbutyrate.13 metabolism. J Nutr. 2004; 134 (10 Suppl): 2775S- In conclusion, clinicians should always con- 2782S. sider the possibility of a child with unexplained 9. Barsotti RJ. Measurement of ammonia in blood. J illness, or without a clear explanation, having Pediatr 2001; 138 (1 Suppl): S11-20. an inborn error of metabolism such as 10. Tuchman M, Yudkoff M. Blood levels of ammonia and nitrogen scavenging amino acids in patients argininosuccinic aciduria. Close collaboration with inherited hyperammonemia. Mol Genet Metab. with the laboratory is potentially life saving. 1999; 66:10–5. 11. Palmer T, Oberholzer VG, Levin B, Burges EA. ACKNOWLEDGEMENT Urinary excretion of argininosuccinic acid. Clin Chim Acta. 1973; 47:443-8 The authors like to thank all the paediatricians 12. Steiner RD, Cederbaum SD. Laboratory evaluation who have referred patients to us, all the patients of urea cycle disorders. J Pediatr. 2001; 138 and their families, and the staff of Metabolic (1 Suppl):: S21-9 Clinic (Ms Balktiah Mat and Ms Norzawani Che 13. Enns GM, Berry SA, Berry GT, Rhead WJ, Brusilow Johari) for assisting in the retrieval of medical SW, Hamosh A. Survival after treatment with phenylacetate and benzoate for urea-cycle disorders. records. The authors also wish to thank Dr N Engl J Med. 2007; 356(22):2282-92. Keng Wee Teik, Dr. Shanti B, Dr Ch’ng Gaik Siew for their clinical support, and the staff of Biochemical Genetics Unit ( Ms Huzaimah bte Sani, Ms Tengku Rosmaliza, Mr Mohd Helmi and Miss Komalam) for their excellent technical assistance.

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