J the Cerebro-Hepato-Renal Syndrome of Zellweger

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LUTGARDE СР. GOVAERTS Electron micrograph of part of a human hepatocyte, showing a number of single-membrane-bound peroxisomes and mitochondrial profiles. Magnification: 50.000 χ THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER Promotor : Prof. Dr. L.A.H. Monnens Co-referent: Dr. Ir. J.M.F. Trijbels THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE GENEESKUNDE AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. J.H.G.I. GIESBERS VOLGENS BESLUIT VAN HET COLLEGE VAN DEKANEN IN HET OPENBAAR TE VERDEDIGEN OP DONDERDAG 29 NOVEMBER 1984 DES NAMIDDAGS TE 2.00 UUR PRECIES

DOOR

LUTGARDE CELESTINE PETRA GOVAERTS

GEBOREN TE HASSELT (BELGIË)

1984 DRUK: STICHTING STUDENTENPERS NIJMEGEN CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG Govaerts, Lutgarde Celestine Petra

The cerebro-hepato-renal syndrome of Zellweger Lutgarde Celestine Petra Govaerts. - (S.l. : s.n.) (Nijmegen: Stichting Studentenpers). - 111. Proefschrift Nijmegen. - Met lit. opg. - Met samenvatting in het Nederlands. ISBN 90-9000739-3 SISO 605.99 UDC 616-098:575.1 Trefw.: peroxisomen; pipecolinezuur.

The investigations described in this thesis were performed in the De partment of Paediatrics (head: Prof.Dr.G. Stoelinga) and in the Labora tory for Paediatrics and Surgery (head: Dr.P. van Munster) of the Sint Radboudhospital of the Catholic University of Nijmegen, The Netherlands.

Financial support was given by the Faculty of Medicine of the K.U.N, and a graduate scholarship of the Rotary Foundation, Evanston, U.S.A. was obtained.

Printing-expenses were partly subsidized by the Jan Dekkerstlchting and Dr.Ludgardine Bouwmanstichting.

CONTENTS

ABBREVIATIONS 14

GENERAL INTRODUCTION 15

CHAPTER I: 17 PEROXISOMES IN MAMMALS AND IN MAN

INTRODUCTION 18

MORPHOLOGY 18

BIOGENESIS OF PEROXISOMES 19

CYTOCHEMISTRY 22

ENZYMATIC COMPOSITION AND METABOLIC PATHWAYS 22 Catalase L-α ^hydroxy acid oxidase and D-amino acid oxidase Glyoxylate metabolism and aminotransferases Urate oxidase Fatty acid g-oxidation Glycerolipid biosynthesis Bile acid synthesis

DEVELOPMENTAL CHANGES 31

PEROXISOMES AND HUMAN METABOLIC DISEASES 32 Acatalasemia Cerebrotendinous xanthomatosis Adrenoleukodystrophy The cerebro-hepato-renal syndrome

REFERENCES 34

7 CHAPTER II: 38 THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

INTRODUCTION 40

HISTORICAL SURVEY 40

NOMENCLATURE AND CLASSIFICATION 41

THE CLINICAL MANIFESTATIONS OF ZELLWEGER SYNDROME 42 Clinical signs Age at death and cause of death Heredity

ROENTGENOLOGIC AND ELECTROPHYSIOLOGIC STUDIES 49 Roentgenologic studies Electrophysiologic studies

HISTOPATHOLOGIC STUDIES 51 Central nervous system Liver Kidneys Skeletal muscle - peripheral nerve Hemato-lymphatic system Adrenal glands Pancreas The eyes Other organs

BIOCHEMICAL STUDIES 63 Introduction Iron overload Pipecolic acid metabolism Liver dysfunction - Abnormal bile acid pattern Immune deficiency Organic aciduria Very long chain fatty acid pattern Deficiency of plasmalogens in tissues Mitochondrial defects in the CHR-S; relation to the deficiency of peroxisomes

8 RELATED DISORDERS 77 Introduction Hyperpipecolic acidemia Trihydroxycoprostanic acidemia

CHAPTER III: 97 CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER: CLINICAL SYMPTOMS AND RELEVANT LABORATORY FINDINGS IN 16 PATIENTS

ABSTRACT 98

INTRODUCTION 99

PATIENTS AND METHODS 99

RESULTS 100 History of the families Gestation and delivery Clinical features Laboratory data Special neurologic examinations

DISCUSSION 106

REFERENCES 108

CHAPTER IV: Ш A NEUROPHYSIOLOGICAL STUDY OF CHILDREN WITH THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

ABSTRACT 112

INTRODUCTION 1|3

SUBJECTS AND METHODS 115 Subjects Methods

9 RESULTS 116 EEC's BAEP's - SSEP's Echo-encephalography - CT scan

DISCUSSION 124

SUMMARY 125

REFERENCES 126

CHAPTER V: 129 DISTURBED ADRENOCORTICAL FUNCTION IN CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

ABSTRACT 130

INTRODUCTION 131

PATIENTS AND METHODS 132 Patients Methods

RESULTS 132

DISCUSSION 134

REFERENCES 136

CHAPTER VI: 139 DISTURBED VERY LONG CHAIN FATTY ACID PATTERN IN FIBROBLASTS OF ZELLWEGER PATIENTS

SUMMARY 140

INTRODUCTION 141

10 PATIENTS AND METHODS 142 Patients Methods

RESULTS 143

DISCUSSSION 146

REFERENCES 148

CHAPTER VII: 153 PIPECOLIC ACID LEVELS IN SERUM AND URINE FROM NEONATES, AND NORMAL INFANTS; COMPARISON WITH VALUES REPORTED IN ZELLWEGER SYNDROME

SUMMARY 154

INTRODUCTION 155

PATIENTS AND METHODS 156 Patients Methods

RESULTS 157

DISCUSSION 164

REFERENCES 166

CHAPTER VIII: 169 GENERAL CONSIDERATIONS

MORPHOLOGIC EVIDENCE FOR THE ROLE OF PEROXISOMES IN CNS 170 AND THE EYES

11 PATHOGENESIS OF BIOCHEMICAL DISORDERS AND ITS CLINICAL SIGNIFICANCE 17 1 Catabollsm of pipecollc acid Pathogenesis of liver dysfunction Deficiency of plasmologens Increased saturated VLCFA levels

BASIC DEFECT IN CHR-S 174

RELATION BETWEEN CHR-S AND THE NEONATAL FORM OF ALD 175 Clinical features Biochemical data Pathologic studies Conclusion

ANTENATAL DIAGNOSIS OF CHR-S 178

REFERENCES 179

SUMMARY 185

SAMENVATTING 187

SAMENVATTING VOOR DE LEEK 189

ACKNOWLEDGEMENTS 193

CURRICULUM VITAE 195

ABBREVIATIONS

ACTH adrenocorticotrophic hormone ALD adrenoleukodystrophy AMN adrenomyeloneuropathy BAEP brainstem auditory evoked potentials CHR-S cerebro-hepato-renal syndrome CNS central nervous system CoA coenzyme A CSF cerebrospinal fluid CTX cerebrotendlnous xanthomatosis DAB d iaminobenz id ine DHAP dihydroxy-acetone-phosphate DHCA dihydroxy coprostanic acid EEG electro-encephalography EMG electro-myo-graphy ER endoplasmic reticulum ERG electro-retino-graphy FAD flavin adenine dinucleotide GABA γ-aminobutyric acid ΗΡΑ hyperpipecolic acidemia НРа hyperpipecolic aciduria ΡΑ pipecolic acid PAS periodic acid-Schiff SSEP somatosensory evoked potentials тз 3, 5, 3' - triiodothyronine T4 thyroxin THCA trihydroxy coprostanic acid VEP visual evoked potentials TSH thyroid stimulating hormone VLCFA very long chain fatty acids

14 GENERAL INTRODUCTION

This study contains clinical, electrophysiologic, morphologic and bio chemical investigations in children with the cerebro-hepato-renal syn drome (CHR-S) of Zellweger. In the first chapter the present knowledge about peroxisomes is presented. This is required to understand the in vestigations reported in Chapter IV, V and VI. Chapter II reviews the literature on the Zellweger syndrome. Special attention is also given to data referring to clinically and/or biochemically related disorders. Chapter III and IV give a survey of clinical symptoms, relevant labora tory findings and neurophysiological data from our patients. As the neo natal form of adrenoleukodystrophy (ALD) has a clinical and biochemical resemblance to CHR-S, the adrenocortical function -disturbed in ALD- was tested in CHR-S (Chapter V). The very long chain fatty acids measured in cultured skin fibroblasts of CHR-S patients demonstrated a constant increased concentration of С., η (Chapter VI). This finding offers the possibility of antenatal diagnosis (Chapter VI and VIII). Pipecolic acid (PA) level in serum and urine, the first reported bioche mical abnormality in CHR-S, remains a valuable tool for the confirmation of the clinical diagnosis of CHR-S, when gestational age and age after birth are taken into consideration (Chapter VII).

CHAPTER I

PEROXISOMES IN MAMMALS AND IN MAN INTRODUCTION

Microbodies were first described in the proximal tubular epithelium of mouse kidney by Rhodin in 1954 and by Rouiller and Bernhard in hepatic parenchymal cells in 1956. They are now considered as ubiquitous subcel lular organelles in eukaryotic cells. The term microbody is only a mor phological description and includes also a percentage of quite unrelated structures such as the hydrogenosome in Trichomonas and the glycosome in protozoan blood parasites. De Duve and Baudhuin, 1966, who developed procedures for isolating microbodies, introduced the name peroxisomes, for cellular organelles characterized by their content of catalase and several oxidative enzymes. The function of peroxisomes involves the production and degradation of hydrogen peroxide. The peroxisomes are a subgroup of the microbodies. They should be distinguished from glyoxyso- mes present e.g. in Tetrahymena, the latter containing in addition at least malate synthetase and isocitrate lyase allowing the functioning of the glyoxylate cycle (De Duve, 1983) . We will limit this short review to the peroxisomes of mammals.

MORPHOLOGY

Peroxisomes are particles ranging in diameter from 0.1 to 1.5 μπι (avera ge about 0.5 um)· The limiting membrane is a single membrane consisting of a triple layered structure. The membrane is thinner than that of lysosomes and plasma membrane. The peroxisomal membrane is highly permea ble to small molecules such as sucrose and hydrogen peroxide. Freeze- fracture replicas of hepatic peroxisomes reveal numerous particles (7-8 nm) often in clusters, on the protoplasmic face of the limiting membra ne, whereas the extracellular face is almost devoid of such particles (Reddy et al, 1974). The matrix has a granular appearance. An electron- dense core (nucleoid), in which a series of parallel membranes or latti ce structure can be observed, is present In the peroxisomes of many ani mal species. The presence of nucleoids is, in most cases, closely rela ted to the occurrence of urate oxidase. The most readily recognizable peroxisomes are those localized in liver and kidney cells.

18 In addition to the peroxisomes with the characteristics just described, a population of smaller "microperoxisomes" has been described in mamma lian cells. These microperoxisomes are roughly circular, elongate in shape with a diameter of 0.15-0.25 μιη and lack a central core. They seem to have frequent continuities of their delimiting membrane with that of the endoplasmic reticulum (ER). They contain catalase while urate oxida se is lacking. The microperoxisomes are widely distributed among mamma lian tissues (Masters and Holmes, 1977, Novikoff and Novikoff, 1982).

BIOGENESIS OF PEROXISOMES

Several models of peroxisome biogenesis have been proposed. In the clas sical model peroxisomal proteins are synthesized on bound ribosomes and channelled through the ER to the growing peroxisomes, budding outgrowths of the ER, which pinch off (De Duve and Baudhuin, 1966). This model has been severely criticized by Lazarow et al (1982). After pulse-labeling of a rat, radioactive catalase was not found in microso mal vesicles. Catalase mRNA was not located on bound polysomes. They proposed an alternative model. The peroxisomal proteins are synthesized on free polysomes and enter preexisting peroxisomes post-translationally from the cell sap; new peroxisomes are thought to be formed by fission from preexisting ones. Their hypothesis is supported by impressive ex perimental evidence. They could prove that the proteins were indeed syn thesized on free polysomes at their mature size rather than as large precursors both in vivo and in vitro.

Fuijke et al (1982) were able to isolate highly purified peroxisomes, mitochondria and rough and smooth microsomes of rat liver. The polypep tide composition of the peroxisomal membrane is very different from that of the ER or mitochondrial membrane, proving that the peroxisomal mem brane is not simple an expansion of the membrane of the ER. The main phospholipids (phosphatidylcholine and phosphatidylethanolamine) are similar in the three membranes. In contrast to the mitochondria, peroxi somal membranes contain no cardiolipin. Crane et al (1982) found a dif ferent relative phospholipid composition in the peroxisomal, microsomal

19 and mitochondrial fraction of mouse liver. The closest similarity in phospholipid composition was between the microsomal and peroxisomal fraction. Lazarow et al (1982) suggested that newly synthesized consti tuents (from cytosol?, from ER?) are incorporated into the preexisting membrane and then further distributed by fission and fusion events. On the basis of their experiments in mouse liver. Masters (1982) propo sed a modification of the theory of Lazarow. In mouse liver catalase exists in five electrophoretic different forms. In the peroxisome cata lase is present as the anodic form, whereas in the cytosol catalase is represented by the more cathodic forms. The cathodic forms show a pro gressive desialation. Masters' working model is represented in Fig. 1. According to this scheme he still accepts that the peroxisomal membrane originates from ER. Newly synthesized catalase apomonomer interacts with receptors on the membrane of the nascent peroxisomes. The hemegroup is inserted and the tetramer form of the enzyme assembled. Sialation of the enzyme takes place. A decreasing degree of association of the active en zyme with the peroxisomal membrane is observed with aging of the peroxi some. Eventual degradation of the peroxisome occurs and catalase is re leased into the cytosol. The cytosol catalase is desialated and heme de graded. The "aged" form of catalase stimulates peroxisomal biogenesis.

20 Fig. 1: A working model of peroxisomal biogenesis and turnover (according to Masters, 1982).

aged catalase binds to receptors on specialized areas of the ER. newly synthesized catalase apoprotein is inserted into the nascent peroxisome and assembled into the active enzyme. 3 catalase activity in membrane associated form. 4,5 catalase converted to a loosely associated form.

oc oc Pool Degradation of Aged Enzyme. Synthesis and Insertion - \ hJ ot New Catalase by Nascent Final Peroxisome Catalase Degradation

E.P.C.

Membrane Bound Catalase (5) Ί4) 'Final Peroxisomal Degradation

• = catalase activity in membrane associated form. о = catalase converted to a loosely associated form. u = aged catalase P. D.E. = peroxisomal detergent extract. P.A.E. = peroxisomal aqueous extract. E.P.C. = extra particulate cytoplasmic catalase.

Permission granted by the New York Academy of Sciences.

21 CYTOCHEMISTRY

Several cytocheraical staining procedures to localize enzyme activities within peroxisomes are reported (Tolbert and Essner, 1981). The most widely used method is the diaminobenzidine (DAB) procedure originally described by Graham and Karnovsky (1966). It is assumed that catalase, acting peroxidatively, oxidizes the DAB to a congregated double-bond structure that binds large amounts of glutaraldehyde and appears opaque by electron microscopy. A selective staining of peroxisomes is possible (Angermüller and Fahimi, 1983). In recent years, immunochemical procedures have attained considerable importance in the localization of proteins because of their high speci ficity. By the protein Α-gold immunochemical procedure Bendayan et al (1982, 1983) were able to localize catalase and heat-labile enoyl-CoA hydratase, the second (3-oxidation spiral enzyme, in the peroxisomal ma trix. The peroxisomal localization of catalase and acyl-CoA oxidase, the first oxidative step of the cyanide-Insensitive ß-oxidation, has also been studied using monospecific Fab fragments labelled with horseradish peroxidase (Yokata and Fahimi, 1981, 1982).

ENZYMATIC COMPOSITION AND METABOLIC PATHWAYS

Many of the enzymes found in the peroxisomal compartment occur in other compartments of the cell. This review is restricted to a consideration of the most characteristic peroxisomal enzymes. In Table 1 the enzymes localized in peroxisomes are presented.

Catalase The association of hydrogen peroxide-producing oxidases with catalase is a constant metabolic feature of peroxisomes. The substrates of this oxi dative capacity include amino acids (D and L), hydroxyacids and fatty acids. Catalase contains 4 heme groups. It consists of four identical subunits

22 with a molecular weight of approximately 60,000. In the reaction mecha nism for catalase (Fig. 2), a stable complex of catalase and Η,,Ο» is first formed; it then reacts with either another molecule of H„0 or R'H„. The stable complex has been monitored in vivo by Chance et al (1979) via its characteristic absorption spectrum. Recent studies indicate that glutathione peroxidase is of minor impor tance relative to that of catalase for the catabolism of H„0 in peroxi somes but of major importance in the ER (Jones et al, 1981).

Fig. 2: The peroxisomal respiratory pathway.

O, Η-,Ο- catalatic activity 2^- ^¿—»·2Η20

^г^ oxidase ^--»Η2θ2 catalase

RH,' R R'H2~ ^-2H 0 2 peroxidatic activity

23 Table 1: Enzymes localized in peroxisomes of mammals.

Catalase Glutathione peroxidase (Jones et al, 1981) ot-hydroxy acid oxidase D-amino acid oxidase Urate oxidase Hydroxypyruvate/glyoxylate reductase Glyoxylate aminotransferases e.g. alanine &-oxidation of fatty acids: acyl-CoA synthetase acyl-CoA oxidase enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase 3-ketoacyl-CoA thlolase (Hashimoto, 1982) 2,4-dienoyl-CoA reductase (Dommes et al, 1981) Carnitine acetyl transferase Carnitine octanyl transferase Acyl-CoA:DHAP acyl transferase (Jones and Hajra, 1977) Alkyl DHAP synthetase Acyl/alkyl DHAP:NADPH oxidoreductase (Hajra and I ishop, 1982) Acyl-CoA-NADPH oxidoreductase (long chain alcohol forming) Glycerol phosphate dehydrogenase Conversion of THCA into cholic acid Gustafsson, 1980) Glutaryl-CoA oxidase (Vamecq and van Hoof, 1984) Polyamineoxidase (Hölttä, 1977) Cytochrome c-reductase (Donaldson et al, 1972)

Adaptation of Table 1 of Masters and Holmes (1977). New literature is added.

24 L-o-hydroxy acid oxidase and D-amino acid oxidase L-a-hydroxy acid oxidase is a flavoprotein that catalyzes the oxidation of L-o-hydroxy acids to produce the corresponding α-keto acids and H-0-. This enzyme exists as two isozymic forms in mammalian tissue. It cataly zes also the oxidative deamination of L-amino acids.

R R I I CHOH + O, • C=0 + Η,Ο, I I COO" COO"

L-a-hydroxy acid • oxygen •a-keto-acid • hydrogen peroxide

D-amino acid oxidase is a flavoprotein, with FAD as prosthetic group, which catalyzes the oxidative deamination of D-amino acids. The enzyme is localized in both the cytosol and peroxisomes.

25 Glyoxylate metabolism and aminotransferases Although microbodies in different life forms may be morphologically similar to animal peroxisomes, they may contain a far greater diversity of enzymes. In leaf peroxisomes glycolate produced by photosynthesis is oxidized to glyoxylate, which is converted to glycine (Tolbert and Ess- ner, 1981). Hydroxy-pyruvate/glyoxylate reductase and several glyoxylate aminotrans ferases (glutamate, serine, histidine, alanine, phenylalanine) are pre sent in rat liver peroxisomes.

CH, H CH, H I I I I CH-NH, C=0 CH-NH, I 2 c=o I I 2 COOH I COOH COOH COOH alanine glyoxylate *= =k pyruvate glycine

Takada and Noguchi (1982) have extensively studied the enzyme alanine- glyoxylate aminotransferase (AGT) in different mammalian species. Mamma lian liver contains two forms of the enzyme, designated AGT. and AGT-, respectively. AGT has a molecular weight of approximately 80,000 and has also serine-pyruvate aminotransferase activity. It is localized in the peroxisomes in human, monkey, rabbit and guinea pig, in the mito chondria in dog and cat, and in both organelles in rat, mouse and ham ster and is not detected in pig and bovine liver. AGT. has a molecular weight of approximately 200,000, is found only in mitochondria and has no serine-pyruvate aminotransferase activity. This study clearly indica tes that it is not possible to infer the subcellular distribution of an enzyme in a species, from experiments in another species.

Urate oxidase This enzyme catalyzes the oxidation of urate with molecular oxygen re sulting in the formation of allantoine. It is present in the liver of many mammalian species but it is absent in microperoxisomes and in man.

26 Although the occurrence of nucleoids in peroxisomes is mostly related to the presence of urate oxidase, an exception is known. In man, lacking this enzyme, the liver is devoid of nucleoids but the kidney exhibits some nucleoid structures (Masters and Holmes, 1977).

Fatty acid B-oxidation ß-oxidation was thought to occur only in the mitochondrial matrix until Lazarow and De Duve (1976) proved the presence of a ß-oxidation system in rat liver peroxisomes. The reaction-scheme can be seen in Fig. 3. The peroxisomal system generates acetyl-CoA through the successive steps of oxidation, hydration, dehydrogenation and thiolytic cleavage (Lazarow, 1982, De Beer and Mannaerts, 1983). The enzymes catalyzing these reac tions have been purified and characterized especially by Hashimoto (1982) and are different from their mitochondrial counterparts. Acyl-CoA synthetase activity is present on the cytosolic side of the peroxisomal membrane indicating that acyl-CoA and not the free acid crosses the peroxisomal membrane. Carnitine is not required for this transport. A fatty acid binding protein could be responsible for a car rier process (Appelkvist and Dallner, 1980). The first enzyme of the ß-oxidation system is not an acyl-CoA dehydrogenase but an acyl-CoA oxi dase and generates H.O-, The enoyl-CoA hydratase and 3-hydroxy-acyl-CoA dehydrogenase reside in a single bifunctional protein.

Hayashi et al (1981) could localize the different enzymes of the ß-oxi dation in rat liver peroxisomes. Thiolase is localized in the matrix, enoyl-CoA hydratase and 3-hydroxy-acyl-CoA dehydrogenase in the core and at least part of acyl-CoA oxidase is weakly associated with the core. Peroxisomes are not active against fatty acids shorter than octanoic acid (Lazarow, 1978). Fatty acids are thus not completely degraded to acetyl-CoA. Peroxisomal ß-oxidation is free of respiratory control. The energy of the desaturation is not used for ATP formation but is dissipated as heat and contributes to thermogenesis. The peroxisomal ß-oxidation system is insensitive to KCN or other inhibitors of mitochondrial electron trans port when assayed in vitro in broken cell systems and in the presence of excess NAD and CoA.

27 The quantitative importance of the peroxisomal ß-oxidation is not exact ly known. It is considered to contribute maximally 10% to the overall hepatic oxidation of the important fatty acids palmitate and oleate. The very long chain fatty acids (>C22) are poorly oxidized by mitochondria and it has been proposed that they are shortened in peroxisomes and then further oxidized in mitochondria. The peroxisomal ß-oxidation seems particularly well suited for the oxi dation of long chain mono-unsaturated fatty acids (Osmundsen and Neat, 1979). Unsaturated fatty acids just as saturated ones are oxidized by ß-oxidation but additional enzymes are necessary to eliminate the inter fering cis-double bonds. The peroxisomes possess the required enzymatic apparatus (Dommes et al, 1981). Dicarboxylic acids are also substrates for the peroxisomal ß-oxidation (Mortensen et al, 1982).

Fig. 3: Fatty acid ß-oxidation sequence in rat liver mitochondria and peroxisomes (according to Thomas, 1979).

MITOCHOND RIAL SYSTEM PEROXISOMAL SYSTEM

R CH; СИ CO SCoA fa'tyacyCoA R CH2-CH2-CO-SCoA

нго ^o2 R-CH = CH-CO-SCoA ? unsaturate atylCoA R-CH-CH-CO-SCoA s-нр нго-> 1 hydratJse 1

R-CHOt-l-CH2-CO-SCoA 3 hydroxy acylCoA R ОЮН CH2 CO SCoA NAD'-*. ^--NAD* respiratory cha π ]dehydrogenase | 3-®* NADH.H*«-^ ^•NADH*H*

н2о io, R-CO-CH2-CO-SCoA 3 oxy асу CoA R-CO-CH2-CO-SCoA COASH-^ ^-COASH | thiolase | * * R-CO-SCoA acylCoA R-CO-SCoA

CH3-CO-SC0A acetyl CoA CH3-CO-SC0A

28 Glycerolipid biosynthesis Dihydroxy-acetone-phosphate (DHAP) is known to be the precursor of gly- cerolipids containing an ether bond. This compound combines with acyl- CoA to 1-acyl-DHAP (Fig. 4). Via an exchange reaction between the acyl group and a long chain alcohol, 1-alkyl-DHAP (containing the ether link) is formed, which is converted in the presence of NADPH to 1-alkylglycerol-3-P. After acylation in the 2 position, the resulting l-alkyl-2- acylglycerol-3-P is hydrolyzed to give the free glycerol derivative, which reacts with either CDP-choline or CDP-ethanolamine to form 1-alkyl, 2-acylglycerol 3-phosphoethanolamine.

Fig. 4: Biosynthesis of ether lipids and plasmalogens.

ACYL-COA 9 R,-(CH,), OH NADPH. H* NADP* г 4?COH V HjC-O-C-R, HjC-0 (CH,)7-R, 1 • Η2Γ Ο (ΓΗ,), R? 0=C ^ »Q-C —^ .. Ю С ^ ' »HO-C H 1 - •=· ACYΛΓνι L Ί Н С О ® * Hf О-© RtUUCIASL н2с-о-® THANbhERASE г 1 Alkylglycerol 3 Ρ O hydroxyAcrtonc 1-Acyldihydroxyacetone 1-Al kyld ι hydroxy acetone phosphate phosphate phosphate ^ACYL CoA I "^- 1 CDP-CHOLINE (COP- ETHANOL- CMP AMINE) Pi H О HJC-O-CHJ-CMJ-R, О H,C-0-(CH,),-R"г. V J \1 £ 0(CHp2-R2 Rj-r-O Γ Η , w -R,-C-0 C-H * ^ •/ R,C О C-H PHOSPHOCHOLINE ti C-0-© CH CI- NH; HjC-O-© p ? г DIACYLGLYCEROL PHOSfHOHYDROLASF TRANSFERASE ' Akyl 2 ясуідіусегоі 3 pnospnoelhdriolarTiine 1 Alkyl 2 acyl glycerol 1 ЛІкуіг acyl glycerol 3 Ρ

NADPH 02 DESATURASE

0 H,C-0-CH-CH-R, ETHER ACYLOLYCEROLS Rj-C-0 С Η ' ETHER PHOSPHOLIPIDS

н2с-о-®-снг-сн2-мн2 1 Alkenyl ^ acyl glycerol 3 phosphoc'hanolannine plasma ogen

Reproduced, with permission, from Martin D Jr. Mayes P, Rodwell V: Harper's Review of Biochemistry, 19th ed. Copywright 1983 by Lange Medical Publications, Los Altos, CA (19-07-1984).

29 Plasmalogens are formed by desaturation of the glycerol ether lipid. Hajra and Bishop (1982) extensively studied the localization of diffe rent enzymes involved in the biosynthesis of plasmalogens. In normal rat liver DHAP-acyltransferase is mainly localized in peroxisomes while alkyl-DHAP-synthase and acyl/alkyl-DHAP-NADPH oxidoreductase had a bimodal distribution pattern between peroxisomes and microsomes. In contrast to the rat aIky1-DHAP-synthase in the guinea pig liver is present mostly in the peroxisomal fraction. Several hypotheses about the role of this pathway in peroxisomes are proposed. It is possible that acyl-DHAP may participate in transporting long chain acyl groups across the peroxisomal membrane (Fig. 5). In this scheme, proposed by Hajra and Bishop (1982), the reducing equivalence of NADH generated inside the peroxisomes can be carried to mitochondria. It is possible to attribute an important role of this pathway in membra ne lipid biogenesis e.g. by supplying lipid precursors such as acyl-DHAP or alkyl-DHAP to ER. This is an important new field of research.

Fig. 5: Possible roles of acyl-DHAP in transporting acyl groups and re ducing equivalents across the peroxisomal membrane (according to Hajra and Bishop, 1982).

PEROXISOME CYTOSOL MITOCHONDRION

AcylCoA · DHAP NADP NADPH î С -DHAP Acyl DHAP ' Acyl G-3-P«^^ Acyl DHAP«- J

CoA FA DH2 »HjO AcylCoA · DHAP r

,NADH FAD OXIDATION ' β-OXIDATION f t I I ^»NAD* I G-3-P- tG-3-P- Acyl G-3-Ρ t FA ι -•Acyl G-3-P-

Permission granted by the New York Academy of Sciences.

30 Bile acid synthesis THCA and DHCA are precursors of cholic and chenodeoxycholic acid (Fig. 6). Pedersen and Gustafsson (1980) found the highest activity for the conversion of THCA to cholic acid in a peroxisomal enriched fraction. In a more detailed study (Kase' et al, 1983) they could conclude that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of THCA. The enzymes involved could be similar but not neces sarily identical with those responsible for 0-oxidation of fatty acids.

Fig. 6: Pathways of cholic acid and chenodeoxycholic acid synthesis (according to Danielsson and Sjövall, 1975).

Cholesterol

7a -hydroxycholesterol 1 7a-hydroxy- 4 -cholesten-3-one 7a 12a-dihydroxy- 4- cholesten- 3-one

5ß-cholestane-3a, 7a,12a-tnol Sß-cholestane-la, 7a-diol 1 i 3a 7al12a-lrihyclroxy-5ß-cholestanoic acid 3a, 7a-dihydroxy-5ß-cholestanoic acid (THCA) (DHCA) cholic acid chenodeoxycholic acid

DEVELOPMENTAL CHANGES

The ontogeny of mouse liver peroxisomal enzymes demonstrates coordinated increases in catalase, L-a-hydroxy acid oxidase and urate oxidase acti vities during the first week of neonatal period. The number of peroxi somes increases also rapidly (Masters and Holmes, 1977). Development of peroxisomes in rat liver and kidney and mouse kidney has also been studied. There is an increase in peroxisomal synthesis and enzyme activity predominantly at birth and during the early neonatal period (Masters and Holmes, 1977). Developmental changes in peroxisomal fatty acid oxidation system have recently been reported in rat liver (Krahling et al, 1979, Horie et al, 1981).

31 PEROXISOMES AND METABOLIC DISEASES IN MAN

Introduction When microscopic and cytochemical studies of peroxisomes are reported in metabolic disorders, it is important to keep in mind the normal varia tion and also abnormalities found in different liver diseases. Various pathologic processes involving the hepatic cytoplasm exert different effects on peroxisomes (Sternlieb and Quintana, 1977). Peroxisomes were more abundant and often concentrated in a perinuclear configuration in cholestatic and cirrhotic liver. Decreased peroxisomal staining was com mon in cholestasis, cirrhosis, hepatitis and in almost all patients with malignancies (Roels et al, 1983).

Acatalasemia (McKusick *20020) This disorder was discovered in 1948 by Takahara in a patient with ulce rating gangrenous lesions of the oral cavity. Addition of H.O. to the operative site did not result in the usual bubbles. It is proposed that H_0. produced by bacteria and not decomposed by catalase, oxidizes hemoglobin, deprives the infected area of oxygen and causes ulceration. There is an almost complete hereditary deficiency of erythrocyte catala se. Not enough information is available about residual catalase activity in tissues. The catalase activity of fibroblasts cultured from Japanese patients was 2-4 percent and from Swiss patients approximately 15 per cent of the activity found in normals. At least five different classes of mutant enzymes can be distinguished in different families indicating considerable genetic heterogeneity.

Cerebrotendinous xanthomatosis (CTX) (McKusick 21370) CTX is a rare familial disorder characterized by progressive neurologic dysfunction (dementia, spinal cord paresis and cerebellar ataxia), tendon xanthomas, cataract and premature atherosclerosis due to deposi tion of cholesterol and cholestanol in many tissues. According to Goldfischer (1982) a peroxisomal defect may be of pathoge netic significance in CTX. An increase in the number and size of peroxi somes was observed. In other patients with CTX hepatocellular peroxiso mes contained greatly increased matrix density and crystalline inclu-

32 slons. Oftebro et al (1980) demonstrated a defect in mitochondrial 26- hydroxylation in a patient with CTX. Studies in subcellular fractions are required before this disease should be included under the head of peroxisomal disorders.

Adrenoleukodystrophy (ALD) Adrenoleukodystrophy is a genetically determined disorder affecting the white matter of the CNS and the adrenal cortex. Four clinical forms of ALD can be distinguished: the childhood form (McKusick *30010), adrenomyeloneuropathy, the symptomatic heterozygote of childhood ALD and the neonatal form (McKusick 20237). It is an X-linked disorder with the ex ception of the neonatal form whose inheritance is autosomal recessive. The different clinical forms are associated with the accumulation of saturated very long chain fatty acids (mainly C-, _, C_. _ and C-, -.). These patients have a defect in the oxidation of the very long chain fatty acids (C.,- and longer) (Singh et al, 1984). It is accepted that this defect may be localized in the peroxisomes (Singh et al, 1984). The relation between the neonatal form of ALD and Zellweger syndrome will be extensively discussed in the general discussion.

The cerebro-hepato-renal syndrome of Zellweger (McKusick *21410) This syndrome is reviewed in the next chapter.

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CHAPTER II

THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER INTRODUCTION

This chapter gives a survey of the literature on the cerebro-hepato- re nal syndrome (CHR-S) of Zellweger. Clinically and/or biochemically simi lar disorders (hyperpipecolic acidemia, trihydroxycoprostanic acidemia and neonatal adrenoleukodystrophy) are also presented in this chapter. The following aspects of the syndrome will be covered: history, clinical symptoms, pathologic findings and biochemical abnormalities.

HISTORICAL SURVEY

In 1964 Bowen, Lee, Zellweger and Lindenberg described "a familial syn drome of multiple congenital defects" in two pairs of sibs (3 girls and 1 boy), one from a Caucasian family and one from a Negro family, both without consanguinity. The prenatal history and delivery were unremarka ble. Hypotonia and congenital anomalies (high forehead, frontal bossing, large fontanels, epicanthal folds, abnormal ears, simian creases, camp- todactyly, hypospady, clitoris hypertrophy) were noted at birth. The children were unable to suck or swallow. Periods of respiratory dis tress, retardation and sudden death occurring before one year of age were reported.

Pathologic findings in the central nervous system (CNS) were described in 3 patients: hypoplasia of the cerebellar hemispheres and partial age nesis of the corpus callosum in each patient, and absent olfactory bulbs in one out of two examined patients. Histopathologic studies of the brains revealed nests of ectopic nerve cells in "some areas of the cere bral cortex, particularly the insula". Pneumonia and/or atelectasis was present in every patient, anomalous ve nous return in two out of three infants (one patient with a communica tion between the left subclavian vein and the left atrium and one infant with the right pulmonary vein entering the right atrium). Multiple sub capsular cysts of both kidneys were mentioned in one infant. Autosomal recessive inheritance was assumed. Unaware of Bowen et al (1964), Smith et al (1965) reported the same disease in 1965 as a "newly recognized syndrome". Subsequently approxi-

40 mately 90 cases of the disorder have been reported (Table I). Our pa tients are reported in Table II (Govaerts et al, 1982). Since 1982, se ven new patients have been diagnosed in our department. The case reports mentioned 9 sibs with a similar clinical history without further de tails. They have been omitted from Table I. On some patients no clinical information is available, and these also have been omitted from Table I (Goldfischer et al, 1972, 1973B, Lott et al, 1979, Hanson et al, 1979, Chalmers et al, 1980, (Endres et al, 1981, Kelley, 1983, Mooi et al, 1983, Björkhem et al, 1984, Moser et al, 1984). A recent review about CHR-S was written by Kelley (1983).

NOMENCLATURE AND CLASSIFICATION

The descriptive term "cerebro-hepato-renal syndrome" was first used in 1967 by Passarge and McAdams. "Zellweger syndrome" was first reported by Opitz et al in 1969. The editor of Birth Defects suggested the designa tion "the cerebro-hepato-renal syndrome of Zellweger", analogous to "the oculo-cerebro-renal syndrome of Lowe" (1969). An "undetected inborn error of metabolism" was first suggested by Pas- sarge and McAdams, 1967. The suggestion of a "neurometabolic defect, possibly in the aminoacid metabolism" was made by Kohn and Mündel in 1969, because of the resemblance of the neuropathologic findings to tho se of maple sirup urine disease. Evidence for an inborn error of metabolism in this syndrome has been ob tained by the establishment of various biochemical abnormalities: iron overloading, (Vitale et al, 1969), a "fault in pipecolic acid metabolism " (Danks et al, 1975, Trijbels et al, 1979), a defect of bile acid syn thesis (Hanson et al, 1979, Monnens et al, 1980), an increased excretion of p-OH-phenyllactate in urine (Chalmers et al, 1980, Govaerts et al, 1982), elevated levels of saturated very long chain fatty acids in fi broblasts (Brown et al, 1982) and serum (Bakkeren et al, 1984), defi ciency of plasmalogens in tissues (Heymans et al, 1983 and 1984), defi ciency of acyl-CoA: DHAP acyltransferase in liver, brain and cultured skin fibroblasts (Schutgens et al, 1984). A review of the various bio chemical abnormalities is given in Table IV.

41 Studies involving mitochondrial and/or peroxisomal dysfunction or absen ce in Zellweger syndrome were published by Goldfischer et al, 1972, 1973A, 1973В, 1979, 1981, 1982, and 1983, Pfeifer and Sandhage, 1979, Müller-Höcker et al, 1981 and 1984, Borchard et al, 1982, Sarnat et al, 1983, Trijbels et al, 1979 and 1983, Borst, 1983. The term "peroxisomes deficiency" was proposed by Pfeifer and Sandhage, 1979, as a name that probably was more closely related to the pathogenetic mechanism than the usual designation cerebro-hepato-renal syndrome. Nevertheless, in many handbooks and textbooks this condition is described in the group of structural (brain) defects (Smith, 1982, Swaiman and Wright, 1982, Nelson, 1983), although the term "inborn error(s) of metabolism" is always noted.

THE CLINICAL MANIFESTATIONS OF ZELLWEGER SYNDROME

Clinical signs The clinical manifestations are summarized in Table IX. Pictures of these infants are given in Chapter III. The CHR-S has been reported in several races. Mostly the Caucasian race was noted, occa sionally other races were described: Negro family (Bowen et al, 1964), Jewish family (Kohn and Mündel, 1969), Indian parents (Poznanski et al, 1970), Mexican parents (Williams et al 1972), Pakistani family (Garner et al, 1982). The gestational period and delivery were usually unremarkable. Two mo thers noticed a poor activity of their baby during pregnancy (Opitz et al, 1969, Sommer et al, 1974). Breech presentation was mentioned in 5 out of 112 patients (patients 7, 10, 11, 18, 95 from Table I). Caesarian section was performed in 4 cases (patients 15, 73, 96, from Table I and patient 12 from Table II). Vertex delivery was reported in the other children. The prominent signs at birth were "mongoloid" appearance with large fontanels and hypotonia. One of our patients presented with the clinical picture of cholestatic icterus; laparotomy was performed.

42 When the children survived the first weeks of life psychomotor retarda tion, seizures, ocular involvement and hearing impairment were frequent ly noted. Feeding difficulties were nearly always present. In 55 out of 90 patients surviving for more than two weeks, failure to thrive was reported despite a normal intake of calories by tube feeding.

Age at death and cause of death Within the group of patients there is a marked variation in the age at death. Most children died within the first year of life, but some pa tients survived for several years (see Tables I and II and Fig. 1). The cause of death is given in Tables I, II and III. Respiratory failure or arrest and sudden death occurred in 35 out of 71 patients.

43 TABLE I: Patients with the cerebro-hepato-renal syndrome of Zellweger reported in the literature.

Case Author Year Sex Age at death Cause of death

1 В owe η 1964 M 3 d not mentioned 2 Bowen 1964 F 5 m sudden death 3 Bowen 1964 F 5 m sudden death 4 Bowen 1964 F 3 m sudden death 5 Smith 1965 M 2 m \ icterus (distended abdomen, intestinal 6 Smith 1965 F 10 w ) haemorrhages 7 Passarge 1967 F 6 m congestive heart failure 8 Passarge 1967 F 1 m respiratory failure 9 Passarge 1967 F 2 w respiratory failure 10 Passarge 1967 F Ih m not mentioned 11 Passarge 1967 F 5 h not mentioned 12 Punnett 1968 M 9 m pneumonia 13 Vitale 1969 F ih m pneumonia 14 Vitale 1969 M 6 m respiratory failure 15 Kohn 1969 F 17 d pneumonia

16 Kohn 1969 F 2 m not mentioned 17 Opitz 1969 F 25 h respiratory failure 18 Opitz 1969 F 7 m respiratory failure 19 Opitz 1969 F 23 d pneumonia? 20 Opitz 1969 M 5 ш respiratory arrest 21 Taylor 1969 M 20 d respiratory arrest 22 Jan 1970 F 1 m sudden death 23 Jan 1970 F 24 d sudden death 24 Poznanski 1970 F 3 m respiratory arrest 25 Poznanski 1970 F 2 m not mentioned 26 Poznanski 1970 F 12 d respiratory arrest 27 Patton 1972 F 18 w pneumonia 28 Patton 1972 F 4 m pneumonia 29 Williams 1972 M 64 m not mentioned 30 Williams 1972 M 5 m not mentioned 31 Stanescu 1972 F 9 not mentioned 32 Volpe 1972 F 4 m sudden death

44 Case Author Year Sex Age at death Cause of death

33 Gotero 1973 M 5 w pneumonia 34 Vincens 1973 F 4 m respiratory failure 35 Goldfischer 1973 M 7 w not mentioned 36 Goldfischer 1973 F 6 w not mentioned 37 S опте г 1974 M 18 d not mentioned 38 Sommer 1974 F 18 d pneumonia 39 Sommer 1974 M 1 d not mentioned 40 Sommer 1974 F 23 d pneumonia 41 Garzuly 1974 M 4 m not mentioned 42 Bernstein 1974 M ? pneumonia 43 Bernstein 1974 F ? pneumonia 44 Bernstein 1974 M ? respiratory failure 45 Gómez 1974 M 5 m pneumonia 46 Danks 1975 F 15 w not mentioned 47 Danks 1975 F 6 w not mentioned 48 Danks 1975 M 9 w not mentioned 49 Danks 1975 M 3 w not mentioned 50 Danks 1975 M 2 w not mentioned 51 Danks 1975 M 9 d not mentioned 52 Danks 1975 F 8 w not mentioned 53 Danks 1975 F 2 d not mentioned 54 Variend 1976 F 7 d pneumonia 55 Agamanolis 1976 M 4 m respiratory failure 56 Haddad 1976 ? 4 d congestive heart failure 57 Liu 1976 M 54 m respiratory failure 58 Escorihuela 1976 M 13 d respiratory failure 59 de León 1977 M 3 m not mentioned 60 de león 1977 M 8 m pneumonia 61 Camera 1977 F 10 w pneumonia 62 Brun 1978 M 14 w respiratory arrest 63 Brun 1978 F 14 w respiratory arrest 64 Carlson 1978 M 16 w pneumonia 65 Evrard 1978 M 5 w not mentioned

45 Case Author Year Sex Age at death Cause of death

66 Bartoletti 1978 M 3 d not mentioned 67 Bartoletti 1978 M 1 d not mentioned 68 Bartoletti 1978 F 2 ш not mentioned 69 Bartoletti 1978 M 2h У not mentioned 70 Agamanolis 1979 M 4 m congestive heart failure 71 Parmentier 1979 F 2 m pneumonia 72 Pfeifer 1979 F 14 У pneumonia 73 Friedman 1980 M 3 m pneumonia 74 Mathis 1980 M 44 m respiratory failure 75 Mathis 1980 M 5 m not mentioned 76 Hong 1981 M 6 m pneumonia 77 Müller-Höcker 1981 F 7 w pneumonia 78 Kretzer 1981 M 3 m respiratory failure 79 Della Giustina 1981 M 6 d respiratory failure 80 Della Giustina 1981 F 12 d not mentioned 81 Hittner 1981 M 7 not mentioned 82 Hittner 1981 F 2h m not mentioned 83 Hittner 1981 F eh m not mentioned 84 Hittner 1981 F ioh m not mentioned 85 Toussaint 1981 F 7 w heart failure 86 Borchard 1982 F 16 d not mentioned 87 Borchard 1982 F 15 m not mentioned 88 Borchard 1982 M 6 m not mentioned 89 Garner 1982 F 13 w pneumonia 90 Sarnat 1983 F 44 m respiratory failure 91 Sarnat 1983 F 1 m not mentioned 92 Sarnat 1983 F 4 m not mentioned 93 Sarnat 1983 M 11 m respiratory failure 94 Gustafsson 1983 M 2 m marasm 95 Gustafsson 1983 F 3 w marasm 96 Müller-Höcker 1984 F 7 w respiratory failure

M - male y » year(s) d = day(s) F = female m « month(s) h = hour(s)

46 TABLE II: Our patients (present situation).

Case Author Year Sex Age at death Cause of death

1 Govaerts 1982 F 54 m pneumonia 2 Govaerts 1982 M 5 m pneumonia 3 Govaerts 1982 M 7 m pneumonia 4 Govaerts 1982 M 1 w sudden death 5 Govaerts 1982 F 5 w pneumonia 6 Govaerts 1982 M 6 ш sudden death 7 Govaerts 1982 M 510/12 y sudden death 8 Govaerts 1982 M born in 1977 alive 9 Govaerts 1982 M 29/12 y pneumonia 10 Govaerts 1982 M 5 w sudden death 11 Govaerts 1982 M 48/12 y sudden death ? 12 Govaerts 1982 F born in 1980 alive 13 Govaerts 1982 M 1 m pneumonia 14 Govaerts 1982 M 3 m sudden death 15 Govaerts 1982 M 2 m sudden death 16 Govaerts 1982 M born in 1981 alive

47 TABLE III: Cause of death in patients with cerebro-hepato-renal syndrome of Zellweger.

Cause of death Number

cardio-resplratory problem 54 - pneumonia 28 - respiratory failure or arrest 22 - congestive heart failure or arrest 4 sudden death 13 intestinal haemorrhages 2 шагает 2 not mentioned 38

N = 109 3 patients are still alive (born 08/06/1977, 08/01/1980, 28/11/1981).

Fig. 1: Age at death of patients suffering from the Zellweger syndrome. N = 104, the age was not mentioned in 5 patients, 3 patients are still alive.

number •

60n

50-

40-

30-

20-

io- 52 31 10 1 1 1^=0 I I"1 0-3 3-6 6-9 9-12 1-2 2-3 3-4 4-5 5-6 age months years

48 Heredity The cerebro-hepato-renal syndrome is inherited as an autosomal recessive trait. In McKusick's "Mendelian Inheritance in Man" the disorder is men tioned as *21410. In twenty out of the eighty families two or more affected children are reported. In nine other families biochemical or pathologic findings we re mentioned in nine sibs with similar clinical history without further description. Consanguinity is known in 13 families. It is not possible to quote the number of normal sibs in the families; generally only the affected sibs were mentioned. In the group of pa tients where the sex is known 55 of them are boys and 56 girls (see Tables I and II). These data on the family history of patients, and the distribution of sex are in accordance with an autosomal recessive inheritance pattern. Biochemical investigations reported by Bakkeren et al, 1984, Heymans et al, 1984 and Schutgens et al, 1984, confirm this mode of inheritance. No structural chromosomal abnormalities have been described. The incidence of this syndrome is estimated to be approximately 1 in 100,000 births in Australia (Danks et al, 1975). In the Netherlands, ba sed on the number of known patients, about the same incidence is found.

ROENTGENOLOGIC AND ELECTROPHYSIOLOGIC STUDIES

Roentgenologic studies Roentgenologic abnormalities were mentioned in the first description of the cerebro-hepato-renal syndrome by Bowen et al, 1964: calcific densi ties overlying the ischium and hip joints bilaterally and in relation to the cervical spine, the epiphyses of the hands and calcific stippling of the patellae (patient 2). This abnormal calcification of the patellae is the most conspicuous roentgenologic feature of the Zellweger syndrome (30 out of 37 patients). Punctate calcifications were described in seve ral bones: os occipitale (1 patient), hyoid bone (2 patients), thyroid cartilages (2 patients), sternum (2 patients), scapula (2 patients), proximal humeral epiphysis (3 patients), radius and ulna (1 patient),

49 spine (3 patients), costovertebral articulations (1 patient), acetabulum (6 patients), hip joint (5 patients), inferior pubic ramus (1 patient), upper femoral epiphysis (4 patients), distal femoral epiphysis (4 pa tients) , tibial epiphysis (1 patient). It is interesting to note that occasionally the diagnosis was first suggested in the radiology depart ment (Bartoletti et al, 1978). A number of authors mentioned retarded bone age (8 patients ). Occasio nally osseous demineralization was reported (2 patients). Six patients with a normal skeletal survey were noted. No consistent abnormalities were found by I.V.P. Cystic changes of the kidneys were suggested by ultrasonography of 3 patients (Garner et al, 1982, Sarnat et al, 1983). Computer tomography and/or pneumoencephalography revealed a slight dila tation of the ventricles. Cerebral arteriography was not mentioned in the literature.

Electrophysiologic studies Electrophysiologic studies were only rarely mentioned: 26 EEC's, 11 EMG's, 8 ERG's, 6 VEP's, no BAEP's or SSEP's. In 3 patients a normal EEG was noted although clinical seizure activity was present. The other EEC's were abnormal, mostly epileptiform activity was seen. High voltage and slow wave activity in the occipital area and vertex was reported by Agamanolis and Patre, 1979, and Mathis et al, 1980. The EMG's were always normal: the motor conduction velocity was normal, no signs of denervation were present. ERG examination revealed normal retinal signs once (Volpe and Adams, 1972); an extinguished ERG was found by the other authors. The VEP's were seriously disturbed or could not be recorded. Garner et al (1982) reported failure of normal maturation and deterioration of the VEP in one patient. The electrophysiologic data of our patients are described in Chapter IV.

50 HISTOPATHOLOGIC STUDIES

Central nervous system (65 patients)

Macroscopic studies. Macroscopic studies of the CNS in the cerebro-hepato-renal syndrome of Zellweger showed consistent alterations. Most conspicuous findings were generalized polymicrogyration and corpus callosum hypoplasia (Gilchrist et al 1976, Evrard et al, 1978, Friedman et al, 1980). Frequently dila tation of the cerebral ventricles and olivary dysplasia were observed (Gilchrist et al, 1976, Evrard et al, 1978, Friedman et al, 1980). Occasionally data concerning the olfactory bulbs and optic nerves were given; in 6 out of 10 patients hypoplasia was noted (Opitz et al, 1969, Volpe and Adams, 1972, Variend et al, 1976, de León et al, 1977, Toussaint et al, 1981).

Dysmyellnatlon was a constant finding (Gilchrist et al, 1976, Evrard et al, 1978, Friedman et al, 1980). No macroscopic abnormalities in spinal cord structure were recorded (Agamanolis and Patre, 1979).

Microscopic studies. The neuropathology showed a striking similarity, with only quantitative differences. Cytoarchitectonic abnormalities with neuronal heterotopia and dysmyell natlon were constant findings (Passarge and McAdams, 1967, Opitz et al, 1969, Volpe and Adams, 1972, Garzuly et al, 1974, Agamanolis et al, 1976 and 1979, Liu et al, 1976, de León et al, 1977, Brun et al, 1978, Evrard et al, 1978). These changes were described as most pronounced in the ce rebral cortex; they were also common in the cerebellum, the brain stem and the spinal cord. Heterotopic Purkinje cells in the white matter of the cerebellar folia and lack of convolution of the dentate nuclei were reported by Volpe and Adams, 1972 and Evrard et al, 1978. Retarded cel lular differentiation was mentioned by Brun et al, 1978 and Garner et al, 1981.

The inferior olivary nuclei were markedly abnormal with laminar discon tinuities in the principal nucleus (Evrard et al, 1978). Agamanolis and

51 Pâtre, 1979, reported ectopic large motor neurons in the subpial white matter of the spinal cord and chromatolysis and vacuolization of many neurons in the dorsal nuclei of Clarke. In contrast to the striking disturbance of the cortex, the architecture of the deep nuclear structures was normal (Volpe and Adams, 1972, Agama- nolis and Patre, 1979). It should be noted that nearly every patient showed subependymal periventricular cysts (Brun et al, 1978). PAS-positive, diastase negative granules in cortical neurons, which were more abundant in the areas of polymicrogyria, were described by Agamano- lis and Patre (1979). Diffuse accumulation of sudanophilic, Sharlach R, oil red 0 and Sudan Black В positive and PAS-negative, non-metachromatic droplets within glial cells and histiocytes scattered in the cortex was frequently des cribed (Passarge and McAdams, 1967, Agamanolis et al, 1976, Liu et al, 1976, de León et al, 1977, Brun et al, 1978, Evrard et al, 1978). Peculiar multinucleated "globoid" cells with partly strongly sudanophi lic cytoplasm and partly PAS-positive granules were reported by de León et al, 1977 and Agamanolis and Patre, 1979. Lipid-loaden histiocytes we re found in periventricular white matter (Volpe and Adams, 1972, Agama nolis and Patre, 1979). Swollen astrocytes with faintly eosinophilic cy toplasm and neurons with ballooned perikaryon were reported by de León et al (1977). Biochemical studies of brain lipids in one patient revealed an extraor dinary increase (14% compared to a normal value of 0.5%) of a neutral lipid, identified as a cholesterol ester by thin-layer and gas chromato graphy, which is the oil red 0-stained material (Goldfischer et al, 1973A). "Siderosis" was mentioned in three out of 65 patients. The decreased volume of white matter, compared with age-matched con trols, is probably caused by hypomyelination and demyelination. Paucity of Oligodendroglia cells, which produce myelin, was noted by Opitz et al, 1969, and Brun et al, 1978. A destructive process is strongly sug gested by the gliosis and the lipid-loaden histiocytes and globoid cells (Agamanolis et al, 1976).

52 Electron microscopie studies. Electron microscopie studies on central nervous system tissue from pa tients with the CHR-S of Zellweger were performed by Goldfischer et al (1972 and 1973A), Agamanolis et al (1976, 1979), Liu et al (1976), de León et al (1977) and Brun et al (1978). The autolytic changes of the brain tissue made accurate account of the ultrastructural details diffi cult; only Goldfischer et al (1972 and 1973A) reported ultrastructural studies on brain biopsy tissue. Goldfischer et al, 1972 and 1973A, payed attention to the structure and function of the mitochondria in CHR-S. Dense matrices and slightly dila- tated intracristal spaces were described in the mitochondria of the CNS. Peroxisomes couldn't be found in cortical astrocytes. On electronmicroscopic examination, the PAS-positive material in neuro nal cytoplasm was composed of closely packed, uniform particles, with an о average diameter of 300 A, identical in all respects with glycogen g- particles (Agamanolis and Patre, 1979). Rarely, small coherent clusters or rosettes corresponding to α-particles were seen in the cytoplasm of neuroglial cells and of nerve cells. In a few instances, the glycogen- filled cells contained also dense or membranous lipid bodies. No other ultrastructural studies reported glycogen accumulation in CNS tissues. The content of the lipid-filled cells consisted of multilamellar myelin bodies and rarely membrane-bound, moderately dense, globular lipid ag gregates (Agamanolis et al, 1976 and 1979, Liu et al, 1976). Longitudi nal or slightly curved fiber-like electron-lucent structures, measuring 90 A, and clusters of short, 250 to 3Ö0 A thick, needle- or cleft-like lucent spaces were visualized. Some lipid bodies contained sligthly cur- o о ved tubules with an overall diameter of 200 to 220 A and a 90 A wide lucent center. Most frequently the lipid bodies were present within the cytoplasm of histiocytes and macrophages, many, however, were adjacent to nuclei of glial cells and rarely of astrocytes (Agamanolis et al, 1976, 1979, Liu et al 1976). Lipids were not seen within the neurons. Agamanolis and Patre, 1979, described degenerative changes of the ecto pic Purkinje cells. Loose concentric membranous cytoplasmic bodies en closing a small amount of dense material, and concentric and parallel arrays of dense lines formed the two types of cytoplasmic inclusions.

53 Liver (80 patients)

Macroscopic studies. In general, macroscopic examination of the liver of patients with CHR-S revealed hepatomegaly with normal gall bladder and extrahepatic bile ducts.

Microscopic studies. Abnormalities in liver tissue were mentioned in all patients investiga ted. Microscopic studies of the liver in Zellweger syndrome showed in general slight or moderate alterations. Hepatic lobular disarray, con sisting of irregular spatial relationship between central and peripheral portions of the lobules, was a common finding (Gilchrist et al, 1976). Frequently (32 patients) intralobular fibrosis was observed. A few au thors mentioned cirrhosis (8 patients). Cholestasis was reported in 13 patients, some of them showed epithelial proliferation of the bile ducts (Monnens et al, 1980). Progression of the intralobular fibrosis into cirrhosis was noted when successive biopsy and autopsy liver specimens were described (Gilchrist et al, 1976, Brun et al, 1978, Carlson and Weinberg, 1978, Pfeifer and Sandhage, 1979, and Mathis et al 1980). Prussian blue stain revealed moderate to heavy iron deposition in hepatocytes and reticuloendothelial cells of liver in 36 children; in 4 other cases no iron could be demonstrated. In eleven infants extramedullary hematopoiesis was found.

Giant cell transformation of liver cells was unusual (3 patients); it decreased during follow up (Carlson and Weinberg, 1978). The presence of PAS-diastase-positive macrophages was reported in three patients of different age (Mooi et al, 1983).

Electron microscopic studies. The first electron microscopic studies on liver tissue of patients with Zellweger syndrome were reported by Goldfischer et al, 1972, 1973A, and 1973B. Increased electron density of the matrix and distortion of the cristae of the mitochondria were generally noted (Goldfischer et al, 1972, Brun et al, 1978, Hanson et al, 1979, Pfeifer and Sandhage, 1979, Monnens et al, 1980, Mathis et al, 1980, Hong et al, 1981, Borchard et

54 al, 1982, Mooi et al, 1983). Borchard et al (1982) and Mooi et al (1983) described giant mitochondria, frequently with paracrystalline inclu sions. Comparison of successive specimens revealed no progression of the mitochondrial abnormalities (Mooi et al, 1983). Peroxisomal abnormalities in CHR-S, demonstrated by enzyme-histochemical electron microscopy, were first mentioned in liver tissue by Goldfischer et al, 1973A and 1973B. After incubation in the alkaline DAB (diaminobenzidine) medium for demonstrating the peroxidatic activity of catalase, a marker enzyme for peroxisomes no reactive peroxisomes could be found. His conclusion was: "if any peroxisomes are present in cerebrohe- pato-renal syndrome our observations indicate that they must be greatly reduced in number or altered in form and enzymatic activity". An asser tion that a structure is absent, is difficult, if not impossible, to ma ke solely on the basis of morphologic studies. A lot of pathologic stu dies failed to visualize peroxisomes: Brun et al, 1978, Carlson and Weinberg, 1978, Pfeifer and Sandhage, 1979, Hanson et al, 1979, Friedman et al, 1980, Monnens et al, 1980, Hong et al, 1981, Müller-Höcker et al, 1981, Borchard et al, 1982 and Björkhem et al, 1984. Müller-Höcker et al (1981) reported absence of peroxisomes in the liver, irrespective of du ration and degree of liver damage. Pfeifer and Sandhage (1979) proposed the term "peroxisomes deficiency" as a name for this syndrome which is more closely related to the pathogenetic mechanism than the usual desig nation cerebro-hepato-renal syndrome.

Some authors reported a subtotal loss of peroxisomes (Carlson and Wein berg, 1978, Mathis et al, 1980, Borchard et al, 1982). Structures, pos sibly resembling microperoxisomes, were occasionally noted (Pfeifer and Sandhage, 1979, Borchard et al, 1982, Mooi et al, 1983), although no catalase positivity of those structures was found. Lysosomal abnormalities in two older patients (12 months and 2 years and 4 months of age, respectively), suffering from the CHR-S were mentioned in two reports. In liver macrophages, lysosomes, filled with fine double lamellae were noted, corresponding to the PAS-diastase positive material observed light microscopically (Pfeifer and Sandhage, 1979, Mooi et al, 1983). The lamellae appeared as pairs of very fine electron-dense lines, slightly less than 10 nra, usually arranged parallel to one another

55 and to the main axis of the lysosome, and determining the angular or fu siform shape of the lysosomes (Mooi et al, 1983). Small numbers of simi lar lysosomes in hepatocytes were reported by Mooi et al (1983).

Kidneys (71 patients)

Macroscopic studies. Macroscopic studies of the kidneys in Zellweger syndrome showed in seve ral cases cortical cysts with a diameter up to 15 mm.

Microscopic studies. Microscopic studies on kidney tissue from patients with the CHR-S revea led cysts throughout the kidney cortex in nearly every patient; occasio nally cysts in the medulla were reported (Patton et al, 1972, Vincens et al, 1973, Bernstein et al, 1974). There was no relationship between the number or size of cysts and the age at death. Exceptionally no abnorma lities of the kidneys were mentioned (Bowen et al, 1964, Gustafsson et al, 1983). No signs of scarring, cartilage or inflammation were found.

Electron microscopic studies. Goldfischer et al, 1973A and 1973B, published the first report of elec tron microscopic studies on kidney tissue from patients with the CHR-S. They reported minimal mitochondrial changes in proximal tubulus cells. No structures resembling peroxisomes could be found by enzyme-histochemical electron microscopy (Goldfischer et al, 1973B, Brun et al, 1978).

Skeletal muscle - peripheral nerve

In several muscles no abnormalities were reported (Passarge et al, 1967, Kohn and Mündel, 1969, Volpe and Adams, 1972, Garzuly et al, 1974, Gil christ et al, 1976) until Carlson and Weinberg (1978) and Müller-Höcker et al (1984) mentioned focal atrophy of skeletal muscle fibers and of fibers of the diaphragm (Müller-Höcker et al, 1984). Sarnat et al (1983) described a "mitochondrial myopathy". Their ultrastructural studies

56 showed large aggregates of mitochondria and increased lipid content in the subsarcolemmal and intermyofibrillar spaces. Müller-Höcker et al, 1984, reported enlarged mitochondria with irregularly oriented cristae in the striated muscle while in the myocardium the number, size and sha pe of the mitochondria appeared normal. Light microscopic studies of the peripheral nerve in the CHR-S revealed "demyelination" (Kohn and Mündel, 1969, Liu et al, 1976), although other authors reported no abnormalities (Volpe and Adams, 1972, Gilchrist et al, 1976). Segmental fusiform enlargement of the axons was demonstrated (Liu et al, 1976). Brun et al, 1978, described axones of the peripheral nerves with irregular swellings of a "torpedo type", though without ob vious concomitant cellular reactions such as inflammatory cells or fi brosis. Electron microscopic study of the peripheral nerve showed many myelina ted nerve fibers, filled with an excess of neurofilaments which assume о a winding pattern. The individual filament measured approximately 80 A and morphologically they did not differ from those seen in normal ner ves.

Hemato-lymphatic system (20 patients)

The weight of the thymic gland was diminished in 4 out of 7 patients (Gilchrist et al, 1976). Diminished numbers of Hassall's corpuscles were mentioned by Opitz et al, 1969, Gilchrist et al, 1974, and Brun et al, 1978. Siderosis of the spleen was mentioned by a few authors (Smith et al, 1965, Kohn and Mündel, 1969, Jan et al, 1970, Goldfischer et al, 1973A, Haddad et al, 1976, Brun et al, 1978, Bartolettl et al, 1978), although no siderosis could be demonstrated in two patients (Liu et al, 1976, Carlson and Weinberg, 1978). Ultramicroscopic studies on spleen, thymus or lymphnode tissue were not reported.

57 Adrenal glands (10 patients)

Only a few reports of histopathologic study on adrenal gland tissue of patients with CHR-S are available. In two patients the microscopic picture of the adrenal gland was consi dered to be normal (Punnett and Kirkpatrick, 1968, Brun et al, 1978). Goldfischer et al, 1983, reported a morphological study of adrenocorti cal tissue of 8 infants who had died of Zellweger syndrome. Histopatho- logical study of the adrenocortical tissue revealed a normal architec ture and no evidence of atrophy. Striated cells, some of which were bal looned, were present in the reticularis-inner fasciculata of 7 from 8 adrenals. Autolysis made interpretation of the eighth case uncertain. There were two types of striated cells. The commonest type of striated cell appeared to be adrenocortical parenchymal cells which occurred within normal trabeculae and were separated by sinusoidal spaces. The second type of striated cell was more eosinophilic with contracted cyto plasm and often an excentric small nucleus.

Ultrastructural examination of these striated adrenocortical cells con firmed the presence of lamellae with 2.5 nm leaflets and lamellar-lipid profiles of very long chain fatty acid-cholesterol esters, lying free in the parenchymal cells and considered to be characteristic of ALD. A lot of non-parenchymal cells contained membrane-bound spicular inclusions.

Pancreas (9 patients)

Hyperplasia of the islands of Langerhans was reported by a few authors (Patton et al, 1972, de León et al, 1977, Brun et al, 1978 and Sarnat et al, 1983). Occasionally acinar tissue cysts were mentioned (Jan et al, 1970, de León et al, 1977),. No ultramicroscopic studies on pancreas tissue were reported.

58 The eyes

Macroscopic studies. Haddad et al, 1976, described an oedematous cornea. The angle of the iris was of normal appearance (Volpe and Adams, 1972, Haddad et al, 1976), the etiology of the glaucoma could not be established in these patients. The lens exhibited early cataractous changes posteriorly (Haddad et al, 1976). Normal optic discs (Volpe and Adams, 1972, Brun et al, 1978), op tic disc pallor (Punnett and Kirkpatrick, 1968, Stanescu et al, 1972, Vincens et al, 1973, de Leon et al, 1977, Agamanolis et al, 1976, Fried man et al, 1980) and macula degeneration (Garner et al, 1982) were re ported. Focal retinal hemorrhages and mottling of the retinal pigment epithelium in the mid and far periphery were noted (Volpe and Adams, 1972, Cohen et al, 1983).

Microscopic studies. Investigation of the sclera (Haddad et al, 1976, Toussaint et al, 1981, Garner et al, 1982), the filtration angle (Volpe and Adams, 1972, Haddad et al, 1976, Garner et al, 1982) and the iris (Toussaint et al, 1981, Garner et al, 1982, Cohen et al, 1983) revealed no abnormalities. An oe dematous cornea (Toussaint et al, 1981, Garner et al, 1982) with irido- scleral adhesions (Haddad et al, 1976) was reported although other pa tients showed normal corneal appearance (Toussaint et al, 1981, Garner et al, 1982). The corpus ciliare was considered to be normal (Toussaint et al, 1981, Garner et al, 1982, Cohen et al, 1983); Haddad et al (1976) however re ported focal necrosis and scattered melanophages within the ciliary body. The lens morphology varied between normal (Garner et al, 1982) and cata ractous abnormalities: subcapsular cortical degeneration (Haddad et al, 1976), mottled lens fibers (Hittner et al, 1981, Kretzer et al, 1981) and subcapsular vacuoles (Kretzer et al, 1981, Cohen et al, 1983) were described.

59 The vitreous was condensed into strands, with many attached spindle-sha ped and mononuclear cells in 2 out of 3 patients (Cohen et al, 1983). Haddad et al, 1976, reported an unremarkable choroid and sclera; Cohen et al, 1983, described an area of extramedullary hematopoiesis within the choroid. Generally, the retinal abnormalities were mainly localized centrally while the periphery revealed pigment epithelium pathology. The following abnormalities were described: subepithelial, lipid-loaden macrophages (Toussaint et al, 1981, Kretzer et al, 1981), photoreceptor cell degene ration, mainly affecting the rods (Haddad et al, 1976, Kretzer et al, 1981, Toussaint et al, 1981, Garner et al, 1982, Cohen et al, 1983), de creased number of the outer nuclear layer cells with normal inner nuclear layer and outer and inner plexiform layer (Haddad et al, 1976, Kretzer et al, 1981), Muller cell proliferation (Kretzer et al, 1981), diastase- sensitive, PAS-positive crystalline material in the peripheral inner re tinal layers (Cohen et al, 1983). There was a marked decrease of ganglion cells (Haddad et al, 1976, Kretzer et al, 1981, Toussaint et al, 1981, Cohen et al, 1983), gliosis of the nerve fiber layer (Haddad et al, 1976, Cohen et al, 1981) with moderate loss of axons (Haddad et al, 1976, Toussaint et al, 1981), while the inner limiting membrane seemed to be thickened (Kretzer et al, 1981).

The optic nerve was moderately atrophic (Opitz et al, 1969, Haddad et al, 1976, Agamanolis et al, 1976 and 1979, de Leon et al, 1977, Tous saint et al, 1981, Hittner et al, 1981, Kretzer et al, 1981, Garner et al, 1982, Cohen et al, 1983) with an increased number of large fibrilla ry astrocytic cells (Opitz et al, 1969, Haddad et al, 1976) and loss of axons (Haddad et al, 1976, Kretzer et al, 1981, Toussaint et al, 1981). Gliosis was mentioned several times (Haddad et al, 1976, Toussaint et al, 1981, Garner et al, 1982) as well as demyelination of the optic ner ve (Opitz et al, 1969, Haddad et al, 1976, Toussaint et al, 1981, Garner et al, 1982).

Iron was demonstrated in the stratified squamous epithelium of the cor nea and the unpigmented epithelium of the ciliary body (Volpe et al, 1972), and also in the choroid (Garner et al, 1982, Cohen et al, 1983). Haddad et al, 1976 and Toussaint et al, 1981 found no iron in the ocular structures.

60 Electron microscopie studies. Transmission electron microscopie studies on the ocular tissues were re ported since 1976. Except for interstitial corneal oedema (Haddad et al, 1976), the cornea, sclera and iris did not reveal ultrastructural abnor malities (Toussaint et al, 1981). The lenticular opacities had ultrastructural analogues, which were inclusion bodies restricted to the cor tical lens fibers and interspersed between osmiophilic dense core granu les. Despite the presence of abnormal mitochondria in the lens epithe lium, the inclusions were not neutral lipid. The lens epithelium showed abnormal mitochondrial proliferation with pale matrix and few cristae, increasing from 18% of the cytoplasmic area at 2.5 months to 23% at 10.5 months. In age-matched controls this was only 5% (Hittner et al, 1981). The retinal pigment epithelium was flattened and contained a variable amount of melanin (Toussaint et al, 1981, Garner et al, 1982). Presumed premelanosomes sometimes were lying in the subretinal space (Garner et al, 1982). Toussaint et al (1981) and Cohen et al (1983) reported bi- leaflet inclusions in the retinal pigment epithelium and in pigmented macrophages. The electron-dense leaflets were 2.2 nm in diameter and were separated by a 2.2 nm electron-lucent space, the leaflet having no membrane and being intermixed with a fine granular background substance (Cohen et al, 1983). If the leaflets were not admixed in loose conglome rates but in densely packed whorls their thickness was 3.7 nm and they were separated by a 3.1 nm electron-lucent space.

Many of the photoreceptor cells were degenerative. They were represented by sparse debris, some forming laminated myelin figures (Garner et al, 1982), others had shed almost all the outer discs (Toussaint et al, 1981, Garner et al, 1982). The inner segment showed considerable loss of mitochondria. Residual mitochondria often were swollen with ring and other abnormal cristae (Garner et al, 1982). The macrophages in the subretinal space contained large round inclusion bodies similar to those of the retinal pigment epithelium (Toussaint et al, 1981, Cohen et al, 1981).

No reports concerning lysosomes and peroxisomes in ocular tissues of Zellweger patients are available.

61 Biochemical analysis of the cholesterol ester fraction of the retinal tissues revealed a more than fivefold Increase in very long chain fatty acids compared with age-matched controls (Cohen et al, 1983). In a biopsy of the conjunctiva. Inclusion bodies, resembling neutral li pids, were noted in the cytoplasm of the vascular endothelial cells and in the Schwann cells (Toussaint et al, 1981).

Other organs

Histopathologic studies of the cardiovascular system in Zellweger syn drome revealed congenital abnormalities in 14 patients, usually patent ductus arteriosus and/or septum defect(s). Abnormalities in lung tissue were reported in 24 patients with the CHR-S Pneumonia was mentioned in 20 patients, eight times atelectasis was found.

62 BIOCHEMICAL STUDIES

Introduction

A survey of the biochemical abnormalities mentioned in the literature about cerebro-hepato-renal syndrome is given in Table VII. The unusual variety of metabolic disturbances is remarkable.

TABLE IV: Biochemical abnormalities described in the cerebro-hepato- renal syndrome of Zellweger.

Biochemical abnormalities Author Year

Liver dysfunction Bowen 1964 Iron overload Vitale 1969 Mitochondrial dysfunction Goldfischer 1972 Peroxisomal dysfunction or absence Goldfischer 1973 Immune deficiency Gilchrist 1974 ΗΡΑ, HPa Danks 1975 Defect in PA catabolism Trijbels 1979 Defect in bile acid synthesis Hanson 1979 Increased p-OH-phenyllactate in urine Chalmers 1980 Dicarboxylic aciduria Chalmers 1980 Increased level of VLCFA in fibro blasts (saturated and С , ) Brown, Moser 1982, 1983 Mitochondrial defect Trijbels 1981, 1983 Deficiency of plasmalogens in tissues Heymans 1983 Increased level of saturated VLCFA in serum Bakkeren 1984 Deficiency of acyl-CoA:DHAP acyltransferase Schutgens 1984

VLCFA = very long chain fatty acids (C,,-C )

63 Iron overload

The first "specific" metabolic derangement described was excessive iron content of several tissues. Deposition of iron in the (reticulum cells of the) spleen (Smith et al, 1968, Kohn and Mündel, 1969) and in the reticulum cells of the liver (Kohn and Mündel, 1969) was described, without determination of the serum iron concentration. Opitz et al (1969) performed studies of the iron metabolism in one pa tient. An unusually high serum iron level was measured. The bone marrow macrophages contained large amounts of stored iron and hepatic hemoside rosis was documented before death. After death at the age of six months, the iron concentration in various tissues was determined. A significant increase of iron was found in the liver and the kidneys. The total body iron concentration was estimated to be 114 mg/kg body weight which re presented a 52% increase over the normal value of 75 mg/kg. An excess of plasma iron was proposed to establish the diagnosis of Zellweger syndro me in a suspected infant. The iron in CHR-S is mostly localized in the reticuloendothelial system rather than in parenchymal cells and corre lates poorly with liver disease (Gilchrist et al,1976, Kelley, 1983, Müller-Höcker et al, 1984).

Normal and increased levels of serum iron and/or iron binding capacity were reported. Gilchrist et al (1976) demonstrated that the iron content of the liver was generally increased, rose to a plateau between 5 and 18 weeks at a level 3-5 times that found in control tissue, and dropped to normal levels in infants living longer than 20 weeks. The pathogenesis of this "early" disturbance in the iron metabolism is unclear.

Pipecolic acid (PA) metabolism

Introduction Mild aspecific amino aciduria in Zellweger syndrome was mentioned by se veral authors (Jan et al, 1970, Poznanski et al, 1970, Volpe and Adams, 1972, Vincens et al, 1973, Sommer et al, 1974, Garzuly et al, 1974) un til Danks et al (1975) reported hyperpipecolic aciduria (HPa) in 4 and

64 hyperpipecolic acidemia (ΗΡΑ) in 2 patients. A disturbance in the meta bolism of pipecolic acid (PA) in CHR-S was shown by Trijbels et al (1979). The range of PA levels in urine, serum and CSF observed in CHR-S is given in Chapter III.

The metabolism of pipecolic acid (PA) Rothstein et al (1953, 1954, 1959) found evidence that L-lysine is con verted to PA in rat liver. Chang et al (1976, 1978A) demonstrated by Γΐ4 1 means of intraventricular injection of С -labeled L- and D-lysine that both isomers (D more effectively than L) were metabolized in rat brain in a considerable amount to labeled L-PA and to a minor amount further into o-amino-adipic acid. The presumed metabolic routes of L- lysine via saccharoplne and via PA are shown in Figure 2. While lysine is mainly metabolized via saccharoplne in most organs, especially the liver, evidence has been obtained that in rat brain, the conversion of lysine to PA is the major metabolic pathway (Chang, 1976, Giacobini et al, 1980). PA can be converted in vivo and in vitro into piperidine in the rat brain (Kasé et al, 1967, 1970). The piperidine levels in Zellweger syndrome are unknown. In 2 Zellweger patients oral loading tests with L-lysine revealed lysine levels comparable to the control values without significant increase of the PA level in serum, urine and CSF (Trijbels et al, 1979). Therefore, the elevated levels of PA in CHR-S are not due to a disturbance in the metabolism of L-lysine via the saccharoplne route resulting in an overflow to the PA pathway. Oral loading tests with (150 mg/kg body weight) DL-PA hydrochloride in controls and in 3 Zellweger patients revealed in the patients a delayed return of the serum PA level to the baseline value as compared to con trol subjects proving that the HPa in CHR-S could be explained by a defect in the catabolism of PA (Trijbels et al, 1979). Dancis and Hutz ier (1982) studied the metabolism of PA in liver and kidney tissue from 10 different animal species. Remarkable differences, in the metabolic rate, to 1000 fold, were noted among the species. The highest activity was found in rabbit and guinea pig kidney. In the adult human kidney PA was also metabolized.

65 Fig. 2: Metabolie routes of L-lysine via saccharopine and via pipecolic acid (according to Trijbels et al, 1979).

о.О Pipendine

СООН I Η,Ν-CH о-с Ι α κ4ο< I ICH,), NADP· NADPH (CK), gluUrate Glutamate (CHJ, Co» • I \t^) Г Ι ν Λ I 4.NAD· СООН ^*—-4 СООН

L α NH2 a dipte NH2 adipe α Keto adipic Δ Piperideine i bemiaJdehyde acid 6 carboxylic acid

66 Blood-brain barrier and distribution of lysine, PA and piperidine The blood-brain barrier transport of L-lysine in the rat was studied by Chang (1978B) through the use of intravenous pulse injection of L С lysine. Chang (1978B) concluded that the L-lysine blood-brain barrier transport system of the rat was not saturated, even at the high dose (30 mg/kg) of L С lysine used. The low level of PA in human urine, compa red with the rat, would indicate a much stronger lysine blood-brain transport barrier in man than in the rat.

Labeled PA administered intracarotidly, intravenously or intraperito- neally, was actively transported in vivo into the mouse and rat brain (Nishio et al, 1981A, 1981B and 1983, Charles et al, 1983). In adult rats the cerebral cortex showed the highest uptake (Charles et al, 1983), while in newborn mice brain the preferential localization of the accumulated radioactivity was the olfactory bulb, the anterior telence phalon and the diencephalon (Nishio et al, 1983). Kinetic studies of L-PA influx revealed a saturable low- and a non-saturable high-capacity uptake mechanism in rats (Charles et al, 1983). An age-dependent difference in blood-brain barrier for PA in mice was sug gested by the age-dependent pattern of accumulation of radioactivity in the CNS after intraperitoneal administration of I С PA (Nishio et al, 1983). In newborn mice the accumulation of radioactivity in CNS reached a peak 24 h after intraperitoneal injection of D, L H PA whereas in adult mice radioactivity in CNS peaked at 5 min, remained approximately constant up to 5 h and then declined slowly to 24 h. The radioactivity in plasma showed essentially the same pattern of accumulation in adult and in newborn mice.

Structurally related substances, such as nipecotic acid, isonipecotic acid, L-proline, piperidine and GABA showed an inhibitory effect on the active blood-brain transport of PA in mice (Nishio et al, 1981В). The regional distribution of labeled piperidine in rabbit brain after intravenous injection of the labeled substance revealed the highest le vels in the nucleus caudatus, the anterior neocortex and the corpora quadrigemina (Abood et al, 1961).

67 The pipecolic acid metabolism and the peroxisomes The exact mechanism of PA metabolism in man, including the subcellular localization of this metabolic route is not known. The association of a disturbance in the metabolism of PA with the deficiency of peroxisomes in the Zellweger syndrome however points to a specific function of peroxisomes in this degradative pathway. Preliminary experiments per formed by our group, have proven that isolated rat liver peroxisomes Γ 14 1 show FAD dependent metabolic activity against 1- CI PA.

Liver dysfunction - Abnormal bile acid pattern

Jaundice persisting after the seventh day of life and/or hepatomegaly and/or gastro-intestinal bleeding called attention to the liver. Eleva ted transaminases, yGT, LDH and direct bilirubin were conspicuous fin dings. Usually hypoprothrombinemia, corrected by Vit К administration, was mentioned. Normal "liver function tests" were occasionally noticed (Taylor et al, 1969, Sommer et al, 1974, Garzuly et al, 1974, Mathis et al, 1980). The presence of abnormal bile acids in CHR-S was first mentioned by Han son et al (1979), and confirmed by Monnens et al (1980) and Mathis et al (1980). An abnormal accumulation of the bile acid precursors trihydroxycoprostanic acid (THCA) and dihydroxycoprostanic acid (DHCA) and some times varanic acid (an intermediate between THCA and cholic acid) in duodenal fluid, serum and urine was documented (see Fig. 6, Chapter I). These compounds are considered to be normal intermediates in the degra dation of the cholesterol side chain in the biosynthesis of cholic acid and chenodeoxycholic acid (Hanson et al, 1979, Danielsson and Gustafs- son, 1981). The presence of moderate amounts of cholic and chenodeoxy cholic acid in the patients indicates that the block in the side chain oxidation is a partial one (Mathis et al, 1980). Hanson et al (1979) and Gustafsson et al (1983) reported varying levels of these precursors during follow up of patients with CHR-S. Absence of DHCA at very young age (2 patients) and a decreasing level of THCA with increasing concen tration of varanic acid (2 patients) were noted. A causal relationship

68 between the abnormal levels of bile acids and the liver disease was sug gested by Hanson et al (1977), Monnens et al (1980) and Borchard et al (1982). Pedersen and Gustafsson (1980) and Kase et al (1983) showed that the peroxisomal fraction of rat liver homogenate is able to catalyse the conversion of THCA into cholic acid. The presence of THCA and DHCA in duodenal fluid, serum and urine is important for the biochemical diagno sis of CHR-S (Monnens et al, 1980, Mathis et al, 1980, Gustafsson et al, 1983). These bile acids were not found in controls with other causes of cholestasis (Boon et al, 1973, Lott et al, 1979).

In serum of one patient with all symptoms of the Zellweger syndrome, Parmentier et al (1979) reported the presence of 3a, 7a, 12a- trihydroxy-50-C29 dicarboxylic bile acid. Small amounts were detected in bile and urine. The same abnormal bile acid was reported in plasma of one Zellweger patient by Björkhem et al, 1984, although specific search for that bile acid by Gustafsson et al (1983) was unsuccessful in serum of one patient. Collins et al (1978) identified 26-hydroxycholesterol in serum of one Zellweger patient. Analysis of serum from 22 other infants with a va riety of liver diseases failed to identify 26-hydroxycholesterol.

Immune deficiency

The pathologic findings of the hemato-lymphatic system have been descri bed previously. Immune deficiency in the CHR-S was suggested by these findings (Gilchrist et al, 1974). The usual assays of cellular immunity (number of lymphocytes, stimulation of the lymphocytes from the periphe ral blood by phytohemagglutinin and pokeweed mitogen, mixed lymphocyte reaction, resetting tests) were found to be normal in 1 patient by Bak keren et al (1976). Camera et al (1977) and Brun et al (1978) reported normal Τ en В cell function. Hong et al (1981) however reported a combi ned Τ en В deficiency in one patient. This patient had a decreased level of serum calcium in association with immunodeficiency. Hypocalcemia was never observed in our patients with CHR-S. No systematic analysis of cellular and humoral immunity in patients with CHR-S has been reported.

69 Organic aciduria

Chalmers et al (1980) listed two siblings with CHR-S manifesting an ab normal dicarboxylic aciduria. No further details were given. Kelley (1983) mentioned also dicarboxylic aciduria in two older patients with CHR-S. This can possibly be related to the carnitine deficiency in their patients (Kelley, 1983). In twelve of our patients no dicarboxylic aci duria could be found by gas chromatography. Increased excretion of p-OH-phenyllactate in urine was first reported by Brun et al, 1978. Chalmers et al (1980) and Govaerts et al (1982) con firmed this observation (in 2 and 7 patients respectively). Seven new patients investigated by our group showed increased excretion of p-OH- phenyllactate in urine (>120 pmol/mmol creatinine). No specific metabo lic defect could be deduced as yet from this observation.

Very long chain fatty acids (VLCFA) pattern

Brown et al (1982) found increased levels of C-.-C», saturated fatty acids in brain white matter, adrenal gland and cultured fibroblasts from Zellweger patients. The increase was similar to that noted in adrenoleukodystrophy (ALD). Increased concentration of hexacosanoic acid (C^^.) was consistently found in the serum of patients with CHR-S (Bakkeren et al, 1984). The ratios С2б С22-0' C25-0/C22-0' and C24-0/C22-0 in serum were also elevated compared to age-matched controls. The concentrations and ratios in serum of heterozygotes were within normal ranges. Further investigation of the saturated VLCFA levels and ratios in cultured fi broblasts of patients, heterozygotes and controls and in amnlocytes from pregnancies not at risk for CHR-S (Moser et al, 1984, Chapter V) reveal ed comparable results. The VLCFA are preferentially degraded by peroxi somal 0-oxidation (Kawamura et al, 1981). Impaired degradation of the VLCFA in cultured skin fibroblasts from Zellweger patients was reported (Moser et al, 1983, Moser et al, 1984). Antenatal diagnosis for this disease by means of determination of the saturated VLCFA in amnlocytes was suggested (Brown et al, 1982, Bakkeren et al, 1984), and performed by Moser et al (1984).

70 Not only the levels of saturated VLCFA but also of hexacosenoic acid (C., .) were increased in plasma and in cultured skin fibroblasts of patients suffering from CHR-S (Moser et al, 1983, Moser et al, 1984, Björkhem et al, 1984). In amniotic fluid from a pregnancy affected by CHR-S similar abnormalities of the VLCFA were reported (Björkhem et al, 1984). The increased VLCFA levels in erythrocyte membranes (Tsuji et al, 1981, Kobayashi et al, 1983, Knazek et al, 1983) and in cholesterol esters of the brain white matter (Brown et al, 1983), mentioned in ALD, have not (yet) been reported in CHR-S.

Deficiency of plasmalogens in tissues

A severe deficiency of plasmalogens in several tissues of Zellweger pa tients was reported by Heymans et al (1983, 1984). Phosphatidylethanola- mine plasmalogen and phosphatidylcholine plasmalogen were nearly absent in tissues of patients with CHR-S. The phosphatidylethanolamine plasma logen concentration in cultured skin fibroblasts was significantly de creased compared to controls and heterozygotes. A general defect in alkyl ether phospholipid synthesis was proposed. Lowered, but substantial amounts of plasmalogens in erythrocytes of patients were an intriguing finding. Schutgens et al (1984) reported a severe deficiency of acylCoA:DHAP acyltransferase, the first enzyme in the pathway leading to plasmalogen biosynthesis in liver, brain and cultured skin fibroblasts from Zellwe ger patients. Acyl-CoA:DHAP acyltransferase is probably localized on the inside face of the peroxisomal membrane (Hajra and Bishop, 1979 and 1982). Cultured amniotic fluid cells from pregnancies not at risk for CHR-S contained an enzymatic activity comparable to that of control fi broblasts. A prenatal test for this disease based on the deficiency of acyl-CoA:DHAP acyltransferase in cultured amniotic fluid cells is now available.

71 Mitochondrial defects in the CHR-S; relation to the deficiency of peroxisomes

Mitochondrial defects and absence of peroxisomes in the cerebro-hepato- renal syndrome were first described by Goldfischer et al (1972, 1973A and 1973В). These authors demonstrated cytochemically, with a diaminobenzidine staining technique, the absence of peroxisomes in hepatocytes and renal proximal tubules. Oxygen consumption by brain mitochondria of a Zellweger patient was severely reduced with several substrates (succi nate, glutamate and malate). Respiration rate was not stimulated by ad dition of ADP. Normal oxygen consumption was found with ascorbate and TMPD (tetramethylphenylenediamine), transferring electrons directly to the cytochromes. Liver mitochondria from a second patient showed similar results. From these observations Goldfischer et al (1973B) concluded that there must be a defect in the electron transport chain before the step involving cytochromes. From histochemical studies in brain, liver and muscle from a Zellweger patient, they postulated that the defect is most probably located between the succinate dehydrogenase flavoprotein and coenzyme Q. A schematic representation of the respiratory chain is given in Fig. 3. Mitochondria in liver were reduced in number and their cristae were irregular.

Mitochondrial and peroxisomal defects were reported by Versmold et al (1977) in a patient with a disorder similar to Zellweger syndrome. Liver of this patient showed no peroxidatic activity of catalase and peroxiso mes were absent. Most mitochondria isolated from patient's liver showed normal appearance. In contrast to the findings of Goldfischer et al (1973) these authors found almost normal succinate and glutamate respi ration in liver mitochondria of their patient. Coupling of respiration to phosphorylation was also normal. Isolated liver mitochondria showed a strongly reduced cytochrome content (one third of normal). Brain mito chondria showed a normal cytochrome content. Versmold et al (1977) did not provide an explanation for the relationship between the defects in mitochondria and peroxisomes.

Considering the studies performed by Goldfischer et al (1973) and Ver smold et al (1977) it can be concluded that mitochondrial defects have been reported by both of them. However, the localization of the defect

72 Fig. 3: Schematic representation of the respiratory chain composed of coenzyme Q (=Q) and cytochromes a, b and c, and its interaction with two dehydrogenases supplying reduction equivalents. The succinate dehydrogenase coupled Q pool is not necessarily separated from the NADH dehydrogenase coupled Q pool. The interaction sites of various non-competitive inhibitors is shown by transverse lines. DCPIP is the artificial acceptor used in the succinate dehydro genase assay. TTFA inhibits the PMS mediated DCPIP reduction in a nearly competitive way.

succinate

succinate dehydrogenase

PMS DCPIP •TTFAЖ Q

NADH NADH- |-*Q •b—J^c, ю •ааз—1-*0 dehydrogenase 2 rotenone antimycinA KCN amytal HONO

DCPIP = dichlorophenol indophenol TTFA = 2-thenoyltrifluoroacetone PMS = phenazine methosulfate HQNO « 2-heptyl-4-hydroxyquinoline-N-oxide

73 in the electron transport chain was different: before the step involving the cytochromes according to Goldfischer et al (1973) and in the cyto chrome regio according to Versmold et al (1977). Goldfischer et al (1979) speculated that the peroxisomal and mitochondrial abnormalities in the CHR-S are different manifestations of a single defect. Biochemical studies in muscle, liver, leucocytes and fibroblasts from patients with the Zellweger syndrome have been performed by Trijbels et al (1983). Oxidation rates of several substrates (pyruvate, malate and 2-oxoglutarate) were strongly reduced in skeletal muscle homogenate, the mean residual activity being 20% of controls. Liver homogenates from Zellweger patients incubated with succinate or glutamate and malate as substrates showed oxidation rates as low as 10% of controls. Oxygen consumption by isolated skeletal muscle mitochondria was not stimulated by addition of substrates (pyruvate and malate, succinate and rotenone), except for ascorbate and TMPD. This finding is in agreement with that of Goldfischer et al (1973) for brain mitochondria from a patient. Cytochrome content in mitochondria from heart muscle and liver was nor mal, in contrast to the strongly reduced values found by Versmold et al (1977) in liver mitochondria from their patient. This finding of a normal cytochrome content agrees with the proposed defect in the electron transport chain being localized prior to the cytochromes according to Goldfischer et al (1973). In order to localize the defect Trijbels et al (1983) investigated the succinate-ubiquinone oxidoreductase activity of a liver homogenate from a CHR-S patient using DCPIP (dichlorophenol indophenol) as electron acceptor. The measured activity was very low. The defect was further elucidated by measuring the succinate-PMS (phenazine methosulphate) oxidoreductase activity as well as the characteristic TTFA (2-thenoyltrifluoroacetone) inhibition of this activity. The rate of reduction of PMS is not affected in the patient but the effect of TTFA is very close to the effect of a competitive inhibitor, in contrast to a non-competitive inhibition in control homogenate. These findings indicate (Trijbels et al, 1983) that the interaction of succinate dehy drogenase with ubiquinone is largely disturbed. Alternatively, a defi ciency of ubiquinone may account for the registered defect. The proposed disturbed interaction between succinate dehydrogenase and ubiquinone might be caused by or related to the deficiency of peroxisomes in the

74 cells of Zellweger patients. Deficiency of peroxisomes can lead to a disturbance in the structure of the inner mitochondrial membrane, as biosynthetic pathways of components of biologic membranes may be pre sent in peroxisomes. The defect in the electron transport chain can thus be caused by the disturbance in the structure of the mitochondrial mem brane. The localization of the defect in the electron transport chain at the level of succinate-ubiquinone oxidoreductase is in agreement with the proposal of Goldfischer et al (1973). Intact leucocytes and fibroblasts from Zellweger patients showed normal Γ 14 1 Γ 14 1 oxidation rates of 1- CI pyruvate and 12- С pyruvate, suggesting a normally functioning electron transport chain in these cells. Indica tions for a similar condition in vivo were obtained by the finding of normal ratios of lactate/pyruvate and ß-hydroxybutyrate/acetoacetate in blood of Zellweger patients (Trijbels et al, 1983). However, the normal ratios do not necessarily exclude a defect in the electron transport chain in vivo because a drastic lowering of the oxidative capacity is required to cause a change in the NADH/NAD or lactate/pyruvate ratio. Kelley and Corkey (1983) investigated the effect of several inhibitors of mitochondrial electron transport in fibroblasts from Zellweger pa tients. Growth velocity of Zellweger cells and control cells was equally inhibited by amytal, rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide, whereas cells from Zellweger patients showed a five-fold greater sensi tivity to antimycin A, an inhibitor of the cytochrome be. complex, as compared to control cells. The authors suggested a primary or secondary functional deficiency at mitochondrial complex III in fibroblasts from Zellweger patients, in contrast with the defect at complex II proposed by Trijbels et al (1983). A mitochondrial myopathy in a case of Zellweger syndrome was reported by Müller-Höcker et al (1984). Enlarged mitochondria with minor structural abnormalities were found in skeletal muscle. Histochemically normal ac tivities of NADH-reductase, succinate dehydrogenase and cytochrome с oxidase were noted. The oxidative phosphorylation was found to be dis turbed. According to the authors a loosely coupled oxidative phosphory lation was present in patient's muscle.

75 Summarizing it can be concluded that several defects have been postu lated in the functioning of the electron transport chain in the Zell weger syndrome. It remains uncertain, however, whether these different defects all originate from the basic defect in this syndrome, being the deficiency of peroxisomes. It cannot be excluded that some observed mi tochondrial defects appear in vitro during manipulation of the patients' specimens owing to a structural defect in the mitochondrial membrane initiated by the peroxisomal deficiency.

76 RELATED DISORDERS

Introduction

A few syndromes described in literature show clinically and/or biochemi cally common features with the CHR-S. These syndromes and their classi fication number in McKusick's "Mendelian Inheritance in Man" are presen ted in Table V.

Table V: Disorders related to CHR-S and their classification.

Name of the disorder Classification according to McKusick

hyperpipecolic acidemia (ΗΡΑ) * 23940 trihydroxycoprostanic acidemia 27565 neonatal adrenoleukodystrophy 20237 infantile phytanic acid storage not classified disease

These four diseases are inherited as an autosomal recessive trait. A review of the literature, concerning the sex of the patients, the age and cause of death is given in Tables VI, VII and VIII. The relation between CHR-S and neonatal ALD is discussed in Chapters V and VIII. A comparison of the clinical features and biochemical abnormalities of ΗΡΑ and neonatal ALD with those of the CHR-S is made in Tables IX and X. The infantile phytanic acid storage disease (Scotto et al, 1982, Yao et al, 1982) shares a lot of clinical and pathologic features with the CHR-S. Thin layer chromatography of serum of four of our CHR-S patients revealed an increased phytanic acid level compared to age-matched con trols (unpublished results). Further biochemical investigation of pa tients with infantile phytanic acid storage disease is required to deli neate whether or not this condition differs from the CHR-S.

77 TABLE VI: Hyperplpecolatemia.

Case Author Year Sex Age at death Cause of death

1 Gatfield 1968 M > ih У not mentioned 2 Thomas 1975 M 2 У pneumonia 3 Roesel 1979 7 ? not mentioned 4 Roesel 1979 ? 2 У not mentioned 5 Burton 1981 M 1% У respiratory insufficiency 6 Burton 1981 M 3*5 У not mentioned 7 Arneson 1982 F 10*5 m intestinal hemorraghe

TABLE VII: Trlhydroxycoprostanlc acidemia - Zellweger-like syndrome.

Case Author Year Sex Age at death Cause of death

1 Eyssen 1972 M 6 m pneumonia 2 Eyssen 1972 F 8% m not mentioned 3 Boon 1973 M 4 m not mentioned * 4 Hanson 1975 M 8 m liver failure 5 Hanson 1975 F 23 m liver failure 6 Parmentier 1979 M 3*5 m pneumonia 7 Parmentier 1979 M born in 1976 alive

* shock during dehydration (personal communication. Boon)

78 TABLE VIII: Patients with neonatal adrenoleukodystrophy.

Case Author Year Sex Age of death Cause of death

1 Ulrich 1978 M 18/12 У not mentioned 2 Manz 1980 M > 6h У not mentioned 3 Man ζ 1980 M 7 У pneumonia 4 Benke 1981 F 28/12 У respiratory arrest 5 Benke 1981 M 23/12 y respiratory arrest 6 Jaffe 1982 M 15/12 У sudden death 7 Jaffe 1982 F 2 У sudden death 8 Brown 1982 M > Ъ1/2 y not mentioned 9 Brown 1982 F 22/12 y not mentioned 10 Mobley 1982 F 27/12 У not mentioned 11 Haas 1982 F 5 У pneumonia 12 Partin 1983 F 22/12 y not mentioned 13 Partin 1983 M ? not mentioned 14 Farrell 1983 M > Us У not mentioned 15 Noetzel 1983 M > lObi y not mentioned 16 Noetzel 1983 F 74 y sudden death

79 TABLE IX: The clinical symptoms of the cerebro-hepato-renal syndrome (CHR-S) and two related diseases as reported in the litera ture.

Neonatal our Clinical symptoms CHR-S ΗΡΑ ALD CHR-S patients

Craniofacial features high forehead 32/32 2/2 3/3 13/15 frontal bossing 11/12 1/1 6/12 large fontanels 38/39 4/4 1/1 12/12 epicanthal folds 18/20 1/1 1/1 10/12 hypertelorism 19/22 0/1 3/4 0/6 abnormal ears 40/41 1/2 2/2 7/13 high arched palate 23/24 1/3 3/4 6/7 shallow supra-orbital ridge 17/17 10/10

Central nervous system hypotonia 75/79 5/5 12/14 15/16 seizures 41/41 4/4 9/10 12/16 poor sucking 45/46 3/3 5/5 16/16 poor activity 31/33 1/1 16/16 absent reflexes 45/47 3/3 5/7 15/16 hearing impairment 5/8 1/1 7/7 5/5 reaction to painstimuli 8/8 1/1 7/7 psychomotor retardation 18/18 4/4 13/13 16/16

Eye symptoms nystagmus 20/20 3/4 2/2 10/11 cloudy cornea 15/15 2/6 cataract-lenticular opacities 17/18 1/1 2/2 0/6 glaucoma 7/9 1/1 0/3 clumping of pigment at the periphery 6/7 4/4 5/5 4/11 optic disc pallor 9/11 4/4 4/4 8/11

Other symptoms hepatomegaly <1/12 y 27/36 2/3 0/15 >1/12 y 30/40 5/5 6/6 15/16 jaundice after 7 days 19/20 8/16 macroscopical gastro-intes tinal bleeding 9/9 1/1 1/16 splenomegaly 11/16 1/4 1/2 3/4 souffle in corde 14/17 0/2 5/16 cryptorchidism 16/17 0/4 1/8 6/13 cliteromegaly 9/12 1/2 pes equinovarus 34/38 0/1 0/2 6/16 ulnar deviation of the hand(s) 6/6 5/16 simian crease(s) 19/21 3/4 1/2 11/16 failure to thrive 52/89 4/5 5/6 14/16

80 TABLE X: The biochemical abnormalities of CHR-S and two related disorders as mentioned in literature.

Neonatal our Biochemical abnormalities CHR-S ΗΡΑ ALD CHR-S patients

Amino-acids ΗΡΑ 4/6 6/6 0/2 10/10 НРа 16/17 5/5 0/2 14/14 increased PA in CSF 1/1 8/8 abnormal DL-PA loading test 3/3 5/5 8/8

Liver dysfunction increased serum transami nases, increased yGT 20/25 3/5 4/4 14/14 increased direct bilirubin 12/17 0/1 14/15 hypoprothrombinemia 11/14 2/3 8/9 THCA in duodenal fluid 1/1 8/8 serum 9/9 urine 4/5 3/3 DHCA in duodenal fluid 7/8 serum 7/9 urine 2/3 1/3

Other albuminuria 14/19 5/14 increased p-OH-phenyllactate in urine 2/2 9/9

81 Hyperpipecolic acidemia (ΗΡΑ) - hyperpipecolic aciduria (НРа)

A number of authors mentioned similarities between the clinical and bio chemical features of the CHR-S and those of the hyperpipecolic acidemia (Trijbels et al, 1979, Burton et al, 1981, Arneson et al, 1982, Govaerts et al, 1982, Kelley, 1983). The resemblance of the clinical and bioche mical abnormalities of the latter disease to CHR-S is striking (see Ta bles IX and X). These patients presented with hypotonia, psychomotor re tardation and hepatomegaly; their facial dysmorphic features were gene rally noted (Thomas et al, 1975, Burton et al, 1981, Arneson et al, 1982).

The PA loading tests of patients with ΗΡΑ and of patients suffering from the CHR-S revealed similar patterns (Gatfield et al, 1968, Thomas et al, 1975, Burton et al, 1981, Arneson et al, 1982, Trijbels et al, 1979). The varying dose of PA and different methodology of the PA determination make quantitative comparison difficult. Lysine loading tests, however, revealed no abnormality in the saccharopine route in both syndromes (Gatfield et al, 1968, Burton et al, 1981, Arneson et al, 1982, Trijbels et al, 1979). Decreasing intake of protein (1 g/kg/d) was accompanied by decreasing serum PA level in two patients (Gatfield et al, 1968, Burton et al, 1981), but no apparent effect on the clinical condition of the patients was noticed. Thomas et al, 1975, mentioned no effect of a diet with 1.25 g protein/kg/d on the serum pipecolic acid concentration. Only Gatfield et al (1968) determined the pipecolic acid level in CSF of one patient with hyperpipecolic acidemia; he noted an increased level of 1-2 ymol/l.

No data concerning the bile acids, organic acids, levels of saturated VLCFA and plasmalogen concentrations in ΗΡΑ are available. An extensive report of pathologic alterations in ΗΡΑ was published by Challa et al (1983). Storage material in astrocytes and satellite cells, but not in neurons, was mentioned by Gatfield (1965) and Challa et al (1983). Accumulation of 1-1.5 μπι granules with PAS positive, diastase resistant, Alcian blue negative, non-lipid, non-fluorescent material was noticed. Electron microscopic examination of brain fragments, obtained after death, revealed accumulation of multiple membrane-bound vesicles of low electron density in the cytoplasm of astrocytes. The origin of

82 these vesicles remains uncertain. The liver tissue of these patients showed distinctive intrahepatocytic accumulation of 0.2-1 urn membrane- bound material of low electron density. The ultrastructural features of these membrane-bound structures were granular material of moderate elec tron density speckled with minute granules of greater density. The che mical nature of the contents of these vacuoles remains unknown. Abundant mitochondria, proliferation of smooth endoplasmic reticulum, normal lysosomes and even normal peroxisomes were reported (2 patients) in liver tissue (Challa et al, 1983). Burton et al (1981) reported normal peroxi somes in one patient. Diffuse moderate tubular dilatation at the corti- co-medullary junction of the kidneys was reported by Gatfield et al, 1968. No cortical cysts were mentioned. No ultramicroscopic studies on kidney tissue were reported.

Further investigations of the biochemical and pathologic abnormalities of infants suffering from ΗΡΑ will be necessary to delineate whether or not this syndrome is identical to Zellweger syndrome. When the presence of peroxisomes in liver tissue is confirmed, then ΗΡΑ is a different disorder.

Trihydroxycoprostanic acidemia

The presence of trihydroxycoprostanic acid (THCA) in the duodenal fluid of two unrelated children with intrahepatic bile duct anomalies was first reported by Eyssen et al (1972). The presence of DHCA was suppo sed. The Zellweger-like appearance of their second patient was mention ed. Hanson et al (1975) studied two children from one family with cholesta sis and increased amounts of THCA in bile, stool, serum and urine. No results were reported about PA or plasmalogen metabolism or VLCFA le vels. The clinical data however, provided by Hanson et al (1975), sug gest a disorder different from CHR-S. Both children died of liver failu re, one at the age of 8 months and the other probably at the age of 23- 24 months. Such a clinical course is unusual in CHR-S. Other symptoms such as facial dysmorphic features, extreme hypotonia and poor sucking were not mentioned.

83 Parmentier et al (1979) described three infants with "coprostanic acide mia" with neurological, dysmorphic and cholestatic symptoms of variable degree. One of the patients had all the symptoms of the Zellweger syn drome. THCA was a major component of the bile acids in bile, serum, uri ne and faeces; also considerable amounts of DHCA were found in bile, se rum, urine and faeces. A small amount of varanic acid was identified in bile. The serum of their three patients contained important amounts of 3a, 7a, 12a-trihydroxy-5 0-C23 dicarboxylic bile acid, small amounts were detected in bile and urine. They noted that the severe clinical expres sion of "trihydroxy-coprostanic acidemia" completely corresponds with the CHR-S. Recent further investigation of their second patient (J.V. II) in our laboratory revealed increased concentration of PA in urine and serum and an increased serum level of the saturated VLCFA, strongly suggesting the biochemical diagnosis of CHR-S. Their patients J.V. I and J.V. II were sibs. Probably the three patients were suffering from the CHR-S.

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CHAPTER III

CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER:

CLINICAL SYMPTOMS AND RELEVANT LABORATORY FINDINGS IN 16 PATIENTS

11 2 1 L. Govaerts , L. Monnens , W. Tegelaers , F. Trijbels , and A. van Raay-Selten

University of Nijmegen, Department of Paediatrics, Nijmegen, The Netherlands. 2 University of Amsterdam, Academical Medical Centre, Department of Paediatrics, Amsterdam, The Netherlands

Eur J Pediatr 139: 125-128, 1982 ABSTRACT

The clinical features of 16 patients suffering from cerebro-hepatorenal syndrome are presented. Five of these children lived beyond 2 years. Four of them are still alive. The increase of plpecolic acid in serum and cerebrospinal fluid (CSF), the abnormality of the bile acids and the increased excretion of p-OH-phenyl lactate were a consistent finding. The concentration of plpecolic acid in urine was not always distinctly elevated. A loading test with DL-pipecolic acid was always abnormal.

KEY WORDS

Zellweger syndrome - Plpecolic acid - Peroxisomes - Cerebro-hepato-renal syndrome

98 INTRODUCTION

The autosomal recessively inherited cerebro-hepato-renal syndrome was first described in 1964 by Bowen et al. (Zellweger syndrome ) (ZS). The clinical features, as reported in the literature, are very consis tent including severe hypotonia, slow or absent feeding, profound psychomotor retardation and a characteristic facial appearance with high forehead, epicanthic folds, and large fontanels. The ZS is considered to be lethal; death occurs during the first year of life. Biochemically the ZS is characterized by disturbances in the metabolism of pipecolic acid (Trijbels et al. 1979) and of bile acids (Monnens et al. 1980). Increa sed excretion of p-OH-phenyl lactate was also noted (Chalmers et al. 1980). A defect in the electron transport chain was present in several organs tested. Probably all these biochemical abnormalities are due to deficiency of peroxisomes in the patients (Goldfischer et al. 1973). The purpose of this paper is to present the clinical data of 16 children seen in the Netherlands. Four of them are still alive and older than 2 years. Information will be given about pipecolic acid concentration in serum, urine and cerebrospinal fluid (CSF), about the urinary excretion of organic acids, and about the bile acids.

PATIENTS AND METHODS

Thirteen boys and three girls have been studied. Five of them are still alive. Ten of the infants died before the age of 8 months. The other child succombed at the age of 2 years and 9 months (see Table 1). D.B. and T.B., as well as J.F. and R.F. and R.v.d.L. and S.v.d.L. are sibs. Pipecolic acid was measured as described previously (Trijbels et al. 1979) with some modifications. The standard procedure for amino acid analysis was used with lithium citrate buffers. Ninhydrin solution was prepared as described but SnCl. was omitted from the solution to prevent precipitations in the reaction coil.

Bile acids were determined as described previously (Monnens et al.1980). Organic acids were measured by gaschromatography.

99 RESULTS

History of the Families The eleven sibs and both parents of the reported patients were complete ly normal. Consanguinity was present in one family. The parents were fourth cousins. Three cousins of B.v.M. died in early infancy. According to information obtained from the parents of B.v.M. autopsy findings re vealed polycystic kidneys.

Gestation and Delivery The gestational period was normal. All children were normally active in utero. The duration of pregnancy was normal. Caesarian section was per formed in T.v.M. because of prolonged labour. Vertex delivery occurred in the other children. Three infants needed active resuscitation after birth. Three children were small for dates; the remainder had a normal birth weight.

Clinical Features (Fig. 1,2) The principal features are summarized in Table 2. Hypertelorism is not mentioned as a clinical symptom. In 6 children considered to have hyper telorism, exact measurement of the inner canthal distance, outer canthal distance and interpupillary distance revealed normal values in 5 (Fein- gold et al. 1974). Hypotonia with hyporeflexia or areflexia was present in all patients except one: CD. was hypertonic with brisk reflexes. The older children become less hypotonic and are able to drink their milk. The nystagmus was horizontal, with a rotatory component in some of them. They did not follow light. In 4 children the pupillary constriction caused by light was present during the first months of life, and disappeared gradually. Fundoscopic examination was performed in 11 children. In 8 optic disc pallor was observed; in 4 retinal pigmentary changes were present. In 2 patients small opacities were present in the cornea. In 5 adequately tested patients a severe impairment of hearing could be demonstrated. Sensory examination revealed no abnormalities. A striking feature was the absence of hepatomegaly at birth. Three out of the four oldest children developed splenomagaly.

100 ig. 2. Note the peculiar Fig facies. I. Patient of 2 months; Fig. note the hypotonia. Table 1. Sex, age of death and cause of death of 16 Zellweger patients

Initials Sex Age of death Cause of death

E.N. F 5.5 months Pneumonia D.B. M 5 months Pneumonia T.B. M 7 months Pneumonia J.F. M 1 week Sudden death R.F. F 5 weeks Pneumonia B.v.M. M 6 months Sudden death J.S. M Still alive (5 years) CD. M Still alive (5 years) M.d.R. M 2 9/12 years Pneumonia T.Ba. M 5 weeks Sudden death B.R. M Still alive (3.5 years) T.v.M. F Still alive (2.5 years) R.v.d.L. M 1 month Pneumonia K.M. M 3 months Sudden death S.v.d.L. M 2 months Sudden death M.G. M Still alive (4 months)

102 Table 2. Clinical symptoms

Our Gatfield Thomas patients et al. et al.

Craniofacial features

High forehead 13/15 Frontal bossing 6/12 + Large fontanel 12/12 + Epicanthal folds 10/12 High arched palate 6/7 Shallow supra orbital ridge 10/10 +

Central nervous system

Hypotonia 15/16 + + Seizures 12/16 + Poor sucking 16/16 + + Psychomotor retardation 16/16 + + Normal sensory examination 7/7 + Severe hearing impairment 5/5

Eye symptoms

Nystagmus 10/11 + + Cataract, cloudy cornea 2/6 Clumping of pigment at the periphery 4/11 + Optic disc pallor 8/11 + +

Other symptoms

Hepatomegaly - at birth 0/15 - during the first year of life 15/16 + + Jaundice after 7 days 8/16 Failure to thrive 14/16 - + Cryptorchidism 6/13 Pes equinovarus 6/16 - Ulnar deviation of the hand(s) 5/16 - Simian crease(s) 11/16 - + Calcific stippling (patellae) 4/10

103 Laboratory Data Initially the liver function tests were always disturbed. As the child ren grew older, the levels of the transaminases mostly decreased. In Table 3 the concentration of pipecolic acid in urine, serum and CSF is presented. The pipecolic acid concentration in serum of controls was always below 10 pmol/l and was never detectable in CSF. In 9 out of 9 tested children we found abnormal bile acids as described earlier (Mon- nens et al. 1980). In 5 out of 5 patients the analysis of the organic acids showed an increased excretion of p-OH-phenyl-lactate (ranging from 120-400 ymol/mmol creatinine). The excretion of this compound by con trols does not exceed 100 ymol/mmol creatinine. No chromosomal aberra tions were detected in 8 out of 8 patients.

Special Neurologic Examinations All the electroencephalograms are considered to be abnormal with spike foci and sharp wave discharges. Echograms showed an enlargement of both ventricles in 5 out of 9 infants. In three infants a normal electromyo- gram was observed.

104 Table 3. The concentration of pipecolic acid In urine, serum and cerebrospinal fluid

Patient Age Urine Urine Serum CSF (umol/l) (pmol/g (umol/l) (ymol/l) creatinine)

E.N. 5 months 698 3357 D.B. 3 months 134 1819 - - T.B. 10 days-7 months 68-306 588-1779 24- 33 2.1 R.F. 0-1 month 143-168 1215 - - B.v.M. 2.5-5 months 81-390 1142-2138 43-384 0.6-2.9 J.S. 23/12-26/12 years 67-344 118-1357 462 6.6 CD. 6 months-4 /12 years 75- 88 476- 541 207 2.1 M.d.R. 3 months 39 86 109 - T.Ba. 3-5 weeks 143-158 1402-1663 21- 23 - B.R. 8 months-2 /12 years 6- 52 9- 220 122-321 2.3-4.7 T.v.M. 2.5-5 months 10- 28 79- 91 84-182 3.2 R.v.d.L. 4 weeks 311 3440 - - S.v.d.L. 2-6 days 39- 82 176- 386 5.1 9.6 M.G. 2 weeks-4 months 75-137 474- 811 10- 80 1.2 DISCUSSION

As yet death is considered to occur invariably during the first year of life in patients with ZS. However, 5 children in our series were older than 2 years and four are still alive. Two older patients described in the literature as hyperpipecolatemia are in our opinion also suffering from ZS (Gatfield et al. 1968; Thomas et al. 1975). Both children were extremely hypotonic, had feeding problems, psychomotor retardation, horizontal nystagmus, abnormal discs and retinal changes, and hepatome galy. Craniofacial abnormalities are not mentioned by Gatfield et al. (1968), frontal bossing was present in the patient reported by Thomas et al. (1975). Autopsy findings showing demyelination and gliosis of the brain and severe fibrosis of the liver in the patient of Gatfield (1968) are In agreement with the findings in ZS (Gilchrist et al. 1975, 1976). Recently 2 male siblings with hyperpipecolic acidemia were described by Burton et al. (1981). The clinical manifestations closely resembled those observed in the ZS. Electron microscopic studies, however, confir med the presence of hepatic peroxisomes. Peroxisomes could be difficult to identify and reduced in number in ZS (Carlson and Weinberg 1978; Pfeifer and Sandhage 1979). It would be very important to demonstrate a biochemical disturbance of the peroxisomes in the 2 male siblings and an analysis of the bile acids would also be required (Hanson et al. 1979; Monnens et al. 1980; Pedersen and Gustafsson 1980).

In 2 infants suffering from cerebro-hepato-renal syndrome, Chalmers et al. (1980) found an increased excretion of 4-hydroxy phenyl-lactic acid and dicarboxylic aciduria. In 5 patients out of 5 we could confirm the increased excretion of 4-hy droxy phenyl-lactic acid. In one of them dicarboxylic aciduria was also present. He was, however, receiving a MCT-diet (medium- chain triglyce- rides-diex), which could explain the dicarboxylic aciduria (Mortensen and Gregersen 1980). Pipecolic acid was found in all urine, serum and CSF samples tested (Danks et al. 1975). A wide range of the pipecolic acid excretion in urine was found in the 13 patients tested: 9-3440 pmol/g creatinine. The excretion also varied markedly within several in dividual patients, whereas others showed a rather constant excretion of pipecolic acid. It is doubtful whether all patients showed increased

106 excretion of plpecollc acid. The values given for patients M.d.R. and T.v.M. are in the normal range (exact values for controls are collected in our clinic). The same holds for patient B.R., who showed once (at the age of 2 /12 years) an excretion of only 9 ymol/g creatinine which can hardly be considered as increased. All serum samples of the patients in vestigated showed increased concentration of plpecollc acid, ranging from 5.1-462 pmol/l. The same is true for CSF. The concentration of pl pecollc acid varies from 0.6-6.6 ymol/l. Therefore, investigation of serum and CSF on pipecolic acid concentration is a better diagnostic criterion for screening of patients suspected from ZS. Loading tests with DL-pipecolic acid in 8 patients were always abnormal (Trijbels et al. 1979).

Bile acid analysis can confirm the diagnosis in suspected infants. The biochemical abnormalities as yet reported in ZS can probably be related to the absence of the peroxisomes or disturbance of the peroxisomal function (Goldfischer et al. 1973; Pfeifer and Sandhage 1979; Müller- Höcker et al. 1981; Trijbels et al. 1981).

Acknowledgements: We are grateful to Dr. H. Kenis, Maaseik, Dr. K. Kwik, Arnhem, Dr. F. Kleijnen, Ottersum, Dr. F. Nabben, 's-Hertogenbosch, Dr.R. Wilms, Doetinchem, Dr. E. Raven, Sittard, Dr. J. Rammeloo. Til- burg, Dr. J. Boeve, Enschede, Dr. F. Rive, Geleen, Dr. P. Barth, Amster dam, Dr. M. Sprangers, Tilburg, for permission to study their patients.

107 REFERENCES

Bowen Ρ, Lee CSN, Zellweger Η, Lindenberg R (1964) A familial syndrome of multiple congenital defects. Bull Johns Hopkins Hosp 114: 402-414

Burton BK, Reed SP, Remy WT (1981) Hyperpipecolic acidemia: Clinical and biochemical observations in two male siblings. J Pediatr 99: 729-734

Carlson BR, Weinberg AG (1978) Giant cell transformation. Cerebro- hepa- to-renal syndrome. Arch Pathol Lab Med 102: 596-599

Chalmers RA, Purkin Ρ, Watts RWE (1980) Screening for organic acidurias and amino acidopathies in newborns and children. J Inherited Metab Dis 3: 27-42

Danks DM, Tippett P, Adams C, Campbell Ρ (1975) Cerebro-hepato-renal syndrome of Zellweger. J Pediatr 86: 382-387

Feingold M, Bessert WH (1974) Normal values for selected physical para meters: An aid to syndrome delineation. Birth Defects 10: 1-14

Gatfield PD, Taller E, Hinton GG, Wallace AC, Abdelnour GM, Haust MD (1968) Hyperpipecolatemia: A new metabolic disorder associated with neu ropathy and hepatomegaly. A case study. Can Med Assoc J 99: 1215-1233

Gilchrist KW, Gilbert EF, Shahidi NT, Opitz JM (1975) The evaluation of infants with the Zellweger (cerebro-hepato-renal) syndrome. Clin Genet 7: 413-416

Gilchrist KW, Gilbert EF, Goldfarb S, Goll U, Spranger JW, Opitz JM (1976) Studies of malformation syndromes of man. XIB. The cerebro- hepa- to-renal syndrome of Zellweger: Comparative pathology. Eur J Pediatr 121: 99-118

Goldfischer S, Moore CL, Johnson AB, Spiro AJ, Valsarais MP, Wisniewski HK, Ritch RH, Norton WT, Rapin I, Gartner LU (1973) Peroxisomal and mi tochondrial defects in the cerebro-hepato-renal syndrome. Science 182: 62-64

Hanson RF, Williams GC, Grabowski G, Sharp HL (1979) Defects of bile acid synthesis in Zellweger's syndrome. Science 203: 1107-1108

Monnens L, Bakkeren J, Parmentier G. van Haelst U, Trijbels F, Eyssen H (1980) Disturbances in bile acid metabolism of infants with the Zellwe ger (cerebro-hepato-renal) syndrome. Eur J Pediatr 133: 31-35

Mortensen PB, Gregersen N (1980) Medium-chain triglyceride medication as a pitfall in the diagnosis of non-ketotic C--C.- dicarboxylic acidurias. Clin Chim Acta 103: 33-37

Müller-Höcker J, Bise К, Endres W, Hübner G (1981) Zur Morphologie und Diagnostik des Zellweger Syndroms. Virchows Arch (Pathol Anat) 393: 103-114

108 Pedersen JI, Gustafsson J (1980) Conversion of 3α, 7α, 12a-trihydroxy- 5ß -cholestanoic acid into cholic acid by rat liver peroxisomes. FEBS Lett 121: 345-348

Pfeifer U, Sandhage К (1979) Licht-und elektronenmikroskopische Leberbe funde beim cerebro-hepato-renalen Syndrom nach Zellweger (Peroxisomen- Defizienz). Virchows Arch (Pathol Anat) 384: 269-284

Thomas GH, Haslam RHA, Batshaw ML, Capute AJ, Neidengard L, Ransom JL (1975) Hyperpipecolic acidemia associated with hepatomegaly, mental re tardation, optic nerve dysplasia and progressive neurological disease. Clin Genet 8: 376-382

Trijbels JMF, Monnens LAH, Bakkeren JAJM, van Raay-Selten AHJ (1979) Biochemical studies in the cerebro-hepato-renal syndrome of Zellweger: A disturbance in the metabolism of pipecolic acid. J Inherited Metab Dis 2: 39-42

Trijbels JMF, Monnens LAH, Bakkeren JAJM, Willems JL, Sengers RCA (1981) Mitochondrial abnormalities in the cerebro-hepato-renal syndrome of Zellweger. In: Busch HFM, Jennekens FGI, Schölte HR (eds) Mitochondria and muscular diseases. Mefar bv. The Netherlands, pp 187-190

109

CHAPTER IV

A NEUROPHYSIOLOGICAL STUDY OF CHILDREN

WITH THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

L. Govaerts (1), E. Colon (2), J. Rotteveel (1), L. Monnens (1)

(1) Institute of Paediatrics (2) Institute of Neurology, Department of Clinical Neurophysiology Sint Radboudhospital, Nijmegen, The Netherlands.

Accepted for publication: Neuropaediatrics, 13th June, 1984 ABSTRACT

The aim of this study was to describe the EEC's, brainstem auditory evo ked potentials and somatosensory evoked potentials obtained in eleven children with the Zellweger syndrome. In six out of the eleven patients BAEP's and in five of them SSEP's were performed. Severe abnormalities could be demonstrated, reflecting diffuse cerebral dysfunction as well as long myelinated fiber tracts dysfunction. In four of six patients no response was obtained by BAEP's and in two the central conduction time was delayed. The specific (until 60 ms) and aspecific (after 60 ms) com plex of the SSEP'S was delayed or the potential could not be elecited. During sleep as well as wakefulness characteristic abnormalities of the EEC's were found in nine patients. These abnormalities consisted of con tinuous negative sharp waves and spikes at the vertex.

KEYWORDS

Cerebro-hepato-renal syndrome of Zellweger (CHR-S), evoked potentials (EP), EEC.

112 INTRODUCTION

The clinical features of the autosomal recessive inherited cerebro-hepa- to-renal syndrome (CHR-S) are very consistent. These symptoms are severe hypotonia, profound psychomotor retardation and a characteristic facial appearance with a high forehead, epicanthic folds and large fontanels (Fig. 1). Most of the patients develop tonic clonic seizures in early infancy. The increase of pipecolic acid in serum and cerebrospinal fluid, the abnormality of the bile acids, the increased excretion of p-OH-phenyllactate, the abnormal long chain fatty acid pattern in plas ma, and a decreased level of plasmalogens in tissues are consistent fin dings in this syndrome (Trijbels et al, 1979, Monnens et al, 1980, Go- vaerts et al, 1982, Heymans et al, 1983, Bakkeren et al, accepted 1984). Although the CHR-S is considered to be lethal during infancy, four of our patients are still alive and in excess of two years. Six patients demonstrated a severe hearing impairment. The pupillary constriction caused by light was present during the first months of life and disap peared gradually for four children. Eight out of eleven patients showed optic disc pallor, and four of them exhibited clumping of pigment at the retinal periphery. Sensory examination, as far as tested, revealed no abnormalities. The severe psychomotor retardation made detailed sensory examination impossible.

The patients explored their surroundings with their hands, and preferred smooth toys. Pain stimuli caused a reaction of crying and withdrawal. Ultramicroscopic and biochemical studies have revealed deficiency of peroxisomes and mitochondrial abnormalities (Goldfischer et al, 1973, Trijbels et al, 1981, Trijbels et al, 1983) in CHR-S. The aim of the study was to investigate the neurophysiologlcal aspects of CHR-S. To our knowledge this area has not yet been reported.

113 Fig. 1: Note the peculiar facies. SUBJECTS AND METHODS

Subjects The initials, age, and different studies as performed in our patients are shown in Table I. An EEG was available in all children. Only in children of recent years, after the technique was well established, were brainstem auditory evoked potentials (BAEP) and somato-sensory evoked potentials (SSEP) performed.

Table I: The patients, their age, and the different studies performed.

Patient Age EEG BAEP SSEP

E.N. 1/12 + - - 4/12 + - - D.B. 3/12 + - - T.B. 4/12 + - - 1/12 + - - B.v.M 2/12 + - - 4/12 + - - J.S. 4 2/12 + + + CD. 6/12 + - - 4 6/12 + + + T.Ba 1/12 + - - B.R. 7/12 + - - 1 9/12 - + - 2 + + + 26/12 - + - T.v.M 6/12 + + - 2 - + - M.G. 3/1 + + + 1 3/12 - - + W.G. 1 4/12 + + -

+ = performed I - - not performed / age in years

115 Methods The EEG-electrodes were positioned according to the international 10-20 system. The recordings were obtained during wakefulness and sleep with photic stimulation (on Elema Schönander mingograf). For the BAEP registration, Nicolet C.A.-1000 was used, with monoaurally click stimulation of 0,1 msec duration, 80 dB, 11,1/s, (± 2000 stimuli). Ag/AgCl discelectrodes preauricular (pA), with F as active and pAj or pA, as reference electrode were used to record the EP, bandpass between 30 and 3000 Hz (3 dB down, rolling off at 12 dB/octave). The SSEP's were obtained by means of 0,1 msec electrical stimulation, twitch level, on the right median nerve at the wrist. Ag-AgCl electrodes were positioned in between C- and P, with the reference electrodes on pA.. For the specific EP components (until 60 ms) 3 Hz regular stimula tion with a bandpass of 5 to 500 Hz (3 dB down, rolling off at 12 dB/oc tave) was used and for the aspecific (after 60 ms) EP components 1/3 Hz at random stimulation with bandpass of 1 to 250 Hz (3 dB down, rolling off at 12 dB/octave) was used. To test the reproducibility each trial was performed twice.

Sample frequency for the specific components was 2560 Hz and for the aspecific components 512 Hz. The resistance of the electrodes was always kept well below 3000 Ohn. For the specific components at least 1000 tra ces and for the aspecific components at least 100 traces were averaged in each trial. Latencies of all EP's were determined by measurements with an electronic cursor.

RESULTS

1) EEC's All the electroencephalograms except one (W.G.), were considered to be abnormal with spike foci and sharp wave discharges (Table II). Discharges at the vertex are seen in nine of eleven children, consisting out of frequent negative spikes or sharp waves during sleep as well as during wakefulness (Fig. 2). No photic driving could be obtained in any of the children (16 EEC's).

116 Table II: EEG findings of the patients at different ages.

Patient Age Abnormal Diffuse Epileptiform Photic years background epileptiform configuration driving activity activity above the vertex

E.N. 1/12 + + 4/12 + + + - D.B. 3/12 + + + - T.B. 4/12 - + + - 1/12 + + + - B.v.M. 2/12 - + + - 4/12 + + + - J.S. 4 2/12 + + + - CD. 6/12 + + - - 4 6/12 + + - - T.Ba. 1/12 + + + - B.R. 7/12 + + - - 2 + + + - T.v.m. 6/12 + + + M.G. 3/12 - + + W.G. 1 4/12 - - - -

117 BORN K-I'T* V 100 uV/e F· 70 Hl TC- 0.6 ·. SPEED- 30 к/>. EFu 13-··7· rp2 - Τ«

Τ4 - 01

Γρ1 - ТЭ \—-\

Τ4 - C4

с. -сзчЛ^Л^-^ ^^ ^^^)/ Д^л/ чА^,/\К^'-

Sit. 'Sb івс

Fig. 2a: An example of EEG findings in children with the Zellweger syndrome. The child is awake. Note the vertex sharp waves and spikes, indicated by an arrow.

118 •ч_

Fig. 2b: An example of EEG findings in children with the Zellweger syndrome. The child is in a quiet sleep. Note the vertex sharp waves and spikes, indicated by an arrow.

119 2) BAEP's - SSEP's The findings of BAEP's and SSEP's are summarized in Table III. An exam ple of a normal and abnormal BAEP is given in Fig. 3 and of a normal and abnormal SSEP is given in Fig. 4. In most of them no response was obtai ned by BAEP (Table III). In two patients the central conduction time (I-V latency) was delayed (CD. and T.v.M.). In five children SSEP's could be applied. The SSEP components after 60 ms were in all patients limited to C,1 area which is in contrast with the findings in normal children (see Fig. 4). There seems to be a interhemispheric propagation disturbance. The specific complex was delayed in four of five registrations indicating a propagation defect, between the wrist and the cortex, (Fig. 3, Table III). Sensory and motor nerve con duction velocity was normal (B.R., T.v.M., W.G.).

3) echo encephalography - CT scan Every patient, except one (E.N.)» had a slightly increased T/D index (Krijgsman, 1977), two (B.v.M. and B.R.) out of three patients showed an increasing T/D index during follow up suggesting increased central atro phy. The defective myelination could not be demonstrated by CAT-scan density abnormalities in CD. (6/12 years old), B.R. (7/12), T.v.M (4/12) and W.G. (1 4/12). Vermis aplasia was noted in CD.

120 Table III: BAEP's - SSEP's.

Patient age B.A .E.P. S.S.E.P.

(years) right left delay specific delay aspecific interhemipheric complex complex propagation defect

J.S. 4 2/12 - - ++ - *

CD. 4 6/12 CCT. CCT. + -(?) * delayed delayed

B.R. 1 9/12 - - N.P. N.P. N.P. 2 - - + + * 2 6/12 - - N.P. N.P. N.P. T.v.M. 6/12 - CCT. N.P. N.P. N.P. delayed

2 - - 'N.P. N.P. N.P. M.G. 3/12 - - - - - 1 9/12 - ? + + * W.G. 1 4/12 - N.P. N.P. N.P.

CCT. » central conduction time » not elicitable + = slightly delayed (p <0,05) ++ = severe delayed (p <0,01) specific complex between median nerve at the wrist and cortex N.P. = not performed * - present Specific complex, aspecific complex and C.C.T.: according to Colon et al, 1983. I II III IV ν t t t t t

Normal

R+R

CHR-S

R + R

Stimulus

Fig. 3: Example of a normal BAEP (upper three traces) and of a BAEP from one of the CHR-S patients, after right ear stimulation. I until V are the different components which can only be seen in the normal traces. R = right preauricular derivation (other on F ). L = left preauricular derivation (other on F J. The BAEP was elicited twice which is represented at the lower traces (R+R).

122 4»«#*»S* 44JTH- ì\

) Normal

StimuluIsH ti P45 PIM P200 F»300

ЦІУ|ЦГ> АУК^^ r^^^^^j*, >CHR-S

сз'ír^Vі q ^ л, 100 ms -< »• Stimulus ffcF P45 PICO Р2Ш P30O

Fig. A: Example of a normal (upper three traces) and an abnormal SSEP derived from С' С ' and С ' (pA reference) after median nerve stimulation of a Zellweger patient (lower three traces). Some components are indicated. Note the very restricted locali zation of the components after 60 ms in the patient.

123 DISCUSSION

The diagnosis of all these patients was based on clinical features and laboratory findings according to Govaerts et al, 1982. Symptoms of the visual system were already present in the neonatal pe riod. These consisted of a horizontal nystagmus (ten of eleven patients) and inability to follow light. In six children who survived to six months of age, the pupillary constriction due to light, which was pre sent during the first months of life, disappeared gradually. In six adequately tested patients a severe impairment of hearing was de monstrated. There was no recognizable reaction by free field audiometry up to 70 dB. An electro-cochleography was not performed because of the high risk of anesthesia in these infants. Profound psychomotor retarda tion was always present. Our oldest patient is six years old and unable to sit without support. Tactile contact is observed. Affected indivi duals smile if they feel comfortable. Sudden death occurred in four of the eleven children and three died due to pneumonia. Four children are alive and older than two years.

The neuropathologic findings consisted of cortical dysplasia (islands of ectopic neurones), dysmyelination throughout the CNS and the spinal cord, and accumulation of neutral fat in histiocytes in the white and gray matter of cerebrum, cerebellum and in the basal ganglia (Passarge et al. 1967, Liu et al, 1976, Agamanolis et al, 1979, Müller-Höcker et al, 1981, Della Giustina et al, 1981). These symptoms are considered characteristic of this syndrome (Agamanolis et al, 1979). The severe hearing impairment prompted us to record the BAEP's. Except in CD. and T.v.M. these were not elicitable. The SSEP's were elicitable but only with a delayed conduction. Nearly every EEG showed abnormal background activity and epileptiform confi guration. These data suggest a severe diffuse cerebral dysfunction, especially of the white matter in the brain. The SSEP's point towards a severe propagation dysfunction over the mye linated pathways. The electrocerebral information from the sense organs (peripheral nerve) was not adequately transported through the central nervous system. We are dealing, therefore, with a form of leukodystrophy

124 (Gloor et al, 1968, Starr et al, 1976, Colon et al, 1979, Gracco et al, 1980, Markand et al, 1982). The CAT-scan however could not reveal the deficient myelination. The clinical neurophyslological studies were more informative. This leukodystrophy is severe since some BAEP's are delayed while SSEP conduction is seriously disturbed, probably of the interhemi- spheral fibers (the interhemispheric SSEP propagation in all examined patients is disturbed). Another interesting finding was the fact that almost all patients with CHR-S show spike or sharp waves above the vertex in the EEG. Sharp vertex activity is described in stage II sleep and as an arousal reac tion after acoustic stimulation (Samson et al, 1961).In our group the EEG phenomena are seen continuously in sleep as well as during wakeful ness. The mechanism producing such midline spikes is uncertain (Nelson et al, 1983). In other forms of leukodystrophy different EEG abnormalities are des cribed. In the metachromatic type the EEG initially remains normal. Later the EEG contains beta activity in the anterior or posterior re gions (Pampiglione, 1968). In Krabbes disease, Pampiglione (1968) found more profound abnormalities, even irregular slow activity with sharp waves and occasional spikes. None of these EEG patterns are comparable which the abnormalities seen in our patient group. Therefore, it is possible that negative spiking (or sharp waves) In the vertex region combined with these EP abnormalities is rather characteristic for the CHR-S.

SUMMARY

EEC's, BAEP's and SSEP's of children with the CHR-S of Zellweger are described. Diffuse cerebral dysfunction could be demonstrated. Rather characteristic abnormalities of the EEC's were found.

125 REFERENCES

Agamanolis, D., Robinson, H., Timmons, D.: Cerebro-hepato-renal syndro me, report of a case with histochemical and ultrastructural observa tions. J. Neuropath, exp. Neurol., 1976, 35: 226-246.

Agamanolis, D., Pâtre, S.: Glygogen accumulation in the central nervous system in the cerebro-hepato-renal syndrome. Journal of the Neurological Sciences, 1979, 41: 325-342.

Bakkeren, J., Monnens, L., Trijbels, J., Maas, J.: Serum very long chain fatty acid pattern in Zellweger syndrome. Clin Chim Acta, 1984, 138: 325-331

Colon, E., Renier, W., Cabreéis, F.: Propagation defects in somato sen sory evoked responses in children. In: H. Lechner and A. Aranilar (Eds): EEG and clinical neurophysiology, Elsevier, North Holland, 1979, pp 306- 313.

Colon, E., Visser, S., de Weerd, J., Zonneveldt, Α.: Evoked potential manual. Martinus Nijhoff Publishers, 1983, pp. 121-128 and 268-270.

Gracco, J., Bosch, V., Gracco, R. : Cerebral and spinal somatosensory evoked potentials in children with CNS degenerative disease. EEG and Clin. Neurophys., 1980, 49: 437-445.

Danks, D., Tippett, P., Adams, C, Campbell, P.: Cerebro-hepato-renal syndrome of Zellweger. J. Pediatr, 1975, 86: 382-387.

Della Giustina, E., Goffinet, Α., Landrieu, P., Lyon, G.: A Golgi study of the brain malformation in Zellweger's cerebro-hepato-renal disease. Acta Neuropathol. (Beri), 1981, 55: 23-28.

Gloor, P., Kalabay, D., Giard, M. : The electroencephalogram in diffuse encephalopathies: electroencephalograhlc correlates of grey and white matter. Brain, 1968, 91: 779-802.

Goldfischer, S., Moore, С, Johnson, Α., Spiro, Α., Valsarais, Μ.: Per oxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science, 1973, 182: 62-64.

Govaerts, L., Monnens, L., Tegelaers, W., Trijbels, J., and van Raay- Selten, Α.: Cerebro-hepato-renal syndrome of Zellweger: clinical symp toms and relevant laboratory findings in 16 patients. Eur. J. Pediatr., 1982, 139: 125-128.

Krijgsman, J.: Echo-encephalographic measurements (Α-scan) of the late ral ventricles in children; its reliability. Clin. Neurol. Neurosurg., 1977, 80: 1-9.

Liu, H., Bangaru, В., Kidd, J., Boggs, J.: Neuropathological considera tions in cerebro-hepato-renal syndrome (Zellweger's syndrome). Acta Neu ropath. (Beri.), 1976, 34: 115-123.

126 Markand, 0., Garg, В., De Meyer, W., Warren, C. and Worth, R.: Brainstem auditory, visual and somatosensory evoked potentials in leukodystro phies. EEG and Clin. Neurophys., 19Θ2, 54: 39-48.

Monnens, L.,Bakkeren, J., Parmentier, G., van Haelst, U., Trijbels, F., Eyssen., H.: Disturbances in bile acid metabolism of infants with the Zellweger (cerebro-hepato-renal) syndrome. Eur. J. Pediatr., 1980, 133: 31-35.

Müller-Höcker, J., Bise, К., Endres, W., Hübner, G.: Zur Morphologie und Diagnostik des Zellweger Syndroms. Virchows Arch. (Pathol, anat.), 1981, 93: 103-114.

Nelson, K., Brenner, R., de la Paz, D.: Midline spikes: EEG and clinical features. Arch. Neurol., 1983, 40: 473-476.

Ochs, R., Markand, 0., De Meyer, W.: Brainstem auditory evoked responses in leukodystrophies. Neurology, 1979, 23: 1089-1093.

Pampiglione, G.: Some inborn metabolic disorders affecting cerebral electrogenesis. In Holt, K.S. and Coffrey, V.P.(Eds): Some recent advan ces in inborn errors of metabolism, Edinburgh, Livingstone, 1968, ρ 89-91.

Passarge, E., McAdams, Α.: Cerebro-hepato-renal syndrome. J. Pediatr., 1967, 71: 691-702.

Samson, D., Samson, M.: Transduction électroencephalographique des reac tions d'éveil chez l'enfant. Exc. Med. Int. Congr. Series, 1961, 37: 60-61.

Starr, Α., Hamilton, Α.: Correlation between confirmed sited of neurolo gical lesions and abnormalities of far-field brainstem responses. EEG and Clin. Neurophys., 1976, 41: 595-608.

Trijbels, J., Monnens, L., Bakkeren, J., van Raay-Selten, Α.: Biochemi cal studies in the cerebro-hepato-renal syndrome of Zellweger: a distur bance in the metabolism of pipecolic acid. J. Inher. Metab. Dis., 1979, 2: 39-42.

Trijbels, J., Monnens, L., Bakkeren, J., Willems, H., Sengers, R.: Mito chondrial abnormalities in the cerebro-hepato-renal syndrome of Zellwe ger. In: Busch, H., Jennekens, F., Schölte, H. (eds). Mitochondria and muscular diseases. Mefar b.v.. The Netherlands, 1981, pp 187-190.

Trijbels, F., Berden, J., Monnens, L., Willems, J., Janssen, Α., Schut gens, R. and van den Broek-van Essen, M.: Biochemical studies in the liver and muscle of patients with Zellweger syndrome. Pediatr. Res., 1983, 17: 514-517.

127

CHAPTER V

DISTURBED ADRENOCORTICAL FUNCTION IN CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

L. Govaerts, L. Monnens, T. Melis, F. Trijbels

University of Nijmegen, Department of Paediatrics, Nijmegen, The Netherlands

Accepted for publication: Eur J Pedlatr, 16th May 1984 ABSTRACT

An ACTH stimulation test was performed in six patients suffering from the cerebro-hepato-renal syndrome of Zellweger. In contrast to controls, no rise in Cortisol was observed. None of these patients showed clinical symptoms of adrenal insufficiency. The sudden death, which occurs in this syndrome, can probably be explained by an impaired stress reaction. In stress situations, such as respiratory infection, corticosteroids should be administered to these patients. A striking resemblance exists between the Zellweger syndrome and the neonatal form of adrenoleukodystrophy.

KEY WORDS cerebro-hepato-renal syndrome (CHR-S), Zellweger syndrome, adrenal func tion, adrenoleukodystrophy (ALD)

130 INTRODUCTION

The autosomal recessively inherited cerebro-hepato-renal syndrome of Zellweger (CHR-S) was first described in 1964 by Bowen et al (5). It is considered to be lethal. Death may occur in early infancy (11,13). The known biochemical abnormalities include: increased concentration of pipecolic acid in serum, urine and cerebrospinal fluid, abnormal bile acid pattern in duodenal fluid, serum and urine, increased excretion of p-OH-phenyllactate in urine (11), disturbances in the electron transport chain (8,24), deficiency of plasmalogens in tissues (12) and an eleva tion of the saturated very long chain fatty acid content in serum (6,2), brain white matter, adrenal gland and cultured fibroblasts (6). The va rious biochemical abnormalities of CHR-S can probably be related to the absence of peroxisomes or disturbance of the peroxisomal function (24). In adrenoleukodystrophy (ALD) similar abnormalities of the very long chain fatty acids were described (16). An elevated content of C26 fatty acids was reported in cultured skin fibroblasts (16), in plasma (17), in adrenal gland (23), in leukocytes (15) and in cultured amniocytes and fetal adrenal gland (18).

The neonatal form of ALD, an autosomal recessive disease, has in addi tion a striking clinical resemblance to CHR-S (4,6). Absence of peroxi somes in the neonatal ALD was reported by Partin and McAdams (19). As the adrenocortical function is disturbed in ALD it was tempting to investigate this function in CHR-S. It is the aim of this report to pre sent these results.

131 PATIENTS AND METHODS

Patients Six patients suffering from the CHR-S were investigated. None of these infants had clinical adrenal insufficiency. Hyperpigmentation of the skin was absent. No electrolyte disturbances were present. The diagnosis Zellweger syndrome was based on the clinical features and biochemical abnormalities (11). Informed consent was obtained from the parents.

Methods The ACTH stimulation test was performed at 9.00 a.m. Synacthen (0.150 mg/m2) was given intravenously and blood samples were collected 0, 30 and 60 min after injection. The Cortisol concentration was determined by radio immuno assay (Clinical Assays, coated tubes).

RESULTS

The Cortisol levels 0, 30 and 60 min after injection of Synacthen into six patients and three controls are given in Table I. No increase in the Cortisol level was observed in CHR-S patients while an adequate reaction of Cortisol was seen in the controls (3).

132 Table I: Cortisol levels (umol/l) 0, 30 and 60 min after intravenous administration of 0.150 mg/m2 Synacthen.

Subjects Time

0 30 60

Patient CD. 0.36 0.30 0.32 B.R. 0.46 0.38 T.v.M. 0.39 0.40 0.41 M.G. 0.36 0.41 0.38 т.е. 0.44 0.50 0.50 J.V. 0.48 0.57 0.56

Controls: 1 0.66 1.06 1.28 2 0.56 1.00 1.18 3 0.34 0.62 0.88

Normal basal Cortisol level at 9.00 a.m.: 0.25-0.70 nmol/l.

133 DISCUSSION

An inadequate response to ACTH injection was observed in all patients investigated, indicating a primary (partial) adrenocortical insufficien cy. Sudden death occurred in 7 out of 17 patients known to us, while 6 of them expired from a pneumonia. Adrenal failure could have been an im portant contributing factor in their death. We recommend the use of cor ticosteroids in stress situations. The same abnormality of adrenocorti cal function is observed in the different forms of ALD (22,4). The pathogenesis of this adrenal insufficiency remains unclear. In his tologic studies striated adreno-cortical cells of the reticularis-inner fasciculata layer were described in patients with CHR-S and in ALD. The se cells contained lamellae and lamellar-lipid profiles of very long chain fatty acids-cholesterol esters (10). The saturated very long chain fatty acids could be cytotoxic and responsable for the adrenocortical dysfunction (20). Measurements of fluorescence polarisation demonstrated a highly significant increase in the microviscosity of erythrocyte mem branes in ALD (14). Experimental data in animals point to a correlation between membrane microviscosity and the number of hormone receptors in target tissues. Therefore Knazek et al (14) proposed that the decrease in responsiveness to trophic hormone is secondary to change in the mem brane microviscosity of the adrenal gland. Cells of the guinea pig adre nal cortex contain numerous small peroxisomes. These peroxisomes possess a high activity for long chain fatty acid g-oxidation. The acetyl-CoA, generated as a result of the peroxisomal g-oxidation, could be directed to the synthesis of cholesterol, the precursor of the adrenal steroids (21). Especially after stimulation by ACTH, the amount of acetyl-CoA could be rate-limiting, when the peroxisomal contribution is absent, as in CHR-S. Several authors have already mentioned similarities between the neonatal ALD and the CHR-S. Both have the same mode of inheritance and the same clinical features. Patients can succumb at early age (4). Pathologic studies reveal the same abnormalities: Agamanolis et al (1) noticed a similarity between the cytoplasmic inclusions (multilamellar bodies) of macrophages, Oligodendroglia! cells and astrocytes of the ce rebrum of Zellweger patients and the inclusions seen in the adrenals,

134 Schwann cells, testis and brain of cases with ALD. The lamellar-lipid profiles, considered to be diagnostic for neonatal ALD were found by Goldfischer et al (10), in the adrenal glands of eight patients with Zellweger syndrome. Ocular histopathologic studies demonstrated that the CHR-S and neonatal ALD are similar with regard to ocular abnormalities (7). In addition increased levels of saturated very long chain fatty acids in fibroblasts (6), serum (6,2), and several tissues (6,10,7) were reported. In CHR-S the adrenal cortical insufficiency is a new feature, already known in ALD. The question arises whether these disorders are different or not. The absence of hepatic peroxisomes in the neonatal form of ALD recently described by Partin and McAdams (19) supports the hypothesis that both diseases have a common cause. To answer this ques tion other biochemical studies are recommended on the neonatal ALD, es pecially analysis of the bile acids, the pipecolic acid level in serum, cerebrospinal fluid and urine and of plasmalogens.

Acknowledgements: We are grateful to Prof.Dr.E. Eggermont and Dr.J. Jaeken from the Department of Paediatrics of the Catholic University of Leuven, Belgium, for permission and cooperation in studying their pa tients (T.C. and J.V.).

135 REFERENCES

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2. Bakkeren J, Monnens L, Trijbels J, Maas J (1984) Serum very long chain fatty acid pattern in Zellweger syndrome. Clin Chim Acta 138: 325-331

3. Bertrand J, Rappaport R, Sizonenko Ρ (1982) Endocrinologie pédiatri- que, physiologie, Physiopathologie, clinique. Payot Lausanne edn. Paris, pp 690-691

3. Benke P, Reyes Ρ, Parker J (1981) New form of adrenoleukodystrophy. Hum Genet 58: 204-208

4. Bowen P, Lee C, Zellweger H, Lindenberg R (1964) A familial syndrome of multiple congenital defects. Bull Johns Hopkins Hosp 114: 402-414

5. Brown F III , McAdams A, Cummins J, Konkol R, Singh I, Moser A, Moser H (1982) Cerebro-hepato-renal (Zellweger) syndrome and neonatal adrenoleukodystrophy: similarities in phenotype and accumulation of very long chain fatty acids. Johns Hopkins Med J 151: 344-361

6. Cohen S, Brown F III, Martyn L, Moser H, Chen W, Kistenmacher M, Punnett H, Grover W, de la Cruz Ζ, Chan Ν, Green W (1983) Ocular histopathologic and biochemical studies of the cerebrohepatorenal syndrome (Zellweger's syndrome) and Its relationship to neonatal adrenoleukodystrophy. Am J Ophthalmol 96: 488-501

7. Goldfischer S, Moore С, Johnson A, Spiro A, Valsamis M, Wisniewski H, Ritch R, Norton W, Rapin I, Gartner L (1973) Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science 182: 62-64

8. Goldfischer S (1979) Peroxisomes in disease. J Histochem Cytochem 27: 1371-1373

9. Goldfischer S, Powers J, Johnson A, Axe S, Brown F, Moser H (1983) Striated adrenocortical cells in cerebro-hepato-renal (Zellweger) syndrome. Virchows Arch (Pathol Anat) 401: 355-361

10. Govaerts L, Monnens L, Tegelaers W, Trijbels J, and van Raay-Selten A (1982) Cerebro-hepato-renal syndrome of Zellweger: clinical symp toms and relevant laboratory findings in 16 patients. Eur J Pediatr 139: 125-128

11. Heymans H, Schutgens R, Tan R, van den Bosch H, Borst Ρ (1983) Seve re plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature 306: 69-70

136 12. Kelley R (1983) Review: The cerebrohepatorenal syndrome of Zellwe ger, morphologic and metabolic aspects. Am J Med Gen 16: 503-517

13. Knazek R, Rizzo W, Schulman J, Dave J (1983) Membrane microviscosity is increased in the erythrocytes of patients with adrenoleukodystrophy and adrenomyeloneuropathy. J Clin Invest 72: 245-248

14. Molzer B, Bernheimer H, Heller R, Toifl K, Vetterlien M (1982) Detection of adrenoleukodystrophy by increased C,, fatty acid levels in leukocytes. Clin Chim Acta 125: 299-305 :

15. Moser H, Moser A, Kawamura Ν, Murphy J, Suzuki K. Schaumburg Η, Kishimoto Y (1980) Adrenoleukodystrophy: elevated C., fatty acid in cultured skin fibroblasts. Ann Neurol 7: 542-549

16. Moser H, Moser A, Frayer К, Chen W, Schulman J, O'Neill В, Kishimoto Y (1981) Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids. Neurology, 31: 1241-1249

17. Moser H, Moser A, Powers J, Nitowsky H, Schaumburg H, Norum R, Migeon В (1982) The prenatal diagnosis of adrenoleukodystrophy. De monstration of increased hexacosanoic acid levels in cultured amniocytes and fetal adrenal gland. Pediatr Res 16: 172-175

18. Partin J, McAdams A (1983) Absence of hepatic peroxisomes in neona tal onset adrenoleukodystrophy. Pediatr Res 17: 294A

19. Powers J, Schaumburg H, Johnson A, Raine С (1980) A correlative stu dy of the adrenal cortex in adrenoleukodystrophy - evidence for a fatal intoxication with very long chain saturated fatty acids. In vest Cell Pathol 3: 353-376

20. Russo J, Black V (1982) Hormone-dependent changes in peroxisomal en zyme activity In guinea pig adrenal. J Biol Chem 257: 3883-3889

21. Schaumburg H, Powers J, Raine С, Suzuki К, Richardson E (1975) Adre noleukodystrophy. A clinical and pathological study in 17 cases. Arch Neurol 32: 577-591

22. Singh I, Moser H, Moser A, Kishimoto Y (1981) Adrenoleukodystrophy: impaired oxidation of long chain fatty acids in cultured fibroblasts and adrenal cortex. Biochem Biophys Res Commun 102: 1223-1229

23. Trijbels J, Berden J, Monnens L, Willems J, Janssen A, Schutgens R, van den Broek-van Essen M (1983) Biochemical studies in the liver and muscle of patients with Zellweger syndrome. Pediatr Res 17: 514-517

137

CHAPTER VI

DISTURBED VERY LONG CHAIN FATTY ACID PATTERN IN FIBROBLASTS OF ZELLWEGER PATIENTS

L. Govaerts, J. Bakkeren, L. Monnens, J.Maas, F. Trijbels, W. Kleijer*

Department of Paediatrics, St.Radboudhospital, Nijmegen, The Netherlands * Department of Clinical Genetics, University Hospital, Rotterdam

Accepted for publication: J Inher Metab Dis, 30rd May 1984 SUMMARY

The very long chain fatty acids in cultured fibroblasts from six pa tients with the cerebro-hepato-renal syndrome of Zellweger, from six of their parents, from three controls, and also in three amniotic fluid control cell lines were analyzed by gas chromatography. Increased concentrations of hexacosanoic acid (C26:0) were consistently found in the Zellweger syndrome. Also the ratios C26:0/C22:0, C25:0/ C22:0, and C24:0/C22:0 were elevated. The very long chain fatty acid le vels and ratios in fibroblasts from the patients' parents were within the normal range. Findings in amniotic fluid cell lines indicate the possibility of antenatal diagnosis for the Zellweger syndrome. The simi larities between neonatal adrenoleukodystrophy and Zellweger syndrome suggest the applicability of this technique also in neonatal adrenoleu kodystrophy.

140 INTRODUCTION

The autosomal recessively inherited cerebro-hepato-renal syndrome of Zellweger (CHR-S) (McKusick * 2Ш0) is characterized by dysmorphic features, several biochemical abnormalities and in 90% of the cases death during infancy (Govaerts et al 1982). The absence of peroxisomes in liver tissue and in proximal tubules of the kidney (Goldfischer, 1973) has been brought into relation with the reported biochemical ab normalities. Such a relationship has been strongly suggested for the abnormal bile acid metabolism (Pedersen and Gustafsson, 1980), the de fect in pipecolic acid metabolism (Trijbels et al, 1979 and 1981), the disturbance in the electron transport chain (Trijbels et al, 1983), the deficiency of plasmalogens (Heymans et al, 1983), and the increased le vels of very long chain fatty acids (C24-C26) in serum (Bakkeren et al, 1984), in brain white matter, adrenal gland and cultured fibroblasts (Brown et al, 1982).

In the X-linked adrenoleukodystrophy (ALD) of childhood (McKusick * 30010), a disorder involving predominantly the white matter of the central nervous system and the adrenal cortex (Schaumburg et al, 1975), similar increased very long chain fatty acid levels were found in plasma (Moser et al, 1981) and in cultured fibroblasts (Kawamura et al, 1978, Moser H et al, 1980A). By determining the very long chain fatty acid levels and ratios in plasma and fibroblasts, identification of female carriers of childhood ALD is possible in 93% of women who are obligate heterozygotes (Tönshoff et al, 1982, Moser et al, 1983). Moser et al (1982) described the possibility of prenatal diagnosis of childhood ALD by application of this method on amniotic fluid cells.

The neonatal ALD (McKusick 20237), an autosomal recessive disease, has a striking clinical resemblance to CHR-S (Benke et al, 1981, Brown et al, 1982). The accumulation of very long chain fatty acids was reported in brain tissue (Manz et al, 1980), in plasma, adrenal gland and in skin fibroblasts (Brown et al, 1982). Absence of peroxisomes in neonatal ALD was reported by Partin and McAdams (1983) in liver tissue. The present study on fibroblasts from Zellweger patients and their pa rents, and on control fibroblasts and amniotic fluid cells, was perfor med to explore the possibilities of antenatal diagnosis and heterozygote detection for this disease.

141 PATIENTS AND METHODS

Patients Six children suffering from the CHR-S were investigated. Six parents of Zellweger patients were included in the study. The clinical and relevant biochemical abnormalities have been described earlier (Govaerts et al, 1982). Patients M.S. and N.v.d.E. fulfilled also all diagnostic crite ria.

Methods Fibroblasts were obtained by skin biopsy and grown in a 5% carbon dioxi de atmosphere at 37°C in TC-199 with Earle's salts and 20% fetal calf serum (FCS) (or 10% fetal plus 10% newborn calf serum in later passa ges). Amniotic fluid cells were obtained by amniocentesis performed in pregnancies not at risk for CHR-S or ALD. Cells were cultured in the same way as the fibroblasts. The cells were harvested with 0.25% trypsin after having reached confluence. After washing twice with phosphate-buf fered saline (PBS) plus 10% FCS and twice with cold PBS, 5 χ 10 cells were disrupted by freezing and thawing once. Heptadecanoic acid (C17:0) (0.2 mol) was added to the homogenate as an internal standard. Lipids were extracted with chloroform/methanol (1:1, v/v), transmethylated with 1.5 mol/1 HCl in methanol at 750C for 16 h, and the methyl esters ex tracted into hexane and analyzed by capillary gas chromatography as described previously (Bakkeren et al, 1984). All peaks corresponding to fatty acids were identified by their retention times and quantitated by integration. Results were expressed as percentages of total fatty acids (C14:0 to C26:0). The identity was confirmed by gas chromatography-mass spectrometry (GC-MS). For GC-MS a 5995 A instrument (Hewlett Packard, Palo Alto, CA, USA) was used, equipped with a 25-m open-tubular fused silica column with chemically bounded phase Sil 8 CB (Chrompack, Middelburg, The Netherlands). The carrier gas was helium. The oven temperature was at first 44° C, after 1 min raised to 180° С and then programmed to 290oC at 8° С per min. Chromatography was carried out in the splitless mode.

142 The ion source temperature was 180° C, the transferline temperature was 300° С The electron energy was 70 eV. Electron impact mass spectra were recorded. Selected ion monitoring was used for identifying the different very long chain fatty acids.

RESULTS

The levels and ratios of the very long chain fatty acids in cultured fi broblasts from Zellweger patients and parents, and from controls, are given in Table I. A striking elevation of the very long chain fatty acids, especially of hexacosanoic acid (C26:0) (about 10 times), was found in the fibroblasts from the Zellweger patients. The proportion of C22:0 in CHR-S fibroblasts was decreased till 50% of the normal level, similar to the results of Rizzo et al (1984) in childhood ALD. If the levels of C26:0, C25:0 and C24:0 are related to those of C22:0, in all patients increased ratios are found.

In a series of six parents of Zellweger patients the levels and ratios of very long chain fatty acids in fibroblasts were in the same range as in fibroblasts from normal individuals (Table I). Table II lists the results of the determination of the very long chain fatty acids in cultured amniotic fluid cells from three pregnancies not at risk for Zellweger syndrome or ALD. The levels of C24:0 and C25:0 were similar to those in normal fibroblasts, whereas the C26:0 level was somewhat higher. Also the ratios of C24:0, C25:0 and C26:0 resp. as com pared with C22:0, appear somewhat higher than was found for normal fi broblasts. However in fibroblasts from Zellweger patients, especially the C26:0/C22:0 ratio, was still considerably higher than in normal am niotic fluid cells.

143 Table I: Very long chain fatty acids and ratios of C24:0/C22:0, C25:0/C22:0, and C26:0/C22:0 in cultured fibroblasts. *

C22:0 C24:0 C25:0 C26:0 C24:0/C22:0 C25:0/C22:0 C26:0/C22:0

Zellweger patients

B.v.M. 0.79* 2.1 0.24 0.44 2.62 0.30 0.55 B.R. 0.74 1.7 0.13 0.31 2.31 0.17 0.42 T.B. 0.97 2.0 0.16 0.47 2.08 0.16 0.49 N.v.d.E. 0.80 1.5 0.12 0.33 1.86 0.15 0.42 M.S. 0.51 1.6 0.12 0.37 3.02 0.24 0.72 J.S. 0.97 1.7 0.17 0.33 1.81 0.18 0.34

Range 0.51-0.97 1.5-2.1 0.12-0.24 0.31-0.47 1.81-3.02 0.15-0.30 0.34-0.72

Parents of Zellweger patients

Father of N.v.d.E. 1.3 2.2 0.068 0.044 1.69 0.051 0.033 Mother of N.v.d.E 1.1 1.5 0.076 0.030 1.35 0.067 0.027 Father of M.d.R 1.6 2.2 0.105 0.038 1.34 0.065 0.023 Mother of M.d.R 1.5 2.5 0.073 0.024 1.67 0.050 0.016 Father of B.R. 1.6 2.2 0.055 0.029 1.37 0.034 0.018 Mother of B.R. 1.4 2.2 0.067 0.049 1.54 0.047 0.034

Range 1.1-1.6 1.5-2.5 0.055-0.105 0.024-0.049 1.34-1.69 0.034-0.067 0.016-0.033

Controls (N=4)

Range 1.0-1.5 1.5-2.3 0.050-0.159 0.030-0.067 1.16-1.79 0.036-0.150 0.023-0.065

л ' ' 1. . - • • * Values are expressed as % of total fatty acids (C14:0-C26:0). Table II: Very long chain fatty acids and ratios of C24:0/C22:0, C25:0/C22:0, and C26:0/C22:0 in cultured amniotic fluid cells. *

C22:0 C24:0 C25:0 C26:0 C24:0/C22:0 C25:0 / C22:0 C26:0 / C22:0

normal amniotic fluid cells

1 0.61* 2.3 0.090 0.115 3.79 0.146 0.188 2 0.85 2.3 0.120 0.107 2.71 0.142 0.126 3 0.64 1.9 0.085 0.047 3.05 0.134 0.074

normal fibroblasts (n=4)

Range 1.0-1.5 1.5-2.3 0.050-0.159 0.030-0.067 1.16-1.79 0.036-0.150 0.023 - 0.065

* Values are expressed as % of total fatty acids (C14:0-C26:0). DISCUSSION

The elevated level of the C26:0 fatty acid in fibroblasts, brain white matter and adrenal gland of Zellweger patients as previously described by Brown et al (1982) and also established in serum (Bakkeren et al, 1984), indicates once again the involvement of the peroxisomal deficien cy in the biochemical disturbances in this syndrome. Very long chain fatty acids are preferably degraded by the peroxisomal ß-oxidation sys tem (Kawamura et al, 1981). In childhood ALD the accumulation of very long chain fatty acids in fibroblasts could be related to an impaired peroxisomal oxidation of these substances (Singh et al, 1984). Partin and McAdams (1983) reported absence of peroxisomes in the liver of neo natal ALD.

Until now three biochemical abnormalities have been found in fibroblasts from CHR-S patients. Besides the abnormal very long chain fatty acid pattern established by Brown et al (1982), an increased sensitivity of Zellweger fibroblasts to antimycin A (Kelley et al, 1983) has been re ported. CHR-S heterozygote fibroblasts however were not investigated by these authors. Recently Heymans et al (1984) showed that fibroblasts of Zellweger patients contain a lower ethanolamine plasmalogen fraction compared to controls. Heterozygotes and controls were not different. The slightly higher level of C26:0 in normal amniocytes compared with normal fibroblasts was already mentioned by Moser et al (1982) and remains unexplained. In Zellweger syndrome the heterozygous parents did not show an elevation of the very long chain fatty acid levels neither in serum (Bakkeren et al 1984) nor in fibroblasts. This fact indicates that this technique can be applicated for the prenatal diagnosis of Zellweger syndrome. In a comparable study on the autosomal recessive neonatal onset form of ALD Jaffe et al (1982) also did not find increased very long chain fatty acid levels in fibroblasts and plasma from the parents of two patients. In the X-linked form of ALD an increased content of very long chain fatty acids has been found in plasma (Moser A et al, 1980) and fibro blasts (Kawamura et al, 1978, Moser H et al, 1980B, Rizzo et al 1984).

146 For 80% of the obligate heterozygotes the C26:0/C22:0 ratio in fibro blasts was more than 2 SD in excess of the control mean (Moser et al, 1980A, O'Neill et al, 1982, Tönshoff et al, 1982, Moser H et al, 1983). This increased ratio can be explained by selection favouring the mutant allele (Migeon et al, 1981).

Acknowledgements: The authors thank Mrs. H. van Lith-Zanders, Mrs. M. Broedelet-Hootzman and Mr. F.v.d. Brandt for their excellent technical assistance.

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150

CHAPTER VII

PIPECOLIC ACID LEVELS IN SERUM AND URINE FROM NEONATES AND NORMAL INFANTS; COMPARISON WITH VALUES REPORTED IN ZELLWEGER SYNDROME

L.Govaerts, F.Trijbels, L.Monnens, A.van Raay-Selten.

University of Nijmegen, Department of Paediatrics, The Netherlands

Submitted for publication. ABSTRACT

Pipecolic acid (PA) excretion from normal neonates, prematures and/or small for dates has been determined and correlated with gestational age, sex and age after birth. Sex influence was not detectable. Only small for dates with a gestational age of > 34-37 weeks had a lower urinary excretion of PA than the appropriate for dates with the same ge stational age. Preterm infants had a higher excretion of PA than term neonates. PA excretion of infants decreases with age after birth. This higher excretion in "younger" infants can be explained by a higher serum concentration and less efficient tubular reabsorption. PA level in serum and urine remains a valuable tool for the confirmation of the clinical diagnosis of Zellweger syndrome when gestational age and age after birth are taken into consideration. No PA was detected in serum or urine of four children suffering from hy perthyroidism.

KEY WORDS

Pipecolic acid - prematurity - cerebro-hepato-renal syndrome of Zellwe ger - hyperthyroidism.

154 INTRODUCTION

Pipecolic acid (PA), a metabolite of lysine, was found in the urine of young infants and neonates (Woody et al, 1970). One of four infants less than 1 year old, two of four term neonates and four of four prematures excreted PA in the urine. PA was not found in urine of four children over 1 year of age (Woody et al, 1970). No serum PA levels were repor ted in that study. Children with hyperlysinemia due to defective functioning of the lysine- saccharopine-a-aminoadipic acid pathway, excreted increased concentra tion of PA in urine. Woody et al (1970) thus suggested a reduced lysine- a-ketoglutarate reductase activity in neonates as an explanation for PA excretion in urine. Hutzler and Dancis (1983) found sligthly elevated plasma levels of PA in newborn infants, compared with older infants. Pipecolic aciduria with increased serum levels is one of the various biochemical abnormalities in the cerebro-hepato-renal syndrome of Zellweger (Danks et al, 1975, Trijbels et al, 1979, Govaerts et al, 1982), and in hyperpipecolatemia (Gatfield et al, 1968, Thomas et al, 1975, Arneson et al, 1982). Pipecolic aciduria was also reported in hyperthyroidism (Sonada et al, 1957) and in kwashiorkor (Schendel et al, 1959, Whitehead et al, 1964).

The aim of this study is to present values for PA excretion in urine and serum levels of preterm infants, term neonates and infants during the first year of life, with a more sensitive method than used by Woody et al (1970). These values will be compared with those published in the cerebro-hepato-renal syndrome of Zellweger, where the PA levels remain a valuable diagnostic tool.

155 PATIENTS AND METHODS

Patients From 224 children (149 boys and 75 girls) 357 24-h collections of urine were analysed. Each urine specimen was emptied into a sterile collection bottle as soon as it was passed and was kept at 4<,C. At the end of the 24-h period, the urine was frozen and stored at -20oC until analysis. Infants delivered before 37 weeks from the first day of the last men strual period were classified preterra (W.H.O.). They were subdivided in three groups: 26-30 weeks, >30-34 weeks, >34-37 weeks of gestation. Small for dates infants had a birth weight

Methods PA was determined by ion-exchange chromatography on an amino acid analy ser (Biotronik LC 2000 or LC 6000) according to a procedure given by the manufacturer. To increase the sensitivity of the method the ninhydrin reagent was prepared in the following way: one volume of methylcello- solve was mixed with 3 volumes of glacial acetic acid. After bubbling oxygen-free nitrogen through this mixture (at least 30 min), ninhydrin (20 g/1) was added and bubbling of nitrogen was continued until ninhy drin was dissolved. No reducing reagent was used. To increase the reac tion time with ninhydrin the reaction coil (internal diameter 0.3 mm) was replaced by a coil with an internal diameter of 0.55 mm. Serum samples of 0.5 ml were required. The detection limit is 3 |ЛЮІ/1.

156 The PA excretion is expressed as ymol/g creatinine. As the collection of 24 h urine samples from young children is notoriously prone to error, the concentrations expressed in vimol/g creatinine and ymol/kg/24 h were correlated from the collections of the boys. The regression coefficients of the different gestational age groups were 26-30 w: r= 0.877; >30-34w: r= 0.827; >34-37w: r= 0.763; >37w: r= 0.787 with a p-value =0.001 in each group of neonates (praol/kg/24 h on the y-axis and pmol/g creatinine on the x-axis). We concluded that the generally used unit ymol/g creati nine is reliable. Further results are given in pmol/g creatinine. As it is assumed that proline, hydroxyproline, glycine and PA share a common renal transport mechanism, the concentration of these compounds was determined by ion exchange chromatography in the urine collections with high and low value of PA.

The statistical analysis of the PA levels was performed according to Koziol et al, 1981, with the Kruskal-Wallis test and with Spearman correlation coefficients in order to determine the significance of the different items. Values of normal infants were compared with those of Zellweger syndrome patients by the Wilcoxon 2 sample test.

RESULTS

I URINE

Significance of the different items in neonates and infants:

Sex: There was no significant sex difference of the PA levels in urine in the different gestational age groups of neonates. The distribution-free test according to Koziol et al (1981) for the four gestational age groups re sulted in a p-value >0.1. The item "sex" was then omitted in the further statistical analysis.

157 Small for dates: Some influence of dysmaturity in the different groups of gestational age was noted. The small for dates (N=12) with a gestational age of>34-37 w had significantly lower levels of PA in urine than the appropiate for dates neonates with the same gestational age (N=43) (P <0.01 with the distribution free-test according to Koziol et al, 1969). Irrespective of the gestational age of the newborns no significant difference in PA ex cretion between appropiate for dates (N=241) and small for dates (N=81) was found with the distribution-free test according to Koziol et al, 1969.

Gestational age: The PA concentration in urine at several ages of the different gestatio nal age groups is presented in Table I. The PA excretion determined at the same age after birth, decreases when the gestational age increases (p <0.01 with the Kruskal-Wallis test).

Age after birth: The influence of the age after birth on the median PA level in urine of the different gestational age groups is also shown in Table I. No diffe rence in the urinary PA level of the prematures of different gestational ages when they have reached the at term age was seen. The PA excretion of infants delivered after a fullterm pregnancy decrea ses with age (Fig. 1). The Spearman correlation coefficient for age is -0.27304 with ρ <0.01 (N=112).

The concentrations of PA, proline, hydroxyprollne and glycine are shown in Table II. As expected, at low urinary PA levels, low proline, hydro xyprollne and glycine excretion was found.

158 Table I: The number of determinations (Ν), the range, and the median level (M) of urinary PA excretion (ymol/g creatinine).

Age Gestational age after birth

26-30 w >30-34 w >34-37 w >37 w

1 d N 8 12 7 22 range 42-392 27-254 31-93 2.6-119 M 142.5 111.5 51 13.5

4 d N 10 25 16 23 range 24-360 22-290 25-202 7-94 M 158.5 88 56 26

8 d N 13 28 17 18 range 23-651 30-255 27-246 13-243 M 135 78 73 27

2 w N 13 19 10 10 range 49-1285 20-229 25-216 13-54 M 255 57 67.5 37

4 w N 12 14 4 4 range 61-1330 19-270 30-130 18-37 M 191 75 103.5 31.5

6 w N 14 9 range 46-393 18-298 M 204 78

8 w N 9 range 53-450 M 107

10 w N 5 range 21-113 M 73

• I d = day(s) w = week(s) Comparison of urinary pipecolic acid levels between normal neonates and patients with the Zellweger syndrome:

When the lowest PA excretion of 14 patients suffering from the Zellweger syndrome (as reported by Govaerts et al, 1982) were compared with the highest PA excretion of neonates with a gestational age of >37w, a sig nificant difference was found (p-value»0.0001 with the Wilcoxon 2-sample test).

Fig. 1: The pipecolic acid excretion of term infants during infancy (N=118).

u,mol/g creatinine-

243

120Ί . j

100

60-

„th ,nd 4th 1st3rd3rd6th6th9th9th12th day week month

160 TABLE II: The excretion of hydroxyprollne, proline and glycine in 24 h urine collections with high and low concentration of PA (pmol/g creatinine).

Pipecolic Hydroxy- Proline Glycine acid (PA) proline

low PA N 8 8 8 8 Range 3-25 25-4526 91-5341 966-13631 Median 11.5 446 295.5 2572.5 Mean 12.9 1017 1078 4323 S.D 7.4 1498 1820 4519

high PA N 8 8 8 8 Range 303-1330 2626-11729 2098-13904 10728-46113 Median 444.5 6025 5628 19946 Mean 650 6067 6293 22163 S.D. 420 3209 3661 12551

161 II SERUM

Pipecolic acid concentration in serum of neonates: The PA level in serum of prematures with a gestational age of 26-30 w is given in Fig. 2. An age dependency in PA serum levels is clear. The great number of sera with a PA concentration below the detection limit made a statistical analysis of these data unreliable. Fifty seven determinations of PA in serum from children with a gestatio nal age of > 30 weeks were performed: only twice a level above 3 pmol/l was found (in the group with a gestational age of >30-34 w: 4.6 pmol/l and 3.5 ixmol/l respectively). When these prematures reached the at term age no PA could be determined in serum.

Fig. 2: The pipecolic acid level in serum of preterm infants with a ge stational age of 26-30 w (N=59).

μ mol /I

la X ie X •Μ

X к Ι 2H 1 о к X X 84 * 5 6 X ft X 44 к X X X

3 3 4 5 6 9 3 2 3 2 —»•аде

.jst 4th gth 2nd4th gth 8th10th12th14th day week

162 Comparison of pipecolic acid concentration in serum of normal infants and children with the Zellweger syndrome: Comparison of the levels of PA in serum of 10 Zellweger patients and normal neonates resulted in a p-value<0.01 (Wilcoxon 2-sample test) for every group of neonates and at every age after birth.

Ill HYPERTHYROIDISM

In four new patients with hyperthyroidism (age between 9 and 13 years) no PA was detectable neither in serum nor in urine. Some relevant infor mation about their thyroid function at the time of urine collection and blood sampling is given in Table III.

TABLE III: Thyroid function of four children with hyperthyroidism due to autoimmune thyroiditis before treatment.

Age TSH Initials h T3 (years) (nmol/1) (ng/100 ml) (uE/ml)

K.d.M. 13 252 598 7.1 F.G.Z. 13 395 600 4.2 E.T. 97/12 244 400 < 2.0 M.L. 13 345 400 < 2.0

Normal values 50-130 85-185 < 2-8

T, = thyroxin T, = triidothyronine TSH = thyroid stimulating hormone

163 DISCUSSION

The urinary excretion of PA is increased in preterm neonates compared with term neonates, and in normal neonates compared with older infants. This increased excretion of PA is due either to elevated filtered load exceeding the reabsorptive capacity or to a maturation of the reabsorp tion in kidney-tubule cell. An elevated serum level of PA contributes to the increased urinary ex cretion of PA. Preterm infants have a higher serum level of PA than term neonates. Our measured values for PA are slightly lower than those reported by Hutzler and Dancis (1983). These authors reported a mean plasma concentration of 2.1 μηοΐ/l ± 1.6 (SD) in their pediatric popula tion (aged 4 months to 17 years). During the first week of life a mean value of 12 ymol/l ±5.1 was calculated (N=9). Their gestational age was not reported. In liver disease a normal, marginally or distinctly eleva ted value was found. The increase of serum PA at younger age can be ex plained by an increased flux of lysine through the PA pathway or by a decreased catabolism of PA. Recent studies in our laboratory have demon strated that PA is metabolized via the peroxisomes in rat liver (Trij- bels et al, in preparation). This might explain the increased PA serum level and the increased excretion of PA in Zellweger syndrome, a disease characterized by the absence of peroxisomes in liver and kidney tissue (Goldfischer et al, 1973). A development of several peroxisomal funtions has been observed in different mammals (Masters and Holmes, 1977). An increase of PA catabolism with age owing to this maturation of peroxiso mal functions is an acceptable hypothesis for the decrease of serum PA level with age.

On the basis of genetic studies in man two modes of transport for the iminoacids (L-proline, 4-hydroxy-L-proline) and glycine are proposed (Scriver, 1968, Lasley and Scriver, 1979). A common system with high capacity and higher affinity for imino acids than for glycine is lack ing in familial renal iminoglycinuria. Two additional systems, each with low capacity, are proposed. One of those systems is shared by pro line and hydroxyproline, the other system has a preference for glycine. The immaturity of the reabsorption of imino acids and glycine during infancy has been well studied (Brodehl, 1978). Hyperiminoaciduria sub-

164 sides during the first three months of life while hyperglycinuria per sists for about six months in the normal infant. If PA is transported by the iminoglycine transport system, decreased reabsorption during the first months of life is expected. Support for this hypothesis can be given by the concordant changes in the excretion of imino acids, glycine and PA (Table II). Ganapathy et al, 1983, studied the transport of proline and PA in purified brush border membrane vesicles from rabbit renal cortex. PA transport was completely inhibited by proline and hydroxyproline and moderate inhibition was observed with glycine. It was concluded that proline and PA share a common transport system in the rabbit kidney.

In one patient with hyperthyroidism, Sonoda (1957) showed the probable presence of PA in urine by paperchromatography. We were unable to con firm his findings in 4 children with hyperthyroidism using a more sensi tive and specific technique. When gestational age and age after birth are taken into consideration, the PA level in serum and urine remains a valuable tool for the confir mation of the clinical diagnosis of Zellweger syndrome.

ACKNOWLEDGEMENTS The extreme care of the nurses for the collection of 24 h samples of urine, is highly appreciated. The expert technical assistance of Mrs.J. Corstiaensen (Laboratory of Paediatrics and Surgery) and of Ir. H. van Lier (Department of Statis tical Consultation) is gratefully acknowledged.

165 REFERENCES

Arneson D, Tipton R, Ward J: Hyperpipecolic acidemia: occurrence in an infant with clinical findings of the cerebrohepatorenal (Zellweger) syn drome. Child Neurology 39 (1982) 713-716

Brodehl J: Renal hyperaminoaciduria. In Edelmann С, Barnett H, Bernstein J, Michael A, Spitzer A (eds), Pediatric kidney disease. Little, Brown and Company, Boston, 1978, pp 1047-1079

Danks D, Tippett P, Adams C, Campbell P: Cerebro-hepato-renal syndrome of Zellweger: J Pediatr 86 (1975) 382-387

Ganapathy V, Roesel R, Howard J, Leibach F: Interaction of proline, 5- oxoproline, and pipecolic acid for renal transport in the rabbit. J Biol Chem 258 (1983) 2266-2272

Gatfield P, Taller E, Hinton G, Wallace A, Abdelnour G, Haust M: Hyper- pipecolatemia: A new metabolic disorder associated with neuropathy and hepatomegaly. A case study. Can Med Assoc J 99 (1968) 1215-1233

Goldfischer S, Moore С, Johnson A, Spiro A, Valsamis M, Wisniewski H, Ritch R, Norton W, Rapin I, Gartner L: Peroxisomal and mitochondrial de fects in the cerebro-hepato-renal syndrome. Science 182 (1973) 62-64

Govaerts L, Monnens L, Tegelaers W, Trijbels J, and van Raay-Selten A: Cerebro-hepato-renal syndrome of Zellweger: clinical symptoms and rele vant laboratory findings in 16 patients. Eur J Pediatr 139 (1982) 125-128

Hutzler J, Dancis J: The determination of pipecolic acid: method and re sults of hospital survey. Clin Chim Acta 128 (1983) 75-82

Kloosterman G: Over intrauterine groei en de intra-uteriene groeicurve. Maandschr Kindergeneeskunde 37 (1969) 209-225

Lasley L, Scriver С: Ontogeny of amino acid reabsorption in human kid ney. Evidence from the homozygous infant with familial renal iminoglycinuria for multiple proline and glycine systems. Pediatr Res 13 (1979) 65-70

Koziol J, Maxwell D, Fukushima M, Colmerau M, Pilch Y: A distribution- free test of tumor growth curve analyses with application to an animal tumor immunotherapy experiment. Biometrics 37 (1981) 383-390

Masters C, Holmes R: Peroxisomes: New aspects of cell physiology and biochemistry. Physiol Rev 57 (1977) 816-882

Scriver С: Renal tubular transport of proline, hydroxyproline and gly cine. III. Genetic basis for more than one mode of transport in human kidney. J Clin Invest 47 (1968) 823-835

166 Schendel H, Antonis A, Hansen J: Increased amino-aciduria in infants with kwashiorhor fed natural and synthetic diets. Pediatrics 23 (1959) 662-675

Sonoda Y: Urinary amino acids of hyperthyroid patient. Proc Japan Acad 33 (1957) 162-165

Thomas G, Haslam R, Batshaw M, Capute A, Neidengard L, Ransom J: Hyper- pipecolic acidemia associated with hepatomegaly, mental retardation, optic nerve dysplasia and progressive neurological disease. Clin Genet 8 (1975) 376-382

Trijbels J, Monnens L, Bakkeren J, van Raay-Selten A: Biochemical stu dies in the cerebro-hepato-renal syndrome of Zellweger: a disturbance in the metabolism of pipecolic acid. J Inher Metab Dis 2 (1979) 39-42

Whitehead R: An unidentified compound in the serum of children with kwashiorkor (protein-calorie malnutrition). Nature 204 (1974) 389

Woody E, Pupene M: Excretion of pipecolic acid by infants and by pa tients with hyperlysinemia. Pediatr Res 4 (1970) 89-95

167

CHAPTER Vili

GENERAL CONSIDERATIONS It is evident that all biochemical abnormalities observed in CHR-S should be related to the absence or near absence of peroxisomes. For most of the biochemical disorders an acceptable explanation can be of fered. In what way the clinical symptoms of CHR-S are induced by this variety of defects, remains a challenge for all investigators involved in this field.

MORPHOLOGIC EVIDENCE FOR THE ROLE OF PEROXISOMES IN THE CNS AND THE EYES

McKenna et al (1976) reported catalase positive microperoxisomes in the spinal cord neurons, oligodendrocytes, ependymal cells lining the fourth ventricle, astrocytes and Schwann cells of the CNS of the adult rat. Neurons of catecholaminergic brain regions and sympathetic ganglia of the rat contain appreciable numbers of microperoxisomes, but the majori ty of neurons of other regions contain few bodies with demonstrable ca talase activity. In dorsal root ganglia only occasional neurons contain appreciable numbers of microperoxisomes (Citkowitz et al, 1973, McKenna et al, 1976). An age-dependent distribution of D-amino acid oxidase-positive and catalase-positive microperoxisomes in rat brain was described by Weimar and Neims (1977) and Arnold and Holtzman (1978). In early postnatal CNS of the rat catalase-positive microperoxisomes were found in all cell types in all regions studied. During the first three postna tal weeks catalase-positive bodies were seen in synaptic terminals in several areas, a location where they are seldom observed in mature tis sue. The varying distribution of these organelles in developing nervous tissue, together with their presence in regions which have few micro peroxisomes in the adult rat, suggest that their functions may differ somewhat in immature and adult nervous tissue (Arnold and Holtzman, 1978). In all regions studied microperoxisomes were found in oligoden drocyte processes immediately adjacent to the outer lamellae of forming myelin sheaths (Arnold and Holtzman, 1978, Holtzman, 1982). Microperoxisomes were found to be abundant in the retinal pigment epi thelium of the human, the rhesus monkey and rodents by ultrastructural histochemistry (Leuenberger and Novikoff, 1975, Robison and Kuwabara, 1975). They were rare in other cells of the retina and choroid (Robi son and Kuwabara, 1975).

170 An extensive DAB-positive tubular system was seen in the synaptic termi nals of the photoreceptor cells in rodents (Leuenberger and Novikoff, 1975). Histopathologic studies of the retina of patients suffering from CHR-S revealed abnormal clumps of pigment epithelial cells in the peri phery (Volpe and Adams, 1972) and an unmistakable loss of photoreceptor cells affecting mainly the rods (Garner et al, 1982) in the outer seg ments of the mldperiphery. No studies concerning (micro-)peroxisomes in these cells in Zellweger syndrome are reported. This particular locali zation of the peroxisomes are in favour of an important metabolic role of the peroxisomes in the eye. In this paper we will further discuss the main biochemical abnormalities seen in CHR-S and speculate about the basic defect. In serum and fibro blasts of ALD almost the same aberrant very long fatty acid pattern is seen as in CHR-S. This fact justifies a separate discussion in this chapter.

PATHOGENESIS OF BIOCHEMICAL DISORDERS AND ITS CLINICAL SIGNIFICANCE

Catabolism of pipecollc acid The role of the peroxisomes in PA catabolism in rats was recently inves tigated in our laboratory. Peroxisomes were isolated by Percoli density 14 gradient centrifugation of rat liver homogenate. The C0„ production rate was measured in the different fractions after incubation with [ 14 1 14 1- СI PA. The peak of the CO. production rate was located in those fractions showing the highest activity of catalase and urate-oxidase, the well-known peroxisomal markers. These experiments indicate that PA catabolism in rat liver is located in peroxisomes. When these findings can be applied to man, the increased concentration of PA in serum, urine and CSF in CHR-S is easily understood. L-PA is a normal constituent of mammalian brain and a major product of L-lysine metabolism in the brain. PA has been shown to have a role in the nervous system function. Intraventricular administration of 500 yg of PA produces ptosis, hypotonia, sedation and hypothermia in mice (Ta- kahama et al, 1982). Administration into the cerebellum and hippocampus of cats with chronically· implanted cannulae and electrodes caused promi nent depression both in EEG and behaviour (Kasé et al, 1980). 171 PA has been found to inhibit the uptake of H GABA into brain slices and also facilitate the release of this compound from the slices. The uptake of PA in glial cell fractions and synaptosomes is inhibited by GABA and vice-versa. Using unit recording and micro-electrophoretic technique Kasé et al (1980) demonstrated a depression of the firing of cortical neurons by PA. These findings suggest that PA is involved in the regulation of synaptic mechanism in the CNS (Charles et al, 1983) by affecting the GABA-ergic transmission i.e. by inhibiting the uptake of endogenous GABA and/or by facilitating the release of the inhibitory transmitter. In the CSF of two of our patients suffering from CHR-S, normal concentration of GABA was measured.

Biosynthesis in vitro of I HI piperidine from D, L Ι H I PA was very low in brain and kidney. No other organs showed any formation of H pipe ridine (Nomura et al, 1983). They questioned the hypothesis of a metabo lic activity of piperidine in brain.

Pathogenesis of liver dysfunction Pedersen and Gustafsson (1980) and Kasé et al (1983) showed that the peroxisomal fraction of rat liver homogenate is able to catalyse the conversion of THCA into cholic acid. Hanson et al (1977) investigated the influence of an intravenous infusion of tauro-THCA on hepatic mor phology in rats. Liver biopsy sections from rats infused for 2h showed dilatation of the rough ER and distortion of the mitochondrial membra nes. Chronic exposure of the liver to tauro-THCA was not tested. This study suggests that the increased level of THCA could be responsible for the liver damage in Zellweger syndrome (Hanson et al, 1977, Monnens et al, 1980, Mathis et al, 1980).

Deficiency of plasmalogens Plasmalogens are ubiquitous membrane constituents in mammals. According to Hajra and Bishop (1982) peroxisomes may supply the lipid precursors acyl-DHAP or alkyl-DHAP to ER for lipid synthesis and provide some kind of substrate level control system for cellular membrane biogenesis. Recent findings of Declercq et al (1984) support their hypothesis. Per oxisomes are not equipped with all enzymes needed for the synthesis of complex ether lipids. The enzymes choline phosphotransferase, ethanolamine phosphotransferase, catalyzing the terminal reactions in the syn-

172 thesis of complex ether lipids, are absent from peroxisomes. The almost complete absence of plasraalogen-phosphatidylethanolamine and plasraalogen-phosphatidylcholine in several tissues of CHR-S (Heymans et al, 1984) suggests the possibility of a disturbance of membrane function in CHR-S. Such an abnormal membrane composition could interfere with e.g. the activity of mitochondrial-membrane-bound enzymes such as succinate- ubiquinone oxidoreductase and the cholesterol side-chain cleavage enzyme of the adrenal cortex (Simpson and Waterman, 1983). As ethanolamine plasmalogen is the most important phospholipid component of human myelin with a changing composition during development (Boggs and Rangarag, 1984), dysmyelination, observed in the CNS of CHR-S, is an expected fin ding.

Increased saturated very long chain fatty acid levels In CHR-S the same abnormalities of VLCFA were observed as in ALD. ALD is a disorder biochemically characterized by increased levels of saturated VLCFA in serum, cultured skin fibroblasts, erythrocyte membranes (Tsuji et al, 1981, Kobayashi et al, 1983, Knazek. et al, 1983), leucocytes (Molzer et al, 1981) and in cholesterolesters of the brain white matter (Brown et al, 1983). One of the apparent effects of this fatty acid accumulation is to in crease the index of microviscosity (Knazek et al, 1983). Measurements of fluorescence polarization demonstrated a highly significant increase in the microviscosity of erythrocyte membranes in ALD (Knazek et al, 1983). Experimental data in animals point to a correlation between membrane viscosity and the number of hormone receptors in target tissues. There fore Knazek et al (1983) proposed that the decrease in responsiveness to ACTH in ALD is secondary to increased membrane viscosity of the adrenal gland cells. On the basis of the same arguments a disturbed thyroid function could be predicted (Bashford et al, 1975). Investigation of the basal levels of T,, T,, and TSH in 5 patients with CHR-S revealed how ever normal values (unpublished results). Studies about the insulin re ceptors are in progress.

Alteration of the lipid fluidity of ER membranes can modify the activity of steroid biosynthetic enzymes (21-hydroxysteroid hydroxylase and 3- B-hydroxysteroid dehydrogenase) (De Paillerets et al, 1984) and in this way influence the action of ACTH.

173 Increased VLCFA in brain gangliosides (Igarashi et al, 1976) and In mye lin fractions of brain white matter from patients with ALD (Brown et al, 1983) were observed. The same biochemical abnormalities are probably present also in CHR-S.

BASIC DEFECT IN CHR-S

The absence of peroxisomes in liver and kidney tissue of CHR-S allows only one hypothesis related to the basis defect: an abnormality in the biogenesis or maintenance of the peroxisomal membrane. In every paper published about CHR-S during recent years the dysfunction of the peroxi somes is proposed as the primary defect. Already in 1979 Pfeifer and Sandhage expressed their view In this way: "The term peroxisome defi ciency is proposed as a name which probably is more closely related to the pathogenetic mechanism than the hitherto usual designation cerebro- hepato-renal syndrome." Although morphologic studies suggest that the membrane of a peroxisome arises from a protusion of the ER, the polypeptide composition of the peroxisomal membranes is very different from that of the ER or mitochon drial membranes. Particularly noteworthy is the absence of cytochrome P450, the most abundant ER membrane protein in peroxisomal membranes (Lazarow, 1982). The biogenesis of the peroxisomal membrane itself is at the present time unclear. Analogous with other Inborn errors of metabolism one enzyme deficiency should exist leading to a defective synthesis of the peroxi somal membrane. The consequences for the activity of the different en zymes normally present inside the peroxisomes can be predicted. Peroxi somal proteins are synthesized on free polysomes, at their mature size rather than as large precursors, and enter peroxisomes without proteo lytic processing. One of the enzymes studied by MIura et al (1984), however, appears to be translated as a large precursor which is proteo- lytically processed to form the mature enzyme. It is not sure that the proteolytic processing is linked with transport into the peroxisome. When we accept a normal formation of the peroxisomal proteins on the free ribosomes in CHR-S, it is quite possible to observe a lowered en-

174 zymatic activity in vitro and in vivo. It is possible that some of the se proteins remain in the precursor form, and lack normal activity. Without "protection" by the peroxisomal membrane catabolism in the cyto plasm could be raised. Finally, and this could be important in vivo, the enzymatic activity is not concentrated and well assembled in peroxiso mes.

RELATION BETWEEN CHR-S AND THE NEONATAL FORM OF ALD

Clinical features Neonatal ALD (McKusick 20237) was first described by Ulrich et al, 1978. Dysmorphic features were first mentioned by Benke et al, 1981, who noti ced clinical similarities with the CHR-S. It should be noted that adre nal insufficiency wasn't clinically apparent in neonatal ALD (Jaffe et al, 1982, Brown et al, 1982) while biochemically evident adrenocortical hypofunction was demonstrated by ATCH stimulation test (Jaffe et al, 1982). The lack of affected individuals in relatives of the mother and the equal severity in both male and female patients suggest autosomal recessive transmission (Benke et al, 1981) in contrast with the X-linked childhood ALD (Table I).

Biochemical data Biochemical analysis of the serum, fibroblasts, and tissues from pa tients with neonatal ALD and CHR-S revealed increased levels of satura ted VLCFA, already known to occur in childhood ALD (Manz et al, 1980, Jaffe et al, 1982, Brown et al, 1982, МоЫеу et al, 1982, Haas et al, 1982, Bakkeren et al, 1984). The increased level of С , in plasma (Moser et al, 1983, Moser et al, 1984) and in cultured skin fibroblasts (Björkhem et al, 1984, Moser et al, 1984) of CHR-S patients is described in cultured skin fibroblasts in neonatal ALD (Moser et al, 1983). Investigation of the adrenal function in CHR-S revealed a primary adre nocortical insufficiency (see Chapter V), one of the conspicious fea tures of ALD. In childhood ALD normal PA levels in plasma were reported (Moser et al, 1984). Analysis of the bile acid pattern and plasmalogens in patients suffering from the neonatal form of ALD is required.

175 Pathologie studies Absence of hepatic peroxisomes in neonatal ALD was reported by Partin and McAdams (1983), while normal peroxisomes were noted in childhood ALD (Moser et al, 1984). Unusual appearing liver mitochondria and lysosomal lamellar crystalline inclusions in hepatocytes and liver macrophages were reported in neonatal ALD (Partin and McAdams, 1983). Striated lamellar bodies, typical for ALD, sometimes surrounded by a single mem brane, were seen in macrophages of brain, liver, thymus and lyniphnodes, in glial cells, adrenocortical cells and hepatocytes in neonatal ALD (Partin and McAdams, 1983, Haas et al, 1982, Ulrich et al, 1978). A few authors mentioned similar abnormalities in ALD and CHR-S. Agamano- lis et al, 1976, noticed a similarity between the cytoplasmic inclusions (multilamellar bodies) of macrophages, Oligodendroglia! cells and astro cytes of the CNS of Zellweger patients and the inclusions seen in the adrenals, Schwann cells, testis and brain of patients with childhood ALD. The lamellar-lipid profiles, considered to be diagnostic for ALD (childhood and neonatal form), were found by Goldfischer et al (1983) in the adrenal glands of 7 patients with CHR-S. Ocular histopathologic and biochemical studies demonstrated that the CHR-S and the neonatal ALD are similar with regard to ocular abnormalities and the presence of satura ted VLCFA (Cohen et al, 1983).

Only one out of four patients with neonatal ALD showed cortical cysts in the kidneys.

Conclusion Neonatal ALD and CHR-S are probably two designations of the same condi tion. The clinical and biochemical abnormalities of childhood ALD are more restricted, and point to another peroxisomal dysfunction; possibly a single peroxisomal enzyme deficiency (Brown et al, 1982, Moser et al 1984).

176 TABLE I: Review of relevant data of CHR-S, neonatal ALD and childhood ALD.

CHR-S Neonatal ALD Childhood ALD

CLINICAL ASPECTS

mode of inheritance autosomal autosomal X-linked recessive recessive dysmorphisms + + - age of onset at birth at birth childhood age at death usually infancy adulthood infancy or childhood adrenal insufficiency + + +

BIOCHEMICAL FINDINGS

hyperpipecolic acidemia + 9 ? increased THCA + 7 ? plasmalogens deficiency + ? - increased VLCFA:-saturated + + + + + 7 - C26:l

ULTRASTRUCTURAL FINDINGS

peroxisomes absent absent normal

+ = present - = absent ? = unknown

177 ANTENATAL DIAGNOSIS OF CHR-S

The abnormal VLCFA pattern in cultured amnlocytes allowed identification on three affected fetuses out of 9 pregnancies (Moser et al, 1984). It is a convenient technique for prenatal detection of CHR-S. The deficiency of plasmalogens and deficiency of acyl-Coa:DHAP acyltransferase offer another possibility for prenatal diagnosis.

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182

SUMMARY

In the first chapter of this thesis a review is given of the present knowledge about peroxisomes in mammals and man. This is required to ful ly understand the Investigations reported in the other chapters. The li terature about clinical, biochemical and pathologic features of the cerebro-hepato-renal (Zellweger) syndrome is summarized in chapter II. The CHR-S is an autosomal recessive inherited disorder, with characte ristic dysmorphisms (high forehead, frontal bossing, large fontanels, epicanthal folds, abnormal ears). It included severe neurological dys function (severe hypotonia, poor sucking, seizures, absent reflexes, severe psychomotor retardation, severe hearing impairment), ocular symp toms (nystagmus, cloudy cornea, lenticular opacities, optic disc pallor and peripheral clumping of pigment), hepatomegaly and failure to thrive. Ninety percent of the patients die before one year of age. Peroxisomes are absent or nearly absent in liver, kidney and brain.

The following relevant biochemical abnormalities are observed: decreased catabolism of pipecolic acid, defect of bile acid synthesis with increa sed concentration of THCA and DHCA in bile, serum and urine, increased urinary excretion of p-OH-phenyllactate, abnormal pattern of very long chain fatty acids in serum and cultured fibroblasts, deficiency of plasmalogens, and mitochondrial disturbance (Chapter II and III). A neurophysiological study of eleven patients revealed diffuse dysfunc tion of the cerebrum and myelinated fiber tracts. The EEC's showed characteristic continuous negative sharp waves and spikes at the vertex (Chapter IV). The striking resemblance of CHR-S and the neonatal form of ALD prompted us to investigate the adrenocortical function in CHR-S as this function is disturbed in adrenoleukodystrophy. A primary adrenocortical insuffi ciency could be demonstrated. This insufficiency might contribute to the unexpected death of infants with CHR-S (Chapter V). An increased level of very long chain fatty acid was measured in cul tured skin fibroblasts. This method is applied for prenatal diagnosis by Moser et al (1984) (Chapter VI).

185 Pipecolic acid (PA) excretion in urine from normal neonates, preterm in fants and/or small for dates has been determined and correlated with ge stational age, sex and after birth (Chapter VII). Preterm infants had a higher excretion of PA than normal neonates. PA excretion decreases with age after birth. The PA level in serum and urine remains a valuable tool for the confirmation of the clinical diagnosis of CHR-S when gestational age and age after birth are taken into consideration. Analogous with other inborn errors of metabolism one enzymatic deficien cy should exist leading to a defective synthesis of the peroxisomal mem brane. This primary deficiency will lead to secondary deficiencies of some enzymes normally located in the peroxisomes. Neonatal adrenoleukodystrophy and CHR-S are probably two designations of the same condition (Chapter VIII). A prenatal diagnosis of CHR-S is feasible.

186 SAMENVATTING

Het eerste hoofdstuk geeft een overzicht van de huidige kennis van peroxisomen in zoogdieren en in de mens. Deze kennis is noodzakelijk om de biochemische en klinische afwijkingen in het Zellweger syndroom, een ziektebeeld gekenmerkt door een peroxisomendeficiëntie, te begrijpen.

Het CHR-S is een autosomaal recessief overervend ziektebeeld waarvan de incidentie geschat wordt op 1/100 000 levend geborenen. Een overzicht van de klinische symptomen, radiologische en electrofysio- logische afwijkingen, pathologische anatomie en biochemische afwijkin gen, gebaseerd op 96 in de literatuur beschreven patiënten en 16 eigen patiënten, wordt weergegeven in hoofdstuk II, III en IV. De meest voorkomende symptomen zijn: dysmorfieën (te wijde fontanellen, hoog gewelfd voorhoofd, epicanthus, abnormale oorschelpvorm en -inplan ting, pes equinovarus en dwarse handlijnen), ernstige hypotonie en psy chomotorische retardatie, convulsles, hyporeflexie tot areflexie, hepa- tomegalie, gehoorsverlies van 70 dB en oogafwijkingen (nystagmus, cornea en lenstroebeling, bleke papillen en beeld van tapetoretinale degenera tie). Er is tactiel kontakt (ze reageren adekwaat op strelen en op pijn- prikkels). Ondanks voldoende kalorieën-intake (meestal via sondevoeding) groeien deze kinderen slecht. Negentig procent van de patiënten over lijdt gedurende het eerste levensjaar, meestal t.g.v. een pneumonie of ze worden onverwacht dood in bed gevonden.

Het Zellweger syndroom wordt gekarakteriseerd door de volgende bioche mische afwijkingen: gestoorde leverfunktie (soms met bloedingsneiging), abnormale galzuursamenstelling (aanwezigheid van de precursors THCA en DHCA) in gal, serum en urine, verhoogde pipecolinezuurspiegel in serum, urine en liquor, verhoogde excretie van p-OH-fenylmelkzuur in urine, verhoogde concentratie van de verzadigde zeer lange ( > C22) vetzuren en van C26:l in serum en gekweekte fibroblasten, plasmalogenendeficiëntie in weefsels, erythrocyten en fibroblasten, deficiëntie van acyl-CoA:DHAP acyltransferase in lever, hersenen en fibroblasten, defect in de ademha lingsketen en een deficiëntie tot afwezigheid van peroxisomen in lever-, hersenen nierweefsel.

187 Het neurofysiologisch onderzoek (EEG, BAEP en SSEP) van elf patiënten met het Zellweger syndroom (hoofdstuk IV) leverde diffuus ernstig ge stoorde registraties op, wijzend op grijze en witte stof aantasting.

Vanwege de klinische (dysmorfieën en neurologisch beeld), biochemische (verhoogde concentratie van verzadigde zeer lange vetzuren in serum en fibroblasten) en anatomopathologische (E.M. van de bijnieren en de ogen) overeenkomsten met de neonatale vorm van ALD hebben we een ACTH stimula- tietest uitgevoerd bij 6 patiënten met het Zellweger syndroom, en drie kontroles van vergelijkbare leeftijd (hoofdstuk V). De patiënten hebben een primaire bijnierschorsinsufficiëntie, welke waarschijnlijk een rol speelt in de onverwachte dood. Vergelijking van alle beschikbare gege vens van kinderen met neonatale ALD en Zellweger syndroom wijst erop dat het hoogstwaarschijnlijk om hetzelfde ziektebeeld gaat dat onder twee verschillende beschrijvingen en namen in de literatuur bekend is.

In hoofdstuk VI worden de gehaltes aan zeer lange vetzuren in gekweekte fibroblasten van Zellwegerpatienten, obligate dragers en gezonde kon troles en in normale gekweekte amnioncellen weergegeven. Antenatale diagnostiek op basis van deze bepaling in vruchtwater en gekweekte am nioncellen is mogelijk indien de ouders dit wensen. De verschillende mo gelijkheden voor prenatale diagnostiek worden aangegeven in hoofdstuk VIII.

Pipecolinezuur, een afbraakprodukt van lysine, is bij Zellwegerpatienten en gezonde prematuren verhoogd in urine en serum. Het normale pipecoli- nezuurgehalte in urine en serum van gezonde prematuren en zuigelingen tot 1 jaar is bepaald (hoofdstuk VII). Er is een significant verschil tussen de pipecolinezuurconcentraties van normale pasgeborenen en zuige lingen enerzijds en van Zellweger patiënten anderzijds.

De (mogelijke) Pathogenese van de verschillende klinische en biochemi sche afwijkingen van het Zellweger syndroom wordt uitvoerig toegelicht in hoofdstuk VIII. Het basisdefect is gelegen in een stoornis in de bio genesis van de peroxisomale membraan.

188 SAMENVATTING VOOR DE LEEK

De samenvatting van dit proefschrift bevat veel "vaktermen", zodat U waarschijnlijk bij woorden als "peroxisomen", "dysmorfieën" en "primaire bijnierschorsinsufficiëntie" de draad verliest. Om die reden wordt deze korte toelichting over de inhoud van dit proefschrift gegeven.

Het "cerebro-hepato-renaal syndroom van Zellweger" is een erfelijk ziek tebeeld. Beide ouders zijn drager van het afwijkende erfelijke mate riaal. Zij hebben geen klachten en weten dan ook niet dat ze drager zijn tot de diagnose Zellweger syndroom bij hun kind gesteld wordt. Bij iede re zwangerschap bestaat een risico van 25% op een kind met het syndroom.

De kinderen hebben uitwendig zichtbare aangeboren afwijkingen (hoog voorhoofd, te laag ingeplante en abnormaal gevormde oorschelpen, afwij kende handlijnen en soms klompvoetjes) en tekenen van hersenbeschadig ing (te zwakke spieren, stuipen, abnormale reflexen, slecht drinken, ernstige ontwikkelingsachterstand, slecht gehoor- en gezichtsvermogen). Ze reageren wel op aanraken, lachen bij op schoot zitten, huilen en ver tonen een afweerreactie bij pijn en laten duidelijk blijken wanneer ze schik hebben. De lever is vergroot en werkt matig; dit kan aanleiding geven tot stollingsstoornissen (vlug blauwe plekken). Negentig procent van deze patientjes overlijdt in het eerste levensjaar; meestal worden ze onverwacht dood in bed gevonden of overlijden ze ten gevolge van een gewone verkoudheid.

Bij laboratoriumonderzoek zijn er vele afwijkingen in bloed, urine, gal, hersenvocht en gekweekte huidcellen. Er zijn specifieke afwijkingen bij weefselonderzoek met de microscoop, vooral in de hersenen, de lever en de nieren. Ze hebben geen last van deze microscopische nierafwijkingen. Een overzicht van deze afwijkingen bij 96 kinderen beschreven in de vak literatuur en 16 patiënten van onze kliniek is weergegeven in hoofdstuk II en III.

189 Gezien de ernstige hersenaantasting zijn er meerdere onderzoeken ver richt om deze aantasting duidelijker te kunnen beschrijven. In hoofdstuk IV staan de resultaten van deze technische onderzoeken. Er is een ern stige algemene aantasting van de verschillende meetbare hersenfunkties. Het Zellweger syndroom lijkt, zowel klinisch als biochemisch, op een ander erfelijk ziektebeeld, namelijk "neonatale ALD". Dit laatste syndroom wordt tevens gekenmerkt door een slechte bijnierfunktie. In hoofdstuk V worden de resultaten van het bijnierfunktie-onderzoek van 6 kinderen met het Zellweger syndroom weergegeven. Ook deze kinderen hebben een onvoldoende reactie van de bijnier. Deze bijnierfunktie is nodig in stress-situaties zoals diarree, koorts, luchtweginfekties. De ze verminderde bijnierfunktie speelt waarschijnlijk een rol bij de plotselinge dood van de Zellweger patientjes. Bij stress-situaties kan men met medicijnen deze onvoldoende bijnierfunktie opvangen. Een uitvoe rige vergelijking van alle gegevens van het Zellweger syndroom en de neonatale ALD (hoofdstuk VIII) geeft aanwijzingen dat het waarschijnlijk eenzelfde ziektebeeld is dat onder twee verschillende namen in de lite ratuur beschreven is.

De biochemische afwijkingen in gekweekte huidcellen bieden de mogelijk heid, wanneer de ouders dit wensen, tijdens een eventuele volgende zwangerschap te bepalen of het kindje de ziekte heeft. Dit wordt afzon derlijk besproken in hoofdstuk VIII.

Een van de vele biochemische afwijkingen in het Zellweger syndroom is een gestoorde afbraak van pipecolinezuur. Deze stof is verhoogd in bloed, urine en hersenvocht. Normale, te vroeg geboren kinderen, kunnen ook een hoge waarde van pipecolinezuur hebben in serum en urine. Van vele te vroeg geboren kinderen is pipecolinezuur in de urine en in het bloed gemeten. Deze uitslagen liggen lager dan de uitslagen van de pa- tienten met Zellweger syndroom (hoofdstuk VII) zodat de meting van het pipecolinezuur voor de diagnose bij een pasgeborene verdacht van Zellweger syndroom zinvol blijft.

190 De oorzaak van het Zellweger syndroom is een tekort (en misschien zelfs een ontbreken) van peroxisomen. In (bijna) alle cellen van zoogdieren en mensen zitten peroxisomen. In deze peroxisomen vinden vele scheikundige reacties plaats (hoofdstuk I). De argumenten om de structurele en bio chemische afwijkingen te verklaren op basis van het tekort aan peroxi somen worden genoemd in hoofdstuk VIII.

191

ACKNOWLEDGEMENTS

My greatest debt is owed to my parents and grandparents, especially my maternal grandmother, for their constant encouragement and advice throughout the thirty years of my life.

Upon completion of this thesis I should like to address a few words of thanks to those who have contributed to its conception and publication. I have been privileged to complete this study in the form of a thesis by the encouragement and guidance given by the promotor and co-referent, their names are noted on page 2, and the other members of the "metabolic group" of the Department of Paediatrics from the Sint Radboudhospital. They should know that I am not only very grateful for their help but that it was a pleasure for me to have been able to work with them. The nursing staff from the Department of Paediatrics merit special thank for their assistance in collecting urine and blood samples. It is impos sible to thank all of them personally.

Furthermore I wish to thank the technicians from the Laboratory of Pae diatrics and Surgery (head: Dr.P. van Munster) and the co-operators from the Department of Submicroscopic Morphology (head: Prof.Dr.A. Stadhou ders) whose contributions for this thesis are without question. I acknowledge the excellent assistance at all stages of this project by the Medical Library (head: Mr.E. de Graaff), the Department of Medical Illustration (head: Mr.M. Bollen) and the Department of Medical Photo graphy (head: Mr.A. Reynen). The expert secretarial help from Mrs C. van den Tempel and Mrs M. Titse- laar is gratefully acknowledged. My gratitude is also due to Prof.Dr.F. Cabreéis, Prof.Dr.U. van Haelst, Dr.A. Pinckers, Prof.Dr.J. Slooff and Prof..Dr.J. Veerkamp for their edi torial contributions.

193 I acknowledge the advice and support given by Mr.R. Wynants and Ir.A. Renard from the Rotary Club Tongeren, Belgium, through which application for the graduate scholarship was made, and the hospitality from the Ro tary Club Nijmegen, especially Prof.Dr.S. Geerts.

I am also very grateful for the cooperation and patience of the families of the Zellweger patients, their house-officers and paediatricians.

To them and all others who have contributed in anyway in the preparation of this thesis, I express my appreciation.

Lutgarde Govaerts

194 CURRICULUM VITAE

Lutgarde C.P. Govaerts was born in Hasselt, Belgium, on 9th July 1954, as daughter from Joseph L. Govaerts and Isabelle M.J. Steegen. After ob taining her final secondary school diploma (sciences) at the Institute Marlavreugde in Borgloon, Belgium, on 30th June, 1972, she started her study of medicine at the Catholic University of Leuven, Belgium, on 5th September, 1972. The diploma of "Candidate in Medical Sciences", cum laude, was obtained on 1st July, 1975. On 30rd June 1979, the degree "Doctor of Medicine, Surgery and Obstetrics", summa cum laude, was obtained at the same uni versity. The following examinations were passed: E.C.F.M.G. = Educational Commis sion for Foreign Medical Graduates on 27th July, 1977, in Brussels, Bel gium; V.Q.E. » Visum Qualifying Examination on 5th and 6th September, 1979, in Paris, France. Due to the cooperation of the senior staff of the Department of Paedia trics of the Catholic University of Leuven, Belgium, she was able to obtain her training for paediatrics under Prof.Dr.E. Schretlen, at the Sint Radboudhospital of the Catholic University of Nijmegen, The Nether lands, from 1st October 1979 to 30th September 1983. From 1st October 1983 to 30th September 1984, she worked at the same de partment to finish this thesis. Since 1st October 1984 she is working at the Institute for Anthropogene- tics (head: Prof.Dr.G. Anders) of the medical faculty of the State Uni versity of Groningen, The Netherlands.

195

STELLINGEN

Behorende bij het proefschrift

THE CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER

In het openbaar te verdedigen op 29 november 1984 des namiddags te 2 uur

door LUTGARDE C.P. GOVAERTS I

De kinderen met de diagnose "Infantile phytanic acid storage disease, a possible variant of Refsum's disease" vertonen meer gelijkenis met het syndroom van Zellweger dan met de ziekte van Refsum. Scotto et al: J Inker Metab Dis 5, 83-90, 1982

Analoog aan de bevindingen bij adrenoleukodystrofie kan een func tionele stoornis van de testosteronesynthese bij het syndroom van Zellweger worden verwacht. Griffin et al: Neurology 27: 1107-1113, 1977

III

Toediening van indocid in combinatie met hydrochlorothiazide is vooral bij zuigelingen en jonge kinderen met nefrogene diabetes in sipidus aan te bevelen. Monnens et al: Clinical Science 66: 709-715, 1984

In tegenstelling met de bevindingen vermeld door Tina et al is de excretie van hydroxylysylglycosiden bij het syndroom van Alport normaal. Tina et al: Pediat Res 13: 774-776, 1979 Schröder et al: Abstract, Interni Ped Nephrol 5: 112, 1984

De benaming "Martin-Bell syndrome" verdient de voorkeur boven "fragile X syndrome". Sherman et al: Ann Hum Genet 48: 21-37, 1984 VI

Prenatale diagnostiek is eerder curatieve dan preventieve geneeskunde.

VII

Moleculaire diagnostiek bij "inborn errors of metabolism" vereist ervaring op een specifiek gebied. Centralisatie van de moleculaire diagnostiek is dus een vereiste.

VIII

Het invoeren van een verplichte (maximaal) veertigurige werkweek (in dienstverband) voor het personeel in de gezondheidszorg zou de tewerkstelling en de gezondheid van het personeel ten goede komen, en hoogstwaarschijnlijk ook de gezondheidszorg van de patiënten.

De aanhef "Cher Monsieur" of "Dear Sir" op overdrukaanvraag- kaartjes is verouderd. "Chère Collègue, Cher Collègue" of "Dear Col league" zijn goede alternatieven.

Het wél of niet getrouwd zijn, of plannen daartoe hebben, is voor de "werkgever" tijdens een sollicitatiegesprek met een vrouwelijke (kin- der-)arts blijkbaar belangrijker dan haar medisch-ethisch denken.

Lutgarde C.P. Govaerts 29 november 1984