syndrome (ZS; McKusick 21410) is the most severe of these lethal peroxisomal disorders.',2 Peroxisome,

Transcription

1 Zeliweger Syndrome in Saudi Arabia and Its Distinct Features M. Al-Essa, MD1 Gursev S. Dhaunsi, PhD2 M. Rashed, PhD2 Pinar T. Ozand, MD, PhD1'2 Z. Rahbeeni, MD1 Summary: Clinical and laboratory findings of Zellweger syndrome (ZS) patients diagnosed at King Faisal Specialist Hospital and Research Center (KFSH & RC), Riyadh, Saudi Arabia over a period of 10 years are presented in this report. Eleven patients (nine females and two males) from 2 to 4 months old were referred to KFSH & RC for evaluation of hypotonia, seizures, and dysmorphic features. The common clinical findings included high forehead, large fontanelle, shallow orbit ridges, micrognathia, upslanting palebral fissures, epicanthal folds, severe hypotonia, hyporeflexia, pigmentary retinopathy, optic nerve atrophy, complete or partial agenesis of corpus callusum, and failure to thrive. We did not observe any Brushfield spots, any renal and brain cysts, or adrenal insufficiency. Some unique clinical findings were the presence of gallstones, club feet, or bilateral knee or hip dislocation in some patients. All patients had markedly elevated plasma levels of very long chain fatty acids (VLCFA). Electron microscopy performed on liver biopsies of two patients revealed absence of peroxisomes. Biochemical studies of dermal fibroblasts from three patients showed deficient 13-oxidation of lignoceric acid and dihydroxyacetone phosphate acyltransferase (DHAPATase) activity. The tribal living in Saudi Arabia and our observation that 10 of the 11 parents in this study were first-degree relatives and, except for families 1 and 3, each family had at least another baby who died of the same disease. This suggests that the incidence of ZS in Saudi Arabia may actually be higher than our experience at KFSH & RC. Clin Pediatr. 1999;38:77-86 Introduction ~lhe disorders of peroxisome biogenesis are a group of genetically heterogeneous diseases; Zellweger syndrome (ZS; McKusick 21410) is the most severe of these lethal peroxisomal disorders.',2 Peroxisome, a cellular organelle that exclusively performs or participates in important cellular functions Departments of 1Pediatrics and 2Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia. Reprint requests and correspondence to: Z. Rahbeeni, MD, Consultant, MBC #58, PO Box 3354, KFSH & RC, Riyadh , Kingdom of Saudi Arabia Westminster Publications, Inc., 708 Glen Cove Avenue, Glen Head, NY 11545, U.S.A. such as beta-oxidation of very long chain fatty acids (VLCFA), plasmalogen synthesis, and bile acid synthesis, is not completely formed in ZS.35 Hence, this syndrome is associated with multiple peroxisomal enzyme deficiencies that lead to accumulation of VLCFA and deficient amounts of plasmalogens. ZS, also called the cerebrohepatorenal syndrome, is well known to exhibit multiple congenital anomalies. Patients with ZS have profound neuro- 1999CLINICAL FEBRUARY PEDIATRICS 77 CLINICAL PEDIATRICS 77

2 Al-Essa, Dhaunsi, Rashed, Ozand, Rahbeeni logic impairment with seizures, severe retardation, and dysmorphic features.67 The main diagnostic features are high forehead, upslanting palpebral fissures, hypoplastic supraorbital ridges, epicanthal folds, severe weakness, hypotonia, and eye abnormalities such as cataracts, glaucoma, corneal clouding, Brushfield spots, pigmentary retinopathy, and optic nerve dysplasia.8 Other clinical findings include hepatomegaly, renal and brain cysts, and skeletal changes. In 1964 Bowen and colleagues9 first reported an apparently similar pattern of multiple malformations in two unrelated pairs of siblings in Iowa. Several reports have appeared in the literature now describing the incidence, clinical features, and pathologic abnormalities of ZS.' Zellweger syndrome has been reported from various parts of the world; however, the existence of this lethal disease in Saudi Arabia and the Middle East had remained unreported, owing partly to inadequate health awareness. In this report we document the ocurrence of ZS in Saudi Arabia and describe its distinctive features. The aim of this study is to increase awareness of this lethal disease and encourage an early referral for complete diagnosis and potential treatment. Materials and Methods Clinical Analysis We reviewed the charts of 11 cases of Zellweger syndrome diagnosed by the inborn errors of metabolism section at King Faisal Specialist Hospital and Research Center, the only tertiary referral center for neurometabolic diseases in Saudi Arabia. This review included the referral diagnosis, symptoms, clinical signs and neurologic and ophthalmologic evaluations. Radiologic examination studies included a skeletal survey of the whole body, ultrasound of abdomen and brain, computerized axial tomogram (CT scan) and/or magnetic resonance imaging (MRI) of the brain. The nutritional status and liver, kidney, and adrenal functions were also evaluated. Neurophysiologic studies included electroencephalogram, visual or auditory evoked potentials, brain stem, nerve conduction studies, and electromyogram. Laboratory Analysis Plasma levels of VLCFA were measured by selected ion monitoring gas chromatography-mass spectrometric assay (SIM-GC/MS) as described earlier'011 with some modifications. The fatty acids were extracted from 0.25 ml plasma by use of the Folch procedurel' with nonadecanoic acid (C19:0) and heptacosanoic acid (C27:0) as internal standards for C22:0, C24:0, and C26:0. Analyses were carried out with a Hewlett Packard Mass Selective Detector (HP, Palo Alto, CA) Model 5970B equipped with an HP Model 5890 gas chromatograph and a DB fused silica capillary column (30m x 0.25mm ID; film thickness 0.25 pm). The temperature was increased from an initial 1500C (held for 1 minute) at a rate of 8 C/min to 270 C (held for 9 min). The injection volume was 1 pl, spitless mode at 2600C. The ion at m/z 74 was used for quantification of all analytes, with appropriate molecular ions of each analyte used for verification of the identity of the peaks of interest. Dermal fibroblasts obtained from the skin biopsies of these patients and nonrelated controls were used for biochemical assays of P3-oxidation of lignoceric acid and dihydroxyacetone phosphate acyltransferase (DHAPATase). f3- oxidation of lignoceric acid was measured in control and patient fibroblasts as described previously.'2 DHAPATase activity was measured in fibroblasts according to the method of Hajra and Bishop.4 Reagents were of highest purity and were obtained from Sigma Chemical Company (St. Louis, MO, USA). Results We reviewed charts of 11 patients diagnosed with ZS. These patients included nine females and two males who were 2-4 months old. These patients presented at birth with the typical dysmorphic features and neurologic impairment. Their referral diagnosis was mostly for evaluation of a seizure disorder, hypotonia, and dysmorphic features. The initial common symptoms in these patients were decreased fetal movement, subsequent hypotonia, poor sucking and feeding problems, seizures with or without deafness and/or blindness, and jaundice. Table 1 lists the referral symptoms of these patients, their positive family history, and consanguinity. Clinical Findings The most common physical findings and their relative frequencies are listed in Table 2 as a comparison to the summary by Heymans of 114 cases reported in literature.'3 One of the central diagnostic features in ZS patients is the typical face (Figure 1), which shows high forehead, upslanting palpebral fissures, hypoplastic supraorbital bridge, and epicanthal folds. Patients had severe weakness, neonatal seizures, he- 78 CLINICAL PEDIATRICS 78 ClJN7CAL PEDIATRICS

3 Zellweger Syndrome in Saudi Arabia patomegaly, and hypotonia (Figure 2). Focal myopathy was found upon muscle biopsy of one patient that might be contributing to hypotonia. Eye abnormalities included pigmentary retinopathy and optic nerve atrophy (Figure 3). Other eye abnormalities observed in these patients were cataracts, clouded cornea, glaucoma, and optic disc pallor; however, we did not observe Brushfield spots. Impaired hearing and nystagmus due to blindness were common. The visual evoked potential and electroencephalogram appeared abnormal. We did not observe any brain cysts; how- FEBRUARY~~~~ 199 ever, complete or partial agenesis of corpus callusum was present in eight patients (Figure 4). All patients in this study were failing to thrive. Nine patients had enlargeof liver, six had jaundice and elevated liver enzymes, and one of the patients had gallstones (Figure 5). Ultrasound studies did ment - CLNCLPDITIS 7 CLIAWAL PEDIATRICS 79

4 Al-Essa., Dhaunsi, Rashed, Ozand, Rahbeeni CLINICAL PEDIATRICS CLUNICAL

5 Zellweger Syndrome in Saudi Arabia Figure 1. A ZS patient showing high forehead, micrognathia, depressed nasal bridge, and epicanthal folds. Figure 2. Hypotonia and hepatomegaly in a ZS patient. not reveal renal cysts in any of our patients. Although stippled calcification of the patella occurs in 50% of Zellweger patients,4 only two patients (Figure 6) had patellar calcification in our study. We also observed bilateral congenital hip dislocation in one patient, and another patient had a bilateral knee dislocation. Uncommonly, one patient had club feet. Distinctive Features We observed that the Saudi ZS patients were different from those in the reported literature in the following respects: (1) unusually high consanguinity and positive family history, (2) absence of Brushfields spots and low incidence of optic disc pallor, (3) absence of renal or brain cysts, (4) absence of adrenal insufficiency, (5) skeletal abnormalities including bilateral congenital hip or knee dislocation and club feet. Besides the aforementioned findings one patient had gallstones. Laboratory Findings Electron microscopy of liver biopsies from two patients revealed absence of peroxisomes CLIMCAL PEDIATRICS~ ~~~~~~~~~~~~~~~~ CLINICAL PEDIATRICS

6 Al-Essa, Dhaunsi, Rashed, Ozand, Rahbeeni Figure 3. patient. Optic atrophy associated with retinal degeneration in a ZS Figure 4. Magnetic resonance image (MRI) of brain in a ZS patient showing the absence of corpus callusum. (Figure 7). Figure 7A (an electron micrograph of liver biopsy from a control subject) shows various subcellular organelles, such as peroxisomes, mitochondria, and endoplasmic reticulum. Figure 7B (an electron micrograph of liver biopsy from one of our ZS patients) shows the absence of peroxisomes. P- oxidation of lignoceric acid and DHAPATase activity (Table 3) were found to be markedly low in dermal fibroblasts from ZS patients as compared with the control dermal fibroblasts. Plasma VLCFA levels were abnormally elevated in all patients in this study (Table 3). Discussion Peroxisomal disorders are a group of inherited metabolic defects that are caused by dysfunction of a single or multiple peroxisomal enzymes. Disorders of peroxisomal biogenesis are genetically autosomal recessive defects in which assembly of the entire organelle is defective thereby causing multiple peroxisomal enzyme deficiencies. Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and hyperpipecolic acidemia are the well-known peroxisomal biogenesis disorders.'5 ZS is the most severe of this lethal group of disorders, is apparent at birth, and results in death within the first year of life. In 1964 Bowen et a19 first described a similar pattern of multiple malformations apparent at birth in two unrelated pairs of siblings in Iowa and Maryland, and since then, several reports have appeared in the literature on the frequency of ZS in various parts of the world. However, the incidence of this lethal disease in middle eastern countries has remained unknown. This is the first report documenting the experience with ZS patients in Saudi Arabia and the distinctive features of this disease in Saudi patients. 82 CLINICAL PEDIATRICS 82 CLINICAL PEDIATR[CS

7 Zellweger Syndrome in Saudi Arabia Figure 5. Abdominal ultrasound of a ZS patient showing a gallstone with posterior shadowing (arrow). Zellweger syndrome, also called the cerebrohepatorenal syndrome, is associated with profound neurologic impairmeat and characteristic dysmorphic features. Patients with ZS show striking and consistent abnormalities that can be easily recognized. The main diagnostic features are high forehead, large fontanelle, shallow orbital ridges, epicanthus, high arched palate, external ear deformity, and micrognathia. Neurologic manifestations of ZS include severe hypotonia, abnormal Moro response, hyporeflexia or areflexia, epileptic seizures, and nystagmus. Other clinical manifestations include eye abnormalities like cataract, optic disc pallor, and retinal degeneration; hepatomegaly; failure to thrive; calcified stippling of patella; and severe psychomotor and sensorial retardation. Patients reviewed in the present study manifested most of the above-mentioned clinical and neurologic abnormalities that are characteristic of classical ZS; however, Saudi patients had some distinct clinical features. The absence of Brushfield spots and low incidence of optic disc pallor were a striking difference from the reported eye abnormali- Figure 6. ties in ZS. Pathologic studies in the survey by Heymans revealed the presence of renal cysts in 78 of 80 patients.13 However, we did not observe any renal or brain cysts in our patients. Other distinctive features such as absence of any adrenal insufficiency, bilateral congenital hip or knee dislocation, and club feet suggest that Saudi patients might have a different mutation and different mechanisms of pathogenesis. ZS has been reported to be associated with hepatomegaly, liver cirrhosis, and cholestasis; however, gallbladder pathology has not been reported.' The formation of gallstones in one patient of our study is a peculiar observation that has not been linked to ZS previously. The exact cause and mechanism of gallstone formation in this ZS patient is not readily available; however, abnormal liver functions and bile acids in ZS X-ray film showing patellar calcification in one of the ZS patients (arrows.) CLINICAL PEDIATRICS 83 CLINICAL PEDIATRICS 83

8 Al-Essa, Dhaunsi, Rashed, Ozand, Rahbeeni A B Figure 7. Electron micrographs showing various subcellular organelles in the liver biopsies from (A) a normal subject and (B) a ZS patient. P, M, L, and rer represent peroxisomes (arrow), mitochondria, lysosomes, and rough endoplasmic reticulum, respectively. patients in our study could partly be responsible for the formation of gallstones, or this could simply be an isolated finding. Bilateral knee or hip dislocations as observed in our study is a distinct feature for the Saudi ZS patients, and the severity of hypotonia in these patients might have facilitated these joint dislocations. The other unique observation in our review is consanguinity and the positive family history. ZS is an autosomal recessive inherited metabolic disease, and all patients in our study fit an autosomal recessive pattern of genetic inheritance. Each parent in this study was married to a first-degree cousin and in each family (except in families 1 and 3) at least another child had died of symptoms similar to ZS. All family members of ZS patients in our study received genetic counseling about 25% risk in each pregnancy. The tribal living that allows marriage between first cousins and inadequate health awareness in Saudi Arabia allow high frequency of inherited diseases in Saudi Arabia. Biochemical abnormalities of ZS include absence or reduced number of peroxisomes, deficient synthesis and reduced tissue levels of plasmalogens, deficient oxidation, and age-dependent accumulation of phytanic acid.16,17 Electron microscopy of liver biopsies 84 CLINICAL PEDIATRiCS 84 CLINICAL PEDIATRICS

9 Zellweger Syndrome in Saudi Arabia first encounters ZS patients be aware of the presence of this disease in the community and its clinical symptoms for an early referral and diagnosis. from ZS patients in our study revealed the absence of peroxisomes, thereby supporting our diagnosis of ZS. Furthermore, abnormally elevated plasma levels of VLCFA in all the patients supported the contention of a peroxisomal dysfunction in our patients. Peroxisomal n-oxidation of fatty acids is defective in ZS; therefore, measurement of the rate of oxidation of VLCFA in cultured skin fibroblastsl8 or measurement of VLCFA levels19 in chorionic villus samples have been used in postnatal and prenatal diagnosis of peroxisomal disorders, respectively. Deficient oxidation of VL- CFA in skin fibroblasts from patients in our study suggests a peroxisomal disorder. Heymans and coworkers20 showed that tissue of five infants who died with Zellweger syndrome contained less then 10% of the normal levels of phophatidylethanolamine plasmalogen, a major phospholipid of cell membranes. The key enzymes in the synthesis of plasmalogens are known to be located exclusively in peroxisomes, and the measurement of dihydroxyacetone phosphate acyltransferase, a peroxisomal enzyme for plasmalogen synthesis, has been used as a diagnostic method in ZS.21 We also ow served deficient DHLAPATase activity in dermal fibroblasts from our patients. In conclusion, it is important that the general pediatrician who REFERENCES 1. Lazarow PB, Moser HW. Disorders of peroxisome biogenesis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. 7th ed. New York: McGraw Hill, 1995: Wilson GN, Holmes RG, Custer J, et al. Zellweger syndrome: diagnostic assays, syndrome delineation, and potential therapy. AmJMed Genet. 1986; 24: Singh I, Moser AE, Goldfischer S, Moser HW. Lignoceric acid is oxidized in the peroxisome: implications for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy. Proc Natl Acad Sci USA. 1984; 81: Hajra AK, Bushop JE. Glycerolipid biosynthesis in peroxisomes via the acyl dihydroxyacetone phosphate pathway. Ann N Y Acad Sci. 1982; 386: Goldfischer S, Moore CL, Johnson A, et al. Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science. 1973;182: Kelley RI. Review: the cerebrohepatorenal syndrome of Zellweger, morphologic and metabolic aspects. AmJ Med Genet. 1983;16: Govaerts L, Monnens L, Tegelaers W, et al. Cerebro-hepato-renal syndrome of Zellweger: clinical symptoms and relevant laboratory findings in 16 patients. EurJPediatr. 1982;139: Schutgens RB, Heymans HS, Wanders RJ, et al. Peroxisomal disorders: a newly recognized group of genetic diseases. EurJ Pediatr. 1986;144: Bowen P, Lee CSN, Zellweger H, Lindberg R. A familial syndrome of multiple congenital defects. BullJohn Hopkins Hosp. 1964;114: Caruso U, Fowler B, Erceg M, Romano C. Determination of very-longchain fatty acids in plasma by a simpli- CLINICAL PEDIATRICS CLINICAL PEDIATRICS

10 Al-Essa, Dhaunsi, Rashed, Ozand, Rahbeeni fled gas chromatographic-mass spectrometric procedure. J Chromatograph. 1991;562: Vallance H, Applegarth D. An improved method for the quantification of very long chain fatty acids in plasma. Clin Biochem. 1994;27: Hashmi M, Stanley W, Singh I. Fatty acid metabolism in cultured skin fibroblasts from patients with peroxisomal disorders: lignoceryl-coa ligase deficiency in childhood adrenoleukodystrophy. Fahimi HD, Seis H, eds. In: Peroxisomes in Medicine and Biology. Springer-Verlag, Heidelberg, 1987; Heymans HS. Cerebro-hepato-renal (Zellweger) syndrome. Clinical and biochemical consequences of peroxisomal dysfunction. Thesis. University of Amsterdam, Poznanski AK, NosanchuckJS, Baublis J, Holt JF. The cerebro-hepato-renal syndrome (CHRS) (Zellweger syndrome). AmJRadiol. 1970;109: Brown FR III, Voigt R, Singh AK, Singh I. Peroxisomal disorders. Neurodevelopmental and biochemical aspects. AmJDis Child. 1993;147p Schrakamp G, Roosenboom CF, Schutgens RB, et al. Alkyl dihydroxyacetone phosphate synthase in human fibroblasts and its deficiency in Zellweger syndrome. J Lip Res. 1985; 26: Wanders RJ, Smit W, Heymans HS, et al. Age-related accumulation of phytanic acid in plasma from patients with the cerebro-hepato-renal (Zellweger) syndrome. Clin Chim Acta. 1987;166: Singh I, Moser HW, Moser AB, Kishimoto Y. Adrenoleukodystrophy: impaired oxidation of long chain fatty acids in cultured skin fibroblasts and adrenal cortex. Biochem Biophys Res Comm. 1981;102: Rocchiccioli F, Aubourg P, Choiset A. Immediate prenatal diagnosis of Zellweger syndrome by direct measurement of very long chain fatty acids in chorionic villus cells. Prenat Diag. 1987;7: Heymans HS, Schutgens RB, Tan R, et al. Severe plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature.1983; 306: Datta NS, Wilson GN, Hajra AK Deficiency of enzymes catalyzing the Biosynthesis of glycerol-ether lipids in Zellweger syndrome. A new category of metabolic disease involving the absence of peroxisomes. N Engl J Med. 1984;31 1: CLINICAL PEDIATRICS