Peroxisomal Disorders: Clinical and BiochemicalStudies in 15 Children and Prenatal Diagnosis in7 Families

Steven J. Steinberg,1 Nursel Elcioglu,1 Christina M. Slade,1 Arun Sankaralingam,2Nicholas Dennis,3 Shehla N. Mohammed,1 and Anthony H. Fensom1*1Division of Medical and Molecular Genetics, United Medical and Dental Schools of Guy’s and St. Thomas’sHospitals (UMDS), London, United Kingdom

2Department of Clinical Biochemistry, Guy’s Hospital, London, United Kingdom3Wessex Clinical Genetics Service, The Princess Anne Hospital, Southampton, United Kingdom

We describe the main clinical and biochemi-cal findings in 15 patients with peroxisomaldisorders, together with the results of 11prenatal investigations for Zellweger syn-drome. The initial laboratory diagnosis de-pended in most cases on demonstration ofelevated very long chain fatty acids inplasma, but follow-up studies using cul-tured fibroblasts were essential for com-plete classification. The patient group com-prises nine cases of Zellweger syndrome,one of neonatal adrenoleucodystrophy, twoof infantile Refsum disease, one of bifunc-tional protein deficiency, and two of rhizo-melic chondrodysplasia punctata. The studyillustrates the clinical and biochemical vari-ability of this group of patients and the de-tailed studies that are required for classifi-cation. Am. J. Med. Genet. 85:502–510, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: Zellweger syndrome; PEXgenes; rhizomelic chondro-dysplasia punctata

INTRODUCTIONPeroxisomal disorders are a heterogeneous group of

metabolic diseases characterised by a deficiency of oneor more functions of peroxisomes [Lazarow and Moser,1995]. The disorders can be classified into two major

groups. In the first there is a defect in peroxisome as-sembly and a consequent impairment of multiple per-oxisomal functions. In the second there is deficiency ofa single peroxisomal enzyme or step within a pathway.The assembly defects can be further subdivided intodisorders in which biogenesis of the whole organelle isdefective, resulting in absence or greatly reduced num-bers of peroxisomes in the tissues and multiple enzymeabnormalities; and disorders in which peroxisomes arepresent but where there is deficiency of a subset ofenzymes that are not imported to the organelle. Thebiogenesis (or generalised) peroxisomal disorders(GPD), which include Zellweger syndrome (ZS), neona-tal adrenoleucodystrophy (NALD), and infantile Ref-sum disease (IRD), are characterised by neurological,hepatic and renal abnormalities, and mental retarda-tion [Lazarow and Moser, 1995]. The failure in theseconditions to form peroxisomes leads to impairment ofmany peroxisomal functions including b-oxidation ofvery long chain fatty acids (VLCFA), plasmalogen bio-synthesis, pipecolic acid oxidation, phytanic acid oxida-tion, and bile acid synthesis.

Rhizomelic chondrodysplasia punctata (RCDP), adisorder of peroxisomal enzyme import, has a pheno-type distinct from that of the biogenesis disorders.There is proximal shortening of the limbs, extensivepunctate calcifications, ichthyosis, cataracts, and se-vere mental retardation. These patients fail to importproteins encoded with the peroxisomal targeting sig-nal-2 (PTS-2) [Slawecki et al., 1995]. Biochemically,there is deficient plasmalogen biosynthesis and phy-tanic acid oxidation. Peroxisomal b-oxidation is normalalthough one of the enzymes, the ketothiolase, is pre-sent in a precursor form.

Among the peroxisomal disorders with a single en-zyme defect are disorders due to deficiency of an en-zyme of the peroxisomal b-oxidation pathway or ofplasmalogen biosynthesis. The former diseases havephenotypes resembling GPDs [Poll-The et al., 1988;Schram et al., 1987; Watkins et al., 1989], whereas inthe latter the phenotypes resemble RCDP [Wanders etal., 1992, 1994]. Ten complementation groups have

Contract grant sponsor: LORS; Contract grant number: 93/07.UMDS is now Guy’s, King’s and St Thomas’s Schools of Medi-

cine, Dentistry, and Biomedical Sciences.*Correspondence to: Anthony H. Fensom, Ph.D., Division of

Medical and Molecular Genetics, Guy’s, King’s, and St. Thomas’sSchools of Medicine, Dentistry and Biomedical Sciences, 8thFloor, Guy’s Hospital Tower, London Bridge, London SE1 9RT,United Kingdom. E-mail: a.fensom@kcl.ac.uk

Received 16 October 1998; Accepted 9 March 1999

American Journal of Medical Genetics 85:502–510 (1999)

© 1999 Wiley-Liss, Inc.

been established for GPDs with use of somatic cell hy-bridisation studies [Brul et al., 1988; Shimozawa et al.,1993; Moser et al., 1995], cells from patients with gen-eralised disorder belonging to nine groups (CG1–CG4and CG6–CG10), and classic RCDP cells belonging toone group (CG11) (Table I). Groups 1 (the predominantgroup of the GPDs), 2, 4, 6, 7, and 8 contain patientswith more than one phenotype, indicating considerableallelic heterogeneity at each locus. Genes associatedwith seven of the complementation groups have nowbeen identified (Table I), much of the work followingfrom studies of yeast and exploiting homology betweenyeast and human peroxisomal proteins. By commonconsent [Distel et al., 1996], proteins involved in per-oxisomal biogenesis are called peroxins and the genesare designated PEX.

Complementation studies have also been used to re-solve the genetic basis of disorders of peroxisomalb-oxidation. In the bifunctional protein (BF) deficiency,which in most, and possibly all, cases is now thought tobe due to defects in the D- rather than the L-bifunctional protein (Suzuki et al., 1997; van Grunsvenet al., 1998a, 1999), there are three complementationsubgroups, dependent on whether either both the hy-dratase and 3-hydroxyacyl-CoA dehydrogenase activi-ties of the BP are deficient, or only one or the other ofthese activities is missing (van Grunsven et al., 1997,1998b). In previous publications from our laboratorywe have detailed our experience using VLCFA analysisas a preliminary screening procedure for some peroxi-somal disorders [Steinberg et al., 1994] and have de-scribed subsequent complementation analysis of pa-tients found to have a GPD [Steinberg and Fensom,1996]. In the present communication we describe indetail the clinical and biochemical findings in 15 pa-tients of varying phenotypes studied in our laboratoryand document the results from 11 prenatal investiga-tions.

MATERIALS AND METHODS

Culture of fibroblasts and measurement of very longchain fatty acids, plasmalogen biosynthesis, peroxi-somal b-oxidation and catalase solubility were per-formed as previously described [Steinberg et al., 1994].Dihydroxyacetone phosphate acyl transferase (DHAP-AT) was assayed in extracts of white blood cells, cul-

tured cells, and chorionic villi, as described by Schut-gens et al. [1984]. Phytanic acid oxidation was mea-sured by using [1-14C]phytanic acid as substrate[Hoefler et al., 1988] in our laboratory. Two RCDP pa-tients were tested in the laboratory of Mrs. A. Moser byusing [2,3-3H]phytanic acid as substrate. Somatic cellcomplementation analysis was performed as described[Steinberg and Fensom, 1996].

RESULTSClinical Reports and Biochemical Results

Using measurement of plasma VLCFA and phytanicacid as preliminary screening protocol and follow-upanalysis with cultured fibroblasts, we confirmed thediagnosis of a peroxisomal disorder in 15 patients.From the biochemical results (Table II) and clinicalphenotype the patients were classified as ZS (9 cases),NALD (1 case), IRD (2 cases), RCDP (2 cases), andbifunctional protein deficiency (1 case). The ZS pa-tients (patients 1–9) shared many features in commonand are described only briefly below; the other patientshad more variable manifestations and are described ingreater detail. There was parental consanguinity infour of the cases (patients 6, 7, 9, and 15).

Patient 1. DI (male) had a wide anterior fontanelleand prominent forehead, low nasal bridge, epicanthicfolds, flattened philtrum, and narrow upper lip, to-gether with bilateral clinodactyly and correctable tali-pes equinus varus. An anal dilatation was performedfor anal stenosis. He was profoundly hypotonic withpoor sucking and required nasogastric feeds. At 5weeks he was found to have coarse lateral nystagmusof equal amplitude in both directions, with no visualfollowing and pale optic discs. He had a soft grade 1/6midsystolic murmur, and the liver was palpable 1 cmbelow the costal margin. Skeletal radiographs showedstippled epiphyses at the distal ends of the femora andthe proximal ends of the tibia. An ultrasound head scanshowed ventricular dilation, and a CT scan demon-strated decreased density in the white matter. He diedat 4 months of age.

Patient 2. KB (female) had typical craniofacial fea-tures of ZS, including a high forehead, bitemporal nar-rowing, anteverted nostrils, flat face, and low-set ears(Fig. 1). She was profoundly hypotonic and had dislo-cated hips and poor vision. She had ventricular dilata-tion and cerebral atrophy with abnormal gyral pattern.

TABLE I. Genes and Peroxins Associated With Different Peroxisomal Complementation Groups

Complementationgroup no. Phenotypes Gene Peroxin characteristics or function Reference

CG 1 ZS, NALD, IRD PEX1 117–127 kD AAA-afamily ATPase Reuber et al. [1997]; Portsteffen et al. [1997]CG 2 ZS, NALD PEX5 PTSI receptorCG 3 ZS PEX12 40–48 kD C3HC4 zinc-binding IMPa Dodt et al. [1995]CG 4 ZS, NALD PEX6 112–127 kD AAA-family ATPase Chang et al. [1997]CG 6 ZS, NALD ? ? Fukuda et al. [1996]CG 7 ZS, NALD PEX10 34–48 kD C3HC4 zinc-binding IMPCG 8 ZS, NALD, IRD ? ? Warren et al. [1998]CG 9 ZS ? ?CG 10 ZS PEX2 35–52 kD C3HC4 zinc-binding IMP Shimozawa et al. [1992]CG 11 RCDP PEX7 PTS2 receptor Braverman et al. [1997]; Motley et al. [1997];

Purdue et al. [1997]

aAAA 4 ATPase associated with diverse cellular activities; IMP 4 integral membrane protein; other abbreviations as in the text.

Peroxisomal Disorders 503

There was calcific stippling of the patellae and re-tarded skeletal maturation. She suffered from fits anddied at 9 months of age after severe pneumonia.

Patient 3. SS (female) was born preterm at 35weeks when polyhydramnios was noted. The baby re-quired a nasogastric tube because of poor feeding. At 18hr of age she had generalised seizures and was treatedwith phenobarbitone. She had a high forehead, hyper-telorism, shallow supraorbital ridges, widely patentanterior and posterior fontanelles, bilateral positionaltalipes equinovarus, and extreme hypotonia. She diedduring the first week from fulminating necrotising en-terocolitis and peripheral circulatory failure.

Patient 4. RW (female) was a premature (28weeks) baby with a phenotype resembling ZS, with hy-potonia, abnormal head ultrasound scan, and conju-gated hyperbilirubinaemia. She died at 2 months ofage.

Patient 5. JS (female) had wide fontanelles, lownasal bridge, upturned palpebral fissures, epicanthus,low-set ears, redundant folding of the neck, limitationof extension of knees and elbows, camptodactyly, sim-ian crease, and hip dislocation. Seizures occurred in theneonatal period, although at that time cerebral ultra-sound and EEG were normal. By 2 months of age,growth and neuromotor retardation were obvious, withglobal hypotonia. She had a transient episode of jaun-dice and high serum levels of BUN and creatinine. In-travenous urography and renal ultrasound resultswere normal. The ophthalmological examination wasalso normal. At the age of 4 months she had no reac-tions to visual or auditory stimuli. Nystagmus and mi-crocephaly were noted, and the fontanelles were stillvery wide. A CT scan disclosed a diffuse cerebral atro-

phy. Her clinical course was marked by severe failureto thrive and no neuromotor development. Episodes ofseizures and bronchopneumonia became frequent,leading to death at age 18 months.

Patient 6. BH (male) was the second child of Bang-ladeshi first-cousin parents. He had profound hypoto-nia, weak Moro response, and he displayed extremelypoor feeding. He had a broad-based nose, low-set ab-normal ears, wide sutures, and large fontanelles. Ab-normal findings included an abnormal EEG, pathologi-cal liver function with hyperbilirubinaemia, unusualcalcific stippling in the patellae and acetabula, andhigh echogenicity on renal ultrasound. He was ex-tremely floppy and died at 2 weeks of age. Prenataldiagnosis was performed in three further pregnanciesof the mother and indicated in each case a normal fetus(see cases 6, 7, and 8 in Table III).

Patient 7. TA (female) was the first child of first-cousin Kuwaiti parents. She had severe hypotonia,large fontanelles, epicanthus, low-set ears, and high-arched palate. Hepatomegaly, hyperbilirubinaemia,and hypoplastic labia majora were observed. There wasno follow-up after 3 months, and although we haveclassified her as ZS, we cannot be sure that she shouldnot be classified as NALD. Fibroblasts from TA werefound to belong to complementation group 2, and mu-tation analysis showed that the patient was homozy-gous for an Asn489Lys substitution in the PEX5 gene[Steinberg and Fensom, 1996].

Patient 8. HP (female) was born normally at termwith large fontanelles, a flat face, slightly prominenteyes, micrognathia, low-set ears, single palmarcreases, and bilateral talipes equinovarus deformities.She was hypotonic and failed to thrive because of poor

TABLE II. Biochemical Results Obtained With Cultured Fibroblasts*

Family no.and patientidentification

VLCFA

DHAP-AT(nmol/2 hr/mg protein)

Plasmalogenbiosynthesis

(3H:14C)

Phytanic acidoxidase (see

footnotesa,b)

Catalasesolubility

(%)

Comple-mentation

groupC26

(mg/mg protein) C26:22

Zellweger syndrome1 Dl ? 0.621 0.847 0.2 10.3 ND 78 ND

fl 1.631 2.513 0.2 6.1 ND 89 12 KB / 0.444 0.795 0.1–0.2 9.4 0.9a 85 13 fS 1.162 1.440 0.4 9.7 ND 77 14 RW / 0.645 1.150 0.3 4.7 ND 92 15 JS / 0.681 0.496 0.2 4.5 ND 81 16 BH ? 0.230 0.270 0.3 6.7 ND ND ND7 TA / 0.842 0.852 ND 1.2 ND 76 28 HP / 1.235 1.570 0.080 1.8 ND 78 ND9 fR 1.145 1.776 0.1 27.7 ND 78 7Neonatal adrenoleucodystrophy10 NP / 0.346 0.783 0.6 3.2 0.4a 78 1Infantile refsum disease11 FW / 0.267 0.578 0.3 7.9 ND 83 112 JG ? 0.320 0.876 0.7 1.2–4.9 0.4a 77 8Rhizomelic chondrodysplasia punctata13 ST / ND ND 0.1 27.7 8.79b ND NA14 CW / ND ND 0.2 6.8 7.00b ND NABifunctional protein deficiency15 MA ? 0.649 1.049 4.7 0.7 ND 8 NANormal range 0.062 ± 0.194 0.026 ± 0.109 0.8–4.5 0.4–0.8 5.3–22.0a 9–33

and mean ± SD (n 4 14);0.11 ± 0.037

(n 4 14);0.055 ± 0.022

(n 4 27);2.21 ± 0.882

(n 4 28);0.55 ± 0.10

31.6 ± 6.9b (n 4 12);17.8 ± 6.5

—

*f preceding an identification indicates a fetal strain; DHAP-AT 4 dihydroxyacetone phosphate acyl transferase; VLCFA 4 very long chain fatty acids;ND 4 not done; NA 4 not applicable.a 14C substrate (results expressed as % substrate oxidised).b 3H substrate (results expressed as pmol/hr/mg protein; data of A. Moser).

504 Steinberg et al.

feeding. Echocardiograms, renal ultrasound, and MRIbrain scans were normal. At the age of 6 months thepatient was very hypotonic and developmentally de-layed but gaining weight. Examination at the age of 11months showed persistent hypotonia, recurrent chestinfections, and poor vision with normal fundi. A firmliver with splenomegaly and prolonged prothrombintime suggested a progressive cirrhosis. The patientdied at age 19 months.

Patient 9. IR (male) was born to first-cousin Paki-stani parents. He died at age 4 months without bio-chemical investigation but with strong clinical suspi-cion of ZS. He had a history of liver disease, metaphy-seal skeletal changes, large anterior fontanelle, and, onnecropsy, renal cysts. Fibroblasts obtained from a posi-tive prenatal diagnosis in a subsequent pregnancy ofthe mother (fR in Table II) had multiple peroxisomalabnormalities consistent with a GPD and belonged tocomplementation group 7 [Steinberg and Fensom,1996].

Patient 10. NP (female) (Fig. 2a) was born to awhite couple by emergency caesarean section for fetaldistress at term. Birth weight was 2,140 g. There wassevere birth asphyxia with Apgars of 1, 6, and 10 at 1,5, and 10 min, respectively. Several physical anomalies

were noted, including a large mouth, small mandible,small head, large fontanelle with separated sutures,and bilateral transverse palmar creases. There wasgeneralised hypotonia and several episodes of diar-rhea, vomiting, and circulatory collapse during the neo-natal period. There was considerable feeding difficultyand initial failure to thrive. At 6 months of age poorvision was noted. On examination bilateral optic atro-phy was found, and absence of visual function was con-firmed by ERG. At this time slight hepatomegaly wasnoted, but this was never a striking or consistent find-ing. From 6 to 12 months there was gross generaliseddevelopmental delay with convulsions that were even-tually controlled with phenytoin. By age 2 years shehad made some slow developmental progress. Shecould sit unaided and would use the word “no” occa-sionally. However, at 4 years she was blind and quad-riplegic with severe developmental delay. From 4 to 16years she was nursed at home by her parents; she wastotally dependent on them for feeding, changing, andmoving. She was unable to talk but was able to swallowliquids and soft foods. There was virtually no commu-nication, but she was able to express pleasure and re-spond to stimulation. After age 16 she gradually dete-riorated and became unable to swallow. She died at 17years during an episode of pneumonia. Biochemicalstudies showed deficient peroxisomal functions in fi-broblasts (Table II) consistent with a GPD. Previouslywe described this patient as a case of long-surviving ZS[Steinberg and Fensom, 1996], but NALD is probably amore correct diagnosis. A younger brother of NP hasessentially the same condition (Fig. 2b). At age 17years he is in long-term residential care. Biochemicalstudies of fibroblasts from NP confirmed multiple per-oxisomal enzyme deficiencies. In addition, she hadmarkedly elevated concentrations of VLCFA, phytanicacid, and pipecolic acid in plasma. Biochemical studieson the younger brother of NP were performed else-where and confirmed a GPD.

Patient 11. FW (female) was born at term after anuneventful pregnancy. The only abnormalities noted atbirth were a large fontanelle and a sacral dimple. Inthe first 4 months there was failure to thrive due topoor feeding, and poor vision because she never fixed orfollowed. At the age of 6 months craniofacial anomalieswere noted: a round face, flat occiput, large fontanelles,high forehead, frontal bossing, epicanthal folds, tele-canthus, depressed nasal bridge, small mouth, protrud-ing tongue, low-set ears, and short neck. In addition,she had bilateral simian creases, overlapping toes, anda coccygeal dimple. There was mild hypotonia, sensoryneural deafness, and rotatory nystagmus. Radiographsurvey was normal without any calcification. A CT scanwas normal. Renal ultrasound, IVP, and cystographyshowed bilateral vesicopelvicalyceal dilatation andvesicourethral reflux (grade II left; grade III right). Inaddition, there was gallbladder lithiasis and mildly de-ranged liver function. Fundoscopic examinationshowed a pigmentary retinopathy (Leber’s congenitalamaurosis). The ERG was extinguished. Brainstem au-ditory evoked potentials showed no reaction up to 65–70 dB. She received treatment for pyelonephritis andwas treated with a low phytanate diet. Some weight

Fig. 1. Facial anomalies in Zellweger syndrome. Note epicanthus, hy-pertelorism, depressed nasal bridge, shallow orbital ridges, and high fore-head (patient KB).

Peroxisomal Disorders 505

gain occurred, but at 1 year of age height and weightwere between the 25th and 50th centiles. She showedmarked motor delay and truncal and limb hypotonia,but with preserved reflexes, the most appropriate clini-cal designation being IRD.

Patient 12. JG (male) has been described before[Steinberg et al., 1994]. Briefly, he was floppy at birthand had rotatory nystagmus but did not require specialcare. He demonstrated signs of development, crawlingat 9 months and walking at 2 years of age. Sensorineu-ral deafness was suspected by 2 years of age, but thiswas not confirmed until 4 years. By the age of 11 yearshe was assessed to have a mental age of 4.5–5.5 years,but this may have been underestimated because of hisauditory and visual (pigmentary retinopathy) deficits.Chronic liver disease was the main clinical problem atthat time; in addition, he had chronic anaemia, yellow-ish skin lesions that appeared around the age of 10years, and an ataxic gait. Nonetheless, he was attend-ing a special school for the deaf and was making prog-ress. He was not screened for a peroxisomal disorderuntil he was 12 years old, when only moderately el-evated VLCFAs were found in plasma [Steinberg et al.,1994]. However, study of cultured fibroblasts (Table II)confirmed that the patient had a GPD. We concludethat IRD is the most appropriate designation. Comple-

mentation analysis showed that he belongs to group 8[Steinberg and Fensom, 1996].

Patient 13. ST (female) was the first child of un-related healthy parents and was strongly suspected tohave RCDP at birth. Characteristic clinical manifesta-tions included shortness of the proximal segments ofthe arms and legs, frontal bossing, and a flat nasalbridge. She had a widespread stippling appearance ofthe skeletal system, particularly of the epiphysis (Fig.3a), and bilateral cataracts. Although she was aware oflight, she did not fix or follow. She was oxygen-dependent and required to be fed mainly by nasogastrictube until she died at 4 months of age. Plasma VLCFAswere normal. Study of cultured fibroblasts (Table II)showed greatly diminished de novo plasmalogen bio-synthesis and deficient phytanic acid oxidase. How-ever, plasma phytanic acid was not elevated at 1 monthof age.

Patient 14. CW (female) was born preterm, thesecond child of unrelated parents whose other daugh-ter is healthy. She had a flat nasal bridge, short nose,and congenital cataracts. Her radiographs showed anepiphyseal punctate appearance with proximal limbshortening (Fig. 3b). At the age of 6–7 weeks her de-velopment was fairly normal for age, but she had anunusual rash involving large areas of the body and ich-

Fig. 2. Appearance of neonatal adrenoleucodystrophy patients. Note deep set eyes, slightly beaked nose, and wasting. (a) Patient NP at 8 years. (b)Patient RP, younger brother of NP, at 17 years.

506 Steinberg et al.

thyosiform erythroderma. She was smiling at 4 weeks.Despite the development of cataracts, she was visuallyalert and responsive. At the age of 3 months she hadsurgery for her cataracts. The severity of her diseaseappears to be at the milder end of the spectrum forRCDP. Her most recent problems at age 18 monthswere failure to thrive, recurrent chest infections, and akyphoscoliosis for which she is being treated with abrace. Biochemically, VLCFAs were normal in plasma,but plasmalogen biosynthesis and phytanic acid oxi-dase were deficient in fibroblasts (Table II). Plasmaphytanic acid was normal at 1 month but elevated at 16months (20 mg/L; normal <6).

Patient 15. MA (male) was the fourth child of first-cousin Bangladeshi parents living in the United King-dom. The three older children are well. He had a phe-notype similar to ZS and developed intractable seizuresat 1 week of age. No developmental progress was ap-parent, and he was probably blind. The baby was lost tofollow-up when the parents took him back to Bangla-desh. Plasma VLCFA were elevated in plasma and fi-broblasts, but further study showed no abnormalitiesin plasmalogen biosynthesis or catalase latency (TableII). A single defect in peroxisomal b-oxidation was con-cluded, and complementation analysis with cells of con-firmed b-oxidation defect showed that the child wasdeficient in the bifunctional protein [Steinberg andFensom, 1996].

Prenatal Diagnosis

Prenatal diagnosis was performed in 10 pregnanciesfrom 6 families by analysis of chorionic villi (CVS) andcultured villous cells, and in 1 pregnancy from a sev-enth family using cultured amniocytes. Biochemical re-sults for all cases are shown in Table III. CVS speci-mens were analysed directly for DHAP-AT activityand, in some cases, for VLCFA content. A portion ofthe villi was also cultured for confirmatory studies,which comprised measurement of de novo plasmalogenbiosynthesis and VLCFA content. The cultured amni-otic cells were analysed for DHAP-AT activity andVLCFAs.

The results of the DHAP-AT measurement in villiwere reported immediately to the referring source. Incases 1 and 5 DHAP-AT was markedly deficient, andthe mothers chose to terminate the pregnancies. Theresults were subsequently confirmed by analysis of thecultured villi (Table III) and fetal fibroblasts culturedfrom termination products (Table II).

Analysis of VLCFA directly in termination villi ob-tained from cases 1 and 5 showed significantly elevatedlevels when compared with villi from normal controls(Table III). In cases 3, 6, 7, and 9 VLCFAs were mea-sured directly in the test biopsy samples, and resultswere normal in agreement with all other parametersmeasured for these cases, but these results were re-

Fig. 3. (a) Widespread stippling appearance of the skeletal system,especially of the epiphysis in rhizomelic chondrodysplasia punctata (pa-tient CW). (b) Epiphyseal punctate appearance with proximal limb short-ening in rhizomelic chondrodysplasia punctata (patient ST).

Peroxisomal Disorders 507

garded as “experimental” and were not reported withthe DHAP-AT results.

DHAP-AT activity in villi from case 8 was below thenormal range although not as deficient as found in theearlier affected pregnancies. However, because simul-taneous normal control villi tested with this samplealso displayed low activity, indicating nonoptimal as-say conditions on the day, the fetus was diagnosed asunaffected; this was confirmed by analysis of the cul-tured villi. Results of the analysis for villi and culturedvilli from all other patients were within the normalranges, and unaffected fetuses were predicted.

Case 11 was late-presenting and was investigatedwith cultured amniocytes. No biochemical data wereavailable for the index case, but the clinical informa-tion strongly suggested that two previous children bornto the couple had ZS. Analysis of DHAP-AT activityand VLCFA accumulation (Table III) indicated that thefetus was affected with a GPD, and a late second-trimester termination was performed. Biochemicalstudy of cultured fibroblasts (Table II) and fetal liver(not shown) from this fetus, together with a detailedpostmortem examination [Steinberg and Fensom,1996], confirmed the diagnosis.

DISCUSSION

The 15 peroxisomal patients have been studied ex-tensively in their clinical presentation and biochemicalaberrations. Most patients had a severe neonatal pre-sentation, and enlarged fontanelles was the most com-mon abnormality. Clinically, the GPD patients could beclassified as ZS, NALD, or IRD. For those presenting inthe neonatal period the clinical picture was dominatedby severe hypotonia and craniofacial anomalies,

whereas in patients older than 6 months the majorsymptoms were psychomotor retardation, visual andhearing deficits, and failure to thrive. In the patientsclassified as ZS, the facial anomalies and hypotoniawere more marked than in the others, and the averageage at death was about 5 months (range 1 week–18months). The severity of the neurological involvementin ZS patients was highlighted by the high percentagewith neonatal seizures and evidence of demyelinationon CT scan. Calcific stippling of the patella or femurhead was noted in four of nine patients. Hepatomegalywas common, and liver disease was noted in all thepatients. Renal cysts were reported in only one autopsycase (fR). There were three normal ultrasound evalua-tions and one with hyperechogenicity, but this may bea reflection of the difficulty in detecting cysts with ul-trasound techniques. Limb problems were common asunilateral or bilateral talipes, hip dislocation, campto-dactyly, and prominent heels. Patient TA was classi-fied as ZS on the basis of the early, severe onset ofhepatitis and neurological dysfunction, and typical fa-cial appearance. However, TA was homozygous for thesame mutations in PEX5 (previously called PXR1) aspatient PBD018 described by Dodt et al. [1995], whowas classified as neonatal adrenoleucodystrophy.Moreover, both TA and PBD018 were born in Kuwaitand probably belong to the same kindred, although thisis not yet confirmed. It is possible that the differentphenotypes of the two patients are determined bymodifier genes that have yet to be identified or thatmore detailed follow-up of TA would have changed thediagnosis to neonatal adrenoleucodystrophy.

The difficulty in assigning patients to the clinicalgroups was further illustrated by the affected fetus fR,

TABLE III. Results of Prenatal Investigations*

CV analysis CVC analysis

Caseno.

Indexcase DHAP-ATa

VLCFAb

Plasmalogenbiosynthesisc

VLCFAb

DiagnosisC26:0 C26:22 C26:0 C26:22

1 DI 0.1 0.34 0.33 8.00 ND ND Affected2 DI 2.7 ND ND 0.60 0.095 0.097 Unaffected3 KB 2.2 0.05 0.02 0.80 0.184 0.156 Unaffected4 KB 2.6 ND ND NDe 0.053 0.111 Unaffected5 BS 0.2 0.55 0.46 27.5 0.995 1.167 Affected6 BH 4.0 0.05 0.04 0.70 0.058 0.052 Unaffected7 BH 1.3 0.05 0.04 0.70 0.122 0.093 Unaffected8 BH 0.4 ND ND 0.60 0.070 0.162 Unaffected9 ASd 2.7 0.03 0.02 0.55 0.162 0.123 Unaffected

10 HDd 5.0 ND ND 0.50 0.177 0.090 UnaffectedNormal ranges and 0.7–4.9 0.045–0.095 0.017–0.054 0.4–1.5 0.029–0.180 0.039–0.185

means ± SD (n 4 21);2.5 ± 1.05

(n 4 13);0.07 ± 0.014

(n 4 13);0.033 ± 0.012

(n 4 17);0.8 ± 0.29

(n 4 37);0.108 ± 0.041

(n 4 37);0.098 ± 0.034

AFC analysis

VLCFAb

DHAP-ATa C26:0 C26:22

11 IR 0.1 2.62/3.03 2.69/6.92 AffectedNormal ranges

(limited data)1.3–1.6 0.081–0.120 0.088–0.136

aDHAP-AT activity is expressed as nmol/2 hr/mg protein.bVLCFA are expressed as mg/mg protein for C26:0 and as a ratio C26:22.cPlasmalogen biosynthesis is expressed as a ratio (3H:14C).dThe index case was diagnosed as ZS elsewhere and is not included in Table II.eIn this case DHAP-AT was measured instead of plasmalogen biosynthesis and yielded a normal result of 2.3 nmol/2 hr/mg protein (control 4 2.0).*ND 4 not done; CV 4 chorionic villi; CVC 4 cultured chorionic villi; AFC 4 cultured amniotic cells.

508 Steinberg et al.

in which hypoplastic adrenal glands were found [Stein-berg and Fensom, 1996]. On the basis of the criteria ofKelley et al. [1986], it would be appropriate to desig-nate this case neonatal adrenoleucodystrophy. How-ever, a previously affected sibling (IR) had metaphyse-al abnormalities on a radiograph that may have beenlike the stippling observed in ZS.

The NALD patient NP and her affected brother sur-vived into the second decade. NP was profoundly hypo-tonic from birth, endured neonatal seizures, and wasquadriplegic at the time of her death. She was referredto previously as long-surviving ZS [Steinberg and Fen-som, 1996], but a review of her records indicates thatNALD is more appropriate, mainly because of apparentabsence of renal cortical microcysts and lack of chon-drodysplasia. Her younger affected brother had minorfacial abnormalities resembling those in Cockayne syn-drome (Fig. 2b), as noted for some NALD patients de-scribed by Kelley et al. [1986].

Patient FW was not classified as ZS because of thelack of calcific stippling and renal cysts. Hypotonia andphysical anomalies were sufficiently subtle not to benoticed in the first few months, and she made slowprogress consistent with designation as IRD.

In general we did not find that the biochemical mea-surements for the GPD patients assisted in assigningthem to the different clinical groups. However, the IRDpatient JG, who had the mildest phenotype, did nothave very significantly elevated plasma VLCFAs, andhis fibroblast levels were at the lower end of the af-fected range. He belonged to complementation group 8,but other patients within this group have not beenfound to have equivocal VLCFAs in plasma [A Moser,personal communication]. Thus, the variation in JG iseither a reflection of the specific mutation causing theGPD, which differs from other group 8 patients, or ofanother modifying factor within his genotype.

In the two RCDP patients ST and CW, cataracts,frontal bossing, nasal hypoplasia, and proximal limbshortening with profound calcific stippling were thepredominant findings. ST had the more classic pheno-type and died at 4 months of age, whereas CW is lessseverely affected and has made some progress. Thenormal plasma phytanic acid level in both patients atonly 1 month of age is not surprising because of thedietary origin of this metabolite; the level in CW wasclearly elevated at 16 months. Biochemical diagnosisrequired culture of fibroblasts for demonstration of de-ficient plasmalogen biosynthesis and phytanic acid oxi-dase.

The only patient with a single enzyme deficiency,bifunctional protein deficiency, had a severe phenotypesimilar to ZS although his facial anomalies were notthought to be entirely typical. The diagnosis was madeby complementation analysis using cells from the pa-tient of Watkins et al. [1989] as reference BF proteindeficiency cell strain. We assume our patient has D-bifunctional protein deficiency (van Grunsven et al.,1999), but we have not established to which comple-mentation subgroup (van Grunsven et al., 1998b) hebelongs. The case illustrates the great importance offollowing up patients with abnormal plasma VLCFA byfurther assessment of peroxisomal function if the accu-

rate information required for offering prenatal diagno-sis in subsequent pregnancies is to be obtained.

A total of 11 prenatal diagnoses in 7 families in preg-nancies at risk for ZS was performed during the courseof our studies. In 5 of these families unequivocal bio-chemical confirmation of a GPD in the previously af-fected child had been made. In one, plasma VLCFAswere grossly elevated in an earlier child, but additionalbiochemical studies were not made. In the other, therewere no biochemical data but strong clinical evidence,and the family chose to have the pregnancy screened,aware that a normal result would not guarantee anunaffected baby. The overall outcome was 3 affectedfetuses that were terminated, and 8 unaffected thatwent to term. There were no false-positive or false-negative results. Measurement of DHAP-AT was usedfor preliminary diagnosis in direct assay of chorionicvilli, a method that is well established to be reliable[Schutgens et al., 1989]. Villi obtained at terminationof 2 affected fetuses were found to contain significantlyelevated concentrations of VLCFAs, in contrast to thenormal levels in villous preparations from four unaf-fected fetuses. This analysis may provide a useful ac-cessory to assay of DHAP-AT in villi but should not berelied on alone, because Lazarow and Moser [1995]noted 2 cases of false-positive results by this method.

In all pregnancies monitored by CVS some villi werecultured for confirmatory analysis, and final results forunaffected pregnancies were not issued until analysisof the culture was completed. We elected to measure denovo plasmalogen biosynthesis in the cultures becauseof the sensitivity of this method. VLCFA concentrationwas also measured for further backup. This analysis isespecially important for any pregnancy in which theremay be doubt in diagnosis of the index case because itwill detect single enzyme defects in peroxisomal b-oxi-dation as well as GPDs. The availability of a culture forbackup was valuable for case 8 (Table III) in whichdirect analysis of DHAP-AT in villi yielded a resultbelow our established normal range.

ACKNOWLEDGMENTSWe thank the physicians who referred cases for di-

agnosis and supplied information about their patients:S. Benton, K.N. Chan, P.T. Clayton, J.E. Collins, R.Giugliani, D.R. Harvey, R.F. Massey, Professor G.Mieli-Vergani, Professor R.F. Mueller, S. Robb andProfessor R.O. Robinson. We are indebted to ProfessorH. and Mrs. A. Moser for supplying cells from patientsof confirmed complementation group. We thank Mrs.Ronayne Grant for assistance with cell culture, ChrisBarnes for prenatal counselling, and Adrienne Knightfor preparation of the manuscript. The work was sup-ported by Locally Organized Research Scheme (LORS),grant number 93/07.

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