Zellweger Spectrum Disorders (Peroxisome Biogenesis Disorders)

Peroxisome biogenesis disorders – Zellweger spectrum disorder (PBD-ZSD): Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and Heimler syndrome, associated with PEX1 and other PEX genes; differential diagnosis with rhizomelic chondrodysplasia punctata and isolated peroxisomal enzyme defects.

Literature and regulatory information updated: 6 October 2026.

0. Initial Reference Table

1. Introduction

Zellweger spectrum disorders provide a clinical model of what happens when a cell loses an entire organelle at once. Peroxisomes participate in very-long-chain fatty acid oxidation, phytanic acid alpha-oxidation, primary bile acid and plasmalogen synthesis, pipecolic acid degradation, glyoxylate detoxification and hydrogen peroxide metabolism. When a protein required for organelle assembly — the peroxins, encoded by PEX genes — is absent or dysfunctional, all these pathways fail simultaneously. The result is a multisystem disease whose severity depends mainly on the residual function permitted by the genotype rather than the gene involved [1, 3, 11].

The entity's history begins with dysmorphological descriptions. In 1964, Bowen, Lee, Zellweger and Lindenberg published a familial syndrome of multiple congenital abnormalities; in 1965, Smith, Opitz and Inhorn described two siblings with multiple developmental defects, polycystic kidneys and intrahepatic biliary dysgenesis. In 1967, Passarge and McAdams introduced “cerebrohepatorenal syndrome”, emphasising sudanophilic leukodystrophy, cirrhosis and renal cysts; the eponym “Zellweger syndrome” was adopted because paediatrician Hans Zellweger contributed two original patients [3, 21–24]. According to OMIM, Bowen and colleagues described two families, each with two siblings, with failure to thrive, absent or poor sucking and swallowing, finger flexion, congenital glaucoma, malformed ears, micrognathia, heart defects, clitoromegaly, hypospadias, corpus callosum agenesis and early death; Passarge and McAdams studied five sisters in a sibship of 13. Opitz and colleagues (1969) proposed the eponym. Chondral calcification, most marked in the patellae, was highlighted by Poznanski and colleagues in 1970 and the neuronal migration defect by Volpe and Adams in 1972 [4].

The transition from dysmorphology to cell biology occurred in 1973, when Goldfischer and colleagues demonstrated by electron microscopy and histochemistry that peroxisomes were unidentifiable in these patients' hepatocytes or proximal renal tubules, also describing mitochondrial abnormalities [25]. Shortly afterwards, in 1978, Ulrich and colleagues described a “conatal” adrenoleukodystrophy case with hypsarrhythmia, seizures, small adrenal glands with lipid inclusions and extensive demyelination, the entity subsequently termed neonatal adrenoleukodystrophy [26, 27]. Benke and colleagues (1981) described a brother and sister with the same presentation, which, together with equal frequency and severity in both sexes and normal parental VLCFA, enabled Moser to distinguish this autosomal recessive form from X-linked adrenoleukodystrophy [5]. Infantile Refsum disease was characterised by phytanic acid accumulation; Jansen and colleagues (2004) considered the designation unfortunate because it suggested an isolated alpha-oxidation defect and proposed abandoning it [5, 11]. Discovery that all three entities shared multiple peroxisomal biochemical abnormalities led to the concept of a single spectrum [3, 11].

From the 1990s, fibroblast complementation studies and cloning of human orthologues of yeast PEX genes identified the responsible genes. In 1997, Reuber and colleagues demonstrated that PEX1 restored peroxisomal protein import in fibroblasts from 30 complementation group 1 patients, representing 51 % of all patients, and that the p.Gly843Asp allele was present in approximately half these patients [28]. Genetic classification of over 600 fibroblast lines by Ebberink and colleagues in 2011 provided the gene frequency distribution still underpinning diagnostic panels [1, 29].

Current terminology reflects these discoveries. GeneReviews and the 2016 North American consensus recommend abandoning ZS, NALD and IRD in favour of “ZSD” with severe, intermediate or mild phenotype, because many patients do not fit historical categories and the same gene, sometimes the same genotype, may produce any presentation [1, 2]. Heimler syndrome, described in 1991 as sensorineural deafness, enamel hypoplasia and nail abnormalities, was recognised in 2015 as the mildest spectrum extreme, caused by hypomorphic PEX1 or PEX6 alleles [3, 30]. Adult Refsum disease, due to phytanoyl-CoA 2-hydroxylase deficiency, is clinically and molecularly distinct from infantile Refsum disease despite similar names [1, 11].

Two recent changes have altered clinical practice. The first was newborn X-linked adrenoleukodystrophy screening by dried blood spot C26:0-LPC, which also detects most children with ZSD and exposes clinicians to asymptomatic newborns with biochemical diagnosis [1–3]. The second was next-generation sequencing, revealing mild forms with normal biochemistry, atypical phenotypes with ataxia or cataracts and, in 2017 and 2023, non-recessive transmission mechanisms in PEX6 and PEX14 [1, 9, 10]. No therapy restores peroxisome biogenesis; treatment is supportive, and evidence for cholic acid, docosahexaenoic acid (DHA), betaine and liver transplantation is limited or negative, as described in section 7 [1–3].

2. Molecular Genetics and Pathophysiology

2.1. The peroxisome and biogenesis machinery

Peroxisomes are single-membrane-bound organelles present in almost all eukaryotic cells, containing over 70 matrix enzymes. They may form by growth and division of pre-existing peroxisomes, the principal pathway, or from an endoplasmic reticulum subdomain. In both cases, matrix proteins are synthesised on free cytosolic polyribosomes and must be imported after translation, already folded, across the organelle membrane [2, 11, 27].

Import depends on two targeting signals. Most matrix enzymes have a carboxy-terminal type 1 signal (PTS1, peroxisomal targeting signal 1), a serine–lysine–leucine tripeptide or conservative variant recognised in the cytosol by the PEX5 receptor. A smaller subset, including alkyl-dihydroxyacetone phosphate synthase (AGPS) and phytanoyl-CoA 2-hydroxylase (PHYH), has a cleavable amino-terminal type 2 signal (PTS2), recognised by PEX7 in collaboration with the long PEX5 isoform (PEX5L), which stabilises PEX7 [11, 27]. The receptor–cargo complex docks at the membrane through PEX13 and PEX14; translocation is mediated by the PEX2, PEX10 and PEX12 ubiquitin ligase complex; monoubiquitinated PEX5 is then extracted from the membrane and recycled to the cytosol by the PEX1–PEX6 AAA ATPase complex, anchored by PEX26. Membrane protein insertion follows a different mechanism, with PEX19 as the cytosolic receptor and PEX3 and PEX16 as docking points; PEX11β participates in organelle division [1, 11, 31].

Without import, matrix enzymes remain cytosolic. Some, such as acyl-CoA oxidase and thiolase, are rapidly degraded; catalase accumulates in the cytosol and is degraded more slowly. Catalase therefore becomes an intracellular localisation marker: in severe disease it is almost entirely cytosolic, whereas in normal individuals up to 65 % sediments with peroxisomal particles [27]. Patient fibroblasts are not entirely devoid of organelles: they contain peroxisomal “ghosts”, membrane structures containing membrane proteins but no catalase or other matrix proteins; in milder forms, some catalase may be present within these structures [27]. In some patients, cells show a mosaic pattern, with one population containing functional peroxisomes and another lacking them. This peroxisomal mosaicism is cellular and should not be confused with somatic genetic mosaicism; in the Amsterdam hepatic series it was associated with better prognosis [3, 35].

2.2. Metabolic consequences and relationship to affected organs

Loss of peroxisomal functions produces a biochemical profile that both underpins diagnosis and partly explains clinical manifestations. The following table assembles the principal pathways, measurable abnormalities and clinical relevance proposed by the sources [3, 11, 27].

Mechanisms connecting these abnormalities to malformations remain incompletely understood. Abnormal neuronal migration producing polymicrogyria, pachygyria and heterotopias is demonstrable in fetal tissue. It does not occur in RCDP or other disorders affecting only plasmalogen synthesis, prompting the proposal that VLCFA accumulation, absent in chondrodysplasias, contributes to migration pathogenesis [27]. C27 bile acid intermediates are less conjugated and less efficiently excreted than C24 bile acids and considered more toxic; they accumulate because absent cholic acid interrupts physiological feedback inhibition of bile acid synthesis, underpinning exogenous cholic acid use [1, 3]. Hyperoxaluria occurs despite alanine–glyoxylate aminotransferase remaining stable and active in these cells' cytosol [36]. DHA levels are reduced in brain, retina, liver and plasma, motivating supplementation; the controlled trial demonstrated no clinical benefit [27, 37].

Biochemical abnormalities are not static. During follow-up of patients reaching adulthood, DHCA, THCA, pipecolic acid and C29-dicarboxylic acid levels fell with age, sometimes normalising while neurological disease continued progressing. Plasma values correlate poorly with severity, limiting their use as surrogate trial outcomes [3, 17].

2.3. Genes, relative frequency and gene-specific correlations

The gene distribution derives from somatic and complementary DNA complementation studies in 810 biochemically confirmed ZSD individuals, 197 from the Kennedy Krieger Institute and 613 from Ebberink and colleagues' series. This distribution describes biochemically and fibroblast-diagnosed patients and may underestimate genes associated with normal biochemistry [1, 29].

GeneReviews emphasises that clinical and biochemical severity overall appears to depend more on genotype than gene. PEX1 and PEX6, the two most frequent genes, are associated with the entire spectrum; the same variability occurs in PEX10, PEX12 and PEX26, and less frequent genes, initially associated only with severe forms, subsequently included mildly affected patients [1].

2.4. Reference variants, nomenclature and genotype–phenotype correlation

All known pathogenic ZSD-PEX variants cause loss of function. Those abolishing activity (large deletions, nonsense, frameshift) are associated with severe phenotype; residual-function missense variants affect peroxisome assembly less, although not all missense variants retain activity [1, 2]. ATLAS describes this relationship by classes: class I variants, with residual protein concentration and function and milder disease, and class II variants, with absent protein and severe phenotype [27].

The two most frequent PEX1 alleles are present on at least one allele in approximately 80 % of people with PEX1-related ZSD; in a series of 168 patients they accounted for over 80 % of abnormal alleles. Their origin on specific haplotypes suggests founder variants rather than recurrent mutation hotspots [1, 27]. The following table gives standardised MANE Select transcript nomenclature, checked in VariantValidator, and ClinVar classification consulted in October 2026 [38–43].

The protein prediction generated by VariantValidator for the PEX6 founder variant (frameshift) differs from the effect observed in patient RNA (internal in-frame deletions). This discrepancy illustrates that protein nomenclature derived from genomic sequence is a prediction and should be replaced by RNA data where available [12, 38]. Similarly, classical literature mentions “c.9097insT” in an ATLAS series of 168 patients, a typographical error for c.2097insT, now c.2097dup [1, 27].

The PEX1 correlation is best studied because hundreds of individuals homozygous or compound heterozygous for the two founder variants have been identified, mainly in laboratory settings rather than systematic natural history studies [1]. Homozygosity for c.2097dup is associated with severe phenotype; p.(Gly843Asp) homozygosity with mild, sometimes intermediate phenotype; compound heterozygosity for both with intermediate phenotype [1, 45]. Correlation is not absolute. In Poll-The's Dutch cohort (31 patients over 1 year), p.(Gly843Asp) homozygosity was generally associated with a more favourable clinical course, but one homozygote had early lethal disease and two phenotypes overlapping the compound heterozygous group [44]. The 2016 consensus adds that even with p.(Gly843Asp) homozygosity the spectrum ranges from intellectual disability to normal intelligence, suggesting unidentified modifier genes [2]. Biochemically, PEX1 p.(Gly843Asp) retains motor activity but has reduced PEX6 affinity and is rapidly degraded by the proteasome; induced overexpression restored import in model cell lines [46].

2.5. Non-recessive mechanisms and diagnosis with a single variant

Two observations require that a single heterozygous variant not be dismissed in patients with a compatible phenotype. In PEX6, Falkenberg and colleagues identified seven unrelated individuals in whom c.2578C>T was overrepresented through allelic expression imbalance: the frequent c.*442_*445del variant on the mutant allele disrupts the most distal polyadenylation site and alters relative transcript abundance. Asymptomatic parents heterozygous for c.2578C>T were homozygous for the 3′ UTR variant and showed no imbalance. Individuals with c.2578C>T on the c.*442_*445del background but without this variant on the opposite chromosome have a 2- to 3-fold increase in abnormal PEX6 and biochemical and clinical ZSD features [1, 9].

In PEX14, Waterham and colleagues described in 2023 two unrelated ZSD patients with different heterozygous de novo variants, both causing abnormal splicing and stable expression of carboxy-terminally truncated PEX14 proteins. Truncated proteins lost import function and increased autophagic peroxisome degradation (pexophagy), with a dominant-negative effect. Autophagy inhibition or NBR1 receptor silencing restored peroxisomal function in patient fibroblasts. This remains a laboratory observation without animal or human studies [10, 31]. These mechanisms are demonstrated exceptions in few patients; autosomal recessive transmission remains the rule [1, 31].

3. Clinical Spectrum and Manifestations by Organ System

3.1. A continuous spectrum with three presentations

The spectrum is continuous, but presentation age permits operational division into three groups: neonatal–infantile, childhood, and adolescent or adult presentation [3, 11, 24]. GeneReviews divides severe from intermediate/mild ZSD and adds atypical forms without sensory loss [1]. Historical designations retain some counselling and prognostic discussion value but should not be forced onto individual patients [1, 3].

Table sources: [1–3, 11, 24, 27].

3.2. Severe phenotype (Zellweger syndrome)

Severely affected newborns present with profound hypotonia, poor sucking and inability to feed, neonatal seizures caused by neuronal migration defects, craniofacial dysmorphism, renal cysts, hepatomegaly with cholestasis and liver dysfunction, and stippled calcification (chondrodysplasia punctata) in patellae and long bones [1, 11]. Smith describes a mean birth weight of 2740 g, frequent breech presentation, failure to thrive, jaundice in some cases and bloody stools possibly related to hypoprothrombinaemia [24]. Most children acquire no developmental milestones and die in the first year from progressive apnoea or infectious respiratory compromise [1, 11]. In a historical ATLAS-cited series, mean age at death was 5.7 months in classical Zellweger syndrome and 15 months in NALD [27].

Craniofacial dysmorphism includes a high prominent forehead, flat occiput, large fontanelles and wide sutures, flat face, hypoplastic supraorbital ridges, broad nasal bridge, anteverted nares, epicanthal folds, hypertelorism, high palate, mild micrognathia, abnormal auricular helices and redundant nuchal skin [2, 11, 24, 27]. Nipples and external genitalia may be hypoplastic [27]. Smith's reference clinical photograph shows three newborns with hypotonia, high forehead, shallow supraorbital ridges, anteverted nares, micrognathia, talipes equinovarus and knee contractures [24]. Klouwer and colleagues' figure 2 illustrates decreasing dysmorphism over time: a 6-month-old infant with typical dysmorphism, a child at 9 and 14 months with less marked features and a boy at 5, 10 and 15 years without evident dysmorphism (Figure 2 of Klouwer 2015) [3].

3.3. Intermediate and mild forms

Intermediate and mild forms lack congenital malformations; disease manifests as progressive peroxisomal dysfunction [1]. Most common presentations are delayed developmental milestones, failed hearing screening or visual impairment. Some children present with severe bleeding caused by vitamin K-responsive coagulopathy, and several had intracranial haemorrhage in their first year [1, 3]. The course varies: many children are very hypotonic, but many learn to walk and speak [1]. In Poll-The's cohort, all 31 patients had cognitive and motor dysfunction, retinopathy, sensorineural deafness and liver involvement; motor ability ranged from supported sitting to normal walking and language from non-verbal expression to reading comprehension; neurological course was stable in some, rapidly declining through leukodystrophy in others, with spinocerebellar syndrome or slow decline [44].

Vision. Retinal dystrophy is almost universal and leads to blindness [47]. In the natural history cohort (66 patients) and review of 119 published cases, median visual acuity was 0.93 logMAR (Snellen 20/320), loss was slow and legal blindness occurred at a mean of 7.8 years. Electroretinograms were reduced in 91 % and extinguished in 46 % of these, without age-related variation; optical coherence tomography (OCT) showed intraretinal schitic changes in 18 of 23 milder patients [47]. GeneReviews mentions cystoid macular oedema on OCT and transient “leopard-skin” pigmentary retinopathy in some children clinically diagnosed with NALD [1]. Cataracts, glaucoma, optic atrophy and Brushfield spots may occur [3, 11].

Hearing. Sensorineural deafness is almost always present and often detected on hearing screening [3]. In a retrospective study of 42 patients and 300 audiograms, most had moderately severe to severe loss, with slow longitudinal change and improved thresholds with amplification; air–bone gaps suggest a mixed component in some patients [48].

Nervous system. Intermediate disease may develop rapidly progressive demyelinating leukodystrophy with loss of acquisitions, corresponding to historical NALD [1, 3]. On sequential imaging, initial abnormalities involve the dentate hilum and superior cerebellar peduncles, followed by cerebellar white matter, brainstem tracts, parieto-occipital white matter, corpus callosum splenium and finally all cerebral white matter; rapidly progressive leukodystrophy beginning after 5 years has not been described [3]. Which patients risk leukodystrophy or how it progresses individually is unknown [1]. Seizures, almost constant in severe patients, were reported in 23 % of less severe patients in one cohort [2]; in a caregiver survey of 79 patients, combined seizure prevalence was 53 % [49]. ATLAS describes refractory myoclonic and generalised tonic–clonic seizures, tremor on light touch, multifocal EEG discharges and, in one case, progression to hypsarrhythmia in NALD [27].

Liver. Liver disease is a frequent cause of death in all forms [35]. In 13 biopsied patients (0.2 to 39 years), all had histological abnormalities ranging from minimal fibrosis to cirrhosis; five progressed to liver failure and early death, others developed silent cirrhosis, one hepatocellular carcinoma at 36 years and another a precancerous lesion at 18 years [35]. A moderately severe ZSD patient treated with cholic acid from 16 months to 19 years developed cirrhosis with severe portal hypertension and hepatocellular carcinoma at 19 years [50]. Oesophageal varices and hepatopulmonary syndrome are described portal hypertension complications [1, 51].

Adrenal. In 24 Dutch patients undergoing Synacthen testing (2.8 to 34.6 years), 7 (29 %) had primary adrenal insufficiency, 4 asymptomatic; insufficiency was associated with plasma C26:0 but not age, sex, genotype or severity [52]. In the US caregiver survey, reported adrenal insufficiency prevalence was 45 % [49]. At autopsy, adrenal glands are small, with cortical atrophy, lipid-containing ballooned cells and lamellar inclusions resembling X-linked adrenoleukodystrophy, explaining “neonatal adrenoleukodystrophy” [27].

Kidney. In 31 Dutch patients surviving beyond one year, urinary oxalate excretion was assessed in 23: hyperoxaluria was present in 19 (83 %) and hyperglycolaturia in 14 of 22 (64 %); five patients had urolithiasis or nephrocalcinosis and one progressed to end-stage kidney disease. Hyperoxaluria correlated with neurological severity, and pyridoxine did not reduce oxalate excretion in six patients [36]. The hyperoxaluria of the Zellweger spectrum is distinguished from the primary hyperoxalurias by its association with elevated VLCFA and the rest of the peroxisomal picture.

Bone and teeth. Thirteen ZSD patients had very low bone mineral density, with mean Z-score −3.2 at lumbar spine and femoral neck, without differences between ambulant and non-ambulant patients or biochemical rickets abnormalities; pathological fractures occurred [1, 53]. Almost all intermediate and mild patients have permanent dentition amelogenesis imperfecta [1, 3].

3.4. Adolescent and adult presentation

The Amsterdam cohort of 19 patients aged 16 to 35 years from 16 families, all of European origin and 16 with at least one PEX1 p.(Gly843Asp) allele, provides the most detailed adult natural history description [17]. Mean follow-up was 16 years. Twelve remained clinically stable and seven had progressive disease, mainly gait disturbance starting in adolescence, resulting from cerebellar syndrome (9/19), pyramidal signs (5/19) and peripheral neuropathy signs (11/19). In two with nerve conduction studies, neuropathy was demyelinating, unlike the usual axonal neuropathy of X-linked adrenoleukodystrophy. All had night blindness and retinopathy, with retinitis pigmentosa in 16, mean visual acuity 14 % and sensorineural hearing loss of 60 to 110 dB; 17 benefited from hearing aids. Nine lived partly independently with supported employment; one had normal cognitive abilities. There was no epilepsy in this cohort. MRI showed T2 hyperintensities in hilar and peridentate regions in 9 of 16, symptomatic leukoencephalopathy in seven and silent leukoencephalopathy in two [17].

The most diagnostically relevant point is biochemical evolution: all had abnormal childhood biomarkers, but in adolescence and adulthood DHCA and THCA fell in 15 of 19, pipecolic acid in 10 of 19, and some normalised completely. At final assessment, plasma analyses alone would have missed diagnosis in two patients [17]. Clinical overlap with Usher syndrome through deafness and retinitis pigmentosa justifies peroxisomal investigation in suspected Usher patients [17, 27].

3.5. Unusual OMIM presentations

OMIM assembles descriptions expanding the spectrum and aiding recognition: a 28-year-old woman with normal cognition, Leber congenital amaurosis, sensorineural deafness, enamel disease and Arnold–Chiari malformation with hydrosyringomyelia, homozygous for PEX1 p.(Gly843Asp) (Majewski 2011); two Israeli Arab infants from consanguineous families whose sole initial manifestation was neonatal cholestasis, diagnosed through VLCFA (Goez 1995); four Amish siblings born to second cousins with IRD and malformed orange-yellow teeth (Bader 2000); and one patient in Moser's series (1995) with deafness and retinopathy from childhood, special schooling and working life in her third decade, developing progressive leukodystrophy at 37 years with language loss and incontinence by 40; in that 173-patient series, 10 had unusually mild manifestations, including survival into the fifth decade or deficits limited to congenital cataracts [4, 5]. In a PEX12 patient (compound heterozygous p.(Arg180Ter)/p.(Leu317Phe)) initially diagnosed with trihydroxycholestanoyl-CoA oxidase deficiency, with ataxia, dysarthria, areflexia and normal fibroblast studies, phytanate restriction reduced plasma phytanate to trace levels and accompanied improved strength and balance [7].

3.6. Heimler syndrome and atypical forms

Heimler syndrome combines sensorineural deafness, amelogenesis imperfecta, nail abnormalities and occasional or late retinal pigmentation. Ratbi and colleagues studied eight families by exome sequencing, identifying biallelic PEX1 or PEX6 variants in six; each family had at least one hypomorphic allele, with very mild peroxisomal dysfunction not suggested by routine blood and fibroblast tests [30]. Individuals may be neurologically and intellectually normal [1, 31].

The 2021 French PNDS, prepared by the reference centre for genetic deafness together with the CARGO and O-Rares centres, describes Heimler syndrome as the mildest end of the spectrum, with an estimated prevalence of the order of 1 in 1,000,000 and fewer than 50 published cases; the first description, by Heimler and colleagues in 1991, combined sensorineural hearing loss, enamel hypoplasia and nail abnormalities in siblings, without cerebral or hepatic dysfunction [14]. Hearing loss is the first sign: congenital or with onset before 3 years, bilateral, severe to profound, sometimes progressive, and usually detected by newborn hearing screening; vestibular function is preserved. Amelogenesis imperfecta, of hypoplastic or hypomineralised type, affects the permanent dentition, with the primary dentition considered normal, and is more marked in the premolars and molars than in the incisors; it therefore goes unnoticed while the child has only primary teeth. The nail abnormalities are Beau lines and leukonychia. Retinal dystrophy appears later and is variable, with described phenotypes of cone–rod dystrophy, macular dystrophy and isolated cone dystrophy; the number of published cases does not allow a detailed description of its course [14].

From a genetic standpoint, the PNDS attributes almost all cases to biallelic PEX1 or PEX6 variants, with more than 20 different variants described, and states that PEX6 appears to be the more frequent gene, although this predominance has not been confirmed. It uses the distinction between class I variants, with residual PEX protein function and mild phenotypes, and class II variants, with loss of function and severe phenotypes; Heimler syndrome belongs to class I and requires at least one hypomorphic allele. The correlation is not strict, because some PEX1 and PEX6 variants have been described in both Heimler syndrome and Zellweger syndrome. Patients with PEX26 variants were initially classified as Heimler syndrome, but follow-up showed involvement of other organs, and the PNDS does not regard PEX26 as a gene of the classic form [14].

A group of atypical forms without sensory loss also exists, with ataxia, peripheral neuropathy and sometimes congenital cataracts, described with PEX2, PEX10, PEX11B, PEX12 and PEX16 variants [1, 2]. In seven with atypical PEX16 disease, classical features (sensory deficits and amelogenesis imperfecta) were absent; all had hypertonia, five dystonia and four of five treated with levodopa/carbidopa had partially improved dystonia and tremor; MRI showed brainstem, cerebellar peduncle, corticospinal tract and splenial hyperintensities [33]. One PEX6-related Perrault syndrome case (deafness with primary ovarian insufficiency) has been described [31]. A boy developing normally until 6.5 years, with school-screen-detected deafness followed by diplopia, clumsiness and cognitive decline, had leukodystrophy and elevated VLCFA suggesting X-linked adrenoleukodystrophy; ABCD1 was normal and final diagnosis was PEX6-related ZSD [54].

4. Clinical Recognition and Red Flags

Recognition depends on age. In newborns, profound central hypotonia with seizures, a large anterior fontanelle and high forehead, prolonged jaundice or hepatomegaly and epiphyseal stippling, particularly patellar, should prompt immediate VLCFA measurement [1, 55]. Oxford Desk Reference includes Zellweger syndrome in approaches to hypotonic infants, large fontanelles, neonatal encephalopathy with intractable seizures, prolonged neonatal jaundice and chondrodysplasia punctata, always recommending VLCFA measurement [55]. In Cowan and colleagues' study (2003), cited there, among 90 newborns over 36 weeks' gestation with seizures in the first 72 hours and without neonatal encephalopathy criteria, referred to a tertiary unit, 31 % had a genetic or congenital metabolic, neurocutaneous, vascular or developmental disorder, including Zellweger syndrome and NALD [55]. Saudubray's paediatric approach chapter places peroxisome biogenesis disorders among severe neonatal hypotonia, gyration abnormality, renal cyst, bile duct abnormality and chondrodysplasia punctata causes, recalling that the most severe forms present at birth with dysmorphism (Zellweger phenotype) and severe neurological dysfunction (NALD phenotype) [56].

Stippling distribution is discriminative. In ZSD, stippling is usually restricted to patellae, knees and hips and disappears in childhood; in RCDP it accompanies symmetrical rhizomelic shortening and vertebral coronal clefts absent in Zellweger syndrome. Patellar stippling, a Zellweger hallmark, is also seen in RCDP [55, 57, 58]. Because stippling disappears after 2 to 3 years, early radiographs should be requested when an older child is referred [55].

5. Diagnosis: Criteria, Tests and Algorithms

5.1. Diagnostic criterion

According to GeneReviews, diagnosis is established in a proband with suggestive clinical and biochemical findings and biallelic pathogenic or likely pathogenic variants in one of 13 ZSD-PEX genes. Two variants of uncertain significance, or one pathogenic and one uncertain variant, neither establish nor exclude diagnosis. PEX6 p.(Arg860Trp) may cause heterozygous disease in the allelic context described in section 2.5 [1]. Because some patients have no biochemical abnormalities in fluids or cultured cells, molecular testing is necessary for confirmation; functional fibroblast studies remain adjuncts for equivocal molecular or biochemical results [1]. Klouwer and colleagues, from a European laboratory perspective, note that even when sequencing is first-line, biochemical blood or fibroblast studies remain necessary to confirm variant pathogenicity and characterise deficiency extent [3].

5.2. Biochemical tests and limitations

Saudubray considers C26:0-LPC analysis the best initial laboratory test, preferably combined with other biomarkers, now measurable in a single assay [11]. The North American consensus recommends that equivocal findings, such as elevated C26:0 with normal ratios and high total lipid content, be repeated after an overnight fast, and that VLCFA alone not be relied on with strong clinical suspicion: PEX2, PEX10, PEX12, PEX16 and PEX11B variants have been identified in patients with minimally or non-elevated VLCFA [2]. Klouwer and colleagues' table 3 summarises profiles distinguishing ZSD from its two closest enzyme-defect mimics [3].

Source: table adapted from Klouwer et al. [3].

5.3. Fibroblast studies

Cultured fibroblasts permit confirmation of fluid abnormalities, assessment of phytanate alpha-oxidation, VLCFA and pristanate beta-oxidation and plasmalogen synthesis, and immunofluorescent localisation of catalase and other matrix proteins, distinguishing ZSD from isolated enzyme defects [2, 61]. With strong suspicion and normal blood and urine biomarkers, Klouwer and colleagues recommend fibroblast studies including culture at 40 °C, because some mild defects are temperature-sensitive and revealed only at this temperature [3, 17]. Complementation studies by cell fusion or PEX complementary DNA transfection identified genes for decades; Ebberink and colleagues' transfection assay rapidly identifies the defective gene [29]. Currently, next-generation sequencing of all PEX genes replaces complementation where impracticable [3].

5.4. Molecular tests and limitations

GeneReviews recommends a peroxisome biogenesis disorder multigene panel including all 13 genes and differential diagnosis genes where clinical and biochemical suspicion is clear; exome or genome sequencing is best when phenotype does not suggest ZSD. Single-gene testing is rarely useful and not recommended. In children with elevated VLCFA but no other ZSD biochemical abnormalities, a broader peroxisomal panel including at least ACOX1 and HSD17B4 should be used [1].

Analytical sensitivity differs by gene and should not be confused with clinical sensitivity. In PEX1, approximately 98 % of pathogenic variants are detected by sequencing and approximately 2 % are deletions detected by deletion/duplication analysis (e.g. MLPA); for other genes, proportions derive from numbers in each complementation group with two sequenced variants, for example 77 of 77 in PEX6, 17 of 18 in PEX10 and 19 of 22 in PEX2. Estimates assume that large deletions, promoter variants and deep intronic variants would be missed, although apparently uncommon [1]. In 2025, Cheung and colleagues described three suspected PEX1-related ZSD patients with non-diagnostic exomes; combining biomarkers, fibroblast import studies, RNA sequencing and RT-PCR revealed “leaky” splicing variants (one intronic in intron 13 with exon 14 skipping, one in intron 22 with intron retention and one synonymous with exon 16 skipping), with very low canonical transcript and residual protein, consistent with non-severe phenotypes [58].

An adequate molecular report should state gene, versioned transcript (e.g. NM_000466.3 for PEX1, NM_000287.4 for PEX6, NM_002617.4 for PEX10), HGVS nomenclature, zygosity and phase where known, ACMG/AMP classification and methodological limitations [1, 38]. For PEX5, GeneReviews recommends the long isoform reference sequence (NM_001131025.1), because the short isoform omits the PEX5L-specific exon [1]. Parental phase confirmation is recommended, also because de novo variants occur in recessive diseases and PEX6 and PEX14 exceptions alter interpretation of isolated heterozygous findings [1, 9, 10].

5.5. Newborn screening

Dried blood spot C26:0-LPC is measured in X-ALD newborn screening in many US states and also detects ZSD [1]. LC-MS/MS method validation identified all affected samples, apart from one attributed to sample misidentification, among 17 neonatal peroxisomal patient samples mixed with over 1000 samples [62]. In New York State, 11 ZSD individuals were confirmed among over 1.4 million screened newborns [1]. Saudubray reports C26:0-LPC screening implementation in the Netherlands [11]. In eight California-screen-identified infants subsequently diagnosed with ZSD, urinary organic acid analysis showed a consistent pattern useful for confirmation [60]. In Japan, a dried blood spot bile acid synthesis disorder method identified THCA (78 % of measured bile acids) in a newborn with PBD1A in a one-case feasibility study [63]. Screening mainly detects clear biochemical abnormalities; mild forms may be missed and future severity in a screened newborn is difficult to predict [1, 3].

5.6. Diagnostic algorithm

Klouwer and colleagues' algorithm begins with blood and urine biochemistry; if abnormal and compatible with ZSD, fibroblast studies follow (to distinguish ZSD from isolated enzyme defects and historically for complementation), with mutation analysis; if normal despite strong suspicion, fibroblast studies including 40 °C culture are performed (Figure 3 of Klouwer 2015) [3]. GeneReviews places the multigene panel or exome centrally in diagnosis [1]. The following sequence integrates both approaches.

In extent assessment, brain MRI may show predominantly perisylvian polymicrogyria, diffuse white matter reduction, delayed myelination, ventricular dilatation and germinolytic cysts, highly suggestive of severe disease; diffusion and diffusion tensor imaging may reveal white matter injury invisible on conventional sequences [1, 3]. Renal ultrasound may show cortical cysts [2, 11].

5.7. Confirmation and initial assessment in Heimler syndrome

In Heimler syndrome, VLCFA and the other peroxisomal metabolites in plasma, erythrocytes and cultured fibroblasts are as a rule within normal limits, and diagnosis depends on molecular testing of PEX1 and PEX6, requested by the clinical geneticist when sensorineural hearing loss is associated with retinal dystrophy or amelogenesis imperfecta. Most published cases were diagnosed by exome sequencing without prior clinical suspicion, which the PNDS attributes to underdiagnosis of amelogenesis imperfecta in the primary dentition and to confusion with Usher syndrome [14, 30]. In France, newborn hearing screening has been mandatory since the ministerial order of 23 April 2012: it is performed in at least one ear on the second day of life, by otoacoustic emissions or automated auditory brainstem responses, repeated before discharge if abnormal and, if the second test is also abnormal, the child is referred to a deafness centre for diagnosis and early intervention [14]. The initial assessment recommended by the PNDS is multidisciplinary (otorhinolaryngology, ophthalmology, dentistry and genetics) and includes the following investigations.

6. Differential Diagnosis

6.1. Hypotonic infant and other non-peroxisomal presentations

In newborns, ZSD is mainly confused with other causes of profound hypotonia, including Down syndrome and other chromosomal abnormalities [1]. The table integrates GeneReviews and Klouwer and colleagues' differentials by dominant symptom [1, 3].

For Heimler syndrome, the PNDS identifies Usher syndrome as the most frequent differential diagnosis, as it is the commonest cause of combined hearing loss and retinal dystrophy, and regards the other syndromes combining congenital hearing loss and amelogenesis imperfecta as exceptional. The document is inconsistent regarding retinal chronology: the summary for the attending physician attributes earlier retinal involvement to Usher syndrome, whereas the main text attributes earlier onset to Heimler syndrome. The stable discriminating features are therefore the enamel of the permanent dentition, vestibular function and the molecular result, rather than the age at onset of retinopathy [14].

In the French PNDS (French National Diagnosis and Care Protocol) on primary bile acid synthesis defects (2019), peroxisomal disorders are listed among the intrahepatic causes of neonatal cholestasis, in the group of hepatocellular and intrahepatic bile duct lesions, alongside alpha-1-antitrypsin deficiency, Alagille syndrome, PFIC and storage disorders. The rationale is biochemical: side-chain modification and conjugation of bile acids take place in enzymes of hepatocyte peroxisomes, so some peroxisomal disorders produce liver involvement similar to that of enzyme deficiencies in that part of the pathway; among them, Zellweger syndrome has the most marked liver involvement. Bile acid synthesis defects as a whole account for about 1% to 2% of childhood cholestasis. The distinctive feature of steroid nucleus defects (3β-HSD and Δ4-3-oxo-reductase) is cholestasis without pruritus, with normal GGT and normal total serum bile acids despite pale stools; in ZSD, the urinary profile shows C27 intermediates (DHCA, THCA) and plasma peroxisomal biochemistry is usually abnormal [1, 18]. 3β-HSD and Δ4-3-oxo-reductase deficiencies, which respond to cholic acid with clinical and biochemical normalisation, are part of the differential diagnosis.

6.2. Isolated peroxisomal enzyme and transporter defects

Approximately 15 % of individuals with a ZSD-like phenotype and elevated VLCFA lack PEX variants but have an isolated peroxisomal beta-oxidation defect, particularly D-bifunctional protein or acyl-CoA oxidase 1 deficiency [1]. The 2016 consensus estimates 10–15 % [2]. These diseases share VLCFA accumulation but retain plasmalogen synthesis and peroxisomal morphology, historically leading to “pseudo-Zellweger” and “pseudo-neonatal adrenoleukodystrophy” [11, 27].

The 2019 French PNDS groups AMACR, ACOX2 and D-bifunctional protein deficiencies among the defects of bile acid side-chain modification, together with sterol 27-hydroxylase deficiency, and adds two conjugation defects, BAAT deficiency (BAAT) and bile acid-CoA ligase deficiency (SLC27A5), both with neonatal cholestasis and fat-soluble vitamin malabsorption. In these defects, liver disease is not usually in the foreground because some primary bile acid synthesis persists, and the presentation is dominated by the neurological picture and by malabsorption of fats and fat-soluble vitamins; in AMACR deficiency, the PNDS describes neonatal liver disease with vitamin malabsorption and late-onset sensorimotor neuropathy, and in ACOX2 deficiency, persistent or isolated elevated transaminases with liver fibrosis, ataxia and intellectual disability [18]. Two points of the PNDS table diverge from other sources. It associates D-bifunctional protein with the EHHADH gene (OMIM 607037), which encodes L-bifunctional protein, whereas the gene for D-bifunctional protein is HSD17B4; and it attributes phytanic acid accumulation to AMACR deficiency, whereas the described marker is pristanate, with normal VLCFA [1, 11, 18].

6.3. Rhizomelic chondrodysplasia punctata and RCDP spectrum

RCDP is the other major peroxisome biogenesis disorder group. Classical RCDP type 1 results from biallelic PEX7 variants, encoding the PTS2 receptor; forms 2 to 5 result from lipid ether synthesis enzyme deficiencies (GNPAT, AGPS, FAR1) or long PEX5 isoform (PEX5L) deficiency [11, 57, 64]. Biochemically, all types have erythrocyte plasmalogen deficiency and types 1 and 5 phytanate accumulation, but VLCFA beta-oxidation is preserved; erythrocyte plasmalogen measurement is a reliable diagnostic test [11, 57].

Smith adds that children surviving beyond 2 months have seizures in over 80 % and scoliosis in most, and recommends cataract surgery for visual stimulation [65]. The PEX7-related RCDP GeneReviews estimates birth prevalence at 0.5 per 100,000 and describes p.(Leu292Ter) as a founder variant in northern European ancestry, with approximately 6 % carriers in a genetically isolated Dutch community [64]. A mild non-classical form also exists, with cataracts, mild chondrodysplasia and lesser intellectual disability, and a Refsum-like presentation [11, 64].

The chondrodysplasia punctata differential also includes X-linked forms (dominant Conradi–Hünermann–Happle due to EBP, with asymmetry and mosaic skin lesions; recessive brachytelephalangic due to ARSE), CHILD syndrome (NSDHL), Greenberg dysplasia (LBR), Smith–Lemli–Opitz syndrome, trisomies 13, 18 and 21, lysosomal diseases and acquired embryopathies from warfarin, phenytoin, maternal vitamin K malabsorption or maternal systemic lupus erythematosus [55, 56, 65].

7. Treatment and Risk Management Strategies

7.1. Principles and initial assessment

No curative therapy or treatment restoring peroxisome biogenesis exists; intervention is supportive, guided by symptoms and surveillance for treatable complications [1, 3, 11]. The North American consensus recalls that some manifestations, particularly brain malformations, develop fetally and are irreversible, requiring realistic expectations for neurological symptoms; others emerge postnatally and may benefit from treatment [2]. Guidance comes from two management reviews, Klouwer and colleagues (Amsterdam, 2015) and Braverman and colleagues' consensus (North America, 2016), synthesised in the 2020 GeneReviews update. These are expert documents rather than randomised-trial-based guidelines; the search identified no subsequent specific consensus from an American or European society or MetabERN [1–3]. GeneReviews recommends the following initial assessment [1].

7.2. Treatment of manifestations

The ATLAS refers to the historical use of oral citrate and generous fluid intake to prevent calcium oxalate stones [27]. The sources consulted give no citrate, hydrocortisone or vitamin A, D and E dose specific to ZSD; in its absence, label dosing for analogous indications is used by extrapolation. In adrenal insufficiency, the European SmPC for Alkindi® (hydrocortisone granules, paediatric use) recommends 8 to 10 mg/m²/day in 3 to 4 doses, with doubling or tripling of the oral dose in mild to moderate illness and parenteral hydrocortisone in severe situations, and that for Plenadren® (modified release, adults) a usual maintenance dose of 20 to 30 mg/day as a single morning dose [69, 70]. For potassium citrate, the FDA label for Urocit-K® (adults with hypocitraturia and calcium oxalate stones) starts at 60 mEq/day in 2 to 3 doses with meals in severe hypocitraturia (urinary citrate <150 mg/day) and 30 mEq/day in mild to moderate hypocitraturia, the Portuguese SmPC for Acalka® starts at 20 mEq three times daily with meals (60 mEq/day), and both advise against doses above 100 mEq/day [71, 72]; there is no labelled paediatric dosing. For vitamin A, the Portuguese SmPC for A-Vite® (50,000 IU capsules) provides for 1 capsule/day in the treatment or prevention of deficiency, including in malabsorption, only above 8 years and for a maximum of 6 to 8 months, and 5000 IU/kg/day from 1 to 8 years in xerophthalmia [73]; for vitamin D, the Portuguese SmPC for Vigantol® (colecalciferol) provides for 3335 to 5336 IU/day for prophylaxis of deficiency due to malabsorption [74]. In ZSD these doses are adjusted according to serum levels, and use in this disease is an off-label extrapolation.

7.3. Cholic acid (Cholbam®)

Rationale. Cholic acid is the principal C24 primary bile acid, formed from THCA through one peroxisomal beta-oxidation cycle. In ZSD, its deficiency interrupts bile acid synthesis feedback inhibition, allowing accumulation of DHCA and THCA, considered hepatotoxic. Exogenous cholic acid restores feedback inhibition, reduces C27 intermediates and may improve fat and fat-soluble vitamin absorption [1, 3, 75].

US prescribing information. Cholbam® (cholic acid, 50 mg and 250 mg capsules) was FDA-approved on 17 March 2015 (NDA 205750). The consulted prescribing information (effective 26 January 2026) indicates treatment of bile acid synthesis disorders due to single enzyme defects and adjunctive treatment of peroxisomal disorders, including ZSD, in patients with manifestations of liver disease, steatorrhoea or fat-soluble vitamin malabsorption complications. Safety and efficacy for extrahepatic manifestations are unestablished [15].

Evidence and limitations. Supporting data derive from two open-label, non-randomised single-arm trials conducted over 18 years: 29 peroxisomal disorder patients in trial 1 and 12 in the extension, mostly under 2 years initially and treated for a mean 4.8 years. In a responder analysis with combined laboratory and clinical criteria, 11 of 24 evaluable patients (46 %) responded; 13 of 31 (42 %) survived beyond 3 years. The prescribing information states that survival improvement versus historical controls was not demonstrated, adherence and response documentation was incomplete, most also received DHA and vitamins, and one patient without prior cholestasis developed cholestasis during treatment and died [15, 76]. The peer-reviewed publication from the same programme, with 85 enrolled patients (22 ZSD), reports improved urinary metabolites and transaminases and favourable tolerance; authors declared financial interests in the product's developer [76].

Berendse and colleagues' Dutch study (2011–2014, ISRCTN96480891) included 19 genetically confirmed ZSD patients in an open pretest/post-test design with 2.5 years' prior observation and 9 months of cholic acid (15 mg/kg/day, adjusted to 10 or 20 mg/kg/day). Bile acid synthesis decreased in most and plasma and urinary C27 intermediates fell. In four with advanced liver disease (elastography ≥15.5 kPa), transaminases, bilirubin and plasma cholic acid increased (up to 250 µmol/L in one), with little intermediate reduction, and parameters returned to baseline after discontinuation; authors advise against treatment in this subgroup [68]. In the extension, 17 completed 21 months: synthesis suppression persisted and intermediates increased again after discontinuation, but liver tests, elastography, coagulation, fat-soluble vitamins and weight did not change significantly [77]. Conversely, three US patients treated for 15 or more years maintained stable liver function without attributed adverse effects [75], whereas another treated from 16 months to 19 years developed cirrhosis and hepatocellular carcinoma [50]. Klouwer and colleagues (2015) considered evidence insufficient to conclude benefit, and GeneReviews states that cholic acid reduces C27 intermediates, which should be measured, but its clinical effect in ZSD is unknown [1, 3].

European Union. In the European Union there is no cholic acid authorised for ZSD. Orphacol® (authorisation of 12 September 2013) is indicated only in 3β-hydroxy-Δ5-C27-steroid oxidoreductase deficiency and Δ4-3-oxosteroid-5β-reductase deficiency; Kolbam® (authorised in 2014 for sterol 27-hydroxylase deficiency, AMACR deficiency and CYP7A1 deficiency) had its marketing authorisation withdrawn on 13 July 2020, at the request of the holder, for commercial reasons [16]. Use of cholic acid in ZSD in Europe is therefore off-label, and the dose used in the Dutch study (15 mg/kg/day) comes from that trial [16, 68]. As a European label reference for the same drug, the Orphacol® SmPC indicates, in the two authorised deficiencies, 5 to 15 mg/kg/day in infants, children, adolescents and adults, with a minimum dose of 50 mg, adjustment in 50 mg steps and a maximum of 500 mg/day in adults; the dose of the Dutch study lies at the upper limit of this range [78]. Saudubray mentions a dose of 100 mg/day, in an incomplete sentence of the text itself, which should not be used as a dosing reference [11].

The 2019 French PNDS on 3β-HSD and Δ4-3-oxoreductase deficiencies is the most detailed guideline on the use of the same drug and serves as a reference by extrapolation, declared as such, for ZSD. In these deficiencies, cholic acid (Orphacol®) is the reference treatment, orally and lifelong: 5 to 15 mg/kg/day from 1 month to 18 years and in adults, with a minimum dose of 50 mg, adjustment in 50 mg steps and a maximum of 500 mg/day in adults, an amount corresponding to physiological adult synthesis, divided into two doses when it includes more than one capsule. The dose is chosen according to serum and/or urinary bile acids measured by gas or liquid chromatography coupled with mass spectrometry, at least every 3 months in the first year and every 6 months in the second, seeking the lowest dose that brings the abnormal metabolites to values close to zero; as there is no correlation between dose and feedback inhibition of cholesterol 7α-hydroxylase, adjustment is individual. Overdose is suspected in the presence of pruritus, diarrhoea or a simultaneous rise in GGT and transaminases and is confirmed by elevated total serum bile acids. Ursodeoxycholic acid does not exert feedback inhibition on 7α-hydroxylase and does not reduce the toxic metabolites, so it serves only as a temporary treatment while awaiting diagnostic confirmation; chenodeoxycholic acid is not preferred because it is more detergent, because its metabolite, lithocholic acid, is hepatotoxic in overdose, because it has no authorisation for these indications in any country and because it is contraindicated in pregnancy. Tolerability is good, with no serious adverse effects in a series of more than 180 patient-years; nine pregnancies in four treated patients were uncomplicated, and vitamin supplementation should be stopped when levels normalise, to avoid overdose [18].

Extrapolation to ZSD has limits that the mechanism itself explains. In the steroid nucleus defects no primary bile acid is formed, and cholic acid fully replaces what is missing, with resolution of jaundice within months, normalisation of liver tests and histological improvement [18]; in ZSD there is residual synthesis, involvement is multisystemic and, in patients with advanced liver disease, cholic acid worsened liver tests [68]. The sources diverge on three points: the dose range (5 to 15 mg/kg/day in the PNDS and the Orphacol® SmPC, 10 to 15 mg/kg/day in the Cholbam® label, 15 mg/kg/day adjusted to 10 or 20 mg/kg/day in the Dutch trial), the monitoring schedule (in the PNDS centred on urinary bile acids by mass spectrometry and clinical examination, in the North American label centred on monthly liver tests in the first 3 months) and the meaning of a rise in transaminases, which the PNDS attributes first and foremost to overdose and which, in ZSD with advanced fibrosis, may reflect hepatotoxicity with an indication for discontinuation [15, 18, 68]. A concomitant rise in GGT and ALT as a sign of overdose is common to the PNDS and the FDA label.

Authorisation does not guarantee marketing or reimbursement in each country; consulted sources did not document cholic acid access for ZSD in Portugal or Brazil.

7.4. Other assessed interventions

Docosahexaenoic acid. Following reports of clinical improvement, including one NALD patient treated with 250 mg/day, not generally accepted [27], Paker and colleagues' randomised double-blind placebo-controlled trial included 50 patients aged 1 to 144 months and DHA 100 mg/kg/day. Two were excluded because they actually had isolated enzyme defects, nine died during the trial and five were lost to follow-up; 34 completed follow-up. There were no group differences in biochemical function, electroretinography or growth, and some improved in both arms. The trial provides class II evidence against DHA benefit [37]. The North American consensus therefore withdrew its previous supplementation recommendation [2, 11].

Lorenzo's oil and low-VLCFA diet. The 4:1 glycerol trioleate/trierucate mixture reduces plasma VLCFA in X-ALD without changing progression; there is no reason to expect benefit in ZSD, and increased monounsaturated fatty acids may be counterproductive in patients already accumulating C26:1. Dietary VLCFA reduction alone does not lower blood levels because most are synthesised endogenously [2, 3].

Plasmalogen precursors. Batyl alcohol (an alkylglycerol) is imported normally; case reports described increased erythrocyte plasmalogens and clinical improvement in some, but no systematic study exists [3]. In RCDP mice, alkylglycerol supplementation corrected plasmalogens in all tissues except brain [11].

Clofibrate. This was used unsuccessfully to induce hepatic peroxisome formation in Zellweger syndrome [27].

Betaine. Fibroblast drug screening identified betaine as a potential chaperone improving peroxisome assembly in PEX1 p.(Gly843Asp) cells [2]. Open-label phase 3 trial NCT01838941 (McGill, 2013–2015) treated 12 patients, 11 aged 18 years or younger (median 4.75 years; range 0.92–31.17), with 6 g/day (<30 kg) or 12 g/day (>30 kg) for 6 months. Mean plasma C26/C22 was 0.180 (range 0.027–0.301) before and 0.188 (0.059–0.286) at 6 months in 10 analysed patients; commonest adverse effects were constipation, altered body odour, fatigue, diarrhoea and infections; one increase in seizure activity occurred. Results are published only in the registry, and investigators emphasise substantial between- and within-individual variability [79].

Hydroxychloroquine. Randomised phase 2 trial NCT03856866 (Hospital for Sick Children, Toronto) assessed pexophagy inhibition with hydroxychloroquine 4 mg/kg/day in three patients, with electroretinogram, plasmalogen, phytanate and VLCFA outcomes at 12 weeks; completed since 2020 without registry-published results [80].

7.5. Liver and cell transplantation

Orthotopic liver transplantation in a 6-month-old and hepatocyte transplantation in a 4-year-old reduced VLCFA and pipecolic acid and improved bile acid profiles, without long-term effects described [3]. In a Belgian series of three mildly affected ZSD patients receiving living-donor transplantation, one died afterwards and two showed neurodevelopmental improvement (17 years and 9 months' follow-up), with normalised phytanic, pristanic and pipecolic acids and stabilised hearing and vision compared with untreated affected siblings [81]. Transplantation was also used for cirrhosis with hepatopulmonary syndrome (section 10) and cirrhosis in a 16-year-old boy subsequently developing subacute polyneuropathy, whose symptoms improved [51, 82]. These reports cannot estimate population risks or benefits, and retinal and neurological progression may continue after biochemical correction [51]. No ZSD bone marrow transplantation reports exist; this might interest patients developing rapidly progressive leukodystrophy, but no predictive markers exist [3].

7.6. Clinical trials and evidence limitations

These studies share limitations preventing indirect intervention comparisons. They are small, heterogeneous in severity and genotype and, with one exception, uncontrolled. Plasma biochemical outcomes fluctuate and decline spontaneously with age, so falling values do not demonstrate benefit; Klouwer and colleagues advise against plasma levels as surrogate outcomes, and Cholbam® prescribing information itself acknowledges incomplete documentation and no survival benefit [3, 15, 17]. Serial electroretinography does not usefully measure visual loss progression [2, 47]. The DHA trial is the only randomised placebo-controlled trial and was negative. Cholic acid interpretation differs between Cincinnati, describing efficacy and safety, and Amsterdam, describing absent clinical benefit and cirrhotic hepatotoxicity; patients, response criteria and duration differ, and FDA prescribing information reflects both dimensions by restricting indication to hepatic manifestations and requiring monitoring and discontinuation without improvement [15, 68, 76, 77].

7.7. Research and developments in the past 12 months

The October 2026 trial search identified no recruiting ZSD-specific interventional trial on ClinicalTrials.gov. Active studies include longitudinal peroxisome biogenesis disorder natural history (NCT01668186, recruiting, 244 planned participants through 2030), prospective ZSD retinopathy (NCT06190626, recruiting since December 2023, 30 participants, 5 years) and Cholbam® postmarketing registry (NCT03115086, active not recruiting, through 2038) [83–85]. A negative search does not exclude studies registered elsewhere.

None of these developments has reached human study. Results in mice homozygous for the p.(Gly843Asp) equivalent cannot be transferred to null genotypes or established lesions, and hepatic correction alone does not address cerebral or retinal manifestations [51, 86, 87]. In 2016, the North American consensus already identified chaperone screening (betaine, arginine), retinal AAV gene therapy and cell therapies as strategies [2].

7.8. Special populations and situations

In severely affected children, seizure control, feeding and respiratory support are central, and invasive ventilation decisions should be shared with families informed about survival and quality of life [2]. During severe acute illness, fever or major surgery, patients with or at risk of adrenal insufficiency should be considered for glucocorticoid stress doses [1, 2]. Antiseizure selection should avoid respiratory depression with respiratory compromise [2]. Mildly affected patients reach adulthood needing auditory, visual, neurological, hepatic and adrenal care and should be followed as patients with slowly progressive chronic disease rather than solely paediatric disease [17]. Pregnancy data in women with ZSD are scarce in consulted sources; Cholbam® prescribing information has no pregnancy studies and maintains a surveillance programme [1, 15]. Cholic acid use below 3 weeks, outside hepatic manifestations in the US, or for any ZSD indication in the European Union is off-label [15, 16].

7.9. Heimler syndrome: sensory rehabilitation, dental care and organisation in France

The HAS PNDS does not provide for specific treatment; the goals are early rehabilitation of hearing and vision and prevention of dental complications. The prognosis is mainly auditory, because the severity and early onset of hearing loss determine language acquisition and schooling, and the later onset of retinal dystrophy creates a dual sensory impairment that limits visual compensation [14].

Hearing. Conventional hearing aids are indicated in mild to severe hearing loss and, in Heimler syndrome, are bilateral from the outset, fitted by audiologists experienced with children and with severe hearing loss, with efficacy checks, hearing aid education and encouragement of daily use. Cochlear implantation follows the 2012 HAS criteria: bilateral profound hearing loss, or severe hearing loss in a child or adult with discrimination below 50 % at 60 dB with a well-fitted hearing aid, or with hearing aid gain insufficient for language development. The indication is confirmed after multidisciplinary assessment (clinical, subjective and objective audiometry, vestibular function, imaging, speech and language therapy and psychology) at an implant centre. Most patients diagnosed early meet these criteria. Implantation may be unilateral or bilateral, simultaneous or sequential, depending on hearing, vestibular function and age; in Heimler syndrome the PNDS favours simultaneous bilateral implantation, in anticipation of visual loss. With a unilateral implant the contralateral hearing aid is maintained, and a second implant is offered if hearing deteriorates. There are no published studies of implant outcomes in this syndrome; the French reference centre describes results that it considers encouraging. Speech and language therapy begins with auditory rehabilitation, at a weekly frequency set by the therapist, and continues after implant activation. The communication plan should favour the oral route, because retinopathy may compromise sign language communication in the long term [14].

Teeth. The approach combines paediatric dentistry, orthodontics, prosthodontics and dental imaging, with support from the O-Rares centres. Because of the increased caries risk, the oral hygiene programme begins with the first primary tooth, around 6 months: brushing performed or supervised by the parents until 6 years, fluoride toothpaste at an age-appropriate concentration and 2 to 4 visits per year, spaced according to caries risk. The first permanent molars, which erupt around 6 years and are decisive for occlusion, should be protected as soon as they show an enamel defect, with plastic restorative materials or preformed crowns for extensive defects; topical fluoride and pit and fissure sealants are also applied, defects of the other permanent teeth are treated, and occlusion and dentofacial deformities are followed with the orthodontist [14]. The European Academy of Paediatric Dentistry recommends supervising brushing and the amount of toothpaste until at least 7 years, one year beyond the limit given in the PNDS, and adjusting fluoride use to the estimated caries risk [89].

Vision. Follow-up is based on visual acuity, visual field and retinal imaging, with attention to early cataract, whose surgical indication is discussed case by case, and to cystoid macular oedema detected on OCT. Refraction is fully corrected; filter lenses and protection against glare, bright light and ultraviolet radiation (including a peaked cap, particularly at the seaside and in the mountains) are recommended, together with adjustment of lighting, early low-vision rehabilitation and training with optical aids (magnifiers, magnifying spectacles, telescopes), non-optical aids (large-print books, talking clocks) and electronic aids. In severe visual impairment, the child is supported by a team or establishment for people with visual impairment, with Braille teaching, orientation and mobility training and, in blindness, the possibility of a guide dog [14].

Disclosure of the diagnosis, pregnancy and transition. The diagnosis should be communicated at a dedicated consultation, at a specialist centre or in liaison with one, by the clinical geneticist or the physician who requested the molecular testing, preferably with a psychologist, with explanation of the disease, follow-up and inheritance, provision of a written document to the family and the attending physician, and information on patient associations. There is no contraindication to pregnancy and no data on worsening of signs during pregnancy or the postpartum period. Transition from paediatric to adult services usually takes place between 16 and 19 years and requires written reports between specialists. At the first adult otorhinolaryngology consultation the history of hearing loss and hearing aids is reviewed, with the audiograms and electrophysiological records, otological examination and audiometry are repeated, images are transferred to avoid duplicate investigations, the situations warranting urgent consultation, practices to avoid and social and university support are explained, and knowledge of the disease is assessed; at the adult genetics clinic the explanation of inheritance and the calculation of recurrence risk are repeated and prenatal or preimplantation diagnosis is discussed [14].

Organisation in France. Treatment and rehabilitation may be covered at 100 % by health insurance, through an application for exemption from the ticket modérateur as a condition outside the ALD list. Conventional hearing aids are reimbursed at 100 % up to 20 years, with two annual maintenance services and reimbursement of earmoulds; after that age, the Maison départementale des personnes handicapées (MDPH) may fund their purchase through the disability compensation benefit, and it grants other compensation in childhood and adulthood. Schooling in a specialised establishment (for children with hearing, visual or dual impairment) is decided case by case, once rehabilitation has been established [14].

8. Reproductive Options and Family Counselling

8.1. Recurrence risks in recessive transmission

ZSD is autosomal recessive in the great majority of families. Parents of an affected child are obligate heterozygotes; after identifying proband variants, parental molecular testing is recommended to confirm heterozygosity and permit reliable risk estimation, because de novo variants occur at a low but non-negligible frequency in recessive disorders [1]. If both parents are heterozygous, each proband sibling has, at conception, a 25 % probability of being affected, 50 % of being an asymptomatic carrier and 25 % of being neither affected nor a carrier. Heterozygotes are asymptomatic and not at risk of developing disease. Each parental sibling has a 50 % carrier probability [1, 3]. Greater clinical variation between affected siblings occurs in milder families [1]. Affected people generally have no offspring; some mildly affected individuals may reproduce and their children are obligate heterozygotes [1].

Carrier detection requires prior identification of the variants in the family: carriers' biochemical markers are normal and cannot be used for this purpose. This is a practical difference from X-linked adrenoleukodystrophy, in which heterozygous women may have elevated VLCFA; in the parents of children with ZSD, plasma and fibroblast VLCFA are normal [1, 27]. In the Saguenay–Lac-Saint-Jean region, where the carrier frequency of the PEX6 founder variant was estimated at 1 in 55, the authors emphasised the implications for carrier screening [12]. In Heimler syndrome, the PNDS considers the risk of an affected adult having affected children to be low, unless the partner has the same disease or is consanguineous; it estimates the frequency of heterozygotes for PEX1 and PEX6 variants in the general population at less than 1 %, so that the risk for healthy relatives is low, and accepts carrier testing in relatives case by case [14]. OMIM records an old description of small lens opacities in heterozygotes (Hittner 1981); this observation has not been incorporated into current guidance, which regards carriers as asymptomatic [1, 4].

8.2. Non-recessive situations

In families with PEX6 c.2578C>T, disease depends on allelic configuration: asymptomatic parents heterozygous for c.2578C>T were homozygous for the 3′ UTR c.*442_*445del variant, whereas affected children had c.2578C>T on this background but lacked it on the opposite chromosome. Counselling therefore requires parental and proband phase and 3′ UTR genotype determination [1, 9]. In two patients with heterozygous de novo PEX14 variants, disease resulted from a dominant-negative effect; consulted sources do not quantify familial recurrence risk [10]. In both situations, a single heterozygous variant in a patient with compatible phenotype and biochemistry should not be dismissed [9, 10].

8.3. Prenatal and preimplantation diagnosis

The ideal time to discuss genetic risk, clarify carrier status and present prenatal and preimplantation diagnostic options is before pregnancy. Counselling should be offered to affected young adults, carriers and those at carrier risk [1]. When both familial variants are identified, molecular prenatal diagnosis by chorionic villus sampling or amniocentesis and preimplantation genetic testing are possible [1–3]. When variants are unknown or proband molecular findings equivocal, biochemical prenatal diagnosis in chorionic villi, cultured villi or cultured amniocytes is possible, provided biochemical abnormalities have been confirmed in index-case fibroblasts; few laboratories perform it [1, 3]. Historical methods include VLCFA measurement, dihydroxyacetone phosphate acyltransferase activity and demonstration of cytosolic catalase; false negatives in uncultured villi prompted recommendations for cultured-cell confirmation, with maternal cell contamination risk [27]. Cultured amniocyte biochemical studies may also be useful with suggestive prenatal ultrasound abnormalities [1].

In France, prenatal or preimplantation diagnosis of Heimler syndrome is possible only after molecular diagnosis in an affected relative. The couple has a joint consultation with the clinical geneticist and a genetic counsellor; the indication is assessed case by case by a multidisciplinary prenatal diagnosis centre (CPDPN), which includes an expert in the disease and must recognise the particular severity and incurability of the condition; testing for the PEX1 or PEX6 variants is performed on chorionic villi or amniotic fluid; the couple is informed of the possibility of termination of pregnancy for medical reasons if the fetus is affected and should receive psychological support [14].

DNA storage should be considered for probands without confirmed molecular diagnoses, for future studies [1]. Prenatal testing cannot reliably predict severity in affected fetuses: genotype–phenotype correlation is approximate and milder families show greater clinical variation between siblings [1, 44]. Views on these tests may differ among professionals and within families; the decision is personal and should be supported by non-directive counselling [1].

9. Special Considerations: Population Diversity and Epidemiology

9.1. Global frequency and estimation limitations

ZSD frequency estimates vary by method and population, with each number describing a different entity and method. The most cited historical estimate, 1 in 50,000 US births, derives from biochemical and fibroblast series [1–3]. New York State newborn screening confirmed 11 cases among over 1.4 million screened with C26:0-LPC, yielding confirmed incidence of 1 in 133,000; GeneReviews emphasises that any biochemical-test-based estimate underestimates frequency because mild forms without a defined biochemical phenotype escape detection [1]. Orphanet records peroxisome biogenesis disorder birth prevalence of 2.0 per 100,000 in the US and 0.2 per 100,000 in Japan [8]. ATLAS reproduces older estimates of 1 in 100,000 for Zellweger syndrome and 1 in 25,000 to 50,000 for peroxisome biogenesis disorders overall, mentioning consanguinity in 17 of 78 in a historical series [27].

Malone and colleagues' population genetic modelling (2025), applying TOPMed, All of Us, UK Biobank, gnomAD and TogoVAR allele frequencies, Hardy–Weinberg assumptions and survival curves, estimated PEX1-only ZSD at approximately 1 in 245,000 US births in the conservative model (known pathogenic variants) and up to 1 in 114,000 in the expanded model (predicted pathogenic variants) [45]. These figures concern PEX1 alone and expected births rather than diagnosed cases.

9.2. Founder effects and allele distribution

Variant distribution partly explains regional differences. The two most frequent Western PEX1 alleles are p.(Ile700TyrfsTer42) and p.(Gly843Asp). In 2025 modelling, p.(Gly843Asp) was frequent throughout continental Europe and less so in the US, whereas p.(Ile700TyrfsTer42) was enriched in northwestern European ancestries, including the United Kingdom; mapping suggested Scandinavian origin for the latter. Neither was detected in available Japanese cohorts, whose commonest known pathogenic variant was PEX1 c.1897C>T, p.(Arg633Ter), also described in Saudi patients [45]. This absence explains lower Japanese incidence, where founder PEX10 c.814_815del was found in all 11 complementation group B patients and apparently arose once on an ancestral haplotype [1, 13]. In the US natural history study, patient concentration in Montana, with two of three homozygous for p.(Gly843Asp), suggests higher local heterozygote frequency or higher diagnosis rates [45].

ATLAS mentions a relatively frequent PEX1 frameshift variant in Australasian patients and PEX26 p.Arg98Trp in 39 % of complementation group 8 alleles, suggesting a founder effect [27]. OMIM also reports unusual Zellweger frequency in Israel's Karaite community (Zung 1990), four Amish siblings with IRD (Bader 2000), consanguineous Israeli Arab families and, for PEX12, homozygous p.(Ser320Phe) in seven patients from consanguineous families [4, 5, 7]. In Quebec, founder PEX6 c.802_815del, identified homozygously in five patients diagnosed between 1990 and 2010, corresponds to the highest described Zellweger incidence worldwide [12]. Within RCDP, PEX7 p.(Leu292Ter) is a northern European founder, with approximately 6 % carriers in a genetically isolated Dutch community [64].

9.3. Europe, Portugal and Brazil

Most European natural history data derive from Amsterdam UMC's Dutch cohort, including Poll-The's cohort (31 patients), hyperoxaluria study (31), adrenal study (24), adult cohort (19, all European without consanguinity) and cholic acid studies [17, 36, 44, 52, 68]. Ebberink and colleagues' 600 cell lines were analysed at the same centre [29]. In Portugal, consulted sources include case reports without epidemiological data: a 2-month-old Zellweger infant presenting with severe malnutrition, opportunistic infections, lymphopenia and a small thymus, initially investigated as primary immunodeficiency, and a newborn MRI description from Braga [90, 91]. No Brazilian ZSD data were found in consulted sources; this does not demonstrate absence of published studies.

9.4. Reference networks, registries and associations

In Europe, peroxisomal disorders fall within MetabERN, the European reference network for inherited metabolic diseases, in its peroxisomal and lipid-related disorders (PD) subnetwork, also maintaining the unified European U-IMD registry [19]. The international longitudinal peroxisome biogenesis disorder natural history study (NCT01668186), coordinated in Montreal, provides the most recent North American vision, hearing and severity data [47, 48, 83, 92]. The Global Foundation for Peroxisomal Disorders brings together families affected by ZSD and single-enzyme peroxisomal diseases and maintains a patient registry; United Leukodystrophy Foundation and National Institute of Neurological Disorders and Stroke are other GeneReviews-listed resources [1, 20]. The family-participating caregiver survey, with 54 living and 25 deceased patients, found seizure and adrenal insufficiency prevalences almost twice those in clinical series, illustrating these sources' value and biases [49].

For Heimler syndrome, the French PNDS defines a specific pathway within the SENSGENE network (filière for rare sensory diseases): the reference centre for genetic deafness, coordinated at Necker–Enfants Malades Hospital (Paris), with constituent sites at Pitié-Salpêtrière and in Lille and a network of competence centres; the CARGO centre (Strasbourg) for ophthalmic genetics; and the O-Rares centre (Strasbourg) for rare oral and dental diseases. The PNDS also appends the lists of French cochlear implant centres and CPDPN. There is no dedicated patient association; the PNDS indicates associations of deaf people, of cochlear implant users and of people with visual impairment (surdi.info, SENSGENE, Retina France) and, at European level, ERN-EYE and ERN CRANIO [14].

For liver disease and the differential diagnosis of cholestasis, the French pathway is that of the reference centre for biliary atresia and genetic cholestasis, coordinated at Bicêtre Hospital (AP-HP, Le Kremlin-Bicêtre), with constituent sites in Lyon (Hôpital Femme-Mère-Enfant) and Paris (Necker–Enfants Malades Hospital) and paediatric and adult hepatology competence centres, within the FILFOIE network (filière for rare liver diseases of children and adults); the patient association indicated is AMFE (Association Maladies Foie Enfants) [18].

10. Illustrative Clinical Case

10.1. Intermediate PEX1-related ZSD with hepatopulmonary syndrome treated by liver transplantation

Tharakan, Rajwal and Schwahn published an intermediate ZSD girl in JIMD Reports in 2025 [51]. She was assessed neonatally after failed hearing screening confirmed sensorineural deafness. Subsequent assessment showed muscle hypotonia, global developmental delay and pigmentary retinopathy with moderate visual impairment; MRI suggested white matter abnormalities. She was referred to metabolic medicine at 19 months: ALT was normal (29 U/L), with mild coagulopathy (PT 18.4 s; aPTT 38.4 s) and elevated VLCFA and phytanic acid (at 1 year 7 months, C26/C22 0.283 versus reference 0.007–0.028; phytanate 101.0 µmol/L versus reference 0–16). Genetic testing showed compound heterozygosity for the two PEX1 founder variants, c.2097dup and c.2528G>A p.(Gly843Asp) [51].

A cochlear implant was fitted at 22 months and supportive vitamin K, ursodeoxycholic acid, chenodeoxycholic acid and dietary phytanate restriction with essential fatty acid supplementation began at 23 months. Coagulopathy improved without normalising; ALT increased from 3.5 years (45–108 U/L). At 4 years, after abnormal Synacthen testing, physiological hydrocortisone began. Phytanate restriction was relaxed because of poor intake and weight loss; she received nasogastric feeding and gastrostomy at 6 years. Chenodeoxycholic acid was stopped at 7 years because of institutional policy changes. Pathological fractures occurred at 6 years 4 months and 7 years 9 months. Development progressed slowly: she walked with a frame, understood words and communicated through signs [51].

At 7 years 2 months, recurrent finger and lip cyanosis and intermittent fever developed. Admission for suspected upper respiratory infection showed dyspnoea, hyperventilation, low pCO₂, transcutaneous saturation 70 % and poor oxygen response; infection markers, viral studies and chest radiography were normal. Echocardiograms showed no significant pulmonary hypertension, but agitated saline rapidly opacified the left heart after the fourth beat without intracardiac shunting, suggesting an extracardiac right-to-left shunt. CT angiography excluded thoracic arteriovenous malformation. Liver ultrasound showed a nodular surface, ductus venosus recanalisation and prominent superior mesenteric vein, indirect portal hypertension signs, with a small spleen (Figure 1 of Tharakan 2025). Technetium-99m-labelled macroaggregated albumin lung perfusion scintigraphy showed brain and kidney uptake, with a calculated right-to-left shunt of 39.8 %, confirming hepatopulmonary syndrome [51].

At the paediatric liver transplant unit, angiography showed free hepatic venous pressure of 7 mmHg and wedged pressure of 22 mmHg. She remained oxygen-dependent (2 L/min via nasal cannula), with saturations around 85 %. After multidisciplinary discussion and parental consent, despite high perioperative risk, living-donor orthotopic transplantation was performed at 7 years 10 months. The explanted liver showed nodular cirrhosis (Figure 2 of Tharakan 2025). Saturation normalised over 2 months. Almost 4 years later, aged 11, she was clinically well without oxygen, with normal liver function and improved biomarkers (C26/C22 0.090, phytanate 1.4 µmol/L and pristanate 0.1 µmol/L at 11 years 4 months), but gradually progressing retinal dystrophy and variable bilateral resting tremor [51].

Clinical interpretation. The case illustrates neonatal deafness and retinopathy presentation, the compound heterozygous c.2097dup/p.(Gly843Asp) intermediate phenotype correlation, stimulation-test-detected adrenal insufficiency, osteopenia with fractures and inconspicuously progressing cirrhosis with moderately elevated transaminases and subtle ultrasound portal hypertension signs [1, 35, 51]. The authors propose considering hepatopulmonary syndrome screening in surveillance of children with chronic liver disease and discuss whether transplantation may attenuate mild and intermediate disease [51]. These are author opinions from one case; available guidance neither includes systematic hepatopulmonary screening nor recommends transplantation for extrahepatic manifestations, and retinal and neurological function progressed despite biochemical correction [1, 3, 51]. Chenodeoxycholic and ursodeoxycholic acid use reflected a centre decision rather than guidance recommendations.

11. Prognosis and Long-Term Follow-Up

11.1. Prognosis

Prognosis depends on spectrum position. Severely affected children usually die in their first year without developmental progress [1, 3]. Childhood or adolescent presenters have better prognosis but may develop progressive liver disease or leukodystrophy, making prognosis poor; life expectancy is reduced and most childhood-presenting patients die before adolescence [3]. Children surviving their first year with a non-progressive course have a 77 % probability of reaching school age [1]. The mildest patients may reach adulthood without progression or with long stability; progression mainly reflects peripheral neuropathy and pyramidal signs with stable cognition [3, 17].

Individual prediction is difficult. Genotype–phenotype correlation is approximate, particularly for PEX1 and PEX26 [3]. Fibroblast studies (DHAPAT activity and C26:0 beta-oxidation) permitted only rough distinction between survival below 1 year or above 5 years [17]. In a review of 107 studies (307 patients) and 136 natural history participants, the number of findings among seizures, abnormal EEG, renal cysts and cardiac abnormalities, together with plasma C26:0, differentiated severity categories; seizures, hypotonia, reduced mobility, feeding difficulties, renal cysts, adrenal insufficiency, deafness, visual loss and lower survival differed between categories [92]. In 2025 modelling, authors applied published-curve-derived mean annual survival of 76 % for severe disease, 96.6 % through 8 years, 99.2 % at 9 to 18 and 98.0 % after 19 for intermediate disease, and 99.4 % for mild disease; these are modelling parameters rather than cohort observations [45]. Hepatic peroxisomal mosaicism was associated with better prognosis, and liver disease frequently caused death in severe and mild forms [35].

In Heimler syndrome no cerebral or hepatic involvement has been described, and functional prognosis depends on the hearing loss and, later, on the retinal dystrophy; dual sensory impairment is the main determinant of educational, social and occupational outcome [14].

11.2. Surveillance

For apparently asymptomatic siblings of mildly affected probands, GeneReviews recommends clarifying genetic status to identify affected individuals early, allowing annual hearing and ophthalmological assessment and coagulation, adrenal and liver monitoring; molecular testing is preferable because mild-form biomarkers vary greatly [1]. Adults need transition to adult teams experienced in metabolic disease and neurological follow-up of gait, neuropathy and leukoencephalopathy [17].

12. Summary and Concluding Remarks

Zellweger spectrum disorders result from usually biallelic variants in one of 13 PEX genes required for peroxisome formation and protein import. PEX1 and PEX6 explain approximately three-quarters of cases, and the two European PEX1 founder variants, c.2097dup and c.2528G>A p.(Gly843Asp), occur in approximately 80 % of PEX1-related ZSD patients. Severity mainly depends on genotype-permitted residual function: null alleles accompany severe neonatal disease with dysmorphism, abnormal neuronal migration, renal cysts, patellar stippling and liver disease, and usual death in the first year; hypomorphic alleles accompany intermediate and mild forms, dominated by retinal dystrophy, sensorineural deafness, hepatic dysfunction, adrenal insufficiency, hyperoxaluria, osteopenia, amelogenesis imperfecta and, in some, leukodystrophy or adult gait disease [1, 3, 17].

Diagnosis begins with peroxisomal biochemistry — C26:0-LPC, VLCFA, phytanate and pristanate, plasmalogens, pipecolate and C27 bile acids — and is confirmed molecularly. Two limitations are decisive: mild forms may have normal biochemistry, particularly in adults, and single heterozygous variants may be causal in PEX6 and PEX14. Fibroblast studies including 40 °C culture and RNA analysis resolve some exome-undetected cases [1, 9, 10, 17, 58]. Principal differentials are isolated peroxisomal beta-oxidation defects (D-bifunctional protein and ACOX1), accounting for approximately 15 % of similar elevated-VLCFA presentations, and RCDP in stippled infants, distinguished by rhizomelia, coronal clefts, low plasmalogens and normal VLCFA [1, 11, 57].

Treatment is supportive and based on expert recommendations (Klouwer 2015, Braverman 2016, GeneReviews 2020), with annual surveillance of hearing, vision, liver, adrenal function from the first year and urinary oxalate, and six-monthly dental surveillance; in Heimler syndrome, the 2021 French PNDS adds six-monthly hearing surveillance until 6 years, dental care from the first tooth, preferential simultaneous bilateral cochlear implantation and an oral communication plan in view of foreseeable visual loss [1–3, 14]. Cholic acid (Cholbam®) is the only authorised medicine with an indication in ZSD, in the United States, as adjunctive treatment of the hepatic manifestations, at a dose of 10 to 15 mg/kg/day; it reduces the C27 bile acid intermediates, but clinical benefit has not been demonstrated and it may worsen advanced liver disease; in the European Union it does not have this indication [15, 16, 68, 77]. DHA was negative in a randomised trial, and betaine did not change the C26/C22 ratio [37, 79]. The most recent developments, hepatic base editing and retinal gene therapy in PEX1 p.(Gly843Asp) models, remain at the preclinical stage [86, 87].

True frequency is unknown: estimates range from 1 in 50,000 to 1 in 133,000 in the US, with 1 in 500,000 in Japan and 1 in 12,191 in a Quebec founder population, and X-ALD newborn screening and genetic modelling suggest many mild forms remain undiagnosed [1, 12, 13, 45]. For geneticists, central tasks are recognising attenuated presentations (deafness with retinopathy, Heimler syndrome, ataxia with cataracts), critically interpreting normal biochemical findings or single variants, reproductive counselling with prenatal and preimplantation diagnosis, and coordinating multidisciplinary surveillance extending into adulthood for some patients [1, 17, 30].

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