Novel PEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBDs) are a group of neuronal migration/neurodegenerative disorders that arise from defects in PEX genes. A major subgroup of the PBDs includes Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). These three disorders represent a clinical continuum with Zellweger syndrome the most severe. Mutations in the PEX1 gene, which encodes a protein of the AAA ATPase family involved in peroxisome matrix protein import, account for the genetic defect in more than half of the patients in this PBD subgroup. We report here on the results of PEX1 mutation detection in an Australasian cohort of PEX1-deficient PBD patients. This screen has identified five novel mutations, including nonsense mutations in exons 14 and 19 and single nucleotide deletions in exons 5 and 18. Significantly, the allele carrying the exon 18 frameshift mutation is present at moderately high frequency (approx. 10%) in this patient cohort. The fifth mutation is a missense mutation (R798G) that attenuates, but does not abolish PEX1 function. We have evaluated the cellular impact of these novel mutations, along with that of the two most common PEX1 mutations (c.2097-2098insT and G843D), in PBD patients by determining the levels of PEX1 mRNA, PEX1 protein, and peroxisome protein import. The findings are consistent with a close correlation between cellular phenotype, disease severity, and PEX1 genotype.     

PEX1 mutations in the Zellweger spectrum of the peroxisome biogenesis disorders

Diseases of the Zellweger spectrum represent a major subgroup of the peroxisome biogenesis disorders, a group of autosomal-recessive diseases that are characterized by widespread tissue pathology, including neurodegeneration. The Zellweger spectrum represents a clinical continuum, with Zellweger syndrome (ZS) having the most severe phenotype, and neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) having progressively milder phenotypes. Mutations in the PEX1 gene, which encodes a 143-kDa AAA ATPase protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The PEX1 mutations identified to date comprise insertions, deletions, nonsense, missense, and splice site mutations. Mutations that produce premature truncation codons (PTCs) are distributed throughout the PEX1 gene, whereas the majority of missense mutations segregate with the two essential AAA domains of the PEX1 protein. Severity at the two ends of the Zellweger spectrum correlates broadly with mutation type and impact (i.e., the severe ZS correlates with PTCs on both alleles, and the milder phenotypes correlate with missense mutations), but exceptions to these general correlations exist. This article provides an overview of the currently known PEX1 mutations, and includes, when necessary, revised mutation nomenclature and genotype-phenotype correlations that may be useful for clinical diagnosis.     

Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), are autosomal recessive diseases caused by deficiency of peroxisome assembly as well as malfunction of peroxisomes, where >10 genotypes have been reported. ZS patients manifest the most severe clinical and biochemical abnormalities, while those with NALD and IRD show the least severity and the mildest features, respectively. PEX1 is the causative gene for PBDs of complementation group I (CG1), the highest incidence PBD, and encodes the peroxin, Pex1p, a member of the AAA ATPase family. In the present work, we found that peroxisomes were morphologically and biochemically formed at 30 but not 37 degrees C, in the fibroblasts from all CG1 IRD patients examined, whereas almost no peroxisomes were seen in ZS and NALD cells, even at 30 degrees C. A point missense mutation, G843D, was identified in the PEX1 allele of most CG1 IRD patients. The mutant PEX1, termed HsPEX1G843D, gave rise to the same temperature-sensitive phenotype on CG1 CHO cell mutants upon transfection. Collectively, these results demonstrate temperature-sensitive peroxisome assembly to be responsible for the mildness of the clinical features of PEX1 - defective IRD of CG1.      

Novel mutations in the PEX12 gene of patients with a peroxisome biogenesis disorder

The peroxisome biogenesis disorders (PBDs) form a genetically and clinically heterogeneous group of disorders due to defects in at least 11 distinct genes. The prototype of this group of disorders is Zellweger syndrome (ZS), with neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) as milder variants. Liver disease, variable neurodevelopmental delay, retinopathy and perceptive deafness are common to PBDs. PBD patients belonging to complementation group 3 (CG3) have mutations in the PEX12 gene, which codes for a protein (PEX12) that contains two transmembrane domains, and a zinc-binding domain considered to be important for its interaction with other proteins of the peroxisomal protein import machinery. We report on the identification of five PBD patients belonging to CG3. Sequence analysis of their PEX12 genes revealed five different mutations, four of which have not been reported before. Four of the patients have mutations that disrupt the translation frame and/or create an early termination codon in the PEX12 open reading frame predicted to result in truncated protein products, lacking at least the COOH-terminal zinc-binding domain. All these patients display the more severe phenotypes (ZS or NALD). The fifth patient expresses two PEX12 alleles capable of encoding a protein that does contain the zinc-binding domain and displayed a milder phenotype (IRD). The three biochemical markers measured in fibroblasts (DHAPAT activity, C26:0 beta-oxidation and pristanic acid beta-oxidation) also correlated with the genotypes. Thus, the genotypes of our CG3 patients show a good correlation with the biochemical and clinical phenotype of the patients.     

Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and beta oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.     

Two novel PEX1 mutations in a patient with Zellweger syndrome: the first Korean case confirmed by biochemical, and molecular evidence

Peroxisome biogenesis disorders (PBD) represent a spectrum of genetic disorders characterized by impaired peroxisome assembly. Zellweger syndrome (ZS) is the most severe form of PBD and is characterized by craniofacial abnormalities, severe hypotonia, neonatal seizures, ocular abnormalities, psychomotor retardation, hepatomegaly and increased levels of very long chain fatty acids (VLCFA). The most common mutation associated with the PBD is PEX1. Here, the first Korean patient with ZS confirmed by clinical, biochemical, and molecular findings is reported. Two novel mutations of the PEX1 gene were identified in the patient with ZS. The patient was a compound heterozygote for c.2034_2035delCA and c.2845C>T mutations of the PEX1 gene. Both mutations are novel findings and were inherited from the patient's parents. In summary, here the first Korean case of ZS is reported that was confirmed by two novel mutations of the PEX1 gene.     

Genetic heterogeneity of peroxisome biogenesis disorders among Japanese patients: evidence for a founder haplotype for the most common PEX10 gene mutation

We, as the only diagnostic center for peroxisome biogenesis disorders (PBD) in Japan, identified a total of 31 Japanese patients with PBD during the last 20 years. They were 27 patients with Zellweger syndrome (ZS), including two sib cases, three with neonatal adrenoleukodystrophy (NALD) and one with rhizomelic type chondrodysplasia punctata (RCDP). No patient with infantile Refsum disease has been detected. These patients were genetically subdivided into complementation group A (five ZS and one NALD), B (11 ZS), C (four ZS), E (five ZS and two NALD), F (two ZS), and R (one RCDP). They were subjected to mutation analysis of PEX1, PEX2, PEX6, PEX7, and PEX10. All the 11 ZS patients with group-B PBD had a common mutation, i.e., a homozygous 2-base-pair deletion in PEX10. To determine whether this highly frequent mutation is due to a founder effect, we analyzed single nucleotide polymorphisms within PEX10 among patients and Japanese controls. The mutation apparently arose once on an ancestral chromosome in the Japanese population. Based on the value of 24 PBD patients identified during the last 10 years, we estimated the prevalence of PBD in Japan to be approximately one in 500,000 births.     

Genotype-phenotype correlations in disorders of peroxisome biogenesis.

Genetically determined human peroxisomal disorders are subdivided into two major categories: disorders of peroxisome biogenesis (PBD), in which the organelle is not formed normally, and those that involve a single peroxisomal enzyme. Twelve PBD have been identified, and the molecular defects have been defined in 10. All involve defects in the import of proteins into the organelle. Factors required for this import are now referred to as peroxins (PEX) and form the basis of a new and preferred classification system. The PBD are associated with four clinical phenotypes, named before their association with the organelle was recognized: Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). The first three are associated with 9 of the 10 PEX defects that have been defined so far, and represent a clinical continuum with variant severity, with ZS the most severe, NALD intermediate, and IRD the least severe. RCDP is associated with PEX7. Genotype-phenotype correlations are complicated by the fact that the clinical manifestations of the ZS-NALD-IRD continuum can be mimicked by disorders that affect single enzymes of peroxisomal fatty acid oxidation, and PEX7 by disorders of plasmalogen synthesis enzymes. Furthermore, clinical manifestations of each of the PEX disorders may vary. Phenotypic expression varies with the nature of the mutation, the milder phenotypes being associated with mutations that do not abolish function completely, or with mosaicism. Definition of the molecular defects is of great value for genetic counseling and may be of aid in establishing prognosis.     

Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.     

Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders

The peroxisomal disorders represent a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided into two subgroups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD group is comprised of four different disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum's disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD the less severe disorders. Studies in the late 1980s had already shown that the PBD group is genetically heterogeneous with at least 12 distinct genetic groups as concluded from complementation studies. Thanks to the much improved knowledge about peroxisome biogenesis notably in yeasts and the successful extrapolation of this knowledge to humans, the genes responsible for all these complementation groups have been identified making molecular diagnosis of PBD patients feasible now. It is the purpose of this review to describe the current stage of knowledge about the clinical, biochemical, cellular, and molecular aspects of PBDs, and to provide guidelines for the post- and prenatal diagnosis of PBDs. Less progress has been made with respect to the pathophysiology and therapy of PBDs. The increasing availability of mouse models for these disorders is a major step forward in this respect.     

Prader-Willi syndrome: causes of death in an international series of 27 cases

The peroxisome biogenesis disorders (PBDs) with generalized peroxisomal dysfunction include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). There is clinical, biochemical, and genetic overlap among the three phenotypes, also known as Zellweger spectrum disorders. Clinical distinctions between the phenotypes are not sharply defined. Only limited sources are available to serve as a background for prognosis in PBD, especially in case of prolonged survival. We delineated the natural history of 31 PBD patients (age 1.2-24 years) through systematic clinical and biochemical investigations. We excluded classical ZS from our study, and included all patients with a biochemically confirmed generalized peroxisomal disorder over 1 year of age, irrespective of the previously diagnosed phenotype. The initial clinical suspicion, age at diagnosis, growth, development, neurological symptoms, organ involvements, and survival are summarized. Common to all patients were cognitive and motor dysfunction, retinopathy, sensorineural hearing impairment, and hepatic involvement. Many patients showed postnatal growth failure, 10 patients displayed hyperoxaluria of whom 4 had renal stones. Motor skills ranged from sitting with support to normal gait. Speech development ranged from non-verbal expression to grammatical speech and comprehensive reading. The neurodevelopmental course was variable with stable course, rapid decline with leukodystrophy, spinocerebellar syndrome, and slow decline over a wide range of faculties as outcome profiles. At the molecular level, 21 patients had mutations in the PEX1 gene. The two most common PEX1 mutations were the G843D (c.2528G-->A) missense and the c.2097insT frameshift mutation. Patients having the G843D/G843D or the G843D/c.2097insT genotypes were compared. Patients homozygous for G843D generally had a better developmental outcome. However, one patient who was homozygous for the "mild" G843D mutation had an early lethal disease, whereas two other patients had a phenotype overlapping with the G843D/c.2097insT group. This indicates that next to the PEX1 genotype other yet unknown factors determine the ultimate phenotype.     

Phenotype-genotype relationships in PEX10-deficient peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBD) are characterized by neural, hepatic, and renal deficiencies, severe mental retardation, and are often lethal. These disorders are genetically and phenotypically heterogeneous and are caused by defective peroxisomal protein import and decreased peroxisomal metabolic function. Mutations in PEX10 have been identified in patients from complementation group 7 (CG7) of the PBDs and we report here an analysis of the genotypes and phenotypes of PEX10-deficient patients. All four PEX10-deficient Zellweger Syndrome (ZS) patients were found to have nonsense, frameshift, or splice site mutations that remove large portions of the PEX10 coding region. In contrast, a more mildly affected PEX10-deficient neonatal adrenoleukodystrophy patient expressed a PEX10 allele with a missense mutation, H290Q, affecting the C-terminal zinc-binding domain of the PEX10 product. These results support the hypothesis that severe, loss-of-function mutations in PEX genes cause more severe clinical phenotypes, whereas mildly affected PBD patients have PEX gene mutations that retain residual function. To quantitate the effects of the PEX10 mutations identified here and elsewhere we employed a functional complementation assay. Surprisingly, we observed that nonsense and frameshift mutations predicted to delete the C-terminal 2/3 (R125X) or 1/3 (c.704insA) of the protein displayed nearly normal PEX10 activity. Even more surprising, we found that the unexpectedly high PEX10 activity displayed by these cDNAs could be eliminated by removing or mutating segments of the PEX10 cDNA downstream of the mutations. Although these results demonstrate serious flaws in the PEX10 functional complementation assay, they do suggest that the C-terminal zinc-binding domain is critical for PEX10 function.     

The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum

Peroxisome biogenesis disorders in the Zellweger syndrome spectrum (PBD-ZSS) are caused by defects in at least 12 PEX genes required for normal organelle assembly. Clinical and biochemical features continue to be used reliably to assign patients to this general disease category. Identification of the precise genetic defect is important, however, to permit carrier testing and early prenatal diagnosis. Molecular analysis is likely to expand the clinical spectrum of PBD and may also provide data relevant to prognosis and future therapeutic intervention. However, the large number of genes involved has thus far impeded rapid mutation identification. In response, we developed the PEX Gene Screen, an algorithm for the systematic screening of exons in the six PEX genes most commonly defective in PBD-ZSS. We used PCR amplification of genomic DNA and sequencing to screen 91 unclassified PBD-ZSS patients for mutations in PEX1, PEX26, PEX6, PEX12, PEX10, and PEX2. A maximum of 14 reactions per patient identified pathological mutations in 79% and both mutant alleles in 54%. Twenty-five novel mutations were identified overall. The proportion of patients with different PEX gene defects correlated with frequencies previously identified by complementation analysis. This systematic, hierarchical approach to mutation identification is therefore a valuable tool to identify rapidly the molecular etiology of suspected PBD-ZSS disorders.     

Genetic and clinical aspects of Zellweger spectrum patients with PEX1 mutations

Objective: To analyse the PEX1 gene, the most common cause for peroxisome biogenesis disorders (PBD), in a consecutive series of patients with Zellweger spectrum. Methods: Mutations were detected by different methods including SSCP analyses as a screening technique on the basis of genomic or cDNA, followed by direct sequencing of PCR fragments with an abnormal electrophoresis pattern. Results: 33 patients were studied. Two common mutations, c.2528G→A, G843D and c.2098_2098insT, I700YfsX42, accounted for over 80% of all abnormal PEX1 alleles, emphasising their diagnostic relevance. Most PEX1 mutations were distributed over the two AAA cassettes with the two functional protein domains, D1 and D2, and the highly conserved Walker motifs. Phenotypic severity of Zellweger spectrum in CG1 depended on the effect of the mutation on the PEX1 protein, peroxin 1. PEX1 mutations could be divided into two classes of genotype–phenotype correlation: class I mutations led to residual PEX1 protein levels and function and a milder phenotype; class II mutations almost abolished PEX1 protein levels and function, resulting in a severe phenotype. Compound heterozygote patients for a class I and class II mutation had an intermediate phenotype. Conclusions: Molecular confirmation of the clinical and biochemical diagnosis will allow the prediction of the clinical course of disease in individual PBD cases.     

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.     

A founder mutation in the PEX6 gene is responsible for increased incidence of Zellweger syndrome in a French Canadian population

ABSTRACT: BACKGROUND: Zellweger syndrome (ZS) is a peroxisome biogenesis disorder due to mutations in any one of 13 PEX genes. Increased incidence of ZS has been suspected in French-Canadians of the Saguenay-Lac-St-Jean region (SLSJ) of Quebec, but this remains unsolved. METHODS: We identified 5 ZS patients from SLSJ diagnosed by peroxisome dysfunction between 1990--2010 and sequenced all coding exons of known PEX genes in one patient using Next Generation Sequencing (NGS) for diagnostic confirmation. RESULTS: A homozygous mutation (c.802_815del, p.[Val207_Gln294del, Val76_Gln294del]) in PEX6 was identified and then shown in 4 other patients. Parental heterozygosity was confirmed in all. Incidence of ZS was estimated to 1 in 12,191 live births, with a carrier frequency of 1 in 55. In addition, we present data suggesting that this mutation abolishes a SF2/ASF splice enhancer binding site, resulting in the use of two alternative cryptic donor splice sites and predicted to encode an internally deleted in-frame protein. CONCLUSION: We report increased incidence of ZS in French-Canadians of SLSJ caused by a PEX6 founder mutation. To our knowledge, this is the highest reported incidence of ZS worldwide. These findings have implications for carrier screening and support the utility of NGS for molecular confirmation of peroxisomal disorders.     

Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms

Zellweger syndrome and its milder variants--neonatal adrenoleukodystrophy and infantile Refsum disease--comprise a clinical continuum of diseases referred to as the Zellweger spectrum. Mutations in the PEX1 gene, which consists of 24 exons and encodes a AAA ATPase protein required for peroxisomal protein import, account for approximately two-thirds of the known Zellweger spectrum patient mutations. In this paper, we report on four novel PEX1 mutations and two polymorphisms in an Australasian cohort. Two of the mutations--c.1108_1109insA and c.2391_2392delTC--that lead to the introduction of a premature termination codon in exons 5 and 14, respectively, are associated with the severe Zellweger phenotype. One patient with a milder disease phenotype was a compound heterozygote for two missense mutations (I989T and R998Q), both affecting amino acids in the second, C-terminal AAA domain of the protein. PTS1 protein import levels in cultured skin fibroblasts from this patient were almost 20% of normal control levels. We have also characterized two co-segregating polymorphisms in the 5' UTR of the PEX1 gene. Based on reporter assays, the c.-137T>C polymorphism leads to reduced PEX1 expression, whereas the c.-53C>G polymorphism leads to increased expression. When present together, these regulatory polymorphisms lead to near-normal PEX1 expression. Altered PEX1 expression due to the presence of either the c.-137T>C or the c.-53C>G variant could impact on residual PEX1 function if another co-allelic mutation was present which did not completely abolish PEX1 function. It also follows that the presence of polymorphisms in the PEX1 promoter region could have implications for patients with mutations in other PEX proteins known to interact with PEX1, such as PEX6. Thus, although not deleterious in control individuals, these polymorphisms could contribute to phenotypic heterogeneity among Zellweger spectrum patients.     

Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive neurodegenerative disorders that affect multiple organ systems. Approximately 80% of PBD patients are classified in the Zellweger syndrome spectrum (PBD‐ZSS). Mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes are found in approximately 90% of PBD‐ZSS patients. Here, we sequenced the coding regions and splice junctions of these five genes in 58 PBD‐ZSS cases previously subjected to targeted sequencing of a limited number of PEX gene exons. In our cohort, 71 unique sequence variants were identified, including 18 novel mutations predicted to disrupt protein function and 2 novel silent variants. We identified 4 patients who had two deleterious mutations in one PEX gene and a third deleterious mutation in a second PEX gene. For two such patients, we conducted cell fusion complementation analyses to identify the defective gene responsible for aberrant peroxisome assembly. Overall, we provide empirical data to estimate the relative fraction of disease‐causing alleles that occur in the coding and splice junction sequences of these five PEX genes and the frequency of cases where mutations occur in multiple PEX genes. This information is beneficial for efforts aimed at establishing rapid and sensitive clinical diagnostics for PBD‐ZSS patients and interpreting the results from these genetic tests. © 2008 Wiley‐Liss, Inc.     

Identification of a common PEX1 mutation in Zellweger syndrome

The Zellweger spectrum of disease, encompassing Zellweger syndrome and the progressively milder phenotypes of neonatal adrenoleukodystrophy and infantile Refsum disease, is due to a failure to form functional peroxisomes. Cell fusion complementation studies demonstrated that these diseases are genetically heterogeneous, with two‐thirds of all patients lying within a single complementation group, CG1. Molecular genetic and cell biology studies have shown that PEX1 is deficient in many CG1 patients. However, previous studies have focused on mildly affected patients and there is still no report of two mutant PEX1 alleles in any Zellweger syndrome patient. Furthermore, mutations in the PMP70 gene have also been identified in two Zellweger syndrome patients from CG1, raising the possibility that CG1 patients may represent a mixture of PEX1‐deficient and PMP70‐deficient individuals. To address the molecular basis of disease in Zellweger syndrome patients from CG1, we examined all 24 PEX1 exons in four patients, including both patients that have mutations in PMP70. PEX1 mutations were detected in all four patients, including a 1‐bp insertion (c.2097insT) in exon 13 that was present in three of the four patients. Subsequent studies demonstrated that this mutation is present in one‐half of all CG1 patients and correlates with the Zellweger syndrome phenotype. As this mutation leads to a loss of protein function its frequency makes it the most common cause of Zellweger syndrome, helping to explain the high percentage of patients that belong to CG1. Hum Mutat 14:45–53, 1999. © 1999 Wiley‐Liss, Inc.     

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor‐2) gene in patients with peroxisome biogenesis disorders

The PEX6 (peroxisome assembly factor‐2, PAF‐2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487–496, 1999. © 1999 Wiley‐Liss, Inc.