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.     

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.     

Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder

The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders and can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe multisystemic disorders. To get insight into the spectrum of PEX gene defects among ZSS disorders and to investigate if additional human PEX genes are required for functional peroxisome biogenesis, we assigned over 600 ZSS fibroblast cell lines to different genetic complementation groups. These fibroblast cell lines were subjected to a complementation assay involving fusion by means of polyethylene glycol or a PEX cDNA transfection assay specifically developed for this purpose. In a majority of the cell lines we subsequently determined the underlying mutations by sequence analysis of the implicated PEX genes. The PEX cDNA transfection assay allows for the rapid identification of PEX genes defective in ZSS patients. The assignment of over 600 fibroblast cell lines to different genetic complementation groups provides the most comprehensive and representative overview of the frequency distribution of the different PEX gene defects. We did not identify any novel genetic complementation group, suggesting that all PEX gene defects resulting in peroxisome deficiency are currently known.     

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.     

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.     

Rational diagnostic strategy for Zellweger syndrome spectrum patients

Zellweger syndrome spectrum (ZSS) comprises a clinically and genetically heterogeneous disease entity, which is caused by mutations in any of the 12 different human PEX genes leading to impaired biogenesis of the peroxisome. Patients potentially suffering from ZSS are diagnosed biochemically by measuring elevated levels of very long chain fatty acids, pristanic acid and phytanic acid in plasma and serum and reduced levels of ether phospholipids in erythrocytes. Published reports on diagnostic procedures for ZSS patients are restricted either to biochemical markers or to defined mutations in a subset of PEX genes. Clarification of the primary genetic defect in an affected patient is crucial for genetic counselling, carrier testing or prenatal diagnosis. In this study, we present a rational diagnostic strategy for patients suspected of ZSS. By combining cell biology and molecular genetic methods in an appropriate sequence, we were able to detect the underlying mutation in various PEX genes within adequate time and cost. We applied this method on 90 patients who presented at our institute, Department of Pediatrics and Pediatric Neurology at Georg August University, and detected 174 mutant alleles within six different PEX genes, including two novel deletions and three new missense mutations in PEX6. Furthermore, this strategy will extend our knowledge on genotype–phenotype correlation in various PEX genes. It will contribute to a better understanding of ZSS pathogenesis, allowing the investigation of the effects of diverse mutations on the interaction between PEX proteins and peroxisomal function in vivo.     

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.     

Clustering of mutations associated with mild Marfan‐like phenotypes in the 3′ region of FBN1 suggests a potential genotype–phenotype correlation

Mutations in the gene for fibrillin‐1 (FBN1) cause Marfan syndrome, a dominantly inherited disorder of connective tissue that primarily involves the cardiovascular, ocular, and skeletal systems. There is a remarkable degree of variability both within and between families with Marfan syndrome, and FBN1 mutations have also been found in a range of other related connective tissue disorders collectively termed type‐1 fibrillinopathies. FBN1 mutations have been found in almost all of the 65 exons of the FBN1 gene and for the most part have been unique to one affected patient or family. Aside from the “hot spots” for the neonatal Marfan syndrome in exons 24–27 and 31–32, genotype–phenotype correlations have been slow to emerge. Here we present the results of temperature‐gradient gel electrophoresis analysis of FBN1 exons 59–65. Six mutations were identified, only one of which had been previously reported. Two of the six mutations were found in patients with mild phenotypes. Taken together with other published reports, our results suggest that a sizable subset (ca. 40%) of mutations in this region is associated with mild phenotypes characterized by the lack of significant aortic pathology, compared with about 7% in the rest of the gene. In two cases, mutations affecting analogous positions within one of the 43 cbEGF modules of FBN1 are associated with mild phenotypes when found in one of the 6 C‐terminal modules (encoded by exons 59–63), but are associated with classic or severe phenotypes when found in cbEGF modules elsewhere in the gene. Am. J. Med. Genet. 91:212–221, 2000. © 2000 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.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia

X-linked hypophosphatemic rickets in humans is caused by mutations in the PEX gene which codes for a protein homologous to neutral endopeptidases. Hyp and Gy mice both have X-linked hypophosphatemic rickets, although genetic data and the different phenotypic spectra observed have previously suggested that two different genes are mutated. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. We now report the cloning of the mouse homolog of PEX which is highly conserved between man and mouse. The 3' end of this gene is deleted in Hyp mice. In Gy mice, the first three exons and the promotor region are deleted. Thus, Hyp and Gy are allelic mutations and both provide mouse models for X-linked hypophosphatemia.      

CNGB3 mutation spectrum including copy number variations in 552 achromatopsia patients

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3 , CNGB3 , GNAT2 , PDE6C , and PDE6H ) or in ATF6 , involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide‐gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3 . We identified a total of 98 different potentially disease‐causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real‐time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray‐based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4?18 was identified, highlighting the importance of CNV analysis for this gene.     

The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6

Human genetic peroxisomal biogenesis disorders (PBDs), such as Zellweger syndrome, comprise 13 different complementation groups (CGs). Eleven peroxin genes, termed PEXs, responsible for PBDs have been identified, whereas pathogenic genes for PBDs of 2 CGs, CG-A (the same CG as CG8 in the United States and Europe) and CG6, remained unidentified. We herein provide several lines of novel evidence indicating that PEX6, the pathogenic gene for CG4, is impaired in PBD of CG6. Expression of PEX6 restored peroxisome assembly in fibroblasts from a CG6 PBD patient. This patient was a compound heterozygote for PEX6 gene alleles. Accordingly, by merging CG6 with CG4, human PBDs are now classified into 12 CGs. Received: December 25, 2000 / Accepted: February 5, 2001     

Delineation of the Marfan phenotype associated with mutations in exons 23–32 of the FBN1 gene

Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24–32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23–32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25–27 of the FBN1 gene in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24–32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. © 1996 Wiley-Liss, Inc.     

Novel genomic techniques open new avenues in the analysis of monogenic disorders

The molecular genetic cause of over 3,000 monogenic disorders is currently unknown. This review discusses how novel genomic techniques like Next‐Generation DNA Sequencing (NGS) and genotyping arrays open new avenues in the elucidation of genetic defects causing monogenic disorders. They will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. These are mainly disorders not amenable to classic linkage analysis, for example, due to insufficient family size. Most monogenic diseases are caused by exonic mutations or splice‐site mutations changing the amino acid sequence of the affected gene. These mutations can be identified by sequencing of all exons in the human genome (exome sequencing) rendering whole genome sequencing unnecessary in most cases. Genotyping arrays containing 10⁵–2×10⁶ single nucleotide polymorphisms (SNPs) and nonpolymorphic markers allow highly accurate mapping of genomic deletions and duplications not detectable by exome sequencing, which are the second most common cause of monogenic disorders. However, several hundred rare, previously unknown sequence variants affecting the amino acid sequence of the encoded protein are found in the exome of every human individual. Therefore, the main challenge will be the differentiation between the many rare benign variants detected by novel genomic techniques and disease causing mutations. Hum Mutat 32:144–151, 2011. © 2011 Wiley‐Liss, Inc.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

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.     

Germline BAP1 alterations in familial uveal melanoma

Uveal melanoma (UM) is the most commonly diagnosed primary intraocular tumor in adults. Familial UM (FUM), defined as two or more family members diagnosed with UM, is rare and estimated at less than 1% of all UM. Currently, BAP1 is the only gene known to contribute significant risk for UM. In this study we aimed to estimate the frequency of BAP1 mutation in FUM and to characterize the family and personal histories of other cancers in these families. We identified 32 families with FUM, including seven families previously reported by our group. BAP1 mutation testing was carried out by direct sequencing of the coding exons and the adjacent untranslated regions of the gene. Germline deletion and duplication analysis of BAP1 was assessed by multiplex ligation‐dependent probe amplification (MLPA). Germline BAP1 mutations were found in 6/32 (19%) families. No deletions or duplications were identified in any of the 24 samples tested by MLPA. Combined with published studies, the frequency of BAP1 mutations was 14/64 (22%) in FUM. FUM families without BAP1 mutations have distinct family histories with high rates of prostate cancer in first‐ and second‐degree relatives. It is likely that additional genes conferring risk for FUM exist. It is important to understand key shared features of FUM to focus future research on identifying these additional tumor predisposition syndromes. Though BAP1 should be tested first in these families, FUM families without BAP1 mutation should be explored for additional predisposition genes. © 2016 Wiley Periodicals, Inc.     

A novel missense mutation (I344K) in the SPG4gene in a Korean family with autosomal-dominant hereditary spastic paraplegia

 Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower extremities. Among eight loci linked with autosomal-dominant (AD)-HSP, the SPG4 locus on chromosome 2p22 accounts for about 40% of all patients. Recently, mutations in a new member of the AAA protein family, called spastin, have been identified as responsible for SPG4-linked AD-HSP. Here, we describe a novel missense mutation (c.1031T>A; I344K) in exon 7 of the SPG4 gene identified in a Korean family with typical clinical features of pure AD-HSP. The mutation affects the third amino acid of the highly conserved AAA cassette domain, which is the most fore part of the domain altered by a missense mutation reported so far. Clinical presentations of affected individuals carrying the I344K mutation were not different from those of pure AD-HSP with SPG4 mutations reported previously. However, it is noteworthy that neither urinary dysfunction nor involvement of upper extremities was noticed in this family. To our knowledge, this is the first report of genetically confirmed AD-HSP in Korea. Received: February 20, 2002 / Accepted: May 21, 2002