NovelPEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients

Abstract

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.

Rational diagnostic strategy for Zellweger syndrome spectrum patients

“…Furthermore, diverse reports of mutations in PEX genes show that such a strategy fails to identify critical molecular defects underlying the clinical manifestation in many ZSS patients. 3,5,10,24,25 As the exact knowledge about the pathogenic mutation in the patient provides valuable information, we were interested in strategies that could detect PEX gene defects in every single ZSS patient within a reasonable time and cost. On applying the published PEX Gene Screen Algorithm, the mutation could not be identified in 21% of the screened ZSS patients 11 .…”

NovelPEX1 coding mutations and 5′ UTR regulatory polymorphisms

“…Procedures for isolation of genomic DNA from cultured fibroblasts, PCR, DHPLC and direct sequence analysis of PCR products have been previously described [Maxwell et al, 2002]. Identified mutations and polymorphisms were confirmed by direct sequencing of a second, independent PCR product.…”

“…Determination of PEX1 protein levels in fibroblast cell extracts by SDS-PAGE and Western analysis, and analysis of peroxisomal PTS1 protein import by immunofluorescence microscopy using an SKL antibody, were carried out as previously described [Maxwell et al, 2002].…”

“…The 5' end of the PEX1 cDNA was mapped using the Primer Extension system (Promega). SDS-PAGE and Western analysis using an anti-PEX1 antibody (BD Biosciences, San Jose, CA) were performed as previously described [Maxwell et al, 2002]. The TnT Coupled reticulocyte system (Promega) was used to generate in vitro transcription/translation products.…”

“…The PEX1 AAA ATPase belongs to subfamily 2 of the AAA ATPases, and contains two AAA cassettes, with the more C-terminal one being the most highly conserved [Beyer 1997;Reuber et al, 1997]. A number of PEX1 mutations have previously been reported from studies of Zellweger spectrum cohorts in Australasia [Maxwell et al, 1999[Maxwell et al, , 2002, Europe [Gärtner et al, 1999;Walter et al, 2001;Preuss et al, 2002], Japan [Imamura et al, 1998;Tamura et al, 1998; and the U.S.A. [Portsteffen et al, 1997;Reuber et al, 1997;Collins and Gould, 1999]. The findings presented in this study result from our continued investigation of the PEX1 gene in an Australasian cohort -we report four further novel mutations and two 5' UTR regulatory polymorphisms.…”