A common PEX1 frameshift mutation in patients with disorders of peroxisome biogenesis correlates with the severe Zellweger syndrome phenotype

Abstract

Peroxisome biogenesis disorders are a heterogeneous group of human neurodegenerative diseases caused by peroxisomal metabolic dysfunction. At the molecular level, these disorders arise from mutations in PEX genes that encode proteins required for the import of proteins into the peroxisomal lumen. The Zellweger syndrome spectrum of diseases is a major sub-set of these disorders and represents a clinical continuum from Zellweger syndrome (the most severe) through neonatal adrenoleukodystrophy to infantile Refsum disease. The PEX1 gene, which encodes a cytoplasmic AAA ATPase, is the responsible gene in more than half of the Zellweger syndrome spectrum patients, and mutations in PEX1 can account for the full spectrum of phenotypes seen in these patients. In these studies, we have undertaken mutation analysis of PEX1 in skin fibroblast cell lines from Australasian Zellweger syndrome spectrum patients. A previously reported common PEX1 mutation that gives rise to a G843D substitution and correlates with the less severe disease phenotypes has been found to be present at high frequency in our patient cohort. We also report a novel PEX1 mutation that occurs at high frequency in Zellweger syndrome spectrum patients. This mutation produces a frameshift in exon 13, a change that leads to the premature truncation of the PEX1 protein. A Zellweger syndrome patient who was homozygous for this mutation and who survived for less than two months from birth had undetectable levels of PEX1 mRNA. This new common mutation therefore correlates with a severe disease phenotype. We have adopted procedures for the detection of this mutation for successful prenatal diagnosis.

Recovery of PEX1-Gly843Asp peroxisome dysfunction by small-molecule compounds

“…To monitor PEX1 function, a primary cell line, heterozygous for the common PEX1 alleles p.G843D and I700fs, was transformed, immortalized, and transfected with a GFP-peroxisome targeting signal 1 (PTS1) reporter. A stable clone was selected; we refer to these cells as PEX1-G843D-PTS1, because the second allele is null (10). We verified that GFP-PTS1 remains predominantly cytosolic in these cells when cultured at 37°C (Fig.…”

“…Walter et al (3) showed markedly reduced levels (5-15%) compared with wildtype PEX1 in fibroblast lysates from patients homozygous for this mutation and a 2-to 3-fold increase at 30°C. Because PEX1 transcript level in these patients is normal (10), it is likely that the PEX1-p.G843D is misfolded and degraded. PEX1 is an AAA (ATPase associated with diverse cellular activities) ATPase that oligomerizes with PEX6 AAA ATPase and when complexed to PEX26, recycles PEX5 from the peroxisome membrane to the cytosol (11).…”

NovelPEX1 coding mutations and 5′ UTR regulatory polymorphisms

“…We screened 25 skin fibroblast cell lines from Australasian patients referred for diagnostic investigation to the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders at the Women's and Children's Hospital, Adelaide. Routine diagnostic tests on cultured fibroblasts, which included determination of very-long-chain fatty acids (VLCFA), dihydroxyacetonephosphate acyltransferase (DHAP-AT), and the proportion of sedimentable catalase, were carried out as previously reported [Paton et al, 1996;Maxwell et al, 1999], and were indicative of a peroxisome biogenesis disorder in each case. Patient cell lines and HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (high glucose), supplemented with 10% fetal bovine serum (FBS) and 100mg/mL penicillin-100U/mL streptomycin (Gibco Invitrogen Corp., Melbourne, Australia).…”

“…The first of these, the c.1108_1109insA mutation in exon 5, leads to the introduction of a PTC upstream of the sequence encoding the first AAA cassette of the protein. Patient 3822, a compound heterozygote for this mutation and the common c.2097_2098insT (also referred to as I700fs or P740X) mutation [Collins and Gould, 1999;Maxwell et al, 1999], is a severely affected patient with a survival age of just 2.8 months. This combination of mutations is analogous to that of a previously reported patient who was a compound heterozygote for the c.904delG (A302QfsX23, also previously named A302fs) mutation and the c.2097_2098insT mutation; PEX1 mRNA levels in cells from this patient were 10% of normal [Maxwell et al 2002].…”

“…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.…”