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

Abstract

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.

Figure 1. Complementation analyses of cells with deleterious alleles in multiple PEX genes

A representative immunofluorescence (IF) image of the indicated fibroblast culture along with a magnification of a boxed region is provided in each panel. Panels A–E depict the analysis of PBD604 fibroblasts with a single PEX1 p. R948Q missense allele and two PEX26 p. R98W alleles. A: PBD604 cells alone, B: PEX1 null cells alone, C: PEX26 null cells alone, D: fused PBD604 and PEX1 null cells, and E: fused PBD604 and PEX26 null cells. Panels F–J depict the analysis of PBD704 fibroblasts with two PEX6 missense alleles (p.R601Q and p.R860Q) and a single PEX12 c.681−2A>C allele. F: PBD704 cells alone, G: PEX6 null cells alone, H: PEX12 null cells alone, I: fused PBD704 and PEX6 null cells, and J: fused PBD704 and PEX12 null cells. Signals were generated using anti-PMP70 (green) and anti-catalase (red) antibodies, as markers of peroxisomal membrane and matrix proteins. Co-localization of PMP70 and catalase, as indicated by yellow signals, indicate proper peroxisome assembly. Nuclei were stained with DAPI (blue). The genotypes of the PEX1, PEX6, PEX12, and PEX26 null cells are provided in Methods.