Ophthalmology Science (Jun 2021)
PEX6 Mutations in Peroxisomal Biogenesis Disorders
Abstract
Purpose: Peroxisomal biogenesis disorders (PBDs) represent a spectrum of conditions that result in vision loss, sensorineural hearing loss, neurologic dysfunction, and other abnormalities resulting from aberrant peroxisomal function caused by mutations in PEX genes. With no treatments currently available, we sought to investigate the disease mechanism in a patient with a PBD caused by defects in PEX6 and to probe whether overexpression of PEX6 could restore peroxisome function and potentially offer therapeutic benefit. Design: Laboratory-based study. Participants: A 12-year-old boy sought treatment with hearing loss and retinopathy. After negative results in an Usher syndrome panel, targeted genetic testing revealed compound heterozygous mutations in PEX6. These included a 14-nucleotide deletion (c.802_815del: p.(Asp268Cysfs∗8)) and a milder missense variant (c.35T→C:(p.Phe12Ser)). Methods: Patient-derived skin fibroblasts were cultured, and a PEX6 knockout cell line was developed using clustered regularly interspaced short palindromic repeats and Cas9 technology in HEK293T cells to emulate a more severe disease phenotype. Immunoblot analysis of whole cell lysates was performed to assess peroxisome number. Immunofluorescence studies used antibodies against components of the peroxisomal protein import pathway to interrogate the effects of mutations in PEX6 on protein trafficking. Main Outcome Measures: Primary outcome measures were peroxisome abundance and matrix protein import. Results: Peroxisome number was not significantly different between control fibroblasts and patient fibroblasts; however, fewer peroxisomes were observed in PEX6 knockout cells compared with wild-type cells (P = 0.04). Analysis by immunofluorescent microscopy showed significantly impaired peroxisomal targeting signal 1- and peroxisomal targeting signal 2-mediated matrix protein import in both patient fibroblasts and PEX6 knockout cells. Overexpressing PEX6 resulted in improved matrix protein import in PEX6 knockout cells. Conclusions: Mutations in PEX6 were responsible for combined hearing loss and retinopathy in our patient. The primary peroxisomal defect in our patient’s skin fibroblasts was impaired peroxisomal protein import as opposed to reduction in the number of peroxisomes. Genetic strategies that introduce wild-type PEX6 into cells deficient in PEX6 protein show promise in restoring peroxisome function. Future studies of patient-specific induced pluripotent stem cell-derived retinal pigment epithelium cells may clarify the role of PEX6 in the retina and the potential for gene therapy in these patients.
