Mutations in Peroxisomal Biogenesis Disorders: An Usher Syndrome Mimic.

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 genes. With no treatments currently available, we sought to investigate the disease mechanism in a patient with a PBD caused by defects in and to probe whether overexpression of 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 . 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 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 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 knockout cells compared with wild-type cells ( = 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 knockout cells. Overexpressing resulted in improved matrix protein import in knockout cells.

Conclusions: Mutations in 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 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 in the retina and the potential for gene therapy in these patients.