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Matthew Benson, Ian M MacDonald; Investigating the Effect of a PEX6 Mutation on Peroxisome Metabolism. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4459.
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© ARVO (1962-2015); The Authors (2016-present)
Peroxisomal biogenesis disorders (PBDs) represent a group of recessive conditions that cause severe vision loss, sensorineural hearing loss, neurologic dysfunction, and other systemic abnormalities due to abnormal peroxisomal function. Appropriate therapies are needed as there are no disease-modifying treatments currently available for these conditions. Our lab identified a PBD in a 12-year old child with compound heterozygous PEX6 mutations resulting in congenital sensorineural hearing loss and retinopathy. Our study seeks to examine the effect of mutations in PEX6 on peroxisome metabolism to aid in identifying potential therapeutic interventions for these patients.
Patient-derived skin fibroblasts were grown in culture and a PEX6 knockout cell line was developed using CRISPR/Cas9 technology in HEK293T cells. The knockout cell line was used to examine the effect of the complete absence of Pex6 and to compare the results with patient fibroblasts. Immunoblot analysis of whole cell lysates examined endogenous Pex14 levels as a marker of peroxisome abundance. Immunofluorescence analysis using antibodies against components of the peroxisomal protein import pathway interrogated the effects of changes in PEX6 on protein trafficking.
Endogenous levels of Pex14 were similar in PEX6 wild-type cells and both mutant fibroblasts and knockout cells, suggesting that there is no gross defect in peroxisome abundance in our patient. Immunofluorescence microscopy demonstrated significantly impaired matrix protein import into mutant cells with a concomitant reduction in the abundance of the cytosolic shuttle Pex5.
Compound heterozygous mutations in PEX6 are responsible for combined sensorineural hearing loss and retinopathy in our patient. In contrast to several previous reports, the primary defect in our patient seems to be one of impaired peroxisomal protein import as opposed to a perturbation in overall peroxisome abundance. Understanding the exact mechanism of disease at an individual patient level will be important in developing a precise therapeutic strategy.
This is a 2020 ARVO Annual Meeting abstract.
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