The Effect of a PEX6 Mutation on Peroxisome Function

• Author / Creator

  Benson, Matthew David

• Purpose. 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 male with compound heterozygous mutations in PEX6 (c.802_815del GACGGACTGGCGCT and c.35T>C (p.Phe12Ser)), resulting in congenital sensorineural hearing loss and retinopathy. Our study sought to examine the effect of mutations in PEX6 on peroxisome metabolism to both enhance our understanding of disease mechanisms in peroxisomal disorders and to aid in identifying potential therapeutic interventions for these patients.

  Methods. Patient-derived skin fibroblasts were grown in culture and a PEX6 knock-out cell line was developed using CRISPR/Cas9 technology in HEK293T cells. The knock-out cell line was used to examine the effect of the complete absence of Pex6, recapitulating a more severe PBD phenotype, and to compare the results with those from the study of a patient’s fibroblasts. Immunoblot analysis of whole cell lysates was performed to examine levels of endogenous Pex14, a marker of peroxisome abundance. Immunofluorescence studies were conducted using antibodies against components of the peroxisomal protein import pathway to interrogate the effects of changes in PEX6 on protein trafficking. Finally, we evaluated whether genetic approaches of over-expressing PEX5, the cytosolic shuttle for cargo destined for the peroxisomal matrix, and over-expressing PEX6 would restore some peroxisome function in cells lacking Pex6.

  Results. Endogenous levels of Pex14 were similar when comparing control fibroblasts to patient fibroblasts, suggesting that there was no gross defect in peroxisome abundance in our patient (P = 0.64). However, endogenous levels of Pex14 were less in PEX6 knock-out cells compared to wild-type cells (P = 0.04), suggesting the presence of fewer peroxisomes. Immunofluorescence microscopy demonstrated significantly impaired PTS1- and PTS2-mediated matrix protein import in both patient fibroblasts and PEX6 knock-out cells when compared to their respective controls. Over-expressing PEX6, but not PEX5, resulted in improved matrix protein import in PEX6 knock-out cells.

  Conclusions. Compound heterozygous mutations in PEX6 are responsible for combined sensorineural hearing loss and retinopathy in our patient. In contrast to 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. We postulate that this may be the mechanism of disease in patients with a milder PBD phenotype as a result of PEX6 missense mutations and residual Pex6 protein. On the other hand, our PEX6 knock-out cell data support a combination of reduced peroxisome abundance and impaired matrix protein import contributing to a severe PBD phenotype. Understanding the precise mechanism of disease at an individual patient level will be important in developing effective therapeutic strategies in the future.

• Subjects / Keywords

  • peroxisome
  • peroxisomal biogenesis disorder
  • pex6
  • retinal dystrophy

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-ey7v-2m50

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.