Aberrant splicing caused by a novel synonymous RPE65 variant, demonstrated in iPSC-derived retinal pigment epithelial cells and on minigene assay

Abstract

Purpose : Genomic studies of our Australian retinal dystrophy cohort are providing molecular diagnoses in >60% of families examined. However, curation can often be challenging when assessing novel variants. This is especially relevant where there are strict inclusion criteria for gene therapy, such as voretigene neparvovec recently approved for use in Australia for retinal dystrophy due to RPE65 mutations. Patient-derived iPSC-RPE cells provide a valuable resource for investigation of genes with retinal-specific expression. This study investigates the pathogenicity of a novel RPE65 synonymous variant detected in trans with a previously reported pathogenic variant on clinical genomic testing in two affected siblings with early onset retinal degeneration.

Methods : iPSC cells were generated from the peripheral blood of a carrier of the RPE65 synonymous variant, and cells were differentiated to RPE. Pluripotency and RPE differentiation were examined with gene expression markers using qRT-PCR (ThermoFisher, USA) and immunohistochemistry. Minigene studies were performed with cloning of the genomic region of interest into a p.ExonTrap vector (MoBiTec, Germany), before transfection into HEK293 cells. RNA was extracted from iPSC-RPE cells and transfected HEK293 cells, and RT-PCR and Sanger sequencing were performed to investigate aberrant transcripts.

Results : Analysis of iPSC and subsequent RPE lines showed gene expression consistent with pluripotency and presence of RPE-specific markers respectively. Studies of iPSCs using SNP microarrays confirmed genomic stability. Analysis of RNA from both patient-derived iPSC-RPE and minigene studies demonstrated exon skipping from the RPE65 transcript, resulting in a mutant RPE65 allele of only 22 amino acids. There was concomitant reduction in RPE65 expression.

Conclusions : Modelling of the RPE65 synonymous variant in both iPSC-RPE and minigene assays demonstrated a RPE65 loss of function allele. These findings facilitate the reclassification of the novel synonymous variant from a variant of uncertain significance to a likely pathogenic variant, in turn providing eligibility for access to voretigene neparvovec gene therapy. This study demonstrates the importance of synergistic stem cell research and diagnostic genomics, especially in the era of gene therapies.

This is a 2021 ARVO Annual Meeting abstract.