A novel intronic RPGR variant in patient-derived iPSC retinal pigment epithelium and retinal organoids reveals abnormal splicing and protein expression

Abstract

Purpose : Novel variants found in the retinal dystrophies require definitive pathogenicity classification for informed patient dialogue and access to current and future clinical trials and therapies. We use patient-derived induced pluripotent stem cells (iPSCs) differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO) to contribute to pathogenicity determination in these cases, and as a platform to test novel therapies. Here, this approach was undertaken in a family with a novel intronic variant in RPGR, which was reported by the diagnostic laboratory as a variant of uncertain significance.

Methods : iPSC patient clonal lines, formed from fibroblast samples, were used to culture iPSC-RPE and iPSC-RO, through proneural induction methods. Fibroblast and iPSC-RPE cells were used to determine presence of aberrant splicing through cDNA sequencing. RPGR gene and protein expression studies were undertaken using qRT-PCR, and immunofluorescence in iPSC-RPE and ROs was utilised to explore protein interactions and localisation.

Results : The novel intronic RPGR variant was shown to alter typical exon 12 splice acceptor site function. Gene expression of RPGR in patient iPSC-RPE and iPSC-ROs was decreased. RPGR protein analysis revealed decreased expression and mislocalisation in primary cilia and the photoreceptor cilium. Additionally, iPSC-ROs showed other photoreceptor protein mislocalisation.

Conclusions : Investigation of this novel patient RPGR variant in iPSC-RPE and ROs has revealed abnormal splicing, with decreased gene and protein expression, and abnormal photoreceptor protein localisations. This work has aided in variant classification to likely pathogenic, providing genetic information and eligibility for future treatments for the affected individuals.

This is a 2021 ARVO Annual Meeting abstract.