Demonstration of the pathogenicity of a common non-exomic mutation in ABCA4 using iPSC-derived retinal organoids, retinal pigment epithelium, and retrospective clinical data

Abstract

Purpose : Mutations in ABCA4 account for more than 17% of patients diagnosed with an inherited retinal degeneration. Despite use of state-of-the art sequencing technologies, molecular etiology remains elusive in a sizable portion of individuals. While often difficult to prove statistically, non-coding sequence variants are emerging as an important cause of disease. The goal of this study was to use patient-derived retinal cells to determine if and by which mechanism a common intronic variant in ABCA4 (IVS30 + 1321 A>G), identified in a cohort of individuals with clinically consistent disease, was pathogenic.

Methods : We generated hiPSCs using standard methods from patients with clinical findings judged to be characteristic of ABCA4-associated disease and screened for the presence of the IVS30 + 1321 A>G variant. iPSCs were directed toward a neural retinal lineage using a stepwise 3D differentiation protocol or retinal pigment epithelium (RPE) using a 2D RPE differentiation protocol. Following differentiation, three-dimensional iPSC-derived retinal organoids and RPE from controls and patients were evaluated immunocytochemically (ICC) for presence of retinal and RPE marker expression. In silico RNA binding protein motif prediction analysis was performed using RBPmap (http://rbpmap.technion.ac.il/). RNA was extracted from retinal organoids and RPE and expression was evaluated via RT-PCR and qRT-PCR. Transcripts were TA-cloned and Sanger sequenced.

Results : ICC and brightfield imaging analyses of retinal organoids and RPE from patients carrying the IVS30 + 1321 A>G variant demonstrated photoreceptors and photoreceptor outer segment development and mature RPE. Sanger sequencing of retinal organoid RT-PCR products revealed inclusion of 345 bp intronic sequence from IVS30 (Exon 30.1) in the proband only. In silico analysis predicted disruption of a Splice Factor 1 (SF1) binding site, which is known to silence alternative splicing. When we quantified Exon 30.1 using qRT-PCR, we observed a 3-fold increase in Exon 30.1 transcript in retinal organoids and RPE from the patient compared to control expression.

Conclusions : Collectively, these data demonstrate a molecular pathogenic phenotype of the common IVS30 + 1321 A>G variant in ABCA4 in photoreceptor cells and RPE from patients with clinically diagnosed ABCA4-associated macular dystrophy.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.