Abstract
Purpose :
Stargardt disease due to bi-allelic variants in ABCA4 (STGD1) is the leading cause of inherited maculopathy, yet ~35% of cases remain genetically unsolved with traditional sequencing methods. We hypothesize that the missing heritability is due to undiscovered RNA defects in ABCA4. We explore this hypothesis through induced pluripotent stem cell (iPSC)-disease modelling to derive a biologically relevant RNA source for long-read RNA sequencing (LRS).
Methods :
iPSCs were generated from two monoallelic [CF1] cases with the complex heterozygous allele c.[5461-10T>C;5603A>T]. Whole genome sequencing (WGS) was conducted to exclude potential phenocopies. Resulting iPSC lines were differentiated alongside a bi-allelic STGD1 case (c.4892T>C and c.4539+2001G>A) and unaffected control using published 3D retinal organoid (RO) protocols to produce a biologically relevant in vitro model of STGD1 (n=2). ROs were characterised during development using immunostaining and quantitative PCR for common markers of the developing retina. ABCA4 expression was confirmed using Western blotting. Fluorescence was quantified using Image J (n=5 images/sample) with statistical analyses performed via two-tailed paired t-test (mean ± SEM).
Results :
WGS on Patient 2 did not report any additional candidate variants in ABCA4 nor in other IRD-associated genes. However, c.5603A>T was identified in trans in Patient 1. All samples produced ROs with the major retinal cells present. A photoreceptor outer segment (POS) brush border formed apically on the RO, which increased from Day 180 onwards. ABCA4 protein was also visualised in the sample at the POS. At Day 120, a proportion of Recoverin+ photoreceptor cells in STGD1 were displaced towards the centre of the RO. Mislocalised expression was mirrored by rod and cone photoreceptors (Rhodopsin+ and OPN1MW/LW+ cells) which did not resolve by Day 230. Immunostaining with Caspase-3 reveals that this phenotype was not correlated with apoptosis.
Conclusions :
We generated STGD1 ROs which develop POS and express ABCA4, validating the use of this model as a source of tissue-specific RNA. We report a novel disease-specific phenotype of mislocalised photoreceptors, occurring independent of apoptosis. Further work is required to characterise this model, but we believe it will aid with the drug-discovery process for STGD1. We are currently applying LRS to unveil the missing inheritance of remaining monoallelic cases in this study.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.