Characterising the disease mechanisms of RP17 structural variants

Abstract

Purpose : We have identified complex structural variants (SVs) at the RP17 locus as a major cause of autosomal dominant RP (adRP). The genomic structure of each SV at the RP17 locus implicates altered topologically associated domain (TAD) structure and ectopic retinal enhancer-gene contact as a potential gain-of-function mechanism, with increased retinal expression of GDPD1. We explored this hypothesis by generating RP17 patient-derived 3D retinal organoids (ROs) and investigated the cellular and retina localisation of GDPD1.

Methods : Cells from RP17 patients were reprogrammed into induced pluripotent stem cells (iPSCs) using episomal vectors or lentiviral transduction, then differentiated into 3D ROs, alongside ROs differentiated from control iPSC. ROs were harvested at D200 and subjected to transcriptomic and lipidomic analyses (Lipotype GmbH), given the reported role of GDPD1 as a lysophospholipase. The GDPD1 gene was cloned into an expression vector for transient transfection in HEK-293 cells, and site-directed mutagenesis was used to create double and single mutants that are predicted to be catalytically inactive. GDPD1 was localized in HEK-293 cells and mouse retina using a commercial antibody to GDPD1 (Proteintech).

Results : iPSCs were developed from two individuals with the NL-SV1 and two individuals with the UK-SV2 SVs. All RP17 iPSC lines differentiated into ROs. The transcriptome of D200 RP17-SV ROs was compared to control ROs using RNAseq, and differentially expressed genes identified. Preliminary lipidomic analysis of RP17-SV vs. control retinal organoids revealed altered phospholipid ratios. The expression of wild-type and mutant GDPD1 was investigated in HEK-293 by immunocytochemistry and Western blotting. Immunohistochemistry demonstrated weak anti-GDPD1 immunoreactivity in photoreceptor inner segments in control ROs and mouse retina.

Conclusions : Our current data support the hypothesis that altered TAD structure leads to ectopic retina- specific enhancer-gene accessibility and interaction with GDPD1, resulting in increased retinal GDPD1 expression. Studies are underway to further test this hypothesis through transcriptomic, epigenetic, lipidomic and morphological analyses.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.