A novel RPE65-related dominant retinopathy caused by founder variant c.1555G>A p.(Glu519Lys) enriched in Belgian patients with a recognizable pattern dystrophy spectrum

Abstract

Purpose : The RPE65 gene has been linked once to a dominant retinopathy in families of Irish descent, due to RPE65 variant c.1430A>G (p.Asp477Gly). Here, we identified and characterized a novel RPE65-related dominant retinopathy caused by a founder variant c.1555G>A p.(Glu519Lys) enriched in Belgian patients with a recognizable pattern dystrophy spectrum.

Methods : The c.1555G>A p.(Glu519Lys) variant was initially discovered in genome sequencing (GS) data (n=3) of an unsolved inherited retinal disease (IRD) cohort, and next identified in RetNet filtered exome data (n=28) and targeted NGS data of IRD cases (n=46) with pattern dystrophy/maternally-inherited diabetes and deafness (MIDD)-like phenotypes. Segregation analysis was done in 44 individuals. Four patients underwent long-read GS (Oxford Nanopore) for haplotype phasing, followed by SNV and microsatellite analysis in a subset of cases. The variant was functionally assessed using an enzymatic RPE65 assay, Western blotting, co-immunoprecipitation (co-IP), a cellular thermal shift assay (CETSA), minigene assays and protein modelling (AlphaFold). Affected cases underwent a thorough ophthalmological evaluation, including multimodal retinal imaging.

Results : We identified 71 index patients and family members of Belgian origin with the rare and highly conserved c.1555G>A RPE65 variant (gnomAD v4, 5x). Haplotype analysis revealed a shared region of 200 kb, supporting a founder effect. While segregation analysis showed complete penetrance, the phenotype ranges from subtle, diffuse mottling of the posterior pole to a macular pattern dystrophy resembling the one found in MIDD with chorioretinal atrophy as a hallmark. Enzymatic activity of mutant Glu519Lys was lowered to ~56%, similar to the Irish Asp477Gly variant. Immunoblotting showed reduced mutant protein expression. Protein modelling and CETSA support a potential shift in protein stability and binding. In silico and minigene assays do not support a splice-effect, unlike Asp477Gly. Further studies in patient-derived iPSC-RPE are ongoing.

Conclusions : We report a novel RPE65-related dominant retinopathy in a Belgian IRD cohort with a recognizable phenotype, reducing the diagnostic gap in dominant IRD and opening new therapeutic perspectives, particularly in light of available gene therapy for recessive RPE65-IRD.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.