A novel dominant RPE65-related retinopathy is caused by p.(E519K), a founder variant of Flemish origin

Background | Recessive RPE65 -retinopathy is an inherited retinal disease (IRD) that is a well-known target for gene therapy. Dominant RPE65 -retinopathy, however, due to the Irish founder variant p.(D477G), is very rare. Here, we present the discovery of a novel dominant RPE65 -retinopathy caused by ultrarare variant c.1555G>A, p.(Glu519Lys), hereafter named p.(E519K). Methods | In addition to genome (n=3), exome (n=28), or targeted sequencing (index: n=14; segregation: n=30) to identify the p.(E519K) variant, patients underwent extensive ophthalmological examinations. Haplotype phasing was based on long-read genome sequencing data in four individuals, combined with microsatellite analysis of six markers in all index cases. The p.(E519K) variant was functionally assessed using an enzymatic RPE65 assay, western blotting, co-immunoprecipitation, cellular thermal shift assay (CETSA), minigene assays and protein modelling (AlphaFold). Results | Using genome, exome, or targeted sequencing in Belgian IRD cases (discovery cohort, n=2,873) and interrogating genomic IRD databases from France, the Netherlands, Germany, United Kingdom, Scotland, Ireland, and Canada (replication cohort, n=18,798) we identified 83 monoallelic p.(E519K)-IRD cases of Flemish ancestry. Long-read sequencing-based haplotyping revealed a shared region of 464 kb, confirming a founder effect. p.(E519K) affects a highly conserved amino acid and lowers RPE65 enzymatic activity to ~56%, in line with reduced protein expression. While no increased interaction with wild type RPE65 or aberrant splicing could be demonstrated in vitro , protein modelling and CETSA supports a shift in protein stability. Segregation analysis revealed dominant inheritance with complete penetrance and phenotypic variability, hallmarked by a characteristic late-onset macula-predominant IRD with two main subtypes. The milder phenotype is characterized by subtle, diffuse mottling of the posterior pole, while the more severe phenotype manifests as a macular pattern dystrophy with chorioretinal atrophy as a hallmark. Conclusions | The discovery of a dominant RPE65 -IRD due to the ultrarare Flemish founder variant p.(E519K) reduces the diagnostic gap in dominant IRD and highlights a novel target for therapy.