Scrutinizing pathogenicity of the USH2A c.2276G>T; p.(Cys759Phe) variant

Abstract

Purpose : USH2A variant c.2276G>T (p.(Cys759Phe)) has been described as a frequent cause of autosomal recessive retinitis pigmentosa (arRP). However, its pathogenicity has been a matter of debate since the description of two a-symptomatic individuals homozygous for this variant. In this study, we therefore aimed to assess pathogenicity of the USH2A c.2276G>T variant using extensive genetic and functional analyses.

Methods : Whole genome sequencing and optical genome mapping were performed for three arRP cases homozygous for USH2A c.2276G>T to exclude alternative genetic causes, including variants in potential regulatory regions of USH2A. A minigene splice assay was designed to investigate the effect of c.2276G>T, in the presence or absence of the nearby c.2256T>C variant, on pre-mRNA splicing. A molecular model of the fifth laminin–epidermal growth factor like domain was generated to observe a putative structural effect. Moreover, an ush2a^{p.(Cys771Phe)} zebrafish knock-in model mimicking human p.(Cys759Phe) was generated and phenotypically evaluated using functional and immunohistochemical analyses.

Results : Besides the homozygous c.2276G>T USH2A variant, no other potentially causative DNA variants could be identified with our genetic approaches. Furthermore, p.(Cys759Phe) is predicted to be detrimental for usherin folding and function, based on molecular modeling. Evaluation of the ush2a^{p.(Cys771Phe)} zebrafish model revealed significantly reduced levels of usherin expression at the photoreceptor periciliary membrane, increased levels of rhodopsin localization to the photoreceptor cell body and decreased ERG b-wave amplitudes as compared to wildtype controls.

Conclusions : We confirmed pathogenicity of USH2A c.2276G>T (p.(Cys759Phe)). Consequently, persons homozygous for c.2276G>T can now receive a definite genetic diagnosis and can be considered eligible for QR-421a-mediated exon 13 skipping therapy in the future.

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