Exploring non-coding variants and evaluation of antisense oligonucleotides for splicing redirection in Usher syndrome.

Exploring non-coding regions is increasingly gaining importance in the diagnosis of inherited retinal dystrophies. Deep-intronic variants causing aberrant splicing have been identified, prompting the development of antisense oligonucleotides (ASOs) to modulate splicing. We performed a screening of five previously described deep-intronic variants among monoallelic patients with Usher syndrome (USH) or isolated retinitis pigmentosa. Sequencing of entire or USH genes was then conducted in unresolved or newly monoallelic cases. The splicing impact of identified variants was assessed using minigene assays, and ASOs were designed to correct splicing. The screening allowed to diagnose 30.95% of the studied patients. The sequencing of USH genes revealed 16 new variants predicted to affect splicing, with four confirmed to affect splicing through minigene assays. Two of them were unreported deep-intronic variants and predicted to include a pseudoexon in the pre-mRNA, and the other two could alter a regulatory -element. ASOs designed for three deep-intronic variants successfully redirected splicing . Our study demonstrates the improvement in genetic characterization of IRDs when analyzing non-coding regions, highlighting that deep-intronic variants significantly contribute to pathogenicity. Furthermore, successful splicing modulation through ASOs highlights their therapeutic potential for patients carrying deep-intronic variants.