Whole genome sequencing revealed causative deep-intronic PCDH15 and de novo USH2A variants in patients with Usher syndrome

Abstract

Purpose : Usher syndrome (USH), the leading cause of deaf-blindness, is an autosomal recessively inherited genetic disease. Exome sequencing has been the primary focus of diagnostic testing and while for the majority of patients the pathogenic variants are known, still 10-15% of all USH cases remain genetically unexplained. Lacking a genetic diagnosis has significant implications for prognosis, genetic counseling and potential future (genetic) therapies. Here, we present the analysis of three initially genetically unsolved USH cases.

Methods : Whole genome sequencing (WGS) was performed for three mono-allelic USH probands. Rare coding and non-coding variants were prioritized based on variant type and effect and selected variants were assessed in detail. Variants with a potential splice-altering effect were investigated using a minigene splice assay.

Results : WGS analysis of the probands revealed three novel deep intronic variants (DIV) predicted to alter pre-mRNA splicing. In proband I with a typical USH2 phenotype, we discovered a de novo DIV in USH2A in trans to a previously described DIV known to impair splicing. The effect of the de novo variant was tested with a minigene splice assay and showed the incorporation of a pseudo-exon. In proband II (typical USH1), we identified a novel PCDH15 DIV in trans with a start-loss variant. Experimental validation of the DIV showed the inclusion of a pseudo-exon. Proband III manifested with atypical USH and carried a missense variant of unknown significance in PCDH15. WGS analysis revealed a novel PCDH15 DIV, which was tested and showed the incorporation of a pseudo-exon. Still, it remains questionable whether the PCDH15 variants in proband III are causative for USH. Diagnostic screening was performed for both this PCDH15 DIV and the novel PCDH15 DIV from proband II for nine monoallelic PCDH15 USH cases. The PCDH15 DIV initially identified in proband III completed the genetic diagnosis for two additional USH1 cases.

Conclusions : Using WGS we identified two novel DIVs in PCDH15 and a de novo USH2A DIV in probands with USH. The results in this study completed the genetic diagnosis for proband I, proband II and two cases that were screened for the PCDH15 DIVs. This underlines the relevance of genetic analysis of the non-coding regions in USH-associated genes. The novel DIVs are suitable candidates for splice-correction RNA studies.

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