Variant effect on splicing regulatory elements, branchpoint usage, and pseudoexonization: Strategies to enhance...

It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect messenger RNA (mRNA) processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 95 exonic variants that impact splicing regulatory elements (SREs) in BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2. We utilized a pre-existing large-scale BRCA1 functional data set to map functional SREs, and assess the relative performance of different tools to predict effects of 283 variants on such elements. We also describe rare examples of intronic variants that impact branchpoint (BP) sites and create pseudoexons. We discuss the challenges in predicting variant effect on BP site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review and analysis highlights the value of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.