Project Summary/Abstract It is currently feasible for small research groups to sequence individual genomes and for larger groups to sequence tens of thousands of individuals. Unfortunately, our ability to identify variants that impact phenotype has not kept pace with our sequencing capacity. This is particularly true of non-coding variants. This proposal presents a pilot screen of more than 32K variants from myCode and ClinVar that suggest 1-2% of exonic mutations affect splicing. The pilot study also revealed that splicing mutations are not uniformly distributed across disease genes or even within genes. This proposal will continue this effort on variants from UK biobank, AllofUs, ClinVar and GTEx that localize to actionable genes. Splicing mutations do not occur uniformly across exons. The proposal seeks to map susceptibility to splicing mutations and identify hotspot exons (exons unusually susceptible to splicing mutations) in the genome. In addition to identifying loss of splicing variants, the little studied problem of gain of splicing variants (i.e. single base mutations that can activate pseudoexons) will also be explored. Preliminary studies locate many intronic regions in pre-mRNA that contain all-but-one of the numerous cis-elements necessary for splicing. In addition to mapping susceptibility to splicing mutations, these efforts will contribute to the basic science of combinatorial signal recognition with a high-level approach that will define mechanisms of splice site selection. Finally, we present a principled method of restoring splicing to mutated exons using antisense oligonucleotides (ASO) that weaken flanking splice sites.