Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans . Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.