Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Among the 309 deep intronic single nucleotide variants, 245 altered core splicing codes, with 38% activating cryptic splice sites, 12% activating cryptic polypyrimidine tracts, and 36% and 12% disrupting authentic polypyrimidine tracts and branchpoints, respectively. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. Previous studies studying mis-splicing mutations were based on exome data and thus our current knowledge is largely limited to exons and the canonical splice sites.
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