Abstract
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. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. 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. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis.
Highlights
Introns account for 42% of the human genome and contain essential splicing codes that control the fidelity of premRNA splicing [1, 2]
The ratio-based identification of abnormal splicing, using a background distribution built from ~4200 normal and cancer samples (Supplementary Table 1), resulted in 1627 mutations (1318 intronic and 366 exonic; Supplementary Tables 2–6), including single nucleotide variants (SNVs) (n = 1469; 90.1%) and small deletions (n = 120; 7.6%) (Supplementary Fig. 1a)
Intronic mutations, which were more than 20 bp away from the nearest exon-intron junction, were defined as deep intronic mutations, because the fraction of the mutations discovered by whole-exome sequencing started dramatically declining at 20 bp from the nearest exon-intron junction (Supplementary Fig. 1b)
Summary
Introns account for 42% of the human genome and contain essential splicing codes that control the fidelity of premRNA splicing [1, 2]. Mis-splicing mutations in tumour suppressor genes (TSGs) frequently introduce premature termination codons (PTCs) in abnormally spliced transcripts and elicit mRNA degradation via nonsense-mediated decay (NMD) [11]. These studies were based on exome data and our current knowledge is largely limited to exons and the canonical SSs. Recent transcriptomic analyses have identified a number of deep intronic mis-splicing variants in the causal genes of Mendelian disorders [19,20,21,22]. We analysed 1134 tumour samples with WGS and RNA-seq data to identify cis-acting somatic mutations associated with abnormal splicing, including those in deep introns
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