TP53 minigene analysis of 161 sequence changes provides evidence for role of spatial constraint and regulatory elements on variant-induced splicing impact
Germline TP53 genetic variants that disrupt splicing are implicated in hereditary cancer predisposition, while somatic variants contribute to tumorigenesis. We investigated the role of TP53 splicing regulatory elements (SREs), including G-runs that act as intronic splicing enhancers, using exons 3 and 6 and their downstream introns as models. Minigene microdeletion assays revealed four SRE-rich intervals: c.573_598, c.618_641, c.653_669 and c.672+14_672+36. A diagnostically reported deletion c.655_670del, overlapping an SRE-rich interval, induced an in-frame transcript {Delta}(E6q21) from new donor site usage. Within intron 6, deletion of at least four G-runs led to 100% aberrant transcript expression. Additionally, assay results suggested a donor-to-branchpoint distance cutoff of <50 nt for complete splicing aberration due to spatial constraint, and >75 nt for low risk of splicing abnormality. Overall, splicing data for 134 single nucleotide variants (SNVs) and 27 deletions in TP53 demonstrated that SRE-disrupting SNVs have weak splicing impact (up to 26% exon skipping), while deletions spanning multiple SREs can have profound splicing effects. Results also provide more data to inform splicing impact prediction for intronic deletions that shorten intron size.