Alternative mRNA splicing can attenuate the pathogenicity of presumed loss-of-function variants in BRCA2

Purpose Current interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants in the canonical splice site sequences of BRCA2, to be associated with high cancer risk. However, some variant alleles produce alternative transcripts that encode (partially) functional protein isoforms leading to possible incorrect risk estimations. For accurate classification of variants it is therefore essential that alternative transcripts are identified and functionally characterized. Methods We systematically evaluated a large panel of human BRCA2 variants for the production of alternative transcripts and assessed their capacity to exert BRCA2 protein functionality. Evaluated variants included all single-exon deletions, various multiple-exon deletions, intronic variants at the canonical splice donor and acceptor sequences, and variants that previously have been shown to affect messenger RNA (mRNA) splicing in carriers. Results Multiple alternative transcripts encoding (partially) functional protein isoforms were identified (e.g., increment [E4-E7], increment [E6-E7], increment E[6q39_E8], increment [E10], increment [E12], increment E[12-14]). Expression of these transcripts did attenuate the impact of predicted LoF variants such as the canonical splice site variants c.631+2T>G, c.517-2A>G, c.6842-2A>G, c.6937+1G>A, and nonsense variants c.491T>A, c.581G>A, and c.6901G>T. Conclusion These results allow refinement of variant interpretation guidelines for BRCA2 by providing insight into the functional consequences of naturally occurring and variant-related alternative splicing events.