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

Abstract

Purpose: Current interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-offunction (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., Δ[E4-E7], Δ [E6-E7], ΔE[6q39_E8], Δ[E10], Δ[E12], ΔE[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.

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction

“…Possibly, one could complement PALB2 KO cells containing DR-GFP with a BAC containing the full length human PALB2 gene. Such a method has previously been described for BRCA1 and BRCA2 (Kuznetsov et al, 2008;Chang et al, 2009;Mesman et al, 2018Mesman et al, , 2020 and would allow for the introduction and functional analysis of splice variants in coding and non-coding regions, further improving their classification.…”

A computational model for classification of BRCA2 variants using mouse embryonic stem cell-based functional assays

“…More recently, BRCA2 variants were expressed in BRCA2-deficent DLD1 cells and their response to multiple PARP inhibitors (PARPi) was used to functionally classify them 18 . We and others have utilized mouse embryonic stem cells (mESC) as a model system for functional evaluation of BRCA1/2 VUS based on the observation that both genes are essential to the viability of mESC [19][20][21][22][23][24][25][26][27][28] .…”

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation

“…For instance, recent reports describe downgrading splicing variants at the ESS in BRCA1 and BRCA2 from P/LP to VUSs or B/LB because of evidence from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium and functional studies showing that some ESS variants result in viable, functional transcript isoforms. 6,[35][36][37] In addition, two clinical laboratories recently reported discordant interpretations of an intronic variant in BRCA2. One lab classified the variant as LP because of its low prevalence, in silico predictions of splicing defects, and RNA analysis demonstrating altered splicing (but without quantification).…”

“…2 Some studies have revealed previously unrecognized variety in RNA transcript isoforms associated with well-studied genes, including BRCA1 (MIM: 113705) and BRCA2 (MIM: 600185), showing that our understanding of naturally occurring alternative splicing of disease gene transcripts is still evolving. [3][4][5][6] Recent studies have also illuminated how differential expression of transcript isoforms can influence whether certain sequence variants are tolerated. 7,8 As a result of this underappreciated complexity, variants that allow biologically viable alternative splicing may be incorrectly classified as disease causing.…”