Abstract
Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.
Highlights
These results suggest that residue p.S1651 is the first position of the tandem BRCT functional and structural unit sensitive to missense changes
All three deletions dramatically affected activity and expression levels of the protein carrying the deletion variants (Fig. 2B). This finding indicates that deletion of as few as 7 amino acid residues from the C terminus leads to protein instability, abrogation of BRCT function, and may be pathogenic
For BRCA1 and BRCA2 variants determination of pathogenicity is based on a multifactorial model incorporating data on segregation analysis, family and personal breast or ovarian cancer history, and co-occurrence [5, 7]
Summary
We tested 102 (99 missense and 3 truncating) VUS in the BRCA1 C-terminal region (aa 1396 –1863) not previously analyzed using the TA assay, with a focus on structural features, divided in five groups: (a) 20 variants located at the border region between the disordered region and the BRCT-[N] at residues p.K1648 (to Gln, Glu, Thr, Arg, Ile, and Asn), p.R1649 (to Gly, Thr, Lys, Ile, and Ser), p.M1650 (to Leu, Val, Thr, Arg, and Lys), and p.S1651 (to Ala, Cys, Pro, and Tyr); (b) 67 variants located in the linker region connecting BRCT-[N] and BRCT-[C]; (c) 9 variants in ␣-helices ␣1 and ␣3 in BRCT-[N] and in ␣Ј1 and ␣Ј3 in BRCT-[C]; (d) 3 deletion mutants to probe the C-terminal border of BRCT domain; and (e) 3 assorted variants documented in the population (p.S1577Y, p.E1609G, and p.G1770E) (Fig. 1) (Table S1). When normalizing against an internal vector control for 20 variants in this group, 19 displayed transcription activity comparable with that of WT BRCA1 protein and variant p.S1651P was the only variant in this set with significantly compromised (Ͻ80%) function (Fig. 2A) These results suggest that residue p.S1651 is the first position of the tandem BRCT functional and structural unit sensitive to missense changes. All three deletions dramatically affected activity and expression levels of the protein carrying the deletion variants (Fig. 2B) This finding indicates that deletion of as few as 7 amino acid residues from the C terminus leads to protein instability, abrogation of BRCT function, and may be pathogenic
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
