Abstract P4-10-01: Edgetic perturbation of BRCT-mediated interactions caused by the BRCA1 H1686Q sequence variant (2012)

Abstract Background: The ever-increasing number of variants of uncertain significance (VUS) that result from BRCA1/BRCA2 genetic testing poses a challenge to healthcare providers, thus impacting clinical decision-making. Though a quantitative posterior probability model, mostly reliant on genetic data, has been developed to estimate the clinical relevance of individual VUS, indirect evidence derived from functional tests continues to be a sought-after adjunct to achieve VUS categorization. Using a multimodal approach, we previously demonstrated the deleterious effect on protein localization and function of the Italian founder sequence alteration BRCA1 V1688del, and the neutral effect of a neighboring variant, BRCA1 V1687I, identified in a patient with early onset triple negative breast cancer. Disease-causality has been suggested for the adjacent BRCA1 H1686Q variant that occurs in the evolutionarily conserved THV motif of the first BRCT repeat and was described in a breast/ovarian cancer family (Giannini et al., 2008). We aimed at substantiating the predicted pathogenicity of BRCA1 H1686Q by assessing its biological significance. Methods: To investigate the structural and functional consequences of the BRCA1 H1686Q sequence alteration, we used a strategy that combined homology modeling with analysis of protein interactions (by immunoprecipitation and GST pull down techniques) and function (by a homologous recombination assay). Results: The three-dimensional model of the BRCA1 H1686Q BRCT domain suggested that the site and nature of the amino acid change could alter the interaction and relative orientation of secondary structure elements in the core region of the protein, thus possibly affecting the overall stability of the protein and triggering a long-range allosteric effect with repercussions on residues involved in phosphopeptide binding. Indeed, in transfected 293T cells, the recombinant BRCA1 H1686Q protein was less abundant than its wild-type counterpart. Moreover, while efficiently heterodimerizing, via its N-terminus, with the binding partner BARD1, the BRCA1 H1686Q protein displayed a peculiar behavior with regards to C-terminal, BRCT-mediated interactions as it bound BRIP1/FANCJ and not CtIP. These results, repeatedly observed in different cell lines, were confirmed by reciprocal immunoprecipitations as well as by GST pull down experiments using a recombinant BRCA1 H1686Q BRCT domain. Notably, the BRCA1 H1686Q protein displayed a homologous recombination defect similar to that observed for the clinically ascertained BRCA1 M1775R mutant protein. Conclusions: Our data suggest that the BRCA1 H1686Q variant, by affecting the BRCT ligand recognition ability, causes an edgetic perturbation (i.e. selective disruption/retention) of BRCT-mediated interactions, which is yet sufficient to impair the homologous recombination function of the encoded protein. These findings provide valuable structure/function insights that could help elucidate the link between BRCA1 DNA repair and tumor suppression functions and aid development of functional tests for VUS screening and classification. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-10-01.