Abstract
BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.
Highlights
Germline predisposing-mutations in the high-susceptibility BRCA1 and BRCA2 genes characterize the hereditary breast and ovarian cancer (HBOC) syndrome (OMIM #604370 and #612555, for BRCA1 and BRCA2, respectively) [1]
We evaluated the functional effect of the p.Pro2767Ser substitution by testing the mutated BRCA2 DNA-binding domain (DBD)together with the nearby nuclear localization sequence (NLS) in in vitro and in living cells assays based on the ability of the mutated or wild-type
The nuclear extracts from the BRCA2-CT P2767S and BRCA2-CT T2766NFs samples (Figure 4A, lanes 3 and 4) had the same intensity as the shifted complexes, similar to the mock-transfected cells, whereas the nuclear extracts from the cells transfected with BRCA2-CT wt affected the single-stranded DNA (ssDNA)-protein complexes of the irradiated cells (Figure 4A, lane 2). These results show that the BRCA2-CT wt protein interfered with the assembly of endogenous protein complexes that were activated upon DNA damage and hampered the binding of BRCA2-RAD51 complexes to ssDNA
Summary
Germline predisposing-mutations in the high-susceptibility BRCA1 and BRCA2 genes characterize the hereditary breast and ovarian cancer (HBOC) syndrome (OMIM #604370 and #612555, for BRCA1 and BRCA2, respectively) [1]. This clinical condition is associated with a young onset age, bilateral and aggressive cancers, and a family history of numerous cases of breast and ovarian cancers [2]. The identification of novel variants and VUSs, at an early age, in relatives of patients and during the screening of non-affected subjects, is a relevant clinical challenge. Much effort is being made to determine the role of BRCA1 and BRCA2 VUSs and of novel variants [9,10,11,12]
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