​Abstract

BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, in order to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1-VUS. Information on both cellular localization and homology-directed DNA-repair (HR) capacity was obtained for 78 BRCT-missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT-domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented based on mutated BRCT-domain solubility, phosphopeptide-binding properties, and VUS HR-capacity. These data suggest that HR-defective variants, which present, in addition, BRCT-domains either insoluble in bacteria or defective for phosphopeptide-binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR-activity and whose BRCT-domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR-activity and defective phosphopeptide-binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis.

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