Combining Homologous Recombination and phosphopeptide-binding assays to predict the impact of BRCA1 BRCT variants on cancer risk


BRCA1 mutations have been identified that increase the risk of developing hereditary breast/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.

We present a high-throughput structural and functional analysis of a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA-repair (HR) capacity was obtained for the 78 BRCT missense variants of the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains.

This extensive 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 that is based on mutated BRCT-domain solubility, phosphopeptide-binding properties as well as VUS cellular localization and HR capacity. We propose that HR-defective variants, which always present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide-binding, lead to an increased cancer risk. On the opposite, variants with a WT HR activity and WT phosphopeptide-binding properties are likely 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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