Abstract 5591: A homologous recombination assay for classifying BRCA2 missense variants

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Abstract Germline mutations in BRCA2 predispose to breast, ovarian and other cancers. Although most mutations in the gene are known to be pathogenic, there is an increasing number of missense variants for which the contribution to cancer risk is unclear. These missense substitutions are referred to as VUS (variants of uncertain significance). Because loss of BRCA2 HR function leads to defective DNA double-strand break repair and an increased risk of cancer, we evaluated the utility of a cell-based Homologous Recombination (HR) assay for determination of the cancer relevance of BRCA2 VUS. Specifically, we studied VUS localized in the BRCA2 DNA binding domain (DBD) for effects on HR. We transfected full length wild-type and mutant BRCA2 constructs into V-C8 BRCA2 deficient cells along with an I-Sce1-expressing plasmid which induces an unique double strand break in an integrated DR-GFP reporter plasmid. We evaluated the ability of each BRCA2 mutant to repair the induced break by quantifying the GFP positive cells, generated by HR repair of the DNA break, with fluorescent activated cell sorting (FACS). To establish the sensitivity and specificity of the HR assay we evaluated a series of known deleterious and known neutral with the HR assay. These mutations of known effect were identified using a multifactorial likelihood approach, based on available family data, with a prior probability approach based on sequence conservation (A-GVGD) algorithm (http://agvgd.iarc.fr/agvgd_input.php). We calculated the total odds of pathogenicity for co-segregation of variants with the disease in affected carrier families, co-occurrence of VUS (in trans) with deleterious mutations and for family history of cancer of VUS and combined this odds with the A-GVGD based prior probabilities to generate the posterior probability of pathogenicity for each variant. Using this approach we successfully classified 31 VUS as either neutral or deleterious. By comparing these data with results from the HR assay we established that the assay is 100% sensitive (95% CI: 75.3% – 100%) and 100% specific (95% CI: 82.4% – 100%) for deleterious mutations in the BRCA2 DBD domain. In summary we evaluated the sensitivity and specificity of the HR assay for VUS in the DBD relative to a probability model based on family data and A-GVGD algorithm and we showed that the assay was highly sensitive and specific. Based on these results we propose that the HR assay can be used as a classifier of the clinical relevance of the VUS in the absence of direct genetic and family data. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5591. doi:10.1158/1538-7445.AM2011-5591