Functional variant assays (FVAs) for predicting breast cancer risks of genetic variants in the DNA double-stranded break repair pathway. H. Ostrer1, A. Pearlman1, K. Upadhyay1, Y. Shao2, J. Loke1 1) Albert Einstein College of Medicine, Bronx, NY; 2) NYU School of Medicine, New York, NY.

   Introduction. Among the 5-10% of women from high-risk breast cancer families, 20% have a mutation in the genes, BRCA1 and 2, and others have mutations in related genes that play a role in repair of DNA double-stranded breaks (DSB). In response to DNA breaks, the proteins encoded by these genes bind to each other, are transported into the nucleus and initiate non-homologous recombination. Mutations in these genes may disrupt any of these processes. Methods and Methods. Lymphoblastoid cells (LCLs) were collected from individuals with three different sets of BRCA1 variants -- known pathogenic mutations, benign variants, and variants of uncertain significance (VUS) and from individuals with known BRCA2, FANCC, and NBN mutations. Functional variant assays (FVAs) were developed to determine whether variants in BRCA1 or in the other DSB repair genes disrupted normal functions, such as binding of BRCA1 to its protein partners, BARD1, PALB2, BRCA2 and FANCD2, the phosphorylation of p53, or the transport of BRCA1 into the nucleus in response to crosslinking drugs, diepoxybutane (DEB) and mitomycin C (MMC), and the DNA breakage drug, bleomycin (Bleo). LCLs for the benign variants and VUS were sequenced via the 1000 Genomes Project and analyzed for the presence of possible genetic modifiers. Results. Mutations in BRCA1 decrease nuclear localization of BRCA1 in response to the DEB (p=9.8X10-32), MMC (p=4.8X10-16), Bleo (p=6.4X10-44), or drug combination (p=4.8X10-20). Mutations in BRCA1 reduce binding to co-factors, PALB2 (p=2.2X10-18), BRCA2 (p=3.0X10-7), and FANCD2 (p=3.1X10-7). Mutations in BRCA1 decrease phosphorylation of p53 (p=4.5X10-23), as do VUS in BRCA1 (p=3.0X10-18). Mutations in BRCA2, FANCC and NBN decrease nuclear localization of BRCA1 in response to the DEB (p=7.7X10-23), MMC (p=4.0X10-18), Bleo (p=7.8X10-43), or drug combination (p=9.2X10-14), reduce binding to co-factors, PALB2 (p=5.6X10-15) and FANCD2 (p=5.8X10-5), and decrease phosphorylation of p53 (p=3.0X10-18). Unsupervised analysis of all of these assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk BRCA1 controls and VUS. Conclusion. FVA assays distinguish BRCA1 mutations from benign variants and categorize most VUS as benign. Mutations in other DSB repair genes produce molecular phenocopies with these assays. FVA assays represent an adjunct to sequencing for categorizing VUS and a possible stand-alone test for assessing breast cancer risk.

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