 Hi, my name is Cindy Lee, and I'd like to tell you about the work that we've been doing on bar one at the Ohio State University. Bar one is the heterodimeric partner of BRCA1, a breast cancer susceptibility gene. Mutations of BRCA1 and BRCA2 confer a 50 to 85% lifetime risk of hereditary breast cancer and 15 to 40% lifetime risk of hereditary ovarian cancer. As penetrant as these mutations are, however, they only account for about 20% of familial breast cancer. So researchers have started looking at other genes in these BRCA pathways to see what could be accounting for the missing heritability. As sequencing technology has become more affordable, we've been able to start looking at these other possible susceptibility genes. Patients can now opt to sequence a panel of 25 genes to see if they have any variants in their DNA sequences. We now have a multitude of information on these genes, but we don't necessarily know what the variants that we're finding could mean. Some variants could be neutral, and some variants could be pathogenic. Now, BRCA1 mutations have been measured in a variety of cellular functions, and the ones that are not able to perform homology directive repair of DNA double-strand breaks have also been found to be pathogenic. So we decided to measure its dimerization partner, BARB1, in the same way in homology directive repair. Using a HeLa-derived cell line with a stable non-functioning GFP construct and a donor GFP sequence, we depleted endogenous BARB1, compressed wild type or a missent substitution BARB1, and induced a double-strand break using ISCE1. The 29 missent substitutions we tested were variants of unknown significance found from breast cancer patient sequencing. These variants span the length of the BARB1 protein. If the BARB1 variant functioned in homology directive repair, the cell would express GFP. If, however, the BARB1 variant did not function in homology directive repair, the cell would have to resort to error-prone repair and would not express GFP. We counted the cells using flow cytometry. The variants could be classified into three categories, functional, intermediate, and defective. The functional variants performed similarly to wild type BARB1, while the defective variants were non-functional in homology directive repair like our empty vector. Intermediate variants were statistically different from both wild type BARB1 and empty. In this 3D structure of the heterodimer, the ring domain of BARB1 is shown in blue, while the ring domain of BRCA1 is shown in pink. You'll notice that crucial BARB1 residues for homology directive repair coordinate zinc ions or are along the interface with BRCA1, and when these residues were altered, cells could not perform homology directive repair. Our strategy can be used to test many more BARB1 variants that have been found in gene sequencing of the population. In this study, we've developed a tissue culture-based method for analyzing variants of unknown significance. We hope that our results will be combined with family history so that patients and their physicians can make a more informed decision about their health care. You can find our full paper in Human Mutation. The study was done by Cindy Lee, Tapashina Banerjee, Jessica Gillespie, Amanda Soravolo, Matthew R. Parvinsmith, Leah M. Starita, Stanley Fields, Amanda E. Toland, and Jeffrey D. Parvin. It was funded by the Ohio State University Comprehensive Cancer Center Molecular Biology Cancer Genetics Program.