My laboratory focuses on the functional analysis of the human breast cancer susceptibility genes, BRCA1 and BRCA2. Breast cancer is the most frequently diagnosed cancer in women in the United States. It has been estimated that about 178,480 new cases of invasive breast cancer were diagnosed and more than 40,400 individuals died from this disease in 2007. Among the various factors responsible for the development of this cancer, a family history of the disease seems to play a major role. Mutations in BRCA1 and BRCA2 are linked to increased risk of early onset familial breast and ovarian cancers. Individuals with mutations in either of these genes are also at risk for developing cancer in other organs as well. The penetrance of the disease in BRCA1 and BRCA2 mutation carriers has been estimated to be 35-80%. In an effort to reduce the mortality from breast cancer through prevention and early diagnosis, BRCA1 and BRCA2mutation carriers are encouraged to undergo intensive screening and, in some cases, prophylactic surgery or chemoprevention. Sequencing based genetic tests are available to identify BRCA1 and BRCA2 mutation carriers. Currently, association analyses in families are used to determine whether a mutation poses a risk. One of the main aims of this project is to understand how deleterious mutations in BRCA1 and BRCA2 result in tumor development. By generating such mutations in mice, we hope to improve our understanding of the role of BRCA1 and BRCA2 as tumor suppressor. To understand the effect of human BRCA1/2 variants, we have generated humanized mouse models where desired mutations are engineered in the BRCA1 or BRCA2 gene in BAC and the phenotypic effect of the mutation is analyzed in transgenic mice that lack both copies of the endogenous gene. We have previously shown that wild type human BRCA1 and the BRCA2 present in BAC clones are able to rescue the lethality of Brca1ko/ko and Brca2ko/ko mice, respectively. Due to the relatively poor nucleotide as well as amino acid sequence conservation between mouse and human BRCA1 and BRCA2, these mice provide an ideal in vivo model system to examine the effect of any mutation identified in the human gene. Over the years we have generated a number of transgenic mice harboring mutations in human BRCA1 (C64G, C61G, A1708E, I26A, R1699Q, M1652I). With the exception of M1652I, which is a neutral variant of BRCA1 in the BRCT domain, all others have resulted in embryonic lethality, consistent with our observation that they are essential for ES cell viability. We examining the effect of these variants on tumor susceptibility in adults. For BRCA1, we have focused on I26A variant, and used M1652I as a control line as described below. We have also generated three knock-in mouse models expressing BRCA2 variants that can rescue the lethality of Brca2ko/ko ES cells but show a defect in DNA repair function and therefore, likely to be hypomorphic. BRCA2 G25R Biochemical studies have shown that G25R BRCA2 has a moderate effect on the binding of BRCA2 with PALB2, which is essential for DNA repair function of BRCA2. Survival of Brca2ko/ko ES cells expressing G25R is comparable to WT BRCA2 expressing cells. However, the G25R expressing cells exhibited mild sensitivity to DNA damaging agents, 50% reduction in HR and accumulation of chromosomal aberrations. There is one reported case of G25R in the BIC database. BRCA2 R3052Q The crystal structure of the C-terminus of BRCA2 depicts arginine at position 3052 in an apparently important position at the interface between two oligonucleotide/ oligosaccharide-binding folds. The BIC database contains two different variants involving this residue; arginine is changed to glutamine (R3052Q) in one variant and to tryptophan (R3052W) in the other. Homology-based modeling revealed R3052W to severely disrupt the structure due to the loss of interaction with surrounding residues while R3052Q had a moderate effect on the structure as this change retainedsome of the interactions. We tested these variants using ES cells and found that R3052W failed to rescue the lethality of Brca2ko/ko ES cells whereas R3052Q expressing cells are viable but exhibit mild to moderate sensitivity to DNA damaging agents. We are generating a knock-in mouse model expressing this variant to determine whether the moderate defect in BRCA2 function associated with the R3052Q variant contributes to cancer development. BRCA2 L2510P In contrast to G25R and R3052Q variants, L2510P results in reduced viability of Brca2ko/ko ES cells and the cells were hypersensitive to DNA-damaging agents, and deficient in IR-induced RAD51 foci formation and HR efficiency. We also observed an increase in chromosomal aberrations, such as breaks, gaps, or radial structures in mutant cells. While we predict homozygous mutant mice expressing this variant may not be viable, one family with two siblings inheriting this variant along with a truncating mutation c.4876GT p.E1550X have been reported to be born alive. These children did not exhibit bone marrow failure but one child developed a Wilms tumor at the age of six months and a year later developed AML. The other child developed T-cell ALL at the age of about 5 years.