Abstract 2182 Characterizing Variants of Undetermined Significance in the BRCA Repair Pathway

Background: "Pathogenic" or "Likely Pathogenic" germline mutations in DNA repair genes such as Brca1, Brca2, and Palb2 in the BRCA repair pathway can lead to an increased risk of developing breast, pancreatic, and ovarian cancers. Other mutations may be found in the patient population, yet whether they play a role in increasing the risk of developing cancer is unknown; these are called Variants of Undetermined Significance (VUS). The impact of these variants' expression on cell proliferation, apoptosis, oxidative stress factors, immune response pathway function and internalization in certain cell types are unclear. Key DNA repair genes such as BRCA1 and BRCA2 have shown to be important for hematopoiesis in animal models, and some studies have observed differential affects among certain progenitor subsets. Further, several questions remain about the prevalence and impact of DNA repair gene variants in certain tissues. Characterizing the phenotypic impacts of these variants in different cell-based models will provide more information about relevant molecular targets. Objective: The aim of this project is to investigate the importance of cellular localization role of DNA repair gene variants using cell-based and functional assays. We hypothesized that the introduction of a mutated and deleterious Palb2 variant would lead to a decrease in its nuclear localization, a decrease in homologous recombination and greater decline in lymphoid progenitor differentiation than megakaryocyte-erythrocyte progenitor differentiation. Methods: To test the hypothesis, mouse HSCs cells were grown in complete culture medium at 37°C. Following expansion, 2.5x105 cells were plated onto culture well-plates. Brca2 and/or Palb2 were overexpressed using an expression vector, or Palb2 was silenced using siRNAs. Cells were maintained for 48 hours and extracted lysates were assessed for expression of wild type Palb2, Brca2, and gamma H2ax. Preliminary Results: Knockdown of Palb2 in HSCs lead to decreased proliferation of certain cell subsets. Conclusion: Palb2 may be involved in facilitating hematopoiesis. Future directions: We will carry out additional cell-based assays in the presence of clinical variants of DNA repair genes, including Brca1, Brca2 and Palb2 in mouse HSCs. We will also perform co-immunoprecipitations to look for resultant binding partners of Palb2 in lymphoid and megakaryocyte-erythrocyte progenitors. Keywords: variants of undetermined significance, Brca2, Palb2, overexpression, genomic stability. We thank the Vice President's Office for Research and Innovation (VPRI) at Prairie View A&M University for support of this research project.