Functional Characterization of RAD51 Paralog Cancer Associated Variants of Unknown Significance

For many individuals harboring a variant of uncertain functional significance (VUS) in a homologous recombination (HR) gene, their risk of developing breast and ovarian cancer is unknown. Integral to the process of HR are BRCA1 and regulators of the central HR protein, RAD51, including BRCA2, PALB2, RAD51C and RAD51D. Due to advancements in sequencing technology and the continued expansion of cancer screening panels, the number of VUS identified in these genes has risen significantly. While variant analysis is improving, there remains a struggle to keep up with demand. Understanding the effects of an HR variant can aid in preventative care and is critical for developing an effective cancer treatment plan. In this dissertation, we functionally screened 27 RAD51C ovarian cancer VUS, identifying two HRD variants. Modeling these and other previously analyzed HRD RAD51C ovarian cancer variants in the newly published BCDX2 cryoEM structure, we found a functionally deficient cluster within the critical Walker B region. Our study of RAD51C also uncovered two active translational start sites located at amino acid 1 and amino acid 10. The resulting protein isoforms and the M1 and M10 patient derived VUS were assessed and found to be functionally similar in their HR capabilities but showed differences in expression and possible cell type specificity. In all, these studies broaden our understanding of RAD51C and how its dysfunction can promote disease progression and response to therapy. These data have the potential to aid in more efficacious pathogenicity classification and improve patient care.