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Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance in Homology-Directed Repair

Adamovich, Aleksandra Igorevna
http://orcid.org/0000-0001-7207-0241
http://rave.ohiolink.edu/etdc/view?acc_num=osu1563316267936516

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
BRCA1 and BARD1 encode proteins that form a heterodimer critical for mediating tumor suppression. Individuals with deleterious germline mutations in BRCA1 and BARD1 are at an increased risk for breast and ovarian cancers associated with poor disease outcomes. Although increased cancer screening has helped identify genetic variants, there is not enough information regarding many of them to ascertain clinical risk. These variants are known as variants of uncertain significance (VUS). Functional assays can be used to predict the clinical predisposition of gene variants. The homology-directed repair (HDR) assay is one such assay that has been developed to examine the function of variants in DNA repair. While BRCA1 has been extensively examined in HDR, with loss of function correlating with pathogenicity, its binding partner BARD1 has not been. In this dissertation, 76 potentially cancer-causing BARD1 truncation and missense variants were identified using whole exome sequencing and examined in the singleton HDR assay. Two were known to be benign variants and found to be functional in HDR, and three were known pathogenic variants found to have loss of function, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. Four variants in the ankyrin repeat domain were found to be non-functional in HDR, indicating that there are DNA repair functions associated with this domain that have not been closely examined. To examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. While over 130 BRCA1 variants have been examined in the HDR assay, more comprehensive analysis of several BRCA1 functional domains, including the repair-mediating BRCT domain, has not been conducted. In this dissertation, all potential missense and truncation variants in amino acids 1577 to 1863 were generated and subjected to multiplex HDR assays. More than 500 variants between amino acids 1577 and 1768 were characterized from an integrated library of over 2000 variants in the multiplex assay, including thirty variants currently classified as VUS. All of the non-functional missense mutants identified were located in the BRCT domain. Results from the multiplex HDR assay also correlated with results from singleton HDR assays. Variants classified as functional by the multiplex HDR assay were also functional in assays examining BRCA1 transcriptional activation (TA) and cell proliferation via saturation genome editing (SGE). However, several known pathogenic variants and variants classified as non-functional in the TA and SGE assays were functional in the multiplex HDR assay. Despite discrepancies between functional assays, the majority of result comparisons were consistent. In addition, variants that were non-functional in the multiplex HDR assay were always non-functional in the TA and SGE assays when comparisons were available. These findings help improve understanding of the BARD1 and BRCA1 functional domains in DNA repair and identify regions and variants of importance for cancer predisposition.
Jeffrey Parvin, M.D., Ph.D. (Advisor)
Michael Freitas, Ph.D. (Committee Member)
Paul Goodfellow, Ph.D. (Committee Member)
Amanda Toland, Ph.D. (Committee Member)
170 p.
Biomedical Research
BARD1; BRCA1; DNA repair

Recommended Citations

Citations

  • Adamovich, A. I. (2019). Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance in Homology-Directed Repair [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1563316267936516

    APA Style (7th edition)

  • Adamovich, Aleksandra. Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance in Homology-Directed Repair. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1563316267936516.

    MLA Style (8th edition)

  • Adamovich, Aleksandra. "Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance in Homology-Directed Repair." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1563316267936516

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.