Logo image
Back
Profiling DNA damage response in lymphocytes from BRCA2 heterozygote carriers: implications for cancer risk assessment
Thesis

Profiling DNA damage response in lymphocytes from BRCA2 heterozygote carriers: implications for cancer risk assessment

Gihan Mohamed
Master of Science (M.S.), Drexel University
Apr 2026
DOI:
https://doi.org/10.17918/00011342
pdf
Mohamed_Gihan_20266.99 MB
PDF Embargoed Access, Embargo ends: 31 May 2028

Abstract

Genetics
Background: Individuals carrying germline pathogenic variants (gPVs) in DNA repair genes, including BRCA2, have a significantly elevated lifetime risk of cancer. Early identification and functional characterization of these risk-conferring variants are essential for accurate cancer risk assessment and the development of targeted prevention strategies. In this study, we measure DNA damage response (DDR) protein activity in lymphocytes from BRCA2 gPV carriers. In the future, these data will be used to define a functional DDR signature associated with BRCA2 pathogenic variation, which we aim to apply to the classification and interpretation of variants of uncertain significance (VUS). Methods: We have generated several EBV-transformed B-cell lines from clinically well-annotated BRCA2 gPV carriers with or without cancer, and healthy non-carrier controls. Select DDR proteins including [gamma]H2AX (Ser139), total ATR, phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), phospho-p53 (Ser15), total MDM2 and total p21 were analyzed in B-cell lysates from carriers and healthy controls at baseline and following genotoxic stress. These DDR proteins were chosen as established components of the DDR machinery, providing readouts of checkpoint activation, DNA damage signaling, and p53-mediated stress responses. Next, cellular proliferation over time was assessed to characterize cellular growth dynamics in BRCA2 carrier immortalized B-cell lines compared to immortalized B-cell lines from healthy controls. Results: Following treatment with different DNA damaging agents (cisplatin, PARP inhibitor (olaparib), irradiation), specific immortalized B-cell lines from BRCA2 heterozygous carriers exhibit impaired and sustained DDR signaling following genotoxic stress compared to immortalized B-cell lines healthy controls, consistent with BRCA2 haploinsufficiency and elevated replication stress. Several DDR proteins ([gamma]H2AX Ser139, total p21 and pCHK1 Ser345) were statistically significant in lymphocytes from specific BRCA2 gPV carriers compared to non-carrier controls at certain time points following genotoxic stress (p < 0.05, Mann-Whitney Test). Notably, the levels of these DDR proteins varied among the BRCA2 gPVs analyzed, indicating differential DDR activity across variants and suggesting that BRCA2 gPVs differ in their DNA repair capacity. Moreover, cellular proliferation assessed over 7 days in the immortalized B-cell lines from BRCA2 carriers demonstrated differential proliferation capacity compared to B-cell lines from healthy non-carriers suggesting a survival advantage (p < 0.05, Mann-Whitney Test). Conclusion: These experiments provide important insights into the DDR activities of distinct BRCA2 gPVs, demonstrating that not all variants respond uniformly to DNA damage. By offering a deeper understanding of lymphocyte DDR profiles, this work has potential applications for evaluating VUS in cancer risk assessments for high-risk individuals. In ongoing work, we are expanding our experiments to include PBMCs from carriers of BRCA2 VUS and development of a computational classifier model using pathogenic variant-derived differential DDR profiles to support VUS interpretation and support cancer risk stratification. Ultimately, these studies could help refine clinical strategies for assessing and managing cancer risk in genetically predisposed populations.

Metrics

1 Record Views

Details

Logo image