Novel Approaches to Studying BRCA Variants of Unknown Clinical Significance

BRCA1;VUS;Breast Cancer;Haploinsufficiency;Centrosome Amplification;Human Genetics and Molecular Biology

Cochran, RoryRiggins, Greg ;

Johns Hopkins University

关键词: BRCA1;    VUS;    Breast Cancer;    Haploinsufficiency;    Centrosome Amplification;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60310/COCHRAN-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Hereditary breast cancer comprises ~7% of the annual breast cancer cases in the United States. The two genes suspected in the majority of cases are the breast and ovarian cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2). Although BRCA genetic testing has aided in the identification of specific individuals at risk for breast and ovarian cancer, the large number of BRCA variants of unknown clinical significance (VUS) continues to complicate prevention strategies. To augment the ongoing VUS classification effort, two novel approaches for studying BRCA VUS were developed. First, an isogenic panel of immortalized human breast cells harboring eight different clinically reported BRCA1 alleles were established to study genomic instability. Consistent with previous findings, cells harboring a deleterious allele (185delAG, C61G and R71G) demonstrate increased ɣ-irradiation sensitivity and genomic instability, as measured by fluorescent in situ hybridization. Interestingly, not all deleterious alleles demonstrated the same level of haploinsufficiency, as each genotype exhibited varying degrees of change from the control cell lines across different assays. Second, an improved approach for studying BRCA variant loss-of-heterozygosity (LOH) within archival tumor tissue was developed. Using a new digital PCR platform, droplet digital PCR (ddPCR), BRCA2 LOH was assessed in formalin fixed paraffin embedded (FFPE) tissues for two related breast cancer patients harboring a rare missense BRCA2 variant of unknown clinical significance (c.6966G>T; M2322I). Conventional PCR followed by Sanger sequencing suggested a change in allelic abundance in the FFPE specimens. However, there was no evidence of LOH as determined by the allelic ratio (wild type:variant) for BRCA2 in both patients’ archival tumor specimens and adjacent normal control tissues using ddPCR. In summary, these experiments demonstrate two novel approaches for studying the clinical significance of uncertain BRCA alleles.