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A minimally invasive assay detects BRCA1 and BRCA2 protein truncations indicative of the presence of a germline mutation

Timothy John Byrne, University of Massachusetts Amherst


Family members with a heritable mutation in the BRCA1 or BRCA2 gene have up to an 85% life-long risk of contracting one or more of the cancers associated with a genetic alteration to these genes. The inheritance risks of a BRCA alteration to first and second-degree relatives of affected individuals are 50% and 25%, respectively. Currently, members from high-risk families can be tested for the presence of BRCA1 or BRCA2 mutations by gene sequencing of blood cell DNA. This test is expensive and generally is not reimbursed by insurance carriers. Over 85% of BRCA1 and BRCA2 mutations are reported to result in the production of a truncated protein, from which the carboxy terminal end sequences of their respective protein is missing. In preliminary studies, using antibodies specific for the amino acid terminal or the carboxy terminal of the BRCA1 protein, we demonstrated that immunohistochemical analysis of surgical specimens, consisting of matched ovarian tumor and normal uninvolved tissue, identified BRCA1 protein truncations in two matched samples. Each BRCA1 protein truncation was shown to be indicative of the presence of a BRCA1 germline mutation. Human buccal cells were then investigated as a minimally invasive cell source for this assay. Buccal cells were shown to express BRCA1 and BRCA2 protein immunohistochemically using distinct antibodies for the BRCA1 and BRCA2 protein terminals. Transcription of the BRCA1 and BRCA2 gene within buccal cells was also shown to occur by RT-PCR. A double blind study was then conducted using blood DNA and buccal cells collected from thirteen high-risk patients for carrying a germline BRCA1 or BRCA2 mutation. Mutation analysis (PCR, SSCP, and gene sequencing) was performed on DNA. Immunohistochemical analysis for the BRCA1 and BRCA2 protein was performed on cytospun deposited buccal cells. The buccal cell assay correctly predicted the presence of six germline BRCA1 mutations, three germline BRCA2 mutations, and three with no mutations. One predicted germline BRCA2 mutation did not correlate with DNA sequencing results. Based on these results, this assay exhibited a 100% negative predictive value and 90% positive predictive value.

Subject Area

Molecular biology|Oncology

Recommended Citation

Byrne, Timothy John, "A minimally invasive assay detects BRCA1 and BRCA2 protein truncations indicative of the presence of a germline mutation" (2000). Doctoral Dissertations Available from Proquest. AAI9988767.