Methylation: a second hit in the two-hit hypothesis.

This case illustrates that methylation of one BRCA1 allele may serve as the second hit in a patient with a diploid locus and missense mutation on the other allele.

Ovarian cancer BRCA1 mutation detection: Protein truncation test (PTT) outperforms single strand conformation polymorphism analysis (SSCP).

Recent studies have shown that the BRCA1 tumor suppressor gene plays a role in the development of both hereditary and sporadic ovarian cancer. Since several different mechanisms may give rise to tumor gene defects, a better understanding of these mechanisms may identify BRCA1 as an attractive therapeutic target in ovarian cancer. Sequencing this large gene is not practical on a population-wide basis. The optimal screening strategy is yet to be determined. The purpose of our study is to compare two common screening techniques: the protein truncation test (PTT) and single strand conformational polymorphism analysis (SSCP). Ninety-four patients with epithelial ovarian cancer and available snap-frozen tissue were screened for BRCA1 mutations by both PTT (five individual PCR reactions with complete translation of the product in the TNT System (Promega, Madison, WI)) and SSCP (41 individual PCR reactions covering the entire coding sequence). All abnormal results were confirmed by sequencing. A paired peripheral blood DNA sample was utilized to determine if the sequence abnormality was a germline mutation. Twenty-three mutations in BRCA1 were found in 22 patients (14 germline, eight somatic, one unknown) including four novel mutations: E489X, 3558delT, 3871delGTCT, del exon 7-10. Although the predictive value of a negative test was close for the two methods (PTT 99.1%, SSCP 99.8%), the comparison of positive predictive value overwhelmingly favored PTT (100.0%, vs. 26.4%, respectively). The specificity for PTT was 100.0% while the sensitivity was 82.6%. While for SSCP, the specificity was 99.0% and the sensitivity was only 60.9%. The concordance rate for the two screening tests was 88.9%. Only SSCP can detect missense mutations. PTT is a superior screening test for truncating BRCA1 mutations that are expected to be of clinical significance.