Abstract
BACKGROUND: Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions. METHODS: We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine. RESULTS: We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile. CONCLUSION: HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).