Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments

Recent studies have revealed a significant proportion of BRCA1 exon deletions or duplications in breast-ovarian cancer families with high probability of BRCA1- or BRCA2-linked predisposition, in which mutations of these genes have not been found. The difficulty of detecting such heterozygous rearrangements has stimulated the development of several new screening methods. Quantitative fluorescent multiplex PCR is based on simultaneous amplification of multiple target sequences under conditions that allow rapid and reliable quantitative comparison of the fluorescence of each amplicon in test samples and in controls. The modified method described here, named quantitative multiplex PCR of short fluorescent fragments (QMPSF), is particularly well suited for large genes. All BRCA1 coding exons were analyzed using four multiplexes in 52 families without point mutations in the exons or splice-sites of BRCA1 and BRCA2, and selected because of high probability of a BRCA1- or BRCA2-linked genetic predisposition. Five distinct BRCA1 rearrangements were detected: a deletion of exons 8–13, a duplication of exons 3–8, a duplication of exons 18–20, a deletion of exons 15–16, and a deletion of exons 1–22—which is the largest deletion found so far within the BRCA1 gene. The method described here lends itself to rapid, sensitive, and cost-effective search of BRCA1 rearrangements and may be included into the routine molecular analysis of breast-ovarian cancer predispositions. Hum Mutat 20:218–226, 2002. © 2002 Wiley-Liss, Inc.