Screening of male breast cancer and of breast-ovarian cancer families for BRCA2 mutations using large bifluorescent amplicons

41 breast cancer or breast-ovarian cancer families, including 12 families w ith at least one affected first-degree male relative, were screened for mut ations in the BRCA2 gene. Mutations had not been found in the BRCA1 gene of these families. Chemical cleavage of Mismatch was used to identify nucleot ide changes within large PCR products (average size 1.2 kb) that carried st rand-specific fluorescent end-labels, 15 amplicons were sufficient to scan 18 exons, including the large exon 11, The remaining 9 small exons were exa mined by Denaturing Gradient Gel Electrophoresis. The high sensitivity of t his approach was documented by the detection, in these 41 patients, of all 9 exonic single nucleotide polymorphisms reported with heterozygosity >0.1. Truncating BRCA2 mutations were found in 7 of the 41 families. 3 of them w ere in the group of 12 families comprising cases of male breast cancer. Sin ce the methods used here have no bias for particular types of mutations, th ese data confirm the high proportion of frameshifts among mutations in BRCA 2. However, relevant single nucleotide substitutions were also found: one r esulting in a stop codon and another one, present in a male patient, was th e previously reported change Asp2723His, that affects a highly conserved re gion of the BRCA2 protein. This study indicates a BRCA2 contribution of 10% (95% CI 2.5-17.5) to our original cohort of 59 breast-ovarian cancer famil ies, whereas the contribution of BRCA1 had been estimated at 46% (95% CI 33 -59). (C) 2001 Cancer Research Campaign.