Multiple copies of mutant BRCA1 and BRCA2 alleles in breast tumors from germ-line mutation carriers

Inactivation of the BRCA1 and BRCA2 breast cancer susceptibility genes has been reported to occur via a germ-line mutation of one allele and a somatic loss of the remaining wild-type allele. We investigated the genetic mechan isms behind the second event in breast tumors from 17 BRCA1 and eight BRCA2 germ-line mutation carriers, as compared with 21 sporadic breast rumors. M icrosatellite markers intragenic or in close proximity to both genes were u sed to analyze imbalances between the mutant and wild-type alleles. The act ual and relative gene copy numbers were scored by fluorescence in situ hybr idization (FISH) analysis of tumor cells using locus and centromere specifi c probes. All but one of the informative BRCA1 and BRCA2 rumors exhibited a llelic imbalance and loss of the corresponding wild type allele. In contras t to sporadic tumors, however, where allelic imbalance at the BRCA1 and BRC A2 loci correlated well with relative copy number losses by FISH, a simple reduction to a single copy (average copy number ratio 2:1) was found in onl y two BRCA1 (12%) and four BRCA2 (50%) tumors. The majority of BRCA1 and BR CA2 tumors showed a copy number reduction (relative to reference probe with ratios 4:2, 3:2, 4:3) at corresponding loci, suggesting that a specific ph ysical deletion of the wild-type BRCA gene allele has been followed by a du plication of the remaining mutant allele via polyploidization. Several tumo rs contained multiple copies of BRCA1 and BRCA2 genes without relative copy number changes, implying that loss of wild-type alleles is executed by alt ernative mechanisms such as mitotic recombination, non-disjunctional chromo somal loss with or without reduplication, or by gene conversion. A paradoxi cal relative copy number gain of the mutant allele was evident in three BRC A1 tumors (18%), which could be of biological relevance if a dominant negat ive or gain-of-function model was ascribed for certain BRCA1 mutants. Our r esults indicate that complex genetic alterations are operational at the BRC A1 and BRCA2 loci in tumors from genetically predisposed individuals. (C) 2 000 Wiley-Liss, Inc.