Cloning, Chromosomal Mapping and Expression Pattern of the Mouse Brca2 Gene

Frances Connor 2 Amanda Smith 2 Richard Wooster 1 Michael Stratton 0 Alistair Dixon 1 Elizabeth Campbell 1 Tere-Michelle Tait 1 Tom Freeman 1 Alan Ashworth 2 0 Section of Molecular Carcinogenesis, The Institute of Cancer Research , Sutton, Surrey, UK 1 Human Genetics Group, The Sanger Centre, Wellcome Trust Genome Campus , Hinxton, Cambs CB10 1SA, UK 2 CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research , Fulham Road, London SW3 6JB, UK A proportion of human breast cancers result from an inherited predisposition to the disease. Mutations in the BRCA2 gene confer a high risk of breast cancer and are responsible for almost half of these cases. The recent cloning of the human BRCA2 gene has revealed that it encodes a large protein having little significant homology to known proteins. Here we describe the mouse Brca2 gene. The gene maps to mouse chromosome 5, consistent with its location on human chromosome 13q12. We have sequenced cDNA for the entire 3329 amino acid Brca2 protein and this has revealed that, like Brca1, Brca2 is relatively poorly conserved between humans and mice. Brca2 is transcribed in a diverse range of mouse tissues, and the pattern of expression is strikingly similar to that of Brca1. Taken together, our data highlight some intriguing similarities between two genes involved in inherited breast cancer susceptibility. - About one in 10 women in the Western world develop cancer of the breast, and an estimated 5% of these cases are thought to result from a hereditary predisposition to the disease, primarily due to a small number of highly penetrant autosomally dominant genes (1,2). Women that carry germ-line mutations in one of these susceptibility genes tend to develop breast cancer at an early age as well as being at elevated risk of bilateral breast cancer and other cancers such as ovarian cancer. Two breast cancer susceptibility (BRCA) genes have been mapped and cloned, and mutations in these genes are responsible for most of these cases in families with large numbers of early-onset breast cancers (16). Analysis of >200 families has indicated that mutations in BRCA1 are responsible for predisposition in the large majority of families with both breast and ovarian cancers, but account for predisposition in only about half of families with breast cancer only (7). The *To whom correspondence should be addressed BRCA2 gene (5) carries a risk of breast cancer similar to that of BRCA1, but is associated with a lower risk of ovarian cancer and a considerably higher risk of male breast cancer. Together, mutations in these genes account for ~ 45% of all breast cancers (1,2). However, no somatic, disease-causing, mutations in either BRCA1 (8) or BRCA2 (911) have been reported in any breast cancer. The BRCA1 gene is composed of 22 coding exons and encodes a protein of 1863 amino acids (4). Although most of the coding region shows no homology to previously described proteins, a clue to its function may be provided by the presence of a zinc finger domain at the N terminus of the protein. This domain is part of the subclass of so-called RING zinc fingers (12). However, the role of this potential proteinprotein interaction or DNA binding motif in BRCA1 has yet to be established. Several contradictory studies have now appeared on the subcellular location of the BRCA1 protein (1315). These include nuclear, nuclear and cytoplasmic, and extracellular locations. Consistent with an extracellular localisation, Jensen et al. (1996) have suggested that the BRCA1 protein contains a granin motif (15). Granins are proteins that are found predominantly in secretory vesicles where they may participate in the processing of proteins whose secretion is regulated by extracellular stimuli (16). Granins can also function extracellularly where cleavage products can function as biologically active peptides (16). However, the significance of the granin motif in BRCA1 has been questioned (17,18) and its role is presently unclear. The BRCA2 gene originally was mapped to an interval of ~ 6 cM on human chromosome 13q (5). Subsequently, a gene was identified that carried independent mutations in several different families (6). This gene (BRCA2) was found to have 27 exons and to encode a protein of 3418 amino acids having an estimated mol. wt of 384 kDa (6,19). One very unusual aspect of the gene structure is the presence of a large coding exon (exon 11) of ~ 5 kb encoding almost half of the BRCA2 protein. The gene appears to be widely transcribed but at a relatively low level (6,19). More than 70 independent mutations have now been found in the BRCA2 gene in breast cancer families (6,911,19; M. Stratton, unpublished). The BRCA2 protein shows no strong homology to known proteins (5,19). However, like BRCA1, BRCA2 has been suggested to contain a sequence which shows some homology to the granin motif (15). In addition, eight copies of a 3080 amino acid repeat have been noted in the part of the protein encoded by exon 11 (20 and Bignell et al., submitted). Here we describe the cloning and sequence analysis of full-length cDNA for mouse Brca2. This reveals that BRCA2, like BRCA1 is relatively poorly conserved between humans and mice. This high rate of divergence should aid the identification of functionally important domains in the protein. Analysis of the expression pattern in mice of Brca1 and Brca2 suggests that the two genes may be co-expressed. Thus we provide evidence for a number of interesting similarities between the two breast cancer susceptibility genes which may be important in understanding their role in normal and cancer cells. Cloning of mouse Brca2 cDNA The human BRCA2 gene has 26 coding exons spread over 70 kb of genomic DNA and produces an mRNA of ~ 11 kb (6,19). However, almost half the coding potential of the gene is contained within exon 11 which is almost 5 kb in length. A fragment of exon 11 [nucleotides 25374396 (ref. 19)] was used to identify homologous sequences in the mouse genome by Southern blotting (data not shown). Using these hybridisation conditions, this DNA fragment was used to screen a mouse l FixII genomic library. Hybridising clones were plaque-purified and l DNA prepared. Hybridising restriction fragments were subcloned and sequenced. DNA sequence analysis revealed homology to the human BRCA2 gene within exon 11. Mouse Brca2-specific oligonucleotides were then designed and utilised to screen a mouse 129 strain genomic BAC library in the vector pBeloBACII by PCR. Positive clones were confirmed by Southern hybridisation. DNA fragments which hybridised to human BRCA2 cDNAs were then subcloned from the BAC and partially sequenced. PCR primers corresponding to mouse Brca2 were then designed and PCR used to amplify fragments of cDNA. These were then sequenced. Part of the 3 end of the cDNA including the polyadenylation site was isolated by screening a mouse testis cDNA library. Using these techniques, sequenc (...truncated)