Towards a general strategy for breast cancer : investigation of germline mutations of BRCA1 and BRCA2 genes in Iranian women with early-onset breast cancer

Breast cancer is the most common female malignancy and a major cause of death in middle-aged women. It results from genetic and environmental factors leading to the accumulation of mutations in essential genes, BRCA 1and BRCA2. To date, germline mutations in the BRCAI and BRCA2 genes in patients with early-onset breast and/or ovarian cancer have not been identified within the Iranian population. This study was set for two main purposes, in first for a cohort study of selected population (Iranian women) with early-onset breast cancer and secondly to evaluate and improve upon existing mutation detection techniques with respect to the BRCA genes. With the collaboration of two main centres for cancer research and treatment in Tehran-Iran, clinical information, family history and peripheral blood were obtained from 96 unrelated families for scanning of germline mutations in the BRCA 1 and BRCA2 genes. These sets of samples consists of 104 women under the age of forty-five, 88 patients affected with early-onset breast cancer or ovarian cancer and 16 unaffected individuals with strong family history of breast and/or ovary cancer. BRCA1 exons 11 and BRCA2 exons 10 and 11 by the Protein Truncation Test (PTT) and BRCAI exons 2, 3, 5, 13 and 20 and BRCA2 exons 9, 17,18 and 23 with the Single Strand Conformation Polymorphism (SSCP)assay were analysed on genomic DNA amplified by polymerase chain reaction. Ten sequence variants were identified: five are frame shift (putative mutations-four novel); three missense changes of unknown significant and two polymorphisms, one seen [BRCA2 (IVSI6-14T>C)] commonly in both Iranian and British population. Identification of these novel mutations suggests that any given population should develop a mutation database for its breast cancer screening. The pattern of mutations seen in the BRCA genes does not appear to differ from other populations studied. Early-onset breast cancer (less than 45 years) and a limited family history is sufficient to justify mutation screening with a detection rate of over 250/0 in this group, whereas sporadic early-onset breast cancer (detection rate less than 5%) is unlikely to be cost-effective. To address the penetrance and mutation spectrum of germline mutation of the contributed genes within Iranian population further studies should be performed. Meta-PCR technique was evaluated for its implication of BRCA genes scanning. Three distinct sets of BRCA gene fragments were selected to assemble with different approach for downstream analysis: the first set consisted ofBReA1 exons 2, 20 and BRCA2 exon 18 and their subsequent analysis by Protein Truncation Test; the second set comprised BRCAI exons 2, 20, 23 and 24 and their subsequent analysis by direct sequencing; and the last one contained six key coding regions from the BRCA genes, the 5' and 3'termini of exon 11 from both BRCAI and BRCA2 genes and exons 2 and 20 from BRCAI. Downstream analysis of Meta-PCR products by Protein Truncation Test was used rather than direct nucleotide sequencing because the total assembled above fragments size (~2.8kb) is sufficiently big to ignore analysing by the latter approach. PTT and direct sequencing were chosen because of their high sensitivity and specificity. These three trials were performed successfully suggesting that it may be possible to assemble the entire of coding regions of BRCAI and BRCA2 genes in a multi-step procedure.