Study Prevalence of BRCA1/2 Common Mutations in East Azerbaijan Province, Iranian Breast CancerPatients Using High Resolution DNA Melting Analysis

Background: Recognition of mutations by DNA sequencing can be simplified by scanning methods to recognize amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are difficult as they require post-PCR management. High resolution melting (HRM) is a cost-effective quick screening approach, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. Materials & Methods: Assays for the analysis of all coding regions and intron-exon borders of BRCA1 and BRCA2 were designed, and optimized. A final set of 50 samples which ran under identical amplification conditions were chosen for BRCA1 and BRCA2. The most common mutations BRCA1 (c.3342_3345 delAGAA), BRCA1 (c.5406+1_5406+3 delGTA), BRCA1 ( c.981_982delAT), BRCA2 (c.3109C.T) were chosen.Results: We have done an extensive blinded validation of the protocol with 50 separate patient DNAs. All DNAs were analyzed for selected mutations, by HRM technique. According to this result there is no variation between HRM and direct sequencing to mutation detect. Prevalence of selected mutations were 11.2%, 5.5%, 3.4% and 0% for BRCA1 (c.3342_3345 delAGAA), BRCA1 (c.5406+1_5406+3 delGTA), BRCA1 ( c.981_982delAT), BRCA2 (c.3109C.T) respectively. Conclusion: This is the first HRM attitude to screen the entire coding region of the BRCA1 andBRCA2 genes using one set of reaction conditions using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 94%. This evidently reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved

Keywords:

High resolution melting (HRM) , BRCA1 and BRCA2 , mutation