Development of new approaches to mutation detection in hereditary breast cancer

Mutation detection sensitivity depends on the method employed and the type of mutation present. Melt-MADGE uses a 96-well plate system for loading DNA samples and a combination of temperature ramp and denaturant to resolve both heteroduplexes and mutant homoduplex bands from PCR amplified alleles. Specifically for examining many subjects in parallel, costs will compare very favourably against SSCP, DGGE, dHPLC and direct sequencing. At the time of developing the technique I have designed an assay for mutation scanning for individual amplimers. I had already optimised long and nested PCRs and fine-tuned melt-MADGE assay for 39 amplimers required for the entire coding region of the BRCA1 gene. I have produced high quality results confirming detection of all the common polymorphisms in the largest exon of the gene (exon 11, 3426bp) as well as in other polymorphic exons. In addition, I have discovered a novel polymorphism. In parallel, I have set out to confirm the detection of mutations by an independent mutation detection method, ARMS assay. I have developed ARMS assay for five SNPs in the BRCA1 gene to distinguish the polymorphic samples obtained from the mutated samples. In a pilot study, a panel of 100 anonymous samples from the West Regional Genetics Laboratory (WRGL), were examined. The samples were screened over the entire coding region and we were able to detect several mutations, subsequently confirmed by direct sequencing. Matching the results from the diagnostic laboratory, we achieved 93% sensitivity for mutation detection. Further work is now being directed towards determining the reasons for the reduced sensitivity of the assay in specific cases.