Detection of the c-kit D816V mutation in systemic mastocytosis by allele-specific PCR

The c-kit D816V activating mutation is found in >80% of cases of systemic mastocytosis (SM) and represents a potential drug target. Furthermore, because D816V is one of the diagnostic criteria for SM, it is clinically relevant to determine whether the mutation is present. Traditional techniques such as DNA sequencing are often not sensitive enough to detect mutations in low-abundance tumour cells, including SM. Here, an allele-specific assay to detect the D816V mutation in DNA from archived formalin-fixed paraffin-embedded tissues is described.

A two-tube PCR format was employed to amplify c-kit exon 17 as a control and an allele-specific reaction to selectively amplify the D816V mutant allele using standard oligonucleotides. A D816V-mutant plasmid control was generated by site-directed mutagenesis of wild-type cells. 14 cases of SM, one D816V-positive seminoma sample, and 35 cases without SM were analysed using the assay.

The assay successfully amplified D816V in the mutant plasmid control, 13/14 cases of SM, and confirmed D816V in a seminoma sample. In addition, D816V was not amplified in 35/35 cases without SM. Serial dilution experiments demonstrated sensitivity down to <1%.

A sensitive, specific and cost-effective assay to detect the D816V mutation in archived formalin-fixed paraffin-embedded tissues from cases of SM has been developed.