c-kit Mutational Analysis in Paraffin Material
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The current WHO classification of mastocytosis defines one major and four minor diagnostic criteria for systemic mastocytosis (SM). One of the minor criteria is the detection of the “gain-of-function” mutation D816V of the c -kit proto-oncogene in extracutaneous organs. The receptor molecule KIT is a potential therapeutic target for tyrosine kinase inhibitors. KIT mutations have been described in more than 80% of SM, but only in the minority of cutaneous mastocytoses (CM). Usually exon 17 amplicons generated by polymerase chain reaction are analyzed for the detection of c -kit mutations. Most frequently the method of restriction fragment length polymorphism (RFLP) analysis using the endonuclease Hinf I is used. Another well-established technique utilizes melting point analysis of amplification products with specific hybridization probes. Recently, also allele-specific PCR assays have been described. The technique used for the detection of c -kit mutations in mastocytosis is dependent on the kind of material to be analyzed and the laboratory equipment available. In this chapter the techniques of PNA-mediated PCR-clamping in combination with melting point analysis for the genotyping of amplification products are described for mutational analysis in total DNA and microdissected cells from formalin-fixed paraffin-embedded bone marrow trephine biopsies.