title: c-kit mutational analysis in paraffin material.
abstract: 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.
authors.affiliation: Institute of Pathology, University of Munich, Munich, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Sotlar
mesh_terms: D000483:Alleles; D001706:Biopsy; D001853:Bone Marrow / Q000473:pathology; D004252:DNA Mutational Analysis; D006801:Humans; D008415:Mastocytosis / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D016612:Paraffin Embedding; D012150:Polymorphism, Restriction Fragment Length; D000090063:Proto-Oncogene Mas; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019009:Proto-Oncogene Proteins c-kit