title: One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.
abstract: The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.
authors.affiliation: Institute of Pathology, University of Tübingen, Tübingen, Germany. klsotlar@med.uni-tuebingen.de
authors.forename: Karl
authors.initials: K
authors.lastname: Sotlar
authors.forename: Luis
authors.initials: L
authors.lastname: Escribano
authors.forename: Olfert
authors.initials: O
authors.lastname: Landt
authors.forename: Stefanie
authors.initials: S
authors.lastname: Möhrle
authors.forename: Sonia
authors.initials: S
authors.lastname: Herrero
authors.forename: Antonio
authors.initials: A
authors.lastname: Torrelo
authors.forename: Ulrich
authors.initials: U
authors.lastname: Lass
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Horny
authors.forename: Burkhard
authors.initials: B
authors.lastname: Bültmann
mesh_terms: D000293:Adolescent; D000328:Adult; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001483:Base Sequence; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D017403:In Situ Hybridization; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D013816:Thermodynamics; D014582:Urticaria Pigmentosa / Q000235:genetics* / Q000473:pathology; D014633:Valine
chemical_list: D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D001224:Aspartic Acid; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit; D014633:Valine