One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes

被引:177
作者
Sotlar, K
Escribano, L
Landt, F
Möhrle, S
Herrero, S
Torrelo, A
Lass, U
Horny, HP
Bültmann, B
机构
[1] Univ Tubingen, Inst Pathol, D-72076 Tubingen, Germany
[2] Hosp Ramon & Cajal, Hematol Serv, Mast Cell Unit, E-28034 Madrid, Spain
[3] Hosp Nino Jesus, Dept Dermatol, Madrid, Spain
[4] TIB Molbiol Inc, Berlin, Germany
[5] Univ Lubeck, Inst Pathol, Lubeck, Germany
关键词
RECEPTOR TYROSINE KINASE; MELTING-CURVE-ANALYSIS; ACUTE MYELOID-LEUKEMIA; RARE MUTANT ALLELES; SYSTEMIC MASTOCYTOSIS; BONE-MARROW; MAST-CELLS; HEREDITARY HEMOCHROMATOSIS; ACTIVATING MUTATION; CATALYTIC DOMAIN;
D O I
10.1016/S0002-9440(10)63870-9
中图分类号
R36 [病理学];
学科分类号
100104 ;
摘要
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 I 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.
引用
收藏
页码:737 / 746
页数:10
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