A novel melting curve-based method for detecting c-kit mutations in acute myeloid leukemia

The c-kit gene encodes a class III tyrosine kinase receptor. Specific somatic mutations in c-kit have been associated with acute myeloid leukemia (AML) and are markers of a poor prognosis in AML. Various methods have been used to detect the c-kit gene mutation; however, the suitability of these methods in the clinical management of AML remains unclear. The current study developed a novel method, using modified hybridization probes and melting curve analysis, for detecting c-kit mutations in exon 17. Dual-labeled self-quenched oligonucleotide probes containing two segments, labeled with carboxyrhodamine or hexachlorofluorescein, were designed to detect sequences around the D816 or N820/N822 mutation hot spots in exon 17 of c-kit. The exon 17 region of c-kit was amplified by polymerase chain reaction using control plasmids carrying wild-type or mutant sequences, or genomic DNA derived from AML patients. Melting curve analysis of the amplification products was performed using a self-quenched probe. The results showed that the detection sensitivity, assayed using mutation-positive control plasmids, was 10% for the N820G mutation and 5% for the six other mutations; N822K(A), N822K(G), D816V, D816Y, D816H and D816F. In addition, c-kit mutations were identified in six of the 12 samples from the core-binding factor (CBF)-AML patients. This demonstrates that the novel method developed in the present study, is simple, rapid, specific and highly sensitive, and may facilitate the diagnosis and treatment of CBF-AML.