title: [Simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia by double PCR].
abstract: Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML. A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3, with the aim of detecting almost all reported mutations. After optimization, a touchdown PCR was chosen for the multiplex PCR procedure, with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively. The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products. The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio. All the positive mutated samples were confirmed by sequencing. The results showed that 17 patients with NPM1 mutation, 15 patients with FLT3-ITD mutation, 6 patients with both NPM1 and FLT3-ITD mutations were found among 93 patents. 7 patients with M₂, 4 patients with M₄, 5 patients with M₅ and 1 patients with M₆ were found out of 17 patients with NPM1 mutation, in which 10 patients were male and 7 patients were female, 15 patients were with type A, 1 patients was with type B and 1 patients was with type Nm, strikingly 1 CML patient in blast crisis was found to carry a type A mutation. Among 15 patients with FLT3-ITD mutation 1 patient with M₁, 8 patients with M₂, 2 patients with M₂, 2 patients with M₃, 1 patient with M₄, 3 patients with M₅ were found, in which 5 patients were male and 10 patients were female. Sequencing results further confirmed the accuracy and reliability of this method. It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated. This method is fast, easy, accurate and capable to calculate the mutant/wild-type ratio.
authors: Fujian Medical University Clinic Teaching Hospital (Xiamen Zhongshan Hospital), Xiamen 361004, Fujian Province, China.
authors: Ze-Chuan
authors: ZC
authors: Zhang
authors: Quan-Yi
authors: QY
authors: Lu
authors: Jiang-Ning
authors: JN
authors: Zhao
authors: Ya-Mei
authors: YM
authors: Chen
authors: Zhi-Peng
authors: ZP
authors: Li
mesh_terms: D005091:Exons; D005260:Female; D005838:Genotype; D006801:Humans; D007621:Karyotyping; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D016133:Polymerase Chain Reaction / Q000379:methods; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3