Simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia by double PCR / 中国实验血液学杂志

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.