Establishment of a Multiplex PCR-Based Procedure for Detection of Most Common Mutations in NPM1, FLT3 in Acute Myeloid Leukemia Patients.

Numerous studies have identified Nucleophosmin 1 (NPM1) and Fms-like tyrosine kinase 3 (FLT3) as the most frequently mutated genes in Acute Myeloid Leukemia (AML) patients. Mutations occurring in these genes, including NPM1+4 (tetranucleotide insertions), FLT3-ITD (internal tandem duplications), and FLT3-TKD (tyrosine kinase domain point mutations) account for approximately 80% of all mutations found in normal karyotype AML (AML-NK) cases. Different methods have been established to detect the three listed mutations, in which, PCR-based techniques are the most widely used due to their efficiency. Past researches on performing multiplex PCR for simultaneous screening were successful for only two out of the three mutations of interest. In this study, from genomic DNA, we amplified three fragments corresponding to the regions containing these mutations in a single PCR reaction, digested the products with EcoRV, and then analyzed the migration pattern using 3% Ultraphor™ agarose electrophoresis. In case of wild types, the results showed three and four distinct bands before and after restriction enzyme treatment, respectively; and in case of mutants, the presence of extra bands indicated suspected length mutations. This method was used to screen 20 samples, identifying a total of 6 possible mutants, including 3 NPM1+4 and 3 FLT3-ITDs, which were verified through established procedures and capillary electrophoresis.