AB141. Multiplex PCR-based procedure establishment for simultaneous detection of two mutations occurring most frequently in FMS-like tyrosine kinase-3

Abstract: Acute myeloid leukemia (AML) is a hematological disorder strongly correlated to several gene mutations which have been used in diagnosis and prognosis of disease. FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase, involving in cell differentiation and proliferation. In AML, abnormal haematopoietic cells carry most commonly two FLT3 mutations, namely internal tandem duplication (ITD) and tyrosine kinase domain (TKD) found in approximately 25% and 5% among patients, respectively. Previously, two separate PCR based procedures was widely used to detect two mutations. In this study, we established a detection procedure for both mutations consisted of two major steps: multiplex PCR for simultaneous amplification of a 329 base-pair amplicon for ITD containing region and a 146 bp amplicon for TKD prone region, and digestion of the PCR products with EcoRV followed by analysis on 8% polyarylamide gel. The procedure was performed with previously confirmed ITDwt/TKDwt, ITDmt/TKDwt, and ITDwt/TKDmt samples and showed consistency with earlier result obtained by using separate procedures for each mutations. Moreover, the procedure is rapid, consuming 6 hours compared to 10 hours and more effective, using less reagents for PCR and polyacrylamide electrophoresis in detection of two mutants. In conclusion, we successfully established more productive multiplex PCR-based method for detection of two common AML-related mutations in FLT3 gene.

Keywords: Multiplex-PCR; FMS-like tyrosine kinase-3-internal tandem duplication (FLT3-ITD); acute myeloid leukemia (AML)