建立一套多對引子聚合酶連鎖反應同時偵測CEBPA、NPM與FLT3/ITD基因突變的方法

Abstract

Acute myeloid leukemias (AML) are clonal disorders that are characterized by acquired somatic mutations in hematopoietic progenitors. Mutations of CCAAT/enhancer binding protein α (CEBPA), nucleophosmin (NPM), and Fms-like tyrosine kinase-3 (FLT3) genes have been reported as the most frequent genetic variations in AML patients, and they have the important value in predicting prognosis, especially in those with normal karyotype. Due to the reappearances of the same CEBPA mutation and NPM mutation at relapse, these mutations are suitable as the biomarkers for monitoring minimal residue disease (MRD) in AML. In this study, we designed a novel, rapid and reproducible method with high sensitivity and specificity for simultaneous screening of the CEBPA mutation, NPM mutation, and FLT3/ITD by multiplex PCR coupled with capillary electrophoresis and fluorescence detection. To verify this novel method, 102 AML patients were studied, and the results were then confirmed by PCR-coupled direct sequencing. In addition to two insignificant mutations, 17 distinct mutations in the CEBPA gene and seven in the NPM gene were found in the thirteen (12.7%) and twenty (19.6%) patients respectively, but none had both. Twenty patients (20.2%) had the FLT3/ITD mutation. The overall sensitivity of multiplex PCR for NPM mutation and FLT3/ITD were up to 100%, and that for CEBPA mutation was 89.5%. This novel method can detect mutant allele percentages down to 5% of total DNA and offer the ability to detect early relapse post-therapy. This simple and reproducible method which shows high sensitivity and apparent accuracy may be used as a screening and disease-monitoring tool for AML patients with CEBPA mutation, NPM mutation, and FLT3/ITD in the future.