Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis.

Abstract

We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2 V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2 V617F (G-->T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non-chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2 V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.