Rapid identification of heterozygous or homozygous JAK2(V617F) mutations in myeloproliferative neoplasms using melting curve analysis

Abstract

Background/purpose: The activating JAK2 mutation with a G-C to T-A transversion at codon 617 (JAK2(V617F)) is associated with myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis. Here, we report a technical advance in the diagnosis of JAK2(V617F) in MPNs by melting curve analysis (MCA).

Methods: From January through December 2006, we prospectively enrolled 78 patients with PV (n = 21), ET (n = 32), myelofibrosis (n = 5), secondary erythrocytosis (n = 4), secondary thrombocytosis (n = 2), acute myelocytic leukemia (n = 4), chronic myelocytic leukemia (n = 8), and myelodysplastic syndrome (n = 2). Mutation analysis for JAK2(V617F) was performed on either bone marrow or peripheral blood cells using allele-specific polymerase chain reaction (AS-PCR) or sequencing and fluorescence resonance energy transfer (FRET) probes with MCA.

Results: For the initial 30 samples, the detection rate of JAK2(V617F) using MCA was comparable to the gold standard of the PCR sequencing methods. However, the turnaround times for MCA and PCR were 2 hours and 2 days, respectively. The detection rate of JAK2(V617F) was 76.2% for PV (homozygous in 14.3%), 46.9% for ET, 80% for myelofibrosis (homozygous in 20%), and 0% for the other conditions. In PV, patients with homozygous JAK2(V617F) presented with significantly longer disease durations than heterozygous patients. In ET, there were no differences in the clinical parameters of patients harboring JAK2(V617F) compared with those with wild-type JAK2.

Conclusion: Heterozygous and homozygous JAK2(V617F) mutations can be identified using the rapid and reliable assay based on FRET probes and MCA. Detection of JAK2(V617F) can be used to assist in the diagnosis of BCR/ABL-negative MPNs.