Simple, Rapid and Sensitive Method for Detection of the JAK2 V617F Mutation

Abstract and Introduction

Abstract

The point mutation 1849 (GAET) V617F in the JAK2gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allele-specific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.

Introduction

Janus kinase 2 (JAK2) is a member of a group of cytoplasmic tyrosine kinases are that involved in the transduction of signals from growth factor receptors. JAK2 is a key player in the JAK–signal transducer and activator of transcription (STAT) pathway. On autophosphorylation following activation via ligand binding, JAK2 recruits STAT molecules, which are then phosphorylated and translocate to the nucleus to act as transcription factors.

It has been recently reported that a somatic point mutation, 1849 (G→T), in the JAK2 gene causing phenylalanine to be substituted for valine in codon 617 (V617F) is present in a number of myeloproliferative disorders such as polycythemia vera (PRV [65%-93%]), essential thrombocythemia (ET [23%-57%]), idiopathic myelofibrosis (IMF [43%-53%]), and, to a smaller extent (~5%), chronic myelomonocytic leukemia, myelodysplastic syndrome, systemic mastocytosis, and chronic neutrophilic leukemia. This mutation disrupts the autoinhibitory J_H2 domain of JAK2, which leads to the constitutive activation of JAK2 and, subsequently, uncontrolled proliferation. No other mutations in JAK2 have been detected, a consistent finding across the 4 independent groups who initially described the V617F mutation.

The incidence of the V617F mutation in patients with bcr-abl-- myeloproliferative disorders varies, but defining the presence or absence of this mutation is now part of clinical diagnostic algorithms, and, in the future, will be part of the assessment for treatment with the potential introduction of JAK2 inhibitors.

Several groups have developed assays to enable detection of the V617F mutation (recently reviewed by Greiner). Some of these methods have been inappropriate for a routine diagnostic laboratory because they are comparatively labor-intensive methods with low detection sensitivities, eg, restriction enzyme (BsaXI) digestion following polymerase chain reaction (PCR) or sequencing. Other methods are relatively complex, such as those approaches with a pyro-sequencer, fluorescence probes, or denaturing high-performance liquid chromatography, considering the monomorphic nature of the V617F mutation.

We developed a diagnostic allele-specific competitive blocker (ACB)-PCR assay to identify the V617F mutation. The sensitivity of the assay allows it to be performed on peripheral whole blood DNA without the need to separate the granulocytes. The ACB-PCR assay is quick, easy, and cost-effective. In addition, it is highly sensitive, permitting detection of the mutation in patients with low mutant cell levels.