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JMD 2007, Vol. 9, No. 1
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Development of a Quantitative Real-Time Polymerase Chain Reaction Assay for the Detection of the JAK2 V617F Mutation

Elizabeth C. Wolstencroft^*, Katy Hanlon^*, Lorna W. Harries^*, Graham R. Standen, Alexander Sternberg and Sian Ellard^*

From the Molecular Genetics Department, * Royal Devon and Exeter National Health Service (NHS) Foundation Trust, Exeter; Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter; Department of Haematology, Bristol Royal Infirmary, Bristol; and Department of Haematology, Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom

Achieving a specific diagnosis of polycythemia vera (PV) and other myeloproliferative disorders (MPDs) is often costly and complex. However, the recent identification of a V617F mutation in the JH2 domain of the JAK2 gene in a high proportion of patients suffering from MPDs may provide confirmation of a diagnosis. This is an acquired mutation and, as such, may only be present in a small number of cells within a sample. There is therefore a clinical need for highly sensitive detection techniques. We have developed a sensitive real-time polymerase chain reaction (PCR)-based approach for both detection and quantification of the JAK2 V671F mutation load, which allows determination of mutation status without the need for prior purification of granulocytes. We have performed a comparison of this assay with two previously published detection methods. Although an amplification refractory mutation system (ARMS) was shown to be slightly superior in terms of sensitivity, our real-time PCR method provides the potential for quantification of the JAK2 V617F mutation, having potential future applications in the monitoring of minimal residual disease or predicting outcome of disease severity.