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JMD 2007, Vol. 9, No. 2
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Quantitative Determination of JAK2 V617F by TaqMan

An Absolute Measure of Averaged Copies per Cell That May Be Associated with the Different Types of Myeloproliferative Disorders

Emma Hammond*, Kathryn Shaw{dagger}, Benedict Carnley{ddagger}, Stephanie P’ng{ddagger}, Ian James* and Richard Herrmann{ddagger}

From the Centre for Clinical Immunology and Biomedical Statistics, * Institute for Clinical Research; Cellular and Tissue Therapies, {dagger} Royal Perth Hospital, Perth; and the Department of Haematology, {ddagger} Pathwest, Royal Perth Hospital, Perth, Western Australia, Australia

We report a novel TaqMan assay for JAK2 V617F that measures averaged copies per cell in absolute terms, as opposed to a ratio of mutant to wild-type alleles. Measurements were obtained by comparing the JAK2 V617F signal generated by the test samples to that generated by a set of external plasmid standards containing the sequence of interest. Specificity of the assay was demonstrated above 36 cycles of amplification, and endpoint titration experiments indicated sensitivity down to 0.05% clinical dilutions. The test measured linearly over a wide logarithmic range and exhibited good reproducibility. Combination of this assay with another TaqMan method for determining cell number allowed identification of 14 cases of myeloproliferative disease with greater than two copies per cell. Mutational frequency was 68% among polycythemia vera (n = 44), 59% (n = 37) among essential thrombocythemia and 46% (n = 13) among idiopathic myelofibrosis. Levels of the mutation were significantly higher in polycythemia vera compared with essential thrombocythemia (P = 0.0005) and correlated with the following jointly significant variables at diagnosis: PRV-1, hemoglobin, white cell count, neutrophil count, and red cell count, using multiple regression analyses (P = 0.015). This method should be useful for assessing the relationship of gene dose to phenotype and possibly for monitoring therapy.




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Copyright © 2007 by the American Society for Investigative Pathology and the Association for Molecular Pathology.