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

Abstract

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.

Figure 1

a: Specificity versus sensitivity. The assay demonstrated sensitivity to 0.05% clinical dilution. Determining the specificity of the assay is hampered by the fact that it is not known whether healthy controls carry low levels of the mutation. However, we can say that amplification of the mutation in controls occurred only after the number of cycles required to detect a 0.05% dilution of the positive sample. b: Measurement of JAK2 V617F copies per reaction in mixtures of DNA from two healthy controls demonstrated a poor relationship between crossing threshold values and dilutions of DNA in water (right) and in DNA from other control sample (left), suggesting nonspecific amplification.

Figure 2

Distribution of JAK2 V617F copies per cell among cases and controls.

Figure 3

Relationship between mutational load and disease phenotype. In terms of a pathogenic model, our data support a significant relationship between mutational load and the different disease phenotypes observed in MPD. Our data have also demonstrated a significant positive relationship between mutational load and myeloproliferative markers and PRV-1 overexpression. Other studies have indicated that the mutation load is also correlated with increases in a number of activation markers of myeloid cells, although exactly how these cellular activations relate to thrombo-hemorrhagic and fibrotic complications is still unclear.