Objective: The somatic JAK2(V617F) mutation is seen in most polycythemia vera (PV) patients; however, it is not clear if JAK2(V617F) is the PV-initiating mutation.
Methods: In order to examine this issue, we developed a novel real-time quantitative allele-specific PCR, in which allelic discrimination is enhanced by the synergistic effect of a mismatch in the -1 position, and a locked nucleic acid (LNA) nucleoside at the -2 position.
Results: Determination of allelic frequencies was reproducible (SD = 1.5%) and sensitive--0.1% mutant allele detected in 40 ng of DNA. The JAK2(V617F) frequency in clonal granulocytes from 3 PV females was less than 50% (27.5 +/- 11) and in 7 females greater than 50% (75 +/- 10.5). We also found that wild-type JAK2 BFU-E colonies from PV patients can grow without erythropoietin. The identification of the primary genetic lesion resulting in PV is essential for the development of novel therapeutic strategies.
Conclusion: Our studies correlating the frequency of JAK2(V617F) mutant allele and clonality, as well as the presence of homozygous wild-type JAK2 erythropoietin-independent erythroid colonies, provide compelling evidence that the JAK2(V617F) is not the PV-initiating mutation. This supports a model wherein the JAK2(V617F) mutation arises as a secondary genetic event. Furthermore, our results indicate that an undefined molecular lesion, preceding JAK2(V617F), is responsible for clonal hematopoiesis in PV. We conclude that development of therapeutic strategies that target the JAK2(V617F) clonal cells may not be sufficient for eradication of PV.