A somatic gain-of-function mutation in exon 14 of the Janus kinase 2 (JAK2) gene is found in Philadelphia-negative myeloproliferative neoplasms, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Hematopoietic cells can be either heterozygous or homozygous for JAK2 (V617F) mutation, producing variable proportions of mutant alleles and a relation was observed between mutation status and clinical phenotype. Moreover, while the vast majority of patients with PV carry the V617F mutation in JAK2 exon 14, mutations in exon 12 have been recently reported in V617F-negative patients with PV. For the evaluation of granulocyte JAK2 (V617F) mutation status, we developed both a quantitative real-time polymerase chain reaction (PCR)-based and a microelectronic chip assay as a model of microarray-based approach. A significant correlation was found between the percentages of JAK2 (V617F) mutant alleles estimated by the two methods. However, the real-time PCR approach proved to be more sensitive in the detection of the minority mutant allele than the microelectronic chip. To further characterize JAK2 exon 12 mutations, we performed analysis in 128 patients with JAK2 (V617F)-negative myeloproliferative neoplasms. Direct sequencing analysis of JAK2 exon 12 led to the identification of five different mutations in cases where the levels of the mutated alleles were higher than 10%
