Allelic Expression Imbalance of JAK2 V617F Mutant Contributes to Phenotypic Variation in BCR-ABL Negative Chronic Myeloproliferative Disorders.

Background: An acquired somatic mutation in the JAK2 gene (V617F) could present the primary causative lesion in BCR-ABL-negative chronic myeloproliferative disorders (CMPD). However, some of the clinical and genetic data implied that the pathophysiological role of JAK2 V617F mutation in the CMPD would be more complex. Quantitative assessment of JAK2 V617F mutation has shown substantial heterogeneity in the genomic DNA. Recently, it has been reported that allelic variation in gene expression is common in the human genome. To test the hypothesis that JAK2 V617F mutant allele could be increased in cDNA, we examined JAK2 V617F mutation status in the genomic DNA and cDNA from a total of 78 patients with BCR-ABL-negative CMPD.

Patients and Methods: Enrolled patients with BCR-ABL-negative CMPD in this study comprised 42 cases of essential thrombocythemia (ET), 26 polycythemia vera (PV), 7 idiopathic myelofibrosis (MF) and 3 unclassifiable (UC) CMPD. A 364-bp PCR product containing JAK2 V617F mutation was bidirectionally sequenced from total BM cells. A quantitative real time PCR-based allelic discrimination assay and pyrosequencing (Pyrosequencer PSQ96) were developed for quantitative analysis of JAK2 V617F mutation status. Homozygous JAK2 V617F mutation was defined if the mutant peak was more than 50% of total peak area.

Results: The proportion of mutant alleles ranged from 36% to 100% in real-time PCR and pyrosequencing analysis. Patients with MF had higher percentages of JAK2 mutant alleles than patients with ET (MF > PV > ET). The prevalence of homozygous V617F mutations was significantly higher in PV patients (73%) than in patients with ET (17%). Allelic expression imbalance of heterozygous JAK2 mutation was common in patients with PV and ET. Interestingly, allelic expression imbalance in patients with MF was not remarkably impaired, but preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with PV and ET.

Conclusion: Allelic imbalance in the gene expression of JAK2 V617F mutant could provide the underlying mechanisms to elucidate phenotypic variation in BCR-ABL negative CMPD.