Role Of JAK2V617F Mutation Research Articles

New advances in the role of JAK2 V617F mutation in myeloproliferative neoplasms.

The JAK2 V617F mutation is the most common driver gene in myeloproliferative neoplasm (MPN), which means that the JAK/STAT signaling pathway is persistently activated independent of cytokines, and plays an important part in the onset and development of MPN. The JAK inhibitors, although widely used in the clinical practice, are unable to eradicate MPN. Therefore, the unavoidable long-term treatment poses a serious burden for patients with MPN. It is established that the JAK2 V617F mutation, in addition its role in the JAK/STAT pathway, can promote cell proliferation, differentiation, anti-apoptosis, DNA damage accumulation, and other key biologic processes through multiple pathways. Other than that, the JAK2 V617F mutation affects the cardiovascular system through multiple mechanisms. Although JAK inhibitors cannot eradicate MPN cells, the combined use of JAK inhibitors and other drugs may have surprising effects. This requires an in-depth understanding of the mechanism of action of the JAK2 V617F mutation. In this review, the authors explored the role of the JAK2 V617F mutation in MPN from multiple aspects, including the mechanisms of non-JAK/STAT pathways, the regulation of cellular methylation, the induction of cellular DNA damage accumulation, and effects on the cardiovascular system, with the objective of providing valuable insights into multidrug combination therapy for MPN.

An investigation of JAK2 mutation in patients with ulcerative colitis with a history of thrombosis

Background/Aim: JAK2 is a gene that provides instructions for making a protein called Janus kinase 2, which is involved in the signaling process that regulates the growth and division of cells. Variations in the JAK2 gene have been associated with several different diseases, including certain blood disorders like myeloproliferative neoplasms (MPNs) and ulcerative colitis (UC). The exact reason for ulcerative colitis is not fully understood. This study aimed to examine the possible role of JAK2 V617F mutation in the etiopathogenesis of ulcerative colitis. Methods: The included patients were selected with UC and with signs of thrombosis. The DNA isolation was carried out from peripheral blood for all included patients. RT-qPCR methods were used to find JAK2 V617F mutations in UC patients with signs of thrombosis. Results: 73.3% of the included patients in this study had 73.3% bloody diarrhea and 80% had abdominal pain. Also, the JAK2 V617F mutation rate was detected in 6.6 % of the patients included in the study. Conclusion: In this study, it was found that the V617F mutation was relatively rare in ulcerative colitis patients and there was no correlation with the JAK2 V617F mutation in most of the ulcerative colitis cases with thrombotic symptoms.

Thrombotic incidents in patients with myelofibrosis suggest to be independent of JAK2 V617f mutational status.

Myelofibrosis (MF) belongs to a group of conditions known as Philadelphia-negative myeloproliferative neoplasms (MPN). Bleeding or various vascular complications could be the main causes of morbidity and mortality in patients with MF. MPNrelated thrombosis is a multifactorial process and in the case of myelofibrosis, little is known. The risk factors for thrombotic complications in MF have been rarely assessed. The purpose of this study was to investigate the incidence of thrombotic events in MF and the role of JAK2 V617F mutation as a risk factor for thrombotic incidents in patients with MF. In our study of 37 patients, 35% had thrombotic events in the past. All patients were admitted to the Clinic of Hematology, St Ivan Rilski University Hospital, Sofia, Bulgaria between 2016 and 2019 and diagnosed based on the WHO criteria of 2016. The majority of patients (23, 62%) proved positive for JAK2 (Janus kinase) V617F mutation carrying one (16, 70%) or two (7, 30%) mutated alleles. Thirteen of the patients (35%) had a thrombotic event in the past and 9 of them (69%) were carriers of JAK2 V617F mutation. Fourteen patients of those without thrombotic history (24, 58%) were also carriers of JAK2 V617F mutation. As a whole, we did not find a statistically significant difference between JAK2 V617F mutation and the frequency of thrombotic events. Rendering an account to the possible life-threatening complications, treatment decisions should be undertaken upon possible antithrombotic prevention in MF.

Molecular biomarkers of thrombosis in myeloproliferative neoplasms

JAK2 mutations define polycythemia vera (PV), CALR and MPL mutations are specific to JAK2 unmutated essential thrombocythemia (ET) and primary myelofibrosis (PMF). We overviewed the current knowledge on the relationship between these phenotypic driver mutations and thrombotic complications that are major cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPN) particularly PV and ET. The JAK2 mutation is found in 50-60% of patients with ET and PMF. The International Prognostic Score for Thrombosis in ET (IPSET-thrombosis) identified JAK2 mutation as an independent risk factor and a 3-tiered prognostic model was devised. IPSET-thrombosis model outperformed the 2-tiered conventional risk stratification that includes age and thrombotic history. PV is usually associated with a JAK2 mutation and studies looking at the role of JAK2V617F allele burden associated with thrombosis are so far inconclusive. In PMF, the rate of major thrombosis is around 2%pt-yr and JAK2 mutation emerged as an independent risk factor for these events. Calreticulin/MPL (CALR) is the second most frequent mutation and occurs in half of JAK2 and MPL wild-type patients with ET and PMF. Despite the fact that these mutations are associated with high platelet counts, the risk of thrombosis compared with JAK2 and MPL mutated cases is significantly lower. The role of MPL in the prediction of thrombosis is of difficult demonstration due to the low frequency in ET and PMF. Therefore, these epidemiologic studies pointed out the role of JAK2V617F mutation as a major contributory factor for the pathogenesis of thrombosis in MPN. Abnormalities of blood cells arising from the clonal proliferation of hematopoietic stem cells may explain the switch to a procoagulant phenotype.

Leukocytosis Can Predict Thrombotic Events in Myelofibrosis

Thrombotic events are the main complication of Philadelphia-negative chronic myeloproliferative neoplasms (MPN). In polycythemia vera (PV) and essential thrombocythemia (ET), risk factors for thrombosis are well established, such as age greater than 60 years and previous thrombosis. However, the role of JAK2 V617F mutation and leukocytosis at diagnosis as risk factor for thrombosis is still controversial. Our aim was to identify factors related to the risk for thrombotic events in the studied population.This study is a retrospective non-interventional cohort. All of the analyses were performed using the database of 142 patients with MPN regularly followed at the Hematology Division (at UNIFESP-SP) from 1992 to 2014. Diagnosis was established according to WHO criteria. We analyzed the JAK2 V617F mutation, hemoglobin (g/dL), hematocrit (%), white blood cells (x109/L) and platelets (x109/L) at the diagnosis and DIPSS-Plus risk score (International Working Group for Myelofibrosis Research and Treatment, 2009). These variables were associated with thrombotic event at any time.Of the 142 patients, 54 had diagnosis of PMF, 28 of PV, 33 of ET and 27 of post-essential thrombocythaemic myelofibrosis (post ET MF) or post-polycythaemic myelofibrosis (post-PV MF). This last group was included in myelofibrosis group for statistical purposes.Thrombotic events were more frequent in PV patients (39.2%), followed by ET (33.3%), and PMF (20.9%).From those which JAK2 mutation was obtained, it was positive in 92.4% of PV patients, 62% of PMF and 50% of ET. In none of the three groups, the presence of JAK2 V617F mutation was related to increased risk of thrombosis.In myelofibrosis, leukocytosis was higher among thrombotic patients (median of 13.7 in thrombotic group versus 9.7x 109/L; p 0.0379). None of the other parameters, hemoglobin, hematocrit, platelets and DIPSS-Plus were statistically significant. In ET, the hemoglobin level at diagnosis was significantly higher in the presence of thrombosis (mean of 14.57 in thrombotic group against 13.03 g/dL in the non-thrombotic one, p 0.0428). The other parameters, hematocrit, white blood cells and platelets were not relevant. The median WBC in the thrombotic group was 9.4 and in the non-thrombotic one 9.3 x109/L. Finally, in polycythemia vera, none of the variables were related to thrombosis. Among the studied population, leukocytosis was increased in patients with thrombotic event in MF. Thus, monitoring leukocyte count in MF is essential to predict thrombosis risk and should be further studied in order to define therapeutic goals in these patients. DisclosuresNo relevant conflicts of interest to declare.

Latent Myeloproliferative Neoplasm as a Cause of Idiopathic Splanchnic Vein Thrombosis: The Role of JAK2V617F Mutation and Bone Marrow Biopsy Evaluation

Latent Myeloproliferative Neoplasm as a Cause of Idiopathic Splanchnic Vein Thrombosis: The Role of JAK2V617F Mutation and Bone Marrow Biopsy Evaluation

Prevalence of JAK2V617F mutation in deep venous thrombosis patients and its clinical significance as a thrombophilic risk factor: Indian perspective.

Venous thromboembolism is known to be a complex interaction of genetic and acquired factors leading to thrombosis. JAK2V617F mutation is believed to contribute to a thrombophilic phenotype, possibly through enhanced leukocyte-platelet interactions in myeloproliferative neoplasms (MPNs). Several studies have focused on the importance of screening for JAK2V617F mutation in patients with splanchnic venous thrombosis (VT) for the detection of nonovert MPNs. The role of JAK2V617F mutation in VT outside the splanchnic region is still widely unsettled. The primary aim of this study was to find out the prevalence of JAK2V617F mutation in patients with deep venous thrombosis (DVT), its clinical significance as a prothrombotic risk factor, and its possible interactions with other genetic thrombophilic risk factors. A total of 148 patients with idiopathic, symptomatic DVT were evaluated. Median age of presentation was 32 years (range 15-71 years) with a sex ratio of 1.3:1. Overall, the most common genetic prothrombotic factor was factor V Leiden mutation, found in 10.8% (16 of 148) of patients who also showed strong association with increased risk of thrombosis (odds ratio 5.94, confidence interval 1.33-26.4, P = .019). Deficiencies in protein C, protein S, and antithrombin were seen in 8 (5.4%), 10 (6.7%), and 8 (5.4%) patients, respectively. It was observed that the frequency of JAK2V617F mutation was lower in Indian patients, and it also showed weaker association with risk of thrombosis, at least in cases of venous thrombosis outside the splanchnic region.

Role of JAK2 V617F mutation and aberrant expression of microRNA-143 in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal myeloid disorders characterized by the overproduction of mature blood cells. The pathogenetic hallmark of MPNs is the dysregulation of JAK-STAT signaling, usually associated with the JAK2 V617F mutation. Multiple additional genetic and epigenetic alterations that constitutively activate the JAK-STAT signaling pathway have been described, including the modulation of the microRNAs (miRs) expression levels. The aims of our study were to investigate JAK2 V617F mutation allele burden and miR-143 expression levels in MPNs patients and to investigate the correlation between these genetic signatures and hematological parameters. In total 78 patients with a clinical diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM), made according to the WHO 2008 criteria, were included in the study. Twenty healthy subjects were checked as controls. Quantification of JAK2 V617F mutation and miR-143 expression levels were determined by real-time quantitative polymerase chain reaction. The miR-143 expression in MPNs patients was 2.97-fold higher than in controls. JAK2 V617F mutation allele burden and miR-143 expression level resulted higher in PV and IM respect to ET patients. Patients who had V617F allele burden >50% displayed a higher miRNA-143 expression level than patients with allele burden <50%. In MPNs patients, a statistically significant positive correlation was observed between JAK2 V617F mutation allele burden and hemoglobin and hematocrit values and between miR-143 expression levels and platelet count. Our findings of aberrant miR-143 expression support the concept that factors other than JAK2 V617F mutation may contribute to the pathogenesis and some clinical signs of MPNs.

Consequences of the Presence of the JAK2V617F Mutation in Endothelial Cells: Towards a Better Understanding of the Increased Angiogenesis in Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, characterized by significant increase in one or more myeloid lineages. A mutation in the Janus kinase 2 (JAK2) gene, JAK2V617F, was identified in half of the patients with Philadelphia chromosome-negative (Ph-) MPNs. This activating mutation causes hypersensitivity to certain growth factors, which explain the increased proliferation of myeloid progenitors. It has been reported that patients with MPNs have an increased risk of thrombosis but also increased microvessel density that may reflect angiogenesis in the spleen and bone marrow (Medinger, Br J Haematol, 2009), with no clear physiopathological explanation. Several recent studies have demonstrated the presence of JAK2V617F not only in blood cells but also in endothelial cells (EC) in these patients (Sozer, Blood, 2009; Teofili, Blood, 2011; Rosti, Blood, 2012).Hypothesis: We hypothesized that the presence of JAK2V617F in EC could change their properties leading to an increased angiogenesis process in MPNs.Methods: To determine whether the presence of JAK2V617F in EC was responsible for increased angiogenesis, we used an in vitro approach with human JAK2V617F ECs and an in vivo mouse model. We first used HUVECs (human umbilical vein endothelial cells) transduced with GFP lentivirus encoding human JAK2V617F. An empty lentivirus encoding only for GFP was used as a negative control. Proliferation of HUVECs was quantified during 3 days culture in EGM-2 medium and tube formation after culture in Matrigel™ was assessed by microscopy. Cell migration was determined by microscopy after scratch assay. Proteins expression level in cell lysate and supernatant was determined using Proteome ProfilerTM Array (R&D). The intensity of dot blots was determined by imageJ. For the in vivo approach, we crossed JAK2Flex/WT mice with PDGFβcreERT2 mice to generate endothelial-specific JAK2V617F knock-in mouse line (PDGFβERT2-JAK2V617F/WT). Our lab was pioneer in developing microCT vascular imaging in order to precisely measure arterial vessel density and organization in 3 dimensions. To analyze neoangiogenesis (in the setting of ischemia), we used the model of mouse hind limb ischemia (Oses, ATVB, 2009): 11 days after ligature of the femoral artery, mice are perfused with latex labeled with barium and the hind limb vascular network is visualized with microCT.Results: We first observed that JAK2V617F HUVECs proliferate more than controls (coefficient rate of 3,53+/-0,18 versus 1,98 +/-0,05), reminding the phenotype of JAK2V617F hematopoietic cells. We then showed that JAK2V617F HUVECs are able to form more tubular structures in Matrigel™. Using an in vitro cell migration assay, we observed that JAK2V617F HUVECs invaded 45% (+/-2.3%) of the total surface area versus 27% (+/-3.3%) for controls. To confirm these results obtained in vitro, we analyzed the vascular network after hind limb ischemia in mice expressing JAK2V617F mutation specifically in endothelial cells (PDGFβERT2-JAK2V617F/WT). Our first results show an increased neoangiogenesis and further results will be presented at the conference. In order to decipher the mechanism responsible for the increased angiogenesis, we then quantified the expression of proteins that regulate angiogenesis, either in transduced HUVECs or in the culture supernatant. Interestingly, we have demonstrated a greater secretion of angiogenin, PDGF-AA, Endostatin, IGFBP-1, MCP-1 and CXCL-16 by HUVEC JAK2V617F.Discussion: In summary, our data suggest that the presence of JAK2V617F mutation in EC modifies their properties toward a pro-angiogenic profile and can explain, at least in part, the reported increase of angiogenesis in MPN patients. Once the role of JAK2V617F mutation in modifying EC properties will be confirmed, further work will be required to identify the molecular mechanisms underlying these phenotypic changes. It will also be particularly important to investigate if ECs are involved in the pathogenesis of increased angiogenesis observed in other diseases. Thus, they could be a new target in the treatment of pathological neo-angiogenesis. DisclosuresNo relevant conflicts of interest to declare.

The Role of JAK2 Mutation in Thrombotic Complications of Chronic Myeloproliferative Neoplasms

Patients diagnosed with myeloproliferative neoplasms (MPNs) often develop thrombotic events as an onset of symptoms or in evolution. The pathogenesis of thrombosis in patients with MPN is multifactorial. There are multiple prognostic score systems, but the presence of JAK2V617F (JAK2) mutation is an independent and strong thrombosis risk factor. Patients with MPN and JAK mutational status usually associate thrombocytosis, increased immature circulating platelets, and leukocytosis, with increased expression of CD62P and CD14, increased levels of circulating microparticles and leuko-platelet microaggregates, and altered endothelial function. This review aims to discuss different factors contributing to the increased thrombotic risk in association with JAK2 mutational status. Also, recent reports incriminate this mutation to have a possible role in spontaneous loss of pregnancy.

Possibility of transformation of primary myelofibrosis to ALL without JAK2V617F mutation

In this letter, we wish to point out the possibility of transformation of myelofibrosis in acute lymphocytic leukemia without the presence of JAK2V617F mutation. Myeloproliferative neoplasms (MPN) are hematologic malignant diseases characterized by a clonal proliferation of one or several lineages [1]. They represent a phenotypically diverse group of chronic myeloid malignancies that are characterized by the presence of clonal hematopoiesis and an excessive production of terminally differentiated myeloid blood cells. Typically, they include four main clinical entities: polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and chronic myeloid leukemia (CML). PV, ET, and PMF are usually subcategorized as bcr-abl-negative MPN. However, sporadic cases of bcr-abl-positive patients with transformation of primary myelofibrosis were reported but without JAK2V617F mutation [2]. The prevalence of JAK2V617F mutation [3] differs between various variants of MF with the higher detection rate for patients with post-PV MF (91 %) if compared to PMF (45 %) and post-ET MF (39 %). Some works emphasize the importance of the predictive role of JAK2V617F mutation for this transformation. JAK2V617F

Chronic Myeloprolipherative Neoplasms (MPNs) and Splanchnic Venous Thrombosis(SVT): Incidence and Risk Factors

AbstractAbstract 5057There is evidence in literature of the clinical and pathogenetic role of JAK2 V617F mutation in MPN, while its contribute as an additional thrombotic risk factor in MPN patients is still under discussion. The jak2 mutation has been identified as occult marker in several patients with splanchnic venous thrombosis (SVT); morover, 45% of patients with Budd-Chiari syndrome (BCS) and 34% of patients with portal vein thrombosis (PVT) have associated MPN. On the other hand, the majority of BCS patients without MPN present additional congenital or acquired thrombosis risk factors.The aim of the present study is to evaluate the incidence of SVT in MPN patients. We also evaluated the presence of other prothrombotic risk factors in MPN patients, in addition to the JAK-2 mutation.Out of the 460 Ph1 negative MPN patients observed in our center from January 2000 to January 2010 and retrospectively evaluated, 9 patients (7 females and 2 males; 2%) presented SVT.Six cases had Essential Thrombocythemia (ET), 2 Primary Myelofibrosis (PMF), and 1 Polycitemia Vera (PV). Five of the 6 cases with ET were females. Among the entire population of ET, SVT incidence was 3%. All the 9 patients diagnosed of SVT were JAK2 V617F positive and they were treated with antiaggregant and anticoagulation therapy; 6 received hydrossiurea.Seven patients had SVT before MPN diagnosis, 2 of them had splenectomy at diagnosis for surgical decision. In these patients developing SVT before MPN diagnosis no other major thrombotic event occurred during follow-up. The remaining 2 MPNs patients presented asymptomatic SVT diagnosed by imaging techniques routinely performed during MPN follow up.Interestingly, 75% of MPNs patients with SVT demonstrated at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had 2 or more associated defects. MPNs patients without SVT (396) had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases white blood cell count, platelets values and the presence of JAK2 V617F mutation correlate with the development of the thrombotic event. Conclusion.Even though SVT has a low incidence in MPNs patients, according to the results of the present retrospective study we suggest the potential benefit of searching for additional prothrombotic risk factors in the whole MPN population in order to prevent and/or properly treat this complication. Disclosures:No relevant conflicts of interest to declare.

JAK2V617F mutation for the early diagnosis of Ph− myeloproliferative neoplasms in patients with venous thromboembolism: a meta-analysis

Recent studies suggested that JAK2V617F mutation is frequent in patients with splanchnic vein thrombosis (SVT) but not in patients with other venous thromboembolic events (VTE). However, whether screening for the JAK2V617F mutation in VTE patients is justified remains unclear. Therefore, we performed a systematic review to assess the frequency of JAK2 mutation in VTE patients and the role of JAK2V617F mutation in the diagnosis of myeloproliferative neoplasms. MEDLINE and EMBASE databases were searched. Two reviewers independently performed study selection and extracted study characteristics. Pooled odds ratios of case-control studies and weighted mean proportion of the prevalence of JAK2V617F mutation of uncontrolled series were calculated. Twenty-four studies involving 3123 patients were included. Mean prevalence of JAK2 mutation was 32.7% (95% confidence interval, 25.5%-35.9%) in SVT patients. JAK2 mutation was associated with increased risk of SVT (odds ratio, 53.98; 95% confidence interval, 13.10-222.45). Mean prevalence of JAK2 mutation in other VTE patients was low (range, 0.88%-2.57%). Presence of JAK2V617F mutation in SVT patients was associated with a subsequent diagnosis of myeloproliferative neoplasm in many patients. JAK2 mutation is strongly associated with SVT, and routine screening of JAK2 mutation appears to be indicated in these patients.

In Vitro and in Vivo Inhibition of JAK2 Signaling by Potent and Selective JAK2 Inhibitor

Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

HLA-G turns off erythropoietin receptor signaling through JAK2 and JAK2 V617F dephosphorylation: clinical relevance in polycythemia vera

HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.

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.