V617F Mutation Of JAK2 Gene Research Articles

Determining the JAK2 V617F mutation at Hue University of Medicine and Pharmacy Hospital

Background: JAK2 V617F mutation is one of the major criteria in diagnosing MPNs. This study aimed to: (1) Establish a procedure and compare Allele specific-PCR and PCR-RFLP techniques in identifying JAK2 V617F mutation; (2) Primarily evaluate the prevalence of V617F mutation of JAK2 gene in patients with MPNs. Materials and methods: DNA extraction was isolated from peripheral blood of 31 patients with MPNs followed by clinicians and 10 healthy individuals. Determining V617F mutation of JAK2 gene by AS-PCR and PCR-RFLP techniques. Sanger sequencing randomly 15% of samples for comparison. Results: The optimal annealing temperature for AS-PCR is 55oC. Conditions of restriction digest in PCR-RFLP uses 300 ng of PCR product. All samples identified by AS-PCR and PCR-RFLP showed similar results. Conclusion: Established a procedure to identify the V617F mutation in JAK2 gene by allele-specific PCR and PCR-RFLP, in which the AS-PCR technique showed more advantages. The frequency of V617F mutation of JAK2 gene in patients was 61.3%. Key words: MPNs, V617F, JAK2, allele-specific PCR, PCR-RFLP

Screening of V617F mutation in JAK2 gene with acute myeloid leukemia in the Saudi population.

Progress in pathogenesis and therapy of acute myeloid leukemia (AML) is presently accelerating. The Janus kinase 2 gene (JAK2) mutations are rare in de novo AML. The gene codes for the tyrosine kinase that has a significant role in the signal transduction in hematopoietic cells. The aim of this study was to induce V617F mutation in the JAK2 gene in the AML patients diagnosed in the Saudi population. In this case-control study, 100 AML patients and 100 healthy controls were recruited. Genotyping was performed with polymerase chain reaction followed with restriction fragment length polymorphism analysis. The mean age of the AML patients and healthy controls was found to be almost similar (p=0.60). In this study, 15% of VF mutation was documented in the AML cases and none of the mutations were documented either in FF mutation in AML cases or VF and FF mutations in the healthy control subjects. VF mutations [VF vs VV; OR-18.79; (95%CIs: 2.442-144.6) and p=0.0001; F vs V; OR-87.76; (95% CIs: 11.76-654.7) and p<0.0001] were found to be significantly associated when compared between AML cases and healthy controls. In conclusion, the V617F mutation showed the positive association in the AML patients diagnosed in the Saudi population.

Development of Molecular Beacon method to detect of JAK2 V617F mutation

V617F mutation of JAK2 gene is a point mutation of Somatic cells leading to permanent phosphorylation and protein kinase activity of the JAK2 protein. This mutation is a valuable marker in the diagnosis of myeloid neoplasms. Development of new techniques can be effective and highly sensitive for the detection of the mutation. The aim of this study is designing and executing a molecular beacon probe-based real-time PCR detect to V617F mutation. Test results were reviewed from a set of wild type and cloned JAK2 exon 12 mutated allele genes into plasmid vector and through amplification by Real-Time PCR system. The sensitivity and specificity of Molecular Beacon probes design were tested. The results showed that the Real-Time PCR system has %100 specificity and % 0.001 sensitivity. Based on the results obtained from the molecular beacon probe-based real-time PCR, it was proved that the system is able to detect normal and mutant alleles of JAK2 V617F position with high accuracy and a short time in a closed tube system

Genetic and Epigenetic Alterations of Ph-Negative Myeloproliferative Neoplasms

Objectives and background: Genetic mutations result in abnormalities of myelopoietic proteins and lie in the basis of Ph-negative myeloproliferative neoplasms (MPNs) development and its subsequent progression. Several somatic mutations in JAK2, MPL, TET2, EZH2, ASXL1, CBL, IDH1, IDH2, IKZF1 genes were detected in chronic and blastic phase MPNs. Recent studies have revealed a number of epigenetic alterations that contribute to Ph-negative MPNs pathogenesis and determine the clinical outcome. Mutations involving the EZH2 gene are thought to result in loss of methyltransferase activity suggesting a potential role of tumor suppressor gene silencing as a mechanism in the disease progression. Decrease in ubiquitin ligase activity caused by mutations CBL gene leads to myeloid proliferation. EZH2, CBL mutations are thought to be of prognostic value in MPN’s at the time of transformation to the blastic phase but data are inconsistent and require the further verification.The goal of our research was to determine the significance of mutations genes EZH2, CBL in the diagnosis and prognosis of Ph-negative MPNs.Methods. We have examined 102 patients with Ph-negative MPNs (45 pts with PV, 30 pts with ET and 27 pts with PMF). For all patients the detection of V617F mutation of JAK2 gene was done. V617F-negative pts with PV and pts with ET or PMF underwent the analysis of mutations in 12-th exon of JAK2 and 515 codone of MPL gene respectively. For 80 pts (30 with PV, 28 with ET and 22 with PMF) cytogenetic analysis and EZH2 mutation status were performed. Identification of CBL mutations was performed in 24 patients with available RNA samples. Mutations in 8, 10, 17, 18, 19 exons of EZH2 and RING-domen of CBL were defined by sequence analysis. V617FJAK2 mutation was detected in 44/45 (97,8%) pts with PV, 16/30 (53,3%) pts with ET and 13/27 (48,1%) pts with PMF. 538-539del-insL in 12-th exon of JAK2 was found in 1/45 (2,22%) patient with PV. W515KMPL mutation was identified in 1/30 (3,33%) pt with ET and 1/27 (3,7%) pt with PMF. 2 mutations of EZH2 gene have been found in 2 individuals with PMF (2/22). Both mutations are located in the 19 exon. The Ile713Thr mutation was detected in the patient with a del(6)(q15) karyotype which is associated with an intermediate cytogenetics risk. This patient subsequently underwent transformation from PMF to myelodysplastic syndrome in 9 months after the disease onset. Another case of mutation harboring (Thr731Asp) was detected in a patient with PMF and poor prognosis karyotype (chromosome 7 monosomy). This patient had transformation PMF to acute myeloid leukemia and died after 20 months. Homozygous mutation Q420R in CBL gene was detected in 1/24 patient with complex karyotype. Disease progression was observed after 16 months from the diagnosis.Conclusion. Mutations in EZH2 and CBL genes could be assessed as additional prognostic markers of unfavourable prognosis in patients with BCR-ABL-negative MPNs with different chromosomal aberrations. The integration of cytogenetic and molecular analyses could be a valuable option for stratification of patients and optimising the treatment strategy.

Incidence of the JAK2V617F Mutation In Mexican Patients with Myeloproliferative Neoplasms (MPNs)

Abstract 5180 Introduction:The V617F mutation in JAK2 gene has been described in approximately 50–90% of patients with ET, MF AND PV [essential trombocythaemia, idiopathic myelofibrosis and policithemia vera]; but has also been reported, albeit at a lower frequency, in patients with other myeloid malignancies such as atypical CML, CMML, AML, MDS, JMML and CNL. A single G>T base substitution in exon 12 results in the conversion of valine to a phenylalanine aminoacid at position 617 of the JAK2 gene. The identification in this study were using techniques such as allele-specific PCR, RFLP-PCR and direct sequencing; for to determine the incidence in Mexican patients with MPNs. Patients and Methods:The JAK2V617F mutation was determined in 88 patients and 5 normal blood samples for healthy individuals as controls. About the patients, 60 were cataloged like MPNs and 28 patients with features suggestive of MPNs vs CML. Samples for bone marrow or peripherical blood were taken either at time of diagnosis of MPNs or during treatment with cytoreductive or anti-thrombotic agents. DNA and RNA were extracted using the QIAamp DNA and RNeasy mini kit (Qiagen) and amplified by the three techniques mentioned for JAK2V617F and by nested RT-PCR for BCR/ABL. Results:The five normal blood samples for controls were negative for JAK2V617F mutation and to BCR/ABL. Patients had median age 65 years (47–85 years old), 46% male and 54% female. In de overall patients: 60 patients with MPNs all were BCR/ABL negative and 20 (33%) had JAK2V617F. In the 28 patients with likely MPNs vs CML, 23 were BCR/ABL positive/JAK2 negative, two had the coexistence of both genetics defects [BCR/ABL+ and JAK2V617F+] and 3 BCR/ABL and JAK2 negative. Finally the patiens with JAK2V617F+, were 12 ET, PV 1, MF 2, CML 2, and 5 continued like MPNs. Discussion:The incidence of the JAK2V617F in this study for MPNs patiens were 33% and the incidence varied between MPNs subtype. Less than ten cases of BCR/ABL+ CML with JAK2V617F have been published; we report two patients with the coexistence and we agree with previous reports that screening for JAK2V617F mutation should be considered in any BCR/ABL+ CML patients and the clinical outcome will be define in long period. Disclosures:No relevant conflicts of interest to declare.

Розробка тест-системи для виявлення мутації V617F гена jak2 у хворих на хронічні мієлопроліферативні захворювання

Розробка тест-системи для виявлення мутації V617F гена jak2 у хворих на хронічні мієлопроліферативні захворювання

Polymorphisms of NOS3 and FCGR2A Genes Contribute to Thrombotic Risk in Essential Thrombocythemia

Thrombosis is the most frequent complication in essential thrombocythemia (ET) that impairs quality of life and contributes to mortality and morbidity of patients. The molecular basis of thrombosis in ET remains unclear. Polymorphisms (SNPs) in coagulation factors (FII-G20210A, FV-Leiden) and the V617F mutation of JAK2 gene (JAK2-V617F) have previously been shown to associate with thrombosis in ET. Since thrombosis occurs frequently in JAK2-V617F negative ET and FII-G20210A and FV-Leiden polymorphisms occur only in up to 5% of patients, we examined the hypothesis that other genetic factors contribute to the thrombotic risk in ET. In addition to FII-G20210A and FV-Leiden we studied thirteen candidate SNPs of genes involved in coagulation (F7, F13A1, VWF, THSD1, ATIII), platelet aggregation (P2Y12, GPIIIA, GPIIB, FCGR2A) and endothelial function (NOS3, ADRA2B) previously implicated in thrombosis in non-MPD patients. We studied 94 patients with the diagnosis of ET retrospectively observed for clinical variables such as venous and arterial thrombosis, stroke, myocardial infarction, deep vein thrombosis (DVT), bleeding, angina pectoris, superficial venous thrombosis (SVT), and pulmonary embolism. SNP genotyping was performed using allele-specific PCR and sequencing. We examined the association of individual SNPs with the presence or absence of clinical variables (adjusted for age, sex, and BMI). In our ET cohort, JAK2-V617F showed significant correlation with the presence of DVT (OR 10.08; 95%CI 1.15–88.49; P=0.01). Both FII-G20210A and FV-Leiden were rare in this cohort (2% and 4%, respectively) and thus did not show any significant associations. Homozygosity for the 298Asp variant of NOS3 gene (encoding the endothelial nitric oxide synthase) showed significant correlation with stroke (OR 46.75, 95%CI 1.38–147, P=0.014). Similarly, homozygosity for the 131Arg variant of Fc receptor gamma 2A (FCGR2A) involved in platelet GPVI signaling correlated with the presence of SVT (OR 9.46, 95%CI 1.48–60.82, P=0.014). Our results indicate that genetic factors other than JAK2-V617F, FII-G20210A and FV-Leiden contribute to thrombotic risk in ET. Furthermore, the nitric oxide and the platelet GPVI signaling pathways are likely to play an important role in the pathogenesis of thrombosis in ET.

Mutation JAK2 V617F in Patients with Myeloproliferative Disorders and Technical Aspects of Its Detection.

Myeloproliferative disorders (MPD) are a heterogeneous group of diseases that involve chronic myeloid leukemia (CML), polycythemia vera (PV), essential trombocythemia (ET), chronic idiopatic myelofibrosis (IMF), hypereosinophilic syndrome and chronic neutrophilic leukemia. Recently, it has been found that in Ph-negative MPD, acquired somatic mutation in JAK2 gene can be identified, causing substitution of valine for phenylalanine at aminoacid position 617 (V617F). Experimental evidence indicates that JAK2 V617F might cause erythropoietin-independence of erythroid colonies and receptor hypersensitivity, thus be implicated in the pathogenesis of PV.The incidence of V617 in PV is high, reaching 96% of cases; in ET or IMF, the mutation is less frequent. Nevertheless, the percentage of JAK2 V617F-positive patients differs greatly among studies, depending on the detection technique used. Sequencing analysis, as one of the options, can underrate the number of positive cases, as its sensitivity in authentic samples with varying proportion of mutant granulocytes is unsatisfactory. The nucleotide context of the V617F site is complicated (direct repeat TGTG/TT), and thus prone to sequencing artifacts.For specific and sensitive detection of V617F mutation in JAK2 gene, we have developed a novel allelic discrimination assay, based on the usage of Real-Time PCR technology and LNA-modified fluorescently labeled probes (Locked Nucleic Acids). This technology is distinguished by 100% discrimination efficiency, and sensitivity detection rate reaching 10% of mutant granulocytes in an authentic sample. LNA-modified probes are extremely powerful tool for precise genotyping, since they are short (12-mers), and due to the interspersed LNA nucleotides, melting temperature (Tm) of the probe/target hybrid is high. Hence, the LNA-based genotyping technology combines the desired features of high sensitivity and absolute specificity.Herein, we present an analysis of JAK2 V617F detection in our group of 157 consecutive patients with the diagnosis of MPD or suspected MPD. Using our novel LNA-based Real-Time PCR genotyping technique, we were able to identify varying proportions of the mutant JAK2 allele in 87 cases (55.4%); in the remaining 70 patients the mutation was not detected.The LNA-based Real-Time PCR technique is extremely precise and efficient in identifying even subtle proportion of JAK2 V617F mutant granulocytes, which in cases of diagnostic uncertainties on the origin of the polycythemia might be of great importance.

Clinical Classification of Myelofibrosis with Myeloid Metaplasia (MMM): Cluster Analysis of 861 Patients Enrolled into a Nationwide Prospective Registry (RIMM).

BACKGROUND: MMM is a rare chronic myeloproliferative disease with heterogeneous clinical presentation, natural history and prognosis. Knowledge about MMM is burdened by limited sample size and selection bias of the available studies.AIM: To get a clinical classification of MMM for biological and clinical studies.METHODS: The database of the RIMM, a population-based prospective cohort started in 1999, was enquired and data of 861 patients at diagnosis were extracted. The EM-algorithm (WEKA software) was used for a non-supervised data-mining based on 11 variables: age, spleen size, constitutional symptoms, hemoglobin level, platelet and blood cell count, LDH and percentages of erythroblasts, immature myeloid cells, blasts, and basophils. Differences among clusters were first tested with ANOVA or n-way Chi-square and subsequently investigated with t-test or 2x2 Chi-square. Survival differences among clusters were tested through Cox univariate and multivariate regression analysis.RESULTS: Five clusters (C) were identified: C1 identified younger patients with thrombocytosis and scarce myeloproliferation. C2 also identified patients with a high platelet count, but older and anemic. C3 identified patients with intense myeloproliferation (marked leukocytosis and splenomegaly), while C4 (5% of the patients) collected patients with an even more severe myeloproliferation, associated with a high number of circulating immature myeloid cells and blasts. C4 was also characterized by frequent chromosome abnormalities, anemia and thrombocythopenia. Finally, C5 identified patients with bilinear/trilinear cytopenias. The clusters also differed for variables not incorporated by the data-mining process, i.e. CD34+ cell count (p=0.028); i.e. the rate of homozygous V617F mutation of JAK2 gene, which was significantly higher in C3 and C4 (32.4% vs 12.5%, p=0.019). The clusters also significantly differed for overall survival (p<0.0001), independently of patients' age. Finally, referral patterns differed among clusters, with C5 and C1 patients being mostly referred to Internal Medicine and Hematology units (p<0.01), respectively.CONCLUSIONS: Thanks to a large and unbiased repository of cases, we identified 5 classes of MMM patients with specific clinical presentation. The biologic and prognostic value of the clusters deserves further investigations at longer follow-up periods.Clinical parameters in the different clusters (means)CLUSTERAGE (yrs) *HB (g/l) */PLT*10(9)*WBC *10(9) *SPLEEN (cm) *BLASTS*(%)CD34 %/μlSEVERITY SCORE§ *156.713.3 /58311.53.50.210.85 /631.25272.110.6 /48218.32.70.821.12 /2972.04365.110.8 /30318.39.91.471.7 /3132.54467.68.4 /11027.513.45.662.72 /2754.30568.28.4 /1344.26.80.782.92 /523.36All65.510.7 /36213.56.51.181.78 /2002.36* p<0.0001 § The score considers HB, WBC, PLT and SPLEEN (Barosi, Leuk Lymph 2002).