| Myeloproliferative neoplasm (MPN) is a clonal hematopoietic disease characterized by sustained proliferation of bone marrow. The classic BCR-ABL-negative MPNs encompass three clonal diseases:polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).Thromboembolism is the key cause of mortality and seriously affects the quality of life of the patients with MPN. Early diagnosis and intervention of thrombosis is essential to reduce the mortality of patients with MPN. JAK2V617F mutation is the characteristic change of the classic BCR-ABL-negative MPNs.In addition,CALR and MPL mutations can be also found in them. Although they were retrospective,there were some studies between JAK2V617F, CALR and MPL mutations and thrombosis in patients with MPN in the country. In this study, the mutations of JAK2V617F, CALR and MPL mutations were detected in all subjets. The incidence of thrombotic complications was observed after 2 years of follow-up.By detecting the expression of pSTAT3,TNF-amRNA and IL-6 mRNA of mononuclear cells of peripheral blood (PBMC) in patients with MPN,we performed further sdudy in the pathogenesis.PART I Study of the incidence of JAK2V617F, CALR andMPL mutation and clinical characteristics in patients with myeloproliferative neoplasmObjective:To investigate the incidence of JAK2V617F, CALR and MPL mutation and clinical characteristics in patients with myeloproliferative neoplasm.Methods:(1) This study selected patients with MPN from September 2013 to April 2014, total 68 patients with MPN but no thromboembolism were enrolled in this study (group case, n=68),65 patients with thromboembolism or phlebothrombosis were selected as group thrombus(group thrombus, n=65).68 individuals in group control included healthy subjects or patients without MPN from hematology department of our hospital(group control, n=68).(2) Record age, sex, place of residence, occupation and past history, smoking history, history of heavy drinking of all subjects, collect the risk factors of thrombosis like blood pressure, fasting blood glucose level, concentrations of blood low-density lipoprotein, high-density lipoprotein concentration, cholesterol concentration, body mass index and routine blood of all subjects.(3) Collect fasting venous blood 3 ml of each subject for detecting JAK2V617F, CALR and MPL mutations. Fasting venous blood of sufficient amount of each subject was collected for fasting glucose, lipid profile, blood coagulation and routine blood test.(4) Use JAK2V617F mutation detection kit to extract DNA from blood cells, and use LightCycler 480 fluorescent PCR gene amplification detector to perform PCR, ABI3730 genetic analyzer was used to perform DNA sequencing and outputted data.Results:(1) 46 in 68 patients with MPN showed JAK.2V617F mutation, the mutation rate was 67.65%, in which 24 patients were diagnosed ET, the mutation rate of ET was 64.86%, 19 patients were diagnosed PV, the mutation rate of PV was 82.61%,3 were PMF, the mutation rate of PMF was 37.5%.1 subjects in 68 controls showed JAK2V617F mutation, the mutation rate was 1.47%. The mutation rate in MPN patients was significantly higher than group control (P<0.05).8 subjects in group thrombus showed JAK2V617F mutation, the mutation rate was 12.31%. The mutation rate in MPN patients was significantly higher than group thrombus (P<0.05). The mutation rate in group thrombus was significantly higher than group control(P<0.05). 12 in 68 patients with MPN showed CALR mutation, the mutation rate was 17.65%, in which 10 patients were diagnosed ET, no patients were diagnosed PV,2 were IMF. None of subjects in 68 controls showed CALR mutation. The CALR mutation rate in MPN patients was significantly higher than group control (P<0.05). None of subjects in 65 group thrombus showed CALR mutation. The CALR mutation rate in MPN patients was significantly higher than group thrombus (P<0.05).3 in 68 patients with MPN showed MPL mutation, the mutation rate was 4.41%, in which 2 patients were diagnosed ET, no patients were diagnosed PV,1 were IMF. None of subjects in 68 controls showed MPL mutation. The MPL mutation rate in MPN patients was significantly higher than group control (P<0.05).None of subjects in 65 group thrombus showed MPL mutation. The MPL mutation rate in MPN patients was significantly higher than group thrombus (P<0.05).There were 7 cases (10.29%) of all three mutations negative in MPN patients, and three mutations did not appear simultaneously.(2) There was no significant difference on blood pressure, fasting blood glucose level, and lipid profile between patients with JAK2V617F mutation and those without JAK2V617F mutation in group case (P>0.05). There was no significant difference on blood pressure, BMI, and lipid profile between patients with CALR mutation and those without CALR mutation in group case (P>0.05). There was no significant difference on blood pressure, BMI, and lipid profile between patients with PML mutation and those without PML mutation in group case (P>0.05).(3) Values of PT [(16.94±2.07)s] in group case were significantly higher than those in group control [(12.26±0.96)s] (P<0.05); values of APTT [(59.26±12.38)s] in group case were significantly higher than those in group control [(30.73±3.13)s] (P<0.05); values of TT [(16.58±1.75)s] in group case were significantly higher than those in group control [(12.53±1.07)s] (P<0.05);values of FIB [(3.02±0.58)g/L] in group case were significantly lower than those in group control [(3.36±0.49) g/L] (P<0.05). Values of PT (17.87±1.56)s] of patients with JAK2V617F mutation in group case were significantly higher than those without JAK2V617F mutation [(15.00±1.60)]s (P<0.05); values of APTT[(64.22±10.82)s] of patients with JAK2V617F mutation in group case were significantly higher than those without JAK2V617F mutation [(48.91±8.49)]s (P<0.05); values of TT[(16.98±1.642)s] of patients with JAK2V617F mutation in group case were significantly higher than those without JAK2V617F mutation [(15.78±1.72)s (P<0.05). There was no significant difference on PT, APTT, TT, FIB between patients with CALR mutation and those without CALR mutation in group case (P>0.05). There was no significant difference on PT, APTT, TT, FIB between patients with PML mutation and those without PML mutation in group case (P>0.05).Conclusion:The mutation rate of JAK2 V617F, CALR and MPL in MPN patients was significantly higher; there was no significant difference on risk factors of thromboembolism between patients with JAK2V617F mutation and those without JAK2V617F mutation in group case; there was great significant difference on blood coagulation between patients with JAK2V617F mutation and those without JAK2V617F mutation in group case.PART II Study of the incidence of thrombosis of MPN patients in two years and the possible pathogenesisObjective:To investigate the correlation between JAK2V617F, CALR and MPL mutation and thrombosis in patients with myeloproliferative neoplasm (MPN) and it's possible pathogenesis.Methods:(1) Subjects of this study were group case and group control of the first part.Since the date enrolled, all subjects of group case and group control were followed up by telephone or outpatient at least twice a month, asking and record a detailed history of all subjects, especially the history related to thrombotic events, as well as blood pressure, body weight and blood test indicators. They were followed for 2 years,no loss of follow-up, the latest follow-up to April 2016.(2) PBMC were isolated from group MPN, the total protein of cytoplasm was extracted and the expression of STAT3 protein was detected by immunoblotting. The mRNA of PBMCs was extracted, and the expression of TNF-amRNA and IL-6 mRNA were detected by RQ-PCR.Results:(1) 26 of 68 patients with MPN were diagnosed thrombus among 2 years, the rate of thrombus was 38.24%.2 in group control were diagnosed thrombus. The rate of thrombus of group case was significantly higher than group control (P<0.05). The rate of thrombus of patients with JAK2V617F mutation in group case was 47.83%(22/46); the rate of thrombus of patients without JAK2V617F mutation in group case was 18.18%(4/22). The rate of thrombus of patients with JAK2V617F mutation was higher than the rate of thrombus of patients without JAK2V617F mutation in group case. The rate of thrombus of patients with CALR and MPL mutation in group case was both of 0.(2) Values of PT [(17.04±2.15)s] in group case were significantly higher than those in group control [(12.18±0.88)s] (P<0.05); values of APTT [(58.15±12.49)s] in group case were significantly higher than those in group control [(30.87±3.00)s] (P<0.05); values of TT [(17.59±2.81)s] in group case were significantly higher than those in group control [(12.47±1.04)s] (P<0.05). Values of FIB [(3.05±0.56)g/L] in group case were significantly lower than those in group control [(3.33±0.44) g/L] (P<0.05).(3) Values of PLT of ET patients with thrombus [(840.15±48.60) ×109/L] were significantly higher than those without thrombus [(809.46±36.98) × 109/L] (P<0.05). Values of TT of patients with thrombus in group case (18.85±2.72)s were significantly higher than those without thrombus (16.81±2.60)s (P<0.05).(4) There was no significant difference on blood pressure (P>0.05), fasting glucose (P>0.05), lipid profile (P>0.05) between group control and group case.(5) There was no significant difference on blood pressure (P>0.05), fasting glucose (P>0.05), lipid profile (P>0.05) between patients with thrombus and patients without thrombus.(6)The expression of p-STAT3 protein of PBMC is significantly higher in MPN patients with JAK2V617F mutation than in those MPN patients without JAK2V617F mutations (P<0.05).The expression of p-STAT3 protein is significantly higher in MPN patients with thrombotic complications than in those without thrombotic complications (P<0.05). The expression of p-STAT3 protein was significantly higher in MPN patients with JAK2V617F mutation and thrombosis complications (P<0.05). (7)The relative value of IL-6 mRNA of PBMC was significantly higher in NPN patients with JAK2V617F mutation than in those MPN patients without JAK2V617F mutation (P<0.05). The relative value of IL-6 mRNA was significantly higher in MPN patients with thrombotic complications than in those MPN patients withouthout thrombotic complications(P<0.05). The relative value of IL-6 mRNA was significantly higher in MPN patients with JAK2V617F mutation and thrombotic complications (P<0.05).Conclusion:MPN patients with JAK2V617F mutation were prone to thrombus; JAK2V617F mutation might be a cause of thrombus of patients with MPN; JAK2V617F mutation could affect results of blood coagulation through some signal transduction pathway, and cause thrombus of patients with MPN. JAK-STAT signal path related to IL-6 through STAT3 activation might be one of the mechanisms of thrombosis complications in MPN patients with JAK2V617F mutation.CREATIVE POINTS IN THIS STUDY(1) The present study examined the mutation rate of JAK2V617F, CALR and MPL genes in all subjects. We performed a prospective study on the relationship of incidence of thrombosis in MPN patients with JAK2V617F, CALR or MPL mutation by setting up a control group and 2 years followed up, found that patients with MPN and JAK2V617F mutation-positive were prone to thrombotic events, CALR and MPL mutations mutation did not increase the incidence of thrombosis in patients with MPN. (2) By detecting expressions of pSTAT3, TNF-a mRNA, IL-6 mRNA in PMBCs with MPN, we suggested JAK-STAT signal path related to IL-6 through STAT3 activation might be one of the mechanisms of thrombosis complications in MPN patients with JAK2V617F mutation. |
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