The Mutation Frenquency Of JAK2 V617F, Expression Of Phosphoted JAK2/STAT5 Protein And Clinical Significance In Chinese Myeloproliferative Disorders Patients

Background:Chronic myeloproliferative disease(CMPDs) are clonal hematopietic stem cell diseases characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased numbers of nature and immature cells in the peripheral blood.The World Health Organization(WHO) category of CMPDs include Chronic Myeloid Leukemia(CML),Chronic Neutrophile Leukemia,CNL), Chronic Eosinophilic Leukemia and the Hypereosinophic Syndrome(HES),Polycythemia vera,(PV),Essential thrombocythemia(ET),Chronic Idiopathic Myelofibrosis(CIMF), Chronic myeloproliferative disease,unclassifiable,(UCMPD)。Although the WHO has a strict catergorical criterion about the subtype of CMPDs,but the accurate classification about CMPDs is also disputable.Futhermore,it is difficult to identify with bone marrow reactive hyperplasia.Only the CML is characterized by a specific moleuclar marker,BCR-ABL fusion gene,and it is known that the fusion gene is primary,may be only one,driving force in the chronic phase of CML.But for the majority of CMPDs,a specific molecular marker is not known.Recently,several groups independently detected a point mutation in highly conserved residue of pseudokinase domain of JAK2 Tyrosine kinase(V617F) in most patients with PV,as well as one-third to one half of patients with either ET or IMF.The JAK2V617F mutation results in constitutive activation of the tyrosine kinase.The activated JAK2 subsepuently phosphorylate the STATs.Phosphorylated STAT5 enter the cellular nucleus and bind to regulatory sequence specificly to activate or up-regulate transcription of target genes.Mutation of JAK2 V617F may explan why the mutated cells are hypersensitive to several kind of different growth factors.In present study,we identified and analyzed the JAK2V617F mutation frenquency of 90 patients with CMPDs(including 38 patients with PV;12 patients with ET;14 patients with IMF;20 patients with CML;1 patient with CNL;5 patients with UMPDs),and detected whether JAK/STATs signal proteins were activated or not,and analysised the realationship between the JAK2V617F mutation and clinical manifestations in patients with CMPDs.The aim was to further reveal the molecule mechanism of chronic myeloid proliferative diseases.PartⅠIdentification the Mutation Frenquency of JAK2V617F in chronic myeloprolifierative disordersObjective:To testify whether JAK2V617F mutation exists in Chinese MPDs patients,and evaluate the frequency of JAK2V617F mutation in MPDs;comparing with DNA sequence,whether the allele-PCR is more sensitive or more specific for detecting JAK2V617F mutation in clinical laboratory.Method:The diagnosis of CMPDs was based on the criteria proposed by World Health Organization.DNA was extracted by standard procedures after isolating total leukocytes from peripheral blood mononuclear cells by density gradient centrifugation over Histopaque 1077.DNA samples were amplified using primers 5′-TCCTCAGAACGTTGATGGCAG-3′(forward) and 5′-ATTGCTTTCCTTTTTCACAAGAT-3′(reverse).Single-stranded biotinylated PCR products were prepared for sequencing.At the same time,in order to testify the realiability of the allele-specific PCR,we also applied the allele-specific PCR using two forward primer and one common reverse primers for identifying the mutation.Results:In 43 of 90 patients with myeloproliferative disorder,JAK2V617F mutation was determined by conventional DNA sequencing-32 of 38 patients with PV,6 of 14 patients with CMIF,5 of 12 patients with ET.But the allele-specific PCR detected 49 patients occurs JAK2V617F mutation--35 of 38 patients with PV,8 of 14 patients with CMIF,6 of 12 patients with ET.And these were testified by DNA sequencing. Conclusion:A-gain -of-function mutation of JAK2V617F occurs in nearly all patients with PV,mutation frenquency is near to 50%both in ET and CMIF,none mutation was found in CML.Allele-specific PCR is a very sencitive and specific method for detecting JAK2V617F mutation. PartⅡPhosphoted JAK2/STAT5 proteins expression in patients with JAK2V617F mutationObsjective:In order to further reveal the molecule mechanism,we compared the expression of JAK2 protein,phospho-JAK2 and phospho-STAT5 in patients with JAK2 V617F-positive and that in patients with JAK2 V617F-negative.Methods:Total cellular protein were extracted from mononuclear cells of pheripheral blood in patient with CMPDs.The Western Blotting Assay is used to measure the expression of JAK2 protein,phospho-JAK2 and phospho-STAT5.Results:There is no significant difference for the express of JAK2 protein both in patients with or without JAK2 V617F mutation.But the expression of phospho-JAK2 and phospho-STAT5 is upregulated obviously on JAK2 V617F-positive patients than that on JAK2 V617F-negative patients.Conclusion:The JAK2 V617F is a constitutively active tyrosine kinase and the phospholation of STAT5,a down-stream target of JAK2 protein,is an important pathogenetic event on BCR/ABL-negative JAK2V617F positive CMPDs. PartⅢThe clinic feature significance of JAK2V617F-positive chronic myeloproliferative disordersObjective:To provide the evidence of clinical features and significance on JAK2V617F mutated patients,,we compared the clinic manifestations of 49 patients with JAK2V617F-positive CMPDs with that of 15 patients with JAK2V617F-negative CMPD.Methods:By comparison the differences of labortary and clinical features between patients with and without the mutation of JAK2V617F,including age,hemoglobin level,platelet count,white cell count,thrombotic events,the itch of skin,the spleen size at the first diagnosed patients. Results:No difference was found on average white cell counts in patients with or without JAK2V617F mutation.However mean hemoglobin concentation was higher on JAK2V617F positive patients than those of JAK2V617F negative;the frenquency of thrombosis event was significantly higher in patients with JAK2V617F mutation,compared with those of without the mutation.But the frequency of the itch of skin is lower in patients with the mutation than of those without JAK2V617F mutation;an enlarged spleen is more obvious on the patients with JAK2V617F positive;average age is older for JAK2V617F mutation patients..Conclusion:Compared with the patients with JAK2V617F negative,the JAK2V617F-positive CMPDs have the following features:older age,higher average hemoglobin level,higher frenquency of thrombosis events,larger spleen volume,but lower frequency of the itch of skin.