| Objective:The classic myeloproliferative tumor(MPN)with Ph chromosome negative is a kind of malignant hematological tumor disease,which is characterized by excessive proliferation of one or more myeloid cells in erythroid,granuloid and megakaryoid lines,mainly including polycythemia vera(PV),essential thrombocythemia(ET)and primary myelofibrosis(PMF).At present,the main molecular causes of MPN include mutations in JAK2,MPL and CALR genes,which are mutually exclusive in MPN diseases.Among them,JAK2 gene mutation is found in 95%of PV patients,and the main types of mutation are JAK2 V617F and JAK2 exonl2.MPLW515 is the most common type of MPL gene mutation,which is only found in 1%-10%of ET and PMF patients.The hot spot of CALR gene mutation is located in exon 9,which is mainly manifested in two mutation types(52-bp deletion and 5-bp insertion),mainly found in ET and PMF patients The remaining MPN that hasn't detected major hot spot mutations of the above three driver genes is called "triple negative" MPN(TN-MPN),which is mainly found in 15%of ET and less than 10%of PMF patients.In recent years,researchers have found double mutations of driver gene in a few MPN patients,most of which is co-mutation of JAK2 V617F and CALR gene,which can be seen in PV,ET and PMF patients.In this study,we found an ET patient with co-mutation of JAK2 exonl2(JAK2 N533S germline mutation)and CALR(52-bp del)gene.Up to now,almost all mutations of JAK2 exon 12 mainly lead to the occurrence of PV,and there are few reports of ET and PMF patients However,the patient we collected has only the clinical features of ET phenotype but lacks PV phenotype,which cannot explain why JAK2 exonl2(JAK2 N533S germline mutation)clone cells didn't cause PV phenotype.In order to elucidate the mechanism of ET occurrence in this patient and increase our understanding of the mechanism of MPN-driven mutation gene,we will analyze the clone relationship of JAK2 N533S and CALR type 1(52 bp del)co-mutation and the function of JAK2 N533S mutation in promoting cell proliferationMethods:I.Analysis of the clonal relationship between JAK2 N533S and CALR type ? co-mutation in patients at clinical levelAccording to the Helsinki Declaration,the patient has signed a written informed consent form.Extraction of fresh peripheral blood mononuclear cells from patients and dilution to prepare single cell suspension(1×102cells/mL),the single cell clone was obtained by limited dilution method,selection of single cell clones,culture of single cell clones with methylcellulose medium containing 3U/ml rhEPO and 100ng/ml rhG-CSF for 15 days respectively to form BFU-E and CFU-G;the DNA of single red line and granulocyte colony were extracted,and JAK2 exon 12 and CALR exon 9 were amplified by PCR with corresponding primers.Then the amplified products were sequenced and the sequencing results were analyzed2.Cell level analysis of the function of JAK2 N533S in promoting cell proliferationTo construct lentivirus expression vectors of JAK2 N533S and its control genes,and transfer JAK2 N533S and its control genes into BaF3 cells through a lentivirus expression system to establish BaF3 cell model with stable expression of each vector;CCK-8 experiment was used to detect the proliferation of BaF3 cells in each group within 0-5 days without IL-3,and analyze the results statistically.To collect the BaF3 cells of each group of JAK2 N533S and its control gene,the total protein of each group was extracted and quantified.The expression levels of phosphorylated STAT3,STAT5,non-phosphorylated STAT3,STAT5 proteins in each group are analyzed by Simple Western methodResults:1.Clinical data of this case at the time of initial diagnosisThe patient was a 75 year old female patient,who had no clinical manifestations such as splenomegaly,hemorrhage,thrombosis and so on.Blood routine examination platelet 707 X 109/L,white blood cell 6.8 X 109/L,red blood cell 4.26 X 1012/L,hemoglobin 132g/L;Blood biochemistry:LDH 297u/L;Myelogram:the morphology and proportion of erythroid and granuloid in bone marrow cells are normal,and the number of megakaryocytes is obviously increased;Bone marrow biopsy shows that megakaryocytes are obviously proliferated without obvious fibrosis;Karyotype analysis showed that:46,XX(20 metaphase cells);MPN related gene mutation detection:JAK2 exon12(JAK2 N533S)and CALR type ? gene mutation are positive.According to the above results,the patient is diagnosed as ET2?Sequence analysis of JAK2 N533S and CALR type ? mutations of BFU-E and CFU-G in this patient?17 clones of red line were selected under microscope,all of them carried JAK2 N533S heterozygous mutation,14 clones(82%)carried CALR type 1 heterozygous mutation,2 clones(12%)were CALR wild type,1 clone(6%)detected CALR type 1 homozygous mutation?The 21 single-grain clones were detected JAK2 N533S heterozygous mutation 20(95%)clones carried CALR type1 heterozygous mutation and 1(5%)clone was Ca1RTY1 wild type? Because JAK2 N533S heterozygous mutation was detected in all clones,we speculated that the mutation might be germline mutation.Therefore,we detected JAK2 N533S and CALR mutations in the hair follicle DNA of the patient.The results showed that JAK2 N533S heterozygous mutation was still positive,while CALR gene was wild type,which confirmed that JAK2 N533Smutation was germline mutation2.Comparison of proliferation and activation of signal transduction pathway in BaF3 cell model stably expressing JAK2 N533S and its control genes(JAK2 WT,JAK2 K539L,JAK2N542-543 Del)?Analysis and comparison of the proliferation ability of Ba/F3 cell model in each groupCCK-8 method was used to detect the growth of Ba/F3 cells independent of IL-3 within 0-5 days in each group.The results showed that cell proliferation didn't occur in JAK2 N533S group,JAK2 WT group,MSCV group and Ba/F3 group(negative control group)at each time point,and there was no significant difference between the groups(P>0.05).The proliferation ability of JAK2 N533S group was compared with the positive control group(JAK2 K539L and JAK2 N542-543del),there was a statistical difference(P<0.001).It is suggested that JAK2 N533S mutation has no effect on cell proliferation?Activation of signal transduction pathway in Ba/F3 cell model of each groupIn the absence of IL-3,the phosphorylation of STAT5(tyr694)and STAT3(tyr705)was increased in JAK2 K539L group and JAK2 N542-543-del group,but not in JAK2 N533S group,JAK2 WT group,MSCV group and Ba/F3 group.It is suggested that the mutation of JAK2 N533S doesn't change the function of JAK2 proteinConclusion:We found a case of ET patient with co-mutation of JAK2 exon 12(JAK2 N533S)and CALR typel.Clonal analysis of hair follicle gene test results confirmed that the atypical mutation of JAK2 exon 12(N533S)was germline mutation and CALR type1 was acquired mutation.Cell function studies have confirmed that the atypical mutation of JAK2 germline line(JAK2 N533S)doesn't have the effect of promoting cell proliferation autonomously.To sum up,the JAK2 exon 12(N533S)mutation carried by the patient doesn't have the ability to generate PV phenotype,while its ET phenotype is caused by the late acquired CALR type1 mutation. |
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