蛋白激酶JAK2V617F点突变在青海省骨髓增殖性疾病中表达的相关性研究
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摘要
目的:对青海省bcr/abl融合基因阴性的骨髓增殖性疾病(myeloproliferativedisorders,MPD)病人进行JAK2V617F点突变定性检测,分析JAK2V617F点突变在我国西部地区MPD病人中的表达率,为临床诊断、治疗提供理论依据。
方法:1.常规提取全基因组DNA、骨髓组织总RNA提取。
2.用逆转录聚合酶链反应(RT-PCR)对140例MPD病人进行bcr/abl融合基因检测。
3.用等位基因特异性聚合酶链反应(AS-PCR)检测140例MPD病人JAK2基因的V617F点突变,对有突变的病例进行测序验证。
4.分析比较青海省与国内部分其它地区MPD病人JAK2基因的V617F点突变表达率的差异。
结果:1、140例典型MPD病人bcr/abl融合基因检测:76例CML病人均为bcr/abl阳性的病人,其余64例典型MPD病人为bcr/abl阴性,其中32例为真性红细胞增多症,20例为原发性血小板增多症,12例为特发性骨髓纤维化病人。
2、AS-PCR检测JAK2V617F突变发生率:bcr/abl阳性的76例慢性粒细胞白血病病人JAK2V617F突变均为阴性;64例bcr/abl阴性MPD病人中的突变率是:32例真性红细胞增多症病人中24例阳性(占75%),20例原发性血小板增多症病人中6例阳性(占30%),12例特发性骨髓纤维化病人中6例阳性(占50%)。
3、青海省与国内部分其它地区MPD病人JAK2基因的V617F点突变率相比存在差别。
结论:JAK2V617F点突变在青海省bcr/abl融合基因阴性的骨髓增殖性疾病的发病中起了重要作用,PV、ET、IMF病人JAK2V617F突变阳性率依次为75%(24/32)、30%(6/20)和50%(6/12),与国内其它部分地区相比存在差别。JAK2V617F可能为PV、ET、IMF的特征性分子事件,结合其临床病理特征可作为一个独立的分子指标用于此三类疾病的临床诊断。
Objective:To detect the JAK2V617F mutation in myeloproliferative disorders(MPD) of Qinghai province by allele-specific primer polymerase chain reaction and evaluate the significance of JAK2V617F in diagnosis and therapy of MPD.
Methods:1.Genomic DNA and total RNA were extracted from white blood cells and marrow respectively by route way.2.The bcr/abl fusion gene was detected in 140 Patients with MPD by the reverse transcription Polymerase chain reaction(PT-PCR).3.The JAK2V617F mutation was detected in 70 patients with MPD by allele-specific polymerase chain reaction(AS-PCR) and the positive cases with the mutation were checked by sequencing.
Results:Between 140 Patients with MPD,the bcr/abl fusion gene is detected in 76 chronic myelocytic leukemia patients,others is negative,including 32 polycythemia vera,20 essential thrombocythemia,12 idiopathic myelofibrosis.The JAK2V617F mutation was not detected in 76 chronic myelocytic leukemia patients,whose bcr/abl fusion is negative.But in 24 of 32(75%) patients with polycythemia vera,6 of 20(30%) with essential thrombocythemia,6 of 12(50%) with idiopathic myelofibrosis.
Conclusion:The difference of mutation frequencies among polycythemia vera,essential thrombocythemia and idiopathic myelofibrosis patients was of no statistical significance(P＞0.05).The JAK2V617F may be a characteristic molecular event in polycythemia vera,essential thrombocythemia and idiopathic myelofibrosis patients.It may serve as an important molecμlar marker for the diagnosis and classification of these three diseases.
方法:1.常规提取全基因组DNA、骨髓组织总RNA提取。
2.用逆转录聚合酶链反应(RT-PCR)对140例MPD病人进行bcr/abl融合基因检测。
3.用等位基因特异性聚合酶链反应(AS-PCR)检测140例MPD病人JAK2基因的V617F点突变,对有突变的病例进行测序验证。
4.分析比较青海省与国内部分其它地区MPD病人JAK2基因的V617F点突变表达率的差异。
结果:1、140例典型MPD病人bcr/abl融合基因检测:76例CML病人均为bcr/abl阳性的病人,其余64例典型MPD病人为bcr/abl阴性,其中32例为真性红细胞增多症,20例为原发性血小板增多症,12例为特发性骨髓纤维化病人。
2、AS-PCR检测JAK2V617F突变发生率:bcr/abl阳性的76例慢性粒细胞白血病病人JAK2V617F突变均为阴性;64例bcr/abl阴性MPD病人中的突变率是:32例真性红细胞增多症病人中24例阳性(占75%),20例原发性血小板增多症病人中6例阳性(占30%),12例特发性骨髓纤维化病人中6例阳性(占50%)。
3、青海省与国内部分其它地区MPD病人JAK2基因的V617F点突变率相比存在差别。
结论:JAK2V617F点突变在青海省bcr/abl融合基因阴性的骨髓增殖性疾病的发病中起了重要作用,PV、ET、IMF病人JAK2V617F突变阳性率依次为75%(24/32)、30%(6/20)和50%(6/12),与国内其它部分地区相比存在差别。JAK2V617F可能为PV、ET、IMF的特征性分子事件,结合其临床病理特征可作为一个独立的分子指标用于此三类疾病的临床诊断。
Objective:To detect the JAK2V617F mutation in myeloproliferative disorders(MPD) of Qinghai province by allele-specific primer polymerase chain reaction and evaluate the significance of JAK2V617F in diagnosis and therapy of MPD.
Methods:1.Genomic DNA and total RNA were extracted from white blood cells and marrow respectively by route way.2.The bcr/abl fusion gene was detected in 140 Patients with MPD by the reverse transcription Polymerase chain reaction(PT-PCR).3.The JAK2V617F mutation was detected in 70 patients with MPD by allele-specific polymerase chain reaction(AS-PCR) and the positive cases with the mutation were checked by sequencing.
Results:Between 140 Patients with MPD,the bcr/abl fusion gene is detected in 76 chronic myelocytic leukemia patients,others is negative,including 32 polycythemia vera,20 essential thrombocythemia,12 idiopathic myelofibrosis.The JAK2V617F mutation was not detected in 76 chronic myelocytic leukemia patients,whose bcr/abl fusion is negative.But in 24 of 32(75%) patients with polycythemia vera,6 of 20(30%) with essential thrombocythemia,6 of 12(50%) with idiopathic myelofibrosis.
Conclusion:The difference of mutation frequencies among polycythemia vera,essential thrombocythemia and idiopathic myelofibrosis patients was of no statistical significance(P＞0.05).The JAK2V617F may be a characteristic molecular event in polycythemia vera,essential thrombocythemia and idiopathic myelofibrosis patients.It may serve as an important molecμlar marker for the diagnosis and classification of these three diseases.
引文
1.Vardiman JW,Hams NL,Brunning RD.World Health Organization (WHO) classification of the myeloid neoplasms.Blood,2002,100: 2292-2302.
2.Nelson ME,Steensma DP.JAK2V617F in myeloid disorders: What do we know now,and where are we headed? Leuk Lymphoma,2006,47: 177-194.
3.Cools J,DeAngelo DJ,Gotlib J,et al.A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hyereosinophilic syndrome.N Engl J Med,2003,348: 1201-1214.
4.Levine RL,Loriaux M,Brian J,et al.The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia,but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia.Blood,2005,106: 3377-3379.
5.Karsten Grote,Maren Luchtefeld,Bernhard Schieffer.JANUS under stress-Role of JAK/STAT signaling pathway in vascular diseases.Vascular Pharmacology,2005,43: 357-363.
6.Manning G,Whyte DB,Martinez R,et al.The protein kinase complement of the human genome.Science,2002,298: 1912-1934.
7.Onishi M,Nosaka T.Cytokine receptors: structures and signal transduction.International Reviews of Immunology,1998,16:617-634.
8.Huang LI,Constantinescu SN,Lodish HE.The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor.Molecular Cell,2001,8: 1327-1338.
9.Kunihiro Yamaoka,Pipsa Saharinen,Marko Pesu,et al.The Janus kinases (Jaks).Genome Biology 2004,5:253-259;
10.Jason S,Rawlings,Kristin M,et al.The JAK/STAT signling pathway.J Cell Sci,2004,117: 1281-1283.
11.Robert A Ortmann,Tammy Cheng,Roberta Visconti,et al.Janus kinases and signal transducers and activators of transcription:their roles in cytokine signaling,development and immunoregulation.Arthritis Research 2000,2:16-32;
12.Leonard WJ,O'Shea JJ.Jaks and STATs:biological implications.Annual Review Immunology .1998,16:293-322;
13.Kralovics R,Passamonti F,Buser AS,et al.A gain-of-function mutation of JAK2 in myeloproliferative disorders.N Engl J Med,2005,352:1779-1790.
14.Levine RL,Wadleigh M.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,essential thrombocythemia,and myeloid metaplasia with myelofibrosis.Cancer Cell,2005,7: 387-397.
15.James C,Ugo V,le Couedic JP,et al.A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.Nature,2005,434:1144-1148.
16.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferarive disorders.Lancet,2005,365:1054-1061.
17.Steensma DP,Dewald GW,Lasho TL,et al.The JAK2 V617F activating tyrosine kinase mutation is an Infrequent event in both'a typical'myeloprolilbrative disorders and myelodysplastic syndrome.Blood,2005,106:1207-1209.
18.McLornan DP,Percy MJ,Jones AV,et al.Chronic neutrophilic leukemia with an associated V617F JAK2 tyrosine kinase mutation.Haematologica,2005,90:1696-1697.
19.Jones AV,Kreil S,Zoi K,et al.Widespread occurrence of the JAK2V617F mutation in chronic myeloproliferative disorders.Blood,2005,106:2162-2168.
20.Jelinek J,Oki Y,Gharibyan V,et al.JAK2 mutation 1849G-T is rare in acute leukemias but can be found in CMML,Philadelphia chromosomenegative CML,and megakaryocytic leukemia.Blood,2005,106:3370-3373.
21.乔娜,杨林花,刘秀娥,康建民,闫文林,张睿娟,张媛,郭志萍,张青 夏.JAK2V617F点突变在原发性血小板增多症中的初步研究.中国药物与临床,2008,8(4):294-295.
22.宫本法,王建祥.JAK2V617F突变与真性红细胞增多症发生.国际输血及血液学杂志.2006,29(5):403-406.
23.Zhao R,Xing S,Li Z,et al.Identification of an acquired JAK2 mutation in polycythemia vera.J Biol Chem.2005,280:22788-22792.
24.AnnZeune,Francesca Pedini,Michele signor,et al.Increased death receptor resistance and FLIP short expression in polycythemia vera erythriod precursor cells.BLOOD,May,2006,107(9):3495-3502.
25.Walz C,Crowley BJ,Hudon HE,et al.Activated JAK2 with the V617F mutation promotes G1/S-phase transition.J Biol Chem,2006,281(26):8177-8183
26.CamPbell PJ,Scott LM,Buck G,et al.Definition of subtypes of essential thrombcythemia and relation to polycythemia vera based on JAK2V617F mutation status:prospective study.Lancet.2005,366:1945-1953.
27.Funakoshi-Tago M,Pelletier S,Matsuda T,tal.Receptors specific downregulation of cytokine signaling by autophosorylation in the FERM domain of jak2.EMBOJ 2006,25:4763-4772.
28.Lu X,Levine R,Wernig G,et al.ExPression of a homodimeric type 1cytokine Receptor is required for JAK2V617F-mediated transformation.Proz Natl Acad Sci USA 2005,102:18962.
29.Lacout C,Pisani DF,Tulliez M,et al.JAK2V617F expression in murine hematopoietic cells lead to MPD mimicking human PV with secondary myelofibrosis.Blood,2006,108(5):1652-1660896.
30.Wenrig G,ercher T,Okaber,et al.Expression of JAK2617F cause a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model.Blood,2006,107:4274-4281.
31.Vannucchi AM,Bianchi L,Paoletti F et al.A pathobiologic pathway linking thrombopoietin,GATA-l,and TGF-beta 1 in the development of myelofibrosis.
32.Mesa RA,Tefferi A,Lasho TS,janus kinase2(V617F) mutation status,signal transducer and activator of transcription-3 phosphorylation and impaired neutrophil apoptosis in myelofibrosis with myloid metaplasia.Leukemia.2006 20(10):1800-8.
33.Pikaman Y,Lee BH,Mercher E,et al.MPLW515L is a noval somatic activating mutation in myelofibrosis with myeloid metaplasia.PLOS Med 2006,3:e270.
34.Steensma DP,De wald GW,Lasho TL,et al.The JAK2V617F activating tyrosine kinase mutation is an infrequent event in both"atypical"myeloproliferative disorders and the myelodysplastic syndrome.Blood.2005,106:1207-1209.
35.Jelinek J,Oki Y,Gharibyan V,et al.JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML,Philadelphia-chromosome negative CML and megakaryocytic leukemia.Blood.2005,106:3370-3373.
36.申徐良,陈方平,魏武,张梅香,史文芝,秦小琪,徐洪亮.等位基因特异性-PCR联合限制性内切酶消化法检测JAK2V617F突变.长治医学院学报.2008,6(22)1:6-8.
37.王冬梅,郭慧梅,王菊美,孙国峰,郭晓玲,潘崚.JA K2V617F点突变与BCR2ABL阴性骨髓增殖性疾病临床关系研究.临床荟萃.2008,23(5):317-321.
2.Nelson ME,Steensma DP.JAK2V617F in myeloid disorders: What do we know now,and where are we headed? Leuk Lymphoma,2006,47: 177-194.
3.Cools J,DeAngelo DJ,Gotlib J,et al.A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hyereosinophilic syndrome.N Engl J Med,2003,348: 1201-1214.
4.Levine RL,Loriaux M,Brian J,et al.The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia,but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia.Blood,2005,106: 3377-3379.
5.Karsten Grote,Maren Luchtefeld,Bernhard Schieffer.JANUS under stress-Role of JAK/STAT signaling pathway in vascular diseases.Vascular Pharmacology,2005,43: 357-363.
6.Manning G,Whyte DB,Martinez R,et al.The protein kinase complement of the human genome.Science,2002,298: 1912-1934.
7.Onishi M,Nosaka T.Cytokine receptors: structures and signal transduction.International Reviews of Immunology,1998,16:617-634.
8.Huang LI,Constantinescu SN,Lodish HE.The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor.Molecular Cell,2001,8: 1327-1338.
9.Kunihiro Yamaoka,Pipsa Saharinen,Marko Pesu,et al.The Janus kinases (Jaks).Genome Biology 2004,5:253-259;
10.Jason S,Rawlings,Kristin M,et al.The JAK/STAT signling pathway.J Cell Sci,2004,117: 1281-1283.
11.Robert A Ortmann,Tammy Cheng,Roberta Visconti,et al.Janus kinases and signal transducers and activators of transcription:their roles in cytokine signaling,development and immunoregulation.Arthritis Research 2000,2:16-32;
12.Leonard WJ,O'Shea JJ.Jaks and STATs:biological implications.Annual Review Immunology .1998,16:293-322;
13.Kralovics R,Passamonti F,Buser AS,et al.A gain-of-function mutation of JAK2 in myeloproliferative disorders.N Engl J Med,2005,352:1779-1790.
14.Levine RL,Wadleigh M.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,essential thrombocythemia,and myeloid metaplasia with myelofibrosis.Cancer Cell,2005,7: 387-397.
15.James C,Ugo V,le Couedic JP,et al.A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.Nature,2005,434:1144-1148.
16.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferarive disorders.Lancet,2005,365:1054-1061.
17.Steensma DP,Dewald GW,Lasho TL,et al.The JAK2 V617F activating tyrosine kinase mutation is an Infrequent event in both'a typical'myeloprolilbrative disorders and myelodysplastic syndrome.Blood,2005,106:1207-1209.
18.McLornan DP,Percy MJ,Jones AV,et al.Chronic neutrophilic leukemia with an associated V617F JAK2 tyrosine kinase mutation.Haematologica,2005,90:1696-1697.
19.Jones AV,Kreil S,Zoi K,et al.Widespread occurrence of the JAK2V617F mutation in chronic myeloproliferative disorders.Blood,2005,106:2162-2168.
20.Jelinek J,Oki Y,Gharibyan V,et al.JAK2 mutation 1849G-T is rare in acute leukemias but can be found in CMML,Philadelphia chromosomenegative CML,and megakaryocytic leukemia.Blood,2005,106:3370-3373.
21.乔娜,杨林花,刘秀娥,康建民,闫文林,张睿娟,张媛,郭志萍,张青 夏.JAK2V617F点突变在原发性血小板增多症中的初步研究.中国药物与临床,2008,8(4):294-295.
22.宫本法,王建祥.JAK2V617F突变与真性红细胞增多症发生.国际输血及血液学杂志.2006,29(5):403-406.
23.Zhao R,Xing S,Li Z,et al.Identification of an acquired JAK2 mutation in polycythemia vera.J Biol Chem.2005,280:22788-22792.
24.AnnZeune,Francesca Pedini,Michele signor,et al.Increased death receptor resistance and FLIP short expression in polycythemia vera erythriod precursor cells.BLOOD,May,2006,107(9):3495-3502.
25.Walz C,Crowley BJ,Hudon HE,et al.Activated JAK2 with the V617F mutation promotes G1/S-phase transition.J Biol Chem,2006,281(26):8177-8183
26.CamPbell PJ,Scott LM,Buck G,et al.Definition of subtypes of essential thrombcythemia and relation to polycythemia vera based on JAK2V617F mutation status:prospective study.Lancet.2005,366:1945-1953.
27.Funakoshi-Tago M,Pelletier S,Matsuda T,tal.Receptors specific downregulation of cytokine signaling by autophosorylation in the FERM domain of jak2.EMBOJ 2006,25:4763-4772.
28.Lu X,Levine R,Wernig G,et al.ExPression of a homodimeric type 1cytokine Receptor is required for JAK2V617F-mediated transformation.Proz Natl Acad Sci USA 2005,102:18962.
29.Lacout C,Pisani DF,Tulliez M,et al.JAK2V617F expression in murine hematopoietic cells lead to MPD mimicking human PV with secondary myelofibrosis.Blood,2006,108(5):1652-1660896.
30.Wenrig G,ercher T,Okaber,et al.Expression of JAK2617F cause a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model.Blood,2006,107:4274-4281.
31.Vannucchi AM,Bianchi L,Paoletti F et al.A pathobiologic pathway linking thrombopoietin,GATA-l,and TGF-beta 1 in the development of myelofibrosis.
32.Mesa RA,Tefferi A,Lasho TS,janus kinase2(V617F) mutation status,signal transducer and activator of transcription-3 phosphorylation and impaired neutrophil apoptosis in myelofibrosis with myloid metaplasia.Leukemia.2006 20(10):1800-8.
33.Pikaman Y,Lee BH,Mercher E,et al.MPLW515L is a noval somatic activating mutation in myelofibrosis with myeloid metaplasia.PLOS Med 2006,3:e270.
34.Steensma DP,De wald GW,Lasho TL,et al.The JAK2V617F activating tyrosine kinase mutation is an infrequent event in both"atypical"myeloproliferative disorders and the myelodysplastic syndrome.Blood.2005,106:1207-1209.
35.Jelinek J,Oki Y,Gharibyan V,et al.JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML,Philadelphia-chromosome negative CML and megakaryocytic leukemia.Blood.2005,106:3370-3373.
36.申徐良,陈方平,魏武,张梅香,史文芝,秦小琪,徐洪亮.等位基因特异性-PCR联合限制性内切酶消化法检测JAK2V617F突变.长治医学院学报.2008,6(22)1:6-8.
37.王冬梅,郭慧梅,王菊美,孙国峰,郭晓玲,潘崚.JA K2V617F点突变与BCR2ABL阴性骨髓增殖性疾病临床关系研究.临床荟萃.2008,23(5):317-321.