Detection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high-resolution melt analysis

Background: Several methods have been established to detect the JAK2 V617F mutation, a frequent event involved in the pathogenesis of myeloproliferative neoplasms (MPNs). High‐resolution melt (HRM) analysis is a newly established technique without the requirement of any gel‐based post‐PCR handling. Methods: An asymmetric PCR with unlabeled specific probe was developed and combined to HRM analysis o screen for JAK2 V617F mutation. Results: Heterozygous mutation was easily distinguished from homozygous JAK2 for the obvious shape change. Homozygous JAK2 mutant can be also well separated from wild‐type JAK2 in the presence of internal temperature calibrators. The easily recognizable and maximal sensitivity of HRM analysis was 5% for the detection of JAK2 V617F mutation, higher than 25% of direct sequencing. In the test of blind screening of 223 samples (111 Ph? MPNs, 60 Ph+ chronic myeloid leukemia, and 52 acute myeloid leukemia), JAK2 V617F mutations were found in 78 (70%) patients with MPNs, but in none with chronic and acute myeloid leukemia. HRM analysis of all cases was fully concordant with the results of PCR‐RFLP and direct sequencing. Conclusions: The HRM method with unlabeled probe could be used as convenient, sensitive and reliable diagnostic test for detection of JAK2 V617F mutation. J. Clin. Lab. Anal. 25:300–304, 2011. © 2011 Wiley‐Liss, Inc.     

高分辨率熔解曲线法检测JAK2基因V617F突变及其应用

摘要:目的：探讨实时定量PCR方法和高分辨率熔解曲线法（HRM）在检测JAK2基因V617F突变中的应用。 方法：以JAK2基因V617F发生纯合突变的人红白血病细胞株（HEL）及不含JAK2基因V617F突变的人白血病细胞株（HL60）为阳性对照和阴性对照,优化HRM法检测条件,分别以优化的HRM法和直接测序法对63例临床疑似骨髓增殖性肿瘤（MPN）患者DNA标本进行JAK2基因V617F突变检测,评价2种检测方法结果的一致性。 结果：HRM法可检出系列混合样本中5%的突变型等位基因突变,且重复性较高。以测序法为金标准,HRM法的敏感性和特异性均为100％,两种方法结果一致。 结论：HRM法能够在单一闭管体系中实现对JAK2基因V617F突变的检测,具有较高的敏感性和特异性,可用于JAK2基因V617F突变的临床检测。     

高分辨熔解曲线法检测骨髓增殖性肿瘤患者 JAK2V617F 基因突变简

本研究探讨高分辨率熔解曲线（High resolution melting,HRM）法检测骨髓增殖性肿瘤（MPN）患者JAK2V617F基因突变的可行性.随机抽取29份2008年1月至2011年1月确诊为MPN患者的骨髓标本,应用HRM法检测JAK2V617F基因突变情况,并与等位基因特异性聚合酶链反应（AS-PCR）和测序法的检测结果比较分析.结果表明,经HRM法检测,在29份MPN患者骨髓标本中检出JAK2 V617F突变阳性11例,突变率为37.9％;与基因测序法比较,结果完全一致,符合率100％.而AS-PCR法与测序法比较,Kappa=0.179,P=0.316,一致性强度较差.结论：HRM方法具有简便、快速、特异性高等优点,可作为临床JAK2V617F基因突变的优选方法.     

Frequency and characteristics of the JAK2 V617F mutation in 23 cerebral venous sinus thrombosis patients with thrombocytosis

ObjectiveTo analyse the frequency and characteristics of the Janus kinase 2 (JAK2) V617F mutation in patients with cerebral venous sinus thrombosis (CVST) with thrombocytosis.MethodsThe study enrolled CVST patients with thrombocytosis that had undergone JAK2 V617F mutation detection to determine the frequency of the JAK2 V617F mutation in this cohort. Correlations between patient demographics, whole blood cell counts, targeted sequencing results and JAK2 V617F mutation status were determined.ResultsA total of 23 patients were enrolled in the study: 11 (47.8%) with the JAK2 V617F mutation and 12 (52.2%) without the JAK2 V617F mutation. The mean platelet count was significantly higher in patients with the JAK2 V617F mutation than in patients without the mutation (478.1 ± 107.4 × 10⁹/l versus 374.4 ± 54.1 × 10⁹/l, respectively). There were no significant differences in age, sex, white blood cell count or haemoglobin level between the two groups. Other than single nucleotide polymorphisms, no hot-spot mutations associated with myeloid tumours other than the JAK2 V617F mutation were detected in four CVST patients that underwent targeted sequencing.ConclusionThe JAK2 V617F mutation was frequently detected in CVST patients with thrombocytosis and it was associated with higher platelet counts.     

Uses and abuses of JAK2 and MPL mutation tests in myeloproliferative neoplasms a paper from the 2010 William Beaumont hospital symposium on molecular pathology

JAK2V617F is sufficiently prevalent in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) to be useful as a clonal marker. JAK2V617F mutation screening is indicated for the evaluation of erythrocytosis, thrombocytosis, splanchnic vein thrombosis, and otherwise unexplained BCR-ABL1-negative granulocytosis. However, the mutation does not provide additional value in the presence of unequivocal morphologic diagnosis, and its presence does not necessarily distinguish one MPN from another or provide useful prognostic information. In general, quantitative cell-based JAK2V617F mutation assays are preferred because the additional information obtained on mutant allele burden enhances diagnostic certainty and facilitates monitoring of response to treatment. JAK2 exon 12 mutation screening is indicated only in the presence of JAK2V617F-negative erythrocytosis that is associated with a subnormal serum erythropoietin level. MPL mutations are neither frequent nor specific enough to warrant their routine use for MPN diagnosis, but they may be useful in resolving specific diagnostic problems. The practice of en bloc screening for JAK2V617F, JAK2 exon 12, and MPL mutations is scientifically irrational and economically irresponsible.     

135例骨髓增殖性肿瘤患者JAK2基因突变的定量研究

目的 研究JAK2基因突变在骨髓增殖性肿瘤(MPN)中的发生率和突变类型.并对突变转录本水平进行定量分析,初步探讨其临床意义.方法 采用突变序列扩增系统PCR(ARMS-PCR)法检测JAK2突变的发生率及其突变类型;采用毛细管电泳法定量分析JAK2突变转录本水平.结果 135例MPN患者共检出95例JAK2V617F阳性,总阳性率为70.4%;真性红细胞增多症(PV)患者JAK2V617F突变发生率为97.4%,原发性血小板增多症(ET)为59.6%,3例特发性骨髓纤维化(IMF)中2例阳性;95例突变患者中纯合突变36例,杂合突变59例,其中PV患者纯合突变发生率为47.3%,高于ET患者的18.1%(P<0.05).纯合突变者其JAK2V617F突变转录本水平高于杂合突变者(P<0.05),杂合型PV患者的JAK2V617F突变转录本水平高于杂合型ET患者(P<0.05);JAK2V617F突变高表达组(转录本水平为70%～100%)的平均年龄高于低表达组(转录本水平为47.3%～70.0%),且两者年龄均较JAK2V617F阴性组为高(P<0.05);年龄<60岁者其JAK2V617F转录本水平低于≥60岁者(P<0.001);PV患者中,JAK2V617F高表达组其白细胞计数高于低表达组(P<0.001),ET患者中,JAK2V617F高表达组其白细胞计数高于低表达组(P<0.05),且二者均较阴性组为高(P<0.05),血红蛋白水平在高表达组与低表达组间差异无统计学意义(P>0.05),但二者均较阴性组为高(P<0.05).101例患者进行了染色体检查,未发现核型异常与JAK2V617F突变之间存在相关性.结论 ARMS-PCR可作为检测JAK2V617F突变较灵敏的方法 ,结合毛细管电泳可用于此突变的定量分析以及临床MPN的诊断和微量残留病的检测.     

JAK2V617F mutation screening as part of the hypercoagulable work-up in the absence of splanchnic venous thrombosis or overt myeloproliferative neoplasm: assessment of value in a series of 664 consecutive patients

The JAK2V617F mutation is recurrent in polycythemia vera and essential thrombocythemia, which are myeloproliferative neoplasms (MPNs) frequently associated with arterial and/or venous thromboembolism. More recently, the JAK2V617F mutation has been identified as a surrogate marker for subclinical or "occult" clonal myeloproliferation in patients with splanchnic venous thrombosis. However, information is limited regarding JAK2V617F-associated thrombosis outside the splanchnic district in patients without overt MPN. To address this issue, we retrospectively studied a consecutive series of 664 such patients who experienced thrombotic events characteristic of an MPN (500 with venous thromboembolism, 136 with stroke, and 28 with myocardial infarction at a young age). The JAK2V617F mutation was detected in only 6 (<1.0%) patients (5 with recurrent venous thromboembolism and 1 with stroke), and the mutant allele burden was low in all instances (range, 2.2%-7.5%). None of these 6 patients developed either overt MPN or recurrent thrombosis after a median follow-up of 40 months. We conclude that the prevalence of the JAK2V617F mutation in patients with nonsplanchnic venous thrombosis in the absence of MPN is too low to warrant mutation screening as part of the hypercoagulable work-up. Our study also suggests that the natural history of a JAK2V617F-positive "occult" MPN might be different from that of a typical MPN.     

骨髓增殖性肿瘤患者活化蛋白C抵抗和凝血功能异常研究

目的 研究骨髓增殖性肿瘤(MPN)患者活化的蛋白C抵抗(APC-R)、凝血、抗凝因子异常及与JAK2V617F突变负荷的相关性.方法 应用在有、无APC存在情况下的部分激活的凝血活酶时间(APTT)比值,即APC敏感比值(APCsr)评价APC-R的情况.检测43例真性红细胞增多症(PV)和32例原发性血小板增多症(ET)患者血浆蛋白C(PC)、蛋白S(PS)、凝血因子Ⅱ(FⅡ)、FV、FⅧ、中性粒细胞表面CD11b表达情况.应用实时定量PCR法检测PV患者JAK2V617F基因突变负荷情况并研究APCsr与凝血异常和JAK2V617F基因突变负荷的相关性.结果 与正常人相比,MPN患者中性粒细胞表面CD11b表达明显升高,APCsr、PS及FV明显减低.且APSsr在血栓栓塞和JAK2V617F基因突变负荷大于75%的PV患者中减低更为明显.MPN患者APCsr与PS表达水平呈正相关,与JAK2V617F突变负荷呈负相关.结论 MPN患者存在中性粒细胞活化增高,PS及FV表达水平减低.伴血栓栓塞的MPN患者存在APCsr减低,即一定程度的APC-R.MPN患者APCsr与PS及JAK2V617F突变负荷存在相关性.

Abstract：

Objective To study the correlation of activated proteinC ( APC ) resistance, coagulation factors and inhibitors abnormality and JAK2V617F mutation burden in patients with myeloproliferative neoplasms(MPN). Methods The APC resistance was defined as the ratio of activated partial thromboplastin time(APTT) in the presence and absence of APC, i. e. APC sensitivity ratio(APCsr). Plasma protein C ( PC ), protein S ( PS), prothrombin ( F Ⅱ ), factor V ( F Ⅴ ), factor Ⅷ levels and CD11b expression on neu trophils were measured. The percentage of mutated JAK2V617F allele ( V617F% ) was evaluated by real time polymerase chain reaction(qRT-PCR). Results Expression of CD11b on neutrophils was significantly elevated in MPN patients compared with that of the control group. APCsr, PS and F Ⅴ levels were reduced in patients with MPN. The APCsr level was decreased mainly in patients with thrombosis and JAK2V617F mutant burden higher than 75%. APCsr was not only positively correlated with PS levels but also inversely correlated with JAK2V617F allele burden in JAK2V617F mutant gene carriers. Conclusion The neutrophil was activated and PS,FⅤ level were reduced in MPN patients. The APCsr level was decreased and the occurrence of relatively acquired APC resistance was found in MPN patients with thrombosis. The APCsr is correlated with the PS level and JAK2V617F mutational furden.     

采用荧光定量PCR检测JAK2基因V617F突变

目的 建立荧光定量PCR检测JAK2基因V617F突变的方法,评估JAK2 V617F突变在诊断骨髓增殖性疾病和白血病中的临床意义.方法 选取71例慢性粒细胞性白血病(CML)、22例原发性血小板增多症(ET)、11例原发性骨髓纤维化(PMF)、9例真性红细胞增多症(PV)、7例嗜酸粒细胞增多症患者,分别采用荧光定量PCR、突变特异性扩增系统(ARMS)对JAK2 V617F突变进行检测,并采用基因测序对结果进行验证.将具有JAK2 V617F纯合子突变的人类红白血病细胞株(HEL)DNA作为阳性对照,比较荧光定量PCR和ARMS对JAK2基因V617F突变的检测敏感度.结果 采用荧光定量PCR检测,野生型的熔解温度(Tm)峰出现于(75.0±0.2)℃处,突变型的Tm峰出现于(76.6±0.2)℃处.JAK2基因V617F突变在PV、ET、PMF等骨髓增殖性疾病中的检出率分别为8例(88.9%)、12例(54.5%)、7例(63.6%),但在71例CML中只检出1例(1.4%).荧光定量PCR与ARMS的结果符合率为100%,结果经过测序证实.使用荧光定量PER法可在每106个正常自细胞中检出低至102个HEL细胞,而使用ARMS方法时要在每106个正常白细胞中HEL细胞达到104个时,方可检测出,前者比后者方法灵敏100倍.结论 成功地建立了荧光定量PCR检测JAK2基因V617F突变的方法,比ARMS更灵敏、简便,适合临床使用.JAK2基因V617F突变在骨髓增殖性疾病中有较高的检出率,可作为骨髓增殖性疾病特异性诊断指标.     

骨髓增殖性肿瘤患者外周血细胞中JAK2V617F突变和p-STAT5蛋白表达及其与临床特征的相关性

本研究旨在探讨骨髓增殖性肿瘤（MPN）患者外周血细胞中JAK2V617F突变和p-STAT5蛋白表达的情况及二者与疾病临床特征之间的相关性,为临床实践及靶向治疗研究提供理论依据。选择山东大学附属省立医院血液科确诊的45名BCR-ABL阴性的MPN患者及15例健康成年人为研究对象,提取外周血单个核细胞的DNA及蛋白质,分别采用实时荧光定量聚合酶链反应和免疫蛋白印迹法定量检测研究对象的朋K2V617F突变比例及p-STAT5蛋白表达量,并结合收集的临床病例资料进行相关性分析。结果表明,MPN患者JAK2V617F突变总体阳性率为73．3％（33／45）,其中真性红细胞增多症（PV）的以K2V617F突变阳性率为83．3％（20／24）,原发性血小板增多症（ET）的阳性率为68．8％（11／16）,特发性骨髓纤维化（IMF）患者的阳性率为40．0％（2／5）;PV、ET、IMF患者的J14K2V617F突变比例分别为0．472±0．245,0．216±0．162,0．435±0．239;p-STAT5蛋白表达灰度值分别为1．396±0．758,0．760±0．623,0．792±0．612;JAK2V617F突变负荷与p-STAT5蛋白表达灰度值呈线性相关（P〈0．05）;在PV患者中,JAK2V617F突变负荷越高,白细胞计数、血红蛋白水平及红细胞压积越高,血小板水平越低;ET患者中JAK2V617F突变负荷越高者,年龄越大,白细胞计数、血红蛋白水平及红细胞压积越高,与血小板水平无明显相关性;IMF患者中JAK2V617F突变负荷越高者白细胞、血小板计数、血红蛋白水平及红细胞压积均较低。JAK2V617F突变阳性者更易发生脾肿大、出血及血栓事件。结论：JAK2V617F突变在BCR-ABL阴性的MPN患者中阳性率较高,突变负荷越高者其下游p-STAT5蛋白的表达量越大,发生脾肿大、血栓事件的几率越高。     

JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology

In early 2005, several groups of investigators studying myeloid malignancies described a novel somatic point mutation (V617F) in the conserved autoinhibitory pseudokinase domain of the Janus kinase 2 (JAK2) protein, which plays an important role in normal hematopoietic growth factor signaling. The V617F mutation is present in blood and marrow from a large proportion of patients with classic BCR/ABL-negative chronic myeloproliferative disorders and of a few patients with other clonal hematological diseases such as myelodysplastic syndrome, atypical myeloproliferative disorders, and acute myeloid leukemia. The JAK2 V617F mutation causes constitutive activation of the kinase, with deregulated intracellular signaling that mimics continuous hematopoietic growth factor stimulation. Within 7 months of the first electronic publication describing this new mutation, clinical molecular diagnostic laboratories in the United States and Europe began offering JAK2 mutation testing on a fee-for-service basis. Here, I review the various techniques used by research groups and clinical laboratories to detect the genetic mutation underlying JAK2 V617F, including fluorescent dye chemistry sequencing, allele-specific polymerase chain reaction (PCR), real-time PCR, DNA-melting curve analysis, pyrosequencing, and others. I also discuss diagnostic sensitivity, performance, and other practical concerns relevant to the clinical laboratorian in addition to the potential diagnostic utility of JAK2 mutation tests.     

骨髓增殖性疾病患者JAK2基因V617F突变频率、磷酸化JAK/STAT蛋白表达水平及临床意义

目的 探讨145例骨髓增殖件疾病患者JAK2基因V617F突变率;并分析JAK2基因V617F突变阳性患者的临床特点及意义.方法 应用PCR产物直接测序和等位基因特异性PCR技术检测145例骨髓增殖性疾病患者JAK2基因V617F突变.并应用Western blot方法测定JAK2基因V617F突变阳性患者JAK2蛋白、磷酸化JAK2蛋白及磷酸化STAT5蛋白表达;对JAK2基因V617F突变阳性与突变阴性的骨髓增殖性疾病患者临床资料进行比较,评价JAK2基因V617F突变阳性的临床意义.结果 ①本组病例真性红细胞增多症(PV)、特发性骨髓纤维化(ET)、原发性血小板增多症(IMF)患者JAK2基因V617F突变率分别为92%(64例中59例)、58%(43例中25例)和50%(38例中19例),等位基因特异性PCR检测较PCR产物直接测序有更高的JAK2基因V617F检出率[PV84%(64例中53例)、MIF 44%(43例中19例)、ET 39%(38例中15例)].②JAK2基因V617F突变阳性患者外周血单个核细胞磷酸化JAK2及磷酸化STAT5蛋白表达较突变阴性者明显增高(P<0.05).③JAK2基因V617F突变阳性骨髓增殖性疾病患者发病平均年龄偏大;平均白细胞计数高于突变阴性患者;血小板计数小于突变阴性患者;脾脏较突变阴性患者小;JAK2基因V617F突变阳性PV、ET和IMF患者血栓性事什发生率分别为17%、32%和16%;而突变阴性PV、ET和IMF患者血栓性事件发生率分别为0、16%和5%.结论 骨髓增殖性疾病患者有较高的JAK2基因V617F突变发生率,且JAK2基因V617F突变阳性患者易发生血栓事件.     

Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases

The discovery of the activating V617F mutation in the JAK2 tyrosine kinase in a high proportion of patients with Ph- chronic myeloproliferative diseases (CMPD) represents a diagnostic breakthrough for these disorders. Trephine bone marrow biopsy is an essential part of the diagnostic workup of CMPD and represents a valuable archival source of DNA. Therefore, we studied 152 paraffin-embedded trephines with CMPD and related disorders for the presence of the V617F mutation, using both allele-specific polymerase chain reaction (PCR) and nested PCR with subsequent digestion with BsaXI. Only 6 of 152 (4%) samples were not evaluable because of poor DNA quality. The V617F mutation was detected in 27 of 28 (96%) cases of polycythemia vera, 17 of 23 (74%) cases of essential thrombocythemia, 28 of 45 (62%) cases of chronic idiopathic myelofibrosis, six of eight (75%) cases of CMPD unclassified, and two of four (50%) cases of myelodysplastic/myeloproliferative syndrome. Ph+ chronic myelogenous leukemia (four cases), reactive (secondary) erythrocytosis (14 cases), and thrombocytosis (one case) as well as normal controls (19 cases) all lacked the V617F mutation. Based on results of BsaXI digestion and sequencing, 24 of 54 (44%) evaluable V617F+ cases were considered homozygously mutated. Thus, detection of the V617F JAK2 mutation is feasible in paraffin-embedded trephine biopsies and represents a major advance in the diagnostic evaluation of CMPD.     

Coexistence of breakpoint cluster region-Abelson1 rearrangement and Janus kinase 2 V617F mutation in chronic myeloid leukemia: A case report

BACKGROUND The Janus kinase 2(JAK2) V617 F mutation is common in patients with breakpoint cluster region-Abelson1(BCR-ABL1)-negative myeloproliferative neoplasms,including polycythemia vera, essential thrombocythemia and primary myelofibrosis, but is rarely detected in BCR-ABL1-positive chronic myeloid leukemia(CML) patients. Here, we report a CML patient with both a BCR-ABL1 rearrangement and JAK2 V617 F mutation.CASE SUMMARY A 45-year-old Chinese woman was admitted to our department with a history of significant thrombocytosis for 20 d. Color Doppler ultrasound examination showed mild splenomegaly. Bone marrow aspiration revealed a karyotype of 46,XX, t(9;22)(q34;q11.2) in 20/20 metaphases by cytogenetic analysis,rearrangement of BCR-ABL1(32.31%) by fluorescent polymerase chain reaction(PCR) and mutation of JAK2 V617 F(10%) by PCR and Sanger DNA sequencing.The patient was diagnosed with CML and JAK2 V617 F mutation. Following treatment with imatinib for 3 mo, the patient had an optimal response and BCRABL1(IS) was 0.143%, while the mutation rate of JAK2 V617 F rose to 15%.CONCLUSION Emphasis should be placed on the detection of JAK2 mutation when CML is diagnosed to distinguish JAK2 mutation-positive CML and formulate treatment strategies.     

骨髓增殖性肿瘤中JAK2V617F突变和TNF-α的表达及两者的相关性研究

本研究旨在探讨骨髓增殖性肿瘤（MPN）患者外周血单个核细胞中JAK2V617F突变和TNF-α因子的表达情况及两者之间的相关性,为临床诊断及治疗提供理论依据.选取山东大学附属省立医院血液科确诊的62名BCR-ABL阴性的MPN患者（35例ET患者,19例PV患者,8例MF患者）及15例健康成年人为研究对象,将患者及正常对照者的外周血单个核细胞分为两部分,分别提取细胞中的DNA及mRNA,并将mRNA反转录为cDNA.采用SYBR Green Ⅰ实时荧光定量PCR分别检测JAK2V617F突变及TNF-α的表达量,并分析两者的相关性.结果表明,MPN患者JAK2V617F基因突变总体阳性率为64.52％ （40/62）,其中ET患者的JAK2V617F突变阳性率为54.28％ （19/35）,PV患者的JAK2V617F突变阳性率为94.74％（18/19）,MF患者的JAK2V617F突变阳性率为37.50％（3/8）;ET、PV、MF患者的JAK2V617F突变比例分别为0.838±0.419、4.417±0.658、2.746±2.009;ET、PV、MF患者TNF-α的表达分别是正常对照的1.7、7.0、8.2倍（P＜0.05）;且JAK2V617F突变负荷与TNF-α的表达水平呈线性相关（Pearson r =0.610,R2 =0.372,P=0.005）.结论：TNF-α在BCR-ABL阴性的MPN发病机制中有重要作用,在ET、PV、MF中表达升高且表达量不同,且与JAK2V617F突变呈线性相关.     

98例原发性血小板增多症Jak2基因突变的定量分析及其临床意义

为研究JAK2V617F在原发性血小板增多症（ET）患者中的发生率和突变类型,定量分析突变转录本水平并初步探讨其临床意义,采用ARMS（amplification—refractorymutationsequencing）PCR法检测JAK2V617F突变的发生率及其突变类型,采用毛细管电泳法定量分析JAK2V617F突变转录本水平。结果显示：98例ET患者中59例JAK2V617F为阳性,其中纯合突变18例。纯合突变及杂合突变患者的平均年龄均较野生型为高（P值均〈0．05）;18例纯合突变患者的白细胞计数高于41例杂合突变者,且二者均高于野生型（P值均〈0．05）。毛细管电泳定量分析显示,纯合突变患者JAK2V617F突变转录本水平为（89．9±6．7）％,高于杂合突变患者的（57．1±6．7）％（P〈0．05）;年龄小于60岁患者的JAK2V617F突变转录本水平为（62．3±16．5）％,低于年龄大于60岁患者的JAK2V617F突变转录本水平为（72．4±15．8）％（P〈0．05）。JAK2V617F阳性组中血栓的发生率高于阴性组,其中纯合突变者高于杂合突变者,发生血栓者的JAK2V617FF转录本水平高于无血栓者（P值均〈0．05）。结论：JAK2V617F阳性与阴性ET患者有着不同的临床特征,分析其突变类型及检测其转录本水平对明确疾病状态、观察疾病进展及指导治疗有重要意义。     

TaqMan探针结合COLD-PCR原理定量检测JAK2 V617F突变率研究

目的建立在外周血基因组DNA中定量检测JAK2V617F突变率的方法。方法应用TaqMan/MGB探针技术,结合较低变性温度下复合扩增聚合酶链反应(COLD-PCR)基因扩增原理,采用Real-Time PCR分析系统,通过JAK2V617F突变率和其循环阈值(Ct值)的标准曲线,建立TaqMan-COLD-PCR测定方法,并对9例骨髓增殖性疾病(MPD)患者外周血基因组DNA中JAK2V617F突变率进行检测,探讨其应用价值。结果建立的TaqMan-COLD-PCR方法检测JAK2V617F突变率的检测下限为0.1%,100%-0.1%的JAK2V617F突变率与其测定的Ct值间呈明显的线性关系。对9例MPD患者外周血基因组DNA中JAK2V617F突变率检测显示,其中6例患者的JAK2V617F突变阳性,突变率范围为18.5%-50.9%,经测序确认全部符合。结论 TaqMan-COLD-PCR方法可高灵敏度、定量检测外周血中JAK2V617F突变率。该方法将明显提高MPD疾病的诊断灵敏度,并有助于疾病治疗过程中治疗效果的监测。     

SOCS超甲基化与经典型慢性骨髓增殖性肿瘤的研究

目的 探讨细胞因子信号转导抑制蛋白(suppressor of cytokine signaling,SOCS)基因超甲基化在经典型骨髓增殖性肿瘤(MPD)中的临床作用及其机制.方法 采用甲基化特异性PCR方法检测SOCS1、2、3基因CpG岛甲基化发生情况,直接测序法检测100例MPD患者JAK2V617F突变情况,用实时定量PCR方法检测SOCS1、2、3的mRNA表达情况.结果 100例MPD患者中有27例(27％)存在SOCS1基因超甲基化,9例(9％)存在SOCS2基因超甲基化,34例(34％)存在SOCS3基因超甲基化.在100例MPN患者中,64例(64％) JAK2V617F突变阳性.MPD患者中SOCS1和SOCS3基因超甲基化组与未甲基化组相比,其SOCSl和SOCS3基因mRNA表达量明显减少(P＜0.05);SOCS1和SOCS3基因JAK2V617F突变组与野生型组相比,其SOCS1和SOCS3基因mRNA表达量明显减少(P＜0.05).结论 MPD患者中存在JAK2V617F突变及SOCS基因超甲基化,且JAK2V617F突变和SOCS基因甲基化导致SOCS mRNA表达水平降低,SOCS超甲基化和JAK2V617F突变可改变JAK-STAT信号通路等的转录活性而最终影响疾病的发展,这些改变可能代表了一种潜在的治疗方向.     

多重位点特异性PCR检测骨髓增殖性疾病五种JAK2基因突变

目的 建立一种同时检测多种JAK2基因突变的多重位点特异性PCR方法,并探讨其对骨髓增殖性疾病(MPD)的临床应用价值.方法 建立同时枪测JAK2 V617F突变和JAK2 exon12中K539L(包括2种基因突变)、N542-E543del和E543-D544del突变的多重位点特异性PCR方法.对115例MPD患者进行检测,包括61例真性红细胞增多症(PV)患者、43例原发性血小板增多症(ET)患者和11例原发性骨髓纤维化(MF)患者.结果 建立的PCR方法可以同时检测上述5种JAK2基因突变,可以检测出1%的JAK2 V617F突变型等位基因,对其他几种突变可检测出0.1%的突变型等位基因.在61例PV患者中,检测出JAK2 V617F突变56例、JAK2 exon12突变3例;在43例ET患者中,检测出JAK2 V617F突变27例,未检测到JAK2 exon12突变;在11例MF患者中,检测到JAK2 V617F突变6例,未检测到JAK2 V617F突变.3例JAK2 exon12突变的患者临床表现为血红蛋白增多,内源性红系集落生成阳性,但白细胞和血小板增多不明显,不伴脾肿大.结论 多重位点特异性PCR方法检测灵敏度高,可联合检测5种JAK2基因突变,能有效提高突变检出率.