JAK2V617F突变分型和相对定量研究——附123例慢性骨髓增殖性疾病检测结果分析

目的 研究JAK2V617F突变在慢性骨髓增殖性疾病(CMPD)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.方法 对2003年1月至2008年5月苏州大学附属第一医院就诊的CMPD患者采用突变特异性扩增系统(ARMS)PCR方法检测JAK2V617F的发生率及其突变类型;采用毛细管电泳方法定量分析JAK2V617F突变转录本水平.结果 123例CMPD患者共检出90例JAK2V617F阳性,其中35例真性红细胞增多症(PV)患者中JAK2V617F阳性率100%,85例原发性血小板增多症(ET)患者中JAK2V617F阳性率62.4%,低于PV患者,差异有统计学意义(P<0.05);3例慢性骨髓纤维化(IMF)患者中JAK2V617F阳性率66.7%.90例JAK2V617F突变患者中共检出纯合突变35例,其中PV患者17例(17/35),占48.6%,ET患者17例(17/85),占20.0%,低于PV患者,差异有统计学意义(P<0.05),IMF患者1例;毛细管电泳定量分析显示,纯合型突变患者JAK2V617F突变转录本水平较杂合型突变患者低,差异有统计学意义(P<0.05);杂合型PV患者JAK2V617F突变转录本水平高于杂和型ET患者,差异有统计学意义(P<0.05).93例患者进行了染色体检查,6例有核型异常,但未发现特异性染色体改变.结论 ARMS-PCR可作为JAK2V617F突变较准确的检测方法,结合毛细管电泳可用于此突变的定量分析以及临床CMPD的诊断.     

JAK2V617F点突变在BCR-ABL阴性骨髓增殖性疾病中的意义

目的研究JAK2V617F点突变在诊断BCR—ABL融合基因阴性的骨髓增殖性疾病（MPD）患者中的意义。方法选择51例BCR—ABL阴性MPD患者,采用等位基因特异PCR法检测各组患者JAK2V617F的突变情况。结果51例BCR—ABL融合基因阴性的MPD患者中,34例JAK2V617F突变阳性,其中原发性血小板增多症（ET）18例（69．23％）,真性红细胞增多症（PV）16例（66．67％）,特发性骨髓纤维化（IMF）1例为阴性;PV与ET患者相比更容易发生肝脾肿大、脑梗死、静脉血栓形成、高尿酸血症等并发症（P〈0．05）。ET患者中,JAK2V617F突变阳性组白细胞计数较阴性组高（P〈0．05）。ET患者中,JAK2V617F突变阳性组白细胞计数较阴性组高（P〈0．05）,ET和PV患者中JAK2V617F突变阳性组都比阴性组更容易发生上述并发症等（P均〈0．05）。结论JAK2V617F点突变在BCR—ABL融合基因阴性MPD中有较高的发生率,具有明确的诊断学意义;ET及PV患者中此突变阳性者更易发生血栓形成等并发症。     

慢性骨髓增殖性疾病JAK2 V617F点突变与ETS2mRNA表达的关系

目的探讨慢性骨髓增殖性疾病（cMPDS）中JAK2V617F点突变的发生率、ETS2mRNA表达情况及其相关性。方法选择BCR／ABL阴性的cMPDs患者62例、慢性粒细胞性白血病（CML）20例、急性白血病（AL）10例和健康志愿者15例为研究对象;用RT—PCR技术检测其ETS2mRNA的表达水平,用AS-PCR筛选各组JAK2V617F点突变,以基因测序验证突变结果,分析ETS2mRNA表达水平在cMPDsJAK2V617F点突变阳性组与阴性组之间的差异。结果62例cMPDs患者中,44例患者检测到JAK2V617F点突变,其余18例及CML、AL、健康志愿者中未检测到该突变。ETS2mRNA表达水平在cMPDs组、CML组和AL组均显著高于健康志愿者。cMPDs组JAK2V617F点突变阳性病例ETS2mRNA表达水平明显高于突变阴性病例,差别有统计学意义（P=0．022）。结论大部分cMPDs患者存在JAK2V617F点突变;JAK2V617F点突变阳性cMPDs患者ETS2mRNA的表达水平高于阴性者。     

141例慢性骨髓增生性疾病中JAK2基因V617F点突变的研究

目的：研究分子遗传学标记JAK2V617F突变在141例慢性骨髓增殖性疾病（cMPD）患者中发生率、突变类型及其与血液学特征的相关性。方法：应用等位基因特异性PCR（AS-PCR）技术进行JAK2V617F点突变的检测,采用PCR-限制性片断长度多态性（RFLP）方法检测JAK2V617F（＋）者的突变类型,然后DNA测序验证。结合临床资料进一步分析。结果：经AS-PCR发现141例cMPD中有73例为JAK2V617F突变阳性样本,其中21例BCR-ABL阳性的CML中未发现突变样本。JAK2V617F（＋）的病例中19例为纯和型突变,54例为杂和型突变。经酶切及测序验证结果相符。结合临床资料进行分析,JAK2V617F突变阳性患者外周血细胞分析中白细胞数和血红蛋白的量较野生型高,而血小板计数较野生型差异无统计学意义。JAK2V617F纯和型突变患者外周血细胞计数中血红蛋白的量较杂合型高,而血小板计数较杂合型低,白细胞计数差异无统计学意义。结论：JAK2V617F点突变可以作为BCR—ABL阴性的MPD诊断的一个参考依据。在突变类型中纯和型突变较为少见。突变类型对血液学特征有一定的影响。     

应用扩增阻碍突变系统检测骨髓增殖性疾病JA砬V617F基因突变的研究

目的探讨JAK2V617F基因突变在恶性血液病中的表达及临床意义。方法运用扩增阻碍突变系统对133例恶性血液病患者进行JAK2V617F基因突变检测分析。结果在133例样本中,60例BCR—ABL融合基因阴性的慢性骨髓增殖性疾病患者存在JAK2V617F基因突变（61．7％）,其余血液病未发现该点突变,差异有统计学意义（P〈0．05）。其中真性红细胞增多症、特发性血小板增多症、原发性骨髓纤维化和嗜酸细胞增多症的阳性率分别为89．5％、53．6％、44．4％和25％。检测JAK2V617F基因突变的灵敏性为2％。结论JAK2V617F基因突变在BCR—ABL融合基因阴性的慢性骨髓增殖性疾病患者中广泛表达,尤其以真性红细胞增多症患者发生率最高。扩增阻碍突变系统检测JAK2V617F基因突变具有很好的特异性和灵敏度,可作为临床筛查的检测方法。     

JAK2基因突变与骨髓增殖性疾病

真性红细胞增多症(PV)、特发性骨髓纤维化(IMF)、原发性血小板增多症(ET)和慢性粒细胞性白血病(CML)是一组临床与骨髓组织学特征相似的疾病,即骨髓增殖性疾病(MPD);但除了CML外,PV、IMF和ET等MPD的发病机制至今尚不十分明了。最近有研究发现染色体9p获得性杂合性丧失(LOH)与JAK2基因突变有密切关系,JAK2基因突变体(JAK2-V617F)在PV、ET和IMF中发生率分别高达65%～97%、23%～57%和35%～57%;并与对细胞因子的高敏感性、PRV1的高表达和内源性红系集落(EEC)的形成等也有密切的联系等等。因此,作为胞浆非受体性酪氨酸激酶是发生于造血干细胞水平克隆性异常的标志,JAK2在多种造血生长因子受体信号转导中发挥关键作用,因而可作为MPD的候选基因。     

骨髓增殖性疾病137例患者JAK2基因突变的研究

目的探讨信号分子JAK2基因突变——JAK2V617F在骨髓增殖性疾病（MPD）患者中的发生情况及诊断价值。方法采用等位基因特异性PCR方法及序列测定,检测137例MPD患者骨髓及外周血单个核细胞JAK2基因第12号外显子,研究JAK2V617F的发生情况,并结合细胞遗传学及分子生物学等检查结果分析其诊断价值。结果137例MPD患者共检出90例JAK2V617F阳性,总阳性率65．7％;其中真性红细胞增多症（PV）57例,阳性率73．7％（42／57）;原发性血小板增多症（ET）68例,阳性率58．8％（40／68）;特发性骨髓纤维化（IMF）12例,阳性率66．7％（8／12）;PV及ET患者突变型平均年龄高于野生型（P〈0．05）。115例进行过染色体检查,其中7例存在核型异常,突变型5例,阳性率为71．4％（5／7）;染色体核型正常者108例,突变型74例,阳性率68．5％;两组差异无统计学意义。结论MPD患者中JAK2V617F发生率较高,其发生与年龄相关,突变型平均年龄高于野生型。     

原发性血小板增多症JAK2V617F点突变研究

2005年3月起多个研究组报道在原发性血小板增多症（ET）等bcr／abl阴性慢性骨髓增殖性疾病（CMPD）中存在JAK2基因点突变即JAK2V617F，为该病的发病机制、诊治带来了革命性进展。由于检测方法不同，检测到ET中该突变发生率为23％～57％。我们收集了45例ET患者的外周或骨髓血DNA，来探讨JAK2V617F阳性率及临床意义。     

慢性骨髓增殖性疾病并发肺动脉高压4例临床分析

目的 加强对慢性骨髓增殖性疾病(CMPD)患者合并肺动脉高压的认识,以期临床早期诊断和治疗,改善预后.方法 回顾性分析4例慢性骨髓增殖性疾病并发肺动脉高压患者的临床及实验室检查资料、治疗及转归.结果 4例患者诊断慢性骨髓增殖性疾病时年龄为44 ～ 72岁,诊断原发病到发现肺动脉高压1～26年.4例患者临床均表现为乏力、活动耐力下降,脾脏明显肿大,均有不同程度的贫血,3例患者有血小板减少.诊断肺动脉高压时肺动脉压力为58～83mm Hg,三尖瓣反流速率3.5 ～4.3 m/s.4例患者诊断CMPD后均接受正规治疗,随诊过程中发现肺动脉高压,3例患者于诊断肺动脉高压1～2年内死亡.结论 肺动脉高压是慢性骨髓增殖性疾病患者常见的心血管并发症,目前治疗方法有限,慢性骨髓增殖性疾病患者病程中出现肺动脉高压提示预后不良.     

常见骨髓增殖性疾病骨髓病理学特征

2008年WHO造血与淋巴组织肿瘤分类（简称2008年WHO分类）将慢性骨髓增殖性疾病（chronic myeloproliferative disease,CMPD）称为骨髓增殖性肿瘤（myeloproliferative neoplasm,MPN）,并在2001年WHo分类的CMPD基础上增加了肥大细胞增生症（mastocytosis,MC）,共8种亚型。慢性中性粒细胞白血病（chronic neutrophilic leukemia,CNL）、慢性嗜酸粒细胞白血病,非特指型（CEL,NOS）、MC和MPN,无法分类（MPN,U）均相对少见,故本文仅对其中常见的真性红细胞增多症（polycythemia vera,PV）、原发性骨髓纤维化（idiopathic myelfibrosis,IMF）和原发性血小板增多症（essential thrombocythemia,ET）的骨髓活检病理诊断与鉴别做一介绍。     

Prevalence of overt myeloproliferative neoplasms and JAK2 V617F mutation in Korean patients with splanchnic vein thrombosis

Introduction: Myeloproliferative neoplasm (MPN) is known to be a major risk factor of splanchnic vein thrombosis (SVT). Recent studies revealed that a significant proportion of patients with SVT harbor a gain‐of‐function mutation in the JAK2 gene (V617F) with or without MPN. In this study, the authors investigated the prevalence of MPN and JAK2 V617F mutation in Korean patients with SVT. Methods: The study subjects were 26 patients diagnosed as having SVT based on Doppler ultrasound and/or computed tomography from January 2008 to January 2010 (16 men and 10 women; mean age 44 years, range 15–75 years). The clinical and laboratory data were reviewed. The JAK2 V617F mutation was detected by allele‐specific polymerase chain reaction and direct sequencing analyses using DNA from peripheral blood leukocytes. Results: Among 26 study patients, 12 had portal vein thrombosis, five had hepatic vein thrombosis, three had mesenteric, and two had splenic vein thrombosis. Four patients had thrombosis involving more than one splanchnic vein. Two patients (7.7%; 2/26) had overt MPN (essential thrombocythemia). JAK2 V617F was detected in three patients (11.5%) including the two patients with overt MPN. Thus, the prevalence of the JAK2 V617F mutation in patients with SVT but without overt MPN was 4.2% (1/24). Conclusion: The prevalence of overt MPN and that of JAK2 V617F were lower in Korean patients with SVT than in previous reports. Data from a larger number of patients with long‐term follow‐up are needed to reveal the clinical relevancy of JAK2 V617F in Korean patients with SVT.     

The JAK2V617F mutation and thrombosis

Since the discovery of the JAK2^V617F mutation, the clinical and pathological consequences of this acquired defect have been extensively investigated to determine whether its presence characterises a distinct subgroup of myeloproliferative disorders (MPD). MPD management remains highly dependent on the patient’s thrombotic risk. Whether the presence of the JAK2^V617F mutation modifies the thrombotic risk is currently contentious, although there is increasing clinical evidence to suggest that the mutation may be variably associated with thrombosis. These observations are further supported by laboratory parameters which suggest that the JAK2^V617F mutation may confer increased activation of leucocytes and platelets in MPD. The role of screening for the JAK2^V617F mutation in patients presenting with thrombosis without overt MPD is unclear, but appears justified in cases of idiopathic splanchnic vein thrombosis.     

The JAK2 V617F somatic mutation, mortality and cancer risk in the general population

JAK2 V617F is present in the majority of patients with myeloproliferative cancer; however, its prevalence and clinical significance in the general population is unknown. We screened for presence of the mutation in 10,507 participants from the Copenhagen City Heart Study with up to 17.6 years of follow up. Prevalence of the mutation was 0.2% (n=18). All 18 mutation positives died during follow up corresponding to a multifactorially adjusted hazard ratio for early death of 3.0 (95%CI:1.9-4.9). Corresponding hazard ratios for men versus women and 1-year age increases were 1.4 (1.1-1.9) and 1.1 (1.1-1.1). Multifactorially adjusted hazard ratios for any cancer, hematologic cancer and myeloproliferative cancer were 3.7 (1.7-8.0), 58 (13-261) and 161 (12-2,197), respectively. Corresponding hazard ratios were 1.2 (0.8-2.0), 2.3 (0.2-25), 1.3 (0.3-5.4) for men versus women, and 1.0 (1.0-1.1), 1.1 (0.9-1.2), 0.9 (0.8-1.1) for 1-year age increases. In the general population, JAK2 V617F is associated with increased morbidity and mortality, although only present in 18 of 10,507 (0.2%).     

Relationship between JAK2V617F mutation,allele burden and coagulation function in Ph-negative myeloproliferative neoplasms

Objectives: Our aim was to explore the relationship between JAK2V617F mutation allele burden and hematological parameters especially in coagulation function in Chinese population.

Methods: This study included 133 Ph-negative myeloproliferative neoplasms (MPNs) patients between 2013 and 2016. All the clinical and experimental data of patients were collected at the time of the diagnosis without any prior treatment, including blood parameters, coagulation function, splenomegaly, vascular events and chromosome karyotype. PCR and qPCR were used to detect JAK2V617F mutation and JAK2V617F mutation allele burden.

Results: In polycythemia vera patients, a positive correlation between the allele burden of JAK2V617F mutation and PLT counts was found; in essential thrombocythemia (ET) patients, WBC counts, RBC counts, HB, and HCT were higher in mutated patients than in wild-type patients. Furthermore, PT-INR was higher in ET and PMF mutated patients. In addition, a positive correlation between the allele burden of JAK2V617F mutation and activated partial thromboplastin time (APTT) was observed in JAK2V617F mutated ET patients.

Conclusions: Higher hematologic parameters including counts of WBC, RBC, and PLT are closely associated with JAK2V617F mutation and its burden in Ph-negative MPNs; importantly, PT-INR, APTT are also related to JAK2V617F mutation and allele burden. Thus, our data indicate that JAK2V617F mutation allele burden might not only represent the burden of MPN but also alter the coagulation function.     

Sex differences in the JAK2V617F allele burden in chronic myeloproliferative disorders

Background

The JAK2^V617F allele burden is a variable measure, determined by the frequency of mitotic recombination events and the expansion of JAK2^V617F clones. Since variability in the JAK2^V617F allele burden is partly responsible for the distinct phenotypes seen in the myeloproliferative disorders, the objective of this study was to identify modifiers of the allele burden.

Design and Methods

Blood samples were obtained between May 2005 and January 2009 from 272 patients with essential thrombocytosis, polycythemia vera, and myelofibrosis. The JAK2^V617F allele burden was measured by an allele-specific quantitative polymerase chain reaction using DNA from purified neutrophils. Repeated measures, on average 2 years apart, were available for 104 patients.

Results

Sex, age at diagnosis, and disease duration all independently influenced the JAK2^V617F allele burden. When considering all patients with myeloproliferative disorders, women had significantly lower allele burdens than men (P=0.04). In those patients with repeated measures, the increase in allele burden per year between the first and second evaluations was significantly less in females than in males. Among those who experienced disease evolution, females were 4.5 times more likely to have evolution from essential thrombocytosis to polycythemia vera, but 0.23 times as likely to have evolution from essential thrombocytosis to myelofibrosis.

Conclusions

Sex is an independent factor accounting for variability in the JAK2^V617F allele burden. We speculate that lower allele burdens in females reflect a lower frequency of mitotic recombination events in females than in males, and should be considered when evaluating the relationship of allele burden to disease phenotype and also in evaluating responses to JAK2^V617F-inhibitors. Because sex may influence genotype and/or clonal expansion, underpinning the variability in JAK2^V617F allele burden, it will be important to explore factors that determine susceptibility to mitotic recombination events.     

Clinical significance of V617F mutation of the JAK2 gene in patients with chronic myeloproliferative disorders

Abstract

Objective: To determine the prevalence of JAK2 V617F mutation and its clinical correlation in patients with chronic myeloproliferative disorders (CMD): polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF).

Materials and methods: Detection of JAK2 V617F mutation by allele specific-PCR.

Results: One hundred and three patients with CMD were included in the study. JAK2 V617F distribution was PV 40/45 (89%), ET 30/43 (69%), and IMF 7/15 (47%). In PV and ET patients only, 18 had thrombosis at diagnosis and 12 during follow-up (these were microvascular: 11, venous: 7 and arterial: 12); of these 28/70 (40%) were JAK2pos versus 2/18 (11%) JAK2neg; P=0·02. In a median of 4 years, two patients with PV JAK2pos evolved to myelofibrosis and one patient with PV presented in leukemic transformation (JAK2pos before and after transformation); six patients died: four patients with IMF and two patients with PV.

Conclusions: We found an association between JAK2 V617F and thrombotic events in patients with PV and ET.