急性髓系白血病患者表观遗传学修饰基因突变的分析

目的探讨急性髓系白血病(AML)患者表观遗传学修饰基因突变(EMMs)的携带率及其临床特征。方法选取2011年5月至2021年2月于连云港市第一人民医院初诊的172例AML患者为研究对象。应用二代测序技术检测42种髓系基因的变异情况, 回顾性分析EMMs患者的临床及分子学特征, 以及去甲基药物(HMAs)对其生存的影响。结果在172例初诊的AML患者中, 71例(41.28%)携带EMMs [EMMs(+)], 变异基因分别为TET2(14.53%, 25/172)、DNMT3A(11.63%, 20/172)、ASXL1(9.30%, 16/172)、IDH2(9.30%, 16/172)、IDH1(8.14%, 14/172)、EZH2(0.58%, 1/172)。EMMs(+)患者的外周血血红蛋白低于EMMs(-)患者(72 g/Lvs. 88 g/L, Z=-1.985, P<0.05)。老年AML患者的EMMs(+)携带率显著高于青年患者[71.11%(32/45)vs. 30.70%(39/127), χ2 = 22.38, P<0.001]。EMMs与NPM1...     

血管免疫母T细胞淋巴瘤患者RHOA、 TET2和DNMT3A基因突变率及临床特征分析

目的探讨血管免疫母T细胞淋巴瘤(AITL)患者RHOA、TET家族羟化酶2(TET2)和甲基化转移酶3A(DNMT3A)基因突变率及临床特征分析。方法应用免疫组化法检测AITL患者组织中RHOA、TET2和DNMT3A的表达。采用多重PCR扩增和测序方法分析RHOA、TET2和DNMT3A突变主要基因型。结果免疫组化结果可知,RHOA阳性表达为68.33%(41/60)、TET2阳性表达为61.67%(37/60)和DNMT3A阳性表达为70.00%(42/60)。RHOA、TET2和DNMT3A阳性表达与血管免疫母T评分明显相关,差异具有统计学意义(P<0.05)。RHOA、TET2和DNMT3A阳性表达与AITL患者性别及年龄不具有突变与血管免疫母T细胞淋巴瘤TNM分期、LDH含量、有无B症状及IPI评分明显相关,差异具有统计学意义(P<0.05)。结论 RHOA、TET2和DNMT3A突变可以作为诊断AITL的分子标志物,RHOA、TET2和DNMT3A突变可能参与调节AITL发生发展。     

一例Bainbridge-Ropers综合征患儿的基因变异分析

目的分析1例Bainbridge-Ropers综合征患儿及其父母ASXL3基因的变异类型,明确其可能的遗传学病因,为其临床诊断和遗传咨询提供依据。方法采集患儿和父母的外周血样,应用全外显子测序的方法对患儿基因进行检测,并采用Sanger测序的方法对变异位点进行验证。结果全外显子测序结果显示,患儿的ASXL3基因c.3106C>T杂合变异(p.Arg1036*),父母外周血ASXL3基因该位点未检测到c.3106C>T变异。该变异为无义变异,会产生截短蛋白。根据美国医学遗传学与基因组学学会变异分类标准与指南,c.3106C>T变异为致病性变异(PVS1+PS2+PP4)。结论ASXL3基因c.3106C>T杂合变异可能为患儿的致病原因,通过ASXL3基因变异分析,可以为其临床诊断和遗传咨询提供依据。     

不明原因复发性流产患者绒毛组织中DNA低甲基化及机制

目的探究不明原因复发性流产(URSA)患者绒毛组织全基因组甲基化水平和甲基化相关酶基因表达情况。方法选取URSA患者31例(URSA组)及同期正常早孕放弃妊娠行人工流产的妇女30例(对照组)。取绒毛组织,采用ELISA测定绒毛组织中总DNA的甲基化水平,实时定量PCR检测DNA甲基化转移酶1(DNMT1)、 DNMT3a、 DNMT3b、去甲基化酶10-11转位酶1(TET1)、 TET2、 TET3的mRNA水平, Western blot法检测DNMT1、 DNMT2、 DNMT3、 TET1、 TET2、 TET3蛋白水平。免疫化学染色检测绒毛组织中DNMT1、 DNMT3a、 DNMT3b、 TET1、 TET2、 TET3的表达和分布。结果与对照组相比, URSA组的全基因组甲基化水平显著降低, URSA组绒毛组织中DNMT1、 DNMT3b的mRNA和蛋白水平显著降低, TET1、 TET2的mRNA和蛋白水平显著增加。结论 URSA妇女的流产绒毛组织呈现出低甲基化水平的状态,可能与DNMT1、 DNMT3b上调,TET1、 TET2下调有关。     

一例伴有t（2；8；21）易位急性粒单细胞白血病患者实验研究

t(8;21)(q22;q22)是急性髓系细胞白血病(AML)N2比较常见一类易位,形成的AML1-ETO融合基因影响髓系细胞的生长和分化.该易位92%见于AML-M2患者,7%见于AML-M4患者,其中只有3%发生复杂变异体.近来我们发现一例少见t(8;21)隐藏复杂变异体M4儿童患者,化疗后骨髓虽然获得完全缓解,但出现持续性骨髓坏死.     

血清铁蛋白与骨髓增生异常综合征危险度分层和预后的关系分析

目的 探讨血清铁蛋白水平在骨髓增生异常综合征(MDS)患者中危险度分层及预后评估的临床意义.方法 对41例初诊MDS患者和30名健康对照者采用电化学发光免疫法测量血清铁蛋白(SF),观察MDS患者SF水平及与国际预后评分系统(IPSS)的关系.对MDS患者进行随访,根据铁蛋白水平和IPSS评分的数值绘制ROC曲线,并将患者分组分为相对低危组(SF＜ 577ng/mL)和相对高危组(SF≥577ng/mL),比较两组间白血病转化率和生存时间.结果 MDS组患者铁蛋白水平明显高于健康对照组(P＜0.05),相对低危组与相对高危组之间铁蛋白水平差异有统计学意义(P＜0.05),随访的39位患者中共12人转化为急性白血病,两组转化率分别为10.5％和45.5％,向白血病转化的中位时间分别为18.5(4 ～46)个月和30.0(6 ～48)个月,差异有统计学意义(P＜0.05),两组间生存时间差异无统计学意义(P＞0.05).结论 铁蛋白在MDS患者中高于正常水平,初诊未输血的患者存在铁过载,高危类型MDS患者的SF水平较高,SF水平与其不良预后有一定的相关性.     

Evi1和MDS1基因

Evil基因在小鼠逆转录病毒诱发的急性髓系白血病(AML)中,是病毒常见的插入位点,在AML发病中起重要作用。MDS基因位于Evil上游,通过选择性剪接,可形成MDS1-Evil基因。本文就Evi-1和MDS1基因的研究近况作一综述。     

血管免疫母细胞性T细胞淋巴瘤75例基因突变特征分析

目的探讨血管免疫母细胞性T细胞淋巴瘤(angioimmunoblastic T-cell lymphoma, AITL)的基因突变特征。方法收集2021年6月至2023年6月经上海交通大学医学院附属瑞金医院病理科诊断为AITL, 并用石蜡包埋组织或新鲜组织进行靶向测序检测的75例病例资料, 分析AITL患者的突变基因分布和突变类型。结果在75例AITL标本中均检测出基因突变, 共检测到492个变异位点, 分别位于84个基因列表中的74个基因。在≥10%的AITL病例中检测到的突变基因依次是TET2(89.3%)、RHOA(57.3%)、IDH2(37.3%)、DNMT3A(36.0%)、KMT2C(21.3%)、PLCG1(12.0%)和KDM6B(10.7%)。TET2和RHOA、TET2和IDH2、RHOA和IDH2基因突变之间均具有显著的共现关系(P<0.05), TET2和KDM6B基因突变之间具有显著的互斥关系(P<0.05)。结论应用二代测序技术可揭示AITL基因突变的独特特征, 为AITL的诊断和靶向治疗提供参考依据。     

芪莲益髓清毒颗粒对低危骨髓增生异常综合征的疗效及其对TET2和IDH1基因的作用

目的:观察芪莲益髓清毒颗粒对低危骨髓增生异常综合征(MDS)患者的疗效,及其对甲基胞嘧啶双加氧酶2(TET2)和异柠檬酸脱氢酶1(IDH1)基因表达及甲基化的影响。方法:选取2018年01月～2019年10月就诊于山东中医药大学附属医院血液病科的IPSS-R低危MDS患者60例,采用随机数字表法将患者分为观察组和对照组,每组各30例。观察组患者给予芪莲益髓清毒颗粒及最佳的支持治疗,对照组患者仅给予最佳的支持治疗,分析两组患者治疗后的临床疗效、中医证候积分、骨髓单个核细胞TET2和IDH1基因的表达水平及甲基化情况。采用液相色谱质谱联用技术筛选芪莲益髓清毒颗粒中相关化学组分。结果:治疗后观察组治疗的总有效率显著高于对照组。观察组中医证候积分的改善优于对照组(P0.05)。观察组和对照组治疗前TET2和IDH1 mRNA的表达水平较正常对照组降低(P0.05);观察组治疗后的TET2和IDH1 mRNA表达水平均显著高于治疗前(P0.05)。对照组治疗后的TET2 mRNA显著高于治疗前(P0.05),而IDH1 mRNA表达水平则与治疗前无显著差异。治疗后,观察组TET2基因甲基化阳性率由30%下降为6.7%,IDH1基因甲基化阳性率由16.7%下降为6.7%。芪莲益髓清毒颗粒中鉴定出了与甲基化相关的21种化合物成分。结论:芪莲益髓清毒颗粒可提高支持治疗对MDS的效果,其机制可能与调控TET2和IDH1基因有关,其中包括去甲基化作用。芪莲益髓清毒颗粒治疗MDS是有效的。     

Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms

Since the discovery of the JAK2V617F tyrosine kinase-activating mutation several genes have been found mutated in nonchronic myeloid leukemia (CML) myeloproliferative neoplasms (MPNs), which mainly comprise three subtypes of "classic" MPNs; polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We searched for mutations in ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 genes in 149 non-CML MPNs, including 127 "classic" MPNs cases. JAK2 was mutated in 100% PV, 66% ET and 68% MF. We found a high incidence of ASXL1 mutation in MF patients (20%) and a low incidence in PV (7%) and ET (4%) patients. Mutations in the other genes were rare (CBL, DNMT3A, IDH2, MPL, SF3B1, SUZ12, NF1) or absent (IDH1).     

Myeloid malignancies with isolated 7q deletion can be further characterized by their accompanying molecular mutations

Deletions in the long arm of chromosome 7 (del(7q)) are recurrent cytogenetic aberrations in myeloid neoplasms. They occur either isolated or as part of a complex karyotype and are associated with unfavorable prognosis in certain disease entities. We performed detailed cytogenetic analysis, molecular analysis, and array comparative genomic hybridization in a cohort of 81 patients with a variety of myeloid malignancies and del(7q) as sole chromosomal alteration. In 70% (57/81) of patients, we identified a commonly deleted region (size: 18 Mb) involving the genomic region 101 912.442 (7q22.1)‐119 608.824 (7q31.31). Furthermore, in 80 patients, we analyzed 17 genes commonly mutated in myeloid neoplasms and identified high mutation frequencies in ASXL1 34% (27/80), TET2 33% (26/80), RUNX1 25% (20/80), DNMT3A 25% (20/80), while TP53 was rarely affected (5%, 4/80). ASXL1 and TET2 showed similar mutation frequencies across all analyzed entities while RUNX1, CBL, and JAK2 were specifically mutated in patients with acute myeloid leukemia (AML), chronic myelomonocytic leukemia, and myeloproliferative neoplasms, respectively. We detected a significantly higher frequency of RUNX1 (42% vs 13%, P = .0001) and ASXL1 (32% vs 14%, P = .008) mutations in AML patients with del(7q) compared to other AML patients in the Medical Research Council unfavorable risk group (n = 464), indicating a cooperative leukemogenic potential. Our data provide further insight into the pathomechanism of this cytogenetic subgroup.     

Clinical features and biological implications of different U2AF1 mutation types in myelodysplastic syndromes

U2AF1 mutations (U2AF1MT) occur commonly in myelodysplastic syndromes (MDS) without ring sideroblasts. The aim of this study was to investigate the clinical and biological implications of different U2AF1 mutation types in MDS. We performed targeted gene sequencing in a cohort of 511 MDS patients. Eighty‐six patients (17%) were found to have U2AF1MT, which occurred more common in younger patients (P = .001) and represented ancestral lesions in a substantial proportion (71%) of cases. ASXL1MT and isolated +8 were significantly enriched in U2AF1MT‐positive cases, whereas TP53MT, SF3B1MT, and complex karyotypes were inversely associated with U2AF1MT. U2AF^S34 subjects were enriched for isolated +8 and were inversely associated with complex karyotypes. U2AF1MT was significantly associated with anemia, thrombocytopenia, and poor survival in both lower‐risk and higher‐risk MDS. U2AF1^S34 subjects had more frequently platelet levels of <50 × 10⁹/L (P = .043) and U2AF1^Q157/U2AF1^R156 subjects had more frequently hemoglobin concentrations at <80 g/L (P = .008) and more often overt fibrosis (P = .049). In conclusion, our study indicates that U2AF1MT is one of the earliest genetic events in MDS patients and that different types of U2AF1MT have distinct clinical and biological characteristics.     

Updates in molecular genetics of acute myeloid leukemia

Acute myeloid leukemia (AML) is a type of cancer caused by aggressive neoplastic proliferations of immature myeloid cells that is fatal if untreated. AML accounts for 1.0% of all new cancer cases in the United States, with a 5-year relative survival rate of 30.5%. Once defined primarily morphologically, advances in next generational sequencing have expanded the role of molecular genetics in categorizing the disease. As such, both the World Health Organization Classification of Haematopoietic Neoplasms and The International Consensus Classification System now define a variety of AML subsets based on mutations in driver genes such as NPM1, CEBPA, TP53, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2. This article provides an overview of some of the genetic mutations associated with AML and compares how the new classification systems incorporate molecular genetics into the definition of AML.     

A Personalized Prediction Model for Outcomes after Allogeneic Hematopoietic Cell Transplant in Patients with Myelodysplastic Syndromes

Allogeneic hematopoietic stem cell transplantation (HCT) remains the only potentially curative option for myelodysplastic syndromes (MDS). Mortality after HCT is high, with deaths related to relapse or transplant-related complications. Thus, identifying patients who may or may not benefit from HCT is clinically important. We identified 1514 patients with MDS enrolled in the Center for International Blood and Marrow Transplant Research Registry and had their peripheral blood samples sequenced for the presence of 129 commonly mutated genes in myeloid malignancies. A random survival forest algorithm was used to build the model, and the accuracy of the proposed model was assessed by concordance index. The median age of the entire cohort was 59 years. The most commonly mutated genes were ASXL1(20%), TP53 (19%), DNMT3A (15%), and TET2 (12%). The algorithm identified the following variables prior to HCT that impacted overall survival: age, TP53 mutations, absolute neutrophils count, cytogenetics per International Prognostic Scoring System–Revised, Karnofsky performance status, conditioning regimen, donor age, WBC count, hemoglobin, diagnosis of therapy-related MDS, peripheral blast percentage, mutations in RAS pathway, JAK2 mutation, number of mutations/sample, ZRSR2, and CUX1 mutations. Different variables impacted the risk of relapse post-transplant. The new model can provide survival probability at different time points that are specific (personalized) for a given patient based on the clinical and mutational variables that are listed above. The outcomes’ probability at different time points may aid physicians and patients in their decision regarding HCT.     

ASXL2 mutations are frequently found in pediatric AML patients with t(8;21)/ RUNX1‐RUNX1T1 and associated with a better prognosis

ASXL2 is an epigenetic regulator involved in polycomb repressive complex regulation or recruitment. Clinical features of pediatric acute myeloid leukemia (AML) patients with ASXL2 mutations remain unclear. Thus, we investigated frequencies of ASXL1 and ASXL2 mutations, clinical features of patients with these mutations, correlations of these mutations with other genetic alterations including BCOR/BCORL1 and cohesin complex component genes, and prognostic impact of these mutations in 369 pediatric patients with de novo AML (0–17 years). We identified 9 (2.4%) ASXL1 and 17 (4.6%) ASXL2 mutations in 25 patients. These mutations were more common in patients with t(8;21)(q22;q22)/RUNX1‐RUNX1T1 (ASXL1, 6/9, 67%, P = 0.02; ASXL2, 10/17, 59%, P = 0.01). Among these 25 patients, 4 (27%) of 15 patients with t(8;21) and 6 (60%) of 10 patients without t(8;21) relapsed. However, most patients with relapse were rescued using stem cell transplantation irrespective of t(8;21). The overall survival (OS) and event‐free survival (EFS) rates showed no differences among pediatric AML patients with t(8;21) and ASXL1 or ASXL2 mutations and ASXL wild‐type (5‐year OS, 75% vs. 100% vs. 91% and 5‐year EFS, 67% vs. 80% vs. 67%). In 106 patients with t(8;21) AML, the coexistence of mutations in tyrosine kinase pathways and chromatin modifiers and/or cohesin complex component genes had no effect on prognosis. These results suggest that ASXL1 and ASXL2 mutations play key roles as cooperating mutations that induce leukemogenesis, particularly in pediatric AML patients with t(8;21), and these mutations might be associated with a better prognosis than that reported previously.     

TET2 mutations in cytogenetically normal acute myeloid leukemia: Clinical implications and evolutionary patterns

Mutations of the Ten‐Eleven‐Translocation 2 (TET2) gene have been identified in patients with various myeloid neoplasms, but the clinical relevance of these mutations and their timing during disease development in cytogenetically normal acute myeloid leukemia (CN‐AML) remain unclear. The total coding region of TET2 was analyzed by direct sequencing in 215 CN‐AML patients younger than 60 years from multicenter treatment trials AML‐SHG 0199 (ClinicalTrials Identifier NCT00209833) and 0295. Associations were analyzed in the context of other molecular markers, such as CEBPA, DNMT3A, NMP1, FLT3, IDH1/2, RAS, and WT1. To investigate the order of appearance of TET2 and concomitant mutations, targeted deep resequencing was performed in six patients. At least one sequence variation with impact on TET2 protein sequence was found in 13 of the 215 CN‐AML patients (6%). Patients with TET2 mutations tended to be older (P = 0.078) and had higher platelet counts (P = 0.041). TET2‐mutated patients were more likely to have concomitant NPM1 (11 of 13; P = 0.047) and DNMT3A (10 of 13; P = 0.001) mutations but were mutually exclusive to partial tandem duplication of the MLL gene (MLL‐PTD) and IDH1/2 mutations. TET2 mutations were identified as subclones in four of the six investigated patients by deep sequencing. Progenitor‐derived colony assays suggest a stepwise acquisition of mutations during disease development, TET2 mutation being later than NPM1 and DNMT3A. The TET2 mutation status did not influence overall or relapse‐free survival. © 2014 Wiley Periodicals, Inc.     

Precursors of acute leukemia: myelodysplastic syndromes and myeloproliferative neoplasms

Myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) represent neoplastic proliferations of hematopoietic stem cells, which may progress to loss of differentiation and acute myeloid leukemia (AML). Transitions between MDSs and MPNs as well as combinations between both disorders occur and MPNs may acquire dysplastic features combined with cytopenia. Myelodysplastic/myeloproliferative neoplasms show dysplastic and myeloproliferative properties and have in common genetic aberrations at the stem cell level (TET2, ASXL?1, CBL, IDH?1, IDH?2, EZH2, p53, Runx1), which may be found in one cell or may affect different hematopoietic stem cells, expanding in parallel. Progress to AML follows a linear clonal evolution only in a subset of cases. Alternatively AML derives from secondary clones, devoid of any marker mutation or originates from a common aberrant progenitor cell which shares other but not the JAK2 ( V617F ) mutation.     

Chromosomal abnormalities in transformed Ph‐negative myeloproliferative neoplasms are associated to the transformation subtype and independent of JAK2 and the TET2 mutations

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more ?7/del(7q) (P = 0.004) and ?5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example ?5/del(5q) and ?17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and ?5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of ?5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild‐type to mutated forms of both genes. The mutated and wild‐type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process. © 2010 Wiley‐Liss, Inc.