PCR分析急性淋巴细胞白血病患者FLT3基因及FLT3/ITD基因突变

目前研究发现约 30 %急性非淋巴细胞白血病(ANLL)患者FLT3基因近膜区发生内部串联重复(ITD)突变 ,称为FLT3/ITD基因突变 ,该突变与ANLL的预后存在密切关系〔1〕。为进一步研究急性淋巴细胞白血病 (ALL)患者FLT3基因及FLT3/ITD基因突变 ,采用PCR技术分析 6 3例ALL患者DNA水平FLT3基因及FLT3/ITD基因突变。材料和方法研究对象 :6 3例经形态学和免疫学确诊ALL患者 ,男 37例 ,女 2 6例 ,中位年龄 2 1岁。免疫分型中B细胞系ALL 4 5例 ,其中前前B细胞ALL 15例 ,前B细胞ALL 18例 ,成熟B细胞ALL 12例 ,T细胞系ALL 14…     

急性白血病患者外周血HMGA1基因的表达

目的:检测急性白血病(AL)患者外周血高迁移率蛋白A1(HMGA1)基因表达水平。方法:采用SYBR Green I RQ-PCR检测30例AL,包括25例AML(M2～M7)和5例ALL(AL组),25例非白血病患者(对照组)外周血HMGA1 mRNA表达水平。结果:AL组HMGA1 mRNA表达明显高于对照组,ALL和AML间HMGA1 mRNA表达水平无显著差异;AML中M7的HMGA1 mRNA表达水平高于M2～M5。结论:HMGA1 mRNA在AL患者外周血高表达,其中M7水平最高,可作为白血病基因治疗的新靶点及M7的鉴别诊断指标。     

急性髓系白血病FLT3-ITD基因突变分析

目的:探讨初发急性髓系白血病(AML)患者FLT3-ITD基因突变情况。方法:选择2015年3月1日~2017年6月1日四川省人民医院血液科收治的初发急性髓系白血病患者207例,取入组患者骨髓标本。采用PCR法检测FLT3-ITD基因的突变情况;采用染色体R显带技术在获得相应染色体后制片、显带,每份标本自动挑选出20个分裂相对清晰的核型完成核型分析,并结合临床资料、随访及预后等进行分析,评价FLT3-ITD基因突变在急性髓系白血病患者中的价值。结果:207例AML患者的标本中42例存在FLT3-ITD基因突变,阳性率为20.29%。FLT3-ITD阳性患者中有3条显示带;42例FLT3-ITD基因突变阳性患者结果表明FLT3-ITD基因突变多依次首尾相接,并且其中插入多个核苷酸,但是所有的突变均为框内突变;根据FAB标准及WHO标准分级,42例FLT3-ITD阳性患者中M0占0.00%,M1占2.38%(1/42)、M2占23.81%(10/42)、M3占0.00%、M4占2.38%(1/42)、M5占69.05%(29/42)、M6占0.00%、M7占2.38%(1/42);FLT3-ITD阳性患者的白细胞(WBC)水平和完全缓解(CR)率均低于FLT3-ITD阴性患者(P0.05)。结论:FLT3-ITD基因突变阳性患者的WBC水平和CR率低于阴性患者,能作为临床危险因素用于AML患者预后的判断。     

慢性粒细胞白血病患者中C/EBPζ基因转录本的定量研究

目的研究慢性粒细胞白血病（chronic myeloid leukemia，CML）患者中CCAAT／增强子结合蛋白ξ（CCAAT／enhancer binding protein，C／EBPξ）的表达及其意义。方法建立实时定量聚合酶链反应对76例不同阶段CML患者和16名正常对照的骨髓单个核细胞中C／EBPξ转录本含量进行检测。结果与正常对照（中位12．20）相比，慢性期、加速期和急变期CML患者中C/EBPξ转录本明显降低，中位含量分别为2，5、3．31和2，22（P〈0，01、〈0，05，〈0，01），8例治疗后达到细胞遗传学缓解者则恢复至正常水平（中位15．43，P〉0．05），而干扰素治疗无效的4例患者中表达水平则进一步下降（中位1．56，P〈0．01）。结论CML患者中C／EBPξ基因表达下降，可能与其发病相关。     

一个母系遗传非综合征耳聋大家系及mtD12SrRNA基因突变

目的 对一个非综合征母系遗传耳聋大家系的分子遗传学进行探讨。方法 采用聚合酶链反应（ＰＣＲ）,扩增ｍｔＤＮＡ与非综合征耳聋相关位点ｎｔ１５５５和ｎｔ７４４５的区域,通过ＰＣＲ－ＳＳＣＰ,ＰＣＲ－ＲＦＬＰ,ＰＣＲ产物克隆序列测定等技术对该家系进行了系统研究。结果 发现该家系中全部患者和４个母系亲属有ｍｔＤＮＡＡ１５５５Ｇ突变,而家系中正常配偶和对照组（１００名正常个体）的ｍｔＤＮＡ１５５５位点均无突     

淋巴细胞白血病细胞分化与基因重排研究

采用ＭｃＡｂｓ免疫酶和ＰＣＲ技术研究３５例淋巴细胞白血病细胞分化起源及ＩｇＨ和ＴＣＲγ、ζ基因重排。结果表明：２０例表达Ｂ细胞表面标记的病例中，１５例Ｂ－ＡＬＬ分别起源于Ｂ祖、前前Ｂ、前Ｂ和成熟Ｂ细胞，５例Ｂ－ＣＬＬ均起源于成熟Ｂ细胞；１７例检测到ＩｇＨ基因重排，８例伴ＴＣＲｒ基因重排，２例伴ＴＣＲξ基因失。８例Ｔ－ＡＬＬ分别起源于幼稚、普通和成熟胸腺细胞，均检测到ＴＣＲｒ基因重排和／或ＴＣＲξ基     

肝细胞癌病人中p53基因突变状况探讨

目的 了解肿瘤抑制基因p53在肝细胞癌中的突变情况,探讨乙型肝炎病毒（HBV）感染与p53基因突变之间的关系。方法 提取50例有乙型肝炎病毒感染史肝癌患者手术样本中的DNA,用聚合酶链式反应（PCR）扩增5－9外显子,作单链构象多态性（SSCP）分析。结果 p53基因突变率超过26％,突变主要分布于5－8外显子,5、6、7、8外显子分别有3、3、4、3例,另仍4个可疑突变。结论 p53基因突变可能是肝细胞癌的病因之一,而乙型肝炎病毒感染在中国肝癌患者p53基因突变中可能起到比较重要的作用。     

再生障碍性贫血骨髓及外周血高水平Flt3配体的来源及意义

目的：研究再生障碍性贫血（AA）患者骨髓及外周血高水平FL（Flt3配体）的来源和影响因素。方法：采用常规ELISA法测定AA患者骨髓和外周血FL含量，应用单色和双色免疫荧光标记流式细胞仪分析FL的分泌细胞及其受体Flt3的表达，并观察AA患者淋巴细胞经PHA激发后环胞菌（CyA)对FL分泌的影响。结果：AA患者骨髓和外周血FL含量显著升高，为正常的25倍以上，外周血和骨髓浓度无区别；正常人CD3^ 细胞内贮存有大量FL，而AA患者CD3^ 细胞以膜型FL为主，胞内基本无FL存在；淋巴细胞经PHA活化后可分泌FL，CyA可抑制FL的分泌，结论：AA患者骨髓和外周血FL水平显著升高，主要由CD^＋细胞产生。     

软骨发育不全FGFR3基因突变的研究

目的了解中国人软骨发育不全患者成纤维细胞生长因子受体３（ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒｒｅｃｅｐｔｏｒ３，ＦＧＦＲ３）基因变异情况。方法应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切方法，分析辽宁地区７例软骨发育不全（ａｃｈｏｎｄｒｏｐｌａｓｉａ，ＡＣＨ）患者外周血ＤＮＡ标本中ＦＧＦＲ３基因第１０外显子区域。结果７例患者均检测到相同的Ｇ３８０Ｒ点突变。结论表明Ｇ３８０Ｒ为中国人ＡＣＨ患者常见突变。应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切的方法检测ＦＧＦＲ３基因突变是产前诊断和早期诊断ＡＣＨ患者的简便、快速、可靠的手段     

KIT and FLT3 receptor tyrosine kinase mutations in acute myeloid leukemia with favorable cytogenetics: two novel mutations and selective occurrence in leukemia subtypes and age groups

Mutations of the receptor tyrosine kinase (RTK) are frequently reported in acute myeloid leukemia (AML) with a normal karyotype. In this study, Southeast Asian AML patients with a favorable karyotype including t(8;21)/AML-ETO, inv(16)(CBFβ/SMMHC), and t(15;17)/PML-RARα were genotyped for KIT and FLT3 RTK mutations by PCR and sequencing. The combined frequency of KIT/FLT3 mutations in patients with t(8;21), inv(16) and t(15;17) was 35%, 18% and 41%. KIT mutations were mainly detected in patients with t(8;21) (23%) and undetectable in patients with t(15;17). Two novel KIT mutations were identified. FLT3 mutations were preferentially found in patients with t(15;17) (41%). Patients with inv(16) had a strikingly low frequency of both KIT and FLT3 mutations (9% each). KIT-mutated patients were older than FLT3-mutated patients and demonstrated a high expression of myeloid antigens and CD56 lymphoid antigen. FLT3 mutation was coexistent with PML-RARα with markedly low or no CD11c and HLA-DR expression. KIT and FLT3 mutations preferentially exist in distinct clinical and genetic AML subtypes, reflecting unique leukemogenetic mechanisms. Targeting therapy with specific RTK inhibitors should provide benefits for a subgroup of AML patients with favorable chromosomes who also carry selective types of RTK mutations.     

Low frequency and variability of FLT3 mutations in Korean patients with acute myeloid leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation.     

Predisposing germline mutations in high hyperdiploid acute lymphoblastic leukemia in children

High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD‐ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer‐relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (<0.01% allele frequency in the Exome Aggregation Consortium, ExAC) and predicted functional (scaled CADD score ≥ 20) in known or potential ALL predisposition genes (SH2B3, CREBBP, PMS2, MLL, ABL1, and MYH9). Three additional patients carried rare and predicted damaging germline mutations in GAB2, a known activator of the ERK/MAPK and PI3K/AKT pathways and binding partner of PTPN11‐encoded SHP2. The frequency of rare and predicted functional germline GAB2 mutations was significantly higher in our patients (2.6%) than in ExAC (0.28%, P = 4.4 × 10^?3), an observation that was replicated in ALL patients from the TARGET project (P = .034). We cloned patient GAB2 mutations and expressed mutant proteins in HEK293 cells and found that frameshift mutation P621fs led to reduced SHP2 binding and ERK1/2 phosphorylation but significantly increased AKT phosphorylation, suggesting possible RAS‐independent leukemogenic effects. Our results support a significant contribution of rare, high penetrance germline mutations to HD‐ALL etiology, and pinpoint GAB2 as a putative novel ALL predisposition gene.     

Examination of the FLT3 and NPM1 mutational status in patients with acute myeloid leukemia from southeastern Poland

Introduction

Acute myeloid leukemia (AML) is a genetically heterogeneous disease at both the cytogenetic and molecular levels. In AML cells many chromosomal aberrations are observed, some of them being characteristic of a particular subtype of patients, and others being less significant. Besides chromosomal abnormalities, the leukemic cells can have a variety of mutations involving individual genes. The aim of this work was to investigate the frequencies of molecular alterations with the focus on FLT3-ITD and NPM1 mutations in AML patients of different age groups living in a southeastern region of Poland.

Material and methods

The study group comprised 50 consecutive AML patients. We analyzed bone marrow samples by conventional cytogenetics. Cytogenetic evaluation in selected cases was complemented by the FISH technique. The internal tandem mutation in the FLT3 gene was identified using polymerase chain reaction (PCR), and the NPM1 mutation was assessed by direct nucleotide sequencing.

Results

The studies using classical cytogenetics showed chromosomal aberrations in 32 (64%) patients. In 18 cases no changes in the karyotype were found by conventional karyotyping. FLT3-ITD mutation was detected in 4 (8%) patients and mutation of NPM1 in 3 patients with AML (6%).

Conclusions

The incidence of both mutations in our study group was lower than described elsewhere. We have confirmed that FLT3-ITD occurred more commonly in older patients and it was associated with shorter overall survival. By contrast, mutation of exon 12 of the NPM1 gene seems to be a good prognostic factor in AML patients with normal karyotype.     

一种同时检测急性髓系白血病FLT3-ITD和NPM1基因突变的快速和简便方法

目的 建立用聚丙烯酰胺凝胶电泳法(polyacrylamide gel electrophoresis,PAGE)同时快速检测正常核型急性髓细胞白血病(cytogenetically normal acute myeloid leukemia,CN-AML)患者FLT3-ITD 和NPM1基因突变的方法.方法 在多重PCR基础上,用毛细管电泳法(capillary electrophoresis,CE)和PAGE凝胶电泳法同时检测117例初发CN-AML患者的FLT3-ITD和NPM1基因突变.结果 突变型双链DNA分子,如突变型FLT3和NPM1的长度均比野生型长(FLT3 -mut:420 bp＞FLT3-wt:327～332 bp,NPM1 -mut:172 bp＞ NPM1 -wt:168 bp),因此,在PAGE凝胶电泳中突变双链DNA比未突变的DNA移动得慢,从而可将突变检出.117例CN-AML患者均通过CE检测得到验证,其结果与PAGE凝胶电泳结果完全一致(FLT3-ITD +/NPM1-患者18例,占15.4％; FLT3 ITD +/NPM1+患者19例,占16.2％;FLT3-ITD -/NPM1+患者25例,占21.4％;FLT3-ITD -/NPM1-患者55例,占47.0％).结论 两种电泳法均可快速、简便地同时检测CN-AML患者FLT3- ITD和NPM1基因突变.CE检测敏感,图像清晰;PAGE凝胶电泳法则操作简单,成本低,结果可靠,更适于在基层医院开展初步筛查.     

Double CEBPA mutations are prognostically favorable in non-M3 acute myeloid leukemia patients with wild-type NPM1 and FLT3-ITD

This study is aimed to investigate the pattern of CEBPA mutations and its clinical significance in Chinese non-M3 acute myeloid leukemia (AML) patients. The entire coding region of CEBPA gene was amplified by PCR and then sequenced in samples from 233 non-M3 AML patients. Fifty mutations were identified in 37 (15.8%) patients with eleven (4.7%) double mutated CEBPA (dmCEBPA) and twenty-six (11.1%) single mutated CEBPA (smCEBPA). dmCEBPA was exclusively observed in M1 and M2 subtypes of FAB classification (P = 0.008), whereas smCEBPA occurred in almost all subtypes (P = 0.401). Patients with dmCEBPA had significantly younger age and higher WBC counts than those with wtCEBPA (P = 0.016 and 0.043, respectively). Both dmCEBPA and smCEBPA were mainly present in cytogenetically normal patients. Patients with dmCEBPA achieved higher rate of complete (CR) than wtCEBPA patients (88% vs. 51%, P = 0.037), whereas smCEBPA and wtCEBPA groups are similar (47% vs. 51%, P = 0.810). Patients with dmCEBPA had a superior overall survival (OS) compared with patients with wtCEBPA (P = 0.033), whereas patients with smCEBPA had a similar OS as patients with wtCEBPA (P = 0.976). dmCEBPA but not smCEBPA was also associated with favorable outcome in patients with wild-type NPM1 and FLT3-ITD (NPM1^wtFLT3-ITD^wt). Our data confirm that dmCEBPA but not smCEBPA is prognostically favorable in NPM1^wtFLT3-ITD^wt AML, and suggest that the entity AML with mutated CEBPA should be definitely designated as AML with dmCEBPA in WHO classification and smCEBPA should be excluded from the favorable risk of molecular abnormalities.     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.