DNMT3A Gene Mutations In Acute Myeloid Leukemia

目的：了解DNMT3A基因在急性髓系白血病（Acutemyeloidleukemia，AML）患者中的突变比例和类型，分析DNMT3A基因突变患者的临床特征和突变在AML种的预后价值，分析该突变在疾病进展中的变化情况。 方法：抽取101例髓系白血病患者的骨髓或外周血，提取RNA逆转录成cDNA，并设计两对引物用于扩增DNMT3A基因，电泳验证产物条带后进行直接测序。提取所有患者的全基因组DNA进行毛细管电泳，检测NPM1和FLT3-ITD两种基因的突变情况。同时检测其它相关危险因素，如外周血白细胞数、免疫表型、染色体核型，并进行统计学分析。 结果：共检测出14例（13.9%）突变患者，其中...Objective: To detect the frequency and forms of DNMT3A gene mutations in patients with acute myeloid leukemia(AML),and to investigate the relationship between DNMT3A mutations and clinical feature of AML.Monitoring mutations in two cases during the clinical course. Methods: DNA and RNA was Extracted from bone marrow or peripheral blood samples.PCR followed by reverse transcriptase was used to amp...学位：医学硕士院系专业：医学院临床医学系_内科学学号：2452009115293

Simultaneous Detection of FLT3-ITD and NPM1Gene Mutations in Acute Myeloid Leukemia by Double PCR

本研究旨在建立一种同时筛查flT3-ITd突变和nPM1突变的检测方法。设计2对引物,分别扩增nPM1基因的外显子12和flT3基因的外显子14、内含子14、外显子15,以覆盖几乎所有已知突变位点。对双重PCr体系的反应程序和引物浓度比例进行优化,将双重PCr产物通过毛细管电泳分离,根据野生型产物和突变型产物的大小差异来判断突变存在与否并利用产物的峰面积对突变比例进行定量,并对突变阳性标本进行测序验证。结果表明,在93例标本中nPM1突变者17例(18.5%),flT3-ITd突变者15例(16.3%),nPM1突变和flT3-ITd突变双阳性6例。17例nPM1突变中7例M2、4例M4、5例M5、1例M6,其中10例男性、7例女性;有15例为A型,1例为b型,1例为nM型;有1例CMl急变为AMl的标本中带有nPM1基因A型突变。15例flT3-ITd阳性中1例M1、8例M2、2例M3、1例M4、3例M5,其中5例男性、10例女性。扩增产物测序结果进一步证明了该检测体系的准确性和可靠性。结论 :建立了同时筛查flT3-ITd突变和nPM1突变的检测体系,该体系以基因组dnA为模板,具有方便、快捷、准确、可定量的优点。Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML.A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3,with the aim of detecting almost all reported mutations.After optimization,a touchdown PCR was chosen for the multiplex PCR procedure,with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively.The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products.The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio.All the positive mutated samples were confirmed by sequencing.The results showed that 17 patients with NPM1 mutation,15 patients with FLT3-ITD mutation,6 patients with both NPM1 and FLT3-ITD mutationts were found among 93 patents.7 patients with M2,4 patients with M4,5 patients with M5 and 1 patients with M6 were found out of 17 patients with NPM1 mutation,in which 10 patients were male and 7 patients were female,15 patients were with type A,1 patients was with type B and 1 patients was with type Nm,strinkingly 1 CML patient in blast crisis was found to carry a type A mutation.Among 15 patients with FLT3-ITD mutation 1 patient with M1,8 patients with M2,2 patients with M2,2 patients with M3,1 patient with M4,3 patients with M5 were found,in which 5 patients were male and 10 patients were lemale.Sequencing results further confirmed the accuracy and reliability of this method.It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated.This method is fast,easy,accurate and capable to calculate the mutant/wild-type ratio

急性髓系白血病NPM1基因突变检测方法的研究

本研究旨在建立检测急性髓系细胞白血病(AML)患者NPM1基因突变的方法。针对NPM1基因突变集中区域设计引物/探针,建立高分辨熔解曲线方法(HRM)和等位基因特异PCR方法(AS-PCR),通过89份AML标本进行了临床评估,并采用毛细管电泳法(CE)和测序法作为对照进行了验证。结果表明,通过上述4种方法共检出阳性标本17例(19.1%),其中A型突变15例,B型和Nm型突变各1例。上述方法中HRM法检测全面,灵敏度为5%,而AS-PCR法有一定的漏检,但灵敏度高达0.01%。结论:考虑到操作的难易程度,同时也结合临床样品的检测情况,HRM在临床上可法用于NPM1突变筛查,而AS-PCR法可用于后续的微小残留病定量检测

Effects of CYP3A4 and MDR1 genetic polymorphism on dosage and concentra- tion of tacrolimus in allogeneic hematopoietic stem cell transplantation patients

目的探讨异基因造血干细胞移植（HSCT）患者中CYP3A4S18B和MDRl C3435T基因多态性与他克莫司血药浓度、稳态剂量和不良反应之间的关系。方法采用直接测序法检测CYP3A4。18B和MDRl C3435T基因型，比较不同基因型患者之间他克莫司初始血药浓度／剂量×体表面积（p0／D0）、稳态剂量／体表面积（D’）及不良反应发生率的差异。结果16例HSCT患者CYP3A4S18B和MDRlC3435T等位基因突变频率分别为34．38％和43．75％。CYP3A4S1／S1基因型患者他克莫司初始风／D0’明显高于％18B等位基因携带者（P〈0．05）；稳态时D’显著低于＊18B等位基因携带者（P〈0．05）。MDRlC3435TTT型患者p0／D0’、D’与C等位基因携带者相比差异无统计学意义（P〉0．05）。CYP3A4S18B和MDRIC3435T基因多态性与急性移植物抗宿主病及他克莫司所致不良反应无关（P〉0．05）。结论HSCT患者CYP3A4＊18B基因多态性与他克莫司的p0／D0、D’相关。AIM To evaluate the effects of CYP3A4 and MDR1 genetic polymorphism on the concentration, dosage and adverse drug reaction of tacrolimus in allogeneic hematopoietic stem cell transplantation patients. METHODS The CYP3A4 ＊ 18B and MDR1 C3435T genotype were detected by direct sequencing method. Thedifferences ofpo/Do＇ ratio, D＇, acute graft versus host disease and adverse drug reaction were compared among dif- ferent genotype groups treated with tacrolimus. RESULTS Totally 16 patients were involved in this study. The frequencies of CYP3A4 ＊ 18B and MDR1 C3435T alleles were 34.38% and 43.75% , respectively. The po/Do＇ of tacrolimus in CYP3A4 ＊ 1/ ＊ 1 genotype groups was significantly higher than that of ＊ 18B allele carriers （P 〈 0.05） , and the D＇ was lower than ＊ 18B allele carriers obviously （P 〈0.05）. No significant association was found between the po/Do＇,D＇ of tacrolimus and MDR1 C3435T genotypes （P 〉 0.05）. There was also no significant correla- tion between CYP3A4 ＊ 18B and MDR1 C3435T gene polymorphisms and acute graft versus host disease and adverse drug reactions induced by tacrolimus （P 〉 0.05）. CONCLUSION CYP3A4 ＊ 18B genetic polymorphism in hemato- poietic stem cell transplantation patients is closely correlated to the po/Do＇ratio and D＇ of tacrolimus.厦门市科技惠民计划（编号3502820144023