卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区甲基化的研究

目的 探讨RASSF1A基因启动子区异常甲基化与卵巢上皮性恶性肿瘤发生、发展的关系。方法 应用甲基化特异性PCR方法,检测80例卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区异常甲基化。结果 80例卵巢上皮性恶性肿瘤组织中,RASSF1A基因启动子区甲基化的发生率为52．5％,而相应痛旁正常组织中,RASSF1A基因启动子区均未发生甲基化（P〈0．05）。浆液性癌、黏液性癌和内膜样癌中,RASSF1A基因启动子区甲基化的发生率分别为54．2％、52．4％和45．5％,差异尤统计学意义。临床Ⅰ期、Ⅱ期卵巢上皮性恶性肿瘤RASSF1A基因启动子区甲基化的发生率分别为21．4％和16．7％,明显低于临床Ⅲ期（66．7％）和Ⅳ期（77．8％）。高分化组和中分化组RASSFlA基因启动子区甲基化的发牛率分别为34．5％和35．0％,均低于低分化组（80．6％）。结论 卵巢上皮性恶性肿瘤组织中存在RASSF1A基因启动子区的异常甲基化,甲基化与卵巢上皮性恶性肿瘤的临床分期和组织学分级有关。     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的：探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系。方法：采用甲基化特异性PCR（methylation—specific PCR,MSP）法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态。结果：胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65．0％（39／60）,艋著高于癌旁组织6．7％（4／60）,及对照组0％（0／30）（P〈0．01）。胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义。结论：胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法。     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的:探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系.方法:采用甲基化特异性PCR(methylation-specific PCR,MSP)法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态.结果:胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65.0%(39/60),显著高于癌旁组织6.7%(4/60),及对照组0%(0/30)(P＜0.01).胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义.结论:胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法.     

RASSF1A 基因在宫颈癌中的甲基化检测及临床意义

 目的 探讨宫颈癌组织中RASSF1A(Ras association domain family 1A)抑癌基因启动子甲基化状态及临床意义以及与高危型HPVs感染的关系. 方法 甲基化特异性聚合酶链反应(MSP)方法检测39例宫颈鳞癌及12例正常宫颈组织中RASSF1A基因甲基化状态.聚合酶链反应(PCR)方法检测宫颈癌组织中HPV16、18型的感染状况. 结果 39例宫颈癌组织中有11例可见异常甲基化(28.2%);12例正常宫颈组织均未见甲基化;宫颈癌组HPV感染率为69.2%;RASSF1A基因甲基化在淋巴结转移组(75.0%)高于淋巴结未转移组(9.1 %),P<0.05.RASSF1A甲基化在患者年龄、肿瘤大小、病理组织学分级、临床分期和HPVs感染组之间差异无统计学意义, P>0.05.结论 RASSF1A基因启动子区5′-CpG岛的高甲基化是导致RASSF1A基因失活的重要机制,可能参与宫颈癌的发生过程.     

鼻咽癌组织中RASSF1A基因甲基化的研究

目的观察RASSF1A基因在鼻咽癌组织和慢性鼻咽炎的甲基化情况。方法采用甲基化特异性PCR技术检测16例鼻咽低分化未角化癌和10例鼻咽黏膜慢性炎组织中RASSF1A基因的甲基化。结果RASSF1A基因的高甲基化率在鼻咽癌组织中为93．75％（15／16）,在慢性鼻咽炎组织中为0,两组病例的RASSF1A基因高甲基化率差别显著,P〈0．005。结论RASSF1A基因在鼻咽癌组织中呈高甲基化,可能是影响鼻咽癌发生发展的抑癌基因之一。     

胃和结直肠腺癌患者血清RASSF1A基因启动子异常甲基化检测及其临床意义

目的 研究胃和结直肠腺癌患者血清RASSF1A基因启动子区域的甲基化状态及其临床意义.方法采用甲基化特异性聚合酶链反应(MSP)技术,检测47例胃腺癌患者、45例结直肠腺癌患者、60例胃肠道良性病变患者及30例健康志愿者血清RASSF1A基因启动子区域的甲基化状态,并同时检测25例进行手术的胃腺癌、结直肠腺癌患者的肿瘤...     

RASSF1A基因甲基化在上皮性卵巢肿瘤中的检测及临床意义

卵巢癌是严重威胁女性健康的一种恶性肿瘤,由于缺乏早期诊断方法,70%的患者就诊时已是晚期,5年生存率仅为20%～30%.因此,如何早期诊断卵巢痛已是当前的研究热点.DNA甲基化是肿瘤发生的早期事件,甲基化表型早于肿瘤恶性表型的出现,且具有基因和肿瘤组织异质性[1],这使其成为目前用于肿瘤诊断最有前景的DNA标志.     

急性髓系白血病儿童DKK3基因启动子高甲基化的临床价值

目的：检测急性髓系白血病（AML）患儿骨髓DKK3基因启动子甲基化情况并研究其临床意义。方法选择急性髓系白血病患儿（AML组）80例和血液系统良性疾病患儿（CON组）40例为研究对象,采用甲基化特异性PCR检测两组患儿骨髓DKK3基因启动子甲基化并比较其差异,分析AML组患儿不同临床病理因素中DKK3基因启动子甲基化的差异,对比DKK3基因启动子甲基化与未甲基化患儿3年生存率及总生存时间的差异。检测髓系白血病细胞株DKK3基因启动子甲基化状态。结果 AML组患儿骨髓DKK3基因启动子甲基化率高于CON组（P﹤0.05）;AML组患儿骨髓DKK3基因启动子甲基化在性别、年龄、初诊发热、外周血白细胞（WBC）、外周血红细胞（RBC）、外周血血小板（PLT）和FAB分型方面比较,差异均无统计学意义（P﹥0.05）,在骨髓原始细胞比例、核型类型和诱导化疗疗效方面比较,差异均有统计学意义（P﹤0.05）;随访3年,AML组DKK3基因启动子甲基化患儿的生存率和生存时间均较DKK3基因启动子未甲基化患儿低（P﹤0.05）;HL-60、KG-1和K562白血病细胞株DKK3基因启动子均处于甲基化状态。结论急性髓系白血病患儿骨髓DKK3基因启动子处于高甲基化状态,在病情及预后评估中具有一定价值。     

RASSF1A基因在宫颈癌中的甲基化检测及

目的　探讨宫颈癌组织中RASSF1A(Ras association domain family1A)抑癌基因启动子甲基化状态及临床意义以及与高危型HPVs感染的关系。方法　甲基化特异性聚合酶链反应(MSP)方法检测39例宫颈鳞癌及12例正常宫颈组织中RASSF1A基因甲基化状态。聚合酶链反应(PCR)方法检测宫颈癌组织中HPV16、18型的感染状况。结果　39 例宫颈癌组织中有11 例可见异常甲基化(28.2%);12例正常宫颈组织均未见甲基化;宫颈癌组HPV感染率为69.2%;RASSF1A基因甲基化在淋巴结转移组(75.0%)高于淋巴结未转移组(9.1 %),P<0.05。RASSF1A甲基化在患者年龄、肿瘤大小、病理组织学分级、临床分期和HPVs感染组之间差异无统计学意义, P>0.05。结论　RASSF1A基因启动子区5′CpG岛的高甲基化是导致RASSF1A基因失活的重要机制,可能参与宫颈癌的发生过程。     

急性髓系白血病中DNA甲基化异常及相关调控基因的研究进展

对急性髓系白血病（AML）全基因组测序及表观遗传学调控的研究,发现了一系列调控基因的突变导致DNA甲基化异常,从而导致AML的发生。其中,已有研究证实某些基因突变导致甲基化发生的作用机制,以及相对应的针对性治疗。这里我们讨论AML中导致DNA甲基化异常的调控基因和针对表观遗传调控的治疗进展。     

Prognostic utility of six mutated genes for older patients with acute myeloid leukemia

Approximately 50% of older patients with acute myeloid leukemia (AML) do not obtain chromosomal abnormalities as an effective risk‐stratification, and present cytogenetically normal AML (CN‐AML). To develop a reliable prediction model for stratifying the risk of these elderly patients, we conducted a study with a discovery and validation design. As a result, we found the top 6 mutated genes in the discovery cohort of 26 case by the whole exome sequencing, and verified as recurrent mutations in the large cohort of 329 patients by Sanger sequencing. The top 6 genes were NPM1, FLT3‐ITD, DNMT3A, CEBPA double allele, IDH1 and IDH2 mutations, and the frequency of each gene in the combining cohort was 36.8%, 19.8%, 20.1%, 5.8%, 14.9% and 22.5%, respectively. In addition, clinical variables such as age, white blood cell counts, genes of IDH1 and DNMT3A mutations, European LeukemiaNet genotype (NPM1 mutations and lacking FLT3‐ITD or CEBPA double allele mutations) and treatment protocols were independent factors for predicting the probabilities of overall and event‐free survival. The prediction nomograms based on these significant factors showed accurate discrimination. In conclusion, we developed a reliable prediction model for stratifying the risk of elderly patients with CN‐AML.     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的:检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法:运用甲基化特异性PCR(MSP)方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果:上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率(58.06%、42.85%)显著高于卵巢良性囊腺瘤组织(13.33%),差异有统计学意义(P<0.05)。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关(P<0.05)而与组织类型和患者年龄及绝经与否无相关性(P>0.05);RASSF1A基因甲基化与其蛋白表达下降一致。结论:卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的：检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法：运用甲基化特异性PCR（MSP）方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果：上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率（58.06%、42.85%）显著高于卵巢良性囊腺瘤组织（13.33%）,差异有统计学意义（P〈0.05）。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关（P〈0.05）而与组织类型和患者年龄及绝经与否无相关性（P〉0.05）;RASSF1A基因甲基化与其蛋白表达下降一致。结论：卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

Multidrug resistance-1 gene polymorphisms associated with treatment outcomes in de novo acute myeloid leukemia

Multidrug resistance-1 (MDR-1) gene single nucleotide polymorphisms (SNPs) have been identified as associated with the treatment outcomes of acute myeloid leukemia (AML) in Caucasians; yet, similar evidence is lacking for Asian populations. A total of 101 AML patients were enrolled in the current study. Two MDR1 SNPs (C3435T and G2677T/A) were analyzed with PCR/RFLP assay. As regards C3435T polymorphism, C/C genotype was significantly correlated with lower functional P-glycoprotein (P-gp) activity in leukemic blasts (7.5%) compared with C/T (10.7%) or T/T genotype (19.9%, p = 0.029). In genotypic analyses, C/C at -3435 (p = 0.05) and G/G at -2677 (p = 0.04) were strongly associated with a higher probability of complete remission (CR). In addition, the 3-year event-free survival (EFS) was higher in G/G genotype at -2677 (60.6%) than nonG/G (21.9%; p = 0.0241), in C/C at -3435 was higher than nonC/C genotype (p = 0.0139), and was higher in GC haplotype homozygote (58.2%) than nonGC homozygote (22.6%; p = 0.0427). In a multivariate analysis, the group without GC haplotype showed worse EFS (p = 0.030), with unfavorable cytogenetic risk (p = 0.008). However, no differences were noted in overall survival according to the MDR1 SNPs (p = 0.491 for C3435T and p = 0.955 for G2677T/A).     

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p = 0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p = 0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.     

急性髓性白血病患者LRP、MRP基因表达

目的：研究急性髓性白血病ＬＲＰ基因、ＭＲＰ基因表达与疗效的关系，方法：用ＲＴ－ＰＣＲ法检测３７例急性髓性白血病ＬＲＰ基因、ＭＲＰ基因表达，按此两种基因表达分成ＬＲＰ－／ＭＲＰ－、ＬＲＰ－ＭＲＰ＋、ＬＲＰ＋／ＭＲＰ－、ＬＲＰ＋／ＭＲＰ＋共４组，统计比较各组疗效。结果：与ＬＲＰ－／ＭＲＰ－组比较，其余３组疗效较低，其中ＬＲＰ＋／ＭＲＰ－组、ＬＲＰ＋／ＭＲＰ＋组存在显著差异，但ＬＲＰ＋／ＭＲＰ＋组与ＬＲ     

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3A^R882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3A^R882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3A^R882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3A^R882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3A^R882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3A^R882/FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3A^R882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3A^R882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.     

急性髓系白血病17种基因异常的检测

目的:探讨应用多重巢式RT-PCR、荧光定量PCR、PCR-SSCP银染技术检测初诊急性髓系白血病(acute myeloid leukemia,AML)中17种基因异常表达及在各亚型的分布情况,为个体化治疗提供依据.方法:采用多重巢式RT-PCR检测融合基因,PCR检测FLT3-ITD,荧光定量PCR检测NPM1的突变类型(A、B、D、I和R)及C-kit/D816V,PCR-SSCP银染技术检测CEBPA.对140例初诊AML患者(APL除外)的骨髓进行17种基因异常分析,并与骨髓染色体检测结果进行对比.结果:在140例初诊AML患者中检出基因异常占69例(49.3％),其中包括FLT3-ITD、NPM1、C-kit/D816V、CEBPA、HOX11、CBFβ/MYH11、AML-ETO、MLL/AF6、MLL/AF10、dupMLL、EVI1.同时对140例初治AML患者采用G显带技术行染色体核型分析,128例获得可供分析的染色体核型,其中57例(44.5％)检出染色体结构和数目异常.PCR在急性髓系白血病基因检测中较染色体核型分析具有更高的检出率.结论:PCR协同染色体核型分析检测初诊AML患者的基因异常,能提高临床诊断率、指导疾病危险度分组,为判断预后及监测微小残留病提供更好的理论依据.