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目的 RASSF1A基因异常甲基化可能参与血液肿瘤的发生,并为微小残留疾病(minimal residual de-sease,MRD)监测、分层、预后评估及靶向治疗提供依据。本研究旨在分析RASSF1A基因启动子区异常甲基化在急性髓系白血病(acute myeloid leukemia,AML)中的临床意义。方法选取2005-01-01-2013-03-01解放军总医院(113例)以及第一附属医院(39例)住院患者和门诊体检患者,共152例AML患者骨髓标本以及15例健康供者骨髓标本纳入本项研究。提取基因组DNA,并进行DNA硫化修饰;设计重亚硫酸盐测序PCR(bisulfite sequencing PCR,BS-PCR)引物以及甲基化特异性PCR(methylation specific PCR,MS-PCR)引物,进行PCR扩增,进而电泳分析以及DNA序列分析。同时对RASSF1A高甲基化组以及低甲基化组的血液学特点、骨髓原始细胞比例、细胞遗传学异常、基因异常、完全缓解率和总生存期进行统计学分析。结果 MS-PCR分析结果显示,RASSF1A基因在15例健康人中呈完全非甲基化状态,在152例AML患者中有38例出现启动子区高甲基化状态,其甲基化阳性率为25%。4例MS-PCR阳性AML患者经BS-PCR测序分析后,显示RASSF1A甲基化率分别为88.2%、85.5%、78.6%和92.7%,而在4例MS-PCR阴性患者RASSF1A基因启动子区甲基化率分别为10%、11.8%、12.7%和6.8%,4例健康供者RASSF1A基因启动子区甲基化率分别为5.0%、9.1%、8.2%和7.3%。进而通过统计学分析发现携带RASSF1A基因高甲基化的AML患者易合并存在ASXL1基因突变或DNMT3A基因突变。携带RASSF1A基因高甲基化的AML患者,其无进展生存期以及总生存期较短。结论 RASSF1A基因启动子区异常甲基化可能参与AML的发生,同时可能为AML分层诊治以及预后评估提供分子理论依据。
Objective The abnormal methylation of RASSF1A gene may be involved in the development of hematological tumors and provide the basis for the monitoring, stratification, prognosis evaluation and targeted therapy of minimal residual desease (MRD). This study aimed to analyze the clinical significance of abnormal methylation of RASSF1A promoter region in acute myeloid leukemia (AML). Methods A total of 152 inpatients and outpatients from PLA General Hospital (113 cases) and the First Affiliated Hospital (39 cases) were recruited from January 2005 to January 2013 on January 15, 2001. A total of 152 bone marrow samples from AML patients and 15 healthy donors Specimens are included in this study. Genomic DNA was extracted and subjected to DNA sulfide modification. Bisulfite sequencing PCR (BS-PCR) primers and methylation specific PCR (MS-PCR) primers were designed for PCR amplification. Further electrophoretic analysis and DNA sequence analysis. At the same time, the hematological characteristics, ratio of bone marrow blast cells, cytogenetic abnormalities, gene abnormalities, complete remission rate and overall survival of RASSF1A hypermethylation group and hypomethylation group were statistically analyzed. Results The results of MS-PCR showed that the RASSF1A gene was completely unmethylated in 15 healthy people. Thirty-eight cases of 152 AML patients showed hypermethylation status and the methylation-positive rate was 25% . The methylation rates of RASSF1A in 4 MS-PCR positive AML patients were 88.2%, 85.5%, 78.6% and 92.7% respectively after BS-PCR sequencing. However, in 4 MS-PCR negative patients, the RASSF1A gene promoter region The methylation rates of RASSF1A promoter in 4 healthy donors were 5.0%, 9.1%, 8.2% and 7.3%, respectively. The methylation rates were 10%, 11.8%, 12.7% and 6.8% respectively. And then through statistical analysis found that patients with AML with high methylation of RASSF1A gene easily combined with ASXL1 gene mutation or DNMT3A gene mutation. Patients with AML with hypermethylation of the RASSF1A gene have a poor progression-free survival and a shorter overall survival. Conclusions Aberrant methylation of RASSF1A promoter may play an important role in the pathogenesis of AML. At the same time, it may provide a molecular basis for stratification diagnosis and prognosis evaluation of AML.