急性髓系白血病RASSF1A基因启动子区异常甲基化临床意义研究

Aberrant DNA hypermethylation of the RASSF1A gene in acute myeloid leukemia

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 Aberrant methylation of the RASSF1A gene may be involved in the pathogenesis of hema- tologic malignancies and provide a basis for monitoring, stratification, prognostic evaluation, and targeted therapy for minimal residual disease. The aim of this study was to evaluate the frequency of aberrant RASSF1A promoter methylation in Chinese patients presenting with acute myeloid leukemia （AML） in an attempt to identify a subset of patients that might be candidates for demethylating agents as a form of targeted therapy. METHODS We studied aberrant RASSF1A promoter methylation levels in 152 newly diagnosed AML patients and 15 apparently healthy donors by bisulfite sequen- cing PCR （BS-PCR） and methylation specific PCR （MS-PCR）. Furthermore, hematological characteristics, cytogenetics, and genetic aberrations were analyzed, followed by an assessment of clinical survivals. RESULTS Fifteen healthy indi- viduals were not detected with RASSF1A hypermethylation using MSP method, however 38 AML patients harbored in- creased methylation levels of the RASSF1A gene. In 4 MSP-positive AML patients, the promter of the RASSF1A gene was hypermethylated by bisulfite sequencing, with methylation rates of 88.2%, 85.5 %, 78.6 %, 92.7%, respectively. While in 4 MSP-negative AML patients and 4 healthy donors, the methylation rates were 10%, 11.8%, 12.7%, 6.8%, and 5.0%, 9. 1%, 8.2%, 7.3%. Hypermethylation of the RASSF1A promoter was associated with ASXLland DN- MT3A mutations. Patients with higher RASSF1A promoter methylation levels exhibited shorter Relapse-free survival （RFS） and overall survival （OS）. CONCLUSIONS We found that hypermethylation of the RASSF1A promoter was a frequent event and may indicate poorer outcomes in AML patients; thus providing the molecular basis for stratified diag- nosis and targeted therapeutic approaches, thus providing the molecular basis for stratified diagnosis and prognosis.