Abstract: Objective To explore the methylation modification of ENO2 gene in peripheral blood of children with autism spectrum disorder (ASD),in order to provide theoretical evidence for early screening of ASD. Method Peripheral blood from ASD children and control children were collected to analyze differential methylation by methylated-DNA immunoprecipitation (MeDIP) chips. One neuron-specific gene,ENO2,was found to be hypermethylated in the autistic samples. In addition,obtained blood samples were collected from 101 autistic children and controls matched with age and sex. This difference was validated by bisulfite sequencing PCR (BSP). The differential expression of ENO2 gene was further analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and ELISA. Results The hypermethylation of ENO2 within the promoter region was confirmed by BSP to be present in 15.8% (16/101) of the autistic samples. The methylation frequency of 16 CpG sites of ENO2 gene promoter was calculated,and it was found that the closer the promoter was to the start site of transcription,the higher the methylation frequency would be. The mean level of ENO2 RNA in these 16 autistic samples was reduced by 30% approximately compared with that in controls. The average level of ENO2 protein expression in 16 autistic samples was (15.15±3.52) μg/L,about half of that in the controls[ (33.78±8.18) μg/L]. Conclusions Hypermethylation of ENO2 gene is found in peripheral blood of 15.8% of ASD children. Moreover,reduced level of ENO2 expression may be a biomarker for a subset of autistic children.

Key words: autism spectrum disorder, ENO2 gene, neurodevelopment, methylation, epegenetics

摘要： 目的 探讨孤独症谱系障碍(ASD)儿童外周血ENO2基因甲基化修饰的水平,为ASD的早期筛查提供理论依据。方法 2018年1-12月在上海市儿童医院儿童保健科收集5对性别和年龄分别匹配的ASD儿童和正常对照儿童的外周血,应用Medip-chip甲基化芯片分析,发现ASD儿童外周血均存在ENO2基因的高甲基化改变,本研究在此基础上进一步扩大样本至101对ASD与正常对照儿童,应用亚硫酸盐变性测序检测ENO2基因甲基化水平,并应用荧光定量PCR和酶联免疫吸附法分别在mRNA和蛋白水平检测ENO2基因的表达。结果 与正常对照组儿童相比,ASD儿童中有16例外周血ENO2基因具有高甲基化改变,频率为15.8%(16/101)。对ENO2基因启动子16个CpG位点甲基化频率进行统计,发现越靠近转录的起始位点,甲基化的频率越高。在16例具有ENO2基因高甲基化改变的ASD儿童中,ENO2基因mRNA的平均水平约为正常对照组的30%。16例高甲基化的ASD儿童ENO2蛋白值为(15.15±3.52)μg/L,约为正常对照组儿童[(33.78±8.18) μg/L]的一半。结论 15.8%的ASD儿童外周血存在ENO2基因的高甲基化改变,ENO2表达的降低有可能成为一部分ASD筛查的标记物。

关键词: 孤独症谱系障碍, ENO2基因, 神经发育, 甲基化, 表观遗传