MECP2基因甲基化和羟甲基化水平的性别差异研究  

Gender differences in methylation and hydroxymethylation levels of MECP2

作　　者：刘亚卉 郑煜芳[1] 刘星[2] 王红艳[1] 蔡春泉[3] 

机构地区：[1]复旦大学生命科学学院遗传工程国家重点实验室--现代人类学教育部重点实验室,上海200433 [2]生物芯片上海国家工程研究中心,上海201203 [3]天津市儿童医院外科,天津300074

出　　处：《中国循证儿科杂志》2016年第2期137-141,共5页Chinese Journal of Evidence Based Pediatrics

基　　金：天津市应用基础与前沿技术研究计划:14JCYBJC25000

摘　　要：目的探讨MECP2在不同性别脑组织中是否有表达差异,从而与孤独症等疾病的性别差异相关。方法利用4例非疾病流产胎儿脑标本,采用酚氯仿方法提取基因组DNA。在Meth Primer在线软件上检测MECP2基因-1 000 bp至+1 200 bp区间的CpG岛。甲基化检测采用亚硫酸氢盐修饰后测序法(使用EZ DNA Methylation-gold^(TM)Kit试剂盒)。对于超声断裂后的基因组DNA,用基因组羟甲基化试剂盒(diagenode,h Me DIP kit)进行ChIP反应。反转录cDNA采用Fast Quant RT Kit(With gDNase)试剂盒,定量PCR检测MECP2表达量采用SuperReal PreMix Plus(SYBR Green)试剂盒。结果标本1男,重量106 g,长度17.4 cm;标本2女,重量100 g,长度19.1 cm;标本3男,重量500 g,长度28.3 cm;标本4女,重量510 g,长度31.5 cm。MECP2表达量男性胚胎(标本1=0.0367,标本3=0.0155)高于女性胚胎(标本2=0.0177,标本4=0.0088)。MECP2的甲基化水平女性个体平均1条X染色体上MECP2的甲基化程度显著高于男性,特别是在启动子的核心区域-309 bp至-179 bp,男性MECP2上几乎没有甲基化,而羟甲基化水平男性高于女性。结论男性MECP2基因的DNA修饰促进其表达,可能提高了男性胚胎对MECP2基因突变的易感性,从而影响MECP2基因突变导致的患病人群的性别差异。Objective To understand whether there are expressional differences of MECP2 between male and female brains,which may contribute to the gender differences in MECP2 associated diseases,e. g. autism. Methods Genomic DNA of brain tissues from four normal aborted fetuses was obtained by phenol-chloroform extraction. By using Meth Primer online software,the Cp G island of MECP2 gene was enriched between-1 000 bp to ＋ 1 200 bp. To detect the level of methylation,bisulfite sequencing method was used（ by using the EZ DNA Methylation-goldTMKit）. For ultrasound fractured genomic DNA,Ch IP assay was performed with genomic hydroxymethyl kit（ diagenode,h Me DIP Kit）. The expression levels of MECP2 in brain tissues were detected with c DNA that was obtained by Fast Quant RT Kit（ With g DNase） and were quantitated using SuperReal Pre Mix Plus（ SYBR Green） kits.Results Sample 1,male,weight 106 g,length 17. 4 cm; sample 2,female,weight 100 g,length 19. 1 cm; sample 3,male,weight500 g,length 28. 3 cm; sample 4,female,weight 510 g,length 31. 5 cm. The expression level of MECP2 in brain of male（ sample1 = 0. 0367,sample3 = 0. 0155） was higher than that in female（ sample2 = 0. 0177,sample 4 = 0. 0088） during early embryonic stage. The methylation level of the core promoter region of MECP2 in male was significantly lower than that in female per X chromosome,especially in the core promoter region-309 bp to-179 bp. Almost no methylation appeared on MECP2 in brain of male while the hydroxymethylation level was the opposite. Conclusion These results indicated that the gene modifications on MECP2 in male may contribute to its expression. This may in turn increase the susceptibility of male to MECP2 mutations in disease such as autism and result in sex ratio changes in patient population.

关 键 词：MECP2基因 甲基化 羟甲基化 

分 类 号：R749.94[医药卫生—神经病学与精神病学]