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中国循证儿科杂志

中国循证儿科杂志 ›› 2016, Vol. 11 ›› Issue (2): 137-141.

• 论著 • 上一篇    下一篇

MECP2基因甲基化和羟甲基化水平的性别差异研究

刘亚卉1 郑煜芳1 刘星2 王红艳1 蔡春泉3   

  1. 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
  • 收稿日期:2016-04-21 修回日期:2016-04-21 出版日期:2016-04-25 发布日期:2016-04-21
  • 通讯作者: 蔡春泉

Gender differences in methylation and hydroxymethylation levels of MECP2

LIU Ya-hui1，ZHENG Yu-fang1，LIU Xing2，WANG Hong-yan1，CAI Chun-quan3   

  1. 1 State Key Laboratory of Genetic Enginering-Ministry of Education Key Laboratory of Contemporary Anthropology,School of Life Sciences,Fudan University,Shanghai 200433；2 National Engineering Center for Biochip at Shanghai,Shanghai 201203； 3 Department of Surgery, Tianjing Children′s Hospital,Tianjing 300074；China
  • Received:2016-04-21 Revised:2016-04-21 Online:2016-04-25 Published:2016-04-21
  • Contact: CAI Chun-quan

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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MethPrimer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-goldTM 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，hMeDIP kit）进行ChIP反应。反转录cDNA采用FastQuant 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。

Abstract:

Objective To understand whether there are al 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 MethPrimer online software, the CpG 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-goldTM Kit ). For ultrasound fractured genomic DNA, ChIP assay was performed with genomic hydroxymethyl kit (diagenode, hMeDIP Kit). The levels of MECP2 in brain tissues were detected with cDNA that was obtained by FastQuant RT Kit (With gDNase) and were quantitated using SuperReal PreMix 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, weight 500 g, length 28.3 cm; sample 4, female, weight 510 g, length 31.5 cm. The 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 . 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.