脆性X综合征二例CGG重复序列及甲基化分析  

Tissue heterogeneity analysis of CGG-repeat mutation in two fragile X affected male fetuses

作　　者：黄文[1] 罗仕玉 杜倩[1] 杨璞[1] 谭虎[1] 邬玲仟[1] 段然慧[1] 

机构地区：[1]中南大学生命科学学院医学遗传学国家重点实验室,长沙410078

出　　处：《中华儿科杂志》2016年第4期287-289,共3页Chinese Journal of Pediatrics

基　　金：国家自然科学基金（81571253、81172513、81071028）;国家重点基础研究发展计划（973计划）（2012CB944600）;教育部新世纪优秀人才支持计划（NCET-10-0832）

摘　　要：目的比较脆性X综合征男性胎儿不同组织中CGG重复扩增和甲基化情况。 方法分别于2013年8月、2012年5月采集1例全突变和1例前突变/全突变嵌合23周终止妊娠男性胎儿的不同组织样本，并提取基因组DNA。联合高GC含量PCR和甲基化敏感Southern印迹技术，测量各组织中CGG重复数、分析甲基化水平，并通过实时PCR和Western印迹分析FMR1表达情况。 结果两例胎儿各组织间CGG重复数目一致，但嵌合胎儿各组织中CGG重复甲基化情况不同，1例脑、皮肤、睾丸和肾组织，均有1个约为540个CGG重复的主要全突变条带，完全甲基化。另1例脑区、皮肤、睾丸、肺、胃、肠道、肝、肾、心脏以及外周中血均检测到160、470和1 100个CGG重复的3条主要条带。检测出脑组织中前突变比例最低，相应FMPR表达最少。 结论明确了CGG重复扩增和甲基化的组织异质性。基于嵌合胎儿母亲为全突变，推测母源全突变CGG重复在三胚层分化前缩减。Objective To compare the CGG-repeat-length and its methylation status in fetal tissues and to explicate the heterogeneity of CGG repeats. Method Multiple tissues from a full mutation （August 2013） and a mosaic aborted fetus of 23-week gestation（May 2012） were collected and genomic DNA from these tissues was extracted. The CGG-repeat-length and methylation status in fetal tissues were determined by a combined strategy of Southern blotting and GC-Rich PCR. FMR1 expression was measured by real time PCR and Western blotting. Result CGG-repeat-length in different tissues of each fetus was similar. A major methylated band in the full mutation range （540 CGG repeats） was detected in the brain, skin, testis and kidney tissues of Case 1. An unmethylated premutation band with 160 CGG repeats, and another two bands with 470 and 1 100 CGG repeats in the full mutation range were shown in the brain, skin, testis, lung, stomach, gut, liver, kidney, heart and blood of Case 2. However, the methylation status of CGG repeats in the mosaic fetus was heterogeneous among different tissues. The lowest premutation ratio was in the brain of the mosaic fetus compared with other tissues, and correspondingly FMR1 expression in its brain was minimum. Conclusion This study clarify the tissue heterogeneity of CGG repeats and provides information for the genetic counseling and clinical diagnosis in fragile X syndrome. Based on the fact that the mosaic fetus＇ mother is a carrier of full mutation, it is speculated that the maternal CGG repeat has contracted before the differentiation of trilaminar germ disc.

关 键 词：脆性X综合征 镶嵌现象 遗传异质性 CGG重复序列 

分 类 号：R714.5[医药卫生—妇产科学] R440[医药卫生—临床医学]