【摘 要】
：
目的：以与唐氏综合征(Down Syndrome,DS)患者神经系统发育相关的PRDM8(PR domain containing 8,正性调节区锌指蛋白8)为研究目标,阐释DS患者外周血中羟甲基化/甲基化修饰对该基因表达的调控作用.方法：首先建立了基于KRu04氧化、亚硫酸盐转化和焦磷酸测序技术的5羟甲基胞嘧啶(5hmC)和5甲基胞嘧啶(5mC)的定量检测方法—oxBS-pyrosequenci
【机 构】
：
上海市儿童医院,上海交通大学附属儿童医院,上海医学遗传研究所 上海市儿童医院,上海交通大学附属儿童
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目的：以与唐氏综合征(Down Syndrome,DS)患者神经系统发育相关的PRDM8(PR domain containing 8,正性调节区锌指蛋白8)为研究目标,阐释DS患者外周血中羟甲基化/甲基化修饰对该基因表达的调控作用.方法：首先建立了基于KRu04氧化、亚硫酸盐转化和焦磷酸测序技术的5羟甲基胞嘧啶(5hmC)和5甲基胞嘧啶(5mC)的定量检测方法—oxBS-pyrosequencing,可进行单碱基分辨率水平的检测.并对氧化剂的浓度、氧化温度和亚硫酸氢盐转化的处理时间进行了优化,在上述技术基础上,对16个DS和19个正常儿童外周血样本中PRDM8不同区域CpG岛的5hmC/5mC进行了定量检测,并分析了其与PRDM8两个转录本表达水平的相关性.随后,采用qRT-PCR对上述样本中的羟甲基化/甲基化修饰酶TET和DNMT(DNAmethyltransferase)的表达进行了定量分析,并探讨了修饰酶的表达与5hmC/5mC水平及PRDM8表达的相关性.结果：通过优化实验条件,本研究中所建立的oxBS-pyrosequencing方法的转换效率(5hmC→U)达到了91.33％.与正常对照相比,DS样本中PRDM8的羟甲基化/甲基化修饰水平明显升高,5hmC/5mC差异修饰区域主要位于PRDM8内部启动子区.内部启动子区5hmC的升高与对应的转录本2表达上调有明显的相关性,基因内部5mC升高则与转录本1的PRDM8表达正相关. DS外周血中5mC修饰差异水平与DNMT3A的表达水平呈正相关,而在正常对照样本中两者之间无相关性.结论：本研究建立了可在单碱基分辨率同时精确检测5hmC和5mC水平的oxBS-pyrosequencing技术平台. DS患儿外周血样本PRDM8内部启动子区存在与基因表达相关的特征性5hmC/5mC差异修饰.
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