先天性小耳畸形全基因组甲基化谱的研究

Experimental research on DNA methylation profile in congenital microtia

目的筛查先天性小耳畸形患者整个基因组存在甲基化水平异常的CpG岛和CpG位点及其相关差异基因，进而探讨基因甲基化水平的异常与先天性小耳畸形发病之间的关系。方法收集2009年7月至2010年12月先天性小耳畸形患者术中废弃的残耳软骨组织为研究对象共50例（实验组），非耳畸形（耳外伤）患者的正常耳软骨组织34例作为对照。应用NimblegenCpG启动子芯片，对实验组3例及对照组3例样本的基因组DNA进行全基因组28226个CpG岛扫描，筛选存在CpG岛甲基化水平差异的基因；应用SpectroCHIP芯片对实验组47例，对照组31例的差异基因的CpG岛进行各个CpG位点的检测，筛选出存在甲基化水平差异的CpG位点。结果实验组与对照组在全基因组范围内存在36个具有甲基化水平差异的CpG岛，其中有29个与已命名的29个基因相关。经t检验分析，在COL18A1、MYH14、RBMY1A1、ZIC3这4个差异基因的6个CpG位点，2组甲基化水平差异具有统计学意义（P〈0．05），实验组和对照组甲基化水平分别为：COL18A1—2-CpG-170．9783±0．0235、0．9526±0．0589；MYH14-CpG-170．9600±0．0414、0．9284±0．0655；RBMY1A1—1-CpG-3．40．9966±0．0055、0．9914±0．0069；RBMY1A1—1-CpG-130．9648±0．0118、0．9757±0．0127；ZIC3_3-CpG-150．0867±0．0212、0．0543±0．0399；ZIC3—2-CpG-270．3775±0．1816、0．4723±0．0439。结论初步建立了先天性小耳畸形甲基化谱，COL18A1、MYH14、RBMY1A1、ZIC3基因甲基化水平差异可能与小耳畸形的发病相关。

Objective To screen for abnormal methylation in CpG islands and CpG sites through whole genome of congenital microtia to identify their associated genes. To discuss the relationship between abnormal methylation level of genes and the etiology of congenital microtia. Methods Residual ear cartilage of 50 patients with microtia was collected with ear cartilage of 34 patients without ear malformations as control. Nimblegen CpG promoter array was chosen to screen the 28 226 CpG islands in the whole genome of both experimental and control groups. The genes with differential methylated CpG islands were selected. SpectroCHIP array was chosen to detect the methylation level of each CpG site in abnormal methyletion CpG islands of both experimental and control groups. The CpG sites with differential methylation level were selected. Results There were 36 CpG islands with differential methylated level in whole genome between experimental group and control group, among which 29 CpG islands were connected with 29 named genes. In the abnormal methylated CpG islands of COL18A1, MYH14, RBMYIA1 and ZIC3, 6 differentially methylated CpG sites were found with statistical significance. The methylation level of these 6 CpG sites in experimental group and control group were COL18A1_2_ CpG_17 0. 978 3 ±0. 023 5 and 0. 952 6 ± 0. 058 9 ; MYH14_CpG_17 0. 960 0 ± 0. 041 4 and 0. 928 4 ± 0. 065 5 ; RBMY1A1_1_CpG_ 3.4 0. 996 6 ± 0. 005 5 and 0. 991 4 ± 0. 006 9 ; RBMY1A1_1_CpG_13 0. 964 8 ± 0. 011 8 and 0. 975 7 ± 0. 012 7 ; ZIC3_3_CpG_15 0. 086 7 ± 0. 021 2 and 0. 054 3 ± 0. 039 9 ;ZIC3_2_CpG_27 0. 377 5 ± 0. 181 6 and 0. 472 3 ± 0. 043 9. Conclusions The DNA methylation profile of the entire genome is initially established. The abnormal methylated CpG islands of COL18A1, MYH14, RBMY1A1 and ZIC3 might be related to the pathogenesis of microtia.