HOXA5基因转录起始点上游区域高甲基化水平与神经管缺陷的关系

Relationship of Methylation within Upper Stream Region of Transcription Starts Site of HOXA5 Gene with Neural Tube Defects

目的探讨同源异形盒基因转录起始点(TSS)上游区域甲基化水平与神经管缺陷(NTDs)的关系。方法采用两阶段病例对照研究设计。第一阶段选择NTDs病例10例和对照8例作为研究对象,运用全基因组甲基化芯片检测其脑或脊髓DNA全基因组甲基化水平。针对HOXA5基因差异甲基化区域,在第二阶段扩大样本量(52例病例和23例对照)进行验证。提取脑或脊髓组织DNA,使用Mass ARRAY系统的基质辅助激光解吸电离飞行时间质谱技术对HOXA5基因差异性甲基化区域进行验证。结果第一阶段在全基因组甲基化芯片结果中筛选出HOXA5基因TSS上游区27个CpG位点在病例组甲基化水平高于对照组(P<0.05)。第二阶段利用Mass ARRAY系统针对上述发现的差异区域进行检测,成功检测到10个可用于分析CpG位点。NTDs病例组及其亚型脊柱裂组和无脑畸形组中甲基化水平高于对照组的CpG位点数各有7、6和2个(P<0.005)。HOXA5基因TSS上游区域病例组平均甲基化水平高于对照组[NTDs病例组:(31.3±13.9)%,对照组:(21.4±9.7)%],调整胎儿性别和孕周后OR值为1.09(1.03~1.16)。结论 HOXA5基因TSS上游区域高甲基化与胎儿神经管缺陷风险增加存在关联性。

Objective To investigate the relationship between the methylation level of transcription starts site（TSS） upper stream of homeobox gene and the neural tube defects（NTDs）.Methods A case-control study of two stages was designed.In the first stage,10 cases and 8 controls were extracted,in whom Illumina Infinium Human Methylation 450 k genome-wide beadchip was used for the quantification of DNA methylation levels of brain and spinal tissue.In the second stage,differentially methylated region within HOXA5 gene was detected with a larger numbers of samples（52 cases and 23 controls）.DNA of brain or spinal tissue was extracted,and Matrix-assisted laser desorption ionization time-of-flight mass spectrometry technique of Mass ARRAY platform was employed for the validation of differentially methylated region of HOXA5 gene.Results In the firststage,27 CpG sites within TSS region of HOXA5 gene were found to be significantly hyper-methylated in case group compared to control group（P〈0.05）.In the second stage,a total of 10 CpG sites were analyzable within the differentially methylated region in the first stage.In the NTD case group,spinal bifida subgroup,and anencephaly subgroup,there were 7,6,and 2 sites with significantly higher methylation levels than that of control group（P〈0.05）.The average methylation level of TSS upper stream region within HOXA5 gene was higher in case group than control group [case group：（31.3±13.9） %,control group：（21.4±9.7） %],and the odds ratio after adjusting gender of fetus and pregnant week was 1.09（1.03-1.16）.Conclusion Hypermethylation within TSS upper stream region of HOXA5 gene in fetus is associated with a higher risk of NTDs.