Genome-Wide Screening of Aberrant Methylation Loci for Nonsyndromic Cleft Lip

The pathogenicity of cleft lip (CL) is pretty complicated since it is influenced by the interaction of environment and genetic factors. The

We detected numerous aberrantly methylated loci in lesion tissues of NSCL fetus. Aberrant gene expression in the REELIN signaling pathway might be related with NSCL. Decreased transcription of DAB1, a member of REELIN signal pathway, resulted from its abnormal high methylation, which might be one of the factors underlying the occurrence of NSCL. 非综合症唇裂的甲基化异常位点筛查摘要背景：唇裂病因尚不清楚，是环境因素和遗传因素相互作用的复杂多基因疾病。本研究的目的是利用甲基化芯片技术筛查单纯性唇裂胎儿局部病变组织的甲基化位点，并利用焦磷酸测序技术进行大样本验证初步候选基因。 方法：搜集了15例引产胎儿正常唇部组织与16例单纯性唇裂引产患儿局部病变组织样本。选择了周龄相匹配的NSCL组和对照组胎儿各3例，取唇部组织提取DNA，采用Illumina公司的Infinium Human Methylation450 BeadChip芯片进行检测。应用R软件的minfi包、GO注释、Pathway代谢通路注释、多样品间表达模式聚类分析等生物信息学方法对差异位点和基因进行功能分析。采用Real-time PCR的方法扩大样本量对筛选出的候选基因在16例患者组和15例对照组中的表达情况进行检测。用焦磷酸测序技术对候选基因位点的甲基化异常进行了验证。 结果：NSCL胎儿的病变组织与正常对照组织相比存在大量的甲基化异常位点，其中高甲基化位点3661个，低甲基化位点1218个。这些甲基化异常位点存在于2849个基因之中。从位点分布的位置来看，在筛选出来的甲基化水平差异位点中，分布于基因本体区、CpG 岛的位点占很大比例。另外，也有一些分布在TSS1500区域和岛前区域。REELIN细胞通路中的两个关键转录因子（DAB1和FYN）在患者组均存在高甲基化的位点。利用Real-time PCR的方法对我们筛选出来的9个候选基因在病变组织中的表达情况进行了检测。结果表明，DAB1、FYN、REELIN基因在NSCL胎儿病变组织中的表达情况与对照组相比，患者组表达下调且有显著差异（P <0.05）。焦磷酸测序验证结果显示，位于DAB1基因体区CpG岛的两个CG位点甲基化水平存在显著差异（P<0.05）。即患者组存在高甲基化现象。 结论：NSCL胎儿的病变组织存在大量的甲基化异常位点。NSCL胎儿的病变组织中DAB1基因异常的高甲基化导致DAB1低表达是NSCL病理改变发生的原因之一。REELIN信号通路基因表达异常可能与NSCL发生有关。.

Fifteen healthy and sixteen NSCL fetal lip tissue samples were collected. The Infinium HumanMethylation450 BeadChip was used to screen aberrant methylation loci in three NSCL and three healthy lip tissues. The differential methylation sites and functions of the annotated genes between NSCL and healthy lip tissues were analyzed using minfi package of R software, cluster analysis, Gene Ontology (GO) annotation, and metabolic pathway annotation. Gene expression was assessed in nine differentially methylated genes by real-time polymerase chain reaction (PCR). The transcriptions mRNA levels of three out of nine candidate genes were downregulated remarkably in NSCL lip tissues, and these three genes' abnormal methylation loci were validated by pyrosequencing in 16 NSCL cases and 15 healthy cases.

In total, 4879 sites in the genes of NSCL odinopoeia fetuses showed aberrant methylation when compared with normal lip tissue genome. Among these, 3661 sites were hypermethylated and 1218 sites were hypomethylated as compared to methylation levels in healthy specimens. These aberrant methylation sites involved 2849 genes and were widely distributed among the chromosomes. Most differentially methylated sites were located in cytosine-phosphoric acid-guanine islands. Based on GO analysis, aberrantly methylated genes were involved in 11 cellular components, 13 molecular functions, and a variety of biological processes. Notably, the transcription of DAB1, REELIN, and FYN was significantly downregulated in lesion tissues of NSCL fetus (P < 0.05). Pyrosequencing results validated that there were two loci in DAB1 with high methylation status in patient tissues (P < 0.05). Conclusions: We detected numerous aberrantly methylated loci in lesion tissues of NSCL fetus. Aberrant gene expression in the REELIN signaling pathway might be related with NSCL. Decreased transcription of DAB1, a member of REELIN signal pathway, resulted from its abnormal high methylation, which might be one of the factors underlying the occurrence of NSCL. 非综合症唇裂的甲基化异常位点筛查摘要背景：唇裂病因尚不清楚，是环境因素和遗传因素相互作用的复杂多基因疾病。本研究的目的是利用甲基化芯片技术筛查单纯性唇裂胎儿局部病变组织的甲基化位点，并利用焦磷酸测序技术进行大样本验证初步候选基因。 方法：搜集了15例引产胎儿正常唇部组织与16例单纯性唇裂引产患儿局部病变组织样本。选择了周龄相匹配的NSCL组和对照组胎儿各3例，取唇部组织提取DNA，采用Illumina公司的Infinium Human Methylation450 BeadChip芯片进行检测。应用R软件的minfi包、GO注释、Pathway代谢通路注释、多样品间表达模式聚类分析等生物信息学方法对差异位点和基因进行功能分析。采用Real-time PCR的方法扩大样本量对筛选出的候选基因在16例患者组和15例对照组中的表达情况进行检测。用焦磷酸测序技术对候选基因位点的甲基化异常进行了验证。 结果：NSCL胎儿的病变组织与正常对照组织相比存在大量的甲基化异常位点，其中高甲基化位点3661个，低甲基化位点1218个。这些甲基化异常位点存在于2849个基因之中。从位点分布的位置来看，在筛选出来的甲基化水平差异位点中，分布于基因本体区、CpG 岛的位点占很大比例。另外，也有一些分布在TSS1500区域和岛前区域。REELIN细胞通路中的两个关键转录因子（DAB1和FYN）在患者组均存在高甲基化的位点。利用Real-time PCR的方法对我们筛选出来的9个候选基因在病变组织中的表达情况进行了检测。结果表明，DAB1、FYN、REELIN基因在NSCL胎儿病变组织中的表达情况与对照组相比，患者组表达下调且有显著差异（P <0.05）。焦磷酸测序验证结果显示，位于DAB1基因体区CpG岛的两个CG位点甲基化水平存在显著差异（P<0.05）。即患者组存在高甲基化现象。 结论：NSCL胎儿的病变组织存在大量的甲基化异常位点。NSCL胎儿的病变组织中DAB1基因异常的高甲基化导致DAB1低表达是NSCL病理改变发生的原因之一。REELIN信号通路基因表达异常可能与NSCL发生有关。.