围产期的早产儿的肠道基因标记的甲基化改变

marked methylation changes in intestinal genes during the perinatal period of preterm
neonates
http://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-15-716

研究对象：围产期的早产儿（用早产猪近似代替）
期刊：BMC Genomics
发表时间：2014年8月26日

摘要

Background
The serious feeding- and microbiota-associated intestinal disease, necrotizing enterocolitis (NEC), occurs mainly in infants born prematurely (5-10% of all newborns) and most frequently after formula-feeding. We hypothesized that changes in gene methylation is involved in the prenatal maturation of the intestine and its response to the first days of formula feeding, potentially leading to NEC in preterm pigs used as models for preterm infants.

Results
Reduced Representation Bisulfite Sequencing (RRBS) was used to assess if changes in intestinal DNA methylation are associated with formula-induced NEC outbreak and advancing age from 10 days before birth to 4 days after birth. Selected key genes with differentially methylated gene regions (DMRs) between groups were further validated by HiSeq-based bisulfite sequencing PCR and RT-qPCR to assess methylation and expression levels. Consistent with the maturation of many intestinal functions in the perinatal period, methylation level of most genes decreased with advancing pre- and postnatal age. The highest number of DMRs was identified between the newborn and 4 d-old preterm pigs. There were few intestinal DMR differences between unaffected pigs and pigs with initial evidence of NEC. In the 4 d-old formula-fed preterm pigs, four genes associated with intestinal metabolism (CYP2W1, GPR146, TOP1MT, CEND1) showed significant hyper-methylation in their promoter CGIs, and thus, down-regulated transcription. Methylation-driven down-regulation of such genes may predispose the immature intestine to later metabolic dysfunctions and severe NEC lesions.

Conclusions
Pre- and postnatal changes in intestinal DNA methylation may contribute to high NEC sensitivity in preterm neonates. Optimizing gene methylation changes via environmental stimuli (e.g. diet, nutrition, gut microbiota), may help to make immature newborn infants more resistant to gut dysfunctions, both short and long term.

关键词

DNA甲基化；早产儿;坏死性小肠结肠炎

研究背景

与严重的喂养和微生物相关的肠道疾病——坏死性小肠结肠炎（NEC），主要发生于早产儿（早产儿占新生儿的5-10%），并且最常见于在配方奶喂养之后。我们猜想基因甲基化的改变与产前成熟的肠道相关，并且在配方喂养的第一天就有反映，在作为早产儿模型的早产猪中可能导致NEC。

研究结果

1、RRBS数据生成和猪小肠甲基化的特征
简化表示重亚硫酸盐测序（Reduced Representation Bisulfite Sequencing (RRBS) ）被用来评估是否肠道的DNA甲基化变化与诱导性的配方饲料使得NEC爆发同年龄从出生前10天到出生后4天相关。

2、四组仔猪的差异甲基化模型
在小组中通过具有差异甲基化基因区域(DMRs)选择的关键基因被进一步认定是有效的，通过基于HiSeq的亚硫酸盐测序的PCR技术和逆转录定量聚合酶链反应（RT-qPCR）去评估甲基化和表达水平。

表一：全球DNA甲基化谱

表二：与产前发育有关的DMRs总结(PN-DMRs, 0d-term vs. 0d-preterm), 新生的响应 (NN-DMRs, 4d-preterm vs. 0d-preterm) 和NEC的发展 (NEC-DMRs, 4d-preterm-NEC vs. 4d-preterm)。
3、不同的产前和新生儿期基因的DNA甲基化降低对肠道成熟的影响
在出生前与出生后时期，大多数基因的甲基化水平下降与提升，同围生期多种肠道功能的成熟一致。

4、结合高通量测序的BSP技术的发展有助于甲基化验证
最大数量的差异甲基化基因区域(DMRs)被发现于新生猪仔和4天早产猪之间。
在受影响的猪和带有NEC的初步证据的猪之间，几乎不存在肠的差异甲基化基因区域(DMRs)的不同。

表三：BSP结合高通量测序HiSeq甲基化验证。

5、主要基因的表达被CGI的超甲基化所抑制
在配方喂养的4天大的早产猪中，肠道代谢相关的四个基因（cyp2w1，gpr146，Top1mt，cend1）显示了显著的超甲基化在其启动子的CGIs，并且因此下调转录。
这些基因的甲基化驱动下调，可能会使不成熟的肠道代谢紊乱和严重的NEC病变。

表四：新生儿的启动子甲基化与基因转录抑制有关。

总结

出生前后肠DNA甲基化的变化，可能有助于高NEC灵敏度在早产儿中。
通过环境刺激（例如，饮食、营养、肠道菌群）去优化基因的甲基化改变，可能更有助于使未成熟的新生短期和长期的抵抗肠功能障碍。

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