Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the <i>VTRNA2-1</i> gene, which was also hypomethylated in cases (FWER <i>p</i> = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as <i>WNT9B, MIR140</i> and <i>LHX8</i>. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

伴或不伴腭裂的唇裂（Cleft lip with or without cleft palate, CL/P）是一种常见的人类出生缺陷，其具体病因仍未完全明确。多项研究已证实，围受孕期补充叶酸可降低子代发生CL/P的风险。本研究旨在验证这一假说：叶酸的预防作用可通过表观遗传修饰实现，具体通过检测DNA甲基化变化是否与CL/P存在关联。为更清晰地观察母体叶酸对子代表观基因组的潜在影响，本研究聚焦于美国强制膳食叶酸强化政策实施前的出生病例（即1997年及更早出生的新生儿）。本研究为纳入182名研究对象的病例对照研究，通过Illumina®人类甲基化450K芯片（Illumina® Human Beadchip 450K array）对存档的新生儿干血斑样本进行全基因组DNA甲基化水平检测。与对照组相比，CL/P病例组呈现出显著的全基因组低甲基化特征：在所检测的CpG位点中，63%在病例新生儿中的甲基化水平更低，这一趋势在按种族分层的亚组中同样存在。共有28个CpG位点达到全基因组表观遗传显著性水平，且所有位点均在病例组中呈现低甲基化状态。与CL/P关联最显著的差异甲基化区域涵盖VTRNA2-1基因，该区域在病例组中同样呈现低甲基化（家族错误率校正p值=0.014）。该区域此前已被鉴定为营养响应性亚稳态等位基因，总体而言，与CL/P相关的甲基化变化在推定的亚稳态等位基因区域及其邻近区域更为显著。对CL/P相关差异甲基化区域（Differentially Methylated Regions, DMRs）进行基因集富集分析后发现，参与腭部发育的基因如WNT9B、MIR140及LHX8存在显著富集现象。CL/P相关的DNA甲基化变化可部分解释口面裂对母体叶酸水平产生响应的分子机制。