Maternal Vitamin C Deficiency and Genetic Risk Factors Contribute to Congenital Malformations through Dysregulation of DNA Methylation. [RNA-Seq E8.5]

Maternal dietary insufficiencies can reshape the offspring epigenome during gestation, contributing to birth defects and developmental disorders. Vitamin C (VitC) is a critical co-factor for Ten-Eleven-Translocation (TET) DNA demethylases, but the impact of its deficiency on embryonic development remains unclear. Here, we show that insufficient maternal VitC combined with genetic susceptibility can give rise to congenital malformations, including neural tube defects (NTDs), through dysregulation of DNA methylation. We previously reported NTDs in Tet1 knockout (KO) mice at low penetrance in C57BL/6J (B6) congenic inbred strains, but at two- to three-fold higher rates when outbred or highly backcrossed (incipient congenic) on a 129S6.B6 background. Similarly, maternal VitC deficiency in L-gulonolactone oxidase (Gulo) KO mice, which like humans are unable to synthesize VitC, resulted in highly penetrant congenital malformations in non-inbred mice, during a vulnerable window coinciding with gastrulation. DNA hypermethylation is a signature of VitC-deprived 129S6.B6-Gulo-/- embryonic headfold tissues, being absent in B6 embryos which are grossly normal. In outbred Gulo-/- embryonic brains, genomic regions harboring hypermethylation hallmarks of TET dysfunction - increased with the severity of embryonic pathologies. A moderate reduction in VitC status is sufficient to induce hypermethylated regions and cause NTDs. Severe embryonic defects in VitC-deprived embryos can be rescued by timely re-supplementation of VitC at the onset of gastrulation, which normalized DNA methylation at most loci. Our results suggest that promoting timely VitC supplementation by at-risk pregnant mothers may prevent many birth defects currently refractory to folic acid supplementation and enhance the health-span of future generations. RNA-seq in E8.5 headfolds, collected from B6 and 129S6.B6-Gulo<-/-> embryos, with or without VitC supplementation.母体膳食摄入不足可在妊娠阶段重塑后代表观基因组，进而引发出生缺陷与发育紊乱。维生素C（Vitamin C, VitC）是十-十一易位（Ten-Eleven-Translocation, TET）DNA去甲基化酶的关键辅因子，但其缺乏对胚胎发育的影响迄今尚未明确。本研究证实，母体VitC缺乏联合遗传易感因素，可通过DNA甲基化失调诱发包括神经管缺陷（Neural Tube Defects, NTDs）在内的先天性畸形。我们此前曾报道，Tet1基因敲除（knockout, KO）小鼠在C57BL/6J（B6）同源近交品系中，神经管缺陷的外显率较低；但当在129S6.B6背景下进行远交或高度回交（初期同源近交）时，畸形发生率提升2至3倍。类似地，在与人类一样无法自身合成VitC的L-古洛糖酸内酯氧化酶（L-gulonolactone oxidase, Gulo）基因敲除小鼠中，母体VitC缺乏会在与原肠胚形成重合的易感窗口期，导致远交小鼠出现高外显率的先天性畸形。对VitC缺乏的129S6.B6-Gulo-/-胚胎头褶组织的分析显示，其存在DNA高甲基化特征，而外观正常的B6胚胎则无此异常。在远交Gulo-/-胚胎脑组织中，携带TET功能失调相关高甲基化特征的基因组区域数量，随胚胎病理严重程度升高而增加。仅需适度降低VitC水平，即可诱导高甲基化区域形成并引发神经管缺陷。在原肠胚形成初期及时补充VitC，可使多数位点的DNA甲基化水平恢复正常，从而挽救VitC缺乏胚胎的严重发育缺陷。本研究结果提示，为存在生育风险的孕妇及时补充VitC，或可预防当前叶酸补充方案难以干预的多种出生缺陷，进而提升后代的健康寿命。本研究的转录组测序（RNA-sequencing, RNA-seq）样本采集自B6及129S6.B6-Gulo+/-胚胎的E8.5头褶组织，分组设置为是否补充VitC。