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. Overall design: RNA-seq in E8.5 headfolds, collected from B6 and 129S6.B6-Gulo<-/-> embryos, with or without VitC supplementation.

母体膳食营养不足可在妊娠期间重塑子代表观基因组，进而引发出生缺陷与发育紊乱。维生素C（VitC）是十-十一易位（TET）家族DNA去甲基化酶的关键辅因子，但目前其缺乏对胚胎发育的影响仍不明确。本研究发现，母体VitC缺乏联合遗传易感因素可通过DNA甲基化调控异常，引发包括神经管缺陷（NTDs）在内的先天性畸形。我们此前曾报道，在C57BL/6J（B6）同源近交系小鼠中，Tet1敲除（KO）小鼠的神经管缺陷发生率较低；但在129S6.B6背景下进行远交或高度回交（早期同源近交）时，其发生率可提升2至3倍。同样，在L-古洛糖酸内酯氧化酶（Gulo）敲除（KO）小鼠（其与人类一样无法自主合成VitC）中，母体VitC缺乏会在原肠胚形成这一易感窗口期，导致远交系小鼠出现高外显率的先天性畸形。VitC缺乏的129S6.B6-Gulo-/-胚胎头褶组织中存在DNA高甲基化特征，而表型完全正常的B6胚胎则无此现象。在远交系Gulo-/-胚胎脑组织中，携带DNA高甲基化（TET功能异常的标志性特征）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，可挽救VitC缺乏胚胎的严重发育缺陷，并使多数位点的DNA甲基化水平恢复正常。本研究结果提示，为存在出生缺陷风险的孕妇及时补充VitC，或可预防目前叶酸补充无法干预的多种出生缺陷，并提升子代的"health-span"。实验设计：对B6及129S6.B6-Gulo±胚胎的E8.5头褶组织进行RNA测序，分组为VitC补充组与非补充组。