孕早期血清叶酸与母亲代谢酶基因多态的交互作用调节子代BDNF甲基化影响婴幼儿神经发育的机制

Folate, forms of which are known as folic acid, plays an important role in nucleotide synthesis and DNA methylation process. Lacking of folic acid is believed to be the main cause for neural tube defects, therefore, it has been recommended by World Health Organization as a daily supplement for pregnant women and couples preparing for pregnancy. Some pregnant women may take excessive folic acid, and this phenomenon emerged in China during recent years. However, over intake of folate could affects offspring's phenotypes by up-regulating their genomic DNA methylation levels. ..Under the catalyzing of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), folic acid can generate S-adenosylmethionine (SAM), which functions as an immediate methyl donor in genomic DNA methylation. In addition, methionine synthase reductase (MTRR) is a cofactor that maintains activation of MTR. More importantly, it had been widely reported that polymorphism of MTHFR, MTR, MTRR gene could affect the efficiency of folate metabolism and generation of SAM. Brain-derived neurotrophic factor (BDNF), a protein encoded by the BDNF gene in mammalian animals, helps to the survival of existing neurons, while promotes the differentiation and growth of new synapses and/or neurons. The transcription level of BDNF factors in human brain is inversely related to methylation levels of BDNF gene in blood and brain. We hypothesize that excessively ingested folate during the early pregnancy period could affect the epigenetic results of offsprings' neurodevelopment through down-regulating BDNF transcriptional level caused by up-regulated methylation level of BDNF gene. For testing this hypothesis, we have collected samples and clinical data from 3000 pregnant women and their babies for a birth cohort study since the early pregnant period of the mother participants, the objectives for this birth cohort study containing blood of early pregnant women (for measuring serum folic acid concentrations and for testing single nucleotide polymorphisms of MTHFR, MTR and MTRR genes), cord blood of newborns (for testing methylation levels of BDNF genes), and follow-up data of infants (scores of infants' early neurodevelopment behaviors). We also plan to use maternal mice given by high-folate supplemented food during the period around conception for testing our hypothesis. ..According to the results of birth cohort study and relevant animal experiment, mechanisms of maternal folic acid metabolism acting on offsprings' epigenetic phenotypes for neurodevelopment could be revealed. Furthermore, the data of this study could contribute to making an initial draft for individual folate supplements depending on maternal genotype.

叶酸参与DNA合成和甲基化影响胚胎神经发育。近年国内外加强围孕期叶酸补充，导致部分孕妇补充过量。叶酸经代谢酶MTHFR、MTR催化生成直接甲基供体SAM参与基因甲基化，MTR酶需经MTRR酶还原。过量SAM可上调DNA甲基化影响子代表观遗传。脑源性神经营养因子BDNF是神经发育的重要物质，BDNF表达水平与血液、脑组织BDNF基因甲基化程度呈负相关。我们推测，孕早期过量摄入叶酸可上调子代BDNF基因甲基化下调子代BDNF表达、不利于婴幼儿神经发育。为验证假说，我们将通过分析出生队列的母婴样本和随访数据，结合动物实验，研究母亲孕早期血清叶酸与MTHFR、MTR、MTRR基因多态的交互作用对脐带血BNDF基因甲基化、婴幼儿神经发育的影响。从孕期营养、母亲遗传背景、基因甲基化、表观遗传多层面深入研究孕早期叶酸调控婴幼儿神经发育的分子机制，为确定适用于我国围孕期妇女的叶酸补充剂量提供理论依据。

叶酸是预防胎儿神经管缺陷（NTDs）的必需维生素，是嘌呤和脱氧核苷酸合成中的主要单碳供体和受体。胚胎发育的早期阶段，细胞分裂和增殖中大量需要叶酸。因此，为预防NTDs，世界卫生组织建议育龄妇女的红细胞叶酸（RBC-FA）浓度高于400 ng/mL。目前，大多数国家检测到的育龄妇女RBC-FA浓度已高于此阈值。此外，在一项评估叶酸补充剂摄入的研究中，约12%的女性在孕前和孕期间服用量超过叶酸耐受上限（1000mg/d）。即便如此，大多科研及医务人员仍然认为，预防妊娠疾病的重要性使叶酸过量带来的潜在风险可以被忽视。有研究揭示叶酸影响神经相关基因甲基化，例如：胚胎脑部基因组甲基化似乎最容易受到孕鼠叶酸补充的影响。但没有提及后代神经发育行为的改变，这不足以支持叶酸过量对神经发育及子代发育结局影响的假设。此外，动物实验中的暴露剂量对评估药物毒理性至关重要，哺乳动物模型复杂的母胎界面代谢不可能量化胎儿叶酸。.血液中叶酸的主要形式是5-甲基四氢叶酸，它是S-腺苷甲硫氨酸（SAM）的前体。SAM是参与众多生化事件的唯一甲基供体，包括胚胎发育和体细胞遗传相关的DNA和组蛋白甲基化。特定CpG基因座的去甲基化可激活基因，基因沉默也归因于关键CpG基因座的甲基化。由此，参与叶酸代谢循环的5-甲基四氢叶酸和富甲基的甲硫氨酸、胆碱和维生素B12，可调控胎儿的表观遗传修饰，并赋予成年后个体对某些疾病易感性。斑马鱼是体外受精的模型生物，适用于观察早期胚胎发育和测试药物或营养素的有效性和毒性，并且具有与人类相似的整体神经和神经递质系统。.本研究将斑马鱼卵暴露于富甲基的营养素（叶酸，蛋氨酸，胆碱和维生素B12），直至孵化。为了证实过量补充叶酸对胚胎和出生后神经发育的影响，我们观测了幼鱼的行为，并通过RNA测序和全基因组亚硫酸氢盐测序得出差异基因。最后，通过实时定量PCR验证和焦磷酸测序验，得出叶酸过量调控的特异性CpG基因座。本研究旨在探索没造成缺陷表型的高剂量叶酸补充带来的副作用，有助于完善叶酸补充的临床指导。

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