叶酸不应答性神经管畸形发生机制及预防新策略

Folic acid supplementation is a great improvement in the prevention and control of major birth defects such as neural tube defects (NTD), but there are still 30 to 50% of NTD populations do not respond to folic acid and the mechanism is not clear. We analyzed the microarray data of GSE51285 which contains the RNA expression profiles of livers from five NTD mouse mutants (heterozygous females) and their corresponding wild type controls. Those five NTD mutants have different responsiveness to FA supplementation. Eight genes differentially expressed between FA-responsive and FA-nonresponsive mutants were identified. Those genes are involved in mitochondrial one carbon metabolism, de novo purine synthesis, and glutathione generation; suggesting that formate and anti-oxidant levels may be effective interventions in FA-resistant NTDs. Knocking out MFT, one of those eight genes, caused NTD phenotype in homozygous mice. Importantly, they do not respond to folic acid supplementation. But formate could significantly reduce the rate of NTD in homozygous mice. Therefore, we propose that the mitochondrial one carbon metabolism is important for folate-nonresponsive NTD. This project intends to identification of mutations in those key enzymes involved in the mitochondrial one carbon metabolism in NTD cohort and examines their functional changes. We will also investigate the role of formate in neural tube closure. The outcome of this project could help to develop a new possible intervention for NTD besides folic acid, which could provide new strategies for reducing overall birth defects.

补服叶酸是神经管畸形（NTD）等重大出生缺陷防治的巨大进步，但30-50%的NTD人群对叶酸不应答且机制不清。申请人基于对叶酸应答和不应答的5个小鼠模型的生信分析发现8个主要差异基因，其参与了线粒体内一碳代谢和细胞的抗氧化过程，提示线粒体一碳代谢的主要产物甲酸盐（formate）可用于叶酸不应答性NTD的预防。进一步敲除上述差异基因之一的MFT建立的小鼠模型确有NTD表型且对叶酸不应答，而补服甲酸盐可极大逆转NTD表型。据此我们提出叶酸不应答性NTD的线粒体代谢假说。本项目拟在NTD队列中鉴定线粒体一碳代谢相关基因的新突变及其功能改变，明确甲酸盐在神经管闭合中的作用及机制，旨在为叶酸不应答人群提供可能的干预药物，丰富对NTD病因和发病机制的理解，为总体降低出生缺陷提供新策略。

补服叶酸是神经管畸形（NTD）等重大出生缺陷防治的巨大进步，但30-50%的NTD人群对叶酸不应答且机制不清。本项目聚焦在线粒体叶酸转运体和纤毛通路上，对叶酸不敏感型的小鼠进行了深入的表型和机制分析。首先，线粒体的甲酸转移体MFT敲除的小鼠有NTD表型，且补服甲酸盐可极大逆转NTD表型。该成果对于叶酸不应答型的NTD的预防策略有重要补充作用。其次，针对纤毛通路，聚焦在动力蛋白中间链组分WDR34和WDR60上。纤毛中间链蛋白WDR34是SHH激活所必需的动力蛋白中间链组件。我们首次发现Wdr34除了参与SHH信号的激活以外，还对PCP信号有促进作用。我们从100例汉族无脑儿（脑部NTD）患者中筛查到WDR34基因的错义突变不仅丧失调控SHH信号的功能，也丧失调控PCP信号的功能。WDR34的突变可能通过影响SHH和PCP信号通路而导致人的NTDs。WDR60除了在胚胎发育中至关重要，还在大脑发育过程中新生神经元的外向迁移中发挥重要作用。这一作用于WDR60与微管组织中心互作相关。我们的发现确认了WDR60的非纤毛功能。最后，我们还成功建立了从人源胚胎干细胞体外培养神经管类器官的系统，并利用该系统检测到高糖可以导致类器官中pH3阳性细胞的极性分布异常，己糖激酶1（HK1）极性分布异常，以及类器官的肌动蛋白Actin内区域减小。这些成果拓宽了在神经管发育中可能由高葡萄糖引起的细胞效应。总之，我们的成果为叶酸不应答人群提供可能的干预策略，并丰富了对NTD病因和发病机制的理解。