父亲叶酸缺乏导致出生缺陷发生的分子机制

Folic acid as supplement for pregnant women has been effective prevention measures of birth defects, including both neural tube defects and congenital heart defects. However, some population is not responsive for folic acid supplementation but the mechanism behind this phenomenon is still elusive. This prevents development on new prevention strategies for birth defects control. The applicant’s previous studies have revealed the importance of elevated homocysteine levels caused by mutations in key enzymes in folate metabolism mutation to metabolites, which increased the risk of birth defects (Circulation, 2012, Cell Res, 2013 Eur Heart J, 2014). Recently, by setting up folate deficiency mouse models, we found that lack of folic acid in the paternal side induced significantly higher percentage of birth defects, especially after two generations (grandfather and father) of folate deficiency. Both the deformity rate in early embryogenesis and the mortality are higher in offsprings from two generations of paternal folic acid deficiency. Those results indicate the importance of folic acid supplement on paternal side to prevent birth defects. In this proposal, we will test and analyze the genome, transcriptome, and methylation changes between deformed and normal offsprings. This study will gain insight understandings on how folate deficiency on paternal side will affect the stability of the genome in both germ cells and somatic cells. Such understanding will provide valuable information on the genetic mechanisms of birth defects induced by folate deficiency in paternal side, which will prompt protection strategy on germ cells and further reduce birth defects.

孕妇补服叶酸是出生缺陷防控的有效措施，但叶酸作用机制长期未明，阻碍了甄别叶酸不应答人群并研发相应的新预防策略。申请人既往揭示出叶酸代谢关键酶变异致叶酸代谢物同型半胱氨酸水平升高而增高出生缺陷风险（Circulation 2012,Cell Res 2013,Eur Heart J 2014）。近期利用叶酸缺乏小鼠模型发现叶酸缺乏除致胚胎发育迟缓外，在两代缺乏后可致第三代畸形率上升，特别是祖父/父亲缺叶酸后第三代胚胎早期畸形率和死亡率更高，提示两代父系缺叶酸严重影响后代发育。据此推测父系叶酸缺乏对出生缺陷发生有重要贡献。拟通过对畸形后代基因组结构、转录组、甲基化的检测和生信分析等，明确父系叶酸缺乏对子代基因组稳定性的影响，分析父源性缺叶酸诱发生殖细胞突变与敏感出生缺陷发生的关系，从而首次阐明父系叶酸缺乏对出生缺陷的影响及遗传学机制，提示始自生殖细胞保护的防控策略有利于总体降低出生缺陷率。

膳食叶酸缺乏（FD）与出生缺陷的发生有关。但关联背后的机制仍然难以捉摸，尽管相关研究已在母体方面广泛开展。特别是，FD如何影响基因组稳定性尚不清楚。为了研究缺乏叶酸的饮食是否会影响基因组稳定性，给予C57BL/6小鼠缺乏叶酸（FA）的合成饮食维持两代。F0小鼠从3周龄开始接受FD饮食，其后代（F1）在断奶后开始FD饮食。喂食FD饲料的雄性和雌性F1小鼠与喂食正常饲料的F1小鼠杂交以产生F2小鼠。在E14.5和E18.5解剖并收集F2胚胎。与喂食正常饲料的F2胚胎相比，喂食FD饲料两代的F2胚胎的畸形率显著增加。对采用FD饮食的F1雄性小鼠的多个家族进行全基因组测序表明，F2胚胎的de novo突变（DNM）率是正常饮食小鼠的三倍。此外，在F2小鼠中观察到的许多DNM的等位基因比率为1:3，而不是2:2，表明这些突变可能以DNA双链错配的形式在配子细胞中累积，而不是在胚胎发育的有丝分裂生长期间发生突变。我们的研究表明，两代FD显著增强了减数分裂过程中DNM的积累，这可能有助于F2小鼠负性出生结局的增加。由于这些DNM发生在精子形成过程中，父亲补充FA对于预防出生缺陷可能是必要且有益的。

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