Impact of Genetic Variation on Folate Metabolism: Etiology of Neural Tube Defect

描述（由申请人提供）：该申请是对四年前授予的一项资助的竞争性更新，该资助旨在支持对叶酸代谢酶编码基因的遗传变异至少在一定程度上导致新生儿神经管缺陷（NTDs）发生的假设进行全面测试。最初的应用程序包括大规模测序组件以及评估这些酶的非同义取代影响的创新策略。为了成功地实施这个项目，我们在分子遗传学、出生缺陷流行病学和儿科方面组建了一个多元化的专家团队。尽管获得的资金比最初批准的要少，但我们的团队基本上完成了所有的目标。因此，我们在几乎整个叶酸通路中编目了一组不同的突变，常见的和罕见的，并为这些数据开发了新的分析策略。具体来说，我们通过基于叶酸通路模型的分析，发现了两个具有挑衅性的复杂遗传特征，一个在西班牙裔中，另一个在白种人中。这些特征显示了将病例与对照区分开来的令人信服的统计证据（p值），并可能有助于解释叶酸的预防作用与被忽视热带病发生之间的机制联系——这种机制联系在科学探究中一直难以捉摸。有趣的是，这些特征在每个种族中都是不同的，这表明潜在的疾病机制也不同：西班牙裔的风险概况指出嘌呤生物合成的改变，而白种人的风险概况则涉及同型半胱氨酸代谢。这些重要的观察结果进一步支持了那些在文献中无法解释的有趣的流行病学观察结果。在当前的更新中，我们努力以三种方式推进这些发现。首先，我们提出了足够有力的重复性研究，以确保这些关联的重要性。其次，这些复杂的特征提供了一个很好的机会来解决被忽视热带病的遗传风险是由母体还是胎儿基因型驱动的。第三，我们提出机制研究，以经验确定这些遗传风险概况的代谢结果。由于西班牙人的特征来源于常见变异的组合，我们将使用具有相关基因型的细胞系作为替代品来代表一系列风险概况。我们建议通过LC/MS定量叶酸途径中间体，并将代谢谱与风险谱相关联。此外，我们将通过精确的基因编辑技术“还原”单个碱基来剖析单个等位基因对风险特征的贡献。最后，白种人的遗传风险特征与同型半胱氨酸代谢有关，这与早期提出的ntd的表观遗传维度相一致。因此，我们建议在ntd的背景下探索全球DNA甲基化，这可能导致特定基因组位点的逻辑扩展。

DESCRIPTION (provided by applicant): This application is a competitive renewal of a grant that was awarded four years ago to support a comprehensive test of the hypothesis that genetic variation in genes encoding folate metabolizing enzymes was responsible, at least in part, for the occurrence of neural tube defects (NTDs) among newborns. The original application included a large-scale sequencing component as well as innovative strategies for assessing the impact of nonsynonymous substitutions in these enzymes. To successfully implement this project, we assembled a diverse team of experts in molecular genetics, birth defects epidemiology, and pediatrics. Despite being awarded less funding than originally approved, our team accomplished essentially all of the aims. As a result, we have catalogued a diverse set of mutations, common and rare, in nearly the entire folate pathway and developed novel analytical strategies for such data. Specifically, we have uncovered two provocative, complex genetic signatures, one in Hispanics and another in Caucasians, through analyses based on folate pathway modeling. These signatures show compelling statistical evidence (P-values) for distinguishing cases from controls, and may help explain the mechanistic link between the preventive effects of folate and the occurrence of NTDs - a mechanistic link that has been elusive to scientific inquiry. Interestingly, these signatures are different in each ethnicity suggesting that the underlying disease mechanisms are also different: the Hispanic risk profile points to alterations in purine biosynthesis whereas that in Caucasians implicates homocysteine metabolism. These important observations further support the intriguing epidemiological observations that exist, unexplained in the literature. In the current renewal we strive to advance these findings in three ways. First, we propose replication studies that are sufficiently powered to secure the significance of these associations. Second, these complex signatures offer an excellent opportunity to resolve whether the genetic risk for NTDs is driven by the maternal or fetal genotype. Third, we propose mechanistic studies to empirically determine the metabolic outcome from these genetic risk profiles. Because the Hispanic signature is derived from a combination of common variants, we will use cell lines with relevant genotypes as surrogates to represent a range of risk profiles. We propose to quantify folate pathway intermediates via LC/MS and correlate metabolic profile to risk profile. In addition, we will dissect the contribution of individual alleles to the risk signature by "reverting" single bases vi precise gene editing techniques. Finally, the genetic risk signature in Caucasians, which implicates homocysteine metabolism, resonates with earlier suggestions of an epigenetic dimension to NTDs. Thus, we propose to explore global DNA methylation in the context of NTDs, which could lead to logical extensions at specific genomic loci.