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

描述（由申请人提供）：本申请是四年前授予的资助的竞争性更新，以支持对以下假设的全面测试，即编码叶酸代谢酶的基因中的遗传变异至少部分地导致新生儿神经管缺陷（NTD）的发生。最初的应用程序包括一个大规模的测序组件，以及用于评估这些酶中非同义取代的影响的创新策略。为了成功实施这个项目，我们组建了一个由分子遗传学、出生缺陷流行病学和儿科学专家组成的多元化团队。尽管获得的资金少于最初批准的资金，但我们的团队基本上实现了所有目标。因此，我们在几乎整个叶酸途径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，我们已经发现了两个挑衅性的，复杂的遗传特征，一个在西班牙裔和另一个在高加索人，通过基于叶酸途径建模的分析。这些特征显示了区分病例与对照的令人信服的统计学证据（P值），并可能有助于解释叶酸的预防作用与NTD发生之间的机械联系-一种科学探究中难以捉摸的机械联系。有趣的是，这些特征在每个种族中是不同的，这表明潜在的疾病机制也是不同的：西班牙裔的风险特征指向嘌呤生物合成的改变，而高加索人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文献中未解释的有趣的流行病学观察结果。 在当前的更新中，我们努力从三个方面推进这些发现。首先，我们提出了复制研究，有足够的动力，以确保这些协会的意义。其次，这些复杂的特征为解决NTD的遗传风险是由母体还是胎儿基因型驱动提供了极好的机会。第三，我们提出了机制研究，以经验确定这些遗传风险的代谢结果。由于西班牙裔特征来自常见变异的组合，因此我们将使用具有相关基因型的细胞系作为替代物来代表一系列风险特征。我们建议通过LC/MS定量叶酸途径中间体，并将代谢特征与风险特征相关联。此外，我们将通过精确的基因编辑技术“恢复”单个碱基来剖析单个等位基因对风险特征的贡献。最后，高加索人的遗传风险特征，这意味着同型半胱氨酸代谢，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的背景下探索全局DNA甲基化，这可能导致特定基因组位点的逻辑延伸。

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.