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

描述(申请人提供)：这项申请是对四年前授予的一项资助的竞争性更新，以支持对以下假设的全面测试：编码叶酸代谢酶的基因的遗传变异至少部分导致新生儿神经管缺陷(NTD)的发生。最初的应用包括大规模测序组件以及评估这些酶中非同义替换的影响的创新策略。为了成功实施这一项目，我们组建了一支由分子遗传学、出生缺陷流行病学和儿科专家组成的不同团队。尽管获得的资金比最初批准的少，但我们的团队基本上实现了所有目标。因此，我们对几乎整个叶酸途径中常见和罕见的一组不同的突变进行了分类，并为这些数据开发了新的分析策略。具体地说，通过基于叶酸途径模型的分析，我们发现了两个具有挑衅性的复杂遗传特征，一个在西班牙裔，另一个在高加索人。这些特征显示了令人信服的统计证据(P值)，可以区分病例和对照，并可能有助于解释叶酸的预防效果与NTDS发生之间的机械联系--这是科学研究一直难以捉摸的机械联系。有趣的是，这些特征在每个种族中都不同，这表明潜在的疾病机制也不同：西班牙裔风险概况指向嘌呤生物合成的变化，而高加索人的风险概况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没有解释的有趣的流行病学观察结果。在目前的更新中，我们努力从三个方面推进这些调查结果。首先，我们建议进行足够有力的复制研究，以确保这些关联的重要性。其次，这些复杂的签名提供了一个很好的机会来解决NTDS的遗传风险是由母亲还是胎儿基因决定的。第三，我们建议进行机械性研究，从这些遗传风险谱中经验性地确定代谢结果。因为拉美裔的特征是从常见变异的组合中衍生出来的，所以我们将使用具有相关基因类型的细胞系作为替代，以代表一系列的风险特征。我们建议通过LC/MS对叶酸途径中间产物进行量化，并将代谢特征与风险特征相关联。此外，我们还将通过精确的基因编辑技术“还原”单碱基，剖析个体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意味着同型半胱氨酸代谢，这与早期关于NTDS的表观遗传学维度的建议产生了共鸣。因此，我们建议在NTDS的背景下探索全球DNA甲基化，这可能导致特定基因组座位的逻辑延伸。

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.