Genetic Risk for Orofacial Clefts in the Folate/Homocysteine Pathway

描述（由申请人提供）：口面裂，特别是唇裂和腭裂，是常见的和昂贵的先天性异常，其病因仍在很大程度上未知。一个最有希望的线索口面裂的原因是，妇女谁使用含有叶酸的维生素在怀孕早期的风险要低得多裂影响怀孕。虽然叶酸有助于降低这些风险的潜在机制尚不清楚，但有证据表明，叶酸摄入量通过补偿叶酸/一碳代谢中的不稳定性来预防裂缝。然而，通过关联研究明确识别这一途径中的遗传决定因素已被证明是难以捉摸的。我们提出的进一步确定口面裂背后的分子遗传机制的方法是基于我们小组以前的两个观察结果。首先，深度测序揭示了叶酸途径基因中大量新的非同义变异（频率<=1%），这些变异对酶功能产生不利影响，但可以通过补充叶酸来补救。第二，在脊柱裂的背景下，所有叶酸途径基因的发现测序研究，一种与孤立的裂缝有许多相似属性的异常，仅通过分析生物学相关的等位基因组合揭示了令人信服的风险特征。这些数据表明，常见和罕见的等位基因的组合被整合到代谢功能中，最终成为疾病风险的基础。因此，我们假设，在一个碳代谢的遗传易感性也可能是裂缝的病因，这些易感性可以赋予低频和常见的等位基因，并可能通过相关的组合之间的协同作用途径变体。为了验证这一假设，我们将对来自约375名受裂婴儿和约375名对照人群的所有叶酸/高半胱氨酸途径基因（N=32）的编码区进行测序。我们将测试酶的变体，单独和组合，其功能 影响和营养补救的基础上，定量细胞为基础的分析，在酵母S。酿酒酵母，并将等位基因分布和功能研究与临床表型相关联。此外，我们还将从两个方面评估口面裂小鼠模型与人类病因学的相关性。首先，我们将对那些对唇/腭闭合有令人信服的贡献的小鼠基因（N=20）的人类直系同源物的编码区进行测序，以鉴定突变的全谱，以测试这些基因/变体是否在人类唇腭裂发育中发挥作用。其次，我们将调查的假设，在小鼠实验的基础上，叶酸通过其控制甲基供体流量和随后的表观遗传变化发挥其预防作用。因此，我们将探讨在裂影响和控制新生儿，这可能会导致在特定的基因组位点的逻辑延伸的整体DNA甲基化。这些研究将更好地确定口面裂的因果关系，以及揭示营养补充剂的治疗机制。这项研究计划利用了一个正在进行的和成功的合作，团结了一个独特的专业知识组合，其执行。

DESCRIPTION (provided by applicant): Orofacial clefts, specifically cleft lip and cleft palate, are common and costly congenital anomalies whose etiologies remain largely unknown. One of the most promising clues to the causes of orofacial clefts is that women who use vitamins containing folic acid in early pregnancy are at much lower risk for cleft-affected pregnancies. Although the underlying mechanisms by which folic acid contributes to these reduced risks are unknown, the evidence suggests that folate intake prevents clefts by compensating for susceptibilities in folate/one-carbon metabolism. However, clear identification of genetic determinants in this pathway through association studies has proven elusive. The approach we propose to further define the molecular genetic mechanisms behind orofacial clefts is based on two previous observations from our group. First, deep sequencing reveals a substantial number of novel, nonsynonymous variants in folate pathway genes (frequencies <=1%) that adversely affect enzyme function, yet are remediable by folate supplementation. Second, a discovery-sequencing study of all folate pathway genes in the context of spina bifida, an anomaly with many similar attributes to isolated clefts, revealed compelling risk signatures only by analyzing biologically relevant allelic combinations. These data suggested that combinations of alleles, both common and rare, are integrated into metabolic function, which ultimately underlies disease risk. Thus, we hypothesize that genetic susceptibilities in one-carbon metabolism may also be etiological for clefts and that these susceptibilities can be conferred by both low-frequency and common alleles, and possibly by synergy between relevant combinations of pathway variants. To test this hypothesis, we will sequence the coding regions in all folate/homocysteine pathway genes (N=32) from a population of ~375 cleft-affected infants and ~375 controls. We will test enzyme variants, individually and in combinations, for their functional impact and nutritional remediation based on quantitative cell-based assays in the yeast S. cerevisiae, and correlate allele distribution and functional studies with clinical phenotype. In addition, we will evaluate the relevance of murine models of orofacial clefts to human etiologies in two ways. First, we will sequence the coding regions of human orthologs of those mouse genes with convincing contributions to lip/palate closure (N=20) to identify the full spectrum of mutation to test whether such genes/variants play a role in human cleft development. Second, we will investigate the hypothesis, based on experiments in mice, that folate exerts its preventive effect through its control of methyl donor flux and subsequent epigenetic changes. Thus, we will explore global DNA methylation in cleft-affected and control newborns, which could lead to logical extensions at specific genomic loci. These studies will better define the causality of orofacial clefts as well as uncover the remedial mechanism of nutritional supplementation. This research plan capitalizes on an ongoing and successful collaboration that unites a unique combination of expertise for its execution.