Genetic Risk for Orofacial Clefts in the Folate/Homocysteine Pathway

描述(申请人提供)：口腔裂隙，特别是唇裂和腭裂，是常见的和昂贵的先天性畸形，其原因仍然很大程度上仍不清楚。关于口腔裂隙病因的最有希望的线索之一是，在怀孕早期使用含有叶酸的维生素的妇女患裂隙妊娠的风险要低得多。尽管叶酸有助于降低风险的潜在机制尚不清楚，但证据表明，叶酸摄入通过补偿叶酸/一碳代谢的易感性来预防唇裂。然而，通过关联研究清楚地确定这一途径中的遗传决定因素已被证明是难以捉摸的。我们提出的进一步定义口裂背后的分子遗传机制的方法是基于我们小组之前的两次观察。首先，深度测序揭示了叶酸途径基因中大量新的、非同义的变异(频率=1%)，这些变异对酶功能有不利影响，但可以通过补充叶酸来补救。其次，在脊柱裂的背景下，对所有叶酸途径基因进行了发现-测序研究，只有通过分析生物相关的等位基因组合，才能揭示令人信服的风险特征。这些数据表明，等位基因的组合，无论是常见的还是罕见的，都整合到代谢功能中，这最终是疾病风险的基础。因此，我们假设单碳代谢中的遗传易感性也可能是裂隙的病因，这些易感性可以由低频率和普通等位基因授予，也可能由相关途径变异组合之间的协同作用所致。为了验证这一假设，我们将对所有叶酸/同型半胱氨酸途径基因(N=32)中的编码区进行测序，这些基因来自约375名受唇裂影响的婴儿和约375名对照婴儿。我们将单独和组合测试酶变体的功能 基于基于细胞的定量分析在酿酒酵母中的影响和营养补救，以及等位基因分布和功能研究与临床表型的相关性。此外，我们将从两个方面评估口面部裂小鼠模型与人类病因的相关性。首先，我们将对那些对唇腭裂闭合有令人信服贡献的小鼠基因的人类同源基因的编码区进行测序(N=20)，以确定全谱突变，以测试这些基因/变体是否在人类唇裂发育中发挥作用。其次，我们将基于在小鼠身上的实验来研究这一假设，即叶酸通过控制甲基供体流量和随后的表观遗传学变化来发挥预防作用。因此，我们将探索在受裂隙影响的新生儿和对照新生儿中的全局DNA甲基化，这可能导致特定基因组座位的逻辑延伸。这些研究将更好地确定口腔裂隙的因果关系，并揭示营养补充的补救机制。这项研究计划充分利用了持续和成功的合作，将独特的专业知识结合在一起进行执行。

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.