Functional tests of non-coding DNA variants associated with risk for orofacial clefting.

口面部裂伤(主要是唇裂和/或腭裂)是一种比较常见的结构性出生。 环境和遗传缺陷对病因学的影响。全基因组关联 研究(GWAS)和连锁研究已经确定了许多基因变异与 孤立性口腔面部裂伤(OFC)的风险增加。然而，我们对 这种疾病的致病机制仍然不清楚，因为，首先，我们还没有 区分直接影响OFC风险的DNA变异(即，因果变异)与那些 仅仅是与它们的联系不平衡，以及两个，监管机构的职能 在颅面发育过程中编码与OFC相关基因的分子很大程度上是未知的。 在每个基因座上，有多个已识别的多个SNP与统计上的 OFC-所有这些都驻留在非编码DNA中。在目标1中，我们将优先考虑 功能测试，通过对GWAS数据进行精细映射，并确定从头开始的突变 800例亲本三联体的新全基因组序列数据。在目标2中，我们建议确定 与OFC相关的功能性(因果)SNP。我们假设致病的SNPs 驻留在推动口腔组织表达增强子中，以及此类SNPs的风险等位基因 定量影响增强剂的活性。为了验证这一假设，我们将扩增基因组 含有风险相关SNPs的DNA，并测试它们的等位基因依赖的增强子活性 体外(以细胞为基础的报告分析)。我们还将测试增强剂的组织特异性。 (斑马鱼和基于老鼠的记者分析)。在目标3中，我们将确定 改变致病SNPs等位基因对相关OFC风险基因表达的影响 (CRISPR/Cas9体外基因组工程)。最后，我们应用染色质免疫。 在口腔上皮细胞系中沉淀和染色质构型捕获以推断 功能性SNP改变OFC风险基因表达的机制。预期中的 拟议实验的结果是确定了遗传风险的机制 变异会导致一种常见的出生缺陷。

Orofacial clefting (primarily cleft lip and/or cleft palate) is a relatively common structural birth defect with environmental and genetic contributions to etiology. Genome wide association studies (GWAS) and linkage studies have identified many gene variants that are associated with elevated risk for isolated oral facial clefting (OFC). However, our understanding of the pathogenic mechanisms underlying this disease remains poor because, one, we have yet to distinguish DNA variants that directly influence risk for OFC (i.e., causal variants) from those that are merely in linkage disequilibrium with them, and two, the functions of the regulatory molecules encoded y OFC-associated genes in craniofacial development are largely unknown. At each locus, there are multiple identified multiple SNPs that are statistically associated with OFC – and all of these reside in non-coding DNA. In Aim 1, we will prioritize the SNPs for functional tests by performing fine mapping of GWAS data, and identifying de novo mutations in new whole genome sequence data from 800 case-parent trios. In Aim 2 we propose to identify the OFC-associated SNPs that are functional (causal). We hypothesize that pathogenic SNPs reside in enhancers that drive expression in oral tissues, and that risk alleles of such SNPs quantitatively affect activity of the enhancers. To test this hypothesis, we will amplify genomic DNA containing risk-associated SNPs and test them for allele-dependent enhancer activity in vitro (cell-based reporter assays). We will also test the tissue specificity of the enhancers (zebrafish and mouse-based reporter assays). In Aim 3, we will determine the effect that altering the allele of pathogenic SNPs has on expression of the relevant OFC-risk gene (genome engineering with CRISPR/Cas9 in vitro). Finally, we apply chromatin immuno precipitation and chromatin configuration capture in the oral epithelium cell line to deduce the mechanism by which functional SNPs change expression of the OFC-risk genes. The expected outcome of the proposed experiments is identification of the mechanisms by which genetic risk variants cause a common birth defect.