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

与口面部裂风险相关的非编码 DNA 变异的功能测试。

基本信息

批准号：
10614747
负责人：
Robert Aaron Cornell
金额：
$ 47.28万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10614747
关键词：
Address Affect Alleles Binding Biological Biological Assay CRISPR/Cas technology Catalogs Cell Line Cells Chromatin Cleft Lip Cleft Palate Cleft lip with or without cleft palate Congenital Abnormality DNA Data Data Analyses Data Set Disease Elements Embryo Engineering Enhancers Epithelial Cells Etiology Face Gene Expression Genes Genetic Genetic Risk Genetic Variation Genome Mappings Genome engineering Genomic DNA Genotype Human In Vitro Lead Linkage Disequilibrium Live Birth Methods Molecular Molecular Analysis Monitor Morphogenesis Mus Oral Outcome Parents Pathogenicity Physiology Precipitation Regulatory Element Reporter Risk Series Signal Transduction Specificity Structural Congenital Anomalies Testing Therapeutic Tissues Transgenic Organisms Untranslated RNA Variant Zebrafish base causal variant chromatin immunoprecipitation cost craniofacial craniofacial development de novo mutation design diagnostic tool experimental study fetal genetic variant genome wide association study genomic locus improved in vitro Assay in vitro activity in vivo oral cavity epithelium oral tissue orofacial cleft promoter rare variant risk variant success transcription factor whole genome

项目摘要

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.

口面裂（主要是唇裂和/或腭裂）是一个相对常见的结构出生与环境和遗传因素有关的缺陷。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，孤立性口面裂（OFC）的风险增加。然而，我们对这种疾病的致病机制仍然很差，因为，一，我们还没有区分直接影响OFC风险的DNA变异（即，因果变量）第二，监管机构的职能，在颅面发育中编码γ OFC相关基因的分子在很大程度上是未知的。在每个基因座处，存在多个鉴定的多个SNP，其与以下各项在统计学上相关：所有这些都存在于非编码DNA中。在目标1中，我们将优先考虑SNP，通过对GWAS数据进行精细映射，并识别来自800个病例父母三人组的新的全基因组序列数据。在目标2中，我们建议确定 OFC相关的SNP是功能性的（因果性的）。我们假设致病性SNP 存在于驱动口腔组织中表达的增强子中，并且存在这种SNP的风险等位基因定量地影响增强子的活性。为了验证这一假设，我们将扩增基因组含有风险相关SNP的DNA，并测试它们在基因组中的等位基因依赖性增强子活性。体外（基于细胞的报告基因测定）。我们还将测试增强剂的组织特异性（基于斑马鱼和小鼠的报告基因测定）。在目标3中，我们将确定改变致病性SNP的等位基因会影响相关OFC风险基因的表达（体外用CRISPR/Cas9进行基因组工程）。最后，我们应用染色质免疫沉淀和染色质构型捕获在口腔上皮细胞系，以推断功能性SNP改变OFC风险基因表达的机制。预期拟议的实验的结果是确定遗传风险的机制，变异导致常见的出生缺陷。