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left裂）是一个相对常见的结构出生对病因的环境和遗传贡献的缺陷。基因组广泛的关联研究（GWAS）和连锁研究已经确定了许多与之相关的基因变异孤立的口腔面部裂口（OFC）的风险升高。但是，我们对这种疾病潜在的致病机制仍然很差，因为，我们尚未区分直接影响OFC风险（即因果变异）的DNA变异型仅在与它们的连锁二极管中进行链接二动，两个调节功能颅面发育中编码的Y相关基因编码的分子在很大程度上尚不清楚。在每个基因座，都有多个与统计相关的多个SNP OFC - 所有这些都驻留在非编码DNA中。在AIM 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从头突变来进行功能测试来自800个案例三重奏的新的全基因组序列数据。在目标2中，我们建议确定功能性的OFC相关SNP（因果）。我们假设该致病性SNP 驻留在驱动口腔组织表达的增强子中，这种SNP的风险等位基因定量影响增强子的活性。为了检验这一假设，我们将放大基因组含有风险相关的SNP的DNA并测试了等位基因依赖性增强子活性体外（基于细胞的记者测定）。我们还将测试增强剂的组织特异性（斑马鱼和基于鼠标的记者分析）。在AIM 3中，我们将确定改变致病性SNP的等位基因在相关的危险基因的表达上具有（在体外使用CRISPR/CAS9的基因组工程）。最后，我们应用染色质免疫在口腔上皮细胞系中捕获降水和染色质构型，以推断出功能性SNP改变OFC风险基因表达的机制。预期提出的实验的结果是识别遗传风险的机制变体会导致常见的先天缺陷。