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：
9924262
负责人：
Robert Aaron Cornell
金额：
$ 46.38万
依托单位：
UNIVERSITY OF IOWA
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 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9924262
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 Diagnostic 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 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 tool 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.

口面部裂（主要是唇裂和/或腭裂）是一种相对常见的结构性出生缺陷与环境和遗传对病因学的贡献有关。研究（GWAS）和连锁研究已经确定了许多与然而，孤立性口颌面裂 (OFC) 的风险增加。这种疾病的致病机制仍然很差，因为，第一，我们还没有区分直接影响 OFC 风险的 DNA 变异（即因果变异）和那些直接影响 OFC 风险的 DNA 变异（即因果变异）只是与它们之间存在联动不平衡，第二，监管的职能颅面部发育中编码 y OFC 相关基因的分子在很大程度上是未知的。在每个位点，有多个已识别的多个 SNP，这些 SNP 与 OFC——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新生突变来进行功能测试在目标 2 中，我们建议鉴定来自 800 个病例亲本三人组的新全基因组序列数据。 OFC 相关的功能性（因果性）SNP。存在于驱动口腔组织表达的增强子中，并且存在此类 SNP 等位基因的风险定量地影响增强子的活性为了检验这个假设，我们将扩增基因组。含有风险相关 SNP 的 DNA 并测试它们的等位基因依赖性增强子活性我们还将测试增强子的组织特异性。（基于斑马鱼和小鼠的报告基因检测）。在目标 3 中，我们将确定其效果。改变致病性 SNP 的等位基因会影响相关 OFC 风险基因的表达（利用 CRISPR/Cas9 进行体外基因组工程）最后，我们应用染色质免疫。口腔上皮细胞系中的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。拟议实验的结果是确定了遗传风险的机制变异导致常见的出生缺陷。