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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：
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.

口面裂（主要是唇裂和/或腭裂）是一种相对常见的结构性出生