Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, China.
Nat Commun. 2017 Mar 13;8:14759. doi: 10.1038/ncomms14759.
Genome-wide association studies (GWAS) do not distinguish between single nucleotide polymorphisms (SNPs) that are causal and those that are merely in linkage-disequilibrium with causal mutations. Here we describe a versatile, functional pipeline and apply it to SNPs at 1p22, a locus identified in several GWAS for non-syndromic cleft lip with or without cleft palate (NS CL/P). First we amplified DNA elements containing the ten most-highly risk-associated SNPs and tested their enhancer activity in vitro, identifying three SNPs with allele-dependent effects on such activity. We then used in vivo reporter assays to test the tissue-specificity of these enhancers, chromatin configuration capture to test enhancer-promoter interactions, and genome editing in vitro to show allele-specific effects on ARHGAP29 expression and cell migration. Our results further indicate that two SNPs affect binding of CL/P-associated transcription factors, and one affects chromatin configuration. These results translate risk into potential mechanisms of pathogenesis.
全基因组关联研究 (GWAS) 无法区分因果单核苷酸多态性 (SNP) 和仅与因果突变连锁不平衡的 SNP。在这里，我们描述了一个通用的功能管道，并将其应用于 1p22 上的 SNP，该基因座在几项非综合征性唇裂伴或不伴腭裂 (NS CL/P) 的 GWAS 中被鉴定出来。首先，我们扩增了包含十个最具风险关联 SNP 的 DNA 元件，并在体外测试了它们的增强子活性，鉴定出三个具有等位基因依赖性增强子活性的 SNP。然后，我们使用体内报告基因检测来测试这些增强子的组织特异性，染色质构象捕获来测试增强子-启动子相互作用，以及体外基因组编辑来显示对 ARHGAP29 表达和细胞迁移的等位基因特异性影响。我们的结果进一步表明，两个 SNP 影响与 CL/P 相关的转录因子的结合，一个 SNP 影响染色质构型。这些结果将风险转化为潜在的发病机制。
