Oral Clefts: Moving from Genome Wide Studies Toward Functional Genomics

描述（由申请人提供）：我们将跟踪来自口裂的全基因组关联研究（GWAS）的信号，该研究目前正在UOl-DE-004425;“International Consortium to Identify Genes & Interactions Controlling Oral Clefts”，2007-2009; TH Beaty的支持下进行。口裂是人类最常见的出生缺陷之一，其病因复杂多样。该项目的基因分型应在2009年初完成，我们的分析将确定直接影响风险的基因，这些基因仅在环境风险因素存在时起作用，和/或显示可测量的原产地影响的基因，这可能代表印记。在对FaceBase倡议（RFA-DE-09-003）的回应中，我们将通过使用基因/区域的高通量测序（HTS）技术来建立我们的GWAS结果，从而产生连锁和不平衡的统计证据（目标1），并对这些相同的基因进行功能基因组研究，以了解它们如何使用斑马鱼模型在体内发挥作用（目标2）。我们将首先关注通过分析GWAS中的单核苷酸多态性（SNP）标记识别的基因，并将使用HTS识别可能导致口腔裂的所有变体（罕见突变和新标记）。然后，我们将对该平台上已知拷贝数变异（CNV）区域中的60，000个标记进行系统分析，以测试和测序可能通过结构变异影响风险的基因。在我们的目标2中，我们将使用斑马鱼对从我们的GWAS中鉴定并在目标1中测序的基因进行表达谱分析和功能丧失分析。我们将进行进一步的功能研究，包括表达阵列，miRNA和/或ChIP-seq分析。这项功能基因组学工作将确定在斑马鱼头部间充质，颅神经嵴细胞，咽弓，脑颅和/或其他相关组织中表达的基因，使用原位杂交和软骨/骨染色方法，在正常胚胎和敲除morphants。我们还将通过全球基因和miRNA表达谱收集进一步的功能信息，以确定可能的网络或途径，涉及相互作用的基因，并探索甲基化/印迹miRNA调控和/或染色质靶向选定的基因。 公共卫生相关性：该项目旨在通过对特定基因进行测序以识别因果变异/突变并使用动物模型更好地定义这些基因在活体动物中的功能，来跟踪一项大型国际人类研究中口腔裂（唇裂，腭裂和唇腭裂）全基因组研究的证据。

DESCRIPTION (provided by applicant): We will follow up on signals from a genome wide association study (GWAS) of oral clefts now being conducted with support from UOl-DE-004425; "International Consortium to Identify Genes & Interactions Controlling Oral Clefts", 2007-2009; TH Beaty. Oral clefts are among the most common human birth defects and have a complex and heterogeneous etiology. Genotyping for this project should be completed in early 2009 and our analysis will identify genes influencing risk directly those acting only in the presence of an environmental risk factor, and/or genes showing measurable parent-of-origin effects which may represent imprinting. In this response to the FaceBase initiative (RFA-DE-09-003) we will build upon our GWAS results by using high throughput sequencing (HTS) techniques of genes/regions yielding statistical evidence of linkage and disequilibrium (Aim 1) and to conduct functional genomic studies of these same genes to understand how they act in vivo using the zebrafish model (Aim 2). We will first focus on genes identified through analysis of single nucleofide polymorphic (SNP) markers from our GWAS and will use HTS to identify all variants (rare mutations and novel markers) that may cause oral clefts. We will then undertake a systematic analysis of 60.000 markers in regions of known copy number variants (CNV) available on this platform, to test and sequence genes that may influence risk through structural variation. In our Aim 2, we will use the zebrafish for expression profiling and loss-of-function analyses of genes identified from our GWAS and sequenced in Aim 1. We will perform further functional studies including expression array, miRNA and/or ChlP-seq analysis. This functional genomics work will identify genes expressed in zebrafish head mesenchyme, cranial neural crest cells, pharyngeal arches, neurocranium and/or other relevant tissues using in situ hybridization and cartilage/bone staining methods in normal embryos and knockdown morphants. We will also gather further functional information via global gene and miRNA expression profiles to identify possible networks or pathways involving interacting genes, and explore methylation/imprinting miRNA regulation and/or chromatin targeting of selected genes. PUBLIC HEALTH RELEVANCE: This project is designed to follow up evidence from a genome wide study of oral clefts (cleft lip, cleft palate & cleft lip and palate) in a large international study of humans by sequencing specific genes to identify causal variants/mutations and using an animal model to better define how these genes function in a living animal.