The role of regulatory variants in FZD6 and interacting genes in nonsyndromic cleft lip and palate

项目摘要 非综合征性唇裂伴或不伴腭裂（NSCLP）是最常见的颅面出生缺陷， 由于胚胎面部发育不完全融合，在唇、原腭和/或 第二腭。每700名新生儿中有1名新生儿的常见出生患病率导致135，000名非神经系统LP新生儿 每年在全球范围内。虽然多学科治疗现在取得了令人满意的结果， 长期健康问题持续存在，造成重大的经济和心理社会负担。家族性 聚集和分离分析表明多基因遗传，但尽管几十年的研究，只有一小部分， 已经确定了一部分NSCLP遗传易感性，留下了很大的知识空白。这个差距可能是 大多数基因组分析的结果，旨在鉴定外显子/编码变体和罕见突变。最近， 调节基因表达的非编码功能变体已被证明在出生缺陷中起作用;我们的研究表明， 这项工作和其他人的工作表明，在颅面发育过程中基因和基因网络的失调， 发展有助于NSCLP。该提议利用了FZD 6，LRP 5， LRP 6和DKK 1，在WNT途径的受体水平上的基因是NSCLP候选基因。他们一起 作用于β-连环蛋白介导的信号传导：FZD 6与LRP 5或LRP 6形成共受体复合物， DKK 1是这种辅助受体复合物的抑制剂。这些基因在颅面发育中起着关键作用 其表达的严格调节对于上唇的正常形成和融合至关重要， 上颚该项目的目标是鉴定FZD 6、LRP 5、LRP 6和LRP 7中的功能性非编码遗传变异。 DKK 1及其对NSCLP的贡献。为了实现这一目标，我们最初优先考虑21名学生， 在FZD 6、LRP 5、LRP 6和DKK 1中发现了10种结合转录因子的变体， 等位基因特异性方式。在这项持续的工作中，荧光素酶报告基因测定在胚胎细胞系和体内 斑马鱼转基因报告基因分析将用于确定这些变异体对基因表达的作用。 表情对于在发育过程中具有细胞特异性和时空效应的变体， 将在我们的大型和特征良好的NSCLP家庭中进行。总之，这项工作的结果将 确定非编码调节变体在基因控制中的个体和组合贡献 口面发育过程中的β-连环蛋白Wnt信号传导。这些结果将增强我们对 NSCLP中的非编码变体，并有可能被翻译到临床环境中，以帮助 NSCLP风险评估。

Project Summary Nonsyndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect resulting from incomplete fusion of the embryonic facial prominences which leaves a gap in the lip, primary palate and/or the secondary palate. The common birth prevalence of 1 per 700 newborns results in 135,000 NSCLP newborns worldwide each year. While multidisciplinary treatments now achieve satisfactory outcomes, there are many long-term health issues that persist, imposing significant economic and psychosocial burdens. Familial aggregation and segregation analyses suggest polygenic inheritance, but despite decades of study, only a small portion of the NSCLP genetic liability has been identified leaving a large knowledge gap. This gap could be the result of most genomic analyses aimed at identification of exonic/coding variants and rare mutations. Recently, noncoding functional variants that regulate gene expression have been shown to play a role in birth defects; our work and that of others demonstrated that dysregulation of genes and gene networks during craniofacial development contributes to NSCLP. This proposal takes advantage of the strong evidence that FZD6, LRP5, LRP6 and DKK1, genes at the receptor level of the WNT pathway are NSCLP candidate genes. Together, they act on the β-catenin mediated signaling: FZD6 forms a co-receptor complex with either LRP5 or LRP6 while DKK1 is an inhibitor of this co-receptor complex. These genes play a critical role in craniofacial development and the tight regulation of their expression is crucial for the normal formation and fusion of the upper lip and palate. The goal of this project is to identify functional noncoding genetic variants in FZD6, LRP5, LRP6 and DKK1 and their contribution to NSCLP. Towards accomplishing this goal, we initially prioritized 21 putatively functional variants in FZD6, LRP5, LRP6 and DKK1 and found 10 variants that bind transcription factors in an allele-specific manner. In this continuing work, luciferase reporter assays in embryonic cell lines and in vivo analysis with transgenic reporters in zebrafish will be used to define the role of these variants on gene expression. For variants with cell-specific and spatiotemporal effects during development, association analyses will be carried out in our large and well-characterized NSCLP families. Together, results from this work will determine both individual and combinatorial contributions of noncoding regulatory variants in genes controlling β-catenin Wnt signaling during orofacial development. These results will enhance our knowledge of the role of noncoding variants in NSCLP and have the potential to be translated into the clinical setting to aid in the assessment of NSCLP risk.