Genomic and Functional Analysis of IRF6 Target Genes in Orofacial Cleft Pathogenesis

项目摘要 唇腭裂(CL/P)等唇腭裂是最常见的 常见的先天性结构异常。大多数OFC病例都是非综合征性的 复杂的遗传机制尚不清楚。大多数可遗传的 OFC风险预计将存在于罕见或从头开始的变异中。随着临床应用的扩大 在产前DNA检测中，迫切需要改进对胎儿的遗传解释 完整的外显子组/基因组序列数据。一个主要的挑战是致病性的分配。 编码和非编码变体。编码转录因子IRF6的基因是 与非综合征性CL/P密切相关，加上IRF6突变导致的 常见形式的综合征CL/P我们和其他人已经证明了IRF6中的基因 通路是影响CL/P风险的罕见变异的很好候选者，例如 GRHL3、ARHGAP29和KLF4。在这项建议中，我们将这一成功的战略扩展到 全面深入分析IRF6基因阐明CL/P的发病机制 下游基因途径。我们采用了严格的基因优先排序策略 利用芯片序列技术从野生型中鉴定了关键的irf6转录靶基因。 胚胎，通过与irf6突变数据集的差减而丰富。目的基因是 然后通过RNA-seq筛选出野生型和irf6突变体之间的差异表达。 然后对该IRF6候选靶基因列表进行时空交叉参考 斑马鱼Wish和Wish与头面部发育相关的表达模式 来自FaceBase项目的小鼠基因表达数据。最后，我们选择了基因 与人类CL/P病理有关的最近800例CL/P病例--亲代三联体WGS 来自加布里埃拉·米勒儿童第一次测序项目的数据集。我们的中心假设 IRF6靶基因对味觉发育至关重要，且罕见且从头开始 这类基因的突变，无论是编码还是非编码，都存在于 非综合征性OFC。为了验证这一假设，我们提出了三个互补的目标 1)从已知的(TFAP2A)和新鉴定的(Dact1)基因中获得新的生物学见解 颅面发育，2)获得从头编码的新的功能和临床认识 OFC的重要基因变异，3)发展方法学和分析非编码基因 与OFC有关的变异。这项工作的预期影响将是弥合差距 在WGS数据和生物学洞察力之间，这是有意义地翻译的关键一步 基因研究数据为临床决策提供信息。

Project Summary Orofacial clefts (OFC) such as cleft lip and/or palate (CL/P) are among the most common congenital structural anomalies. Most OFC cases are non-syndromic with complex genetic mechanisms that are yet to be elucidated. The majority of heritable OFC risk is expected to reside in rare or de novo variations. With expanding clinical use of prenatal DNA tests, there is a pressing need to improve the genetic interpretation of whole exome/genome sequence data. A major challenge is assignment of pathogenicity to both coding and non-coding variants. The gene encoding transcription factor IRF6 is strongly associated with non-syndromic CL/P, plus mutations in IRF6 cause the most common form of syndromic CL/P. We and others have shown that genes in the IRF6 pathway are good candidates to harbor rare variants that influence risk for CL/P, such as GRHL3, ARHGAP29 and KLF4. In this proposal, we extend this successful strategy to elucidate CL/P pathogenesis with a comprehensive and deep analysis of the IRF6 downstream gene pathway. We employed a rigorous gene prioritization strategy where critical IRF6 transcriptional target genes were identified via ChIP-seq from wild type embryos, enriched by subtraction against irf6 mutant dataset. The target genes were then selected for differential expression between wild type and irf6 mutants via RNA-seq. This IRF6 candidate target gene list was then cross-referenced for spatiotemporal expression patterns relevant for craniofacial development with zebrafish WISH and mouse gene expression data from the FaceBase project. Finally we selected genes associated with human CL/P pathology from a recent 800 CL/P case-parent trios WGS dataset from the Gabriella Miller Kids First sequencing project. Our central hypothesis is that IRF6 target genes are critical for palate development, and that rare and de novo mutations in such genes, whether coding or non-coding, are present in patients with non-syndromic OFC. To test this hypothesis, we propose three complementary aims to 1) gain new biological insight from known (Tfap2a) and newly identified (Dact1) genes in craniofacial development, 2) gain new functional and clinical insight of de novo coding gene variants important for OFC, 3) develop methodology and analyze non-coding gene variants implicated for OFC. The expected impact of this work will be to bridge the gap between WGS data and biological insight, an essential step to meaningfully translate genetic research data to inform clinical decisions.