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

项目摘要 口面裂（OFC），如唇裂和/或腭裂（CL/P）是最常见的 常见的先天性结构异常大多数OFC病例为非综合征型， 复杂的遗传机制尚未阐明。大多数遗传 OFC风险预计存在于罕见或新发变异中。随着临床应用的不断扩大 产前DNA测试，迫切需要改善遗传解释， 全外显子组/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IRF 6的基因是 与非综合征性CL/P密切相关，加上IRF 6突变导致最 我们和其他人已经表明，IRF 6中的基因 途径是隐藏影响CL/P风险的罕见变异的良好候选者，例如 GRHL 3、ARHGAP 29和KLF 4。在本提案中，我们将这一成功策略扩展到 通过对IRF 6的全面深入分析阐明CL/P发病机制 下游基因通路。我们采用了严格的基因优先级策略， 通过ChIP-seq从野生型中鉴定关键的IRF 6转录靶基因 胚胎，通过针对IRF 6突变体数据集的减法富集。靶基因 然后通过RNA-seq选择野生型和irf 6突变体之间的差异表达。 然后将该IRF 6候选靶基因列表交叉引用用于时空分析。 与斑马鱼WISH颅面发育相关的表达模式， 小鼠基因表达数据来自FaceBase项目。最后我们选择了 与来自最近800例CL/P病例-父母三人组WGS的人类CL/P病理学相关 来自Gabriella米勒儿童第一测序项目的数据集。我们的核心假设 IRF 6靶基因对腭发育至关重要， 这些基因中的突变，无论是编码还是非编码，都存在于患有以下疾病的患者中： 非综合征型眶额肌痛。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（Tfap 2a）和新发现的（Dact 1）基因中获得新的生物学见解， 颅面发育，2）获得从头编码的新功能和临床见解 对OFC重要的基因变异，3）开发方法学并分析非编码基因 与OFC有关的变体。这项工作的预期影响将是弥合差距 WGS数据和生物学洞察之间的联系，这是有意义地翻译 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.