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

项目概要 口面裂 (OFC)，例如唇裂和/或腭裂 (CL/P) 是最常见的裂痕之一。 常见的先天性结构异常。大多数 OFC 病例都是非综合征性的 复杂的遗传机制尚未阐明。大部分可遗传 OFC 风险预计存在于罕见或新的变异中。随着临床用途的不断扩大 产前 DNA 检测中，迫切需要改进基因解释 整个外显子组/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 突变是最常见的原因 综合征 CL/P 的常见形式。我们和其他人已经证明 IRF6 中的基因 途径是包含影响 CL/P 风险的罕见变异的良好候选者，例如 GRHL3、ARHGAP29 和 KLF4。在本提案中，我们将这一成功的策略扩展到 通过全面深入分析 IRF6 阐明 CL/P 发病机制 下游基因途径。我们采用了严格的基因优先级策略，其中 通过 ChIP-seq 从野生型中鉴定出关键的 IRF6 转录靶基因 胚胎，通过对 irf6 突变体数据集进行减法富集。目标基因是 然后通过 RNA-seq 选择野生型和 irf6 突变体之间的差异表达。 然后将该 IRF6 候选靶基因列表进行时空交叉引用 斑马鱼 WISH 与颅面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 来自最近 800 个 CL/P 病例-亲本三人组 WGS 的人类 CL/P 病理学相关 来自 Gabriella Miller Kids First 测序项目的数据集。我们的中心假设 IRF6 靶基因对于味觉发育至关重要，而且罕见且从头开始 这些基因的突变，无论是编码基因还是非编码基因，都存在于患有以下疾病的患者中： 非综合征性 OFC。为了检验这一假设，我们提出了三个互补的目标 1) 从已知 (Tfap2a) 和新鉴定的 (Dact1) 基因中获得新的生物学见解 颅面发育，2) 获得从头编码的新功能和临床见解 对 OFC 很重要的基因变异，3) 开发方法并分析非编码基因 与 OFC 相关的变体。这项工作的预期影响将是弥合差距 WGS 数据和生物学见解之间的联系，这是有意义转化的重要一步 遗传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.