Functional analysis of ESRP1/2 and CTNND1 gene variants in orofacial cleft

技术摘要 口面裂（orofacial clefts，OFC）的遗传学研究是先天性结构性唇腭裂遗传学的基础 出生缺陷大多数OFC病例是非综合征型的，涉及复杂的遗传学。 机制尚未完全阐明。目前，唇腭裂的基因诊断 两个关键的知识缺口阻碍了异常现象的研究。第一，基因对于 上颚形成不完全确定。第二，即使裂缝风险基因是 相关的算法，用于从良性基因变异中估算有害基因， 计算和统计方法仍然不可靠。迫切需要 将基因组测序转化为临床可操作的数据， 提供最高级别的证据来推断致病性。我们发现Esrp 1和 它的同源体Esrp 2（以下简称Esrp 1/2）在小鼠和斑马鱼的外周血中起作用， 调节颅面发育。Esrp 1/2介导RNA选择性剪接 转录，产生在口腔上皮和牙周膜中起作用的上皮同种型。这 该提案测试了Esrp 1/2是产生上皮细胞所必需的这一中心假设。 Ctnnd 1的亚型，其维持颅面所必需的牙周膜完整性 形态发生 我们将估算ESRP 1/2和CTNND 1人类基因变体的致病性 与OFC有关。利用已完成的基因组测序项目和 我们收集了大量的ESRP 1、ESRP 2和CTNND 1基因变体， 确定其功能。我们采用互补的斑马鱼esrp 1/2突变体 测定和体外Esrp 1/2鼠Py 2 T细胞系以优化方法的严谨性。我们 还将利用斑马鱼发现和功能验证Esrp调控基因 上皮转基因报告细胞系。我们发现，Esrp 1/2调节替代的 CTNND 1的剪接，并将在功能上询问人CTNND 1基因变体， 斑马鱼ctnnd 1突变体在拯救试验中的作用。 该项目的预期成果是通过以下方式获得机械见解： 利用与口面裂队列相关的基因组测序数据， 分析ESRP 1/2和CTNND 1基因变异在颅面发育中的功能。 我们还将鉴定并功能验证在细胞凋亡中起作用的Esrp调控基因。 围产期。这项工作将有更广泛的影响，通过阐明如何调控RNA 选择性剪接和细胞信号传导机制在围产期是重要的， 颅面形态发生

Technical abstract Genetic study of orofacial clefts (OFC) is foundational to genetics of congenital structural birth defects. Most OFC cases are non-syndromic and involve complex genetic mechanisms that are yet to be fully elucidated. Currently, genetic diagnosis for cleft anomalies is hampered by two critical knowledge gaps. First, genes essential for palate formation are incompletely identified. Second, even when the cleft risk genes are associated, algorithms used to impute deleterious from benign gene variants via computational and statistical methods remain unreliable. There is a critical need to translate genome sequencing to clinically actionable data, where functional studies provide the highest-level evidence to impute pathogenicity. We showed that Esrp1 and its paralog Esrp2 (hereafter Esrp1/2) operate in the periderm of mouse and zebrafish to regulate craniofacial development. Esrp1/2 mediates alternative splicing of RNA transcripts, creating epithelial isoforms that function in oral epithelium and periderm. This proposal tests the central hypothesis that Esrp1/2 is required to generate epithelial isoform of Ctnnd1, which maintains periderm integrity necessary for craniofacial morphogenesis. We will impute pathogenicity of ESRP1/2 and CTNND1 human gene variants associated with OFC. Using completed genome sequencing projects and projects in progress, we curate large numbers of ESRP1, ESRP2 and CTNND1 gene variants to ascertain their function. We employ complementary in vivo zebrafish esrp1/2 mutant assay and in vitro Esrp1/2 murine Py2T cell lines to optimize rigor of approach. We will also discover and functionally validate Esrp-regulated genes, using zebrafish epithelial transgenic reporter lines. We discovered that Esrp1/2 regulates alternative splicing of CTNND1 and will functionally interrogate human CTNND1 gene variants in the zebrafish ctnnd1 mutant in a rescue assay. The expected outcome of this project is to gain mechanistic insights by leveraging genome sequencing data associated with orofacial cleft cohorts, to functionally analyze ESRP1/2 and CTNND1 gene variants in craniofacial development. We will also identify and functionally validate Esrp-regulated genes acting in the periderm. This work will have broader impact by elucidating how regulation of RNA alternative splicing and cell signaling mechanisms are important in periderm and craniofacial morphogenesis.