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

ESRP1/2和CTNND1基因变异在口面裂中的功能分析

基本信息

批准号：
10565102
负责人：
Eric Chien-Wei Liao
金额：
$ 67.59万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-15 至 2028-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10565102
关键词：
Address Algorithms Alternative Splicing Anterior Benign Biological Assay Biology Birth Candidate Disease Gene Cell Line Cellular Assay Complex Computing Methodologies Contracts Data Defect Development Embryo Epithelium Face Genes Genetic Genetic study Healthcare Heritability Human Human Genetics In Vitro Injections Knock-out Knowledge Large-Scale Sequencing Level of Evidence Mediating Mesenchymal Mesenchyme Messenger RNA Modeling Morphogenesis Mus Mutation Outcome Palate Pathogenesis Pathogenicity Patients Periderm Phenotype Protein Isoforms Regulation Reporter Genes Research Support Risk Scheme Signal Transduction Statistical Methods Structural Congenital Anomalies Structure Surface Testing Tissues Transcript Transfection Transgenic Organisms Translating Variant Work Zebrafish clinically actionable cohort craniofacial craniofacial development de novo mutation exome genetic disorder diagnosis genetic variant genome sequencing in vitro Model in vivo insight mutant oral cavity epithelium orofacial cleft overexpression paralogous gene rare variant response risk variant screening transcriptome sequencing transcriptomic profiling

项目摘要

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.

技术摘要口面裂 (OFC) 的遗传学研究是先天性结构遗传学的基础出生缺陷。大多数 OFC 病例都是非综合征性的，并且涉及复杂的遗传因素机制尚未完全阐明。目前，唇裂的基因诊断异常现象受到两个关键知识差距的阻碍。首先，基因必不可少上颚的形成尚未完全确定。其次，即使裂解风险基因是相关的算法用于通过以下方式从良性基因变异中推断出有害基因变异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，其中功能研究提供最高级别的证据来估算致病性。我们证明了 Esrp1 和其旁系同源物 Esrp2（以下简称 Esrp1/2）在小鼠和斑马鱼的周皮中发挥作用调节颅面发育。 Esrp1/2 介导 RNA 选择性剪接转录物，产生在口腔上皮和周皮中发挥作用的上皮亚型。这该提案测试了中心假设，即 Esrp1/2 是生成上皮细胞所必需的 Ctnnd1 亚型，维持颅面所需的周皮完整性形态发生。我们将估算 ESRP1/2 和 CTNND1 人类基因变异的致病性与 OFC 相关。使用已完成的基因组测序项目和项目随着进展，我们整理了大量的 ESRP1、ESRP2 和 CTNND1 基因变体确定它们的功能。我们采用互补的体内斑马鱼 esrp1/2 突变体测定和体外 Esrp1/2 鼠 Py2T 细胞系，以优化方法的严谨性。我们还将利用斑马鱼发现 Esrp 调节基因并对其进行功能验证上皮转基因报告系。我们发现 Esrp1/2 调节替代 CTNND1 的剪接，并将在功能上询问人类 CTNND1 基因变异救援试验中的斑马鱼 ctnnd1 突变体。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，对颅面发育中的 ESRP1/2 和 CTNND1 基因变异进行功能分析。我们还将鉴定并在功能上验证 Esrp 调控的基因周皮。这项工作将通过阐明 RNA 的调控方式产生更广泛的影响选择性剪接和细胞信号传导机制在周皮和颅面形态发生。