Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo

体内非编码变异引起肢体畸形的破译机制

• 批准号: 10538362
• 负责人: Ethan W Hollingsworth
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538362
• 关键词: 3-Dimensional; Address; Affect; Anterior; Architecture; Binding; Biological Assay; Biological Models; Birth; Cell Culture Techniques; Cells; Chromatin; Chromatin Loop; Chromatin Structure; Competence; Congenital Abnormality; DNA; DNA Sequence; Data; Defect; Detection; Disease; Ectopic Expression; Enhancers; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Goals; Higher Order Chromatin Structure; In Vitro; Knock-in Mouse; Lead; Limb Bud; Limb Development; Limb structure; Link; Live Birth; Maps; Mediating; Microscopy; Modeling; Mus; Mutation; Nucleic Acid Regulatory Sequences; Organoids; Pathogenesis; Pathogenicity; Patients; Phenotype; Predisposing Factor; Predisposition; Reporter; Reporting; Reproducibility; Resolution; Role; SHH gene; System; Technology; Testing; Tissues; Transgenic Mice; Untranslated RNA; Variant; base; cell type; clinical predictors; clinically significant; cohesin; developmental disease; disease phenotype; epigenomics; gain of function; human disease; in vivo; insight; malformation; multiple omics; novel; promoter; rare variant; transcription factor

PROJECT SUMMARY/ABSTRACT Limb malformations are the second most common congenital abnormality, occurring in 1 in every 500 live births. Mounting evidence implicates rare noncoding mutations to underlie non-syndromic (isolated) limb malformations. Many of these variants map to transcriptional enhancers, regions of regulatory DNA that tune gene expression. However, a fundamental gap remains in our understanding of the mechanisms by which these variants alter enhancer activity and their role in causing limb defects. The most frequently affected noncoding loci is the limb-specific enhancer of Sonic hedgehog (Shh). With over 30 independent rare variants linked to limb malformations, the Shh limb enhancer is particularly susceptible to so-called Gain-Of-Function (GOF) variants. GOF variants cause enhancer overactivity that leads to ectopic expression of their target genes. However, why GOF variants only cause ectopic gene expression in specific cell types and why only a small subset of enhancers are susceptible to GOF variants are both unknown. GOF variants are among the least understood enhancer mutations that cause human disease. Much of our lack of understanding of how GOF variants contribute to disease is owed to a lack of suitable model systems. In vitro cell culture and organoid-based systems fail to recapitulate ectopic expression from GOF variants nor model their phenotypic consequences. Thus, it is essential to use in vivo systems to determine the functional and clinical significance of GOF variants. To address this major need, our group recently developed a novel mouse enhancer reporter assay that enables highly-reproducible detection of ectopic gene expression in the cells of the anterior limb domain where Shh is normally not expressed. The overall goal of this proposal is to determine the genetic factors mediating the unique susceptibility of anterior limb bud cells and the Shh limb enhancer to GOF variants. I will test the hypothesis that susceptibility to GOF variants is dictated by the regulatory landscape of anterior limb bud cells and a unique, stable higher-order chromatin structure of the Shh locus. To identify the genetic factors that mediate ectopic Shh expression, I will characterize the regulatory landscapes and local chromatin architecture of anterior limb bud cells in which Shh is ectopically active at single-cell resolution. To determine genetic factors that predispose specific enhancers to pathogenesis, I will test the requirement of higher-order chromatin structure for limb malformations resulting from GOF variants. By identifying targetable genetic factors mediating ectopic gene expression, these studies will provide mechanistic insights into how GOF variants in the limb-specific Shh enhancer contribute to limb malformations. Findings resulting from this proposal can also be applied to predict the clinical significance of noncoding variants from patient sequencing data and will have implications for other developmental disorders linked to GOF variants.

项目概要/摘要 肢体畸形是第二常见的先天性异常，每 500 人中就有 1 人发生肢体畸形 活产数。越来越多的证据表明罕见的非编码突变是非综合征（孤立）肢体的基础 畸形。其中许多变体映射到转录增强子，即调节 DNA 的区域 基因表达。然而，我们对这些机制的理解仍然存在根本差距。 变异改变了增强子的活性及其在导致肢体缺陷中的作用。最常受影响的非编码 位点是 Sonic Hedgehog (Shh) 的肢体特异性增强子。拥有超过 30 个与肢体相关的独立稀有变异 由于畸形，Shh 肢体增强子特别容易受到所谓的功能获得（GOF）变异的影响。 GOF 变异会导致增强子过度活跃，从而导致其靶基因异位表达。然而，为什么 GOF 变异仅在特定细胞类型中引起异位基因表达，为什么只有一小部分 增强子是否容易受到 GOF 变异的影响尚不清楚。 GOF 变异是人们最不了解的导致人类疾病的增强子突变之一。很多 我们对 GOF 变异如何导致疾病缺乏了解是由于缺乏合适的模型系统。 体外细胞培养和基于类器官的系统无法重现 GOF 变体的异位表达，也无法重现 模拟它们的表型后果。因此，有必要使用体内系统来确定功能 GOF 变异的临床意义。为了解决这一重大需求，我们小组最近开发了一种新颖的 小鼠增强子报告基因检测能够高度重复地检测异位基因表达 前肢区域的细胞通常不表达Shh。该提案的总体目标是 确定介导前肢芽细胞和Shh肢独特易感性的遗传因素 GOF 变体的增强子。 我将检验以下假设：对 GOF 变异的易感性是由监管环境决定的 前肢芽细胞和 Shh 基因座的独特、稳定的高阶染色质结构。为了识别 介导异位Shh表达的遗传因素，我将描述调控景观和局部 前肢芽细胞的染色质结构，其中 Shh 在单细胞分辨率下具有异位活性。到 确定使特定增强子易于发病的遗传因素，我将测试以下要求 GOF 变异引起的肢体畸形的高阶染色质结构。通过识别目标 遗传因素介导异位基因表达，这些研究将为 GOF 如何发生提供机制见解 肢体特异性 Shh 增强子的变异会导致肢体畸形。该提案得出的结论 还可用于根据患者测序数据预测非编码变异的临床意义 将对与 GOF 变异相关的其他发育障碍产生影响。