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.

项目摘要/摘要 肢体畸形是第二个最常见的先天性异常，每500次发生1例 活产。越来越多的证据暗示罕见的非编码突变是非综合（孤立）肢体的基础 畸形。这些变体中的许多映射到转录增强子，调节DNA的区域 基因表达。但是，我们对这些机制的理解仍然存在根本差距 变体改变增强子活性及其在引起肢体缺陷中的作用。最常见的非编码 基因座是Sonic刺猬（SHH）的肢体特异性增强子。与肢体相关的30多个独立稀有变体 畸形，SHH肢体增强子特别容易受到所谓功能获得（GOF）变体的影响。 GOF变体会导致增强子过度活跃，从而导致其靶基因异位表达。但是，为什么 GOF变体仅在特定细胞类型中引起异位基因表达，以及为什么仅一小部分 增强剂容易受到GOF变体的影响。 GOF变体是引起人类疾病的增强子突变之一。大部分 我们缺乏对GOF变体如何促进疾病的理解，这是由于缺乏合适的模型系统。 体外细胞培养和基于器官的系统无法从GOF变体中概括异位表达 建模其表型后果。因此，必须在体内系统确定功能 GOF变体的临床意义。为了满足这一主要需求，我们的小组最近开发了一本小说 小鼠增强剂报告基因测定器，可实现对异位基因表达的高度可再生检测 通常未表达SHH的前肢结构域的细胞。该提议的总体目标是 确定介导前肢体芽细胞和SHH肢体独特敏感性的遗传因素 GOF变体的增强剂。 我将检验以下假设，即对GOF变体的敏感性取决于 前肢芽细胞和SHH基因座的独特，稳定的高阶染色质结构。识别 介导异位SHH表达的遗传因素，我将表征调节景观和局部 前肢芽细胞的染色质结构，其中SHH在单细胞分辨率下异位活跃。到 确定使特异性增强子易于发病机理的遗传因素，我将测试 GOF变体引起的肢体畸形的高阶染色质结构。通过确定目标 介导异位基因表达的遗传因素，这些研究将提供机械洞察力，以了解GOF 肢体特异性SHH增强子中的变体导致肢体畸形。该提议产生的发现 也可以应用于预测患者测序数据和 将对与GOF变体相关的其他发育障碍有影响。