Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo

项目概要/摘要 肢体畸形是第二常见的先天性畸形，每500人中有1人发生 活产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。 畸形这些变异中的许多都映射到转录增强子，即调节DNA的区域， 基因表达然而，在我们对这些机制的理解方面， 变体改变增强子活性及其在引起肢体缺陷中的作用。最常受影响的非编码 基因座是Sonic hedgehog（Shh）的肢体特异性增强子。有超过30个独立的罕见变异与肢体 在畸形中，Shh肢体增强子特别容易受到所谓的功能获得（GOF）变体的影响。 GOF变体引起增强子过度活性，导致其靶基因的异位表达。可是为什么 GOF变异体只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GOF变异体在特定细胞类型中引起异位基因表达？ 增强子对GOF变体的敏感性都是未知的。 GOF变异是引起人类疾病的增强子突变中最不为人所知的。大部分 我们对GOF变异体如何导致疾病缺乏理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不能重现GOF变体的异位表达， 模拟它们的表型结果。因此，必须使用体内系统来确定功能性的 以及GOF变异的临床意义。为了满足这一主要需求，我们的团队最近开发了一种新的 小鼠增强子报告基因测定，其能够高度可重复地检测在细胞中的异位基因表达， Shh通常不表达的前肢区细胞。本提案的总体目标是 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。 我将检验这样一个假设，即GOF变异的易感性是由以下监管环境决定的： 前肢芽细胞和一个独特的，稳定的高阶染色质结构的Shh基因座。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 为了确定遗传因素，使特定的增强子易于发病，我将测试 GOF变体导致的肢体畸形的高级染色质结构。通过识别目标 遗传因素介导异位基因表达，这些研究将提供机制的见解GOF如何 肢体特异性Shh增强子的变异导致肢体畸形。本提案得出的结论 也可用于从患者测序数据预测非编码变体的临床意义， 将对其他与GOF变异相关的发育障碍产生影响。

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.