Deciphering Mechanisms of Limb Malformations Caused by Noncoding Variants In Vivo

项目摘要/摘要 肢体畸形是第二常见的先天性畸形，每500人中就有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上仍然存在一个根本性的差距 变异体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 Loci是Sonic Hedgehog(Shh)的肢体特异性增强子。有30多个独立的稀有变异与肢体相连 Shh肢体增强器特别容易受到所谓的功能增益(GOF)变体的影响。 Gof变异体导致增强子过度活性，从而导致其目标基因的异位表达。然而，为什么 GoF变异体只会导致特定细胞类型的异位基因表达，而且为什么只有一小部分 增强子对GOF变种敏感，这两种变种都是未知的。 Gof变异是最不为人所知的导致人类疾病的增强子突变之一。大部分 我们缺乏对GOF变异如何导致疾病的了解，这是由于缺乏合适的模型系统。 体外细胞培养和基于有机物的系统无法概括GOF变异体的异位表达 对它们的表型后果进行建模。因此，使用体内系统来确定功能是必不可少的。 以及GOF变异的临床意义。为了满足这一主要需求，我们团队最近开发了一部小说 小鼠增强子报告实验，能够高度重复性地检测异位基因在 正常情况下Shh不表达的前肢区域细胞。这项提案的总体目标是 确定介导前肢芽细胞和Shh肢体独特易感性的遗传因素 GOF变体的增强器。 我将测试这样一个假设，即对GOF变体的易感性是由以下监管环境决定的 前肢芽细胞和Shh基因座独特、稳定的高阶染色质结构。要确定 介导异位Shh表达的遗传因素，我将描述调控景观和局部 前肢芽细胞染色质构筑，其中Shh在单细胞分辨率下是异位活跃的。至 确定使特定增强子致病的遗传因素，我将测试 GOF变异所致肢体畸形的高阶染色质结构。通过确定目标 遗传因素介导异位基因表达，这些研究将提供对GOF如何 肢体特异性Shh增强子中的变异导致肢体畸形。根据这项提案得出的结果 还可以应用于从患者测序数据和 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.