Multiscale functional characterization of genomic variation in human developmental disorders

项目摘要/摘要 大规模研究已经确定了数千种与发育缺陷有关的基因变异， 以及含有这些变体的调控元件以及这些变体可能所处的细胞类型 功能。变异体、调控元件和细胞类型的多样性表明，多种机制 会导致发育缺陷。我们理解这些机制的一个关键挑战是 每个变异体的分子、细胞和功能表型在很大程度上仍未确定。直到这些关键时刻 解决了知识差距、发育疾病的潜在分子和细胞决定因素 易感性仍将是不完整的。为了弥合这些差距，我们建议建立“德克萨斯大学西南分校 调控元素变异和功能中心“。该中心的主要目标是系统地 对人类发育中疾病相关增强子的分子和细胞表型进行分类，并提供 专注于对非规范人类遗传学和基因调控机制的洞察。 为了建立一个可概括的框架来理解人类基因变异对功能的影响， 我们提出了一个具有三个主要目标的高吞吐量扰动平台：(1)有助于 与人类发展疾病有关的变异/要素/表型目录，重点是要素 基因与先天性心脏病(心肌细胞)、自闭症(神经元)和胎盘缺陷有关 (滋养层细胞)；(2)对非规范人类遗传学的变异/要素/表型目录作出贡献， 专注于人类遗传学中两个未被研究的主题：多效性效应和非细胞自主效应；以及 (3)与基因调控机制相关的变异/成分/表型目录，重点 在增强子RNA上。该中心将利用基因组工程方面的最新技术创新， 单细胞基因组学和高含量筛选，以实现基因组的多尺度功能表征 人类发育障碍的变异。其中几种技术是由研究人员首创的 对该项目作出贡献，包括：开发用于增强扰动和耦合的新工具 单细胞RNA-Seq读出的内源性增强子扰动(Mosaic-Seq)。 影响和意义：该项目的努力将产生一些关键成果和 可交付成果，包括(1)更好地理解序列变异和基因组之间的关系 功能，(2)社区的广泛变体/元素/表型目录，(3)生成工具 社区的预测模型，以及(4)支持未来功能基因组学研究的资源。一起， 我们的多方面和组合方法将为理解监管的影响开辟新的视野 发育疾病表型的变异。

Project Summary/Abstract Large-scale studies have identified thousands of genetic variants linked to developmental defects, together with the regulatory elements harboring these variants and the cell types in which these variants likely function. This diversity of variants, regulatory elements, and cell types indicates that multiple mechanisms contribute to developmental defects. One key challenge to our understanding of these mechanisms is that the molecular, cellular, and functional phenotypes of each variant remain largely uncharacterized. Until these critical gaps in knowledge are addressed, the underlying molecular and cellular determinants of developmental disease susceptibility will remain incomplete. To bridge these gaps, we propose to establish the “UT Southwestern Center for Regulatory Element Variation and Function”. The primary goal of this Center is to systematically catalog molecular and cellular phenotypes for disease-associated enhancers in human development, with a focus on gaining insights into mechanisms of non-canonical human genetics and gene regulation. To build a generalizable framework to understanding the impact of human genetic variation on function, we propose a high throughput perturbation platform with three primary goals: (1) Contribute to a variant/element/phenotype catalog with relevance to diseases of human development, focusing on elements genetically associated with congenital heart disease (cardiomyocytes), autism (neurons), and placental defects (trophoblasts); (2) Contribute to a variant/element/phenotype catalog for non-canonical human genetics, focusing on two understudied topics in human genetics: pleiotropic effects and non-cell autonomous effects; and (3) Contribute to a variant/element/phenotype catalog with relevance to mechanisms of gene regulation, focusing on enhancer RNAs. The Center will take advantage of recent technological innovations in genome engineering, single-cell genomics, and high content screening to enable the multiscale functional characterization of genomic variation in human developmental disorders. Several of these techniques have been pioneered by investigators contributing to this project, including: the development of novel tools for enhancer perturbation and the coupling of endogenous enhancer perturbations with a single-cell RNA-Seq readout (Mosaic-Seq). Impact and Significance: The efforts on this project will lead to a number of key outcomes and deliverables, including (1) greater understanding of the relationships between sequence variation and genome function, (2) an extensive variant/element/phenotype catalog for the community, (3) tools for generating predictive models for the community, and (4) resources to enable future functional genomics studies. Together, our multifaceted and combinatorial approaches will open new horizons to understanding the impact of regulatory variants on developmental disease phenotypes.