Single-cell human genetics: effects of structural variants and mutations during embryonic development at single cell resolution.

罕见遗传病通常是严重的多系统疾病，具有广泛的表型，在受影响的个体之间可能有很大的差异，从相对轻微到严重。罕见儿科遗传病的诊断率目前在35%至75%之间。然而，基因型-表型相关性是极其困难的，因为即使在同一基因突变的个体中，这些疾病的严重程度也可能不同。我们假设，在小鼠胚胎发生过程中，患者特异性突变和结构变异的整个生物体单细胞谱可以为理解罕见的多系统疾病的表型变异性提供重要的新见解。研究体内胚胎发生的一个基本挑战是，目前缺乏足够的通量和分辨率的技术来获得快速多样化和扩大数量的细胞类型的分子状态和轨迹的全局视图。为了解决这些挑战，我们将采用三种主要的实验方法：AIM 1：我们的目标是通过分析携带患者特异性突变的小鼠突变体，在单细胞水平上研究胚胎发育过程中严重多系统疾病的多效性效应。我们将使用全胚胎sci-RNA-seq方法对Runx2缺陷小鼠、锁骨颅骨发育不良模型和科涅利亚·德·兰格综合征小鼠模型进行表型分析。我们预计，整个生物体的sci-RNA-seq方法将能够发现分子程序中的细微缺陷或特定细胞类型的相对比例。AIM 2：我们的目标是在单细胞分辨率下研究更复杂的遗传变异，例如微缺失综合征和21三体。这些变异的具体挑战在于它们包括许多基因和调控序列，这些基因和调控序列在它们独特的组合中有助于表型。我们将通过分析16p11.2微缺失综合征（16p11.2+/−小鼠）和人类21三体小鼠模型（Hsa21小鼠）来研究结构变异。我们的数据将促进我们对人类疾病中sv的认识，并建立单细胞rna -seq作为转基因小鼠中sv的表型分析工具。目的3：我们旨在研究与先天性疾病相关的染色质可及性和非编码调控环境的变化。我们将利用单细胞ATAC-seq技术建立小鼠器官发生（E9.5-E13.5）过程中染色质可及性的单细胞图谱，这将为胚胎基因调控的研究提供重要资源。我们还将分析CdLS的小鼠模型。单细胞方法是非常雄心勃勃和及时的，从未在人类遗传学领域应用。我们将产生大量的数据，这些数据本身就是值得发布的宝贵资源。我们的目标不亚于将发育生物学中的复杂过程转化为可以通过算法而不是通过湿的实验室分析来研究的计算问题。

Rare genetic diseases are often severe multisystem disorders with a wide range of phenotypes and may vary widely among affected individuals ranging from relatively mild to severe. Diagnostic yields for rare pediatric genetic diseases are currently between 35 and 75%. Yet genotype-phenotype correlations are extremely difficult, since the severity of these disorders can differ even in individuals with mutations in the same gene. We hypothesize, that whole organism single cell profiling of patient specific mutations and structural variants during mouse embryogenesis can provide important new insights to understand phenotypic variability of rare multisystem disorders.A fundamental challenge to study in vivo embryogenesis, is the lack of current technologies with sufficient throughput and resolution to obtain a global view of the molecular states and trajectories of a rapidly diversifying and expanding number of cell types. To address these challenges, we will apply three main experimental approaches: AIM 1: We aim to investigate the pleiotropic effects of severe multisystem disorders during embryonic development at single cell level by analyzing mouse mutants harboring patient specific mutations. We will use the whole embryo sci-RNA-seq approach for phenotyping of Runx2 deficient mice, a model for cleidocranial dysplasia and a mouse models for Cornelia de Lange syndrome. We anticipate that the whole organism sci-RNA-seq approach will enable the discovery of subtle defects in the molecular programs or the relative proportions of specific cell types.AIM 2: We aim to study more complex genetic variants, e.g. microdeletion syndromes and trisomy 21 at single cell resolution. The specific challenge of these variants is that they include many genes and regulatory sequences, which in their unique combination contribute to the phenotype. We will study structural variants by analyzing the 16p11.2 microdeletion syndrome (16p11.2+/− mice) and a mouse models for human trisomy 21 (Hsa21 mice). Our data will advance our knowledge about SVs in human disease and establish single cell-RNA-seq as phenotyping tool for SVs in transgenic mice. AIM 3: We aim to investigate changes in chromatin accessibility and the non-coding regulatory landscape associated with congenital disease. We will use single cell ATAC-seq and create single cell atlas of chromatin accessibility during mouse organogenesis (E9.5-E13.5) that will serve as an important resource for the study of embryonic gene regulation. We will also analyze a mouse models for CdLS.The single cell approach is extremely ambitious and timely and has never been applied in the field of human genetics. We will generate an enormous amount of data that will be by itself a valuable resource worth publishing. We aim at nothing less than transforming complex processes in development biology into computational problems that can be investigated by algorithms rather than by wet lab-based assays.