Single cell transcriptome profiling of aqueous humor outflow development for discovery of novel childhood glaucoma genes

项目摘要/摘要 原发性先天性青光眼(PCG)是一种影响婴儿的破坏性眼病，表现为 总体上对男性的性别偏见(1.5：1)。它是由眼压升高引发的，这会导致 眼睛疼痛的扩大和严重的组织损伤，通常会导致失明和眼球摘除 相当大比例的儿童。所有已知的原因都是由于未能正确发展房水。 流出(Aho)结构对体液排出的调节很重要--主要是小梁网(TM) Schlemm管(SC)。目前已知有4个基因的突变，即CYP1B1、LTBP2、TEK和ANGPT1 在不同的人群中造成25%的PCG。仅CYP1B1突变就占了20%的病例，而且影响更大 女性(1：2)。这些已知基因对TM和/或SC的发育都很重要。尽管是全球性的 外显子组测序工作到目前为止，其余75%病例背后的机制仍未被发现 而与男性性别相关的基因还没有确定。我们假设那些难以捉摸的基因 PCG对AHO途径的发展至关重要，因此在AHO途径的发展过程中表达，但我们 目前缺乏对这些重要基因形成过程中的基因表达的全面了解 眼部结构。我们认为，识别这些重要途径将使我们能够发现 PCG难以捉摸的分子机制，这反过来将使治疗的发展成为可能 针对潜在的疾病生物学。直到最近，对Aho进行表情剖析的可行性 由于其复杂的结构和多样的细胞类型，通路受到限制。现在，单细胞RNA测序 (ScRNAseq)技术通过对不同细胞群体的分子分离实现了这类研究 在单细胞水平上的表达谱。为了解决我们的中心假设，我们将分析转录本 在其整个发育过程中利用scRNAseq从AHO途径内的所有细胞类型。老鼠的组织会 被利用是因为它们与人类分享AHO的所有主要阶段和形态特征 途径发展，可以繁殖到特定的发育年龄，并将提供显著更大的组织 对小鼠进行scRNAseq分析。我们将在6点从男性和女性组织中生成单独的数据集 不同年龄(P4、P8、P16、P48、P80和P180)，能够识别差异表达的基因和 每种细胞类型、发育阶段和性别特有的途径。然后我们将利用这个资源来揭示 来自分子上未解决的PCG的现有外显子组测序数据中可能的致病基因变异 案子。在发育中的AHO结构中的候选疾病基因的表达将通过原位确认 杂交(RNAScope)研究。最后，将创建野生型和变异型微基因，以测试 每一个候选的致病变异基因的功能使用各种基于细胞的分析。

Project Summary / Abstract Primary congenital glaucoma (PCG) is a devastating eye disorder that affects infants and demonstrates an overall gender bias towards males (1.5:1). It is triggered by elevated intraocular pressure (IOP) which leads to painful enlargement of the eye and severe tissue damage, often resulting in blindness and eye enucleation in a significant proportion of children. All known causes are due to failure to correctly develop the aqueous humor outflow (AHO) structures important for regulation of fluid drainage - primarily the trabecular meshwork (TM) and Schlemm’s canal (SC). Mutations in 4 genes, CYP1B1, LTBP2, TEK, and ANGPT1, are currently known to cause 25% of PCG in diverse populations. CYP1B1 mutations alone account for 20% of cases and effect more females (1:2). These known genes are all important for development of the TM and/or SC. Despite global exome sequencing efforts to date, the mechanisms underlying the remaining 75% cases remain undiscovered and male sex-linked genes have yet to be identified. We hypothesize that the elusive genes underlying PCG are critical for and therefore expressed during the development of the AHO pathway, but we currently lack a comprehensive knowledge of gene expression during the formation of these important ocular structures. We propose that the identification of these vital pathways will enable discovery of the elusive molecular mechanisms of PCG, which in-turn will permit the development of treatments aimed at the underlying disease biology. Until recently, the feasibility of expression profiling the AHO pathway was limited due to its complex architecture and variety of cell types. Now, single-cell RNA sequencing (scRNAseq) technology has enabled such studies by molecular separation of different cell populations based on expression profiles at the single-cell level. To address our central hypothesis, we will profile transcriptomes from all cell types within the AHO pathway throughout of its development utilizing scRNAseq. Rat tissues will be utilized because they share with humans all major stages and morphological characteristics of AHO pathway development, can be bred to defined developmental ages, and will supply significantly larger tissues versus mice for scRNAseq profiling. We will generate separate datasets from male and female tissues at 6 different ages (P4, P8, P16, P48, P80, and P180), enabling identification of differentially expressed genes and pathways specific to each cell type, developmental stage, and gender. We will then use this resource to reveal likely disease-causing gene variants within existing exome sequencing data from molecularly unsolved PCG cases. Candidate disease gene expression within the developing AHO structures will be confirmed by in situ hybridization (RNAscope) studies. Finally, wild-type and variant mini-genes will be created to test the effect of each candidate disease-causing variant on the gene’s function using a variety of cell-based assays.