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

房水流出发育的单细胞转录组分析以发现新的儿童青光眼基因

• 批准号: 10510279
• 负责人: Stuart William James Tompson
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510279
• 关键词: ANGPT1 gene; Address; Affect; Age; American; Animals; Aqueous Humor; Architecture; Atlases; Biological Assay; Biology; Blindness; CYP1B1 gene; Candidate Disease Gene; Cells; Characteristics; Child; Childhood; Clinical Management; Complex; Cornea; Data; Data Set; Development; Developmental Biology; Diagnostic tests; Disease; Drainage procedure; Expression Profiling; Eye; Eye Development; Eye Enucleation; Eye diseases; Failure; Family; Female; Gender; Gene Expression; Gene set enrichment analysis; Genes; Genetic; Genetic Counseling; Genome; Glaucoma; Goals; Human; In Situ Hybridization; Inbreeding; Individual; Infant; Knowledge; Link; Liquid substance; Melanins; Modeling; Molds; Molecular; Molecular Disease; Morphology; Mus; Mutation; Ocular Hypertension; Operative Surgical Procedures; Optic Nerve; Outcome; Pain; Pathogenesis; Pathogenicity; Pathway interactions; Pharmacologic Substance; Physiologic Intraocular Pressure; Population; Population Heterogeneity; Positioning Attribute; Preparation; Proteins; RNA; Rattus; Regulation; Research; Resource Sharing; Resources; ST13 gene; Sampling; Sex Bias; Stretching; Structure; Structure of sinus venosus of sclera; Technology; Testing; Text; Tissue Differentiation; Tissue-Specific Gene Expression; Tissues; Trabecular meshwork structure; Transcript; Variant; Visual; albino rat; anterior chamber; base; cell type; cohort; convict; design; differential expression; disability; effective therapy; exome; exome sequencing; experimental group; gene function; genetic variant; improved; in silico; male; molecular marker; nerve damage; novel; overexpression; population based; primary congenital glaucoma; public health relevance; selective expression; sex; single-cell RNA sequencing; therapy development; transcriptome

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.

项目概要/摘要 原发性先天性青光眼 (PCG) 是一种毁灭性的眼部疾病，会影响婴儿并表现出一种 总体性别偏向男性（1.5:1）。它是由眼内压（IOP）升高引发的，导致 眼睛疼痛性增大和严重的组织损伤，通常导致失明和眼球摘除 儿童所占比例很大。所有已知的原因都是由于房水未能正确发育所致 流出 (AHO) 结构对于调节液体引流很重要 - 主要是小梁网 (TM) 和施累姆氏管 (SC)。目前已知 CYP1B1、LTBP2、TEK 和 ANGPT1 4 个基因的突变会导致 不同人群中 25% 的 PCG 均由其引起。仅 CYP1B1 突变就占病例的 20%，且影响更大 女性（1:2）。这些已知基因对于 TM 和/或 SC 的发育都很重要。尽管全球 迄今为止，外显子组测序工作尚未发现其余 75% 病例的潜在机制 男性的性连锁基因尚未确定。我们假设潜在的难以捉摸的基因 PCG 对于 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基因表达的全面了解 眼部结构。我们建议识别这些重要途径将有助于发现 PCG 难以捉摸的分子机制，这反过来将允许治疗方法的开发 针对潜在的疾病生物学。直到最近，AHO 表达谱分析的可行性 由于其复杂的结构和多种细胞类型，该途径受到限制。现在，单细胞RNA测序 (scRNAseq) 技术通过基于不同细胞群的分子分离来实现此类研究 单细胞水平的表达谱。为了解决我们的中心假设，我们将分析转录组 利用 scRNAseq 从 AHO 途径的整个开发过程中的所有细胞类型中提取数据。大鼠组织将 被利用是因为它们与人类共享 AHO 的所有主要阶段和形态特征 途径发育，可以培育到规定的发育年龄，并且将提供更大的组织 与小鼠进行 scRNAseq 分析。我们将在 6 时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。然后我们将使用此资源来揭示 来自分子未解的 PCG 的现有外显子组测序数据中可能致病的基因变异 案例。正在发育的 AHO 结构内的候选疾病基因表达将通过原位证实 杂交（RNAscope）研究。最后，将创建野生型和变异型小基因来测试其效果 使用各种基于细胞的检测来分析基因功能的每个候选致病变异。