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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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要