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

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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.