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

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2022 · $233,250

## Abstract

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.

## Key facts

- **NIH application ID:** 10510279
- **Project number:** 1R21EY034251-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Stuart William James Tompson
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $233,250
- **Award type:** 1
- **Project period:** 2022-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10510279

## Citation

> US National Institutes of Health, RePORTER application 10510279, Single cell transcriptome profiling of aqueous humor outflow development for discovery of novel childhood glaucoma genes (1R21EY034251-01). Retrieved via AI Analytics 2026-07-27 from https://api.ai-analytics.org/grant/nih/10510279. Licensed CC0.

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