Leveraging Single-Cell Transcriptomics of Developing Rat Ocular Outflow Tissues for Prioritization of Congenital Glaucoma Candidate Genes.

UNLABELLED

Abnormal development of the intricate trabecular meshwork (TM) or Schlemm's canal (SC) structures in the eye can result in reduced aqueous humor fluid drainage and elevated intraocular pressure. If left untreated, these processes can lead to retinal ganglion cell loss, damage to the optic nerve, and infant-onset vision loss, termed congenital glaucoma. To identify gene expression important for development of these specialized aqueous humor outflow pathway (AHOP) structures, single-cell RNA sequencing was performed on rat AHOP tissues during three major periods of growth. High-quality transcriptomic profiles of 29,626 genes from 86,653 limbal cells were obtained and clustered into 13 general cell types. Refined identification of cell subtypes revealed 10,037 TM- and 546 SC-related cells that together showed enhanced expression of 395 genes compared to other limbal tissues. Interrogation of 44 genes known to be associated with Mendelian forms of childhood glaucoma revealed 36/44 (82%) were more highly expressed by SC/TM cells, whilst only 29/372 (8%) genes associated with complex later-onset glaucoma were specifically enriched. We propose prioritization of these selectively expressed genes during genome analyses of congenital/childhood glaucoma patients as they represent strong novel candidates for disease-causing variants. The transcriptomic dataset derived from these critical developing eye structures will also aid the generation of polygenic risk score estimates tailored toward early-onset glaucoma with underlying complex genetics.

SIGNIFICANCE STATEMENT

This study presents the first single-cell RNA sequencing dataset of the developing aqueous humor outflow pathway (AHOP), offering insight into the genes important for forming the tissues primarily affected in congenital/childhood-onset glaucoma. By profiling limbal cells from developing rat eyes, key AHOP cell populations, which constitute the trabecular meshwork (TM) and Schlemm's canal (SC), were identified via cell type-specific expression of known markers. This enabled examination of gene expression associated with childhood- and adult-onset glaucoma in these developing cell types and revealed 395 selectively expressed genes. These findings provide a foundational resource to understand early AHOP development, identify novel candidate disease genes, and reveal molecular targets for glaucoma prevention and therapy.