Integrative ‘omics analyses to investigate trabecular meshwork changes in glaucoma

Abstract

Purpose : The trabecular meshwork (TM) is the primary site of resistance to aqueous humor outflow, yet its molecular changes associated with dysregulated outflow in glaucoma are unknown. To investigate and characterize TM subtypes that may be implicated in glaucoma, we applied single nuclei RNA-sequencing on non-glaucomatous and glaucomatous human donor limbal ring tissue.

Methods : Limbal ring tissues were collected and flash-frozen within 6 hours post-mortem from 6 glaucomatous and 5 non-glaucomatous human donors. Samples were thawed and lysed to isolate nuclei, then barcoded using 10X Genomics Single Cell 3’ gene expression kit (v2 chemistry). Libraries were sequenced on Illumina NovaSeq 6000. Sequencing data were demultiplexed and aligned to the GRCh38 genome using 10X’s Cell Ranger software.
Single nuclei RNA-seq quality control, normalization, clustering, and differential expression analysis were run using the Seurat R package. Pathway activity scores were calculated using the AUCell R package. Deconvolution of an internal limbal ring glaucoma bulk RNA-seq study was run using the MuSiC R package. Bulk RNA-seq differential expression analysis was run using edgeR and limma R packages.

Results : By clustering our single nuclei data by pathway activity scores, we identified 6 putative TM subtypes, including a subtype (TM2) which expands in proportion with glaucoma. Genes upregulated in this “glaucoma-associated” TM subtype were related to extracellular matrix (log2FC ≥ 0.25; FDR ≤ 0.05). These genes showed stable expression in this TM population across most donors. Using our single nuclei data, we deconvolved a larger, independent glaucoma limbal ring bulk RNA-seq study (10 glaucoma, 7 non-glaucoma donors) and observed a reproducible expansion of this TM2 subtype in glaucoma.

Conclusions : Our data demonstrates that by applying novel clustering approaches using pathway activity scores, we can identify and characterize a TM subtype which may be implicated in glaucoma. Integrative snRNA-seq and bulk RNA-seq analysis reinforces our findings and facilitates target identification efforts. Further studies are needed to determine whether this identified trabecular meshwork subtype is causative or correlated to glaucoma and enable identification of new therapeutic pathways.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.