Discovering novel glaucoma neural repair genes using computational approaches based on single-cell RNA-seq data

Abstract

Purpose : To identify novel genes involved in neural repair mechanisms in glaucoma, leveraging computational analyses of single-cell RNA sequencing (scRNA-seq) data, and to uncover potential therapeutic targets for glaucoma.

Methods : We generated a single-cell RNA-seq database of mice retina and extracted RGCs using THY1 antibody-coated beads. We sequenced 7,594 cells from glaucomatous and healthy retinal samples through HiSeq 3000 and SMART-Seq v4 technologies and employed advanced computational algorithms to analyze gene expression profiles (Figure 1). Steps 1-5 primarily included preprocessing and steps 6-9 included graph-based clustering, cluster scoring and annotation based on type-specific markers to exclude non-RGC clusters, and then re-clustering the RGCs using the Louvain algorithm. We selected 30 top genes in each cluster and assessed these genes by eleven evaluation factors including Pearson and Spearman correlations of genes with strong Pan-RGC marker (Rbpms/Rbpms2), genes expressed in glaucomatous samples and not expressed in control samples, expression level of genes in retina tissue/eye, checking literature, gene summaries, gene functions, phenotype of knockout/mutation, diseases related to genes, and investigating genes by ChatGPT4, to discover candidate genes associated with glaucoma (Figure 2).

Results : We discovered 32 retina clusters of which 4 were identified to be RGC types and subtypes. We identified Reep1, Hint2, Jazf1, Meis3, and Vax2 as novel potential markers for N-RGC subtype and Apoc1 and Fcgrt as novel markers for the F-RGC subtypes that are associated with glaucoma. The summary results in Figure 2 demonstrate the potential of this approach to discover novel genes in these and other retinal cells linked to cellular reliance, plasticity, and repair.

Conclusions : The findings of this study may provide novel understanding of the genetic underpinnings of neural repair in glaucoma. The identified genes not only enhance our understanding of glaucoma pathogenesis but may also offer promising avenues for the development of targeted gene therapies. Results highlight the important role of single-cell technologies in uncovering complex disease mechanisms.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1. Workflow of the pipeline accompanied by visualization.

 

Figure 2. Candidate genes associated with glaucoma and various approaches to validate findings.