Transcriptomic profiling reveals neural–immune–stromal dysregulation and risk-associated gene signatures in advanced keratoconus

This study aimed to characterize transcriptomic alterations and the effects of demographic and behavioral risk factors on disease-associated gene expression patterns in late-stage keratoconus (KC). Corneal stromal tissues from 31 KC patients and four normal donors underwent bulk RNA sequencing. Differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were used to identify key molecular changes. Subgroup analyses based on sex, allergy status, eye rubbing intensity (ERI), body mass index (BMI), and childhood socioeconomic status (SES) were conducted using gene set enrichment analysis (GSEA) and WGCNA. Key genes from WGCNA modules were validated by quantitative real-time PCR (qRT-PCR) and three public transcriptomic datasets. Enrichment analyses based on the 4469 identified DEGs (Padj < 0.05) revealed suppression of extracellular matrix (ECM) organization, axon guidance, synaptic signaling, and immune activity (Padj < 0.05). Subgroup GSEA demonstrated that high ERI correlated with ECM pathways, elevated BMI correlated with mitochondrial dysfunction, and low childhood SES correlated with upregulated mitochondrial metabolism and immune activation (Padj < 0.05). Chromatin regulation and antioxidant activity were enriched in females (Padj < 0.05). No transcriptomic associations were detected for allergy status. WGCNA revealed ERI-related modules involving Wnt and PI3K-Akt signaling and SES-related modules implicating cholinergic synapse and axon guidance pathways (Padj < 0.05). Validation using qRT-PCR and public datasets confirmed the downregulation of all 15 selected genes in KC (Padj < 0.05). qRT-PCR also confirmed differential expression of five subgroup-associated genes across KC subgroups (P < 0.05). These findings emphasize the neural-immune-stromal axis dysregulation in KC and elucidate the contribution of common risk factors to disease progression.