Identification of microglia-associated hub genes and a CX3CR1/P2RY12 two-gene diagnostic model for cross-subtype epilepsy-associated cortical malformations: integrated transcriptomic analysis with external validation

Abstract

Focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC) are major causes of drug-resistant epilepsy, yet shared molecular mechanisms across these structurally distinct malformations remain poorly characterized. We performed an integrated transcriptomic analysis of RNA-seq data from GSE256068 [85 samples: 64 cortical (FCD2a, FCD2b, TSC, controls) and 21 hippocampal (TLE-HS, controls)]. Differential expression analysis with limma identified 865, 1,915, 2,772, and 3,749 DEGs in FCD2a, FCD2b, TSC, and TLE-HS versus controls, respectively, with 49 shared DEGs across all four subtypes. Weighted gene co-expression network analysis (WGCNA) identified the brown and red modules as key disease-associated modules. By intersecting WGCNA hub genes (|GS| > 0.5, |MM| > 0.8) with shared DEGs, we identified 15 hub genes, all functionally associated with microglia/myeloid lineage, including IRF8, TLR7, CX3CR1, P2RY12, C3AR1, and CCR1. Gene set enrichment analysis (GSEA) revealed 15 KEGG pathways consistently enriched across all subtypes, converging on Toll-like receptor, NF-kB, chemokine, and complement signaling. Immune deconvolution using MCP-counter and xCell confirmed elevated monocytic lineage and T cell infiltration in disease cortex, with monocytic scores showing a positive trend with seizure frequency. A two-gene logistic regression model (CX3CR1 + P2RY12), selected by LASSO and random forest, achieved a training AUC of 0.979 and a nested five-fold cross-validation AUC of 0.862 (95% CI: 0.693–1.000), with an external validation AUC of 0.865 (95% CI: 0.681–1.000) in GSE213488. Drug repurposing via Enrichr identified vorinostat (HDAC inhibitor) as a top candidate, and IRF8 was predicted as the master transcriptional regulator. In vitro , mTOR activation with MHY1485 preserved P2ry12 expression in BV-2 microglia, whereas LPS stimulation induced its loss, supporting a distinct microglia/myeloid-associated program driven by mTOR hyperactivity. These findings demonstrate that cross-subtype transcriptomic convergence on microglial innate immune activation represents a unifying molecular mechanism in FCD and TSC, and that CX3CR1 and P2RY12 together constitute a two-gene diagnostic classifier with encouraging but preliminary performance with potential clinical utility.