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

Overview

Authors: Hongliang Zeng¹, Bin Li¹

Ganzhou Hospital-Nanfang Hospital, Southern Medical University (Ganzhou People’s Hospital), Ganzhou, China

Journal: Frontiers in neuroscience, volume 20, article 1954412

Dates: received 31 July 2026; accepted 15 September 2026; published online 8 October 2026

Type: Research article · Language: English

License: CC BY

Identifiers: DOI · PMCID PMC13687088

Status: data only (how the code is found and verified)

Categories: genetics / omics (modality), epilepsy (population)

Methods: Statistics, Smoothing, state filtering, decompositions, Machine learning, Connectivity, Spectral & time-frequency

Keywords: CX3CR1, diagnostic model, focal cortical dysplasia, microglia, neuroinflammation, P2RY12, tuberous sclerosis complex, WGCNA

Citations: not cited yet (Europe PMC); 28 references in the paper

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.

Reproduced under the paper's license (CC BY), from the paper cited above.

Code

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Data

Datasets cited

geo:GSE256068, at NCBI GEO; found in “Data availability statement”

Data availability statement

The data are publicly available in the GEO repository under accession numbers GSE213488 (https://ncbi.nlm.nih.gov/nucleotide/GSE256068) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE256068) and GSE213488 (https://ncbi.nlm.nih.gov/nucleotide/GSE213488) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213488).

Reproduced under the paper's license (CC BY), from the paper cited above.

Versions

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Version 1, 24 October 2026: the first record

Recorded: type, language, journal, volume, pages, dates, 2 authors, 8 keywords, 28 references.

Cite

This paper

Zeng, H., & Li, B. (2026). 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. Frontiers in neuroscience, 20, 1954412.

BibTeX

@article{zeng2026identification, author = {Zeng, Hongliang and Li, Bin}, title = {{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}}, journal = {Frontiers in neuroscience}, year = {2026}, month = oct, volume = {20}, pages = {1954412}, publisher = {Frontiers Media SA}, issn = {1662-4548}, pmcid = {PMC13687088} }

RIS

TY - JOUR AU - Zeng, Hongliang AU - Li, Bin TI - 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 T2 - Frontiers in neuroscience J2 - Front Neurosci PY - 2026 DA - 2026/10/24 VL - 20 SP - 1954412 SN - 1662-4548 PB - Frontiers Media SA LA - en ER -

CSL-JSON

{ "id": "pmcid:PMC13687088", "type": "article-journal", "title": "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", "container-title": "Frontiers in neuroscience", "author": [ { "family": "Zeng", "given": "Hongliang" }, { "family": "Li", "given": "Bin" } ], "container-title-short": "Front Neurosci", "volume": "20", "page": "1954412", "PMCID": "PMC13687088", "ISSN": "1662-4548", "publisher": "Frontiers Media SA", "language": "en", "issued": { "date-parts": [ [ 2026, 10, 24 ] ] } }

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