Multistage Genetic, Transcriptomic, and Single-Cell Evidence Prioritizes MAP1LC3A among Ferroptosis-Related Genes in Glioblastoma.

Abstract

Glioblastoma (GBM) remains a highly aggressive malignancy, and the contribution of ferroptosis-related genes to disease susceptibility remains incompletely understood. A genetically anchored, multistage framework was applied to prioritize ferroptosis-related genes associated with GBM. Among 483 genes curated from FerrDb V2, 315 had candidate cis-expression quantitative trait loci (cis-eQTLs) in eQTLGen, 250 retained at least three independent instruments after linkage disequilibrium clumping, and 226 yielded valid inverse-variance weighted (IVW) Mendelian randomization estimates using a GBM genome-wide association study comprising 6,183 cases and 18,169 controls. Thirty-four genes met the exploratory discovery criteria of P < 0.05 and a Benjamini-Hochberg false discovery rate (BH-FDR) < 0.20, with directionally concordant Bayesian weighted Mendelian randomization (BWMR) estimates. Replication-stage Mendelian randomization using GTEx V10 whole-blood cis-eQTLs supported four genes: ATG7, RPTOR, MAP1LC3A, and CHMP6. Evaluation across three independent tumor-control transcriptomic cohorts demonstrated that MAP1LC3A was consistently downregulated in tumor tissue and showed a significant random-effects pooled estimate (log&#x2082; fold change, -1.273; 95% confidence interval, -1.625 to -0.920; false discovery rate = 0.016), whereas the other three genes lacked comparable cross-cohort statistical support. Single-cell virtual knockout analysis was subsequently performed in a patient-balanced subset of 2,400 malignant cells selected from 4,916 eligible cells across 20 adult IDH-wild-type GBM tumors. Across five independently seeded runs, 3, 15, 4, and 7 robust downstream genes were identified for ATG7, RPTOR, MAP1LC3A, and CHMP6, respectively. The resulting consensus sets comprised 17 unique genes, with RND3 shared across all four targets. Gene Ontology analysis indicated enrichment of cell-adhesion and cell-surface processes, whereas no KEGG or Reactome pathways remained significant after multiple-testing correction. Collectively, these findings prioritize MAP1LC3A for future experimental investigation while distinguishing genetic association, tumor-expression concordance, and computational perturbation from definitive evidence of causality or mechanism.