Background: Methotrexate-induced neurotoxicity (MTX-NTX) is a severe complication during childhood acute lymphoblastic leukemia (ALL) treatment, yet its underlying pharmacogenetic determinants remain incompletely understood. Methods: A multicenter retrospective nationwide case–control study was performed including 71 pediatric ALL patients (34 MTX-NTX cases and 37 controls). Targeted next-generation sequencing of 17 genes involved in MTX transport, intracellular metabolism and folate pathways was performed. Results: Transport-related genes accounted for 61.4% of all detected variants and showed the highest number of nominally significant variants. Pathway-level burden analysis demonstrated a significant enrichment of transporter-related variants in patients with MTX-NTX compared with controls (FDR-adjusted p = 0.004), whereas the analysis for folate/metabolism-related genes did not remain significant after multiple-testing (FDR-adjusted p = 0.096). Similar findings were observed in the stroke-like syndrome (SLS) subgroup (FDR-adjusted p = 0.014 and 0.108, respectively). Gene-level burden analysis identified ABCG2 and ABCC4 as the predominant contributors, harboring both 18.4% of significant variants in the overall MTX-NTX analysis and 20.6% and 17.6%, respectively in the SLS phenotype. Moreover, the distribution of significant variants was strongly correlated between the overall MTX-NTX and SLS analyses (Spearman’s ρ = 0.549, p = 0.022). Volcano plot analysis revealed ABCG2 as the gene showing the most prominent pattern of nominal associations, containing both the variants associated with a protective direction (e.g., c.1728-46G>A, p = 2.6 × 10⁻⁴) and variants associated with increased risk, including c.34G>A and c.203+36A>G (p = 0.002) and c.263+10A>G (p = 0.0097). Phenotype-specific analyses revealed distinct genetic signatures across neurological manifestations, with transporter genes predominating in focal neurological deficits. Conclusions: Our findings suggest that genetic variability in ABC transporter genes may contribute to susceptibility to MTX-NTX, whereas folate metabolism genes may represent complementary phenotype-modifying factors. These findings are exploratory and require validation in larger, independent cohorts and functional studies.

Background: Methotrexate-induced neurotoxicity (MTX-NTX) is a severe complication during childhood acute lymphoblastic leukemia (ALL) treatment, yet its underlying pharmacogenetic determinants remain incompletely understood. Methods: A multicenter retrospective nationwide case-control study was performed including 71 pediatric ALL patients (34 MTX-NTX cases and 37 controls). Targeted next-generation sequencing of 17 genes involved in MTX transport, intracellular metabolism and folate pathways was performed. Results: Transport-related genes accounted for 61.4% of all detected variants and showed the highest overall variant burden. Pathway-level burden analysis demonstrated a significant enrichment of transporter-related variants in patients with MTX-NTX compared with controls (FDR-p = 0.004), whereas the analysis for folate/metabolism-related genes did not remain significant after multiple-testing (FDR-p = 0.096). Similar findings were observed in the stroke-like syndrome (SLS) subgroup (FDR-p = 0.014 and 0.108, respectively). Gene-level burden analysis identified ABCC4 as the predominant contributor, harboring 20.0% of significant variants in the overall MTX-NTX analysis and 22.8% in the SLS phenotype, followed by ABCC1 and MTHFR. Moreover, the distribution of significant variants was strongly correlated between the overall MTX-NTX and SLS analyses (Spearman’s ρ = 0.616, p = 0.011). Volcano plot analysis revealed ABCG2 as the main susceptibility locus, containing both the variants with the strongest protective associations (e.g., c.1728-46G>A, p = 2.6×10⁻⁵) and the highest-risk variants, including c.34G > A and c.203+36A > G (p = 0.002) and c.263+10A > G (p = 0.009). Phenotype-specific analyses revealed distinct genetic signatures across neurological manifestations, with transporter genes predominating in focal neurological deficits. Conclusion: Our findings suggest that genetic variability in ABC transporter genes may contribute substantially to susceptibility to MTX-NTX, whereas folate metabolism genes would act as complementary phenotype-modifiers factors.