Phenotype-Specific Pharmacogenetic Patterns of Methotrexate Neurotoxicity in Pediatric Acute Lymphoblastic Leukemia

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.