Early Erythrocyte Methotrexate Polyglutamate-4 Predicts Treatment Response and Is Influenced by GGH 401C>T Genotype in Patients with Rheumatoid Arthritis

Background/Aims: This study aimed to examine the ability of methotrexate polyglutamate (MTXPG) concentrations in erythrocytes to predict methotrexate (MTX) treatment response and evaluate the association between MTXPG concentrations and single nucleotide polymorphisms (SNPs) involved in MTX metabolism. Materials and Methods: Methotrexate polyglutamate (MTXPG) concentrations in erythrocytes were measured in 76 MTX-naive patients utilizing liquid chromatography-tandem mass spectrometry. Real-time polymerase chain reaction was conducted to identify solute carrier family 19 member 1, folylpolyglutamate synthase, and gamma-glutamyl hydrolase (GGH) genotypes. Associations between MTXPG concentrations and MTX treatment response and between MTXPG concentrations and SNPs were analyzed. Results: This study identified 67 MTX responders and nine non-responders at 24 weeks. MTXPG4 concentrations at four weeks were significantly greater in responders than in non-responders (P < .01). Receiver-operating characteristic analyses for detecting MTX responders at 24 weeks using MTXPG4 at four weeks revealed an area under the curve of 0.77 at a cut-off level of 0.46 nmol/L. The rate of achieving MTXPG4 of ≥0.46 nmol/L at four weeks was significantly higher in patients with CC genotype than those with CT and TT genotypes in GGH 401C>T (P = .032). The adjusted odds ratio of achieving MTXPG4 of ≥0.46 nmol/L at four weeks was significantly greater in patients with the CC genotype than those with the CT and TT genotypes in GGH 401C>T (2.99 [1.16-7.96]). Conclusions: This study indicates that MTXPG4 concentrations at four weeks after MTX initiation predict treatment response at 24 weeks. Further, patients with the CC genotype in GGH 401C>T achieve higher MTXPG4 concentrations. Cite this article as: Takahashi M, Takahashi S, Mori A, et al. Early erythrocyte methotrexate polyglutamate-4 predicts treatment response and is influenced by GGH 401C>T genotype in patients with rheumatoid arthritis. ArchRheumatol. Published online March 3, 2026. doi:10.5152/ ArchRheumatol.2026.25173.