MTX pathway gene variants, erythrocyte methotrexate polyglutamates, and treatment outcomes in rheumatoid arthritis

Rheumatoid arthritis (RA) exhibits significant inter-patient variability in response to and toxicity from methotrexate (MTX). The clinical utility of erythrocyte methotrexate polyglutamates (MTXPGs) and MTX-pathway pharmacogenetics remains uncertain. This study investigates the relationships between MTX-pathway gene polymorphisms, erythrocyte MTXPG levels, and MTX treatment outcomes in RA. In a single-center, cross-sectional cohort study conducted in southern Fujian from 2017 to 2020, we analyzed 140 Han Chinese RA patients who had been receiving stable low-dose oral MTX (7.5-15 mg/week) for at least three months. Genotyping was performed using MassARRAY, and MTXPG levels 1-6 were quantified in red blood cells via LC-MS/MS. Data on treatment efficacy (measured by ACR20 and clinical scales) and MTX-related adverse drug reactions (ADRs) were collected, with associations analyzed through univariate and multivariable models. MTXPG levels 1-3 were detectable in all patients, while longer-chain MTXPGs were infrequent. The SLCO1B1 521T>C polymorphism was independently associated with lower levels of MTXPG1 (B=-1.119), MTXPG2 (B=-0.924), and total MTXPG (B=-0.849), all with P-values ≤0.045. However, MTXPG levels did not correlate with MTX efficacy or ADRs. The GGH 401C>T polymorphism was associated with a reduced ACR20 response (OR=0.421, p=0.021) and higher visual analog scale (VAS) and patient global assessment (PGA) scores. Additionally, the variants SLCO1B1 521T>C and ABCB1 3435C>T were linked to higher scores in the Patient Health Global Assessment (PHGA) and Health Assessment Questionnaire (HAQ). In this low-dose MTX cohort, erythrocyte MTXPGs did not predict clinical outcomes. However, variants in SLCO1B1, GGH, and ABCB1 emerged as exploratory candidate markers for MTX response, warranting validation in larger prospective cohorts.