Effect of methotrexate and genotype on RBC folate concentrations and polyglutamate distribution in JIA

Abstract

Purpose: The mechanism of action of methotrexate (MTX) remains incompletely understood. As a potent antifolate drug, MTX has several enzymatic targets in the folate pathway. We measured intracellular folate redox states and polyglutamates to better understand the effect of drug and genotype upon intracellular folate pools in JIA. Method(s): This single center cross-sectional study evaluated 101 JIA patients on stable doses of MTX, and 94 JIA patients who were not currently on MTX. After obtaining informed consent, blood was obtained during routine lab monitoring. Genotyping for 34 SNPs in 19 genes within the MTX metabolic pathway was performed. Folates were extracted from RBCs and analyzed in a semi-quantitative relative fashion (i.e. without standards) using a similar method as for MTX polyglutamates, involving an ion-pairing chromatographic procedure with mass spectrometric detection. Result(s): Measured intracellular folate isoforms included: 5,10 methenyl tetrahydrofolate (5,10-MTHF) which included the isoforms 5 formyl THF, 10 formyl THF and 5,10 methenyl THF; 5 methyl tetrahydrofolate (5-MTHF); and 5-MTHF polyglutamates (5-MTHF-PG%3-10). Subjects on MTX had expectedly lower folate isoform concentrations than those not on MTX including 5-MTHF (678.6 +/- 281.2 nmol/L vs. 1022.2 +/- 489.3 nmol/L, p<0.0001) and 5,10-MTHF (68.4 +/- 77.0 nmol/L vs. 91.7 +/- 106.3 nmol/L, p=0.04). 5-MTHF-PG% distribution revealed higher proportions of long chain polyglutamates in patients receiving MTX. Of all clinical variables tested (including the use of folate supplementation) only MTX dose (in mg/kg) was inversely related to 5-MTHF concentrations (p=0.0009). Study participants with active arthritis had higher concentrations of summed RBC folates than those without active arthritis (p=0.01). Variations in folate pathway genes were further investigated in the individuals on MTX. Homozygote variant (var/var) subjects in MTHFD1 (rs2236225) had significantly higher concentrations of 5-MTHF than heterozygotes (wt/var, p=0.009) and wild type patients (wt/wt, p=0.016). MTHFR (rs1801133) var/var subjects had significantly lower concentrations of 5-MTHF than wt/var individuals (p=0.017). On the contrary, MTHFR (rs1801133) var/var subjects had significantly higher concentrations of 5,10-MTHF than both wt/var and wt/wt patients (p<0.0001). Two distinct SNPs in the ATIC gene had opposite effects upon 5,10-MTHF concentrations. ATIC (rs12995526) var/var subjects had significantly higher concentrations of 5,10-MTHF than wt/wt subjects (p=0.01), and ATIC (rs4673990) wt/var subjects had significantly lower concentrations than wt/wt subjects (p=0.015). Conclusion(s): Variation in intracellular folate isoforms may be predictive of drug response in JIA. Genotypic differences beyond MTHFR may explain differential intracellular folate concentrations. (Table Presented).