Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that causes loss of joint function and significantly reduces quality of life. Plasma metabolite concentrations of disease-modifying anti-rheumatic drugs (DMARDs) can influence treatment efficacy and toxicity. This study explored the relationship between DMARD-metabolising gene variants and plasma metabolite levels in RA patients. DMARD metabolite concentrations were determined by tandem mass-spectrometry in plasma samples from 100 RA patients with actively flaring disease collected at two intervals. Taqman probes were used to discriminate single-nucleotide polymorphism (SNP) genotypes in cohort genomic DNA: rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Mean plasma concentrations of methotrexate (MTX) and MTX-7-OH metabolites were higher (p < 0.05) at baseline in rs4149081 GA genotype patients. Patients with rs1476413 SNP TT or CT alleles have significantly higher (p < 0.001) plasma poly-glutamate metabolites at both study time points and correspondingly elevated disease activity scores. Patients with the rs17421511 SNP AA allele reported significantly lower pain scores (p < 0.05) at both study intervals. Genotyping strategies could help prioritise treatments to RA patients most likely to gain clinical benefit whilst minimizing toxicity.

Keywords: DMARD; SNP; methotrexate; pharmacogenomics; rheumatoid arthritis.

Figure 1

Endpoint polymorphism genotype assay. Genotype were determined for the listed polymorphisms in the RADAR study cohort (n = 100) from the ratio of fluorescent intensities (nm) of the two-allele specific Taqman probes (VIC and FAM) at the end of PCR amplification. Clusters in upper and lower quadrants represent groups of individuals with a homozygous genotype for either allele; the middle quadrant represents individuals with heterozygous genotype. (A) rs246240, (B) rs717620, (C) rs1476413, (D) rs17421511, (E) rs2231142. (F) rs3740065, (G) rs4149081, (H) rs4846051, (I) rs10280623, (J) rs16853826. Genotypes were also confirmed by pyrosequencing in selected individuals. Genotype frequency is summarized in Table 1.

Figure 2

rs4149081 and rs1476413 genotype associations. Statistically significant associations between two polymorphisms, plasma drug metabolite concentration, other laboratory and clinical outcome measures are shown for individual genotypes in methotrexate treated participants (n = 66). Each symbol represents an individual participant of the genotype indicated on the x axes. Data grouped by rs4149081 genotypes: (A) baseline plasma concentration of unmetabolised methotrexate, (B) baseline plasma concentration of 7-hydroxy-methotrexate, (C) baseline bilirubin blood concentration, (D) weekly plasma concentrations (log scale) of listed methotrexate metabolites (PGs: polyglutamate subtypes) of a rs4149081 GA genotype participant. Data grouped by rs1476413 genotypes: (E) 6-week follow-up plasma concentration of 7-hydroxy-methotrexate, (F) baseline and (G) 6-week follow-up plasma concentrations of long-chain methotrexate 4-glutamate, (H) baseline and (I) 6-week follow-up disease activity (DAS28ESR) scores. Statistically significant differences between genotype group means are indicated by horizontal bars and an asterisk used to summarise p values adjusted by Bonferroni’s multiple comparison test: (*) p < 0.05; (**) p < 0.005 (descriptive statistics data shown in Table 3). Red horizontal bar represents genotype group mean; error bars represent standard deviation. MTX: methotrexate; BL: baseline; 6w: 6-week follow-up.

Figure 3

rs2231142 and rs17421511 genotype associations. Statistically significant associations between two polymorphisms and other laboratory and clinical outcome measures are shown for individual genotypes in methotrexate treated participants (n = 66). Each symbol represents an individual participant of the genotype indicated on the x axes. Data grouped by rs2231142 genotypes: (A) baseline and (B) 6-week follow-up red blood cell (RBC) count, (C) baseline blood alkaline phosphatase (ALP) concentration, (D) baseline and (E) 6-week follow-up lymphocyte count. Data grouped by rs17421511 genotypes: (F) baseline and (G) 6-week follow-up patient assessed pain (PgPain) levels, (H) baseline disease activity (DAS28ESR) scores, (I) baseline blood alanine aminotransferase (ALT) concentration. Statistically significant differences between genotype group means are indicated by horizontal bars and an asterisk used to summarise p values adjusted by Bonferroni’s multiple comparison test: (*) p < 0.05; (**) p < 0.005 (descriptive statistics data shown in Table 3). Red horizontal bar represents genotype group mean; error bars represent standard deviation. BL: baseline; 6w: 6 weeks follow-up.