The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

Abstract

Objective. The response to and toxicity of methotrexate (MTX) are unpredictable in patients with juvenile idiopathic arthritis (JIA). Intracellular polyglutamation of MTX, assessed by measuring concentrations of MTX polyglutamates (MTXGlu), has been demonstrated to be a promising predictor of drug response. Therefore, this study was aimed at investigating the genetic predictors of MTXGlu variability and associations between MTXGlu and drug response in JIA.Methods. The study was designed as a singlecenter cross-sectional analysis of patients with JIA who were receiving stable doses of MTX at a tertiary care children's hospital. After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing. Clinical data were collected by chart review. Genotyping for 34 single-nucleotide polymorphisms (SNPs) in 18 genes within the MTX metabolic pathway was performed. An ion-pair chromatographic procedure with mass spectrometric detection was used to measure MTXGlu 1-7 .Results. Analysis and genotyping of MTXGlu was completed in the 104 patients. K-means clustering resulted in 3 distinct patterns of MTX polyglutamation. Cluster 1 had low red blood cell (RBC) MTXGlu concentrations, cluster 2 had moderately high RBC MTXGlu 1 ؉ 2 concentrations, and cluster 3 had high concentrations of MTXGlu, specifically MTXGlu 3-5 . SNPs in the purine and pyrimidine synthesis pathways, as well as the adenosine pathway, were significantly associated with cluster subtype. The cluster with high concentrations of MTXGlu 3-5 was associated with elevated liver enzyme levels on liver function tests (LFTs), and there were higher concentrations of MTXGlu 3-5 in children who reported gastrointestinal side effects and had abnormal findings on LFTs. No association was noted between MTXGlu and active arthritis.Conclusion. MTXGlu remains a potentially useful tool for determining outcomes in patients with JIA being treated with MTX. The genetic predictors of MTXGlu variability may also contribute to a better understanding of the intracellular biotransformation of MTX in these patients.The response to methotrexate (MTX) in patients with juvenile idiopathic arthritis (JIA) is variable, with no identified predictors of response or toxicity (1-3). The mechanism of action of the drug is complex and incompletely understood. However, over the past several decades, we have gained increasing knowledge regarding specific sites of MTX action within the cellular folate cycle. Due to its inhibitory effect on several target enzymes, we know that MTX disrupts the folate cycle, which results in decreased production of DNA precursors, disruption of methyl-dependent reactions, and accumulation of adenosine, the latter of which has antiinflammatory properties (4-6).

Using Fluorescence Polarization Immunoassay for Determination of Erythrocyte Methotrexate Polyglutamates, a Quick and Easy Test?

“…3 However, MTX polyglutamates (MTX-PGs) accumulate intracellularly, which are related to clinical response and have been proposed to be of use for therapeutic drug monitoring purposes. [4][5][6][7] In low-dose MTX treatment, the pentaglutamate (MTX-PG5) is the highest order of glutamylation detected, although the triglutamate form (MTX-PG3) of MTX predominates. 5,8 Polyglutamylation retains MTX in the cell because MTX-PGs are a poor substrate for the MTX efflux proteins.…”

Role of methotrexate in juvenile idiopathic arthritis: where we have been and where we are going

“…Furthermore, in a longitudinal Dutch cohort of 287 JIA patients on MTX, multivariate analysis corrected for the disease duration prior to therapy, the dose of MTX and the PGA at baseline revealed that SNPs in two transporter genes (ABCB1 SNP rs1045642 or ABCC3 SNP rs4793665) increased the likelihood of achieving an ACR Pedi 70 response within the first year, and the presence of a SNP in SLC19A1 SNP rs1051266 diminished the likelihood two-to three-fold [54]. The membrane transporter SLC19A1 transports MTX into the cell and has been shown to have an effect upon response in RA [55,56]; however, this association was not seen with JIA in prior studies [49,53], and in the current work by de Rotte et al, the association did not remain significant after Bonferroni correction, thus requiring further replication and validation despite a potential physiologic explanation. Although there are variable reports supporting or refuting the association of these SNPs and drug effect in the literature [54], efflux transporters ABCB1 and ABCC3 could affect MTX response by resulting in cellular retention Red dotted lines and squares denote known enzymes inhibited by MTX.…”

“…Subsequently, the list of MTX target genes has been extended to include aminoimidazole carboxamide ribonucleotide (AICAR) transformylase (gene name ATIC ), which inhibits de novo purine synthesis and promotes the accumulation of AICAR that inhibits AMP deaminase and results in a build up of intracellular AMP, with subsequent increase in extracellular adenosine [43][44][45][46]. Adenosine has Role of methotrexate in juvenile idiopathic arthritis Review been thought to be a large contributor to the site specific anti-inflammatory effects of MTX [45,47,48], and furthermore, pharmacogenomic studies in RA and JIA have provided additional support for the importance of the purine synthesis and adenosine pathways with MTX response [46,49,50]. g-glutamyl hydrolase (GGH), the enzyme responsible for glutamate removal from MTX, transforms MTX into a form that can be effluxed from the cell by the ATP-binding cassette (ABC) family of transporters (Figure 2) [39].…”

“…The polyglutamated forms of MTX (MTXGlu n ) are important for cellular retention of the drug and target enzyme inhibition, and are more stable than rapidly cleared serum levels of MTX, thus have been explored as more reliable cellular MTX biomarkers [40]. Although an association between red blood cell (RBC) long-chain MTXGlu concentrations and the effectiveness of MTX in R A has been reported [46,64,65], these findings have not been consistently replicated in RA or JIA [49,66,67]. In approximately 100 JIA patients on stable doses of MTX, RBC MTXGlu 1-7 concentrations and proportions were found to vary 40-100-fold.…”

Therapeutic advancements in juvenile idiopathic arthritis

“…Serum levels of cellular proteins indicative of subclinical disease activity such as calprotectin (MRP 8/14) have been identified as plausible predictors of successful MTX withdrawal [20]. Polyglutamated forms of MTX and folate have been studied for correlation with drug response, and may be useful in the future to predict optimal or poor responders to the drug [21][22][23]. The effect of genotype upon drug response has been extensively studied in adults with RA and recently more in JIA.…”