Background/Purpose: Methotrexate (MTX) intolerance is common in juvenile idiopathic arthritis (JIA) and poses the risk of premature termination of an effective treatment. MTX intolerance is a compound concept of stomachache, nausea, vomiting and behavioral symptoms associated with low-dose MTX and covers anticipatory and associative symptoms. Large inter-individual variation in the level of MTX intolerance exists and genetic factors may play a role. The objective is to investigate if MTX intolerance is associated with selected single nucleotide polymorphisms (SNPs) in MTX transporter proteins in children with JIA treated with low-dose MTX.

Methods: The local research ethics committee approved this observational study. Eligible children were diagnosed with JIA (ILAR criteria), aged ³9 years and treated with low-dose MTX. The enrolment period was December 2013 – July 2016. MTX intolerance was assessed by the parentsÕ completion of the MTX intolerance severity score (MISS) (1). A child was categorized MTX intolerant if the MISS score ³6 and min. 1 point in an anticipatory, associative or behavioral symptom. At enrolment a blood sample was drawn for the SNP analysis. DNA was extracted from EDTA blood using salt precipitation. The SNP analysis was performed by PCR amplifying an amplicon containing the selected SNP and then Sanger Sequencing (Eurofins Genomics¨). Statistic analyses for the SNPs were performed using PLINK 1.9, logistic regression of additive effect of alleles, filtering for Hardy-Weinberg equilibrium and accounting for more than two possible alleles present in the study population for one SNP.

Results: The selected SNPs within genes encoding the MTX transporter proteins were: SLCO1B1 (rs4149056; rs4149081), SLCO1B3 (rs2117032), SLC19A1 (rs1051266), ABCC2 (rs2273697; rs3740066; rs717620), ABCB1 (rs2032582; rs1045642). The study population consisted of 121 JIA patients, 82 girls and 39 boys, with a median age of 13.3 years (IQR: 11.3-15.1). The median MTX dose was 9.7 mg/m²/week (IQR: 9.0-10.9) and the median MTX treatment duration was 340 days (IQR: 142-766). A completed MISS and SNP analysis was available for 118 patients (the MISS: 1 missing; the SNP analysis: 2 missing) and 72 children were categorized as MTX intolerant.

Table 1

OR; Odds Ratio. Chr; Chromosome. BP; Base Position.

*SNP variant not available for 1 child

Conclusion: MTX intolerance was not significantly associated with any of the selected SNPs in the MTX transporter proteins. This could indicate that focus in future should be directed at other genetic targets, possibly SNPs in genes encoding other MTX relevant factors.

(1) Bulatovic M, Heijstek MW, Verkaaik M, van Dijkhuizen EH, Armbrust W, Hoppenreijs EP, et al. High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: development and validation of a methotrexate intolerance severity score. Arthritis Rheum 2011 Jul;63(7):2007-2013