European Journal of Clinical Pharmacology

, Volume 64, Issue 11, pp 1057–1068 | Cite as

Genetic determinants of methotrexate toxicity in rheumatoid arthritis patients: a study of polymorphisms affecting methotrexate transport and folate metabolism

  • Petra Bohanec Grabar
  • Dušan Logar
  • Boris Lestan
  • Vita DolžanEmail author



Methotrexate (MTX) is a disease-modifying antirheumatic drug used in the treatment of rheumatoid arthritis (RA). Genetic polymorphisms of reduced folate carrier (RFC1 A80G), P-glycoprotein (MDR1 G2677T>A/C and C3435T), 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TS 2R→3R), methionine synthase (MS A2756G) and methionine synthase reductase (MTRR A66G) modify MTX transport and metabolic effects and may influence the treatment response.


A genotyping approach was used to determine the studied polymorphisms in 213 RA patients.


We observed that 56 (26.3%) patients discontinued MTX treatment due to poor response and/or toxicity. RFC1 A80G and MDR1 C3435T polymorphisms increased the risk for overall MTX toxicity (P = 0.039, OR = 3.574, 95% CI = 1.065–11.993 and P = 0.032, OR = 7.801, 95% CI = 1.194–50.960 respectively), while MTHFR A1298C polymorphism had a protective effect on overall MTX toxicity (P = 0.027, OR = 0.170, 95% CI = 0.035–0.820).


Our results suggest that genetic polymorphisms in the folate metabolic pathway and MTX transporters modify the toxicity but not the efficacy of MTX treatment.


Folate metabolic pathway Genetic polymorphism Methotrexate Rheumatoid arthritis 



The authors wish to thank Prof. Emilio Martínez de Victoria Muñoz, PhD, and Prof. Mariano Mañas Almendros, PhD, from Instituto de Nutrición y Tecnología de los Alimentos (INYTA), University of Granada, Spain, for kindly providing the program Alimentación y Salud 2.0. The authors wish to acknowledge Blaž Rozman, MD, PhD, Matija Tomšič, MD, PhD, Mojca Kos-Golja, MD, Sonja Praprotnik, MD, PhD, Aleš Ambrožič, MD, PhD, Alenka Šipek-Dolničar, MD, MSc, Martina Plešivčnik-Novljan, MD, MSc, Cvetka Kastelic-Klasinc, MD, and Nataša Gašperšič MD, MSc, from the Department of Rheumatology, University Medical Centre Ljubljana, Slovenia, for referring the patients. We thank Ms. Milena Pavić-Nikolić for her excellent technical support. We are especially grateful to Snežna Sodin-Šemrl, MS, PhD, for a critical review of the manuscript and the language corrections. This work was financially supported by The Slovenian Research Agency (ARRS Grant No. PO-0503–0381).

Conflict of interest statement

The authors declare no conflict of interest.


  1. 1.
    Brzezinska A, Winska P, Balinska M (2000) Cellular aspects of folate and antifolate membrane transport. Acta Biochim Pol 47:735–749PubMedGoogle Scholar
  2. 2.
    Antony AC (1992) The biological chemistry of folate receptors. Blood 79:2807–2820PubMedGoogle Scholar
  3. 3.
    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG et al (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 10:111–113PubMedCrossRefGoogle Scholar
  4. 4.
    van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK et al (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 62:1044–1051PubMedCrossRefGoogle Scholar
  5. 5.
    Kaneda S, Takeishi K, Ayusawa D, Shimizu K, Seno T, Altman S (1987) Role in translation of a triple tandemly repeated sequence in the 5′-untranslated region of human thymidylate synthase mRNA. Nucleic Acids Res 15:1259–1270PubMedCrossRefGoogle Scholar
  6. 6.
    Horie N, Aiba H, Oguro K, Hojo H, Takeishi K (1995) Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5′-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 20:191–197PubMedCrossRefGoogle Scholar
  7. 7.
    Leclerc D, Campeau E, Goyette P, Adjalla CE, Christensen B, Ross M et al (1996) Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet 5:1867–1874PubMedCrossRefGoogle Scholar
  8. 8.
    Chango A, Emery-Fillon N, de Courcy GP, Lambert D, Pfister M, Rosenblatt DS et al (2000) A polymorphism (80G->A) in the reduced folate carrier gene and its associations with folate status and homocysteinemia. Mol Genet Metab 70:310–315PubMedCrossRefGoogle Scholar
  9. 9.
    Drescher S, Schaeffeler E, Hitzl M, Hofmann U, Schwab M, Brinkmann U et al (2002) MDR1 gene polymorphisms and disposition of the P-glycoprotein substrate fexofenadine. Br J Clin Pharmacol 53:526–534PubMedCrossRefGoogle Scholar
  10. 10.
    Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A et al (2000) Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA 97:3473–3478PubMedCrossRefGoogle Scholar
  11. 11.
    Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV et al (2007) A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science 315:525–528PubMedCrossRefGoogle Scholar
  12. 12.
    Johne A, Kopke K, Gerloff T, Mai I, Rietbrock S, Meisel C et al (2002) Modulation of steady-state kinetics of digoxin by haplotypes of the P-glycoprotein MDR1 gene. Clin Pharmacol Ther 72:584–594PubMedCrossRefGoogle Scholar
  13. 13.
    Kim KA, Park PW, Park JY (2007) Effect of ABCB1 (MDR1) haplotypes derived from G2677T/C3435T on the pharmacokinetics of amlodipine in healthy subjects. Br J Clin Pharmacol 63:53–58PubMedCrossRefGoogle Scholar
  14. 14.
    Chowbay B, Cumaraswamy S, Cheung YB, Zhou Q, Lee EJ (2003) Genetic polymorphisms in MDR1 and CYP3A4 genes in Asians and the influence of MDR1 haplotypes on cyclosporin disposition in heart transplant recipients. Pharmacogenetics 13:89–95PubMedCrossRefGoogle Scholar
  15. 15.
    Aletaha D, Smolen JS (2002) The rheumatoid arthritis patient in the clinic: comparing more than 1,300 consecutive DMARD courses. Rheumatology (Oxford) 41:1367–1374CrossRefGoogle Scholar
  16. 16.
    Maetzel A, Wong A, Strand V, Tugwell P, Wells G, Bombardier C (2000) Meta-analysis of treatment termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic drugs. Rheumatology (Oxford) 39:975–981CrossRefGoogle Scholar
  17. 17.
    Bannwarth B, Pehourcq F, Schaeverbeke T, Dehais J (1996) Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clin Pharmacokinet 30:194–210PubMedCrossRefGoogle Scholar
  18. 18.
    Kumagai K, Hiyama K, Oyama T, Maeda H, Kohno N (2003) Polymorphisms in the thymidylate synthase and methylenetetrahydrofolate reductase genes and sensitivity to the low-dose methotrexate therapy in patients with rheumatoid arthritis. Int J Mol Med 11:593–600PubMedGoogle Scholar
  19. 19.
    Urano W, Taniguchi A, Yamanaka H, Tanaka E, Nakajima H, Matsuda Y et al (2002) Polymorphisms in the methylenetetrahydrofolate reductase gene were associated with both the efficacy and the toxicity of methotrexate used for the treatment of rheumatoid arthritis, as evidenced by single locus and haplotype analyses. Pharmacogenetics 12:183–190PubMedCrossRefGoogle Scholar
  20. 20.
    Wessels JA, de Vries-Bouwstra JK, Heijmans BT, Slagboom PE, Goekoop-Ruiterman YP, Allaart CF et al (2006) Efficacy and toxicity of methotrexate in early rheumatoid arthritis are associated with single-nucleotide polymorphisms in genes coding for folate pathway enzymes. Arthritis Rheum 54:1087–1095PubMedCrossRefGoogle Scholar
  21. 21.
    Weisman MH, Furst DE, Park GS, Kremer JM, Smith KM, Wallace DJ et al (2006) Risk genotypes in folate-dependent enzymes and their association with methotrexate-related side effects in rheumatoid arthritis. Arthritis Rheum 54:607–612PubMedCrossRefGoogle Scholar
  22. 22.
    Kager L, Cheok M, Yang W, Zaza G, Cheng Q, Panetta JC et al (2005) Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics. J Clin Invest 115:110–117PubMedGoogle Scholar
  23. 23.
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS et al (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–324PubMedCrossRefGoogle Scholar
  24. 24.
    Trumbo P, Schlicker S, Yates AA, Poos M (2002) Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein and amino acids. J Am Diet Assoc 102:1621–1630PubMedCrossRefGoogle Scholar
  25. 25.
    Cockcroft DW, Gault MH (1976) Prediction of creatinine clearance from serum creatinine. Nephron 16:31–41PubMedCrossRefGoogle Scholar
  26. 26.
    van Riel PL, van Gestel AM (2000) Clinical outcome measures in rheumatoid arthritis. Ann Rheum Dis 59(Suppl 1):i28–31PubMedCrossRefGoogle Scholar
  27. 27.
    Aupperle KR, Alsalameh S, Stock KP, Burmester GR, Kalden JR (1996) Comparison of rheumatoid test procedures–value and critical interpretation of sensitivity and specificity and their effect on pre-test and post-test probability. Z Rheumatol 55:158–167PubMedGoogle Scholar
  28. 28.
    Prevoo ML, van ‘t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL (1995) Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 38:44–48PubMedCrossRefGoogle Scholar
  29. 29.
    Bruce B, Fries JF (2003) The Stanford health assessment questionnaire: dimensions and practical applications. Health Qual Life Outcomes 1:20PubMedCrossRefGoogle Scholar
  30. 30.
    Fries JF, Spitz P, Kraines RG, Holman HR (1980) Measurement of patient outcome in arthritis. Arthritis Rheum 23:137–145PubMedCrossRefGoogle Scholar
  31. 31.
    Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215PubMedCrossRefGoogle Scholar
  32. 32.
    Shaw GM, Lammer EJ, Zhu H, Baker MW, Neri E, Finnell RH (2002) Maternal periconceptional vitamin use, genetic variation of infant reduced folate carrier (A80G), and risk of spina bifida. Am J Med Genet 108:1–6PubMedCrossRefGoogle Scholar
  33. 33.
    Matsuo K, Suzuki R, Hamajima N, Ogura M, Kagami Y, Taji H et al (2001) Association between polymorphisms of folate- and methionine-metabolizing enzymes and susceptibility to malignant lymphoma. Blood 97:3205–3209PubMedCrossRefGoogle Scholar
  34. 34.
    Jacques PF, Bostom AG, Selhub J, Rich S, Ellison RC, Eckfeldt JH et al (2003) Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study. Atherosclerosis 166:49–55PubMedCrossRefGoogle Scholar
  35. 35.
    Iacopetta B, Grieu F, Joseph D, Elsaleh H (2001) A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil. Br J Cancer 85:827–830PubMedCrossRefGoogle Scholar
  36. 36.
    Kurzawski M, Pawlik A, Gornik W, Drozdzik M (2006) Frequency of common MDR1 gene variants in a Polish population. Pharmacol Rep 58:35–40PubMedGoogle Scholar
  37. 37.
    Cascorbi I, Gerloff T, Johne A, Meisel C, Hoffmeyer S, Schwab M et al (2001) Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 69:169–174PubMedCrossRefGoogle Scholar
  38. 38.
    Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinf 1:47–50Google Scholar
  39. 39.
    Dervieux T, Furst D, Lein DO, Capps R, Smith K, Walsh M et al (2004) Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum 50:2766–2774PubMedCrossRefGoogle Scholar
  40. 40.
    Dervieux T, Kremer J, Lein DO, Capps R, Barham R, Meyer G et al (2004) Contribution of common polymorphisms in reduced folate carrier and gamma-glutamylhydrolase to methotrexate polyglutamate levels in patients with rheumatoid arthritis. Pharmacogenetics 14:733–739PubMedCrossRefGoogle Scholar
  41. 41.
    Relling MV, Fairclough D, Ayers D, Crom WR, Rodman JH, Pui CH et al (1994) Patient characteristics associated with high-risk methotrexate concentrations and toxicity. J Clin Oncol 12:1667–1672PubMedGoogle Scholar
  42. 42.
    Takatori R, Takahashi KA, Tokunaga D, Hojo T, Fujioka M, Asano T et al (2006) ABCB1 C3435T polymorphism influences methotrexate sensitivity in rheumatoid arthritis patients. Clin Exp Rheumatol 24:546–554PubMedGoogle Scholar
  43. 43.
    Dervieux T, Greenstein N, Kremer J (2006) Pharmacogenomic and metabolic biomarkers in the folate pathway and their association with methotrexate effects during dosage escalation in rheumatoid arthritis. Arthritis Rheum 54:3095–3103PubMedCrossRefGoogle Scholar
  44. 44.
    Drozdzik M, Rudas T, Pawlik A, Gornik W, Kurzawski M, Herczynska M (2007) Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis. Pharmacogenomics J 7:404–407CrossRefGoogle Scholar
  45. 45.
    van Ede AE, Laan RF, Blom HJ, Huizinga TW, Haagsma CJ, Giesendorf BA et al (2001) The C677T mutation in the methylenetetrahydrofolate reductase gene: a genetic risk factor for methotrexate-related elevation of liver enzymes in rheumatoid arthritis patients. Arthritis Rheum 44:2525–2530PubMedCrossRefGoogle Scholar
  46. 46.
    Taniguchi A, Urano W, Tanaka E, Furihata S, Kamitsuji S, Inoue E et al (2007) Validation of the associations between single nucleotide polymorphisms or haplotypes and responses to disease-modifying antirheumatic drugs in patients with rheumatoid arthritis: a proposal for prospective pharmacogenomic study in clinical practice. Pharmacogenet Genomics 17:383–390PubMedCrossRefGoogle Scholar
  47. 47.
    Kurzawski M, Pawlik A, Safranow K, Herczynska M, Drozdzik M (2007) 677C>T and 1298A>C MTHFR polymorphisms affect methotrexate treatment outcome in rheumatoid arthritis. Pharmacogenomics 8:1551–1559PubMedCrossRefGoogle Scholar
  48. 48.
    Berkun Y, Levartovsky D, Rubinow A, Orbach H, Aamar S, Grenader T et al (2004) Methotrexate related adverse effects in patients with rheumatoid arthritis are associated with the A1298C polymorphism of the MTHFR gene. Ann Rheum Dis 63:1227–1231PubMedCrossRefGoogle Scholar
  49. 49.
    Friedman G, Goldschmidt N, Friedlander Y, Ben-Yehuda A, Selhub J, Babaey S et al (1999) A common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentrations. J Nutr 129:1656–1661PubMedGoogle Scholar
  50. 50.
    Berkun Y, Abou Atta I, Rubinow A, Orbach H, Levartovsky D, Aamar S et al (2007) 2756GG genotype of methionine synthase reductase gene is more prevalent in rheumatoid arthritis patients treated with methotrexate and is associated with methotrexate-induced nodulosis. J Rheumatol 34:1664–1669PubMedGoogle Scholar
  51. 51.
    Drozdzik M, Rudas T, Pawlik A, Kurzawski M, Czerny B, Gornik W et al (2006) The effect of 3435C>T MDR1 gene polymorphism on rheumatoid arthritis treatment with disease-modifying antirheumatic drugs. Eur J Clin Pharmacol 62:933–937PubMedCrossRefGoogle Scholar
  52. 52.
    Bohanec Grabar P, Jazbec J, Dolzan V (2007) Gene-gene interactions in the folate metabolic pathway influence the risk for acute lymphoblastic leukemia in children. Leuk Lymphoma 48:786–792CrossRefGoogle Scholar
  53. 53.
    Tiilikainen A, Fischer G, Grubic Z, Gyodi E, Ivaskova E, Jungerman M et al (1997) Anthropological features of the East European region. In: Charron D (ed) HLA: genetic diversity of HLA. Functional and medical implications. Proceedings of the 12th International Hhistocompatibility Workshop and Conference. EDK, Paris, pp 307–314Google Scholar
  54. 54.
    Vidan-Jeras B, Jurca B, Dolzan V, Jeras M, Breskvar K, Bohinjec M (1998) Caucasian Slovenian normal. In: Gjertson D, Terasaki P (eds) HLA 1998. American Society for Histocompatibility and Immunogenetics, Lenexa, KSGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Petra Bohanec Grabar
    • 1
  • Dušan Logar
    • 2
  • Boris Lestan
    • 2
  • Vita Dolžan
    • 1
    Email author
  1. 1.Institute of Biochemistry, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Department of RheumatologyUniversity Medical Centre LjubljanaLjubljanaSlovenia

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