Methotrexate (MTX) is the most widely used disease-modifying anti-rheumatic drug (DMARD) for rheumatoid arthritis (RA). Many studies have attempted to understand the genetic risk factors that affect the therapeutic outcomes in RA patients treated with MTX. Unlike other studies that focus on the populations of Caucasians, Indian and east Asian countries, this study investigated the impacts of six single nucleotide polymorphisms (SNPs) that are hypothesized to affect the outcomes of MTX treatment in Malaysian RA patients. A total of 647 RA patients from three ethnicities (NMalay = 153; NChinese = 326; NIndian = 168) who received MTX monotherapy (minimum 15 mg per week) were sampled from three hospitals in Malaysia. SNPs were genotyped in patients using TaqMan real-time PCR assay. Data obtained were statistically analysed for the association between SNPs and MTX efficacy and toxicity. Analysis of all 647 RA patients indicated that none of the SNPs has influence on either MTX efficacy or MTX toxicity according to the Chi-square test and binary logistic regression. However, stratification by self-identified ancestries revealed that two out of six SNPs, ATIC C347G (rs2372536) (OR 0.5478, 95% CI 0.3396–0.8835, p = 0.01321) and ATIC T675C (rs4673993) (OR 0.5247, 95% CI 0.3248–0.8478, p = 0.008111), were significantly associated with MTX adequate response in RA patients with Malay ancestry (p < 0.05). As for the MTX toxicity, no significant association was identified for any SNPs selected in this study. Taken all together, ATIC C347G and ATIC T675C can be further evaluated on their impact in MTX efficacy using larger ancestry-specific cohort, and also incorporating high-order gene–gene and gene–environment interactions.

1. Smolen, J. S. et al. Rheumatoid arthritis. Nat. Rev. Dis. Primers 4, 18001. https://doi.org/10.1038/nrdp.2018.1 (2018).
2. Arthritis Foundation. Rheumatoid arthritis https://www.afm.org.my/2011/07/31/rheumatoid-arthritis/ (accessed 31 July 2011).
3. Department of Statistics Malaysia. Demographic Statistics Second Quarter 2021, Malaysia https://www.dosm.gov.my/v1/index.php?r=column/ctwoByCat&parent_id=115&menu_id=L0pheU43NWJwRWVSZklWdzQ4TlhUUT09 (2021).
4. Singh, J. A. et al. 2015 American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Rheumatol. 68, 1–26. https://doi.org/10.1002/art.39480 (2016).
5. Schrezenmeier, E. & Dörner, T. Mechanisms of action of hydroxychloroquine and chloroquine: Implications for rheumatology. Nat. Rev. Rheumatol. 16, 155–166. https://doi.org/10.1038/s41584-020-0372-x (2020).
6. Weinblatt, M. E. et al. Efficacy of low-dose methotrexate in rheumatoid arthritis. N. Engl. J. Med. 312(13), 818–822. https://doi.org/10.1056/NEJM198503283121303 (1985).
7. Visser, K. & van der Heijde, D. Optimal dosage and route of administration of methotrexate in rheumatoid arthritis: A systematic review of the literature. Ann. Rheum. Dis. 68(7), 1094–1099. https://doi.org/10.1136/ard.2008.092668 (2009).
8. Molina, J. T. et al. Recommendations for the use of methotrexate in rheumatoid arthritis: Up and down scaling of the dose and administration routes. Reumatol. Clin. 11(1), 3–8. https://doi.org/10.1016/j.reuma.2014.02.012 (2015).
9. Kinder, A. J. et al. The treatment of inflammatory arthritis with methotrexate in clinical practice: Treatment duration and incidence of adverse drug reactions. Rheumatology 44(1), 61–66. https://doi.org/10.1093/rheumatology/keh512 (2005).
10. Aletaha, D. & Smolen, J. S. The rheumatoid arthritis patient in the clinic: Comparing more than 1,300 consecutive DMARD courses. Rheumatology 41(12), 1367–1374. https://doi.org/10.1093/rheumatology/41.12.1367 (2002).
11. Maetzel, A. et al. Meta-analysis of treatment termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic drugs. Rheumatology 39(9), 975–981. https://doi.org/10.1093/rheumatology/39.9.975 (2000).
12. Albrecht, K. & Müller-Ladner, U. Side effects and management of side effects of methotrexate in rheumatoid arthritis. Clin. Exp. Rheumatol. 28(Suppl. 61), S95–S101 (2010).
13. Alarcón, G. S. Early rheumatoid arthritis: Combination therapy of doxycycline plus methotrexate versus methotrexate monotherapy. Nat. Clin. Pract. Rheumatol. 2, 296–297. https://doi.org/10.1038/ncprheum0195 (2006).
14. Sulaiman, W., Toib, A., Chandrashekhar, G. & Arshad, A. The trends of DMARDS prescribed in rheumatoid arthritis patients in Malaysia. Oman. Med. J. 24(4), 260–263. https://doi.org/10.5001/omj.2009.53 (2009).
15. Ministry of Health, Malaysian Society of Rheumatology, & Academy of Medicine Malaysia. Clinical Practice Guidelines 2019: Management of Rheumatoid Arthritis. https://www.moh.gov.my/moh/resources/Penerbitan/CPG/2)_CPG_Management_of_Rheumatoid_Arthritis.pdf (accessed 9 Oct 2020).
16. Tian, H. & Cronstein, B. N. Understanding the mechanisms of action of methotrexate: Implications for the treatment of rheumatoid arthritis. Bull NYU Hosp. Jt. Dis. 65(3), 168–173 (2007).
17. Wessels, J. A. M., Huizinga, T. W. J. & Guchelaar, H. J. Recent insights in the pharmacological actions of methotrexate in the treatment of rheumatoid arthritis. Rheumatology 47(3), 249–255. https://doi.org/10.1093/rheumatology/kem279 (2008).
18. Kremer, J. M. Toward a better understanding of methotrexate. Arthritis Rheum. 50(5), 1370–1382. https://doi.org/10.1002/art.20278 (2004).
19. van der Straaten, R. J. et al. Exploratory analysis of four polymorphisms in human GGH and FPGS genes and their effect in methotrexate-treated rheumatoid arthritis patients. Pharmacogenomics 8(2), 141–150. https://doi.org/10.2217/14622416.8.2.141 (2007).
20. Dervieux, T. et al. Contribution of common polymorphisms in reduced folate carrier and gamma-glutamylhydrolase to methotrexate polyglutamate levels in patients with rheumatoid arthritis. Pharmacogenetics 14(11), 733–739. https://doi.org/10.1097/00008571-200411000-00004 (2004).
21. Padovan, M. et al. Adenosine and adenosine receptors in rheumatoid arthritis. Int. J. Clin. Rheumatol. 8(1), 13–25. https://doi.org/10.2217/ijr.12.76 (2013).
22. Kurzawski, M. et al. ATIC missense variant affects response to methotrexate treatment in rheumatoid arthritis patients. Pharmacogenomics 17(18), 1971–1978. https://doi.org/10.2217/pgs-2016-0125 (2016).
23. Lee, Y. C. et al. Investigation of candidate polymorphisms and disease activity in rheumatoid arthritis patients on methotrexate. Rheumatology 48(6), 613–617. https://doi.org/10.1093/rheumatology/ken513 (2009).
24. Iannaccone, C. K. et al. Using genetic and clinical data to understand response to disease-modifying anti-rheumatic drug therapy: Data from the Brigham and Women’s Hospital Rheumatoid Arthritis Sequential Study. Rheumatology 50(1), 40–46. https://doi.org/10.1093/rheumatology/keq263 (2011).
25. Lima, A., Bernardes, M., Azevedo, R., Medeiros, R. & Seabra, V. Pharmacogenomics of methotrexate membrane transport pathway: Can clinical response to methotrexate in rheumatoid arthritis be predicted?. Int. J. Mol. Sci. 16(6), 13760–13780. https://doi.org/10.3390/ijms160613760 (2015).
26. Owen, S. A. et al. Genetic polymorphisms in key methotrexate pathway genes are associated with response to treatment in rheumatoid arthritis patients. Pharmacogenom. J. 13(3), 227–234. https://doi.org/10.1038/tpj.2012.7 (2013).
27. Yanagimachi, M. et al. Influence of polymorphisms within the methotrexate pathway genes on the toxicity and efficacy of methotrexate in patients with juvenile idiopathic arthritis. Br. J. Clin. Pharmacol. 71(2), 237–243. https://doi.org/10.1111/j.1365-2125.2010.03814.x (2011).
28. Shahrir, M. et al. Multicentre survey of rheumatoid arhritis patients from Ministry of Health Rheumatology Centers in Malaysia. Int. J. Rheum. Dis. 11, 287–292 (2008).
29. R Core Team. R: A language and environment for statistical computing (2013). http://www.R-project.org.
30. Wickham, H. ggplot2: elegant graphics for data analysis. Springer (2016). https://ggplot2.tidyverse.org.
31. Muralidharan, N. et al. Folyl polyglutamate synthethase (FPGS) gene polymorphisms may influence methotrexate adverse events in South Indian Tamil Rheumatoid Arthritis patients. Pharmacogenom. J. 20(2), 342–349. https://doi.org/10.1038/s41397-019-0097-x (2020).
32. Sivadas, A., Salleh, M. Z., Teh, L. K. & Scaria, V. Genetic epidemiology of pharmacogenetic variants in South East Asian Malays using whole-genome sequences. Pharmacogenom. J. 17(5), 461–470. https://doi.org/10.1038/tpj.2016.39 (2017).
33. The HUGO Pan-Asian SNP Consortium et al. Mapping human genetic diversity in Asia. Science 326(5959), 1541–1545 (2009). https://doi.org/10.1126/science.1177074.
34. Karczewski, K. J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581(7809), 434–443. https://doi.org/10.1038/s41586-020-2308-7 (2020).
35. 1000 Genomes Project Consortium A global reference for human genetic variation. Nature 526(7571), 68–74 (2015). https://doi.org/10.1038/nature15393.
36. Chen, Y., Zou, K., Sun, J., Yang, Y. & Liu, G. Are gene polymorphisms related to treatment outcomes of methotrexate in patients with rheumatoid arthritis? A systematic review and meta-analysis. Pharmacogenomics 18(2), 175–195. https://doi.org/10.2217/pgs-2016-0158 (2017).
37. Lima, A. et al. Prediction of methotrexate clinical response in Portuguese rheumatoid arthritis patients: Implication of MTHFR rs1801133 and ATIC rs4673993 polymorphisms. Biomed. Res. Int. 2014, 368681. https://doi.org/10.1155/2014/368681 (2014).
38. Dervieux, T. et al. 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(9), 2766–2774. https://doi.org/10.1002/art.20460 (2004).
39. Dervieux, T. et al. Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: Results of a multicentred cross sectional observational study. Ann. Rheum. Dis. 64(8), 1180–1185. https://doi.org/10.1136/ard.2004.033399 (2005).
40. Dervieux, T. Methotrexate pharmacogenomics in rheumatoid arthritis: Introducing false-positive report probability. Rheumatology 48(6), 597–598. https://doi.org/10.1093/rheumatology/kep060 (2009).
41. University of Washington. NHLBI Exome Sequencing Project (ESP), Exome Variant Server. http://evs.gs.washington.edu/EVS/ (accessed 9 Oct 2020).
42. Sharma, S. et al. Purine biosynthetic pathway genes and methotrexate response in rheumatoid arthritis patients among north Indians. Pharmacogenet. Genom. 19(10), 823–828. https://doi.org/10.1097/fpc.0b013e328331b53e (2009).
43. Muralidharan, N., Mariaselvam, C. M., Jain, V. K., Gulati, R. & Negi, V. S. ATIC 347C> G gene polymorphism may be associated with methotrexate-induced adverse events in south Indian Tamil rheumatoid arthritis. Pharmacogenomics 17(3), 241–248. https://doi.org/10.2217/pgs.15.170 (2016).
44. Lee, Y. H. & Bae, S.-C. Association of the ATIC 347 C/G polymorphism with responsiveness to and toxicity of methotrexate in rheumatoid arthritis: A meta-analysis. Rheumatol. Int. 36(11), 1591–1599. https://doi.org/10.1007/s00296-016-3523-2 (2016).
45. Qiu, Q. et al. Polymorphisms and pharmacogenomics for the clinical efficacy of methotrexate in patients with rheumatoid arthritis: A systematic review and meta-analysis. Sci. Rep. 7(1), 1–24. https://doi.org/10.1038/srep44015 (2017).
46. Dervieux, T. et al. Gene–gene interactions in folate and adenosine biosynthesis pathways affect methotrexate efficacy and tolerability in rheumatoid arthritis. Pharmacogenet. Genom. 19(12), 935–944. https://doi.org/10.1097/FPC.0b013e32833315d1 (2009).
47. Dervieux, T. et al. Patterns of interaction between genetic and nongenetic attributes and methotrexate efficacy in rheumatoid arthritis. Pharmacogenet. Genom. 22, 1–9. https://doi.org/10.1097/FPC.0b013e32834d3e0b (2012).
48. Fales, K. R. et al. Discovery of N-(6-Fluoro-1-oxo-1, 2-dihydroisoquinolin-7-yl)-5-[(3 R)-3-hydroxypyrrolidin-1-yl] thiophene-2-sulfonamide (LSN 3213128), a potent and selective nonclassical antifolate aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) inhibitor effective at tumor suppression in a cancer xenograft model. J. Med. Chem. 60(23), 9599–9616. https://doi.org/10.1021/acs.jmedchem.7b01046 (2017).
49. Hoffmeyer, S. et al. 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. U.S.A. 97(7), 3473–3478. https://doi.org/10.1073/pnas.97.7.3473 (2000).
50. Takatori, R. et al. ABCB1 C3435T polymorphism influences methotrexate sensitivity in rheumatoid arthritis patients. Clin. Exp. Rheumatol. 24(5), 546–554 (2006).
51. Malik, F. & Ranganathan, P. Methotrexate pharmacogenetics in rheumatoid arthritis: A status report. Pharmacogenomics 14, 305–314. https://doi.org/10.2217/pgs.12.214 (2013).
52. Ranganathan, P. & McLeod, H. L. Methotrexate pharmacogenetics: The first step toward individualized therapy in rheumatoid arthritis. Arthritis Rheum. 54, 1366–1377. https://doi.org/10.1002/art.21762 (2006).
53. van Ede, A. E. et al. The C677T mutation in the methylenetetrahydrofolate reductase gene: A gene risk factor for methotrexate-related elevation of liver enzymes in rheumatoid arthritis patients. Arthritis Rheum. 44, 2525–2530. https://doi.org/10.1002/1529-0131(200111)44:11%3c2525::aid-art432%3e3.0.co;2-b (2001).
54. Purcell, S. et al. PLINK: A tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81(3), 559–575. https://doi.org/10.1086/519795 (2007).