Original Article

Association of rs3135500 and rs3135499 Polymorphisms in the MicroRNA-binding Site of Nucleotide-binding Oligomerization Domain 2 (NOD2) Gene with Susceptibility to Rheumatoid Arthritis

NOD2 Gene Polymorphisms and Rheumatoid Arthritis


The nucleotide-binding oligomerization domain 2 (NOD2) is the key regulator of inflammatory responses and has been involved in the pathogenesis of rheumatoid arthritis (RA). Laboratory and in silico evaluations have demonstrated that some polymorphisms in 3ˊUTR of NOD2 gene could influence the secondary structure of this region and similarly thermodynamic features of hybridization site and finally deregulate the expression of NOD2. In the current study, for the first time, we evaluated the possible association between single nucleotide polymorphisms (SNPs) rs3135500 and rs3135499 in the NOD2 gene with RA risk in the Iranian population.
One hundred and fifteen patients with RA and 120 healthy subjects were recruited in this case-control study. Genotyping of rs3135500 and rs3135499 polymorphisms were accomplished using the real‑time polymerase chain reaction high resolution melting (HRM) method.
We found a substantial association of AA and AG genotypes in rs3135500 with the risk of RA (AA vs GG; OR=5.547; 95%CI [2.564-11.999]; p<0.001 and AG vs GG; OR=2.179; 95%CI [1.145-4.147]; p=0.017). Moreover, in the patient group, there was a significant relationship between the increased concentration of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) with rs3135500 (A allele) (p<0.05). However, there were no important associations between rs3135499 with the risk of RA (p>0.05). However, we found a noteworthy association of the C allele in rs3135499 with an increased level of CRP in patients (p>0.05).
Our findings propose a considerable association between NOD2 polymorphisms with increased risk of RA and disease activity.

1. Aletaha D, Smolen JS. Diagnosis and management of rheumatoid arthritis: a review. JAMA. 2018;320(13):1360-72.
2. van der Woude D, van der Helm-van AH. Update on the epidemiology, risk factors, and disease outcomes of rheumatoid arthritis. Clin Rheumatol. 2018;32(2):174-87.
3. Guo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018;6(1):1-14.
4. Gibofsky A. Epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis: A Synopsis. Am J Manag Care. 2014;20(7 Suppl):S128-35.
5. Gibofsky A. Overview of epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis. Am J Manag Care. 2012;18(13 Suppl):S295.
6. Cross M, Smith E, Hoy D, Carmona L, Wolfe F, Vos T, et al. The global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis. 2014;73(7):1316-22.
7. Zhang M, Mu H, Lv H, Duan L, Shang Z, Li J, et al. Integrative analysis of genome-wide association studies and gene expression analysis identifies pathways associated with rheumatoid arthritis. Oncotarget. 2016;7(8):8580-9.
8. McAllister K, Eyre S, Orozco G. Genetics of rheumatoid arthritis: GWAS and beyond. Open Access Rheumatol. 2011;3:31-46.
9. Al-Koofee DA, Mubarak SM. Genetic Polymorphisms. Genetic Polymorphisms: IntechOpen; 2019.
10. Simonian M, Mosallaei M, Khosravi S, Salehi R. rs12904 polymorphism in the 3'-untranslated region of ephrin A1 ligand and the risk of sporadic colorectal cancer in the Iranian population. J Cancer ResTher. 2019;15(1):15.
11. O'Brien J, Hayder H, Zayed Y, Peng C. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol. 2018;9:402.
12. Mosallaei M, Simonian M, Esmaeilzadeh E, Bagheri H, Miraghajani M, Salehi AR, et al. Single nucleotide polymorphism rs10889677 in miRNAs Let-7e and Let-7f binding site of IL23R gene is a strong colorectal cancer determinant: Report and meta-analysis. Cancer Genet. 2019;239:46-53.
13. Evangelatos G, Fragoulis GE, Koulouri V, Lambrou GI. MicroRNAs in rheumatoid arthritis: From pathogenesis to clinical impact. Autoimmun Rev. 2019;18(11):102391.
14. Churov AV, Oleinik EK, Knip M. MicroRNAs in rheumatoid arthritis: altered expression and diagnostic potential. Autoimmun Rev. 2015;14(11):1029-37.
15. Karimzadeh MR, Zarin M, Ehtesham N, Khosravi S, Soosanabadi M, Mosallaei M, et al. MicroRNA binding site polymorphism in inflammatory genes associated with colorectal cancer: literature review and bioinformatics analysis. Cancer Gene Ther. 2020:1-15.
16. Yuan Y, Weidhaas JB. Functional microRNA binding site variants. Mol Oncol. 2019;13(1):4-8.
17. Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, LeBourhis L, Philpott DJ, et al. Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis. Proc Natl Acad Sci. 2008;105(26):9017-22.
18. Franca R, Vieira S, Talbot J, Peres R, Pinto L, Zamboni D, et al. Expression and activity of NOD1 and NOD2/RIPK2 signalling in mononuclear cells from patients with rheumatoid arthritis. Scand J Rheumatol. 2016;45(1):8-12.
19. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278(11):8869-72.
20. Caruso R, Warner N, Inohara N, Núñez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014;41(6):898-908.
21. Ospelt C, Brentano F, Jüngel A, Rengel Y, Kolling C, Michel BA, et al. Expression, regulation, and signaling of the pattern‐recognition receptor nucleotide‐binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts. Arthritis Rheumatol. 2009;60(2):355-63.
22. Kim HW, Kwon YJ, Park BW, Song JJ, Park YB, Park MC. Differential expressions of NOD-like receptors and their associations with inflammatory responses in rheumatoid arthritis. Clin Exp Rheumatol. 2017;35(4):630-7.
23. Chuang AY, Chuang JC, Zhai Z, Wu F, Kwon JH. NOD2 expression is regulated by microRNAs in colonic epithelial HCT116 cells. Inflamm Bowel Dis. 2014;20(1):126-35.
24. Ye J, Gillespie KM, Rodriguez S. Unravelling the roles of susceptibility Loci for autoimmune diseases in the Post-GWAS Era. Genes. 2018;9(8):377.
25. Ebrahimiyan H, Mostafaei S, Aslani S, Jamshidi A, Mahmoudi M. Studying the association between STAT4 gene polymorphism and susceptibility to rheumatoid arthritis disease: an updated meta-analysis. Iran J Immunol. 2019;16(1):71-83.
26. Negroni A, Pierdomenico M, Cucchiara S, Stronati L. NOD2 and inflammation: current insights. J Inflamm Res. 2018;11:49-60.
27. Goethel A, Croitoru K, Philpott DJ. The interplay between microbes and the immune response in inflammatory bowel disease. J Physiol. 2018;596(17):3869-82.
28. Dugan J, Griffiths E, Snow P, Rosenzweig H, Lee E, Brown B, et al. Blau syndrome–associated NOD2 mutation alters expression of full-length NOD2 and limits responses to muramyl dipeptide in knock-in mice. JImmunol. 2015;194(1):349-57.
29. Kanazawa N, Okafuji I, Kambe N, Nishikomori R, Nakata-Hizume M, Nagai S, et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-κB activation: common genetic etiology with Blau syndrome. Blood. 2005;105(3):1195-7.
30. Yu S-L, Wong C-K, Wong PT-Y, Chen D-P, Szeto C-C, Li EK, et al. Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production. PLoS One. 2011;6(8):e23855.
31. Vanaki N, Golmohammadi T, Jamshidi A, Akhtari M, Vojdanian M, Mostafaei S, et al. Increased inflammatory responsiveness of peripheral blood mononuclear cells (PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis. Immunopharmacol Immunotoxicol. 2018;40(5):393-400.
32. Diler SB, Polat F, Yaraş S. The P268S and M863V Polymorphisms of the NOD2/CARD15 Gene in Crohn’s Disease and Ulcerative Colitis. Cytol Genet. 2019;53(5):424-9.
33. McGovern D, Van Heel D, Ahmad T, Jewell D. NOD2 (CARD15), the first susceptibility gene for Crohn's disease. Gut. 2001;49(6):752-4.
34. Tuupanen S, Alhopuro P, Mecklin JP, Järvinen H, Aaltonen LA. No evidence for association of NOD2 R702W and G908R with colorectal cancer. Int J Cancer. 2007;121(1):76-9.
35. Tian Y, Li Y, Hu Z, Wang D, Sun X, Ren C. Differential effects of NOD2 polymorphisms on colorectal cancer risk: a meta-analysis. Int J Colorectal Dis. 2010;25(2):161-8.
36. Angeletti S, Galluzzo S, Santini D, Ruzzo A, Vincenzi B, Ferraro E, et al. NOD2/CARD15 polymorphisms impair innate immunity and increase susceptibility to gastric cancer in an Italian population. Hum Immunol. 2009;70(9):729-32.
37. Landi D, Gemignani F, Naccarati A, Pardini B, Vodicka P, Vodickova L, et al. Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer. Carcinogenesis. 2008;29(3):579-84.
38. Chaleshi V, Tajali R, Savabkar S, Zali N, N Mojarad E, Haghazali M, et al. Lack of association between NOD2 rs3135500 and IL12B rs1368439 microRNA binding site SNPs and colorectal cancer susceptibility in an Iranian population. Microrna. 2016;5(2):152-6.
39. Ahangari F, Salehi R, Salehi M, Khanahmad H. A miRNA-binding site single nucleotide polymorphism in the 3′-UTR region of the NOD2 gene is associated with colorectal cancer. Med Oncol. 2014;31(9):173.
40. Weidinger S, Klopp N, Rümmler L, Wagenpfeil S, Baurecht H, Gauger A, et al. Association of CARD15 polymorphisms with atopy‐related traits in a population‐based cohort of Caucasian adults. Clin Exp Allergy. 2005;35(7):866-72.
41. Cao B, Chen Y, Zhou Q, Zhang L, Ou R, Wei Q, et al. Functional variant rs3135500 in NOD2 increases the risk of multiple system atrophy in a Chinese population. Front Aging Neurosci. 2018;10:150.
42. Icduygu FM, Erdogan MO, Ulasli SS, Yildiz HG, Celik ZS, Unlu M, et al. Is There an Association Between NOD2 Gene Polymorphisms and Chronic Obstructive Pulmonary Disease Progression? Int J Hum Genet. 2017;17(2):86-96.
43. Enevold C, Oturai AB, Sørensen PS, Ryder LP, Koch-Henriksen N, Bendtzen K. Polymorphisms of innate pattern recognition receptors, response to interferon-beta and development of neutralizing antibodies in multiple sclerosis patients. Mult Scler J. 2010;16(8):942-9.
44. Cai X, Xu Q, Zhou C, Zhou L, Dai W, Ji G. The association of nucleotide‐binding oligomerization domain 2 gene polymorphisms with the risk of asthma in the Chinese Han population. Mol. Genet. Genomic Med. 2019;7(6):e00675.
45. Dessein PH, Joffe BI, Stanwix AE. High sensitivity C-reactive protein as a disease activity marker in rheumatoid arthritis. J Rheumatol. 2004;31(6):1095-7.
46. Rosa Neto NS, Carvalho JFd. O uso de provas de atividade inflamatória em reumatologia. Rev Bras Reumatol. 2009;49:413-30.
IssueVol 20 No 2 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v20i2.6051
Inflammation Polymorphism Rheumatoid arthritis

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Ehtesham N, Alani B, Mortazavi D, Azhdari S, Kenarangi T, Esmaeilzadeh E, Pakzad B. Association of rs3135500 and rs3135499 Polymorphisms in the MicroRNA-binding Site of Nucleotide-binding Oligomerization Domain 2 (NOD2) Gene with Susceptibility to Rheumatoid Arthritis. Iran J Allergy Asthma Immunol. 20(2):178-187.