eISSN: 1896-9151
ISSN: 1734-1922
Archives of Medical Science
Current issue Archive Manuscripts accepted About the journal Special issues Editorial board Abstracting and indexing Subscription Contact Instructions for authors
SCImago Journal & Country Rank
vol. 15
Basic research

Lack of association of –863C/A (rs1800630) polymorphism of tumor necrosis factor-α gene with rheumatoid arthritis

Tayyaba Sadaf, Peter John, Attya Bhatti, Javaid M. Malik

Arch Med Sci 2019; 15, 2: 531–536
Online publish date: 2018/07/05
Article file
- Lack.pdf  [0.25 MB]
Get citation
JabRef, Mendeley
Papers, Reference Manager, RefWorks, Zotero


Among several inflammatory autoimmune disorders rheumatoid arthri­tis (RA) is one of the most common, characterized by synovial membrane inflammation and immune cell-mediated joint destruction. It primarily affects the joints, resulting in painful swollen joints to severe polyarthritis with progressive articular cartilage destruction. The worldwide prevalence of RA is about 0.24% [1]. According to Cross et al., RA continues to remain a source of modest global infirmity, with serious consequences in affected individuals. Collected data from studies suggested that the disease occurrence is inconsistent in different racial groups [2]. In Pakistan, disease prevalence is still unspecified, but it has been perceived that it is more common in the country’s northern parts than the southern part [3]. It depends on the combination of various environmental as well as a number of genetic factors [4]. It has also been observed that RA occurs in individuals having multiple common genetic factors and these genetic risk factors are expected with 60% heritability [5]. Multiple genetic risk factors of various candidate genes incline the patient towards disease susceptibility by the development of different clinical symptoms following exposure to unknown environmental factors [6]. Microsatellite mapping across the HLA region suggested that a nearby class III region possibly contributes to disease susceptibility or severity [7–9]. Evidence emphasized that within the class III region the tumor necrosis factor-α (TNF-α) gene is a major candidate for RA [10].
Single nucleotide polymorphisms (SNPs) in the promoter regions are likely to have a potential to cause differential expression of proteins and possibly have an association with disease [11]. TNF gene promoter polymorphisms may influence the transcriptional activity by transforming the transcription factor binding site [12]. Various SNPs have been reported for the TNF gene promoter region. Among them SNP at –863 position is involved in NF-κB binding affecting the transcriptional regulation [13]. In HLA-DR4+ individuals TNF –863A is found to be prognostic for severe joint disease [14]. Moreover, some studies have also found a positive association of TNF –863A allele and RA outcome [14]. Conversely, inconsistency among the different studies exists regarding TNF-α promoter polymorphisms’ association with RA outcome [14, 15]. There are limited data available concerning SNPs and RA disease susceptibility association in Pakistan [16, 17]. Still, there is a great need to explore the association studies of RA susceptible genes’ polymorphism not only in our study group but also in different ethnic groups due to conflicting results. Thus, considering the above statements, in this study we further evaluated the TNF-α –863C/A variant in Pakistani patients with RA to assess any potential association.

Material and methods

The study included 268 human subjects. Among them 134 individuals were patients with RA and 134 individuals were ethnically matched controls. In this study, the physical parameters of the RA patients in a Pakistani population were amassed to investigate any association of these clinical features with the patients. The percentage of males and females and the average age of all the included individuals in the study were calculated for the analysis of SNP. All the included patients were diagnosed by a rheumatologist from Rehmat Noor Clinic, Rawalpindi, working in collaboration with our institution.
Inclusion criteria: patients included in this study fulfilled the American College of Rheumatology (ACR) criteria 2011 for RA diagnosis and classification. Exclusion criteria: individuals overlapping with any other rheumatic or autoimmune disease were excluded from the study. To diagnose RA different inflammatory markers including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (anti-CCP) were used. Anti-CCP of all the patients was analyzed by the fully automated chemiluminescent immunoassay system COBAS ELECSYS 411. Reference values used for detection of markers were: anti-CCP (negative < 17 U/ml; positive > 17 U/ml), RF (up to 20 IU/ml), C-reactive protein (normal range < 6 mg/l), ESR (normal range for males: 0–9 mm; for females: 0–15 mm).
Research was carried out in compliance with the Helsinki Declaration. Written informed consent was obtained from the entire study group. The study was approved by the ethical committee of the Institutional Review Board (IRB) of ASAB-NUST.
Clinical features of RA patients and healthy individuals collected are shown in Table I.

Sampling and DNA extraction

Blood samples of all the enrolled individuals were collected in an ethylenediaminetetraacetic acid (EDTA) vacutainer. The samples were immediately dispatched to the Functional Genomics and Immuno-genetics laboratory (IGL) of Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan. From the blood cells of collected EDTA blood samples genomic deoxyribonucleic acid (DNA) was extracted using the organic method. The extracted DNA was qualitatively analyzed on 1% agarose gel and quantitatively analyzed by a bio-photometer. The extracted DNA was then stored at 4°C before any further processing.

Design of primers

SNP selected for the association study was located in the promoter region of the TNF-α gene. The sequence of the TNF-α promoter region was taken from National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) with the accession number rs1800630. Forward and reverse primers for SNP were then manually designed and their properties were calculated by the oligonucleotide properties calculator Oligo- Calc [18]. The specificity was checked on Primer-BLAST. One common forward (TNF-F-5’TGTGTGTGTGTGTCTGGGAGTGAGAA3’) and two reverse (TNF-R1-5’TCTACATGGCCCTGTCTTCGTTAAGG3’ and TNF-R2-5’TCTACATGGCCCTGTCTTCGTTAAGT3’) primers resulting in a 389 bp fragment were designed for studying the TNFA –863A>C polymorphism. The prerequisite Internal Amplification Control forward (IAC-F-5’ATGGTCTTAGTATAGCTTGCAGCCTTGT3’) and reverse (IAC-R-5’TGCAGATACCATCATCCTGGCTTCAAG3’) primers resulting in a 144 bp fragment were also designed considering β-globin as the standard in order to validate the reaction.


The RA patients and healthy individuals were examined for the occurrence of the polymorphism by allele specific ARMS-PCR. Denatured DNA templates at 95°C for 5 min were hybridized with primers at 54°C for 45 s, subsequently amplified in a 96-well thermocycler 2,720 (Applied Biosystems) with 35 cycles of PCR. The PCR product and loading dye (0.25% bromophenol blue in 40% sucrose solution) was mixed carefully for loading the mixture in the wells of 2% (w/v) agarose gel. The gel was ethidium bromide stained. After running gel electrophoresis at 120 V in 1XTBE buffer for about 0.5 h, the gel was analyzed on the Dolphin-Doc plus gel documentation system (Wealtech). Length of the PCR product was determined by comparing the PCR product size with a 50 bp DNA ladder.

Statistical analysis

Data were statistically examined for any association of a variant with disease by GraphPad Prism 6 software. Hardy-Weinberg equilibrium (HWE) of genotypes was confirmed by the 2 test (1 df). All binomial variables were assessed by χ2/Fisher exact test for association analysis of the variant with disease in our study group. To estimate the degree of association of each allele, the odds ratio (OR) and its 95% confidence interval (CI) were calculated.


The study included a total of 268 individuals. In the case group 86.86% were female and 13.14% were male, with a mean age of 44.15 ±12.21 years, and in the control group 80.59% were female and 19.40% were male, with a mean age of 35.74 ±7.1 years. Out of 134 patients, 58 (43.3%) individuals were > 45 years of age while 76 (56.7%) patients were ≤ 45 years of age. All the 134 patients were positive for the anti-CCP test and 133 patients were positive for the RF factor.
All possible allele combinations distributed in our subjects were studied. The allele counting method for TNF-α –863C/A (rs1800630) was employed to compute the allele and genotype frequencies, and the polymorphism was verified for any deviation from Hardy-Weinberg equilibrium (HWE). Both sets were suitable for auxiliary examination and association studies. The p-value estimated for RA patients’ was 0.8968 with χ2 = 0.02, while the p-value was 0.8299 with χ2 = 0.05 for the control group. Genotype and allele frequency distribution calculated and matched with the frequency of arbitrary healthy people (Figure 1). The value of association was found to be 2.771 with the probability of error (p-value) of 0.2502 (Table II). Allele frequency distribution considered using two-tailed analysis shows no substantial difference between cases and controls with the odds ratio (95% CI) of 0.7490 (0.5317–1.055).


Rheumatoid arthritis is an inflammatory auto­immune disease affecting humans throughout the world that causes joint inflammation, swelling and pain. A wide variety of factors together with environmental and genetic elements contribute to the disease pathogenesis and progression. Genome scanning indicated a strong genetic component [19], revealing that there is more than one region that is linked to the disease [20–22]. Cumulative studies also counsel that patients whose genetic history includes several common elements are more susceptible to RA. Genetic susceptibility contributed by these genetic risk elements or alleles was estimated to be approximately 30% [23]. According to our investigation in a Pakistani populace the rate of occurrence of RA is higher in females than in males. In our study, we found that a higher percentage of females (86.86%) were affected than males (13.14%), which is consistent with findings from Taiwan [24] and a North Indian cohort [25], implying that gender may have a substantial consequence for RA susceptibility. Studies show that promoter regions of RA candidate genes are extremely polymorphic, which has been accompanied with disease susceptibility as well as severity in different populations [26]. Among them, TNF-α plays a central role in disease pathogenesis, increased levels having been reported in inflamed joints [27]. High genetic variability with several SNPs has been determined in the promoter area of the TNF-α gene [28]. These SNPs could possibly alter transcription factor binding sites, affecting promoter activity and ultimately leading to altered mRNA and protein levels [29]. TNF-α is biologically active in two distinctly different forms, i.e., soluble form (solTNF) and transmembrane form (tmTNF). Both forms are specified for their roles; solTNF has an imperative, perhaps the dominant, role in the inflammatory response, while tmTNF plays a fundamental role in maintaining innate immunity against infections [30]. It is a multifunctional pro-inflammatory cytokine that is involved in different pathological processes including autoimmunity and neurodegenerative diseases [31].
Studies have focused on the association of TNF-α gene polymorphisms with RA. The role of TNF-α in pathogenesis of autoimmune diseases has been discussed in a number of studies [11, 32]. Despite some contradictory views, so far available data reveal that the variants of the TNF gene have the potential for disease progression and could act as potential genetic risk factors [33, 34]. Despite this, auxiliary studies are needed to ascertain whether the formerly recognized TNF-α gene promoter variations act as potential markers of RA. TNF-α gene polymorphisms variants have been found associated with several autoimmune diseases [35–37], inflammatory arthritis like psoriatic arthritis (PsA) [38–41], juvenile rheuma­toid arthritis [42], and systemic juvenile rheuma­toid arthritis [43]. In some studies, TNF-α promoter variants were found with aggressive disease [44]. According to Skoog et al., TNF-α –863 variant rs1800630 was found to be associated with the disease severity. A SNP at position –863 is involved in NF-κB binding affecting the transcriptional regulation [13]. Tumor necrosis factor –863A allele lessens the NF-κB p50/p50 binding that directs the enhanced TNF production in human monocytes [12]. Moreover, some studies found a positive association of TNF –863A allele and RA outcome [14]. In HLA-DR4+ individuals, TNF –863A is found to be prognostic for severe joint disease [14]. In our study the frequency of allele distribution at position –863 of TNF-α was comparable with a North Indian population in a previous report [15], but different from that of Udalova et al. [12].TNF-α –863A allele frequency distribution revealed no significant difference between our study groups when our RA patients (39.93%) were compared with the control group (47.01%) with p-values of 0.0978 and 0.7490 (95% CI: 0.5317–1.055), which is consistent with the North Indian population with no association of TNF-α –863 with the disease. Results with lack of a positive association were also consistent with the findings of Uglialoro et al. [45]. In our study groups, TNF-α –863A minor allele frequency was comparable with Japanese data [46]. Our data do not support the TNF-α –863A allele as a genetic component contributing to RA disease susceptibility in our population as the distribution of the TNF-α –863A allele was not different between the study groups. However, these allelic polymorphisms explain comparatively incomplete clinical intervention as RA implicates multiple genes. Thus, to describe the relationship of TNF-α alleles with RA, independent larger population study validation is required to confirm the association.
In conclusion, in our study group, the TNF-α –863A allele was not found to be a genetic risk susceptibility element in RA progression. None­theless, information related to TNF promoter variants is imperative to gain a better insight into RA genetics. However, in order to endorse and extend the results, further investigations on larger populations are required.


This research was supported by Higher Education Commission (HEC) Pakistan.

Conflict of interest

The authors declare no conflict of interest.


1. Cross M, Smith E, Hoy D, et al. The global burden of rheumatoid arthritis: estimates from the Global Burden of Disease 2010 study. Ann Rheum Dis 2014; 73: 1316-22.
2. Alamanos Y, Drosos AA. Epidemiology of adult rheumatoid arthritis. Autoimmun Rev 2005; 4: 130-6.
3. Farooqi A, Gibson T. Prevalence of the major rheumatic disorders in the adult population of north Pakistan. Br J Rheumatol 1998; 37: 491-5.
4. Klareskog L, Padyukov L, Rönnelid J, Alfredsson L. Genes, environment and immunity in the development of rheumatoid arthritis. Curr Opin Immunol 2006; 18: 650-5.
5. Begovich AB, Carlton VE, Honigberg LA, et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004; 75: 330-7.
6. Pawlik A, Herczyńska M, Kurzawski M, et al. IL-1beta, IL-6, and TNF gene polymorphisms do not affect the treatment outcome of rheumatoid arthritis patients with leflunomide. Pharmacol Rep 2009; 61: 281-7.
7. Ota M, Katsuyama Y, Kimura A, et al. A second susceptibility gene for developing rheumatoid arthritis in the human MHC is localized within a 70-kb interval telomeric of the TNF genes in the HLA class III region. Genomics 2001; 71: 263-70.
8. Zanelli E, Jones G, Pascual M, et al. The telomeric part of the HLA region predisposes to rheumatoid arthritis independently of the class II loci. Hum Immunol 2001; 62: 75-84.
9. Jawaheer D, Li W, Graham RR, et al. Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis. Am J Hum Genet 2002; 71: 585-94.
10. Fonseca JE, Cavaleiro J, Teles J, et al. Contribution for new genetic markers of rheumatoid arthritis activity and severity: sequencing of the tumor necrosis factor-alpha gene promoter. Arthritis Res Ther 2007; 9: R37.
11. Bayley JP, Ottenhoff TH, Verweij CL. Is there a future for TNF promoter polymorphisms? Genes Immun 2004; 5: 315-29.
12. Udalova IA, Richardson A, Denys A, et al. Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region. Mol Cell Biol 2000; 20: 9113-9.
13. Skoog T, van’t Hooft FM, Kallin B, et al. A common functional polymorphism (C → A substitution at position− 863) in the promoter region of the tumour necrosis factor-alpha (TNF-alpha) gene associated with reduced circulating levels of TNF-alpha. Hum Mol Genet 1999; 8: 1443-9.
14. Udalova IA, Richardson A, Ackerman H, Wordsworth P, Kwiatkowski D. Association of accelerated erosive rheumatoid arthritis with a polymorphism that alters NF-kappaB binding to the TNF promoter region. Rheumatology (Oxford) 2002; 41: 830-1.
15. Gambhir D, Lawrence A, Aggarwal A, Misra R, Mandal SK, Naik S. Association of tumor necrosis factor alpha and IL-10 promoter polymorphisms with rheumatoid arthritis in North Indian population. Rheumatol Int 2010; 30: 1211-7.
16. John P, Bhatti A, ul Ain N, Iqbal T, Sadaf T, Malik JM. Case-control study of vitamin D receptor gene polymorphism in Pakistani rheumatoid arthritis patients. Rev Bras Reumatol Engl Ed 2017; 57: 633-6.
17. Sadaf T, John P, Bhatti A, et al. Lack of tumor necrosis factor alpha gene polymorphism-857c/t (rs1799724) association in Pakistani rheumatoid arthritis patients. Int J Rheum Dis 2016; 19: 1119-25.
18. Kibbe WA. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 2007; 35 (Suppl 2): W43-6.
19. MacGregor AJ, Snieder H, Rigby AS, et al. Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins. Arthritis Rheum 2000; 43: 30-7.
20. Jawaheer D, Seldin MF, Amos CI, et al. Screening the genome for rheumatoid arthritis susceptibility genes: a replication study and combined analysis of 512 multicase families. Arthritis Rheum 2003; 48: 906-16.
21. MacKay K, Eyre S, Myerscough A, et al. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum 2002; 46: 632-9.
22. Fisher SA, Lanchbury JS, Lewis CM. Meta-analysis of four rheumatoid arthritis genome-wide linkage studies: confirmation of a susceptibility locus on chromosome 16. Arthritis Rheum 2003; 48: 1200-6.
23. Vries ND, Tijssen H, Riel PL, Putte LB. Reshaping the shared epitope hypothesis: HLA-associated risk for rheumatoid arthritis is encoded by amino acid substitutions at positions 67-74 of the HLA-DRB1 molecule. Arthritis Rheum 2002; 46: 921-8.
24. Yen JH, Chen CJ, Tsai WC, et al. Tumor necrosis factor promoter polymorphisms in patients with rheumatoid arthritis in Taiwan. J Rheumatol 2001; 28: 1788-92.
25. Raina P, Matharoo K, Kumar A, Sarangal P, Sharma R, Bhanwer AJS. Association of tumor necrosis factor-alpha –308 G>A polymorphism with rheumatoid arthritis in two north Indian cohorts. Arch Rheumatol 2014; 29: 241-9.
26. Fonseca JE, Cavaleiro J, Teles J, et al. Contribution for new genetic markers of rheumatoid arthritis activity and severity: sequencing of the tumor necrosis factor-alpha gene promoter. Arthritis Res Ther 2007; 9: R37.
27. Kirkham BW, Lassere MN, Edmonds JP, et al. Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort). Arthritis Rheum 2006; 54: 1122-31.
28. Richardson A, Sisay-Joof F, Ackerman H, et al. Nucleotide diversity of the TNF gene region in an African village. Genes Immun 2001; 2: 343-8.
29. Bayley JP, Ottenhoff TH, Verweij CL. Is there a future for TNF promoter polymorphisms? Genes Immun 2004; 5: 315-29.
30. Lis K, Kuzawińska O, Bałkowiec-Iskra E. Tumor necrosis factor inhibitors – state of knowledge. Arch Med Sci 2014; 10: 1175-85.
31. Zheng X, Zhou J, Xia Y. The role of TNF-alpha in regulating ketamine-induced hippocampal neurotoxicity. Arch Med Sci 2015; 11: 1296-302.
32. El-Tahan RR, Ghoneim AM, El-Mashad N. TNF-alpha gene polymorphisms and expression. Springerplus 2016; 5: 1508.
33. De Vries N, Tak PP. The response to anti-TNF-alpha treatment: gene regulation at the bedside. Rheumatology (Oxford) 2005; 44: 705-7.
34. Fonseca JE, Carvalho T, Cruz M, et al. Polymorphism at position –308 of the tumour necrosis factor α gene and rheumatoid arthritis pharmacogenetics. Ann Rheum Dis 2005; 64: 793-4.
35. Sashio H, Tamura K, Ito R, et al. Polymorphisms of the TNF gene and the TNF receptor superfamily member 1B gene are associated with susceptibility to ulcerative colitis and Crohn’s disease, respectively. Immunogenetics 2002; 53: 1020-7.
36. Li H, Groop L, Nilsson A, Weng J, Tuomi T. A combination of human leukocyte antigen DQB1* 02 and the tumor necrosis factor alpha promoter G308A polymorphism predisposes to an insulin-deficient phenotype in patients with type 2 diabetes. J Clin Endocrinol Metab 2003; 88: 2767-74.
37. Correa PA, Gomez LM, Cadena J, Anaya JM. Autoimmunity and tuberculosis. Opposite association with TNF polymorphism. J Rheumatol 2005; 32: 219-24.
38. Höhler T, Grossmann S, Stradmann-Bellinghausen B, et al. Differential association of polymorphisms in the TNFalpha region with psoriatic arthritis but not psoriasis. Ann Rheum Dis 2002; 61: 213-8.
39. Balding J, Kane D, Livingstone W, et al. Cytokine gene polymorphisms: association with psoriatic arthritis susceptibility and severity. Arthritis Rheum 2003; 48: 1408-13.
40. Rahman P, Siannis F, Butt C, et al. TNFalpha polymorphisms and risk of psoriatic arthritis. Ann Rheum Dis 2006; 65: 919-23.
41. Murdaca G, Gulli R, Spanò F, et al. TNF-alpha gene polymorphisms: association with disease susceptibility and response to anti-TNF-alpha treatment in psoriatic arthritis. J Invest Dermatol 2014; 134: 2503-9.
42. Jiménez-Morales S, Velázquez-Cruz R, Ramírez-Bello J, et al. Tumor necrosis factor-alpha is a common genetic risk factor for asthma, juvenile rheumatoid arthritis, and systemic lupus erythematosus in a Mexican pediatric population. Hum Immunol 2009; 70: 251-6.
43. Modesto C, Patiño-García A, Sotillo-Piñeiro E, et al. TNF- alpha promoter gene polymorphisms in Spanish children with persistent oligoarticular and systemic-onset juvenile idiopathic arthritis. Scand J Rheumatol 2005; 34: 451-4.
44. González S, Rodrigo L, Martínez-Borra J, et al. TNF-alpha -308A promoter polymorphism is associated with enhanced TNF-alpha production and inflammatory activity in Crohn’s patients with fistulizing disease. Am J Gastroenterol 2003; 98: 1101-6.
45. Uglialoro AM, Turbay D, Pesavento PA, et al. Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-alpha gene promoter. Tissue Antigens 1998; 52: 359-67.
46. Seki N, Kamizono S, Yamada A, et al. Polymorphisms in the 5′-flanking region of tumor necrosis factor-alpha gene in patients with rheumatoid arthritis. Tissue Antigens 1999; 54: 194-7.
Copyright: © 2018 Termedia & Banach. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
Quick links
© 2019 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe