| Abstract|| |
Context: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the bowel (IBD) whose causes are not fully known. Emerging data indicate that alterations in cytokine synthesis may play a role in IBD pathogenesis. Aims: We aimed to determine the association between tumor necrosis factor-alfa (TNFα) promoter polymorphisms (at positions − 308 and − 1031) and susceptibility to IBD among Iranian Azari Turkish patients. Settings and Design: One hundred and one patients with IBD and 100 healthy subjects were analyzed. Materials and Methods: Both polymorphisms in the promoter region of the TNFα gene at positions -1031T/C and -308G/A were detected by polymerase chain reaction-restriction fragment length polymorphism assay. All statistical analyses were calculated with SPSS for Windows 16.0. The Fisher's exact test was used to test for departure from Hardy-Weinberg equilibrium of the genotype frequencies (P > 0.05). Results: The allele frequency of the TNFα-308G and -1031T were higher in IBD patients but did not reach statistical significance. However, the homozygous TT genotype for the SNP-1031 T > C was significantly higher in UC patients than in healthy controls (P = 0.01) and the heterozygous CT genotype for the SNP -1031 T > C was significantly lower in UC patients than in healthy controls (P = 0.03). Conclusions: The TNFα-1031 T allele confers a significant risk for developing UC in Iranian Azeri Turkish patients. Also the frequency of TNFα-1031 C allele was considerably low among patients with UC and it may have protective role among them (OR = 0.43; P = 0.01).
Keywords: Genetics, inflammatory bowel disease, single nucleotide polymorphisms, tumor necrosis factor alfa
|How to cite this article:|
Bonyadi M, Abdolmohammadi R, Jahanafrooz Z, Somy MH, Khoshbaten M. TNF-alpha gene polymorphisms in Iranian Azari Turkish patients with inflammatory bowel diseases. Saudi J Gastroenterol 2014;20:108-12
|How to cite this URL:|
Bonyadi M, Abdolmohammadi R, Jahanafrooz Z, Somy MH, Khoshbaten M. TNF-alpha gene polymorphisms in Iranian Azari Turkish patients with inflammatory bowel diseases. Saudi J Gastroenterol [serial online] 2014 [cited 2019 Oct 13];20:108-12. Available from: http://www.saudijgastro.com/text.asp?2014/20/2/108/129475
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic condition that involves inflammation of the digestive tract. , It has been the most common health problem worldwide, particularly in the developed countries. UC and CD affect different parts of the digestive tract. UC causes swelling and the formation of ulcers on the surface lining the top layer of the large intestine. Patients with CD experience thickening of the intestinal wall, most frequently in the last part of the small intestine and the first part of the large intestine. , Although IBD can appear at any age, it is rare in babies and very young children. It has been reported that incidence rates were 0.70 per 100,000 person-years for CD and 1.85 per 100 000 person-years for UC worldwide. 
The cause of the IBD-UC and CD- is not well known. Studies have suggested that IBD is a multifactorial disease that has arisen from both environmental risk factors and genetic susceptibility. ,, There is multiple evidence in favor of a genetic role in the pathogenesis of IBD. These include increased rates of IBD among identical twins compared with fraternal twins and among siblings compared with spouses of affected individuals, the differences in prevalence in diverse ethnic groups and the increased familial incidence. Although there is strong evidence to suggest a genetic basis for IBD, no specific gene has been identified thus far. , The main genetic associations in IBD can be divided into genes that contribute to innate and adaptive immune responses.  TNFα is a multifunctional cytokine involved in the promotion of inflammatory responses and plays a critical role in the pathogenesis of inflammatory, autoimmune, and malignant diseases. ,, TNFα is also known to play a key role in cell-mediated immunity and elevated serum levels of TNFα have been demonstrated in IBD.  These facts suggest a direct role for TNFα in IBD pathogenesis. The TNFα has a large number of polymorphisms-most of them located in its promoter region-of which some of them have effect on the expression level of the gene.  We postulated that differences in cytokine levels as a result of gene polymorphisms may have an important role in the inflammatory response and thus influence the pathophysiology of IBD.
In this study, for the first time, the association between TNFα promoter polymorphisms (at positions -308 and -1031) and susceptibility to IBD among Iranian Azari Turkish patients was investigated.
| Materials and Methods|| |
Our case-control study included 101 Iranian Azeri Turkish IBD patients (21 CD patients and 80 UC patients) and 100 healthy, unrelated, age- and gender-matched individuals from the same geographical area were selected as a control group. The diagnosis and extent of IBD was made on the basis of clinical symptoms, endoscopic, radiological, and histopathological findings, according to conventional criteria.  Genomic DNA was extracted from peripheral blood cells using simple salting out procedure.  Both polymorphisms in the promoter region of the TNFα gene at positions -1031T/C and -308G/A were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. For the amplification of -1031T/C polymorphism site via PCR we used the forward (5'-GGGGAGAACAAAAGGATAAG-3') and reverse (5'-CCCCATACTCGACTTTCATA-3') primer pair. Initially, the PCR reaction was subjected to denaturation for 5 min at 95°C, followed by 30 cycles of amplification (30 s at 95°C, 30 s at 55°C and 30 s at 72°C). A final elongation step (5 min at 72°C) was applied at the end of the 30 cycles. The PCR product is 270-bp. Then PCR followed by an overnight digestion with the restriction enzyme BbsI (C allele, 159 and 111 bp; T allele, 270 bp) at 37°C. Digested PCR fragments were separated by a 10% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. Primers forward (5'-AGGCAATAGGTTTTGAGGGCCAT-3') and reverse (5'-TCCTCCCTGCTCCGATTCCG-3') were used to amplify the 107-bp DNA fragment of the TNFα-308A/G polymorphism. PCR conditions were 5 min for initial denaturation at 95°C; 35 cycles at 95°C for 1 min for denaturation, 30 s at 65°C for annealing and 30 s at 72°C for extension, followed by 5 min at 72°C for final extension. After amplification, PCR products were digested (at 37°C) by restriction endonuclease NcoI (G allele, 87 and 20 bp; A allele, 107 bp) for 16 h. Digested PCR products were electrophoresed in a 10% polyacrylamide gel and visualized by ethidium bromide staining.
Allelic and genotypic association between patients and healthy controls was carried out using Chi-square test with Yates' correction or Fisher's exact test, where appropriate. The Fisher's exact test was used to test for departure from Hardy-Weinberg equilibrium of the genotype frequencies (P > 0.05). Allele frequencies were determined by gene counting. Odds ratios and corresponding 95% confidence intervals were estimated by cross-tabulation. Statistics were calculated using SPSS 16.0.
| Results|| |
In this study, 101 patients with IBD, 21 patients with CD and 80 patients with UC (21-69 years old, 51 males (51%), 50 females (50%) as well as 100 healthy controls (16-74 years old, 49 males (49%), 51 females (51%) were included. No statistically significant differences were observed between patients with IBD and controls regarding age and gender. Clinical analysis of IBD patients showed that 20.8% (21/101) had fever, 34.7% (35/101) had diarrhea, and 18.8% (19/101) had vomiting. For TNFα we genotyped two SNPs in the promoter region: -1031 T > C and -308 A > G. The allelic and genotypic frequencies are shown in [Table 1]. We found no significant difference of -1031T/C and -308 G/A allele/carrier frequency between IBD patients and healthy controls. The frequency of the TNFα-308G and -1031T alleles were higher in IBD patients but did not reach statistical significance (at -308G allele frequency 92.58% vs 90.5%, P = 1 and at -1031T allele frequency 89.1% vs 83%, P = 0.08). However, when the UC patient groups were compared with controls separately, we observed significant difference for the SNP -1031 T > C with the allele C present in 8% of UC and 17% of controls and allele T present in 92% of UC and 83% of controls. The heterozygous CT genotype for the SNP-1031 T > C was significantly lower in UC patients (16.2%) than in healthy controls (30.0%), and reached statistical significance (P = 0.03; OR = 2.31; 95% CI = 1.11-4.93) [Table 2]. For the SNP-308 A > G, with the allele G present in 92.5% of UC and 90.5% of controls, was although not statistically significant. We also did not find homozygous CC genotype for the SNP-1031 T > C in UC patients.
|Table 1: TNF-alfa genotype and allele frequencies at position -308 and position -1031 in patients with IBD and in healthy control|
Click here to view
|Table 2: TNF-alfa genotype and allele frequencies at position -308 and position -1031 in patients with UC and in healthy controls|
Click here to view
For assessing the relationship between genotype and clinical parameters, the allelic and genotypic frequencies of these polymorphisms between controls and patients with/without symptoms were evaluated. These data showed no significant difference in genotype distribution and allelic frequency among groups.
| Discussion|| |
CD and UC are chronic inflammatory diseases of the bowel, whose causes are not fully known. Emerging data indicate that alterations in cytokine synthesis may play a role in IBD pathogenesis.  The single nucleotide polymorphisms in gene promoter regions result in differential production of cytokines and may influence disease activity as well as more intense inflammatory activity in both forms of IBD.  Tumor necrosis factor (TNF)-alfa is an important proinflammatory cytokine that has been implicated in the pathogenesis of IBD. Overexpression of TNFα have been observed in inflamed and normal intestinal mucosa and in the serum of patients with IBD.  In 2003, Gonzalez et al., showed that the TNFα-308A polymorphism was associated with enhanced TNFα production and an increased risk for susceptibility to arthritis in CD patients.  In the present study, we determined the allelic and genotypic distribution of two SNPs (at positions -308 and -1031) in the TNFα gene among IBD patients and healthy controls in the Iranian Azari Turkish ethnic group. In our study, we found a nonstatistically significant reduction of -308A and -1031C allele frequencies in IBD patients compared with controls. However, the comparison of UC group with healthy controls showed a significant variation in TNFα-1031 polymorphism allele and genotype frequency [Table 2]. The homozygous TT genotype for the SNP-1031 T > C was significantly higher in UC patients (83.8%) than in healthy controls (68%) (P = 0.01; OR, 2.42; 95%CI, 1.17-5.02). Also, the heterozygous CT genotype for the SNP -1031 T > C was significantly lower in UC patients (16.2%) than in healthy controls (30.0%), and reached statistical significance (P = 0.03; OR, 0.45; 95%CI, 0.21-0.94). These observations suggest that the TNFα-1031 T allele confers a significant risk for developing UC and the SNP-1031 T > C polymorphism may have a functional effect in IBD pathogenicity and could explain the reason for high levels of TNF-α production observed in IBD patients. Nevertheless, until now, there has been no available molecular genetic mechanism to explain how the -1031T > C allele influences TNFα production. The polymorphic -1031T > C site may either bind to unknown regulatory elements or alter the secondary structure of DNA to affect accession of cis-acting transcription factors to the promoter/enhancer region of the TNFα gene.  Previous studies regarding the association of TNFα gene polymorphism and IBD susceptibility have reported conflicting results. Depending on studied populations, some reports show positive association and the others failed to show any significant association with TNFα gene polymorphisms and IBD. ,,,,, Bouma et al. reported small but statistically significant association between TNFα-308 polymorphism and UC. They found a lower frequency of the TNFα-308 A allele in UC patients compared with healthy controls.  Also Vatay et al. reported the frequency of TNFα-308 A allele to be less in IBD patients (both UC and CD) compared with controls.  The results of our study are consistent with some previous reports. , In the study by Zipperlen et al., no association between TNFα promoter polymorphisms and CD was observed.  Conversely, in a study carried out by Balding and colleagues, there was a significant variation in TNFα-308 polymorphism genotype frequency when comparing the control group with the UC group. The UC group had a lower frequency of the TNFα-308A allele.  A meta-analysis by Lu et al. showed that the TNFα-308 A allele confers a significant risk for developing UC in East Asians but there is no association between the polymorphism of TNFα-308 gene promoter and UC in Europeans.  The associations between IBD and the TNFα polymorphism have already been discussed. Genetic variations among different populations and ethnic variability might be responsible for these discordant results in the frequency of -308 and -1031 TNFα gene polymorphisms between the present study and others' studies. ,,
In conclusion, the homozygous TT genotype for the TNFα-1031 T > C was significantly associated with UC and the TNFα-1031 T allele confers a significant risk for developing UC in Iranian Azeri Turkish patients. Also the frequency of TNFα-1031 C allele was considerably low among patients with UC and may have a protective role among them (OR = 0.43; P = 0.01). Finally, one of the limitations of our study is that a relatively small number of patients are available for study. Because of the small sample size, this report should be considered as exploratory and further studies are required to confirm these genetic associations with IBD.
| Acknowledgments|| |
The authors express their appreciation to all the participants for their contribution to this study. This work is financially supported by Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences.
| References|| |
|1.||Ferguson L, Huebner C, Peterman I, Gearry R, Barclay M, Demmers P, et al. Han Single nucleotide polymorphism in the tumor necrosis factor-alpha gene affects inflammatory bowel diseases risk. World J Gastroenterol 2008;14:4652-61. |
|2.||Balding J, Livingstone W, Conroy J, Mynett-Johnson L, Weir D, Mahmud N, et al. Inflammatory bowel disease: The role of inflammatory cytokine gene polymorphisms. Mediators of Inflamm 2004;13:181-7. |
|3.||Baumgart D. The diagnosis and treatment of Crohn's disease and ulcerative colitis. Dtsch Arztebl Int 2009;106:123-33. |
|4.||Fan W, Maoqing W, Wang Yang Ch, Fulan H, Dandan L, Jiaojiao R, et al. Relationship between the polymorphism of tumor necrosis factor-α-308 G4A and susceptibility to inflammatory bowel diseases and colorectal cancer: A meta-analysis. Eur J Hum Genet 2011;19:432-7. |
|5.||Van Heel D, Udalova I, De Silva A, McGovern D, Kinouchi Y, Hull J, et al. Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF-κB transcription factors. Hum Mol Genet 2002;11:1281-9. |
|6.||Sanchez R, Levy E, Costea F, Sinnett D. IL-10 and TNFA promoter haplotypes are associated with childhood Crohn's disease location. World J Gastroenterol 2009;15:3776-82. |
|7.||Vries H, Morsche R, Oijen M, Nagtegaal I, Peters W, Jong D. The Functional 2765GRC polymorphism of the COX-2 gene may reduce the risk of developing Crohn's Disease. PLoS One 2010;11:e15011. |
|8.||Bouma G, Crusiusc G, Koutroubakis B, Blomberg B, Meuwissen S, Pena A. Distribution of four polymorphisms in the tumor necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD). Clin Exp Immunol 1996;103:391-6. |
|9.||Meijer M, Mieremet-Ooms M, Hogezand R, Lamers C, Hommes D, Verspaget H. Role of matrix metalloproteinase, tissue inhibitor of metalloproteinase and tumor necrosis factor-α single nucleotide gene polymorphisms in inflammatory bowel disease. World J Gastroenterol 2007;13:2960-6. |
|10.||Shih D, Targan S. Recent advances in IBD pathogenesis: Genetics and immunobiology. Curr Gastroenterol Rep 2008;10:568-75. |
|11.||Cao Q, Zhu Q, Wu ML, Hu WL, Gao M, Si JM. Genetic susceptibility to ulcerative colitis in the Chinese Han ethnic population: Association with TNF polymorphisms. Chin Med J 2006;119:1198-203. |
|12.||Hampe J, Shaw H, Saiz R, Leysens N, Lantermann A, Mascheretti S, et al. Linkage of Inflammatory Bowel Disease to Human Chromosome 6p. Am J Hum Genet 1999;65:1647-55. |
|13.||Tremelling M, Waller S, Bredin F, Greenfield S, Parks M. Genetic variants in TNFA but not DLG5 are associated with inflammatory Bowel Disease in a Large United Kingdom Cohort. Inflamm Bowel Dis 2006;12:178-84. |
|14.||Hajeer AH, Hutchinson IV. TNFA gene polymorphism: Clinical and biological implications. Microsc Res Tech 2000;50:216-28. |
|15.||Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol Suppl 1989;170:2-6. |
|16.||Miller SA, Dynes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:12-5. |
|17.||Cantor MJ, Nickerson P, Bernstein CN. The role of cytokine gene polymorphisms in determining disease susceptibility and phenotype in inflammatory bowel disease. Am J Gastroenterol 2005;100:1134-42. |
|18.||Sykora J, Subrt I, Didek P, Siala K, Schwarz J, Machalova V, et al. Cytokine tumor necrosis factor-alpha A promoter gene polymorphism at position -308 G->A and pediatric inflammatory bowel disease: Implications in ulcerative colitis and Crohn's disease. J Pediatr Gastroenterol Nutr 2006;42:479-87. |
|19.||Zipperlen K, Peddle L, Melay B, Hefferton D, Rahman P. Association of TNFA Polymorphisms in Crohn Disease. Hum Immunol 2005;66:56-9. |
|20.||Han Z, Li C, Han S, Han Y, Qiu J, Shi Y, et al. Meta-analysis: Polymorphisms in TNF-a gene promoter and Crohn's disease. Aliment Pharmacol Ther 2010;32:159-70. |
|21.||Gonzalez S, Rodrigo L, Martinez-Borra J, Lopez-Vazquez A, Fuentes D, Nino P, 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. |
|22.||Kawasaki A, Tsuchiya N, Hagiwara K, Takazoe M, Tokunaga K. Independent contributions of HLA-DRB1 and TNF-a promoter polymorphisms to the susceptibility of Crohn's disease. Genes Immun 2000;1:351-7. |
|23.||Fowler EV, Eri R, Hume G, Johnstone S, Pandeya N, Lincoln D. TNF-a and IL10 SNPs act together to predict disease behavior in Crohn's disease. J Med Genet 2005;42:523-8. |
|24.||Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP. Cytokine (TNF-α, LTα and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: Differential effects on production and allele frequencies. Genes Immun 2000;1:185-90. |
|25.||Vatay A, Bene L, Kovacs A, Prohaszka Z, Szalai C, Romics L, et al. Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease. Immunogenetics 2003;55:247-52. |
|26.||Louis E, Satsangi J, Roussomoustakaki M, Parkes M, Fanning G, Welsh K, et al. Cytokine gene polymorphisms in inflammatory bowel disease. Gut 1996;39:705-10. |
|27.||Lu Z, Chen L, Li H, Zhao Y, Lin L. Effect of the polymorphism of tumor necrosis factor-alpha-308 G/A gene promoter on the susceptibility to ulcerative colitis: A meta-analysis. Digestion 2008;78:44-51. |
Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz
Source of Support: This study was supported by the Centre of Liver and Gastrointestinal Disease Research Centre, University of Medical Sciences, Tabriz, Iran., Conflict of Interest: None
Clinical trial registration 5/4/9469
[Table 1], [Table 2]