|Year : 2015 | Volume
| Issue : 1 | Page : 16-20
Tumour necrosis factor-alpha promoter polymorphism and its association with viral dilated cardiomyopathy in Indian population: A pilot study
B Mishra1, M Sharma1, S Sarkar1, A Bahl2, UN Saikia3, RK Ratho1
1 Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Cardiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||30-Jul-2013|
|Date of Acceptance||02-Dec-2013|
|Date of Web Publication||5-Jan-2015|
Department of Virology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
Purpose: Tumour necrosis factor-α (TNF-α), a pro-inflammatory cytokine has been implicated in the pathophysiology of several viral infections. TNF-α promoter gene polymorphism is thus believed to play the modulating role in this disease pathogenesis. Several studies have shown the increased level of TNF-α in dilated cardiomyopathy (DCM). However, the role of the TNF-α promoter polymorphism is yet to be delineated in this regard. The present study for the first time tried to explore the association of TNF-α gene polymorphism with DCM of viral aetiology.
Materials and Methods: Eighteen histopathologically proven DCM cases with viral genome positivity and 17 healthy controls were genotyped using polymerase chain reaction of TNF-α promoter gene followed by restriction fragment length polymorphism to determine the SNPs of -238G/A, -308G/A, -857C/T and -863C/A. Results: Of the 18 DCM cases 4 (22.2%) were positive for adenovirus (AdV), 2 (11.1%) for enterovirus (EV) and 12 (66.7%) had co-infection. Six of the 18 DCM cases (35.3%) had -238G/A polymorphism, and 10 (55.5%) had -863 homozygous AA genotype. The association of these polymorphisms was statistically significant as compared to controls (P < 0.05). Conclusions: The present pilot study suggests the possible association of TNFα -238G/A and -863C/A polymorphism with DCM of viral aetiology.
Keywords: Adenovirus, dilated cardiomyopathy, enterovirus, TNF-α, polymorphism
|How to cite this article:|
Mishra B, Sharma M, Sarkar S, Bahl A, Saikia U N, Ratho R K. Tumour necrosis factor-alpha promoter polymorphism and its association with viral dilated cardiomyopathy in Indian population: A pilot study. Indian J Med Microbiol 2015;33:16-20
|How to cite this URL:|
Mishra B, Sharma M, Sarkar S, Bahl A, Saikia U N, Ratho R K. Tumour necrosis factor-alpha promoter polymorphism and its association with viral dilated cardiomyopathy in Indian population: A pilot study. Indian J Med Microbiol [serial online] 2015 [cited 2020 Dec 5];33:16-20. Available from: https://www.ijmm.org/text.asp?2015/33/1/16/148370
| ~ Introduction|| |
Myocarditis is the representative of myocardial inflammation of diverse aetiologies where viruses are the most common aetiological factor, predominantly enteroviruses (EV) and adenoviruses (AdV). Viral myocarditis accounts for up to one-third of the cases of dilated cardiomyopathy (DCM) characterised by cardiac dilation, diminished contraction of the left or both the ventricles which may further progress to heart failure and sudden cardiac death.  Acute myocarditis occurs as the first phase of disease pathogenesis followed by the phase of immune response or sub-acute myocarditis and finally to cardiac remodeling phase leading to chronic myocarditis and DCM.  However, it is not yet explored whether this entire process is modulated by any host genetic factor or related immune response may be responsible for susceptibility to DCM. 
Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which has been described as a "mixed blessing for the higher organism". , Indeed the controlled self-limited expression of TNF plays a critical role in activating host defense mechanisms and in homeostatic tissue repair, but uncontrolled overexpression of TNF accounts for the devastating consequences for the host organism, consequently leading to diffuse inflammation, multiorgan dysfunction, haemodynamic collapse and death.  In various studies, TNF-α is implicated in pathogenesis of atherosclerosis, myocardial infarction and chronic heart failure.  Raised TNF levels have been found in patients with DCM as compared to the controls thus implicating its role in disease pathogenesis.  However, the pathophysiological role of endogenous TNF-α in DCM with viral aetiology is still enigmatic and remains to be explored.
The role of host immune response in viral pathogenesis is well established. Thus, recently research is being focused on clinical manifestation-related cytokines which exhibit polymorphisms and influence susceptibilities and disease outcome.
Till date 621 SNPs in TNF-α gene have been reported in the NCBI website, with more than 10 SNPs in the promoter region, including -238G/A, -244A/G, -308G/A, -376A/G, -575A/G, -857C/T, -863C/A and -1031T/C. , Among them some SNPs regulate the production of TNF-α. , There are few studies from different geographical areas where the association between the idiopathic DCM and the SNPs in TNF promoter region have been studied. ,, The objective of the present study was to explore the role of TNF-α promoter gene polymorphism at various loci in DCM cases with viral aetiology.
| ~ Materials and Methods|| |
The endomyocardial biopsies from 21 of histopathologically proven DCM cases were tested for the presence of adenovirus (AdV) and enterovirus (EV) genome by polymerase chain reaction (PCR) as described below. Of these 18 cases that were positive for AdV/EV genome were finally included as study group as viral DCM cases. Seventeen healthy individuals were included as control groups.`
Three millilitres of blood was collected aseptically in a vial containing anticoagulant from both cases (n = 18) and controls (n = 17). The endomyocardial biopsies (EMBs) were collected for histopathological examination and viral genome detection. The tissue collected for molecular study were stored in RNA later (Ambion, USA) at -80°C till further use. Informed consent was taken from all the patients before EMB. The study protocol was approved by Institute's Ethics Committee.
Viral genome detection
Custom Taqman assays containing the primers and an FAM-labeled probe targeting hexon gene of AdV and 5'NTR of EV genome were developed from ABI, Foster City, CA, USA. The EMB was subjected to nucleic acid extraction using RNAqueous Isolation kit (Ambion). One half of the nucleic acid was treated with RNase enzyme to obtain DNA while the other half was treated with DNase I enzyme to get RNA. RNA (1 μg) extracted from the myocardial tissues was reverse transcribed to cDNA using high-efficiency cDNA synthesis kit (Ambion) followed by Taqman Real Time PCR. The reaction mixture (25 μl) contained 1X TaqMan PCR master mix, 1.25 μl AdV or EV-specific primer-probe assay (ABI) and 500 ng of extracted DNA or 2 μl of cDNA. Real-time PCR assays were performed in ABI 7500 following the universal thermal profile. For each run no template control and positive controls were included.
Study of TNF-α promoter polymorphism by PCR-RFLP
Genomic DNA was extracted from the whole blood using the QIAGEN DNA extraction kit as per the manufacturer's protocol and stored at -20°C until further use. The SNPs of -238G/A, -308G/A, -857 C/T and -863C/A at the TNF-α promoter region were determined by the gene-specific PCR followed by restriction fragment length polymorphism (PCR-RFLP). The PCR was carried out using the specific primers as described earlier.  The amplified products were visualised in 2% agarose gel stained with ethidium bromide. These PCR products were further subjected to restriction digestion by allele-specific restriction enzymes (MBI Fermentas, USA) by overnight incubation at 4°C. Cleaved DNA fragments were separated by 12% polyacrylamide gel electrophoresis and visualised by ethidium bromide staining. The allelic type was determined according to the presence or absence of the digested product of the desired length [Table 1].
|Table 1: Primers, restriction enzymes, digestion pattern and genotypes of TNF promoter at different sites|
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The data was analyzed using SPSS software version 16.0. The allele and genotype frequencies were examined by Hardy Weinberg Equilibrium test. The TNF-α promoter gene polymorphism in cases and controls was evaluated using χ2. SPSS was used to calculate odds ratio (OR) and 95% confidence intervals (CI) for assessing the risk associated with particular allele and genotype. The 'P value' of < 0.05 was taken as significant.
| ~ Results|| |
EMBs from the clinically and histopathologically confirmed DCM cases were tested for viral genome detection. Viral genome was detected by Real-time PCR using EV and AdV specific Taqman assay. Of the 18 DCM cases positive for viral genome, 4 (4/18; 22.2%) were positive for AdV genome, 2 (2/18; 11.1%) had EV positivity and 12 (12/18; 66.7%) cases had co-infection of AdV and EV.
The DCM cases with viral positivity were analyzed for polymorphism at TNF-α promoter position. The genotypic and allele frequency distribution for the four polymorphisms of -238G/A, -308G/A, -857C/T and -863C/A between the two study groups is listed in [Table 2]. The allelic and genotypic frequencies were observed to be in Hardy-Weinberg equilibrium. The frequency distribution of the heterozygous GA genotype at -238 (χ2 = 8.264; P =0.008; OR = 2.545; 95% CI = 1.606- 4.034) and the frequency distribution of homozygous AA genotype at -863 were found to be statistically significant (χ2 = 5.381; P =0.035; OR = 5.833; 95% CI = 1.231- 27.632) in comparison to controls. However, no statistical difference was observed in the genotypic and allelic frequency distribution of -308G/A and -857C/T between the cases and controls [Table 3]. [Table 4] shows the possible association of viral DCM with TNF-α polymorphism.
|Table 2: Genotype distribution and allelic frequency of TNF-α polymorphism between Viral positive DCM cases and controls|
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|Table 3: Statistical analysis of genotype frequency of TNF-α polymorphism in DCM and control groups|
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|Table 4: Association of viral genome positivity in DCM cases with TNF-α polymorphism|
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| ~ Discussion|| |
Myocarditis is the inflammation of myocardium which can progress to DCM and heart failure in upto 30% of the patients.  Viruses have been implicated predominantly in the genesis and evolution of viral myocarditis and DCM. ,, Although 90% of the people get infected with one of the cardiotropic viruses in their life but only few develop clinical symptoms of myocarditis and progress to DCM.  This may be attributed to host genetic make-up which induces immune response alterations or increased susceptibility to viral infections.  The study on family background has shown an inherited gene mutation to be the cause of approximately 25-30% of idiopathic DCM.  However, the knowledge pertaining to genetics risk factors for DCM is yet to be explored.
Pro-inflammatory cytokines, such as TNF-α has been found to produce various biological effects which include immune activation, stimulation of gene expression involved in inflammatory response and cellular alteration through nitric oxide production.  Several studies suggest that TNF play a crucial pathophysiological role in the development of viral myocarditis and its progression to DCM. The effect of TNF-α on cardiac function depends on the amount and duration of TNF-α expression. As demonstrated in in-vitro models, TNF-α can affect heart by cardiomyocyte loss, through necrosis or apoptosis. Both animal model and human studies have shown upregulation of TNF-α mRNA and protein levels in hearts of patients during myocarditis and DCM. During the chronic stage of viral myocarditis, the persistence of TNF-α expression in the cardiovascular system is responsible for the infiltration of leucocytes which leads to the myocardial damage. It has been observed that myocarditis is aggravated by exogenous administration of TNF whereas its neutralisation can attenuate myocarditis. ,,, The human TNF-α gene lies within the class III region of the major histocompatibility complex (MHC) in between HLA-B and DR. TNF has potent physiological actions and the 5' flanking region of TNF-α gene regulates TNF-α production at transcriptional level.  Therefore, it is speculated that polymorphism in this locus may attribute to infectious or autoimmune diseases. The polymorphism of cytokines in DCM have been studied previously which included particularly TNF-α, IL-10 and IL-17. The anti-inflammatory cytokines may influence the function of pro-inflammatory cytokines and raised levels of IL-10 have been found in serum of patients with congestive heart failure though Ito et al., reported no significant difference of IL-10 allele frequency at -1082 position in DCM cases as compared to control group.  Similarly, study conducted by Peng et al., explored the associations of IL-17 gene polymorphisms in Chinese patients with DCM and stated that rs2275913 and rs763780 polymorphisms in IL-17 genes were not responsible for disease outcome of DCM. However, polymorphisms of TNF-α promoter region in idiopathic DCM have been studied in different populations across the globe.  TNF-α promoter polymorphism has been studied at -308, -238, -857, -863, -1031 positions in DCM but none of these previous studies have simultaneously studied polymorphism at four loci (-308, -238, -857, -863) of TNF-α promoter region. ,, Thus, in the present study a possible association of TNFα -308, -238, -857, -863 polymorphism with viral DCM was explored.
Among the various aetiological factors of DCM, cardiotropic viruses like enterovirus and adenovirus, have been implicated as the most common cause. However, it has been observed from previous studies the predominance of virus and rate of co-infection vary with the geographical origin. The rate of enterovirus positivity has been reported from 1.2% to 27.3%, where as adenovirus positivity ranges from 2% to 12% in various studies. ,, The high positivity of enterovirus and adenovirus in DCM patients in our study as compared to previous studies can be due to the use of more sensitive technique i.e. Real-time PCR for positivity of cardiotropic viruses, difference in the inclusion criteria of DCM patients and different geographic origin of DCM patients studied. In the present study, 18 DCM cases positive for AdV or EV genome were further subjected to analysis for the association with TNF-α -238G/A,-308G/A, -863C/A and -857C/T polymorphism. The A allele frequency was significantly high in DCM cases as compared to the controls (P < 0.05) thus, suggesting association of A allele polymorphism with DCM of viral aetiology. Few studies have demonstrated the polymorphism of TNF gene at various loci in idiopathic DCM cases. ,, The present study for the first time demonstrated the association of TNF-α -238G/A and -863C/A polymorphism with DCM of viral aetiology.
The point mutation of G to A at position -308 and -238 of TNF promoter has been reported previously to predispose to higher TNF production.  Polymorphism at -863C/A, has been found to inhibit the binding of the NFkB p50-p50 homodimer to its domain in TNF promoter, where NF-kB acts as the transcriptional repressor. This inhibits down-regulation of TNF-α expression and thereby leading to increased TNF production.  Thus, from the results of the present study, it may be hypothesised that polymorphism at -238 and -863 possibly increases the TNF-α expression and susceptibility to viral infection which together may have attributed to the disease progression to DCM [Figure 1]. However, additional studies including large number of samples are required to explain the proposed hypothesis in pathogenesis of viral DCM.
|Figure 1: Hypothetical model describing possible mechanism of TNF-α polymorphism leading to increased TNF-α expression on viral infection in DCM. In the case of wild type alleles at -238 and -863 position of TNF-α promoter gene as shown in Figure 1A, regulated production of TNF-α occurs after viral infection which may lead to the viral clearance but in the case of hosts having mutant alleles of TNF-α promoter gene i.e., 238GA and 863AA as shown in Figure 1B, after viral infection TNF-α over-expression may lead to disease progression to dilated cardiomyopathy|
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| ~ Conclusions|| |
TNFα -238G/A and -863C/A polymorphism was significantly associated with DCM cases of viral aetiology suggesting the possible association of TNF-α polymorphism with susceptibility to viral infection and risks of DCM.
| ~ References|| |
Tavares PS, Rocon-Albuquerque R Jr, Leite-Moreira AF. Innate immune receptor activation in viral myocarditis: Pathophysiological implications. Rev Port Cardiol 2010;29:57-78.
Mason JW. Myocarditis and dilated cardiomyopathy: An inflammatory link. Cardiovasc Res 2003;60:5-10.
Dennert R, Crinjs HJ, Heymans S. Acute viral myocarditis. Eur Heart J 2008;29:2073-82.
Beutler B, van Huffel C. Unraveling function in the TNF ligand and receptor families. Science 1994;264:667-8.
Mann DL. Tumor necrosis factor and viral myocarditis: The fine line between innate and inappropriate immune responses in the heart. Circulation 2001;103:626-9.
Liang WB, Lv ML, Su XW, Gao LB, Fang WL, Luo HB, et al
. Association of tumor necrosis factor gene polymorphisms with susceptibility to dilated cardiomyopathy in a Han Chinese population. DNA Cell Biol 2010;29:625-8.
Yamada T, Matsumori A, Sasayama S. Therapeutic effects of anti-tumor necrosis factor-a antibody on the murine model of viral myocarditis induced by encephalomyocarditis virus. Circulation 1993;89:846-51.
Baena A, Leung JY, Sullivan AD, Landires I, Vasquez-Luna N, Quiñones-Berroca J, et al
. TNF-alpha promoter single nucleotide polymorphisms are markers of human ancestry. Genes Immun 2002;3:482-7.
Higuchi T, Seki N, Kamizono S, Yamada A, Kimura A, Kato H, et al
. Polymorphism of the 5'- flanking region of the human tumor necrosis factor (TNF)-alpha gene in Japanese. Tissue Antigens 1998;51:605-12.
Heesen M, Kunz D, Wessiepe M, van der Poll T, Zwinderman AH, Blomeke B. Rapid genotyping for tumor necrosis factor alpha (TNF-alpha) 863C/A promoter polymorphism that determines TNF-alpha response. Clin Chem 2004;50:226-8.
Pujhari SK, Ratho RK, Prabhakar S, Mishra B, Modi M. TNF-α promoter polymorphism: A factor contributing to the different immunological and clinical phenotypes in Japanese encephalitis. BMC Infect Dis 2012;26:12-23.
Ito M, Takahashi H, Fuse K, Hirono S, Washizuka T, Kato K, et al
. Polymorphisms of tumor necrosis factor-alpha and interleukin-10 genes in Japanese patients with idiopathic dilated cardiomyopathy. Jpn Heart J 2000;41:183-91.
Alikasifoglu M, Tokgozoglu L, Acil T, Atalar E, OTO MA, Sirri Kes S, et al
. Tumor necrosis factor-alpha polymorphism in Turkish patients with dilated cardiomyopathy. Eur J Heart Fail 2003;5:161-3.
Du T, Guo XH, Zhu XL, Li JH, Lu LP, Gao JR, et al
. Association of TNF -alpha promoter polymorphisms with the outcomes of hepatitis B virus infection in Chinese Han population. J Viral Hepat 2006;13:618-24.
Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss HP, McCarthy R, et al
. Detection of viruses in myocardial tissues by polymerase chain reaction. Evidence of adenovirus as a common cause of myocarditis in children and adults. J Am Coll Cardiol 2003;42:466-72.
Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassener D, et al
. High prevalence of viral genomes and multiple viral infections in the myocardium of adults with "idiopathic" left ventricular dysfunction. Circulation 2005;111:887-93.
Matsumori A, Shimada T, Chapman NM, Tracy SM, Mason JW. Myocarditis and heart failure associated with hepatitis C virus infection. J Card Fail 2006;12:293-8.
Andréoletti L, Leveque N, Boulagnon C, Brasselet C, Fornes P. Viral causes of human myocarditis. Arch Cardiovasc Dis 2009;102:559-68.
Grunig E, Tasman JA, Kucherer H, Franz W, Kubler W, Katus HA. Frequency and phenotypes of familial dilated cardiomyopathy. J Am Coll Cardiol 1998;31:186-94.
Bristow MR. Tumor necrosis factor alpha and cardiomyopathy. Circulation 1998;97:1340-1.
Matsumori A, Yamada T, Suzuki H, Matoba Y, Sasayama S. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J 1994;72:561-6.
Satoh M, Tamura G, Segawa I, Tashiro A, Hiramori K, Satodate R. Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy. Virchows Arch 1996;427:503-9.
Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, et al
. Dilated cardiomyopathy in transgenic mice with cardiac specific overexpression of tumor necrosis factor-alpha. Circ Res 1997;81:627-35.
Haren MT, Malmstrom TK, Miller DK, Patrick P, Perry HM 3 rd
, Herning MM, et al
. Higher C-reactive protein and soluble tumor necrosis factor receptor levels are associated with poor physical function and disability: A cross-sectional analysis of a cohort of late middle-aged African Americans. J Gerontol A Biol Sci Med Sci 2010;65:274-81.
Peng Y, Zhou B, Wang YY, Shi S, Zhang K, Zhang L, et al
. Analysis of IL-17 gene polymorphisms in Chinese patients with dilated cardiomyopathy. Human Immunol 2013;74:635-9.
Grumbach IM, Heim A, Pring-Akerblom P, Vonhof S, Hein WJ, Muller G, et al
. Adenoviruses and enteroviruses as pathogens in myocarditis and dilated cardiomyopathy. Acta Cardiol 1999;54:83-8.
Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195-9.
Udalova IA, Richardson A, Denys A, Smith C, Ackerman H, Foxwell B, 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.
[Table 1], [Table 2], [Table 3], [Table 4]