Journal of the Practice of Cardiovascular Sciences

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 5  |  Issue : 1  |  Page : 35--43

Tumor necrosis factor-alpha gene polymorphisms and complex disorders: A study among mendelian population with East Asian Ancestry


Salam Kabita1, Priyanka Rani Garg1, Masan Kambo Newmei1, Kallur Nava Saraswathy1, Huidrom Suraj Singh2,  
1 Department of Anthropology, University of Delhi, Delhi, India
2 Department of Anthropology, Manipur University, Imphal, Manipur, India

Correspondence Address:
Dr. Huidrom Suraj Singh
Department of Anthropology, Manipur University, Canchipur, Imphal - 795 003, Manipur
India

Abstract

Background and Objectives: Tumor necrosis factor-alpha (TNF-α)-238G/A and -308G/A single-nucleotide polymorphisms (SNPs) in the promoter region of the gene have been implicated in numerous diseases. The present study aims to investigate the frequency of two TNF-α polymorphisms among Mendelian population of India and to assess their association with various cardiovascular risk variables and outcomes (hypertension [HTN], metabolic syndrome [MetS], and type 2 diabetes mellitus). Materials and Methods: A total of 1142 unrelated individuals aged 35–75 years belonging to Meitei community of Manipur were included in the study. Height, weight, waist and hip circumferences, blood pressures (systolic and diastolic), and lipid profile were measured. Polymerase chain reaction was done using standard protocols. Further, HTN, MetS, diabetic, and healthy individuals were identified, and a nested case–control study design was formulated. Results: The mutant allele frequency was found to be similar (4%) for both the polymorphisms among the Meiteis of Manipur. No mutant homozygote of TNF-α-308G/A polymorphism was observed. No significant risk of the two polymorphisms was found with any of the disease groups in nested case–control analysis. However, TNF-α-308G/A polymorphism, but not TNF-α-238G/A polymorphism, was found to be associated with total cholesterol (TC), triglyceride (TG), and very-low-density lipoprotein (VLDL) in overall population only with TG and VLDL among the HTN cases. Conclusion: The association of TNF-α-308A allele with metabolic risk factors such as TC, TG, and VLDL suggests that though it may not increase the development of any of the diseases considered in the present study, it could possibly enhance the risk of human metabolic disorder. The absence of mutant homozygote among cases is suggestive of lethality of TNF-α-308A allele in double dose coupled with other environmental factors or in the presence of haplotype pairing with TNF-α-238A allele.



How to cite this article:
Kabita S, Garg PR, Newmei MK, Saraswathy KN, Singh HS. Tumor necrosis factor-alpha gene polymorphisms and complex disorders: A study among mendelian population with East Asian Ancestry.J Pract Cardiovasc Sci 2019;5:35-43


How to cite this URL:
Kabita S, Garg PR, Newmei MK, Saraswathy KN, Singh HS. Tumor necrosis factor-alpha gene polymorphisms and complex disorders: A study among mendelian population with East Asian Ancestry. J Pract Cardiovasc Sci [serial online] 2019 [cited 2019 Dec 12 ];5:35-43
Available from: http://www.j-pcs.org/text.asp?2019/5/1/35/257605


Full Text



 Introduction



Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that plays a crucial role in the regulation of inflammatory and immune responses.[1] TNF-α is predominantly produced by monocytes/macrophages and, in turn, is a strong activator of phagocytic cells.[2],[3] The TNF-α-gene is located on the short arm of chromosome 6, in the class III region, within the major histocompatibility complex in a position defined as 250 kb centromeric to HLA-B locus and about 850 kb telomeric to HLA-DR locus [Figure 1].[4]{Figure 1}

Several TNF promoter polymorphisms have been identified and implicated in the regulation of TNF transcription.[5],[6] Single-nucleotide polymorphisms (SNPs) at the positions -308 (rs1800629) and -238 (rs361525) of the promoter region of the TNF gene have been commonly studied. Both polymorphisms are G→A substitutions, and changes they introduce can alter the transcription-binding site and affect the transcription rate.[6] Indeed, -308 polymorphism affects gene expression with a rare allele, resulting in higher in vitro TNF production.[7] As well, the rare TNF-α-238A allele has been associated with high TNF production.[8] Because both promoter polymorphisms have been associated with the transcriptional enhancement rate, it is possible that when acting in cis, these two markers show even stronger interaction.[6],[9] The G to A SNPs, at position -308 and -238 in the promoter of the TNF-α gene, have now been independently correlated with numerous complex diseases.[10],[11] The purpose of the present study is to investigate the frequency of the two mentioned TNF-α polymorphisms among a Tibeto-Burman-speaking Mongoloid population of India and to investigate their association with various cardiovascular risk variables (waist circumference [WC], body mass index [BMI], waist-to-hip ratio [WHR], total cholesterol [TC], triglyceride [TG], low-density lipoprotein [LDL], high-density lipoprotein [HDL], very LDL [VLDL], and fasting glucose level) and outcomes (hypertension [HTN], metabolic syndrome [MetS], and type 2 diabetes mellitus [T2DM]).

 Materials and Methods



In this cross-sectional study, a total of 1142 (625 males and 517 females) unrelated individuals aged 35–75 years were included. All included people were Meiteis of Manipur, Northeast India [Figure 1]. Meitei is a major ethnic nontribal group of Manipur and a Tibeto-Burman-speaking (Grierson, 1903) Mongoloid population. Each individual gave written informed consent before participating in the study, and the present study was approved by the Departmental Ethics Committee, Department of Anthropology, University of Delhi. Anthropometric, physiological, and clinical data were collected through a household survey. An additional fasting 5 ml intravenous blood sample was collected for biochemical tests and genomic DNA isolation. Genomic DNA was isolated using salting out method.[12] Polymerase chain reaction analysis was used to identify G/A at position -308 and -238 in the promoter region of TNF-α using a standard protocol.[13],[14] A total of 1075 samples could be genotyped for TNF-α-308G/A, and 1086 samples could be genotyped for TNF-α-238G/A polymorphisms. Of a total 1142 individuals, HTN, MetS, and diabetic (T2DM) individuals were identified, and a nested case–control study design was formulated: 323 had HTN, 128 had metabolic syndrome, 83 had diabetes, and 397 were healthy controls (without any kind of complex disease).

Anthropometric measurements such as height, weight, WC, and hip circumference were collected using the method described by Singh and Bhasin.[15] BMI was calculated as weight in kilograms divided by height in meter squared. WHR was calculated as WC divided by hip circumference. Blood pressure (BP) was measured using a mercury column sphygmomanometer. BP was measured twice after 5 min of rest in the sitting position, and the average of the measurements was recorded. HTN was defined as systolic BP ≥140 mmHg and/or diastolic BP ≥90 mmHg or those who are on anti-HTN medications.[16] T2DM was diagnosed by a history of previously known disease. High TC was defined as ≥200 mg/dl; TGs ≥200 mg/dl; LDL cholesterol as >150 mg/dl; HDL cholesterol as <35 mg/dl for males and <45 mg/dl for females; VLDL as >40 mg/dl, and fasting blood glucose >105 mg/dl.

Diagnosis of the metabolic syndrome in the present study was made using the International Diabetes Federation definition,[17] and the presence of metabolic syndrome was confirmed when a person has central obesity: WC ≥90 cm for men and ≥80 cm for women plus any of the two following four factors – (1) TG level ≥150 mg/dl or specific treatment with this lipid abnormality; (2) HDLC: <40 mg/dl for men and <50 mg/dl for women or specific treatment for this lipid abnormality; (3) raised BP: arterial BP ≥130/85 mmHg or on anti-HTN medication; and (4) raised fasting plasma glucose ≥100 mg/dl or previously diagnosed T2DM.

Statistical analysis

Genotype and allele frequencies were calculated by gene counting method using POPGENE software. Linkage disequilibrium and haplotype frequencies were determined using SNPstats. Comparisons between genotypes for both TNF-α-308G/A and -238G/A polymorphisms were carried out using t-test. The prevalence of cardiovascular risk was assessed from genotype groups, and the associated relative risk was calculated for both the polymorphisms. Genotype and allele frequencies for both cases (HTN, MetS, and T2DM) and controls were calculated. Haplotype frequency, linkage disequilibrium (LD), odds ratio (OR), and 95% confidence interval (CI) were calculated for both the polymorphisms among HTN, MetS, and T2DM cases and controls using freely available software SNPstats [18] and OR calculator,[19] respectively. P < 0.05 was considered statistically significant. Multivariate analysis was performed controlling confounding factors. Data analysis was done using SPSS software version 15.0 SPSS for Windows (SPSS Inc., Chicago, IL, US).

 Results



Both the TNF-α polymorphisms (-308G/A and -238G/A) were found to be polymorphic in the population under study. Both the polymorphisms were found to follow Hardy–Weinberg equilibrium. Frequencies of heterozygote were found to be almost similar for the two polymorphisms (7.54% for -308G/A and 7.27 for -238G/A). Mutant homozygotes of -238G/A were relatively high (0.28%) as compared to -308G/A (0.09%). However, mutant allele frequency of both the polymorphisms was found to be similar (4%) [Table 1]. The two SNPs were found to be in LD though not statistically significant (D′ = 0.847, P ≥ 0.05).{Table 1}

When the mean values of various cardiovascular variables were assessed with respect to the genotypes of the two polymorphisms, it could be seen that there was a significant increase in WC (in males), WHR (borderline significance in females), TC, TGs, and VLDL in the individuals carrying “A” allele of TNF-α-308G/A polymorphism. However, no such difference was observed for TNF-α-238G/A polymorphism [Table 2]. A similar trend could be noted for the prevalence of cardiovascular risk factors such as TC and TG, which were found to be significantly high in individuals carrying “A” allele of TNF-α-308G/A.{Table 2}

The prevalence of other cardiovascular risk factors such as WC, BMI, LDL, VLDL, MetS, and HTN was found to be relatively higher among individuals carrying the mutant allele of either -308G/A or -238G/A polymorphism than those carrying the normal allele. Individuals carrying mutant allele “A” of -238G/A polymorphism had relatively higher blood sugar level as compared to those carrying normal allele [Table 3].{Table 3}

Further, a nested case–control design was framed where the cases of HTN, MetS, and T2DM were segregated on the basis of available disease profile of the individuals and were compared with healthy individuals (free from any established diseases). The role of the two TNF-α polymorphisms in the pathogenesis of these complex diseases was assessed.

For TNF-α-308G/A polymorphism, heterozygote GA was found to be higher in HTN (7.82%) and metabolic syndrome (8.87%) cases than the controls (6.37%); the difference was not statistically significant. No mutant homozygotes of TNF-α-308G/A could be observed among cases; however, 0.27% were found among controls. The heterozygote GA of TNF-α-238G/A polymorphism was found to be higher in cases of HTN (9.18%), MetS (9.68%), and diabetes (7.5%) than that of controls (6.63%), but the difference was not found to be statistically significant. The mutant homozygote AA was completely absent in HTN and metabolic syndrome cases, whereas 1.25% could be observed among diabetic cases and 0.53% among controls. The mutant allele frequency of both the TNFs was found to be similar in all the cases (4% for -308G/A and 5% for -238G/A) [Table 4].{Table 4}

On calculating the OR for TNF-α-308G/A polymorphism, one-fold increased risk was observed with HTN – 1.19 (95% CI: 0.67–2.14) and metabolic syndrome – 1.37 (95% CI: 0.65–2.87), though not significant, and OR for diabetes was 0.92 (95% CI: 0.34–2.49). For TNF-α-238G/A polymorphism, ORs for HTN, metabolic syndrome, and diabetes were 1.31 (95% CI: 0.75–2.27), 1.06 (95% CI: 0.52–2.16), and 1.24 (95% CI: 0.52–2.96), respectively [Table 5]. No significant increased risk could be attributed to the two polymorphisms with respect to the disease groups even in the multivariate analysis (after controlling for probable confounding variables).{Table 5}

On assessing the association of various cardiovascular variables with the two polymorphisms, significantly high levels of TG and VLDL were found among HTN individuals carrying “A” allele of -308G/A polymorphism. Further, we calculated OR to evaluate the risk of these lipid variables in 308A allele carrying HTN individuals. More than two-fold significant increased risk could be attributed to TG and VLDL in HTN individuals carrying 308A allele (OR: 2.65; 95% CI: 1.03–6.82 and OR: 2.58; 95% CI: 1.00–6.61 for TG and VLDL, respectively). Among the MetS individuals, men carrying mutant allele “A” were found to have higher WC showing borderline significance (P = 0.06) [Table 6]. TNF-α-238G/A polymorphism was not found to be significantly associated with any of the selected parameters with respect to HTN, metabolic syndrome, or T2DM [Table 7].{Table 6}{Table 7}

On calculating the haplotype frequencies of both the polymorphisms with respect to HTN, MetS, and T2DM, one-fold increased risk with mutant allele combination of either of the SNPs was observed, though the risk was not significant [Table 8]. Haplotype combination with both A (mutant) alleles of the two SNPs was found to be in the lowest frequency among HTN (0.0016) and MetS cases (0.0021), but was absent among the controls.{Table 8}

 Discussion



TNF-α-308A and -238A alleles are normally found throughout the world in very low frequencies. In the present study, the frequency of mutant A allele for both polymorphisms was found to be 4%. For TNF-α-308G/A polymorphism, the present study documents slightly higher frequency of A allele as compared to Mongoloid population of Japan (0.8% among Japanese).[20] However, the frequency of -308A allele in Asians is considerably lower than that in European Caucasians ranging from 12% to 18.9% (Finnish populations had 12%; French had 15.7%; and British had 18.9%).[21],[22],[23] In India, North Indian populations are reported to have a frequency of 8.5%,[24] which is higher than that found in the present study, whereas South Indian populations are reported to have a comparatively low frequency of 2.95%.[24] For -238A allele, the frequency was found to be lower than that of North India (6.25%) and South India (17.08%).[24]

The present study demonstrated that TNF-α-308G/A polymorphism is significantly associated with high WC (in males), WHR (borderline significance in females), TC, TG, and VLDL in general. The association of TNF-α-308G/A polymorphism with obesity in the present population is in accordance with the findings of Chang et al.[25] in the Taiwanese population. Significantly high levels of biochemical markers in individuals carrying A allele accord with the findings of Antonicelli et al.,[26] who also reported high plasma levels of biochemical ischemic markers among individuals carrying AA + AG TNF-α-308 genotype. The A allele of the TNF-α-308G/A gene increases TNF-α transcription activity and also serum TNF-α levels. This allele has also been associated with obesity, T2DM, coronary artery disease, serum C reactive protein, and insulin resistance.[27] In a meta-analysis,[27] the authors suggested that A allele of the TNF-α-308G/A gene increases essential HTN susceptibility. The present study did not find any association of this polymorphism with any of the complex disorders under study. Therefore, this polymorphism did not contribute to the development of neither HTN nor T2DM or MetS in the Meitei community of Manipur. However, TNF-α-308G/A polymorphism is found to be associated with other environmental factors such as WC (in males), and TG and VLDL in general and also with respect to MetS and HTN, respectively. Moreover, both HTN and MetS are complex disorders and their expression is, therefore, controlled by both genetic and environmental factors. The inconsistent association of this polymorphism with these diseases may be due to the reason that most of the studies do not consider most of the environmental factors, and therefore, there are possibilities that variation at this locus might have modest effects on the complex conditions considered in the study, but environmental factors may mask the effects of this variation. Moreover, this polymorphism may not have a direct role in the pathogenesis of HTN, MetS, or T2DM; however, its association with high WC, TG, and VLDL is indicative of its effect when combined with these environmental factors. The absence of mutant homozygote (AA) of this polymorphism among cases is likely suggestive of its lethality in double dose coupled with other environmental factors. In a recent meta-analysis, Feng et al.[28] have reported no association between TNF-α-308G/A polymorphism and T2DM. However, they have pointed out that it is still unknown whether the lifestyle characteristics of different population influence the association between genotypes and T2DM. They realized that the unconsidered factors mixed together may cover the role of TNF-α-308G/A polymorphism and that even if the variation has a causal effect on T2DM, it may take a long time to be observed. In addition, interaction of the TNF gene with other pro- and anti-inflammatory cytokine genes plays an integrated role in the destruction of pancreatic beta cells and cytokines in the circulation interact with each other in the pathogenesis of diabetes. Therefore, it is not surprising that no influence of the TNF-α-308G/A polymorphism was found in susceptibility to T2DM as suggested by Feng et al.[28]

A meta-analysis on relation between TNF-α-308G/A polymorphism and metabolic syndrome indicated that individuals carrying TNF A allele had significantly higher fasting insulin level, systolic arterial BP, higher risk of developing obesity, but no significant association with BMI, WHR, and glucose and plasma leptin levels, suggestive of the increased risk of MetS with TNF A allele. Findings of the present study reveals that MeTS individuals carrying TNF-α-308A allele had abnormal lipid profile level, significantly higher WC (in males), high BMI and lower glucose levels while compared with individuals carrying TNF-α-308G allele. TNF A allele also associated significantly with high TG and VLDL among HTN cases in the present study. Such interaction of TNF-α-308 G/A polymorphism with other risk variables may be the reason for not finding the mutant homozygotes among patients as it is likely that individuals carrying this mutation, if also have abnormal metabolic risk profile, might not be able to survive, and therefore, it may be hard to capture such individuals and, hence, the mutation.

For TNF-α-238G/A polymorphism, Shiau et al.[29] found that participants carrying mutant genotype had significantly lower and higher plasma high-density lipoprotein-c and cholesterol levels, respectively. In a meta-analysis by Feng et al.,[30] they included a total of seven populations – Japanese, German, Korean, Finn, Chinese, Chilean, and UK.[29],[31],[32],[33],[34],[35],[36] In a study on TNF-α-238G/A in relation to the risk for T2DM, they did not detect any significant association of TNF-α-238G/A polymorphism on T2DM susceptibility, though there have been relatively few studies of the relationship between the -238G/A variation and T2DM. Walston et al.[37] also reported that TNF-α-238G/A polymorphism does not associate with traits related to obesity and insulin resistance. A case–control study by Sheu et al.[38] concluded that TNF-α promoter gene polymorphism at position -238 does not play a major role in the pathogenesis of insulin resistance in Chinese individuals with or without HTN. Another study from the Japanese population showed that polymorphism at -238 of the TNF-α promoter region did not relate to the insulin resistance syndrome.[39] In the present study, no significant association of this polymorphism with HTN, metabolic syndrome, or T2DM could be observed. This polymorphism was not associated even with any of the environmental risk factors. However, unlike TNF-α-308G/A polymorphism, individuals carrying minor allele of TNF-α-238G/A polymorphism had a higher prevalence of T2DM than those with normal allele, and T2DM patients had relatively higher minor allele frequency than the controls as also shown by Boraska et al.[40] in patients with T1DM.

In accord with the present findings, independent groups from the Japanese populations and one from Taiwanese population reported no association of these polymorphisms with insulin resistance syndrome.[39] A study by Sheu et al.[38] also showed that the two polymorphisms were not associated with HTN in the Taiwanese population.

In haplotype analysis of the two selected SNPs of the TNF, haplotypes of 308A and 238 A were very rare in HTN and metabolic syndrome individuals and were absent in T2DM patients, as also reported by Boraska et al.[40] in a study on T1DM patients from South Croatia. They provided a possible explanation that-308A -238A haplotype could produce some great impairment that could be lethal or semilethal to all individuals who carry this haplotype and that could be a reason for not observing it in the combined dataset. Moreover, the possibility of not observing -308A -238A haplotype could also be due to a simple chance event. Further, haplotype analysis showed that individuals with haplotypic combination having mutant allele of either or both the polymorphisms (i.e., either 308A 238G or 308G 238A haplotype) had increased risk of HTN, MetS, and T2DM, though not significant. Boraska et al.[40] also showed, with limited significance, the association of 308A and 238G haplotype in T1DM patients. The two polymorphisms are in weak LD in the present study as also shown by Boraska et al.[40] who showed no LD between these SNPs. Studies conducted till date have not looked into this aspect of the two polymorphisms with respect to the selected complex disorders.

 Conclusion



No association of the two TNFs with HTN, MetS, or T2DM could be found. However, the association of TNF-α-308A allele with other metabolic risk factors suggests that though it may not increase the development of any of the diseases considered in the present study, it could possibly enhance the risk of human metabolic disorder and other autoimmune diseases conferred by various cardiovascular risk factors. The absence of homozygous mutant genotype among cases indicates that the susceptibility may be increased when TNF-α-308A allele is present in double dose. Therefore, TNF-α-308G/A polymorphism may not have a direct role in the pathogenesis of the complex diseases, but the risk may exaggerate when TNF-α A allele is present in double dose coupled with other environmental factors or when present in haplotype pair with TNF-α-238A allele. Future studies are warranted to confirm these findings in larger numbers of participants considering the confounding risk agents.

Acknowledgments

We are very thankful to the University Grants Commission for providing funds to carry out this work. We are also very thankful to the participants and people of the Meitei community in Manipur.

Financial support and sponsorship

The study was funded by the University Grants Commission, Delhi, India.

Conflicts of interest

There are no conflicts of interest.

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