|Year : 2018 | Volume
| Issue : 1 | Page : 12-17
Association of proinflammatory cytokine tumor necrosis factor-α gene promoter -308 and -238 polymorphism with psoriasis in North Indian population
Wani Aadil1, Bashir Ahmad Ganai2, Tahseena Akhtar3, Tarun Narang4, Rajinder Kaur3
1 Department of Human Genetics, Punjabi University, Patiala, Punjab; Department of Biochemistry, University of Kashmir, Srinagar, Jammu and Kashmir, India
2 Center of Research for Development (CORD), University of Kashmir, Srinagar, Jammu and Kashmir, India
3 Department of Human Genetics, Punjabi University, Patiala, Punjab, India
4 Department of Dermatology, Venereology and Leprology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||10-May-2017|
|Date of Acceptance||11-Oct-2017|
|Date of Web Publication||12-Mar-2018|
Department of Human Genetics, Punjabi University, Patiala, Punjab
Source of Support: None, Conflict of Interest: None
Background/objective Tumor necrosis factor (TNF) is an important proinflammatory cytokine that plays a role in the pathogenesis of psoriasis. The aim of the present study was to investigate the role of TNF-α −308G/A and TNF-α −238G/A polymorphism, haplotype, and serum level in the pathogenesis of psoriasis.
Materials and methods A total of 200 psoriatic patients and 200 controls were genotyped for TNF-α −308G/A and TNF–α −238G/A polymorphism by using polymerase chain reaction. In addition, serum levels of TNF-α were measured by enzyme-linked immunosorbent assay.
Results Polymorphism of TNF–α −308 was found to be associated with a decreased risk for psoriasis odds ratio=0.29; (95% confidence interval=0.14–0.62), and polymorphism of TNF–α −238 was associated with an increased risk for psoriasis odds ratio=37.81; (95% confidence interval=12.77–112.01). HT2 GA haplotype was found to be associated with an increased risk for psoriasis. Moreover, the serum TNF-α level increased in patients compared with controls, with a significant correlation between serum TNF-α and psoriasis severity.
Conclusion Our findings suggested that TNF-α polymorphisms imparted significant risk toward the development of psoriasis in north Indian population.
Keywords: cytokine, polymorphism, psoriasis, tumor necrosis factor
|How to cite this article:|
Aadil W, Ganai BA, Akhtar T, Narang T, Kaur R. Association of proinflammatory cytokine tumor necrosis factor-α gene promoter -308 and -238 polymorphism with psoriasis in North Indian population. Egypt J Dermatol Venerol 2018;38:12-7
|How to cite this URL:|
Aadil W, Ganai BA, Akhtar T, Narang T, Kaur R. Association of proinflammatory cytokine tumor necrosis factor-α gene promoter -308 and -238 polymorphism with psoriasis in North Indian population. Egypt J Dermatol Venerol [serial online] 2018 [cited 2018 Jul 19];38:12-7. Available from: http://www.ejdv.eg.net/text.asp?2018/38/1/12/227093
| Introduction|| |
Psoriasis is known as chronic immune-mediated inflammatory skin disease affecting about 3% of the world’s population ,. Previous studies suggest that psoriatic skin is recognized by clinical presentation of red, scaly plaques containing numerous and dense infiltrates of immune cells that can produce many cytokines and inflammatory molecules ,,. Increasing knowledge about disease-specific immunologic abnormalities and the genetic control of immunoregulatory mechanisms such as the production of protective or harmful cytokines has been of great advantage in studying genetic factors associated with diseases like allergic asthma, rheumatoid arthritis, and lupus erythematosus ,. Such an approach may also be considered when investigating the genetics of psoriasis, the psoriatic inflammatory process being characterized by a disturbance of the local cytokine network with overexpression of proinflammatory mediators, such as tumor necrosis factor-α (TNF-α) ,. The genetic landscape of psoriasis is dominated by major histocompatibility complex HLA-Cw*0602 , and the genes encoding TNF-α ,. The TNF-α gene is located on the major histocompatibility complex region, which is only 250 kb centromeric from the human leukocyte antigen-B, showing its presence on the short arm of chromosome 6 (6p21.3). This region is polymorphic, with up to 44 polymorphisms reported . Two single-nucleotide polymorphisms involving GA transitions in the promoter region at the −238 and −308 sites have been shown to influence TNF-α expression and have been found associated with the development of psoriasis ,,,. On the basis of the above studies, we designed this study to investigate whether the polymorphisms of TNF-α −308G/A and TNF-α −238G/A and serum levels may be risk factors for the development of psoriasis in north Indian psoriatic patients and to establish possible allelic and genetic differences between them and healthy controls and its relation to TNF-α serum levels with the severity of psoriasis.
| Materials and methods|| |
This hospital based case–control study was conducted after approval by the institutional ethical committee and the participants were included only after they willingly decided to become part of the study. An informed consent form was filled and signed by all the participants. The study was conducted over the period of 15 months starting from July 2014 to October 2015 and included 200 clinically diagnosed (by dermatologist) psoriatic patients. Age (±7 years) and sex matched healthy participants from the same geographical region were included as controls.
Psoriasis Area and Severity Index calculation
Psoriasis Area and Severity Index (PASI) is a widely used tool for the measurement of severity of psoriasis. PASI score was calculated as described in the study by Langley and Ellis . A higher PASI score represents a greater degree of psoriatic severity .
A volume of 5 ml of venous blood were taken by trained laboratory technician, out of which 2 ml were taken in a sterile EDTA coated vials for genomic DNA extraction and the remaining 3 ml was centrifuged at 4000 rpm for 5 min. The serum was taken out and stored at −80°C till analysis.
Genomic DNA extraction
Genomic DNA was isolated from the blood samples by using the phenol–chloroform method  and the isolated DNA was stored at −20°C for future use.
Genotyping of tumor necrosis factor-α 308
For the detection of SNPs in TNF the amplification refractory mutation system-PCR method was applied, as described by Karma et al. . The primers forward: 5′-CTGCATCCCCGTCTTTCTCC-3′, reverse 1: 5′-ATAGGTTTTGAGGGGCATCG-3′, and reverse 2: 5′-ATAGGTTTTGAGGGGCATCA-3′ were used. PCR was performed at at 96°C for 3 min (initial denaturation) followed by 30 cycles at 96°C for 45 s (denaturation), 55°C for 80 s (annealing), and 72°C for 2 min (extension). A final extension step was carried out at 72°C for 3 min. PCR products (836 bp) were separated on 2% agarose stained with ethidium bromide and analyzed by using ultraviolet transilluminator. Positive amplification of one allele indicates homozygosity for that allele, whereas positive amplification of both alleles indicates heterozygosity of both alleles. To assess the success of PCR amplification in both reactions, an internal control was amplified using a pair of primers − forward primer: 5′-CCTTCCAACCATTCCCTTA-3′ and reverse primer: 5′-TCACGGATTTCTGTTGTGTTTC-3′ − from the nucleotide sequence of the human growth hormone.
Genotyping of tumor necrosis factor-α 238
The primer pair used for the amplification of TNF-α 238 was 5′-AAACAGACCACAGACCTGGTC-3′ (forward primer) and 5′-AGGATACCCACACTCCCCATCCTCCCGGATC-3′ (reverse primer) to assess polymorphism. The conditions for the TNF-α −238 polymorphism were as follows: denaturing step at 95°C for 5 min followed by 35 cycles at 95°C for 40 s, 61°C for 1 min, 72°C for 40 s, and finally 72°C for 10 min. The final PCR product of 165 bp was obtained and was digested by a restriction enzyme (BamHI at 37°C for 3 h; fermntas). Digested products were separated on 3% agarose gel. The digestion of the fragments (165 bp) revealed products of 123 bp and 42 bp (allele G) and 165 bp (allele A).
Measurement of serum tumor necrosis factor concentration
TNF concentration in the serum was determined by using a double antibody sandwich ELISA kit provided by Krishgen Biosystem (Whittier, California, USA), followed the manufacturer’s instructions by using standard curve.
Odds ratio (OR) with 95% confidence limits, calculated by using logistic regression, was used to assess the relationship between TNF-α genotypes and risk for psoriasis. A P value of less than 0.005 was taken as statistically significant. All the statistical calculations were done using SNPstat (China) software and VassarStats (USA).
| Results|| |
Out of 200 cases recruited in the present study, 130 (65%) were male patients and 70 (35%) were female patients. Their ages ranged from 18 to 70 years with a mean age of 38.61±13.713 years among the psoriatic patients, whereas among controls, the age ranged from 19 to 65 years with a mean age of 36.695±11.4765 years. Thirty-five (16%) psoriatic patients showed a positive family history of psoriasis. The majority of the cases, 57.5%, were from rural areas whereas 42.5% were from urban areas. The mean disease duration was 9.88±8.19 years. The mean BMI of psoriatic patients and controls was 26.94±4.17 and 24.80±4.28 kg/m2, respectively. The mean PASI score for clinical assessment in psoriatic patients was 10.654±9.09 ([Table 1]).
The allele and genotype frequencies of TNF-α −308 gene in patients and controls are summarized in [Table 2]. The allele A frequency was found to be higher in controls [25.75% (103/400)] as compared with cases [14% (56/400)] OR=0.469; [95% confidence interval (CI)=0.3273–0.6732]. It was observed that AA genotype was higher in controls [15.5% (31/200)] than in cases [7% (14/200)] OR=0.29; (95% CI=0.14–0.62). The polymorphism of TNF-α −308 was found to be associated with a decreased risk for psoriasis (P=0.0012). However, cases had a higher number of G allele, as well genotype GG, which indicates that carriers of this GG genotype are more likely to develop psoriasis. The dominant 0.40 (95% CI=0.23–0.68) and recessive 0.24 (95% CI=0.16–0.72) models show significant association of mutant allele A with a decreased risk for psoriasis. However, over dominant model does not show any association with the risk for psoriasis ([Table 2]).
|Table 2 Genotype and allele distribution of tumor necrosis factor-α −308 allele in psoriasis cases and controls|
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The allele and genotype frequencies of TNF-α −238 gene in patients and controls are summarized in [Table 3]. The allele A frequency was found to be higher among patients [51.50% (208/400)] as compared with controls [26.25% (105/200)] OR=3.043; (95% CI=2.162–4.049) P=0.001. It was observed that AA genotype was higher among patients [38.5% (77/200)] than among controls [2.5% (5/200)] OR=37.81; (95% CI=12.77–112.01) P=0.001, confirming that TNF-α −238 G/A is associated with an increased risk for psoriasis. The dominant OR=2.34 (95% CI=1.44–3.79) and recessive OR=39.63 (95% CI=13.83–113.54) models also showed significant association of mutant allele A with an increased risk for psoriasis ([Table 3]). SNP–SNP interaction did not show any modulating effect as the test of interaction was statistically nonsignificant (P=0.74) ([Table 4]). The haplotype frequencies of psoriasis patients and controls are summarized in [Table 5]. The pair-wise LD matrix demonstrated that the nearly complete LD (D>0<1) (D, between 0.20 and 0.45) existed between the polymorphism of position −308 and 238 within the TNF-α gene. The presence of four haplotypes with a frequency of more than or equal to 1 was estimated. The haplotypes accounted for 90% of all haplotypes in the pooled samples. Psoriasis patients had a significantly increased frequency of HT2 GA haplotype (P=0.001) OR=2.52 (95% CI=0.28–0.79), which confirms that HT2 GA haplotype is associated with an increased risk for psoriasis, and haplotype HT3 AG OR 0.47 (95% CI: 1.30–3.70; P=0.0045) shows a decreased risk for psoriasis among patients hailing from North India.
|Table 3 Genotype and allele distribution of tumor necrosis factor-α −238 allele in psoriasis cases and controls|
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|Table 4 SNP–SNP interaction between tumor necrosis factor-α 308 and tumor necrosis factor-α 238 polymorphism of tumor necrosis factor-α gene|
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|Table 5 Possible haplotype frequencies of tumor necrosis factor–α gene in cases and controls|
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TNF-α serum level in psoriasis patients were significantly elevated (225±22.82 pg/ml) as compared with controls (69.80±10.02 pg/ml). Statistically significant differences were observed after data analysis (P=0.02). In addition, we observed a significant correlation between the TNF-α serum level and disease severity PASI score (P=0.03) ([Table 6]).
|Table 6 Correlation between serum levels of psoriasis patients and control and its relation with Psoriasis Area and Severity Index|
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| Discussion|| |
Psoriasis is an inflammatory dermatosis that is multifactorial and has a strong polygenic genetic basis ,. TNF gene has been recognized as candidate gene in immune-mediated disorders including psoriasis ,. In the present study we determined the association of TNF-α −308 and TNF-α −238 cytokine polymorphisms and their serum levels with the severity of psoriasis in the North Indian population. Transition of single nucleotide from guanine (G) to adenine (A) at position −308 is the most common in general populations. This change has impact on the expression of TNF-α gene in which there is about six-fold increased transcriptional activity and higher levels of TNF-α . Therefore, the genetic polymorphisms in TNF-α gene can have some effects on the hosts’ susceptibility to psoriasis by the changed TNF-α expression. As regards TNF-α −308, our results demonstrated increase in the G allele and GG genotype in psoriasis patients compared with the control group. On the other hand, the increased frequency of A allele and AA genotype in the control group compared with the psoriasis patients’ group confirmed that this mutant genotype plays a protective role in psoriasis. In agreement with our results, some earlier studies have found a higher frequency of TNF-α −308 GG in moderate to severe psoriasis patients  or a higher frequency of the TNF-α −308G allele in patients with early-onset psoriasis ,. However, other investigators reported no difference in the distribution of TNF-α alleles or genotypes between patients and controls ,,. Our results substantiate early findings in which it has been reported that TNF-α −308 G/A polymorphism is associated with a decreased risk for psoriasis in both Caucasian and Asian populations ,. We observed that the mutant allele TNF-α −238A occurred more frequently in patients with psoriasis compared with controls, which confirms that TNF-α −238 G/A polymorphism is associated with an increased risk for psoriasis in north Indian population. In their study, Rahman et al.  revealed that TNF-α −238G/A polymorphism as a risk factor for psoriasis in Egyptian population. A meta-analysis by Zhuang et al.  suggested that TNF-α −238G/A is associated with a risk for psoriasis in different populations. In contrast to our results, some previous studies concluded that the −238GG genotype is more frequent among individuals with more severe forms of psoriasis . Some studies have observed these differences only in patients with early-onset psoriasis  or only in male patients . Our study also revealed elevated serum level of TNF-α in psoriasis patients when compared with the control group. Our findings are in agreement with many previously conducted studies ,,. Jacob et al.  found no differences in TNF-α levels between patients and controls. The difference in severity level (PASI) was found to be significantly correlated with serum TNF-α level (P=0.03), proving the role of these cytokines as main parameters for disease severity. The study by Takahashi et al.  revealed that serum level of TNF-α was markedly elevated in psoriatic patients as compared with healthy controls; elevated levels were also correlated with PASI. Interestingly, the HT2 GA haplotype of TNF gene was associated with an increased risk for psoriasis, which substantiates early finding by Weissensteiner et al. . In summary, TNF-α 308 G/A polymorphism plays a protective role in psoriasis and TNF-α 238 G/A polymorphism is associated with an increased risk for psoriasis; furthermore, the serum level was significantly increased in psoriasis patients compared with controls, with a correlation between serum levels and disease severity. Highlighting the role of cytokines in the pathogenesis of psoriasis is important for the development of a database, maintenance, and resolution of lesions. Therefore, anti-TNF therapy may be helpful in controlling the pathogenesis of psoriasis. However, because of a small sample size (a limitation of the study) we recommend future studies with a larger size to ascertain our findings.
The authors thank the Indian Council of Medical Research, New Delhi, India for providing financial support in the form of major research (No. 61/13/2011-BMS), sanctioned to Dr Rajinder Kaur, Department of Human Genetics, Punjabi University, Patiala, Punjab.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]