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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 34  |  Issue : 1  |  Page : 5-9

Tumor necrosis factor-α promoter gene polymorphism (308 G/A) in the Egyptian patients with systemic lupus erythematosus


1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Clinical and Chemical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Rheumatology and Rehabilitation, Faculty of Medicine, Benha University, Benha, Egypt

Date of Submission17-Feb-2016
Date of Acceptance20-Feb-2016
Date of Web Publication24-May-2017

Correspondence Address:
Walid A Abdel Halim
Department Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha 13512
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-208X.206895

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  Abstract 


Background
Tumor necrosis factor-α (TNF-α) is a very important cytokine having different physiological and pathogenic effects that lead to tissue destruction. The polymorphism of the TNF-α promoter gene at position –308 has been thought as a genetic risk factor for systemic lupus erythematosus (SLE). Thus, we aimed in this study to evaluate the association between TNF-α gene polymorphism (308 G/A) and SLE in the Egyptian population and its relation to the activity and the clinical manifestations of SLE.
Participants and methods
A case–control study was performed on 50 female patients with SLE (mean age, 32.4 ± 8.6 years) and 50 female healthy volunteers (mean age, 31.9 ± 8.3 years) served as controls. Genotyping was carried out by PCR. The PCR products were digested by NcoI restriction enzyme.
Results
TNF-α promoter gene polymorphism (rs1800629) GA and AA genotypes and A allele were significantly increased in the studied SLE patients when compared with healthy controls (P = 0.023 and 0.007, respectively). TNF-α genotypes showed significant differences between the various Systemic Lupus Erythematosus Disease Activity Index activity grades (P = 0.012). Moreover, GG genotype had a significantly lower incidence of renal disorders and persistent proteinuria when compared with AG or AA genotypes (P = 0.006 and 0.001, respectively). However, AA genotype showed a significantly higher incidence of neurologic disorders when compared with GG and AG genotypes (P < 0.001 and P = 0.026, respectively).
Conclusion
Our results indicate that TNF-α (–308 G/A) polymorphism may have a role in the susceptibility and pathogenesis of SLE in the Egyptian population.

Keywords: Egyptians, PCR–RFLP, polymorphism, systemic lupus erythematosus, tumor necrosis factor


How to cite this article:
Rageh IM, Sharaawy AA, Fouda AI, Abdelgawad ER, Abdel Halim WA. Tumor necrosis factor-α promoter gene polymorphism (308 G/A) in the Egyptian patients with systemic lupus erythematosus. Benha Med J 2017;34:5-9

How to cite this URL:
Rageh IM, Sharaawy AA, Fouda AI, Abdelgawad ER, Abdel Halim WA. Tumor necrosis factor-α promoter gene polymorphism (308 G/A) in the Egyptian patients with systemic lupus erythematosus. Benha Med J [serial online] 2017 [cited 2017 Sep 20];34:5-9. Available from: http://www.bmfj.eg.net/text.asp?2017/34/1/5/206895




  Introduction Top


Systemic lupus erythematosus (SLE) is a chronic and systemic autoimmune disease with a complex pathogenesis involving multiple genetic and environmental factors. The disease is characterized by autoantibody production, abnormalities of immune inflammatory system function, and inflammatory manifestation in several organs. However, the complete etiology of SLE is still unknown [1],[2]. Consistent with the systemic nature of SLE, the clinical manifestations of this disease are diverse, with the skin, joints, kidneys, nervous system, serosal surfaces, and blood elements prominently involved. These manifestations occur to a variable extent in the individual patient and their activity can change over time [3].

The association between SLE and inflammation emphasize the importance of cytokine network genes. Tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine, exerts a variety of physiological and pathogenic effects, including the activation of a cascade of inflammatory events, which lead to tissue destruction in autoimmune diseases [4],[5],[6]. It stimulates cytokine production, enhancing expression of adhesion molecules and neutrophil activation, and acts as a costimulator for T-cell activation and antibody production [7]. Several studies have analyzed the association of TNF-α genetic variants with susceptibility to and outcome of SLE, showing variable results in most of the cases [8].

A change in G-to-A single nucleotide polymorphism at position − 308, directly affects gene regulation and has been associated with altered transcriptional activity of TNF-α in various disorders [9],[10].

Thus, we aimed to study the association between TNF-α gene polymorphism (−308 G/A) and SLE in the Egyptian population and evaluate the relation among this polymorphism with the activity and the clinical manifestations of SLE.


  Participants and methods Top


We studied 50 Egyptian female patients with a mean age of 32.4 ± 8.6 years referred to the Rheumatology and Rehabilitation Department at Benha University Hospital between March and November 2014, and 50 healthy female volunteers with a mean age of 31.9 ± 8.3 years. The patients were diagnosed by a consultant rheumatologist according to the American College of Rheumatology (ACR) criteria. Disease activity was evaluated according to the Systemic Lupus Erythematosus Disease Activity Index (Modified SLEDAI 2k) [11]. The study was performed according to the principles approved by the local ethics committee of Faculty of Medicine, Benha University.

Genomic DNA was extracted from peripheral blood leukocytes by QIAamp DNA Mini Kit (Cat No. 51104; Qiagen, Valencia, California, USA), according to the manufacturer's protocol. Genotyping of the single nucleotide polymorphism in the promoter region of the TNF-α gene (−308 G/A) was carried out by PCR Taq PCR Master Mix Kit (Cat No. 201443; Qiagen), using primer sequences (forward primer: 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and reverse primer: 5′-TCCTCC CTGCTCCGATTCCG-3′). PCR was performed on an automated DNA thermal cycler (PERKIN ELMER-ConeAmp 2400, USA) under the following conditions: initial denaturation at 94°C for 3 min, 30 cycles of amplification consisting of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1 min and in the last cycle, extension was prolonged to 10 min. The PCR products were digested using 2 U/μl of NcoI restriction enzyme (Invitrogen, Carlsbad, San Diego, California, USA) in a total volume of 20 μl, containing 10 μl of PCR product in supplied buffer. The mixture was incubated at 37°C for 60 min. The NcoI restriction enzyme only digest the PCR product in the presence of G allele into two fragments (87, 20 bp), while leaving the A allele undigested (107 bp). The digested PCR product was fractionated on 3% agarose gel and visualized after staining by ethidium bromide.

Statistical analysis

The statistical analysis of data was done using Microsoft Excel program (Microsoft Office 2013) and statistical package for social science program, version 20 (SPSS Inc., Chicago, Illinois, USA). χ2-Test was used to compare groups. Deviations from Hardy–Weinberg equilibrium (HWE) expectations were determined using the χ2-test. GG genotype and G allele were considered as references. Odds ratio (OR) and 95% confidence interval (CI) were calculated.


  Results Top


The frequencies of the genotypes of −308 G/A of TNF-α in the SLE and control groups are shown in [Table 1] and [Figure 1]. This sample of individuals was selected randomly from the population in Kaliobyia Governorate in Lower Delta, Egypt. Applying HWE equation, revealed that TNF-α −308 (rs1800629) genotypes in both the cases and controls were independent (i.e. they are in HWE). In the SLE group, 64% (32/50) of the patients had the TNF-α G/G genotype, 26% (13/50) had the G/A genotype, and 10% (5/50) had the A/A genotype. In the control group, 84% (42/50) had the G/G genotype, 14% (7/50) had the G/A genotype, and 2% (1/50) had the A/A genotype. The statistical analyses showed that TNF-α (rs1800629) GA+AA genotypes and A allele were significantly increased in the studied SLE patients when compared with healthy controls (P = 0.023 and 0.007, respectively), with a higher risk to develop SLE (OR = 1.959, 95% CI = 1.097–3.501; OR = 3.020, 95% CI = 1.319–6.914, respectively). On the contrary, GA and AA genotypes did not show any significant differences between SLE patients and controls.
Table 1 Genotypic distribution and allelic frequencies of the TNF-α promoter gene polymorphism (−308 G/A) in patients with SLE and controls

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Figure 1 Distribution of TNF-α promoter gene polymorphism (−308 G/A) genotypes in SLE patients and healthy controls. SLE, systemic lupus erythematosus; TNF-α, tumor necrosis factor-α.

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When we analyzed the genotypic distribution among the different degrees of activity of SLE, TNF-α genotypes showed significant differences between various SLEDAI activity grades (P = 0.012). Severe cases had significantly lower GG and significantly higher AA genotypes (P = 0.008 and 0.010, respectively).

In our study, we compared between TNF-α genotypes regarding ACR criteria in studied SLE group. Patients with GG genotype had a significantly lower incidence of renal disorders and persistent proteinuria when compared with those with AG or AA genotypes (P = 0.006 and 0.001, respectively). However, patients with AA genotype showed a significantly higher incidence of neurologic disorders when compared with those with GG and AG genotypes (P < 0.001 and P = 0.026, respectively). No statistical differences were found between TNF-α genotypes regarding other ACR criteria for SLE (P > 0.05 for each).

We applied age, disease duration, anti-dsDNA, albumin/creatinine ratio, C3, C4 concentrations, and TNF genotypes as covariates. In univariate analysis, anti-dsDNA, albumin/creatinine ratio, TNF-α GA, and AA genotypes were significantly associated with a risk of increased SLEDAI score, whereas higher C3, C4 concentrations were significantly associated with a lower SLEDAI score. In multivariate analysis, taking those variables that showed significant associations in univariate analysis, anti-dsDNA, albumin/creatinine ratio, and TNF-α AA genotype were significantly associated with a risk of increased SLEDAI score, whereas higher C3 concentration was significantly associated with a lower SLEDAI score ([Table 2],[Table 3],[Table 4]).
Table 2 Comparison between TNF-α promoter gene polymorphism (–308 G/A) genotypes regarding SLEDAI grades in studied SLE group

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Table 3 Comparison between TNF-α promoter polymorphism (–308 G/A) genotypes regarding ACR criteria in studied SLE group

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Table 4 Ordinal regression analysis for prediction of activity of SLE (assessed by SLEDAI score)

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  Discussion Top


TNF-α, a proinflammatory cytokine, is known to be involved in the severity of various immune-regulated diseases as autoimmune, infectious, and malignant diseases [12]. Both TNF-α and lymphotoxin-α alleles have been related to SLE susceptibility in different ethnic groups [13].

The TNF-α (−308) A allele was reported in a higher frequency (up to 48%) among SLE European patients [14]. Furthermore, a meta-analysis confirmed the association of the A/A risk genotype (recessive model) and G/A+A/A genotypes (dominant model) with SLE susceptibility in the North-Europeans [15]. On the contrary, the TNF-α (−308) A allele was not associated with SLE susceptibility in the Asians [12], Black-Africans [15], African-Americans [16], Mexicans [17], and Italians [18].

Thus, in this study, we analyzed the TNF-α promoter gene (−308) polymorphism in the Egyptian SLE patients. TNF-α (rs1800629) GA+AA genotypes and A allele were significantly increased in studied SLE patients when compared with healthy controls (P = 0.023 and 0.007, respectively), with a higher risk to develop SLE (OR = 1.959, 95% CI = 1.097–3.501; OR = 3.020, 95% CI = 1.319–6.914, respectively). This was similar to that found by Angelo et al.[19], in the Brazilian population, who reported a statistical significant differences in the distribution of the TNF-α gene polymorphism between SLE patients and the control groups (P = 0.0001). Individual carriers of the variant allele A had a 3.29 (95% CI = 1.7738–6.1325)-fold increased risk for SLE. On the contrary, Lin et al.[20], in a study on Taiwanese patients, found that the allele and genotype frequencies of the polymorphisms at −308 were not significantly different (P = 0.749).

In our study, when we analyzed the genotypic distribution among the different degrees of activity of SLE, TNF-α genotypes showed significant differences between various SLEDAI activity grades, and these results were in accordance with a study performed by Santos et al.[21] in the Portuguese population who reported that SLE disease activity, measured by the SLEDAI, was higher among patients carrying the TNF-α (−308) A allele (P = 0.01). However, these results were in contrast to those of study performed by Angelo et al.[19], in the Brazilian population, who found no significant difference (P = 0.4131), when they analyzed the genotypic distribution among the different degrees of activity of SLE.

Various studies suggest an association between certain polymorphisms and specific SLE clinical features [22],[23],[24]. For instance, malar rash, discoid rash, photosensitivity, oral ulcers, serositis, and hematological disorders were found to be associated with the TNF-α (−308 G/A) polymorphism in the Taiwanese population [20]. Moreover, Santos et al.[21] demonstrated a significant increase in the development of nephritis in carrying A allele in Portuguese White patients. Angelo et al.[19], while studying Brazilian patients, observed an association between TNF-α (−308 G/A) gene polymorphism and serositis. In the present study, we found an association of the TNF-α (−308 G/A) gene polymorphism with both neurological and renal disorders. In contrast, many studies could not confirm the association between the TNF-α gene polymorphism (−308 G/A) and the development of various clinical manifestations in SLE patients [7],[13],[21],[25]. These controversial results could be due to the genetic heterogeneity of SLE in different ethnicities.

Thus, our findings suggest that the (TNF)-α −308 polymorphism may play an important role in the susceptibility and pathogenesis of SLE in the Egyptian population.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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