key: cord-0002271-mae0rxi2
authors: Al-Kholy, Wfaa; Elsaid, Afaf; Sleem, Aml; Fathy, Hend; Elshazli, Rami; Settin, Ahmad
title: TNF-α − 308 G > A and IFN-γ + 874 A > T gene polymorphisms in Egyptian patients with lupus erythematosus
date: 2016-06-04
journal: Meta Gene
DOI: 10.1016/j.mgene.2016.06.002
sha: 0a1f43c04e0e22fb6efbd94611920bc9680d7ae3
doc_id: 2271
cord_uid: mae0rxi2

BACKGROUND: Systemic lupus erythematosus (SLE) is associated with immunogenetic factors. This study was planned to test for the association of TNF-α − 308 and IFN-γ + 874 gene polymorphisms with susceptibility and severity of SLE in Egyptian cases. SUBJECTS AND METHODS: This is a case controlled study including 125 Egyptian cases with SLE in addition to 112 healthy unrelated individuals from the same locality. For all participants, TNF-α − 308 G > A and IFN-γ + 874 A > T genetic polymorphisms were characterized using the PCR technique. RESULTS: Cases with SLE showed a significantly higher TNF-α − 308 A allele carriage rate (AA + GA genotypes) compared to controls (26.4% vs. 12.5%, p = 0.009, OR = 2.51, 95% CI = 1.26–4.99). These cases showed also a significantly higher carriage rate for the IFN-γ + 874 T allele (AT + TT genotypes) compared to controls (47.2% vs. 32.1%, p = 0.02, OR = 1.89, 95% CI = 1.11–3.21). Comparing age, gender, and disease severity presented by nephritis class, activity and chronicity indices in cases carrying the TNF-α − 308 A allele and in cases carrying IFN-γ + 874 T allele versus others showed no significant difference (p > 0.05). CONCLUSIONS: TNF-α − 308 A and IFN-γ + 874 T allele carriage are associated with susceptibility but not severity of SLE in Egyptian subjects.

Systemic lupus erythematosus (SLE) is the prototypic autoimmune complex disease which is characterized by excessive production of autoantibodies against a broad range of self-antigens (Gurevitz et al., 2013) . Vital organs and tissues are often implicated, including kidney, brain, cardiovascular, joint and skin (Sánchez et al., 2006) . One of its most severe manifestations is lupus nephritis (LN), which remains a cause of substantial morbidity and mortality, either secondary to kidney disease, or as a result of intense immunosuppressive drug toxicity (Sestak et al., 2007; Hiraki et al., 2012; Pons-Estel et al., 2011) . Genes most likely contribute to the development of lupus have been identified particularly of the major histocompatibility complex (MHC) types HLA-A1, B8, and DR3 (Wakeland et al., 2001; Walport et al., 1982) . In addition, there is a strong evidence supporting the role of cytokine genes in the pathogenesis of SLE (Lin et al., 2009 ). One of the major cytokines that has been intensely investigated is the tumor necrosis factor alpha (TNF-α), an important pro-inflammatory cytokine that plays an important role in the inflammatory and immune responses (Aggarwal et al., 2012) . High serum levels of TNF-α have been reported in SLE that correlated well with the renal inflammatory activity (Studnicka-Benke et al., 1996; Aringer et al., 2002; Maury and Teppo, 1989; Postal and Appenzeller, 2011) . Effects of TNF-α were found to be related to activation of a cascade of inflammatory events, enhancing expression of adhesion molecules, activation of neutrophils in addition to acting as a costimulator for T cell activation and antibody production (Serrano et al., 2006) . The single-nucleotide polymorphism (SNP) TNFα −308 GN A (rs1800629) is located on chromosome 6p21.3, within the class III region of MHC. The TNF-α −308 A allele has been reported to be a stronger transcriptional activator in vitro than the common G allele. Therefore, the A allele frequency has been associated with the risk of autoimmune disorders (Zou et al., 2011; Santos et al., 2012) .

Interferon-gamma (IFN-γ) is one of the main cytokines secreted by activated T lymphocytes and has antiproliferative, antiviral and immunomodulatory activities as it controls the development of T helper 1 (Th1) cells (Silva et al., 1992; Torrico et al., 1991) . Several studies have indicated that expression of the IFN-γ level has significant effects into the susceptibility of various autoimmune diseases. There is a report showing significant increasing IFN-γ messenger ribonucleic acid expression in peripheral blood mononuclear cells of patients with SLE that might trigger inflammatory responses (Baccala et al., 2005; Meta Gene 9 (2016) 

Meta Gene j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m g e n e Csiszár et al., 2000) . The human IFN-γ gene located on chromosome 12q24.1 has a SNP in the first intron at its 5′ end, adjacent to a CA repeat region (+ 874 A NT, rs2430561) which was known to influence the secretion of IFN-γ and had been implicated in numerous autoimmune and chronic inflammatory conditions (Chong et al., 2006; Pravica et al., 2000) . Analysis of the biological role of this SNP suggested that + 874 T allele carriers showed higher production of IFN-γ (Pravica et al., 2000; López-Maderuelo et al., 2003) . The aim of this study is to investigate the association between TNF-α − 308 G N A and IFN-γ + 874 A NT gene polymorphisms with the SLE susceptibility and severity among Egyptian subjects.

This is a case controlled study involving 125 adult patients with SLE all suffering from LN (72 females and 53 males, age mean ± SD = 23.4 ± 4.4 years) and 112 healthy unrelated subjects (46 males and 66 females, age mean ± SD = 28.3 ± 6.2 years). Patients were recruited from the Urology and Nephrology Center, Mansoura University, Egypt. The diagnosis of SLE in patients was according to the American College of Rheumatology (1982) revised criteria for SLE (Tan et al., 1982) . Assessment of the severity of SLE was based upon the classification of LN (Weening et al., 2004) where class I: normal glomeruli (0 cases), class II: purely mesangial disease (8 cases), class III: focal proliferative glomerulonephritis (25 cases), class IV: diffuse proliferative glomerulonephritis (88 cases), and class V: membranous glomerulonephritis (4 cases). The study was approved by the ethical and scientific committees of Mansoura University. An informed consent was also obtained from all participants before the start of the study.

For all participants, genomic DNA from a peripheral blood was extracted and purified using the kit provided by Gentra Systems, USA. Typing of the TNF-α − 308 G NA (rs1800629) SNP and IFN-γ + 874 ANT (rs2430561) were performed using the polymerase chain reaction with amplification refractory mutation system (PCR-ARMS) as described by Perrey et al. (Perrey et al., 1999) and Pravica et al. (Pravica et al., 2000) respectively. The PCR amplification was carried out using Veriti® thermal cycler (Applied Biosystems, USA) in a 20 μl total volume containing about 100 ng of genomic DNA template, 2 pM of each primer, 2 mM of each dNTP, 1.5 mM of MgCl 2 , 1 × PCR buffer and 0.6 U of Taq DNA polymerase. TNF-α Primer sequences were as follows: TNF-α G forward primer: 5′-ATA GGT TTT GAG GGG CAT GG-3′; TNF-α A forward primer: 5′-AATA GGT TTT GAG GGG CAT GA-3′; and reverse primer, 5′-TCT CGG TTT CTT CTC CAT CG-3′. Reaction conditions were carried out in a thermocycler at 95°C for 1 min followed by 10 cycles of 95°C for 15 s, 65°C for 50 s, and 72°C for 40 s and then 20 cycles of 95°C for 20 s, 56°C for 50 s, and 72°C for 50 s. On the other hand, primer sequences for INF-γ were as follows: reverse primer: 5′-TCA ACA AAG CTG ATA CTC CA-3′, INF-γ A forward primer: 5′-TTC TTA CAA CAC AAA ATC AAA TCA-3′, INF-γ T forward primer: 5′-TTC TTA CAA CAC AAA ATC AAA TCT-3′. The PCR conditions consisted of an initial denaturation step at 95°C for 2 min, 10 cycles of incubation at 95°C for 15 s, 62°C for 50 s and 72°C for 40 s, followed by 20 cycles of incubation at 95°C for 20 s, 56°C for 50 s and 72°C for 50 s, with a final extension at 72°C for 5 min. The amplified PCR products were analyzed by 2% agarose gel and ethidium bromide staining followed by ultraviolet visualization. The PCR product for TNF-α − 308 was detected at 184 bp ( Fig. 1) while that of IFN-γ + 874 was detected at 263 bp (Fig. 2) .

Data were analyzed using the statistical package of social science (SPSS, version 17.0) software program. The frequencies of studied genotypic and allelic polymorphisms among cases were compared to those of controls using Fisher's exact test and odds ratio (OR) with the 95% confidence interval (95% CI). For statistical analysis, the significance of clinical parameters related to age, gender, SLE classes, severity index and chronicity index was determined by Fisher's exact test. Conformity with the Hardy Weinberg law of genetic equilibrium was tested by Chi square test comparing the observed versus expected genotype frequencies in both groups of cases and controls. A minimum level of statistical significance was considered at a p level of b0.05.

Cases with SLE showed a significantly higher TNF-α −308 (GA + AA genotypes) compared to controls (26.4% vs. 12.5%, p = 0.009, OR = 2.51, 95% CI = 1.26-4.99) (Table 1) . Similarly, cases showed a significantly higher carriage rate for the IFNG-γ + 874 (AT + TT genotypes) compared to controls (47.2% vs. 32.1%, p = 0.02, OR = 1.89, 95% CI = 1.11-3.21) ( Table 2) . This was mainly due to the higher frequency of the heterozygous TNF-α GA and INF-γ AT genotypes, consistent with the dominant mode of inheritance. On other hand, comparing the homozygous genotype frequency between cases and control (recessive model) did not yield any significant difference. The frequencies of TNF-α and IFN-γ genotypes were in agreement with Hardy-Weinberg equilibrium in both groups of SLE cases and controls (p N 0.05) (Tables 1 and 2). Cases of TNF-α GA + AA genotypes vs. those with the GG genotype (1 and 2) represent genotype GA, lanes (3 and 4) represent genotype GA, lanes (5 and 6) represent genotype GA, lanes (7 and 8) represent genotype GA, lanes (9 and 10) represent genotype GA, TNF-α −308 GNA was detected at 184 bp. and those having the INF-γ AT + TT genotypes vs. those with the AA genotype revealed no significant difference regarding their age, gender and nephritis class, activity and chronicity indices (p N 0.05) ( Table 3) .

This study tested the association of two important gene polymorphisms with SLE presenting mainly with nephritis. It showed clearly IFN-γ + 874 T and TNF-α − 308 A alleles are risk alleles for SLE in Egyptian subjects although apparently not affecting the clinical severity of the disease. In another study done among Egyptian SLE patients recruited from Upper Egypt, Ahmed et al. have found a high frequency of the TNF-α −308 A allele in the SLE patients but not in patients with nephritis (Ahmed et al., 2014) .

Recent meta-analysis has shown also that individuals carrying TNF-α −308 A risk allele were significantly associated with SLE in European, South American and Mexican subjects but was not associated with SLE in Asian and African subjects (Pan et al., 2012) . Similar conclusion regarding the association of TNF-α − 308 A allele was also found among Brazilian (Angelo et al., 2012) , Colombian (Guarnizo-Zuccardi et al., 2007) , Mexican (Jiménez-Morales et al., 2009) , North American (Parks et al., 2004) , Spanish (Suárez et al., 2005) , Asian (Lin et al., 2009) and Caucasian (van der Linden et al., 2001) patients. This might be explained by the fact that TNF-α is a pro-inflammatory cytokine involved in different immune-regulated diseases including autoimmune, infectious, and malignant ones (Lee et al., 2006) .

Regarding the other polymorphism, we could find that a few number of articles were done to analyze the relationship between the IFN-γ +874 ANT SNP and susceptibility to SLE. For instance, Kim et al. found an association between the + 874 A N T IFN-γ polymorphism and SLE patients in Korean patients (Kim et al., 2010) . This might be also explained by the fact that IFN-γ is an important cytokine produced in response to inflammatory stimuli to regulate the human immune response. It was believed that the pathogenesis of SLE involves the over-production of IFN-γ (Bennett et al., 2003) . It has been postulated that genetic polymorphisms affecting cytokine transcription exist in the regulatory regions of pro-inflammatory cytokine genes, and that such polymorphisms may control the level of inflammation (Baechler et al., 2003) . In addition, the positive association was found between the amino acid polymorphism (Val14Met) within the IFN-γ receptor 1 gene and SLE. Several studies have indicated that alterable expression of the IFN-γ level has significant effects to the susceptibility of various autoimmune diseases, particularly in SLE (Csiszár et al., 2000; Tanaka et al., 1999) . Nonetheless, other studies found contradictory results of no association of TNF-α − 308 G NA polymorphism with SLE among Argentinean (Muñoz et al., 2014) , Mexican (Zúñiga et al., 2001) , Japanese (Takeuchi et al., 2005) , Colombian (Tobón et al., 2005) , African Americans (Parks et al., 2004 ), Italian (D'Alfonso et al., 1996 , Chinese (Fong et al., 1996) , Thai and Portuguese (Santos et al., 2012) patients. In spite of the above evidence, we have encountered two studies stating a non-association of IFN-γ +874 T allele and SLE among Thai and Brazilian subjects (Tangwattanachuleeporn et al., 2007; da Silva et al., 2014) . (1 and 2) represent genotype AT, lanes (3 and 4) represent genotype TT, lanes (5 and 6) represent genotype AT, lanes (7 and 8) represent genotype AT, lanes (9 and 10) represent genotype TT, lanes (11 and 12) represent genotype AT, IFN-γ +874 ANT was detected at 263 bp. ⁎ p b 0.05 = significant. ** p b 0.01 highly significant. ⁎ p b 0.05 = significant. ** p b 0.01 highly significant.

These contradictory results might be attributed to different ethnic origins in addition to diverse genetic and epigenetic interactions with different environmental effects. The relatively small sample size might also present other limiting factor. Lastly, we can conclude that TNF-α − 308 G N A and IFN-γ +874 ANT gene polymorphisms might play a role in the susceptibility but not the severity and activity of lupus in Egyptian cases. However, taking into consideration the limiting factors, we recommend undertaking a wide-scale multicenter study into the entire genome sequence studying all cytokine gene polymorphisms and their expression status in cases of SLE.

Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey

Association between TNF promoter −308 GNA and LTA 252 ANG polymorphisms and systemic lupus erythematosus

Tumor necrosis factor alpha promoter polymorphism −308 G/A in Brazilian patients with systemic lupus erythematosus

Increased bioactive TNF in human systemic lupus erythematosus: associations with cell death

Interferons as pathogenic effectors in autoimmunity

Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus

Interferon and granulopoiesis signatures in systemic lupus erythematosus blood

The interferon gamma gene polymorphism + 874 A/T is associated with severe acute respiratory syndrome

Increased interferon-gamma (IFN-gamma), IL-10 and decreased IL-4 mRNA expression in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE)

Interferon gamma and Interleukin 10 polymorphisms in Brazilian patients with systemic lupus erythematosus

Association between polymorphisms in the TNF region and systemic lupus erythematosus in the Italian population

Polymorphism of the regulatory region of tumour necrosis factor alpha gene in patients with systemic lupus erythematosus

Cytokine gene polymorphisms in Colombian patients with systemic lupus erythematosus

Systemic lupus erythematosus: a review of the disease and treatment options

Prevalence, incidence, and demographics of systemic lupus erythematosus and lupus nephritis from 2000 to 2004 among children in the US Medicaid beneficiary population

Genetic susceptibility to SLE is associated with TNF-alpha gene polymorphism −863, but not −308 and −238, in Thai population

Tumor necrosis factor-alpha is a common genetic risk factor for asthma, juvenile rheumatoid arthritis, and systemic lupus erythematosus in a Mexican pediatric population

Interferon-gamma gene polymorphisms associated with susceptibility to systemic lupus erythematosus

Meta-analysis of TNF-alpha promoter −308 A/G polymorphism and SLE susceptibility

Association of TNF-alpha gene polymorphisms with systemic lupus erythematosus in Taiwanese patients

Interferon-gamma and interleukin-10 gene polymorphisms in pulmonary tuberculosis

Tumor necrosis factor in the serum of patients with systemic lupus erythematosus

4G/5G plasminogen activator inhibitor-1 and −308 A/G tumor necrosis factor-α promoter gene polymorphisms in Argentinean lupus patients: focus on lupus nephritis

Association of TNF-α promoter-308 A/G polymorphism with susceptibility to systemic lupus erythematosus: a meta-analysis

Genetic polymorphisms in tumor necrosis factor (TNF)-alpha and TNF-beta in a population-based study of systemic lupus erythematosus: associations and interaction with the interleukin-1alpha-889 C/T polymorphism

ARMS-PCR methodologies to determine IL-10, TNF-alpha, TNF-beta and TGF-beta 1 gene polymorphisms

Epidemiology and management of refractory lupus nephritis

The role of tumor necrosis factor-alpha (TNF-α) in the pathogenesis of systemic lupus erythematosus

A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma production

Association study of genetic variants of pro-inflammatory chemokine and cytokine genes in systemic lupus erythematosus

TNF promoter −308 GNA and LTA 252 ANG polymorphisms in Portuguese patients with systemic lupus erythematosus

Non-HLA associations with autoimmune diseases

Current status of lupus genetics

Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection

Tumour necrosis factor alpha and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus

Differential effect of IL10 and TNF{alpha} genotypes on determining susceptibility to discoid and systemic lupus erythematosus

Genetic contribution of the tumour necrosis factor (TNF) B + 252*2/2 genotype, but not the TNFa,b microsatellite alleles, to systemic lupus erythematosus in Japanese patients

The 1982 revised criteria for the classification of systemic lupus erythematosus

Association of the interferon-gamma receptor variant (Val14Met) with systemic lupus erythematosus

Association of interferon-gamma gene polymorphism (+ 874A) with arthritis manifestation in SLE

Lack of association between TNF-308 polymorphism and the clinical and immunological characteristics of systemic lupus erythematosus and primary Sjögren's syndrome

Endogenous IFN-gamma is required for resistance to acute Trypanosoma cruzi infection in mice

Six microsatellite markers on the short arm of chromosome 6 in relation to HLA-DR3 and TNF-308A in systemic lupus erythematosus

Delineating the genetic basis of systemic lupus erythematosus

The immunogenetics of SLE

The classification of glomerulonephritis in systemic lupus erythematosus revisited

Metaanalysis of TNF-α promoter − 308A/G polymorphism and SLE susceptibility in Asian populations

Tumor necrosis factor-alpha promoter polymorphisms in Mexican patients with systemic lupus erythematosus (SLE)

Authors are grateful for the Staff members of Rheumatology Department, Mansoura University Hospital, Egypt for their help in selecting cases under the study.

The authors declare that they have no conflict of interest. 3.7 ± 0.6 3.5 ± 0.4 0.48 3.7 ± 0.5 3.9 ± 0.7 0.72 Activity index (M ± SD) 10.7 ± 4.6 11.4 ± 10.8 0.73 11.1 ± 4.2 17.2 ± 25.9 0.12 Chronicity index (M ± SD)1.2 ± 0.9 2.8 ± 13.6 0.47 1.6 ± 1.2 1.1 ± 0.9 0.23