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A promoter polymorphism in rheumatoid arthritis
The Egyptian Rheumatologist (2015) 37, 49–54
H O S T E D BY
Egyptian Society of Rheumatic Diseases
The Egyptian Rheumatologist www.rheumatology.eg.net www.elsevier.com/locate/ejr
ORIGINAL ARTICLE
Clinical significance of serum TNFa and -308 G/A promoter polymorphism in rheumatoid arthritis Tamer A. Gheita a, Ghada S. Azkalany a b
a,*
, Wafaa Gaber a, Abeer Mohey
b
Rheumatology Department, Faculty of Medicine, Cairo University, Egypt Clinical Pathology Department, Faculty of Medicine, Cairo University, Egypt
Received 15 June 2014; accepted 1 July 2014 Available online 4 November 2014
KEYWORDS Rheumatoid arthritis; TNF-a -308 G/A promoter polymorphism; Tumor necrosis factor (TNFa)
Abstract Aim of the work: To evaluate the clinical significance of serum levels of tumor necrosis factor alpha (TNFa) and -308 G/A promoter polymorphism in rheumatoid arthritis (RA) patients. Patients and methods: We studied 43 RA patients and 30 controls. Demographic, clinical and serological data were prospectively evaluated. Disease activity score (DAS28) and the Health Assessment Questionnaire (HAQ) were assessed. Serum TNF-a level was measured and promoter (-308 G/A) genotyped. Results: Serum TNF-a level was significantly higher in the RA patients compared to controls ( p = 0.036) and was significantly higher in those with AA promoter polymorphism who had a significantly younger age at disease onset. In the multivariate analysis, disease duration would predict the TNFa level ( p = 0.006) while age at disease onset, DAS28 and HAQ would predict the TNFa polymorphism ( p = 0.004, p = 0.04 and p = 0.03 respectively). A significant negative correlation was present between TNFa level with age ( p = 0.001) and age at disease onset ( p < 0.0001) while in those with GA genotype a significant negative correlation was present with DAS28 (r = 0.66, p = 0.038). Conclusion: Serum TNFa levels and -308 G/A promoter polymorphism were higher in RA patients than in controls and could be predicted by disease activity and HAQ. 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.
1. Introduction Cytokine unbalance is responsible for the pathogenesis of diverse inflammatory, autoimmune and infectious diseases [1]. Rheumatoid arthritis (RA) is a model of multigenic * Corresponding author. E-mail address: [email protected] (G.S. Azkalany). Peer review under responsibility of Egyptian Society of Rheumatic Diseases.
inflammatory disorder in which tumor necrosis factor-alpha (TNF-a) plays an important and central role in its etiology and pathogenesis [2,3]. Overproduction of TNF-a is thought to be the main contributor to increased ROS release in RA patients, leading to tissue damage associated with the inflammation [4]. Genetic factors may be implicated in the susceptibility to disease initiation and in the severity of its course [2,3]. TNF-a and gene expression are the key regulators of inflammation and critical for RA [5]. It is substantiated that RA is favored by the presence of the -308 TNF promoter
http://dx.doi.org/10.1016/j.ejr.2014.07.001 1110-1164 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.
50 polymorphism which is responsible for the increased TNF production [2]. There are, however, controversial reports that TNF-a promoter -308 A/G polymorphism may be an independent marker of RA susceptibility and severity [6–8]. The TNF gene is located on chromosome six within the major histocompatibility complex (MHC). Mutation affects the -308 promoter region of the TNF gene which defines the TNF1 and TNF2 alleles, determining low and high levels of TNF expression, respectively [1,2]. The TNF-a -308 polymorphism in the promoter region with the TNF2 allele was found in almost a quarter of RA patients and 10% of normal subjects [9]. It has been linked to increased susceptibility and severity in a variety of autoimmune and inflammatory disorders, including RA, systemic lupus erythematosus (SLE) and inflammatory bowel disease. Significant impact of -308 promoter polymorphism is the increased TNF production and this difference does not appear to be due to other genes lying within the MHC region [1,2,9]. Moreover, there is a great deal of evidence that points to the association of the TNF-a and/or its gene -308 G/A promoter polymorphism with pathogenesis and involvement in the disease susceptibility and severity of SLE [9,10] and JRA [11–13]. Polymorphisms affecting the TNFa gene and its regulatory regions are associated with RA prevalence and course. There is a possible association between these polymorphisms and the clinical response to the use of anti-TNFa antibodies. The possibility that the determination of genotype -308 may have prognostic and therapeutic consequences was argued [14]. The introduction of anti-TNF agents has improved the outcome for many patients with RA and the only replicated genetic predictor of anti-TNF response is the -308 G/A single-nucleotide polymorphism in the TNF promoter region. The presence of -308 TNF G/G genotype appears to be a marker of good response to anti-TNF treatment [15]. Meanwhile, a meta-analysis showed that the TNF-a -308 A/G polymorphism is not associated with responsiveness to TNF blockers in RA patients [16]. The aim of the present study was to evaluate the clinical significance of serum levels of tumor necrosis factor alpha (TNFa) and -308 G/A promoter polymorphism in rheumatoid arthritis (RA) patients and find any association with age at disease onset, clinical and laboratory features and disease activity. 2. Patients and methods Forty-three rheumatoid arthritis patients with definite RA diagnosed according to the 2010 ACR/EULAR RA classification criteria [17] were consecutively recruited from the Rheumatology outpatient clinic and the department of Cairo University Hospitals. Full history taking, thorough clinical examination, laboratory and plain X-ray investigations of the affected joints were performed for all the patients. All patients were regularly receiving methotrexate with or without corticosteroids or chloroquine. Disease activity score in 28 joints (DAS-28) was calculated [18]. The Health Assessment Questionnaire-II (HAQ-II) was used [19]. Radiological grading of the hands and feet of the studied RA patients was assessed and percentage damage was calculated according to the modified Larsen score [20]. The study was approved by the local university ethics committee and the study conforms
T.A. Gheita et al. to the provisions of the Declaration of Helsinki in 1995. All patients gave their informed consent prior to their inclusion in the study. Serum TNF-alpha was assayed using a Human TNF-alpha ELISA kit (quantitative sandwich enzyme immunoassay technique), provided by Ray Biotech, Inc. 2.1. Determination of TNF-a gene (-308) promoter region polymorphism Promoter region polymorphism of TNF-alpha gene (-308 G/A) was determined by the polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) technique. 2.2. DNA isolation and TNF-alpha genotyping Genomic DNA was isolated from white blood cells (WBCs) of peripheral blood using a Biospin Whole Blood Genomic DNA Extraction Kit (Bioflux Corporation, Arrow Plaza, Tokyo, Japan). Enzymatic amplification was performed by a PCR using the Master Taq polymerase enzyme and Hybaid thermal cycler (Promega Corporation, 2800 Woods Hollow Road Madison, W 153711-5399 USA). Amplification of the promoter region (-308 G/A) of the TNF-alpha gene was performed as proposed by Dalziel et al. [21] using 2 primers which were purchased from Operon Biotechnologies (GmbH/ Biocampus, Germany). Forward: Primer 50 -AGGCAATAGGTTTTGAGGGCCA T-30 , and Reverse Primer: 50 -ACACTCCCCATCCTCCCTG CT-30 . The PCR reaction mixture (25 ll) contained 12.5 ll 2· PCR Master Mix {10· PCR buffer, 4 mM MgCl2, 0.5 Taq DNA polymerase/ll, and 0.4 mM dNTPs (dATP, dCTP, dGTP, dTTP)}, 1 ll of each primer (25 pmol), 3 ll of genomic DNA and 7.5 ll sterilized nuclease-free water. The reaction was carried out with the following cycles: 95 C for 2 min; 35 cycles of 95 C for 30 s, 60 C for 15 s and 74 C for 15 s; and 74 C for 10 min for final extension. Then amplified products were digested with 5 units Fast Digest NcoI restriction enzyme at 37 C for 10 min (supplied by Fermentas, LT02241 Vilnius, Lithuania).The digested products were then detected in 3.5% agarose gel containing ethidium bromide by performing E/P on the gel electrophoresis apparatus and visualized by UV transillumination (Promega, USA) for the identification of TNF-alpha gene (-308 G/A) promoter polymorphism. A single band at 107 bp identified AA homozygous individuals, two bands at 87 and 20 bp identified GG homozygous individuals, and three bands at 107, 87 and 20 bp indicated a heterozygote at the TNF-a -308 locus, while the 20 bp band was very small to be viewed on the gel. Statistical analysis: Data were collected, tabulated and analyzed by SPSS package version 15 (SPSS corporation, USA). Data were summarized as mean ± SD. Mann–Whitney test was used for the comparative analysis of 2 quantitative data. Non-parametric analysis of variance (Kruskal–Wallis) was used for the comparison of more than two groups; Spearman’s correlation analysis was used for the detection of the relation between 2 variables. Logistic regression analysis was applied to detect the predictors for the elevated TNF-a level and promoter polymorphism. Results were considered significant at p < 0.05.
Clinical significance of serum TNFa and -308 G/A promoter polymorphism
51
3. Results There were 43 RA patients (40 females and 3 males) and 30 age and sex matched healthy controls with a mean age of 43.53 ± 3.23 years and were all females with 2 males. The mean TNFa level for controls (5.57 ± 3.84 pg/ml) was significantly lower than the level in patients ( p = 0.036). The demographic, clinical and laboratory features of and medications used by the RA patients are presented in Table 1. All the patients were irregularly receiving diclofenac sodium (50–100 mg) while only three were receiving full dose of sulfasalazine (2000 mg/day). Three of the patients were diabetic and hypertensive with two of them having HCV which was also present in another two patients. Anti-cyclic citrullinated peptide (Anti-CCP) was positive in 74.42% of the patients. None of the patients gave family history for any rheumatic disease. There were 62.8% with GG TNFa (-308 G/A) gene promoter polymorphism, 23.2% with GA and 14% with AA while it was GG in all the controls. The TNFa was significantly higher in patients with AA (480 ± 621.6 pg/ml) compared to those with GG (9.98 ± 23.63 pg/ml) and GA (92.81 ± 48.58 pg/ml), ( p = 0.000) (Fig. 1). Furthermore, there was a significant difference in age at disease onset according to the three gene promoters ( p = 0.007) being older in those with GG (37.06 ± 15.21 years) compared to those with GA (24.7 ± 11.2 years) and AA (19.33 ± 11.93 years). There were no other significant differences in the characteristic features of the patients according to the promoter genes. A significant negative correlation was present between the TNFa level with age of the patients (r = 0.49, p = 0.001) and age at disease onset (r = 0.58, p < 0.0001). On using multiple linear regression analysis including age, age at disease onset and disease duration, only the disease duration would predict the TNFa level ( p = 0.006) while age at disease onset would highly predict the TNFa (-308 G/A) promoter polymorphism
Table 1 Demographic, clinical and laboratory features of the rheumatoid arthritis patients. Feature
RA patients (n = 43) Mean ± SD
Age (years) Disease duration (years) Morning stiffness (min) No. of swollen joints No. of tender joints VAS (mm) ESR (mm/1st h) Hemoglobin (g/dl) Platelets (·103/ml) TNFa (pg/ml) Modified Larsen score DAS28 HAQ
40.9 ± 15.5 9.2 ± 7.2 25.4 ± 37.6 2.1 ± 3.3 6.8 ± 8.1 36.2 ± 34 53.2 ± 24.04 11.2 ± 1.7 305.9 ± 90.2 94.8 ± 269.6 38.5 ± 16 4.6 ± 1.7 0.8 ± 0.8
Medications: Methotrexate (35/43) Prednisolone (19/43) Leflunomide (9/43) Chloroquine (22/43)
15.4 ± 8.4 3.8 ± 6.7 4 ± 7.9 125 ± 133.6
VAS, visual analog scale; ESR, erythrocyte sedimentation rate; TNFa, tumor necrosis factor alpha; DAS28, disease activity score in 28 joints; HAQ, health assessment questionnaire.
Figure 1 Box plot of the serum TNF-a levels in the RA patients and controls according to the TNFa (-308 G/A) promoter polymorphism type.
( p = 0.004). DAS28, HAQ or steroid dose could not predict the serum TNFa level while both DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism ( p = 0.04 and 0.03 respectively). No significant correlation was present with the other clinical and laboratory features or disease activity. However, in those patients with GA genotype of TNFa polymorphism there was a significant negative correlation of the serum TNF-a level with the DAS28 (r = 0.66, p = 0.038) (Fig. 2) which was only a tendency in those with AA genotype (r = 0.81, p = 0.05). There was a tendency for an association between the TNFa level and the modified Larsen score in the RA patients with GG genotype (r = 0.48, p = 0.058). There was a tendency for a higher TNFa in the four patients with HCV (463.03 ± 833.5 pg/ml) compared to those without (57.06 ± 98.7 pg/ml).
Figure 2 Correlation of the serum TNFa level and DAS28 in RA patients with GA -308 promoter polymorphism.
52 4. Discussion In the present study, there was a significant elevation in the serum TNF-a levels in the RA patients and this was associated with the increased polymorphisms in its gene promoter region. None of the controls in the present study had any promoter polymorphism. This is in agreement with the results of another study that found elevated TNF-a in the plasma and synovial fluid from RA patients and was associated with polymorphisms in the promoter region of the TNF-a gene. However, in disagreement to our results was their results that found no differences in genotype distribution and allelic frequencies of -308 G/A TNF-a promoter polymorphism between RA patients and controls [3,6]. Interindividual variation in the expression of TNF suggests the existence of functionally distinct TNF alleles including -308 promoter polymorphism that could play a role in RA susceptibility [22]. All the RA patients in the present study were Egyptian and the TNFa -308 A/G promoter polymorphism was considered as an obvious risk factor compared to the controls especially those with juvenile onset. None of the controls had the A allele and all had the G allele. In the present RA patients, the A allele was heterozygous in 23.26% and homozygous in 13.95%. It may be thus thought of the G allele as a protective from the development of RA in Egyptians. In a meta-analysis study, TNF-a -308 A/G polymorphism represented a significant risk factor for RA in Latin Americans, but not in Europeans which favors that different ethnic populations influence this association [7]. The -308 G/A polymorphism was significantly associated with a poor outcome in the Turkish juvenile idiopathic arthritis (JIA) patients ( p = 0.005) but there was no association in the Czech patients. This genetic variation may accompany the phenotypic differences found [23]. Again, analysis of the TNFa -308 AA allele showed increased frequency in severe RA when compared to nonsevere disease and to healthy controls. This association might be used as a prognostic marker for severe RA [24]. Contrarily, TNF -308A itself or a neighboring gene may be a protective factor for the development of RA in some of the population in Taiwan [25]. In another study, polymorphism TNF -308A was less prevalent among RA patients (1.7%) than controls and found that the A allele at -308 locus provides protection against RA in North Indians [26]. Again, another study found that the distribution of TNF-genotypes in RA patients did not differ from that in controls, suggesting that TNF-a -308 promoter polymorphism is not a genetic risk factor for RA susceptibility and severity [6,8,27]. A meta-analysis study showed that the TNF-a -308 A/G polymorphism may represent a significant risk factor for RA in Latin Americans but not in the European, Arab, or Asian populations [28]. Considering the relevance of TNF-a in the pathophysiology of JIA, it is likely that polymorphisms in its promoter area may be relevant in disease susceptibility and activity. Twenty-four percent of JIA Portuguese patients presented the -308 GA/AA genotypes and 76% the GG, similar to findings in controls thus conferring no association with disease susceptibility [29]. However, the -308 G/A polymorphism tends to be more frequent in the systemic onset rather than the oligoarticular JIA patients [30]. In the present study, there was no association of the TNF-a -308 promoter with the other clinical or laboratory
T.A. Gheita et al. manifestations of the RA patients. In agreement, the TNF promoter polymorphisms at position -308, were not related to the clinical manifestations of RA patients [25]. Moreover, there was also no correlation between TNF-a polymorphism and disease activity measures, including ESR, CRP, number of swollen and tender joints, and morning stiffness duration [6]. In the present study, a significant negative correlation was present between the TNFa level with age of the patients (r = 0.49, p = 0.001) and age at disease onset (r = 0.58, p < 0.0001). On using multiple linear regression analysis including age, age at disease onset and disease duration, only the disease duration would predict the TNFa level ( p = 0.006) while age at disease onset would highly predict the TNFa (-308 G/A) promoter polymorphism ( p = 0.004). There was no significant association of TNFa promoter variation at positions -308 and -238 with susceptibility to RA. However, a significant difference in the mean age at disease onset was found between -238 TNFa genotypes. In addition, a difference in the presence of nodular disease was observed between -308 TNFa genotypes suggesting that TNF a gene may play a role in the disease profile of RA patients [31]. Others found no association between TNF-a genotypes, age at disease onset, disease activity, and joint and extra-articular involvement [6]. In the present study, both DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism. Patients with GA genotype showed a significant negative correlation of the serum TNF-a level with the DAS28 (r = 0.66, p = 0.038) while there was a tendency for this association in those with AA genotype (r = 0.81, p = 0.05). TNF-a -308 GA/AA genotypes were found to be related to higher inflammatory activity and worse measures of disease activity in rheumatoid patients compared to those with GG genotype [29]. TNF promoter gene polymorphisms and TNF receptor gene heterogeneity play a significant role in non-response to biologic agents and disease course/severity [32]. In a study on active Chilean RA patients genotyped for -308 TNFa polymorphism who were treated by an anti-TNF-a agent, 88.2% of G/G and 68.4% of G/A genotypes were DAS28 responders and the serum TNFa levels of the G/A patients were lower than those of the G/G patients. A negative relationship occurred between DAS28 improvement and increased circulating TNFa levels [33]. The mean TNF-a level increased significantly with respect to basal levels in most of patients after anti-TNF-a therapy and was inversely related to the improvement in disease activity [34]. In JIA patients with the genotype -308 GG achieved an obvious improvement in disease activity at month 6 more frequently than those with the genotype -308 GA or AA [35]. Patients not responding to anti-TNF therapy showed an increased frequency of the A allele. The significant association between the TNF-a promoter -308 A/G polymorphism and responsiveness to anti-TNF therapy, suggests that RA patients who carry the A allele have a poorer response to therapy than those with the G allele [36,37]. Patients with a TNFa -308 G/G genotype are better responders to anti-TNFa treatment than those with A/A or A/G genotypes irrespective of the treated rheumatic disease (RA, psoriatic arthritis or ankylosing spondylitis) [38]. In a previous study including another TNF gene -863A allele it was suggested that it can be used as a genetic marker for SLE susceptibility and was associated with high TNF-a
Clinical significance of serum TNFa and -308 G/A promoter polymorphism production, Raynaud phenomenon, and nephritis in juvenile SLE patients [39]. Almost three quarters of the patients in the present study had positive anti-CCP. The TNF- 308A allele is less frequently found in rheumatoid factor negative but not in rheumatoid factor positive polyarthritis and may therefore be associated with a more severe disease, while the more common TNF 308G allele may be protective [12]. In this study, there was a tendency for an association between the TNFa level and the modified Larsen score in the RA patients with GG genotype. Although the introduction of anti-TNF antibodies has contributed to the slowing and arrest of RA progression, destruction itself has been generally acknowledged as an irreversible process with little hope in resolution [40]. Another study reported that erosive damage in patients with recent-onset RA is not influenced by the -308 TNF-a genotype [41]. In conclusion, serum TNFa levels and -308 G/A promoter polymorphism were higher in RA patients than in healthy controls and could be predicted by the disease activity and HAQ. Only the disease duration would predict the TNFa level while age at disease onset, DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism. To confirm our results we propose that larger scale, multicentre studies with longer evaluation periods are needed in selected RA patients using direct sequencing of the genotype.
[10]
[11]
[12]
[13]
[14]
[15]
Conflict of interest None.
[16]
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The Egyptian Rheumatologist www.rheumatology.eg.net www.elsevier.com/locate/ejr
ORIGINAL ARTICLE
Clinical significance of serum TNFa and -308 G/A promoter polymorphism in rheumatoid arthritis Tamer A. Gheita a, Ghada S. Azkalany a b
a,*
, Wafaa Gaber a, Abeer Mohey
b
Rheumatology Department, Faculty of Medicine, Cairo University, Egypt Clinical Pathology Department, Faculty of Medicine, Cairo University, Egypt
Received 15 June 2014; accepted 1 July 2014 Available online 4 November 2014
KEYWORDS Rheumatoid arthritis; TNF-a -308 G/A promoter polymorphism; Tumor necrosis factor (TNFa)
Abstract Aim of the work: To evaluate the clinical significance of serum levels of tumor necrosis factor alpha (TNFa) and -308 G/A promoter polymorphism in rheumatoid arthritis (RA) patients. Patients and methods: We studied 43 RA patients and 30 controls. Demographic, clinical and serological data were prospectively evaluated. Disease activity score (DAS28) and the Health Assessment Questionnaire (HAQ) were assessed. Serum TNF-a level was measured and promoter (-308 G/A) genotyped. Results: Serum TNF-a level was significantly higher in the RA patients compared to controls ( p = 0.036) and was significantly higher in those with AA promoter polymorphism who had a significantly younger age at disease onset. In the multivariate analysis, disease duration would predict the TNFa level ( p = 0.006) while age at disease onset, DAS28 and HAQ would predict the TNFa polymorphism ( p = 0.004, p = 0.04 and p = 0.03 respectively). A significant negative correlation was present between TNFa level with age ( p = 0.001) and age at disease onset ( p < 0.0001) while in those with GA genotype a significant negative correlation was present with DAS28 (r = 0.66, p = 0.038). Conclusion: Serum TNFa levels and -308 G/A promoter polymorphism were higher in RA patients than in controls and could be predicted by disease activity and HAQ. 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.
1. Introduction Cytokine unbalance is responsible for the pathogenesis of diverse inflammatory, autoimmune and infectious diseases [1]. Rheumatoid arthritis (RA) is a model of multigenic * Corresponding author. E-mail address: [email protected] (G.S. Azkalany). Peer review under responsibility of Egyptian Society of Rheumatic Diseases.
inflammatory disorder in which tumor necrosis factor-alpha (TNF-a) plays an important and central role in its etiology and pathogenesis [2,3]. Overproduction of TNF-a is thought to be the main contributor to increased ROS release in RA patients, leading to tissue damage associated with the inflammation [4]. Genetic factors may be implicated in the susceptibility to disease initiation and in the severity of its course [2,3]. TNF-a and gene expression are the key regulators of inflammation and critical for RA [5]. It is substantiated that RA is favored by the presence of the -308 TNF promoter
http://dx.doi.org/10.1016/j.ejr.2014.07.001 1110-1164 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.
50 polymorphism which is responsible for the increased TNF production [2]. There are, however, controversial reports that TNF-a promoter -308 A/G polymorphism may be an independent marker of RA susceptibility and severity [6–8]. The TNF gene is located on chromosome six within the major histocompatibility complex (MHC). Mutation affects the -308 promoter region of the TNF gene which defines the TNF1 and TNF2 alleles, determining low and high levels of TNF expression, respectively [1,2]. The TNF-a -308 polymorphism in the promoter region with the TNF2 allele was found in almost a quarter of RA patients and 10% of normal subjects [9]. It has been linked to increased susceptibility and severity in a variety of autoimmune and inflammatory disorders, including RA, systemic lupus erythematosus (SLE) and inflammatory bowel disease. Significant impact of -308 promoter polymorphism is the increased TNF production and this difference does not appear to be due to other genes lying within the MHC region [1,2,9]. Moreover, there is a great deal of evidence that points to the association of the TNF-a and/or its gene -308 G/A promoter polymorphism with pathogenesis and involvement in the disease susceptibility and severity of SLE [9,10] and JRA [11–13]. Polymorphisms affecting the TNFa gene and its regulatory regions are associated with RA prevalence and course. There is a possible association between these polymorphisms and the clinical response to the use of anti-TNFa antibodies. The possibility that the determination of genotype -308 may have prognostic and therapeutic consequences was argued [14]. The introduction of anti-TNF agents has improved the outcome for many patients with RA and the only replicated genetic predictor of anti-TNF response is the -308 G/A single-nucleotide polymorphism in the TNF promoter region. The presence of -308 TNF G/G genotype appears to be a marker of good response to anti-TNF treatment [15]. Meanwhile, a meta-analysis showed that the TNF-a -308 A/G polymorphism is not associated with responsiveness to TNF blockers in RA patients [16]. The aim of the present study was to evaluate the clinical significance of serum levels of tumor necrosis factor alpha (TNFa) and -308 G/A promoter polymorphism in rheumatoid arthritis (RA) patients and find any association with age at disease onset, clinical and laboratory features and disease activity. 2. Patients and methods Forty-three rheumatoid arthritis patients with definite RA diagnosed according to the 2010 ACR/EULAR RA classification criteria [17] were consecutively recruited from the Rheumatology outpatient clinic and the department of Cairo University Hospitals. Full history taking, thorough clinical examination, laboratory and plain X-ray investigations of the affected joints were performed for all the patients. All patients were regularly receiving methotrexate with or without corticosteroids or chloroquine. Disease activity score in 28 joints (DAS-28) was calculated [18]. The Health Assessment Questionnaire-II (HAQ-II) was used [19]. Radiological grading of the hands and feet of the studied RA patients was assessed and percentage damage was calculated according to the modified Larsen score [20]. The study was approved by the local university ethics committee and the study conforms
T.A. Gheita et al. to the provisions of the Declaration of Helsinki in 1995. All patients gave their informed consent prior to their inclusion in the study. Serum TNF-alpha was assayed using a Human TNF-alpha ELISA kit (quantitative sandwich enzyme immunoassay technique), provided by Ray Biotech, Inc. 2.1. Determination of TNF-a gene (-308) promoter region polymorphism Promoter region polymorphism of TNF-alpha gene (-308 G/A) was determined by the polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) technique. 2.2. DNA isolation and TNF-alpha genotyping Genomic DNA was isolated from white blood cells (WBCs) of peripheral blood using a Biospin Whole Blood Genomic DNA Extraction Kit (Bioflux Corporation, Arrow Plaza, Tokyo, Japan). Enzymatic amplification was performed by a PCR using the Master Taq polymerase enzyme and Hybaid thermal cycler (Promega Corporation, 2800 Woods Hollow Road Madison, W 153711-5399 USA). Amplification of the promoter region (-308 G/A) of the TNF-alpha gene was performed as proposed by Dalziel et al. [21] using 2 primers which were purchased from Operon Biotechnologies (GmbH/ Biocampus, Germany). Forward: Primer 50 -AGGCAATAGGTTTTGAGGGCCA T-30 , and Reverse Primer: 50 -ACACTCCCCATCCTCCCTG CT-30 . The PCR reaction mixture (25 ll) contained 12.5 ll 2· PCR Master Mix {10· PCR buffer, 4 mM MgCl2, 0.5 Taq DNA polymerase/ll, and 0.4 mM dNTPs (dATP, dCTP, dGTP, dTTP)}, 1 ll of each primer (25 pmol), 3 ll of genomic DNA and 7.5 ll sterilized nuclease-free water. The reaction was carried out with the following cycles: 95 C for 2 min; 35 cycles of 95 C for 30 s, 60 C for 15 s and 74 C for 15 s; and 74 C for 10 min for final extension. Then amplified products were digested with 5 units Fast Digest NcoI restriction enzyme at 37 C for 10 min (supplied by Fermentas, LT02241 Vilnius, Lithuania).The digested products were then detected in 3.5% agarose gel containing ethidium bromide by performing E/P on the gel electrophoresis apparatus and visualized by UV transillumination (Promega, USA) for the identification of TNF-alpha gene (-308 G/A) promoter polymorphism. A single band at 107 bp identified AA homozygous individuals, two bands at 87 and 20 bp identified GG homozygous individuals, and three bands at 107, 87 and 20 bp indicated a heterozygote at the TNF-a -308 locus, while the 20 bp band was very small to be viewed on the gel. Statistical analysis: Data were collected, tabulated and analyzed by SPSS package version 15 (SPSS corporation, USA). Data were summarized as mean ± SD. Mann–Whitney test was used for the comparative analysis of 2 quantitative data. Non-parametric analysis of variance (Kruskal–Wallis) was used for the comparison of more than two groups; Spearman’s correlation analysis was used for the detection of the relation between 2 variables. Logistic regression analysis was applied to detect the predictors for the elevated TNF-a level and promoter polymorphism. Results were considered significant at p < 0.05.
Clinical significance of serum TNFa and -308 G/A promoter polymorphism
51
3. Results There were 43 RA patients (40 females and 3 males) and 30 age and sex matched healthy controls with a mean age of 43.53 ± 3.23 years and were all females with 2 males. The mean TNFa level for controls (5.57 ± 3.84 pg/ml) was significantly lower than the level in patients ( p = 0.036). The demographic, clinical and laboratory features of and medications used by the RA patients are presented in Table 1. All the patients were irregularly receiving diclofenac sodium (50–100 mg) while only three were receiving full dose of sulfasalazine (2000 mg/day). Three of the patients were diabetic and hypertensive with two of them having HCV which was also present in another two patients. Anti-cyclic citrullinated peptide (Anti-CCP) was positive in 74.42% of the patients. None of the patients gave family history for any rheumatic disease. There were 62.8% with GG TNFa (-308 G/A) gene promoter polymorphism, 23.2% with GA and 14% with AA while it was GG in all the controls. The TNFa was significantly higher in patients with AA (480 ± 621.6 pg/ml) compared to those with GG (9.98 ± 23.63 pg/ml) and GA (92.81 ± 48.58 pg/ml), ( p = 0.000) (Fig. 1). Furthermore, there was a significant difference in age at disease onset according to the three gene promoters ( p = 0.007) being older in those with GG (37.06 ± 15.21 years) compared to those with GA (24.7 ± 11.2 years) and AA (19.33 ± 11.93 years). There were no other significant differences in the characteristic features of the patients according to the promoter genes. A significant negative correlation was present between the TNFa level with age of the patients (r = 0.49, p = 0.001) and age at disease onset (r = 0.58, p < 0.0001). On using multiple linear regression analysis including age, age at disease onset and disease duration, only the disease duration would predict the TNFa level ( p = 0.006) while age at disease onset would highly predict the TNFa (-308 G/A) promoter polymorphism
Table 1 Demographic, clinical and laboratory features of the rheumatoid arthritis patients. Feature
RA patients (n = 43) Mean ± SD
Age (years) Disease duration (years) Morning stiffness (min) No. of swollen joints No. of tender joints VAS (mm) ESR (mm/1st h) Hemoglobin (g/dl) Platelets (·103/ml) TNFa (pg/ml) Modified Larsen score DAS28 HAQ
40.9 ± 15.5 9.2 ± 7.2 25.4 ± 37.6 2.1 ± 3.3 6.8 ± 8.1 36.2 ± 34 53.2 ± 24.04 11.2 ± 1.7 305.9 ± 90.2 94.8 ± 269.6 38.5 ± 16 4.6 ± 1.7 0.8 ± 0.8
Medications: Methotrexate (35/43) Prednisolone (19/43) Leflunomide (9/43) Chloroquine (22/43)
15.4 ± 8.4 3.8 ± 6.7 4 ± 7.9 125 ± 133.6
VAS, visual analog scale; ESR, erythrocyte sedimentation rate; TNFa, tumor necrosis factor alpha; DAS28, disease activity score in 28 joints; HAQ, health assessment questionnaire.
Figure 1 Box plot of the serum TNF-a levels in the RA patients and controls according to the TNFa (-308 G/A) promoter polymorphism type.
( p = 0.004). DAS28, HAQ or steroid dose could not predict the serum TNFa level while both DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism ( p = 0.04 and 0.03 respectively). No significant correlation was present with the other clinical and laboratory features or disease activity. However, in those patients with GA genotype of TNFa polymorphism there was a significant negative correlation of the serum TNF-a level with the DAS28 (r = 0.66, p = 0.038) (Fig. 2) which was only a tendency in those with AA genotype (r = 0.81, p = 0.05). There was a tendency for an association between the TNFa level and the modified Larsen score in the RA patients with GG genotype (r = 0.48, p = 0.058). There was a tendency for a higher TNFa in the four patients with HCV (463.03 ± 833.5 pg/ml) compared to those without (57.06 ± 98.7 pg/ml).
Figure 2 Correlation of the serum TNFa level and DAS28 in RA patients with GA -308 promoter polymorphism.
52 4. Discussion In the present study, there was a significant elevation in the serum TNF-a levels in the RA patients and this was associated with the increased polymorphisms in its gene promoter region. None of the controls in the present study had any promoter polymorphism. This is in agreement with the results of another study that found elevated TNF-a in the plasma and synovial fluid from RA patients and was associated with polymorphisms in the promoter region of the TNF-a gene. However, in disagreement to our results was their results that found no differences in genotype distribution and allelic frequencies of -308 G/A TNF-a promoter polymorphism between RA patients and controls [3,6]. Interindividual variation in the expression of TNF suggests the existence of functionally distinct TNF alleles including -308 promoter polymorphism that could play a role in RA susceptibility [22]. All the RA patients in the present study were Egyptian and the TNFa -308 A/G promoter polymorphism was considered as an obvious risk factor compared to the controls especially those with juvenile onset. None of the controls had the A allele and all had the G allele. In the present RA patients, the A allele was heterozygous in 23.26% and homozygous in 13.95%. It may be thus thought of the G allele as a protective from the development of RA in Egyptians. In a meta-analysis study, TNF-a -308 A/G polymorphism represented a significant risk factor for RA in Latin Americans, but not in Europeans which favors that different ethnic populations influence this association [7]. The -308 G/A polymorphism was significantly associated with a poor outcome in the Turkish juvenile idiopathic arthritis (JIA) patients ( p = 0.005) but there was no association in the Czech patients. This genetic variation may accompany the phenotypic differences found [23]. Again, analysis of the TNFa -308 AA allele showed increased frequency in severe RA when compared to nonsevere disease and to healthy controls. This association might be used as a prognostic marker for severe RA [24]. Contrarily, TNF -308A itself or a neighboring gene may be a protective factor for the development of RA in some of the population in Taiwan [25]. In another study, polymorphism TNF -308A was less prevalent among RA patients (1.7%) than controls and found that the A allele at -308 locus provides protection against RA in North Indians [26]. Again, another study found that the distribution of TNF-genotypes in RA patients did not differ from that in controls, suggesting that TNF-a -308 promoter polymorphism is not a genetic risk factor for RA susceptibility and severity [6,8,27]. A meta-analysis study showed that the TNF-a -308 A/G polymorphism may represent a significant risk factor for RA in Latin Americans but not in the European, Arab, or Asian populations [28]. Considering the relevance of TNF-a in the pathophysiology of JIA, it is likely that polymorphisms in its promoter area may be relevant in disease susceptibility and activity. Twenty-four percent of JIA Portuguese patients presented the -308 GA/AA genotypes and 76% the GG, similar to findings in controls thus conferring no association with disease susceptibility [29]. However, the -308 G/A polymorphism tends to be more frequent in the systemic onset rather than the oligoarticular JIA patients [30]. In the present study, there was no association of the TNF-a -308 promoter with the other clinical or laboratory
T.A. Gheita et al. manifestations of the RA patients. In agreement, the TNF promoter polymorphisms at position -308, were not related to the clinical manifestations of RA patients [25]. Moreover, there was also no correlation between TNF-a polymorphism and disease activity measures, including ESR, CRP, number of swollen and tender joints, and morning stiffness duration [6]. In the present study, a significant negative correlation was present between the TNFa level with age of the patients (r = 0.49, p = 0.001) and age at disease onset (r = 0.58, p < 0.0001). On using multiple linear regression analysis including age, age at disease onset and disease duration, only the disease duration would predict the TNFa level ( p = 0.006) while age at disease onset would highly predict the TNFa (-308 G/A) promoter polymorphism ( p = 0.004). There was no significant association of TNFa promoter variation at positions -308 and -238 with susceptibility to RA. However, a significant difference in the mean age at disease onset was found between -238 TNFa genotypes. In addition, a difference in the presence of nodular disease was observed between -308 TNFa genotypes suggesting that TNF a gene may play a role in the disease profile of RA patients [31]. Others found no association between TNF-a genotypes, age at disease onset, disease activity, and joint and extra-articular involvement [6]. In the present study, both DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism. Patients with GA genotype showed a significant negative correlation of the serum TNF-a level with the DAS28 (r = 0.66, p = 0.038) while there was a tendency for this association in those with AA genotype (r = 0.81, p = 0.05). TNF-a -308 GA/AA genotypes were found to be related to higher inflammatory activity and worse measures of disease activity in rheumatoid patients compared to those with GG genotype [29]. TNF promoter gene polymorphisms and TNF receptor gene heterogeneity play a significant role in non-response to biologic agents and disease course/severity [32]. In a study on active Chilean RA patients genotyped for -308 TNFa polymorphism who were treated by an anti-TNF-a agent, 88.2% of G/G and 68.4% of G/A genotypes were DAS28 responders and the serum TNFa levels of the G/A patients were lower than those of the G/G patients. A negative relationship occurred between DAS28 improvement and increased circulating TNFa levels [33]. The mean TNF-a level increased significantly with respect to basal levels in most of patients after anti-TNF-a therapy and was inversely related to the improvement in disease activity [34]. In JIA patients with the genotype -308 GG achieved an obvious improvement in disease activity at month 6 more frequently than those with the genotype -308 GA or AA [35]. Patients not responding to anti-TNF therapy showed an increased frequency of the A allele. The significant association between the TNF-a promoter -308 A/G polymorphism and responsiveness to anti-TNF therapy, suggests that RA patients who carry the A allele have a poorer response to therapy than those with the G allele [36,37]. Patients with a TNFa -308 G/G genotype are better responders to anti-TNFa treatment than those with A/A or A/G genotypes irrespective of the treated rheumatic disease (RA, psoriatic arthritis or ankylosing spondylitis) [38]. In a previous study including another TNF gene -863A allele it was suggested that it can be used as a genetic marker for SLE susceptibility and was associated with high TNF-a
Clinical significance of serum TNFa and -308 G/A promoter polymorphism production, Raynaud phenomenon, and nephritis in juvenile SLE patients [39]. Almost three quarters of the patients in the present study had positive anti-CCP. The TNF- 308A allele is less frequently found in rheumatoid factor negative but not in rheumatoid factor positive polyarthritis and may therefore be associated with a more severe disease, while the more common TNF 308G allele may be protective [12]. In this study, there was a tendency for an association between the TNFa level and the modified Larsen score in the RA patients with GG genotype. Although the introduction of anti-TNF antibodies has contributed to the slowing and arrest of RA progression, destruction itself has been generally acknowledged as an irreversible process with little hope in resolution [40]. Another study reported that erosive damage in patients with recent-onset RA is not influenced by the -308 TNF-a genotype [41]. In conclusion, serum TNFa levels and -308 G/A promoter polymorphism were higher in RA patients than in healthy controls and could be predicted by the disease activity and HAQ. Only the disease duration would predict the TNFa level while age at disease onset, DAS28 and HAQ would predict the TNFa (-308 G/A) promoter polymorphism. To confirm our results we propose that larger scale, multicentre studies with longer evaluation periods are needed in selected RA patients using direct sequencing of the genotype.
[10]
[11]
[12]
[13]
[14]
[15]
Conflict of interest None.
[16]
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