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Tumor Necrosis Factor-Alpha Promoter Gene Polymorphism (308
G/A) in Egyptian Patients with Systemic Lupus Erythematosus
Ibrahim M. Rageh1, Ahmed A. Sharaawy2, Ali I. Fouda3, Eman R. Abdelgawad1 &
Walid A. Abdelhalim1
1
Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha
University, Egypt.
2
Department of Clinical and Chemical Pathology, National Liver Institute, Menoufiya
University, Egypt.
3
Department of Rheumatology and Rehabilitation, Faculty of Medicine, Benha
University, Egypt.
Background: Tumor Necrosis Factor-α (TNF-α), is very important
cytokine, having different physiological and pathogenic effects which
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
Egyptian population and its relation to the activity and the clinical
manifestations of SLE.
Subjects 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 polymerase chain reaction, 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 studied SLE
patients when compared to healthy control subjects (p=0.023, 0.007
respectively). TNF-α genotypes showed significant differences between
various SLEDAI activity grades (p=0.012). Moreover, GG genotype had
significantly lower incidence of renal disorders and persistent proteinuria
when compared to AG or AA genotypes (p=0.006, 0.001 respectively).
While, AA genotype showed significantly higher neurologic disorder
when compared to GG and AG genotypes (<0.001, 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 Egyptian
population.
Key words: SLE, TNF, polymorphism, Egyptians, PCR-RFLP
1
Introduction
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 emphasise to the
importance of cytokine network genes. Tumour necrosis factor (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 [4–6]. TNF-α stimulates cytokine production, enhancing
expression of adhesion molecules and neutrophil activation, and acts as a
co-stimulator 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
cases [8].
A change G-to-A single nucleotide polymorphism (SNP) 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 Egyptian population and evaluate
the relation among this polymorphism with the activity and the clinical
manifestations of SLE.
Subjects and methods
We studied 50 Egyptian female patients with mean age of 32.4±8.6
years referred to Rheumatology and Rehabilitation department at Benha
University Hospital between March and November 2014 and 50 healthy
female volunteers with mean age 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.
2
Genomic DNA was extracted from peripheral blood leukocytes by
QIAamp DNA Mini Kit (Qiagen,USA) (Cat # 51104), according to
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 polymerase chain reaction Taq PCR Master Mix Kit
(Qiagen,USA), (Cat # 201443), using primers 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 1min and in
the last cycle, extension was prolonged to 10 min. The PCR products
were digested using 2 unit/µl of NcoI restriction enzyme (Invitrogen,
China) 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 minutes. 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 SPSS (statistical package for social
science) program (SPSS, Inc, Chicago, IL) version 20. Chi square test
was used to compare groups. Deviations from Hardy–Weinberg
equilibrium expectations were determined using the chi-squared test. GG
genotype and G allele were considered as references. Odds ratio and 95%
confidence interval were calculated.
Table (1): Genotypic distribution and allelic frequencies of the TNF-α promoter gene
polymorphism (–308 G/A) in patients with SLE and controls.
TNF-α
SLE patients % (n = Controls % (n = P value
OR
95% CI
Promoter 50)
50)
Genotype
N
%
N
%
GG
32
64
42
84
–
–
GA
13
26
7
14
0.069
3.119
0.914-10.646
AA
5
10
1
2
0.086
1.743
0.925-3.283
GA
+
18
36
8
16
1.959
1.097-3.501
0.023
AA
Allele
G
77
77
91
91
3.020
1.319-6.914
0.007
A
23
23
9
9
OR: odds ratio; CI: confidence interval
3
Table (2): Comparison between TNF-α promoter gene polymorphism (–308 G/A)
genotypes regarding SLEDAI grades in studied SLE group.
SLE
N=50
SLEDAI
GG
GA
AA
P1
(n=32)
(n=13)
(n=5)
Score; median (range)
5.5 (0-30) 17 (0-25) 28 (24-32) <0.001
Inactive
11 (34.4) 2 (15.4) 0
(0)
Grades;
Mild to moderate
14 (43.8) 4 (30.8) 0
(0)
0.012
N (%)
Severe
7
(21.9) 7 (53.8) 5
(100)
P2
0.401
0.008
0.010
P1, significance between genotypes.
P2, significance within each genotype.
Figure (1): Distribution of TNF-α promoter gene polymorphism (–308 G/A)
genotypes in SLE patients and healthy control subjects.
Table (3):Comparison between TNF-α promoter polymorphism (–308 G/A)
genotypes regarding ACR criteria in studied SLE group.
SLE
N=50
ACR criteria
GG
GA
AA
(n=32)
(n=13)
(n=5)
P
N
%
N
%
N
%
Malar rash
22 68.8 8
61.5
3
60
0.904
Discoid rash
9 28.1 1
7.7
3
60
0.074
Photosensitivity
14 43.8 4
30.8
2
40
0.824
Oral ulcers
8 25.0 3
23.1
3
60
0.286
Alopecia
15 46.9 6
46.2
3
60
0.917
Arthritis
28 87.5 10 76.9
4
80
0.511
Serositis
13 40.6 10 76.9
4
80
0.055
Renal disorder
8
25
9
69.2
5
100
<0.001
Persistent Proteinuria
8
25
9
69.2
5
100
<0.001
Cellular casts
7 21.9 6
46.2
2
40
0.207
Neurologic disorder
4 12.5 3
23.1
4
80
0.005
4
Hematological disorder
Anemia
Leucopenia
Thrombocytopenia
Pancytopenia
Immunologic disorder
Positive Anti ds DNA
Positive ANA
17
15
15
9
7
32
19
32
53.1
46.9
46.9
28.1
21.9
100
59.4
100
8
7
6
3
2
13
8
13
61.5
53.8
46.2
23.1
15.4
100
61.5
100
3
2
3
2
1
5
5
5
60
40
60
40
20
100
100
100
0.907
0.917
0.917
0.796
1
0.234
-
Table (4): Ordinal regression analysis for prediction of activity of SLE (assessed by
SLEDAI score).
Age (years)
Disease duration (years)
Anti ds DNA
Albumin /Creatinin ratio
C3 (mg/dL)
C4 (mg/dL)
GG
TNF-α
GA
AA
SE: standard error.
Univariate
estimate
0.002
0.014
1.212
3.174
-0.047
-0.074
Reference
1.687
5.190
SE
0.029
0.055
0.601
0.761
0.013
0.022
P
0.932
0.800
<0.001
<0.001
<0.001
0.001
0.628
1.209
0.007
<0.001
Multivariate
estimate SE
2.336
0.644
3.274
0.974
-0.035
0.016
-0.019
0.023
Reference
0.501
0.775
4.523
1.383
P
<0.001
0.001
0.025
0.407
0.518
0.001
Results:
The frequencies of the genotypes of -308 G/A of TNF-α in the SLE
and control groups are shown in Table (1), Figure (1). This sample of
individuals was selected randomly from population in Kaliobyia
Governorate in Lower Delta, Egypt. Applying Hardy Weinberg equation,
revealed that TNF-α -308 (rs1800629) genotypes in both cases and
control subjects were independent [i.e., they are in HW equilibrium
(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 to healthy control subjects
(p=0.023, 0.007 respectively), with 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 other hand, GA and AA genotypes did not show
significant differences between SLE patients and control subjects.
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
5
(p value = 0.008, 0.010 respectively).
In our study when we compared between TNF-α genotypes
regarding ACR criteria in studied SLE group. Patients with GG genotype
had significantly lower incidence of renal disorders and persistent
proteinuria when compared to those with AG or AA genotypes (p=0.006,
0.001 respectively). While, patients with AA genotype showed
significantly higher neurologic disorder when compared to those with GG
and AG genotypes (<0.001, 0.026 respectively). No statistical differences
were found between TNF-α genotypes regarding other ACR criteria for
SLE (p>0.05 for each).
When we applied age, disease duration, anti-dsDNA,
albumin/creatinin ratio, C3, C4 concentrations and TNF genotypes as
covariates. In univariate analysis, anti-dsDNA, albumin/creatinin ratio,
TNF-α GA and AA genotypes were significantly associated with risk of
increased SLEDAI score, while higher C3, C4 concentrations were
significantly associated with lower SLEDAI score. In multivariate
analysis, taking those variables that showed significant associations in
univariate analysis, anti-dsDNA, albumin/creatinin ratio, TNF-α AA
genotype were significantly associated with risk of increased SLEDAI
score, while higher C3 concentration was significantly associated with
lower SLEDAI score.
Discussion
TNF-α, a pro-inflammatory 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 metaanalysis confirmed the association of the A/A risk genotype (recessive
model) and G/A + A/A genotypes (dominant model) with SLE
susceptibility in North Europeans [15]. On the contrary, the TNF-α (-308)
A allele was not associated with SLE susceptibility in Asians [12], Black
Africans [15], African Americans [16], Mexican [17], and in Italians [18].
Thus, in this study we analyzed the TNF-α promoter gene (-308)
polymorphism in Egyptian SLE patients. TNF-α (rs1800629) GA+AA
genotypes and A allele were significantly increased in studied SLE
patients when compared to healthy control subjects (p=0.023, 0.007
respectively), with 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 and associates (2012) [19], in Brazilian
population, who reported a statistical significant differences in the
distribution of the TNF-α gene polymorphism between SLE patients and
6
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 other
hand, Lin and co-workers (2010) [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 done by Santos and colleagues
(2011) [21] in 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). While these results were on the
contrary with a study done Angelo and associates (2012) [19], in
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 Taiwanese [20]. Moreover, Santos et al.
[21] demonstrated a significant increase to the development of nephritis
in carrying A allele in Portuguese Caucasian 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 [13,21,25,26]. 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.
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