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Serum Tumor Necrosis Factor Alpha and Gene

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Journal of Global Pharma Technology

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RESEARCH ARTICLE

Serum Tumor Necrosis Factor Alpha and Gene Polymorphisms in

Rheumatoid Arthritis Patients in Babylon Province, Iraq
Ahmed K. Alanzy, Abdulsamie H. Altaee*, Sabah J. Alrubiae

College of Medicine, University of Babylon, Postal Code 51002, Hilla P.O. Box 473, Iraq.

*Corresponding author: Abdulsamie H. Alta'ee

Abstract

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease affects 0.5-1% of the
worldwide population, of unknown etiology, characterized by chronic inflammation of the synovial
joints, hyperplasia, and overgrowth of synoviocytes, with the destruction of articular cartilage that can
cause serious weakness and inability to work. Objective: The present study aims to investigate the
possible association between tumor necrosis factor alpha (TNF-α) levels and (-308 G/A) TNF-α promoter
polymorphism in patients with RA in Babylon Province. Patients and Methods: This study was
designed as a case control. Forty-five (10 males, 35 females) patients with RA and forty-five (9 males,
36 females) apparently healthy persons as control with the compatible age and sex were enrolled in this
study. Serum level of TNF-α and anti-cyclic citrullinated peptide antibodies (ACCPA) were measured
using enzyme-linked immunosorbent assay (ELISA) method. Disease activity of RA patients was
assessed using the Disease Activity Score-28 (DAS28). The frequency of TNF-α -308 G/A gene
polymorphism was determined using polymerase chain reaction, restriction fragment length
polymorphism (PCR-RFLP) technique. Results: Present study finds significant high levels of serum
TNF-α and ACCPA in patients with RA in comparison with healthy controls. The genotype of (-308 G/A)
TNF-α gene promoter polymorphisms the GG genotype was 60% in RA patients and 42.2% in control
group, while the GA genotype was 40% in RA patients and 53.3% in control group, whereas, the AA
genotype was 0% in RA patients and 4.4% in control group. Correlation between TNF-α levels with both
of DAS-28 and ACCPA in RA patients found to be a significant positive correlation proposes a probable
role of TNF-α in RA pathogenesis. Statistical analysis showed no significant difference in the genotype
frequency of the TNF-α (-308 G/A) gene promoter polymorphisms between two groups. Conclusions: The
results of the present study were shown that TNF-α (-308G/A) gene polymorphism had not associated
with a risk factor of RA in Babylon patients and this SNP do not affect the serum level of TNF-α in RA
patients.

Keywords: Rheumatoid arthritis, TNF-α, -308 G/A gene polymorphism, ACCP Antibody, PCR-RFLP ,
Iraq.
Introduction
Rheumatoid arthritis is persistent systemic
inflammatory is the most common type of
autoimmune sickness causes morning joint
stiffness, pain, fever and damage to
function. thus lead to the transformation in
the joint as joint inflammatory that
resemble rheumatic fever [1].
The RA is initially associated with
inflammation of synovial joints. RA mostly
effected all peripheral joints although the
most commonly affected joints are included
hands, feet, and knees [2]. RA may develop
the risk of several other diseases such as
intestinal pathologies, pulmonary
©2009-2018, JGPT. All Rights Reserved
dysfunction, cardiovascular disease, and
kidney disorders with a significant rise in
the risk of early death [3].
The epidemiology of RA is affecting 0.5-1 %
of the worldwide and affects females more
frequently than male in the ratio (3:1) [4, 5].
RA may appear at any age. RA has a
prevalence of 20-50 cases each 100.000
persons / year [6].
Arthritis is the most common health
problem in the United States population,
affected over than 46 million people and
387
 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395
resulting in disability for
populations [7].
The peak age of onset worldwide is between
65 to 75 years in men and 55-64 years in
women [8]. There is no description of the
reason for the geographic variation [9].
There are no predictions for the future
incidence of rheumatoid arthritis, although
some studies are reported a decrease in
incidence and a rise in the average age of
onset [10,11]. A proposed reason for the
decrease in RA is hygiene [10]. There is
multi-factorial that causes RA but they still
consider unidentified etiology. Genetic,
environmental factors and deregulated
immune responses in the body found to play
a part in the stimulation of the disease
activity [12].
The disease is defined by increased
expression of proinflammatory cytokines
such as Interleukin (IL)-1, IL-2, IL-6, IL-17,
Interferon-gamma (INF-g), and Tumor
necrosis factor-alpha (TNF-α). Most of these
cytokines can be detected in synovial fluid
from RA patients [13]. TNF-α is an
inducible cytokine with a broad range of
proinflammatory and immunostimulatory
actions. This cytokine plays an important
role during the pathogenesis of RA. It
initiates the inflammatory response leading
to edematous joint and subsequent bone
destruction during the development of
disease [14].
The location of TNF-α gene on chromosome
number 6p21.3, TNF-α is a cytokine that is
produced by many cell types, but
macrophages and monocytes are the
primarily associated with the synthesis of
this TNF- α protein [15].When the TNF-α
polymorphisms in promoter region occur,
this lead to increase the TNF-α production,
which in turn may have an impact on
inflammatory responses, disease expression
and response to therapy. There is many of
SNPs in TNF- α region are reported to be
associated with RA susceptibility in various
populations [16, 17].
The present study aims to estimate the
levels of TNF-α and ACCPA in patients
with RA and healthy controls in the case-
control study, as well as to investigate the
relationship among TNF-α and ACCPA with
DAS-28 in RA patients. The present study
also aims to investigate the possible
©2009-2018, JGPT. All Rights Reserved
19 million association between TNF-α levels and TNF-
α (-308 G/A) gene promoter polymorphism
in patients with RA in Babylon Province,
Iraq.
Materials and Methods
Ethical Issues
The ethical issues in the present study are
based on the following:
 The acceptance of the Scientific Committee
of Department of Biochemistry at the
College of Medicine University of Babylon.
 The Ethical Committee in College of
Medicine, University of Babylon.
 The acceptance of the Ethical Committee of
Babylon General Directory of Health.
All persons participate in this study were
understood the objectives of study and signed
an informed consent.
Patients
The sample size of patients group in the
present study was selected according to
sample size equation. This case control study
was included 45 patients (10 males and 35
females) with RA with mean age (47.75
±7.67) years, diagnosed by a specialist
physician when they attended to Rheumatoid
Unit at Merjan Teaching Medical City in
Hilla City, and according to the American
College of Rheumatology (ACR) criteria in
the year 1987 and according to 2010 /
European League Against Rheumatism
(EULAR) classification criteria for RA [18].
The activity of RA disease was assessed by
using DAS-28 measurement. The complete
history was reported and a comprehensive
questionnaire has been filled. Control group
consist of 45 (9 males and 36 females)
apparently healthy persons with mean of age
(47.46 ± 7.76) years. They were selected from
medical staff of the same medical city. The
period of this study was extended from first
of August / 2016 to the first of July / 2017.
Exclusion Criteria
The persons with any type of acute or chronic
disease as well as persons who were obese,
smokers and pregnant women were excluded
from the present study.
Inclusion Criteria
Individuals were established with RA within
age from 35-60 years were included in the
388
 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395
present study as patients group, in addition
to apparently healthy persons as control
group within age from 35-59 years.
Methods
Measurement of Disease Activity Score
(DAS-28)
The DAS-28 of RA patients were estimated
by Van Riel formula [19].
Measurement of Serum Tumor Necrosis
Factor Alpha (TNF-α)
Quantitative determination of TNF-α in the
current study was done by using Bio base®
ELISA kit.
Measurement of Serum
Antibodies
Quantitative determination of ACCPA in the
current study was done by using Bio base®
ELISA kit.
Measurement of Blood Rheumatoid
Factor (RF)
The RF in the study groups was measure by
using Spin react® RF-latex agglutination test
kit.
Measurement of Blood C - reactive
Protein (CRP)
The CRP in the current study was
determined by using Agappy® latex-promoted
nephelometry kit.
Determination of (-308) Promoter Codon
Polymorphism of TNF-α Gene
The PCR/ RFLP was used to investigate the
(-308 G/A) TNF-α gene polymorphism
promoter codon in RA patients and control
groups.
DNA Extraction and TNF- α Genotyping
Figure: 1 Distribution of Study Group According to Gender
©2009-2018, JGPT. All Rights Reserved
The Favorgen- Taiwan Mini Kit for Genomic
DNA gives a good method for the purification
of DNA from blood samples. Enzymatic
amplification was done by PCR using Master
Taq polymerase enzyme and hybrid thermal
cycler. The amplification process was done as
proposed by E. F. Karray et al. [20] by
utilizing two primers (Promega®).
The forward and reverse primers are
5'AGGCAATAGGTTTTGAGGGCCAT-3'
5'TCCTCCCTGCTCCGATTCCG-3'
The reaction mixture of PCR (25 µl ) consist
of 12.5 µl, PCR Master Mix [10 × PCR buffer,
4 mM MgCl2, 0.5 Taq DNA polymerase 0.4
ACCP mM dNTPs (dTTP, dCTP, dATP and dGTP)],
1 µl of both primer, 3 µl of extracted DNA
and 7.5 µl sterilized nuclease-free water.
The reaction was performed with the
following cycles
One cycle 5 min at 94 °C, 30 seconds at 94°C
for denaturation, 30 cycles 35 seconds at
60°C for primer annealing, 1 minute at 72 °C
for template elongation, 10 minutes at 72°C
for final elongation. After that, the amplified
products were digested with 5 units Fast
Digest NCOI restriction enzyme at 37°C for
about (2-4 hours). The digested products
were then detected on (6%) of polyacrylamide
gel electrophoresis technique (PAGE) and
visualized on (UV transilluminator) [21].
Results
Results of the present study and other study
were shown no difference between males and
females in the most parameters involved
[13]. Therefore, categorization of participants
did not depend on the gender, as shown in
Figure 1.
389
 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395

Also, this study showed no significant between patients with RA and healthy
differences in the mean age when compared control group, as shown in Table (1).
Table 1: The Mean of Age of Study Group
Study group No. Male Female Mean ± SD P value
Patients 45 10 35 47.75 ±7.67
Age (years) 0.86
Control group 45 9 36 47.46 ± 7.76

The DAS-28 in the present study was shown DAS-28 according to gender among patients
no significant differences between means of with RA, as shown in Table (2).

Table 2: DAS-28 of Patients with Rheumatoid Arthritis

Gender No. Mean ± SD T-test P value
Male 10 4.02 ±0.89
DAS-28 0.037 0.971
Female 35 4.00 ± 0.86

The serum TNF-α concentration of RA patients groups significantly elevated than control
groups, as shown in Table (3).
Table 3: Tumor Necrosis Factor Alpha of Patients and Control Group

Study group No. Mean ± SD P value

Patients 45 81.31 ±28.14

TNF-α (pg/ml) < 0.001*
Control group 45 24.52 ± 6.64

In the present study, serum ACCPA found to be significantly greater than

concentrations of patients with RA were control groups, as shown in Table (4).

Table 4: Anti-Cyclic Citrullinated Peptide Antibody in Patients with RA and Healthy Control
Study group No. Mean ± SD P value
Patients 45 27.31 ±11.2
ACCPA (U/ml) < 0.001*
Control group 45 5.66 ± 2.23

The Correlations among TNF-α levels and
DAS-28, TNF-α levels and ACCPA, and DAS-
28 and ACCPA in RA patients were
investigated and found to be significant
positive correlations. In the present study,
CRP of patients with RA found to be positive
(50% of males and 100% of females). The
present study was found RF of patients with
RA positive in 69.3% and negative in 30.7 %
in all cases with RA patients. After that, the
amplification products by PCR were
separated by 3% agarose gel electrophoresis
technique and stained with ethidium bromide
dye. The PCR product length was (107 bp), as
shown in Figure (2).

Figure 2: The Amplification and PCR Product (107bp) on 3% Agarose Gel Electrophoresis.
Lane M, DNA ladder 50bp (128ng/µl) and other lanes represent PCR products (107bp)

©2009-2018, JGPT. All Rights Reserved 390

 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395

The yields of amplification after that digested
by using a specific restriction enzyme NcoI of
-308 G/A TNF-α gene promoter codon via
PCR-RFLP method were shown two alleles
(G and A) with three genotypes:
They appear one band with 107 bp and AA
genotype mean the digestion not occur, GG
was digested with restriction enzyme NcoI
into two bands 87 and 20 bp, and AG has
three bands 107, 87, and 20 bp, as shown in
Figure (3).

Figure (3) Amplification and Digestion by Restriction Enzyme of -308 G/A TNF-α Gene
Promoter Codon using 6% Polyacrylamide Gels Electrophoresis. The Lane (M) DNA Ladder 50
bp(128ng/µl) and Lanes (1-11) for Patients and (12-14) for Control

Results of current study found no significant 53.3% in control group, the AA genotype
association of TNF-α (-308 G/A) gene frequency 0% in RA group and 4.4% in
promoter polymorphisms of susceptibility to control group, the GG more genotype
RA, the current study also found genotype of frequency, while the AA fewer genotype
GG 60% in RA and 42.2% in control group, frequency in all subjects of present study, as
the GA genotype frequency 40% in RA and shown in Table (5).

Table 5: The Single-Nucleotide Polymorphism (SNP) of (-308 G/A) TNF-α Gene Promoter Polymorphism
Model Genotype Control Patients OR (95% CI) P-value
Codominant G/G 19 (42.2%) 27 (60%) 1.00 0.12
G/A 24 (53.3%) 18 (40%) 0.53 (0.23-1.23)
A/A 2 (4.4%) 0 (0%) 0.142
(0.006-3.12)
Dominant G/G 19 (42.2%) 27 (60%) 1.00 0.13
G/A-A/A 26 (57.8%) 18 (40%) 0.49 (0.21-1.13)
Recessive G/G-G/A 43 (95.6%) 45 (100%) 1.00 0.49
A/A 2 (4.4%) 0 (0%) 0.191
(0.009-4.097)
Over dominant G/G-A/A 21 (46.7%) 27 (60%) 1.00 0.29
G/A 24 (53.3%) 18 (40%) 0.58 (0.25-1.35)

Results of current study concerning allele genotype and allelic frequencies in Babylon
association of TNF-α (-308 G/A) people after comparing between RA patients
polymorphism of patients with RA were and healthy control group, as shown in Table
shown that no significant difference in (6).

Table 6: The Allelic Association of (-308 G/A) TNF-α Gene Promoter Polymorphism in RA Patients and Healthy Group
Control Patients OR (95% CI) P-value
Allele Count Proportion Count Proportion
G 62 0.69 72 0.8 1.8 (0.913-3.575) 0.08
A 28 0.31 18 0.2 0.55 (0.280-1.096)

©2009-2018, JGPT. All Rights Reserved 391

 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395
The result of the present study was found a
negative non-significant association between
TNF-α levels and genotypes in RA patients
the P-value = (0.6). Also, the result of the
present study was shown a negative non-
significant association between DAS-28
activity and TNF-α (-308 G/A) gene promoter
polymorphism in patients with RA the P-
value = (0.73). The results of the current
study were also found a negative non-
significant correlation between ACCP
antibody and TNF-α (-308 G/A) gene
promoter polymorphism of patients with RA
the P-value = 0.56.
Discussion
The present study was found the serum TNF-
α concentration of RA patients groups
significantly, elevated than control groups,
this result is agreed with Hadinedoushan et
al. study that recorded the levels of serum
TNF-α concentration in cases of RA were
significantly greater than the healthy control
group [21, 22]. TNF-α has the vital key role
in pathogenesis events of RA. This cytokine is
formed in elevated concentration through
many of cells, for example, monocytes and T
cells in case of RA infection [23]. An elevated
level of TNF-α is present in the synovial fluid
and serum of RA patients [24]. Therefore, it
has been established that countervail
monoclonal antibody versus TNF-α lead to
decrease the production of another's pro-
inflammatory cytokine, for example, IL-1 and
GM-CSF in the synovial fluid of RA [25].
The result of the current study disagreed
with Beyazal et al. study which recorded the
mean serum IL-17 and TNF-α concentration
was not significantly different among RA
patients and healthy group [26]. The
correlation between TNF-α and ACCP levels
in the present study were investigated and
shown a positive and significant correlation
in patients with RA groups (P= < 0.001, r =
0.542) the result of the current study is
agreed with Clavel et al.[27] study that
reported ACCPA contain immune complexes
stimulate TNF-α production through human
macrophages via binding of Fc γ RII α at the
surface of macrophage, this means that when
ACCP levels are elevated this will lead to
induce TNF-α production [28].
The current study was found a positive and
significant correlation between serum TNF-α
levels and DAS-28 in patients with RA
disease. The DAS-28 is usually used in the
©2009-2018, JGPT. All Rights Reserved
measure of disease activity in RA and help
patients in how controlled his RA and
whether therapy is effective or not [29]. This
result of the current study is agreed with
Nemec et al. [30] study which found the
elevated level of TNF-α in synovial fluid of
RA patients indicated a positive correlation
between the serum TNF-α level and disease
activity.
Results of current study found no significant
association of TNF-α (-308 G/A) gene
promoter polymorphisms of susceptibility to
RA also, the current study found genotype of
GG 60% in RA and 42.2% in control group,
the GA genotype frequency 40% in RA and
53.3% in control group, the AA genotype
frequency 0% in RA group and 4.4% in
control group, the GG more genotype
frequency, while the AA fewer genotype
frequency in all subjects of present study is
agreed with Hadinedoushan et al.[22] study,
that found GG more genotype frequency and
the AA fewer genotype frequency at Iran
population. The current study is also agreed
with Deepali G. et al.[31] study that reported
no significant association of TNF-α -308 bp
G/A polymorphism with susceptibility to RA
in North Indian people. There are no
associations have also been found in South
East Asians [32].
As well as agreed with Song et al. [33] study
that reported the TNF-α (-308 A/G)
polymorphism positive risk factor for RA in
Latin Americans, but not in the European,
Arab, or Asian populations. The present
study disagreed with Camelia A et al. [34]
study that carried out on Egypt population
that reported the TNF-α -308 G/A gene
polymorphism positive association in
development of RA. Results of current study
concerning allele association of TNF-α (-308
G/A) polymorphism of patients with RA were
shown that no significant difference in
genotype and allelic frequencies in Babylon
people after comparing between RA patients
and healthy control group.
The allele frequency in the current study of A
allele found no significant difference and low
frequency of this allele in patients, where the
A allele count was 18 and proportion 20%
while the A allele in control was 28 and
proportion 31% (P=0.08). Results were shown
that the allele frequency of G allele was the
major allele frequency in patients where it
was found that the G allele counts 72 and
392
 Abdulsamie H. Altaee et. al.| Journal of Global Pharma Technology| 2018; 10(03):387-395
proportion 80%, while the G allele in control
group was count 62 and proportion 69%
(P=0.08). The current study is agreed with
Nemec et al.[35] study that found no
significant difference in genotype and allelic
frequencies of TNF-α (−308) polymorphism in
Czech population with and without RA. As
well, the current study disagreed with
Hussein et al.[36] study that found a sig-
nificant association of TNF-α -308 alleles
with RA. The exact cause of the difference in
the distribution of alleles and genotypes of
TNF-α (−308) polymorphism in RA patients
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