The polymorphism of Interleukin 4 in Chronic Hepatitis B Virus Infection in Hemodialysis Patients at Khartoum State, Sudan

Abstract

Background: Interleukin-4 is best known as an important mediator and modular of immune and inflammatory responses to several diseases conditions such as hepatitis B, which is a significant health concern worldwide. Objectives: To estimate the frequency of different genotypes in IL 4 polymorphism gene-590 C/T in chronic Hepatitis B Virus patients and the control group. To correlate the different genotype of IL4 gene polymorphism with age, gender and location in Khartoum state. Method: IL4-590C/T polymorphism was examined in 56 patients with chronic hepatitis B and 30 non-hepatitis B infected controls, using the polymerase chain reaction –restriction length polymorphism method.

Full text

Arch Microbiol Immunology 2017; 1 (1): 33-40 33
Volume 1, Issue 1 Research Article
The polymorphism of Interleukin 4 in Chronic Hepatitis B Virus Infection in
Hemodialysis Patients at Khartoum State, Sudan
Yousif N1, Khair O2, Abdel Rahim M El Hussein2, Isam M Elkhidir3 and Khalid A Enan2*
1Department of Microbiology, Faculty of Medical Laboratories, Al Neelain University, Khartoum, Sudan
2Department of Virology Central Laboratory - The Ministry of Higher Education and Scientific Research,
Khartoum, Sudan
3Department of Microbiology and Parasitology, University of Khartoum, Khartoum, Sudan
*Corresponding Author: Khalid A Enan, Department of Virology Central Laboratory - The Ministry of Higher
Education and Scientific Research, Khartoum, Sudan, Tel: +249912651103; E-mail: khalid.enan@gmail.com
Received: 23 March 2017; Accepted: 03 April 2017; Published: 07 April 2017
Abstract
Background: Interleukin-4 is best known as an important mediator and modular of immune and inflammatory
responses to several diseases conditions such as hepatitis B, which is a significant health concern worldwide.
Objectives: To estimate the frequency of different genotypes in IL 4 polymorphism gene -590 C/T in chronic
Hepatitis B Virus patients and the control group. To correlate the different genotype of IL4 gene polymorphism with
age, gender and location in Khartoum state.
Method: IL4-590C/T polymorphism was examined in 56 patients with chronic hepatitis B and 30 non-hepatitis B
infected controls, using the polymerase chain reaction –restriction length polymorphism method.
Result: Our results showed a significant difference between the C/C, T/C, and T/T genotypes and the C and T
alleles of the -590 region of IL-4 in chronic hepatitis B patients compared with the controls.
Conclusion: Results of this study indicate that the functional gene polymorphisms of IL-4 may play an important
part in the development of chronic hepatitis B.
Keywords: HBV; IL4; Khartoum; Sudan

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1. Introduction
Hepatitis B virus (HBV) is a member of the Hepadnaviridae family of viruses, and has a double-stranded circular
DNA and a DNA polymerase enzyme. It has two major proteins: hepatitis B surface antigen (HBs Ag), and hepatitis
B core antigen, an inner protein. A third protein is hepatitis B e antigen (HBe Ag) [1], The virion envelope contains
three protein species designated as small (S), medium (M) and large (L).
The M and L proteins are longer versions of the S Protein and each envelope protein has one or more glycosylation
sites [2]. Chronic HBV infection is defined as the presence of HBsAg in serum for at least 6 months or the presence
of HBsAg and the absence of anti-HBC immunoglobulin M (IgM). The risk of developing chronic infection varies
inversely with age and is highest (up to 90%) for infants infected in the perinatal period [3]. HBV affects more than
200 millions people around the world causing an estimated 600000 deaths per year. Hepatits B remains as one of the
major causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [4] HBV is present in blood and
bodily secretions. The virus is most commonly spread through sexual contact, but it may also spread from mother to
child at birth, through contaminated needles and transfusion (extremely rare). The incubation period of HBV
infection averages 75 days (range 1-6 months) [5]. The cellular and humoral immune responses to HBV infection
are complex.
Most research in the field suggests that HBV is not directly damaging to infected liver cells and that the cellular
immune response to viral proteins correlates with the severity of clinical disease and viral clearance [6]. Cytokine
are small proteins secreted by immune system cells and other cells responsible for immune response. These soluble
proteins play their role by binding specific cell receptors that either induce or inhibit cytokine regulatory genes
during viral infection [7].
Interleukin-4 (IL-4), mainly produced by activated T helper 2 (Th2) cells, function major roles as a mediator and
modulator of immune and inflammatory responses [8]. It is also a survival and growth factor for lymphocytes.
Although it was recognized as a B cell differentiation and stimulatory factor (3), It also has a crucial role in
regulating T cell differentiation during the immune response [9]. The IL-4 gene in humans is situated on
chromosome 5q31, within 25 Kbp of the proximal portion [10].
The polymorphism -590C/T (rs2243250) in the IL-4 gene promoter region is the most commonly reported variation
of this gene [11]. In this region, the T allele of the 590C/T polymorphism of the IL-4 promoter gene was correlated
with increased IL-4 gene promoter activity [12]. IL-4 gene polymorphisms may have a role in hepatitis-related HCC
as -590C/T and 233C/T which are linked with increased risk for HBV related HCC and IL-4 -590C/T which has
been described to be linked with HBV progression to cirrhosis and HCC [13].
The present study was conducted to estimate the frequency of different genotype in IL 4 polymorphism gene in
CHB Virus infection and also aimed to correlate the different genotype of IL-4 gene polymorphism with age, gender
and location in Sudan.

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Arch Microbiol Immunology 2017; 1 (1): 33-40 35
2. Materials and Methods
Ethical approval for this study was obtained from Sudan Ministry of Health. Only patients agreeing
to participate were recruited in this study and informed written consent was obtained regarding the data and the
collection of blood samples. The demographic data such as name, age, gender, period and place of dialysis, history
of jaundice, date of infection with CHBV and the results for various previous investigations were collected using a
structured questionnaire.
3. Sample Collection
Whole blood samples were collected from Hemodialysis patients who are chronically infected with HBV during the
period of September 2016 to February 2017. Fifty six samples were collected from hemodialysis patients from
different hospitals at Khartoum state (Ibin Sina hospital, Omdurman teaching hospital, Al-now hospital, Al-Amal
hospital). In addition, 30 control samples were collected from healthy (Non HBV infected hemodialysis) donors in
Khartoum.
A volume of 5 ml of whole blood was collected from each patient through venipunctures technique using
Ethylenediaminetetraacetic acid (EDTA) container.
3.1 DNA extraction
Genomic DNA was extracted By using the standard saturated sodium chloride method from 300 µl whole blood.
3.2 PCR amplification
Conventional PCR (Thechni England) was performed to detect genomic 589C/T in IL-4 by amplification of this
region. The reaction was performed in a total was performed in a total Volume of 20 µl in the first PCR reaction,
containing 13 µL of water and 5 µl of DNA mixed with 2 µl of each primer Forward: 5’AACACCTAAACTTGGG
AGGA3’ Reverse: 5’CTGTCATGGAAAAGCTGATCT3’ (Sang on Biotech Company (Shanghai, China). 5 µL of 2
mM dNTP mix, 2 µL of 25 mM MgCl2, 2.5 U Tag DNA Polymerase (Promega Corporation, Madison, WI, USA),
The amplification was conducted using 35 cycles of PCR reaction (denaturation at 95°C for 1 Minute, annealing at
58 – 54 0C (touchdown) for 1 minute and extension at 72°C for 45 seconds).
The amplicons were resolved and screened using a 2% agarose gel electrophoresis method. Only 56 bands with the
best optimum resolution were selected for Restriction fragment length polymorphism (RLFP).
3.3 Genotyping
A total of 5 µl of PCR product in 8 µl of sterile water was added to 1 µl of NE Buffer and 0.5 µl of BSA, 0.5µL
BsmFI (500 unit) (sib enzyme Russia) to give a final volume of 15 µl. The restriction digests mixture was then
incubated at 37°C for one hour. The reaction was stopped by the addition of 5 µl to 2.5% agarose gel loading buffer
(5xTBE, 0.5 mol/l EDTA, 10% (v/v) glycerol, 0.05% (w/v) ethidium bromide.

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Arch Microbiol Immunology 2017; 1 (1): 33-40 36
3.4 Statistical Analysis
Variances in both groups were calculated, the frequencies of different variables in both test and controls were
calculated and cross tabulation of genotypes and alleles. Binary logistic regression analysis was used Ypred = a +
b1X1 + B2X2 … + BnXn. IL4 polymorphism pred= a+ b1 x age + b2 x gender + b3 x central*east + b4 x central *
west + b5 HBV infection. Odds ratios (OR) 80% Confidence Intervals (CI) were used to estimate the association
between individual polymorphisms a age, gender, locality and chronic HBV. P-values of less than 0.05 were
considered statistically significant. All statistical analyses were performed using the SPSS 16.0 software.
4. Results
In this research the case group consisted of 56 chronic HBV patients, including 40 male and 16 female individuals,
and the control group consisted of 30 healthy individuals with 23 males and 7 females. The results of the RFLP
assay are given in Figure 1. Table 1 summarizes the frequencies between ages, gender and locality. The cross
tabulation of genotype and allelic frequencies of IL-4 polymorphism gene variation of the patient and control groups
are presented in Table 2. The frequencies of genotypes in the case group were 7.1%, 73.2%, and 19.6% for, CC, CT
and TT genotypes, respectively, while in the control group these frequencies were 2%, 3.3%, and76.7%. However,
after A binary logistic regression analysis was conducted to predict IL4 polymorphism from five predictors based on
the equation. IL4 polymorphism pred = 5.979+ -0.02 age+ 0.346 gender + -0.346 central and east + 0.262 central
and west + -4.147 HBVinfection (Table 3). The model was statistically significant (p = 0.000). HBV infection had
the highest statistically significant contribution of -4.247 with a P-value of 0.000. Age, gender and locality 1&2
were contributing less for -0.02, 0.346, 0.346 and -4.147 respectively, with a P-value of 0.347, 0.631, 0.638 and
0.835 respectively, which was statistically insignificant. They are significantly found in the CT genotype between
chronic hepatitis B and gene polymorphism (OR: 0.014, P = 0.000).
Figure 1: PCR-RFLP assay for analyzing the IL4-590C/T polymorphism of the IL4 gene. PCR product was
digested by restriction enzyme and visualized under agarose gel.

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Arch Microbiol Immunology 2017; 1 (1): 33-40 37
Variable Number %
Gender (n=86)
Control (n=30)
Female 7 23.3
Male 23 76.7
Patient (n=56)
Female 16 28.6
Male 40 71.4
Age in years (n=86)
Control (n=30)
Mean (Sd) 33.7 (12.3)
Range (Min-Max) 18-62
Patient (n=56)
Mean (Sd) 36.7 (13.9)
Range (Min-Max) 14-70
locality (n=86)
Control (n=30)
Central 24 80
West 6 20
Patient (n=56)
West 42 75
Central 10 17.9
East 4 7.1
Table 1: Study participants (patients and controls) distribution by gender, age and Locality (n =86).
Genotype Control % control Patient %patients Total
CC 6 20.0 4 7.1 10
CT 1 3.3 41 73.2 42
TT 23 76.7 11 19.6 34
Total 30 100.0 56 100.0 86
Table 2: Distribution of the genotype and alleles in controls and HBV patients (n=86).
Variable
B
Wald(Chi squre)
Df
p-value
Odd ratio
Age
-0.02
0.885
1
0.347
0.98
Gender(1)
0.346
0.231
1
0.631
1.413
Locality
-
0.317
2
0.853
-

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Arch Microbiol Immunology 2017; 1 (1): 33-40 38
locality(1)
-0.356
0.221
1
0.638
0.7
locality(2)
0.262
0.043
1
0.835
1.299
HBVinfection
-4.247
13.438
1
0.000
0.014
Constant
5.979
14.292
1
0.000
394.92
Table 3: Logistic regression Predicting IL4 polymorphism from age, gender, locality and HBV infection (n=86).
5. Discussion
From the model a IL4 polymorphism (CC& TT was considered as a normal alleles of IL4 gene, while CT was
considered as abnormal allele) was negatively affected with HBV infection. Exploring the IL-4 590C/T
polymorphism and its susceptibility to liver disease including HBV infection, HCV infection, liver cirrhosis, etc.,
significant associations between the IL-4 590C/Tpolymorphism and increased chronic hepatitis B was found in
Sudanese populations (Ahmed et al 2016 unpublished data), Cytokines play a critical role in immune and
inflammatory responses. However, cytokine coding genes are highly polymorphic, which means some of these
polymorphisms can affect the expression of cytokines. Single nucleotide polymorphisms (SNPs) are the most
frequent types of genetic variations. In this regard, SNPs within cytokine genes can affect the gravity and
progression of immune-mediated and chronic inflammatory diseases [14].
Polymorphisms in the regulatory regions of the cytokine genes may influence their expression [15]. IL-4 is
synthesized mainly in Th2 lymphocytes and of induces the expression MHC particles class I and II on the
lymphocytes, which facilitates recognition of the viral antigens. IL-4 lso stimulates the cytotoxicity and
phagocytosis of monocytes and macrophages.
This may provoke the incidence of the auto immunological processes found in HCV infection [16]. Dendritic cells,
as a group of antigen-presenting cells, can build a bridge between pathogens and the T-cell system. This mechanism
also has been described for viral diseases such as Hepatitis B infection [17, 18]. The natural outcome of HBV
infection varies dramatically among individuals. Infection is usually self-limited in the majority of cases, while a
minority of subjects develops persistent infections [19, 20]. The HBV infected individuals who carry the low-
activity genotypes of major Th1 cytokines and/or the high-activity genotypes of major Th2 cytokines may be at
extraordinarily high risk for HBV [21]. However, Some of the previous data revealed that there was no relation or
no significant differences were observed regarding the IL-4 2590C/T and 233C/T polymorphisms genotypes, alleles,
or haplotypes between the patient groups and the healthy controls [8]. While other studies suggest that
polymorphisms in some cytokine genes influence persistent HBV and HCV infection [22]. Roli Saxena, Indo Verma
reported that the significant positive association between IL-4,IL-2, IL-12B [23]. The difference in genotype and
alleles distribution might be due to difference in study design, sample size and different selection criteria adopted for

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Arch Microbiol Immunology 2017; 1 (1): 33-40 39
patients and controls in particular clinical manifestation, ethnicity and environmental risk factors may also be
possible confounders.
IL-4 polymorphism haplotypes demonstrated by some previous experimental data to be associated with respiratory
syncytial virus [24], multiple sclerosis [25], oral cancer [26], and systemic lupus erythematosus (SLE) [27],
suggesting that certain polymorphisms could affect the regulation of this cytokine [8]. In this study, the results
indicated that the CT genotype of the (rs2243250) could significantly be correlated with HB chronic infection in our
patients. To our knowledge this represents the first study performed to correlate the polymorphisms in the -590
region of IL-4 gene with chronic hepatitis B among Sudanese patient. It is recommended that cytokines be used as
markers for diagnosis in Sudan, especially in the high risk groups such as hemodialysis patients.
6. Conclusion
Results varied considerably between different populations studied This may be due to differences in sample size, the
ethnicity of the study population, the disease stage, and even the genotyping method, and it is difficult to conclude
definite associations based on the available data. Moreover, a thorough understanding of host virus interactions,
which may or may not end in chronic hepatitis B infection, is still in progress.
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