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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 373-378

Contribution of the STAT4 gene single nucleotide polymorphism to systemic lupus erythematosus


1 Clinical Pathology Department, Menoufiya University, Shebein El-kom, Menoufiya, Egypt
2 Clinical Pathology Department, Ain Shams University, Shebein El-kom, Menoufiya, Egypt
3 Clinical Pathology Department, Tanta University, Faculty of Medicine, Menoufiya University, Shebein El-kom, Menoufiya, Egypt

Date of Submission02-Sep-2014
Date of Acceptance16-Nov-2014
Date of Web Publication18-Oct-2016

Correspondence Address:
Ahmed S Hasab El-Naby
Tanta University, Tanta, 31511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192430

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  Abstract 

Objectives:
We investigated the prevalence of the STAT4 G > C (rs7582694) polymorphism in patients with systemic lupus erythematosus (SLE) and controls in a sample of the Egyptian population.
Background:
STAT4 has been found to be a susceptible gene in the development of SLE in various populations.
Participants and methods:
The presence of the STAT4 G>C (rs7582694) polymorphism was determined by PCR-RFLP.
Results:
There was an insignificant difference between SLE patients and controls in their phenotypes (P> 0.05). Also, the distribution of the polymorphism among the SLE patients in terms of different symptoms and the anti-dsDNA titer showed an insignificant difference (P> 0.05).
Conclusion:
There is no association between the STAT4 G>C (rs7582694) polymorphism and susceptibility for SLE in the population of Egypt that may be different from other populations in geographic location, together with the racial and ethnic differences, and also environmental factors, with differences in lifestyle.

Keywords: polymorphism, systemic lupus erythematosus, STAT4


How to cite this article:
El Bassuonia MA, Mohamed DF, Radwana WM, Hasab El-Naby AS. Contribution of the STAT4 gene single nucleotide polymorphism to systemic lupus erythematosus. Menoufia Med J 2016;29:373-8

How to cite this URL:
El Bassuonia MA, Mohamed DF, Radwana WM, Hasab El-Naby AS. Contribution of the STAT4 gene single nucleotide polymorphism to systemic lupus erythematosus. Menoufia Med J [serial online] 2016 [cited 2024 Mar 28];29:373-8. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/373/192430


  Introduction Top


Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the development of an immune response directed against any part of the host body. The course of SLE is unpredictable, with periods of remission and flare-ups. Moreover, this autoimmune disorder is highly heterogeneous, with various clinical manifestations and biosynthesis of a broad array of autoantibodies. The occurrence of SLE is nine times more frequent in premenopausal women than in men [1].

It is known that environmental factors together with genetic components are involved in the abnormal immune responses and pathogenesis of SLE [2],[3]. Flare-ups of SLE can be triggered by various environmental components, such as exposure to ultraviolet light, drugs, chemicals, and viral infections [4]. Candidate gene and genome-wide association studies showed numerous susceptibility genes of SLE, and the association of some of these genes has been confirmed in distinct populations [3].

The immune cells from patients with SLE show many abnormalities, including reduced T-cell cytotoxicity, abnormal function of CD4 T cells, abnormal activation of B cells, and alterations in cytokine biosynthesis [5],[6].

The STAT4 (signal transducer and activator of transcription) gene is expressed in T and B cells, monocytes, macrophages, natural killer cells, and dendritic cells. STAT4 is a transcription factor and a member of the STAT family. Its expression may support the differentiation of immune cells to inflammatory subsets, production of inflammatory cytokines and autoantibodies, prevention of apoptosis, and presentation of autoantigens, which may promote the development of autoimmune diseases [7].


  Participants and Methods Top


This study was carried out on 57 SLE patients, two males and 55 females, age range 11–37 years old. Fifty-four age-matched and sex-matched individuals were selected as a control group.

All patients were subjected to the following:

  1. Full assessment history and a thorough clinical examination.
  2. Routine laboratory investigations including complete blood picture, renal function tests, ESR, urine analysis, and anti-dsDNA titer.
  3. STAT4 G>C (rs7582694) polymorphism by PCR-RFLP.


Detection of the STAT4 G>C (rs7582694) polymorphism by PCR-RFLP

DNA extraction

Genomic DNA was extracted from whole blood cells using the GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, San Jose, California, USA).

PCR amplification of STAT 4 gene

The STAT4 gene was amplified from the extracted DNA using DreamTaq Green PCR Master Mix (2×) (Promega, USA) and STAT primers (Thermo Scientific). Promega DreamTaq Green PCR Master Mix (2×) is a ready-to-use solution containing DreamTaq DNA polymerase, optimized DreamTaq Green buffer, MgCl2, and dNTPs. The master mix is supplemented with two tracking dyes and a density reagent that allows for direct loading of the PCR product on a gel.

Primers

Two pairs of primers were utilized to evaluate the STAT4 G>C (rs7582694) polymorphism by PCR-RFLP. The lyophilized primers were reconstituted by the addition of sterile-distilled water to a final concentration of 100 pmol/µl, distributed in aliquots, and stored at −20°C.

Forward primer sequence: 5 ′ ATCCAACTC TTCTCAGCCCTT 3 ′.

Reverse primer sequence: 5 ′ TCATAATCAG GAGAGAGGAGT 3 ′.

Determination of the STAT 4 genotype

The amplified PCR products were digested using the fast digest TAA I restriction enzyme.

Detection of the digested fragment

Fifteen microliter of the digested fragment was then resolved on a 3% agarose gel using TBE buffer. The gel was run at 80 V for 35 min. A 100 bp ladder was used as a reference marker. U/V was used for visualization of DNA bands and they were photographed. The STAT4 C allele was cleaved into 258 and 80 bp, whereas the STAT4 G allele remained uncut.

The clinical and immunological criteria used in the Systemic Lupus International Collaborating Clinics (SLICC) Classification criteria are presented in [Table 1] [8].
Table 1: Clinical and immunological criteria used in the SLICC classification criteria

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  Results Top


This study was carried out on 57 SLE patients, two males and 55 females, age range 11–37 years, attending the Rheumatology department, Ain Shams University Hospitals. Fifty-four age-matched and sex-matched individuals were selected as a control group. We evaluated the STAT4 G>C (rs7582694) polymorphism distribution in SLE patients and its association with various clinical manifestation of SLE and anti-dsDNA titer.

STAT4 genotype distribution among patients and controls

Comparison of patients and controls in their STAT4 genotypes considering the GG genotype as the reference indicated an insignificant difference between them (P > 0.05). Also, comparison between them in the prevalence of the C allele considering the G allele as the reference indicated an insignificant difference between them (P > 0.05) ([Table 2]).
Table 2: STAT4 genotype distribution among patients and controls

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Association of STAT4 genotypes with various clinical manifestations of the patients included

Comparison of the distribution of polymorphisms among the patients in terms of different symptoms such as neurogenic manifestations, malar rash, arthritis, nephritis, photosensitivity, respiratory symptoms, and oropharyngeal ulcer indicated an insignificant difference between different types of polymorphisms (P > 0.05) ([Table 3]).
Table 3: Association of STAT4 genotypes with various clinical manifestations of the patients included

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Distribution of the polymorphism among the patients in terms of hematological disorders

Comparison of the distribution of polymorphisms among the patients in terms of hematological disorders, with P value more than 0.05 indicating an insignificant difference between different types of polymorphisms, is shown in [Table 4].
Table 4: Distribution of the polymorphism among the patients in terms of hematological disorders

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Distribution of the polymorphism among the patients in terms of the anti-dsDNA titer

Comparison of the distribution of polymorphism among the patients in terms of the anti-dsDNA titer, with P value more than 0.05 indicating an insignificant difference between different types of polymorphisms, is shown in [Table 5].
Table 5: Distribution of the polymorphism among the patients in terms of the anti-dsDNA titer

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Distribution of the polymorphism among the patients in terms of renal disorders

Comparison of the distribution of polymorphism among the patients in terms of renal disorders, with P value more than 0.05 indicating an insignificant difference between different types of polymorphisms, is shown in [Table 6].
Table 6: Distribution of the polymorphism among the patients in terms of renal disorders

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Agarose gel electrophoresis: the PCR-amplified fragments of STAT4 gene were 338 bp in length

The PCR-amplified fragments of the STAT4 gene were 338 bp in length and among PCR products after enzyme digestion, the STAT4 C allele was cleaved into 258 and 80 bp fragments, whereas the STAT4 G allele remained uncut ([Figure 1]).
Figure 1: Agarose gel electrophoresis: the PCR-amplified fragments of the STAT4 gene were 338 bp in length

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PCR product after enzyme digestion: The STAT4 C allele was cleaved into 258 and 80 bp fragments, whereas the STAT4 G allele remained uncut.


  Discussion Top


SLE is a chronic autoimmune disorder characterized by the development of an immune response directed against any part of the host body. The course of SLE is unpredictable, with periods of remission and flare-ups. Moreover, this autoimmune disorder is highly heterogeneous, with various clinical manifestations and biosynthesis of a broad array of autoantibodies. The occurrence of SLE is nine times more frequent in premenopausal women than in men [1].

It is known that environmental factors together with genetic components are involved in the abnormal immune responses and pathogenesis of SLE [2],[3]. Flare-ups of SLE can be triggered by various environmental components, such as exposure to ultraviolet light, drugs, chemicals, and viral infections [4]. Candidate gene and genome-wide association studies reported numerous susceptibility genes of SLE, and the associations of some of these genes have been confirmed among distinct populations [3].

The immune cells from patients with SLE show many abnormalities, including reduced T-cell cytotoxicity, abnormal function of CD4 T cells, abnormal activation of B cells, and alterations in cytokine biosynthesis [5],[6].

The STAT4 gene is expressed in T and B cells, monocytes, macrophages, natural killer cells, and dendritic cells. STAT4 is a transcription factor and a member of the STAT family. Its expression may support the differentiation of immune cells into inflammatory subsets, production of inflammatory cytokines and autoantibodies, prevention of apoptosis, and presentation of autoantigens, which may promote the development of autoimmune diseases [7].

STAT4 is essential for signal transduction by interleukin-12 (IL-12), IL-23, and type 1 interferon (IFN) in T cells and monocytes [7]. IL-12 induces the STAT4-dependent NK cell activation and differentiation of naive CD4 lymphocytes into Th1 effector cells and IFNγ production [9].

STAT4 also mediates the IL-23-dependent expansion of Th17 cells, contributing toward autoimmune diseases [0]. It has been reported that STAT4-deficient mice show reduced manifestation of T cell-linked experimental autoimmune diseases including encephalomyelitis, arthritis, myocarditis, colitis, and autoimmune diabetes [7]. Moreover, STAT4 deficiency results in a reduction in IFNγ biosynthesis in immune cells [7]. Accordingly, an association between disease activity in SLE patients and activation of the type 1 IFN system has been observed [1].

This study was carried out to evaluate the distribution of the STAT4 G>C (rs7582694) polymorphism in SLE patients and its association with various clinical manifestations of SLE and anti-dsDNA titer in an Egyptian population.

We found an insignificant difference between SLE patients and controls in their phenotypes (P > 0.05). Also, the distribution of polymorphisms among SLE the patients in terms of different symptoms and DNA showed an insignificant difference (P > 0.05).

The above-mentioned results are in agreement with the results of Zervou et al. [2], who reported the same findings in a Turkish population. Numerous studies have been carried out on the role of the STAT4 in the genetic predisposition to SLE; the data presented here show that the distribution of the STAT4 G>C (rs7582694) polymorphism is not associated with an increased susceptibility for SLE in the population of Egypt, indicating that the geographic location of both populations (Egypt and Turkey) may be implicated in the lack of susceptibility to the disease observed [13],[14].

In addition to racial and ethnic differences that may affect the causes, expression, and prevalence of the disease, environmental factors together with the extensive differences in lifestyle (diet, alcohol, smoking, etc.) may play a major role in the development of some diseases to the same degree as genetic factors [9]. Thus, the same disease may have different contributing factors in one ethnic group compared with another as certain polymorphisms may exist only in one ethnic group.

Recent studies carried out by Luan et al. [5] reported a statistically significant contribution of STAT4 G/C (rs7582694) toward the incidence of SLE in the Mainland Chinese female population. The contribution of the STAT4 G>C (rs7582694) polymorphism toward the incidence of SLE was also observed in large groups of patients of European origin, among them a Finnish family cohort as well as Spanish, Swedish, and other populations [16],[17],[18],[19].

The different effects of the STAT4 G>C (rs7582694) on clinical manifestations in various ethnicities may result from different sizes of the groups studied, genetic heterogeneity, or patient interaction with several environmental factors [0]. STAT4 gene variants have also been found to be risk factors for other autoimmune diseases including rheumatoid arthritis, Crohn's disease, asthma, systemic sclerosis, and Sjogren's syndrome [1].

In conclusion, here, we report that there was no association between the STAT4 G>C (rs7582694) polymorphism and susceptibility for SLE in a population of Egypt that is different from other populations in terms of the geographic location, together with the racial and ethnic differences, and also environmental factors with differences in lifestyle.


  Conclusion Top


Here, we found that there was an insignificance difference between SLE patients and controls in the STAT4 G>C (rs7582694) polymorphism in a population of Egypt. In addition, our study showed that there was an insignificance difference between SLE patients in the STAT4 G>C (rs7582694) polymorphism and its association with various clinical manifestations of SLE and anti-dsDNA titer.

Conflicts of interest

There are no conflicts of interest.[21]

 
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Luan H, Li P, Cao C, Li C, Hu C, Zhang S, et al. A single-nucleotide polymorphism of the STAT4 gene is associated with systemic lupus erythematosus (SLE) in female Chinese population. Rheumatol Int 2012; 95:1251–1255.  Back to cited text no. 15
    
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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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