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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 17  |  Issue : 2  |  Page : 182-189

Study of the effect of interleukin-6 gene polymorphism in patients with rheumatoid arthritis


1 Department of Medical Biochemistry, Faculty of Medicine for Girls, Al Azhar University, Egypt
2 Department of Internal Medicine, Faculty of Medicine for Girls, Al Azhar University, Egypt

Date of Submission18-Mar-2019
Date of Decision19-Apr-2019
Date of Acceptance02-Jun-2019
Date of Web Publication23-Oct-2019

Correspondence Address:
Marwa H Sedira
Department of Medical Biochemistry, Faculty of Medicine (Girls) Al Azhar University, Cairo, 11751
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_55_19

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  Abstract 


Background Rheumatoid arthritis (RA) is an autoimmune disease that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. Although the clear etiology of RA remains to be elucidated, it is widely accepted that RA is a complex disease, and both environmental and genetic factors can contribute to disease initiation as well as its evolution. The −174G/C polymorphism of interleukin-6 (IL-6) gene has been reported to be responsible for changes in the expression of IL-6, which could in turn lead to greater inflammation and thus affect the clinical status of patients with RA.
Aim To determine the association of IL-6 gene polymorphism with patients with RA by studying the frequency of the polymorphism of IL-6 (−174G/C) gene in those patients compared with healthy participants to predict the risk of development of RA.
Patients and methods The study was conducted on 200 (150 patients with RA and 50 age-matched and sex-matched healthy control) adult Egyptian patients. IL-6 (−174G/C) genotypes were determined by PCR-restriction fragment length polymorphism analysis.
Results The prevalence of polymorphic genotype of GC and CC and C allele was statistically significantly increased in patients with RA than in control (P<0.001).
Conclusion The GC and CC genotype and C allele of IL-6 (−174G/C) may represent a significant genetic molecular marker to predict the risk of RA.

Keywords: IL-6 (−174G/C) polymorphism, rheumatoid arthritis, PCR-RFLP


How to cite this article:
Sedira MH, Seoudy OA, Ahmed LA, El Attar S, Mohamed EF. Study of the effect of interleukin-6 gene polymorphism in patients with rheumatoid arthritis. Al-Azhar Assiut Med J 2019;17:182-9

How to cite this URL:
Sedira MH, Seoudy OA, Ahmed LA, El Attar S, Mohamed EF. Study of the effect of interleukin-6 gene polymorphism in patients with rheumatoid arthritis. Al-Azhar Assiut Med J [serial online] 2019 [cited 2019 Dec 7];17:182-9. Available from: http://www.azmj.eg.net/text.asp?2019/17/2/182/269763




  Introduction Top


Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammatory synovitis and progressive joint destruction, which are associated with severe disability and increased mortality [1]. Soft synovial joint swelling is the key clinical feature of RA and is typically accompanied by morning stiffness and tenderness on examination [2].

It is 2–3 times more prevalent in women than men and affects ∼1% of the worldwide population [3]. Although the cause of RA is not clear, it is believed to involve a combination of genetic and environmental factors [4]. The pathophysiology of RA is complex and involves an interactive network of autoantibodies, T and B cells, cytokines, and other inflammatory mediators [5].

Interleukin-6 (IL-6) has a pivotal role in the pathogenesis of RA, and its level correlates with both disease activity and articular destruction [6]. IL-6 is a glycoprotein with 26 kD as a molecular weight, whose gene is found on chromosome 7. It consists of five exons and four introns as shown in [Figure 1],[Figure 2],[Figure 3] [7]. It is produced by various cell types such as T cells, B cells, monocytes, fibroblasts, and osteoblasts [8].
Figure 1 Interleukin-6 gene.

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Figure 2 Agarose gel electrophoresis showing PCR product-based restriction fragment length polymorphism analysis of interleukin-6 gene amplification guided by marker in the first lane.

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Figure 3 Agarose gel electrophoresis showing PCR product-based restriction fragment length polymorphism analysis of interleukin-6 (−174G/C) gene polymorphism digested by NLa III guided by marker in the first lane, performed on 3% agarose, being visualized by ultraviolet transillumination.

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Elevated IL-6 promotes chronic synovitis and bone and cartilage erosion through degradation of cartilage by activating fibroblast-like synoviocytes and chondrocytes to release matrix metalloproteinases; stimulates osteoclastogenesis and osteoclast activity through receptor activator of nuclear factor-kappa B ligand expression, leading to structural damage through bone resorption; and stimulates macrophages/monocytes to produce IL-1 and tumor necrosis factor-α, which stimulate effector T-cell proliferation and activation, leading to tissue damage [9].

The genomic sequence of IL-6 is highly polymorphic, and the promoter (−174G/C) is one of the most frequently evaluated variants that includes GG, GC, and CC genotypes and G and C alleles [10]. IL-6 (−174G/C) promoter polymorphism, representing G to C base substitution at 174 bp upstream of the transcriptional site, has been identified. It has been observed that polymorphism in the promoter region of the IL-6 gene may be responsible for changes in the expression of IL-6, which could in turn lead to greater inflammation and thus affect the clinical status of patients with RA [11].


  Aim Top


The aim is to determine the association of IL-6 gene polymorphism with patients with RA, by studying the frequency of the polymorphism of IL-6 (−174G/C) gene in those patients compared with healthy participants.


  Patients and methods Top


This study was done during the period from July 2017 till January 2018. Samples were collected from Al-Zahra University Hospital (Rheumatology Outpatient Clinic and Internal Medicine Department), and investigations were carried out at Medical Biochemistry Department, Faculty of Medicine for Girls, Al Azhar University. The protocol of this study was approved by the medical ethics committee in the faculty, and oral informed consent was obtained from all patients and healthy controls. It included a total of 200 participants who were classified into two groups.

Control group had 50 (31 females and 19 males) apparently healthy participants with no personal or family history of autoimmune diseases. Their mean age was 47.18±5.68 years. The study included 150 (115 female and 35 males) patients with RA. They were with definite diagnosis of RA, according to American College of Rheumatology and the European League Against Rheumatism criteria [12], supported by clinical examination and plain radiograph of joints. Their mean age was 48.02±6.26 years.

Patients with clinical evidence of other autoimmune disease or arthritis of other causes were excluded from the study.

The patients and control were subjected to full history taking, clinical examination, and routine laboratory investigations, which included the following: complete blood count, fasting blood glucose level, serum urea and creatinine, serum lipid profile, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP).

Estimation of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) was also done. For only patients with RA, plain radiograph of the affected joints and disease activity score for 28 joints (DAS28) were performed.

Specimen collection

Morning venous blood samples (10 ml) were withdrawn under complete aseptic conditions from all participants. Five millilitres was collected in a sterile ‘EDTA’ vacutainer and was used for molecular testing of IL-6 gene by lymphocyte separation and extraction of DNA for PCR. DNA was extracted and stored at −20°C till time of assay. Five ml was collected in plain vacutainer and left for 10 min to clot and then centrifuged at 3000 rpm for 10 min. The serum was separated and used for measurement of serum glucose, lipid profile, urea, creatinine, ACPA, CRP, and RF.


  Methods Top


Determination of fasting serum glucose level [13], serum lipid profile (total cholesterol [14] and triglyceride [15]), serum urea [16], serum creatinine [17], serum RF [18], and serum ACPA [19] was done. Genomic DNA extraction from blood leucocytes was analyzed for IL-6 (−174G/C) gene polymorphism using PCR-restriction fragment length polymorphism technique.

Genomic DNA analysis for determination of IL-6 genotype was done by detecting single nucleotide polymorphism at position −174G/C in the promoter region of the IL-6 gene using PCR technique and restriction fragment length polymorphism. The test was done in five main steps:
  1. Extraction of genomic DNA from peripheral blood leucocytes of EDTA anti-coagulated blood included the following:
    1. Lymphocyte separation.
    2. DNA extraction: DNA was extracted using Whole Blood Genomic DNA Purification Miniprep Kit #D3024.
  2. Amplification of the extracted DNA (PCR) by primers for IL-6 (−174G/C): The IL-6 (−174G/C) genotype was determined by PCR according to the protocol published by Gaber et al. [20]: amplification was performed using Taq Red PCR Master Mix (#K1081; Thermo-Scientific, Ampliqon A/S, Stenhuggervej 22, 5230 Odense M, Denmark).
  3. Primer sequence:
    1. Forward primer (5′-GCC TCA ATG ACG ACC TAA GC-3′).
    2. Reverse primer (5′-TCA TGG GAA AAT CCC ACA TT-3′).
      • The amplified fragment was 163 base pair (bp).
      • For amplification of −174G/C region of IL-6, the computerized thermo-cycler (TechneProgene, Cole-Parmer, Beacon Road, Stone, Staffordshire, ST15 OSA, UK) was programmed for the following conditions:
        1. An initial cycle of 95°C for 5 min (for initial denaturation), followed by 35 cycles under the following conditions: denaturation at 95°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 30 s. A final extension cycle at 72°C for 10 min was done.
  4. Detection of PCR amplification products was done using 2% agarose gel electrophoresis stained by ethidium bromide and ultraviolet light transillumination as bands at 163 bp.
  5. IL-6 (−174G/C) gene polymorphism was determined using specific restriction enzyme: the amplified products were digested with Fast Digest NLa III restriction enzyme (Thermo-Scientific), then the digested products were separated by 3% agarose gel electrophoresis stained with ethidium bromide and visualized using ultraviolet transilluminator. The wild type (GG) produced one band (163 bp long), the homozygote genotype (CC) produced 2 bands (111 and 52 bp long), and the heterozygote genotype (GC) produced 3 bands (163, 111, and 52 bp long).


Statistical methods

Statistical presentation and analysis of this study was conducted using the mean, SD, unpaired Student t-test, analysis of variance, and χ2-tests by statistical package for the social sciences (SPSS, SPSS Inc., Chicago, Illinois, USA), version 20.0. Unpaired Student t-test was used to compare between two groups in quantitative data. χ2-Test is used to test the hypothesis that the row and column variables are independent, without indicating strength or direction of the relationship. Pearson chi-square and likelihood-ratio χ2 were used. Fisher’s exact test and Yates’ corrected χ2 are computed for 2×2 tables. analysis of variance test was used for comparison among different times in the same group in quantitative data.

P values more than 0.05 were considered statistically nonsignificant, P values up to 0.05 were considered statistically significant, and P values less than 0.001 were considered statistically highly significant [21].


  Results Top


Regarding clinical data of the studied groups, our study showed that no statistically significant differences were found among patients with RA and control group regarding age (P=0.402), whereas statistically significant differences were found among them regarding sex (P=0.043; [Table 1]).
Table 1 Clinical data of the studied groups

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Regarding descriptive data for RA group, this study showed that the duration of illness ranged from 2 to 17 years, with a mean of 9±3.56 years. Duration of morning stiffness ranged from 15 to 80 min, with a mean of 34.56±15.66 min. The number of tender joints ranged from 4 to 15 joints, with a mean of 8.83±2.82, and the number of swollen joints ranged from 1 to 6 joints, with a mean of 3.47±1.10. DAS28 of patients ranged from 3.31 to 5.79, with a mean of 4.67±0.51 ([Table 2]).
Table 2 Descriptive data for patients with rheumatoid arthritis

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Regarding laboratory data of the studied groups, our study showed that there were statistically significant differences among patients with RA and control group regarding their ESR, CRP, hemoglobin (Hb), and platelets (P<0.001), whereas no statistically significant differences were found regarding cholesterol, triglyceride, glucose, urea, creatinine, and red blood cells (RBCs) in comparison of the two groups (P≥0.05; [Table 3]).
Table 3 The laboratory data of the studied groups

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This study showed statistically significant differences among patients with RA and control group regarding RF and ACPA (P<0.001; [Table 4]).
Table 4 Comparison between rheumatoid factor and anti-citrullinated protein antibodies in the studied groups

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In this study, a statistically significant increase in the frequency of GC and CC polymorphic genotypes versus GG genotype (wild type) was observed among patients with RA when compared with control group (P<0.001). Moreover, a statistically significant difference was observed on comparing the frequency of C allele and T allele in both groups (P<0.001; [Table 5]).
Table 5 Interleukin-6 (−174G/C) genotypic and allelic frequencies in the studied groups

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There was a statistically significant difference on comparing the different IL-6 genotypes (GG, GC and CC) together regarding number of tender joints, number of swollen joints, and DAS28 (P<0.001). Regarding sex, there was a statistically significant difference on comparing GG with GC genotypes (P2=0.012), whereas no statistically significant difference on comparing GG with CC or GC with CC genotypes (P1 and P3=0.605 and 0.512, respectively). Regarding disease duration, there was a statistically significant difference on comparing GG with GC genotype (P2=0.040), whereas no statistically significant difference on comparing GG with CC or GC with CC genotypes (P1 and P3=0.207 and 0.903, respectively). Regarding morning stiffness, there was a statistically significant difference on comparing GG with GC genotype (P2=0.017), whereas there was no statistically significant difference on comparing GG with CC or GC with CC genotypes (P1 and P3=0.122 and 0.851, respectively). Regarding age, there was no statistically significant differences among the three groups together (P1, P2, and P3 values were 0.311, 0.584, and 0.572, respectively; [Table 6]).
Table 6 Comparison between interleukin-6 (−174G/C) genotypes regarding clinical data of the studied groups, and its P value

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There was a statistically significant difference on comparing the different IL-6 genotypes (GG, GC and CC) together regarding CRP and platelet count (P<0.001). There was no statistically significant difference on comparing the three genotypes together regarding serum cholesterol, triglyceride, fasting glucose, urea, creatinine, and total leukocytic count (TLC) (P>0.05). Regarding ESR, there was a statistically significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2≤0.001), whereas there was no statistically significant difference on comparing GC with CC genotype (P3=0.200).

Regarding Hb, there was a statistically significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2=0.028 and <0.001, respectively), whereas there was no statistically significant difference on comparing GC with CC genotypes (P3=0.735). Regarding RBCs, there was a statistically significant difference on comparing GG with GC genotypes, (P2=0.014), whereas there was no statistically significant difference on comparing GG with CC or GC with CC genotypes (P1 and P3=0.425 and 0.999, respectively; [Table 7]).
Table 7 Comparison between interleukin-6 (−174G/C) genotypes regarding laboratory findings of the studied groups, and its P value

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Moreover, on comparing IL-6 genotypes with RF and ACPA of the studied groups, regarding RF, there was a statistically significant difference on comparing GG with CC and GG with GC genotypes, (P1 and P2<0.001), whereas there was no statistically significant difference on comparing GC with CC genotype (P3=0.172). Regarding ACPA, there was a statistically significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2<0.001), whereas there was no statistically significant difference on comparing GC with CC genotype (P3=0.058; [Table 8]).
Table 8 Comparison between interleukin-6 (−174G/C) genotypes regarding rheumatoid factor and anti-citrullinated protein antibodies of the studied groups, and its P value

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  Discussion Top


RA is a serious, progressive disease characterized by chronic inflammation of synovial joints that frequently leads to functional disability, shortened life expectancy, and increased mortality [22].

IL-6 is a multifunctional cytokine with well-defined proinflammatory and anti-inflammatory properties. Although only small amounts in the picogram range can be detected in healthy humans, IL-6 expression is highly and transiently up-regulated in nearly all pathophysiological states [23].

In immunity, IL-6 has the ability to cause an imbalance between two types of T cells: Th17 and T regulatory [24]. Th17 is involved in the pathophysiology of RA, whereas the role of T regulatory is to inhibit T-cell activation and suppress proinflammatory cytokine production [5].

High concentrations of IL-6 in both synovial fluid and serum of patients with RA suggested a major role of this cytokine in the pathogenesis of RA, which is further confirmed by the recent introduction of the drug tocilizumab, a humanized monoclonal antibody targeting IL-6R [9]. IL-6 has a vital role in mediating inflammation, auto-antibody production, joint destruction, bone resorption, and also systemic manifestations of RA like anemia and thrombocytosis [25].

In this study, an association between a single nucleotide polymorphism at IL-6 (−174G/C) was found with patients with RA. The frequency of polymorphic genotypes GC and CC was statistically significantly increased in patients with RA compared with control group (P<0.001). However, the frequency of GG genotype was significantly increased in control group compared with patients with RA. This agree with Gaber et al. [20] who reported that the polymorphic genotypes GC and CC were statistically significantly increased in patients with RA compared with control group (P<0.001). Moreover, Jancic et al. [26] agree with our results and reported a statistically significant increased genotypic distribution of IL-6 (−174G/C) polymorphism among patients with RA in comparison with the control group (P<0.001).

In addition, Li et al. [27] agree with our results and found that GC and CC genotypes were statistically significantly increased in patients with RA than control group (P<0.001). In accordance with our results, Li et al. [28] revealed that the frequency of GC and CC genotypes was higher in patients with RA in comparison with the control group (P=0.049). The same results were found by Mosaad et al. [29] who found that IL-6 (−174CC) genotype has been reported to represent a marker of increased susceptibility, whereas GG genotype can be considered a low-risk genotype (P<0.001). Moreover, Lee et al. [30] showed that IL-6 (−174G/C) polymorphism may confer susceptibility to RA in Europeans (P=0.004), and this agrees with this study. Pawlik et al. [31] disagree with this study, who reported that the distribution of IL-6 genotypes in patients with RA did not differ from that in control group (P>0.05).

On studying the alleles frequency of IL-6 (−174G/C), C allele was higher in patients with RA compared with control group (P<0.001). Li et al. [27] agree with our results and found that C allele was statistically significantly increased in patients with RA than control group (P<0.001). The same results were found by Li et al. [28] reported that the C allele was statistically significant higher in patients with RA than control group (P=0.016). Contrary to this study, Marinou et al. [32] reported that the frequency of C allele was lower in patients with RA than control group and G allele was higher in patients with RA than control group (P=0.005).

In addition, Pascaul et al. [33] disagree with this study, who revealed that no statistically significant difference was observed in the distribution of IL-6 promoter genotype or allele frequency between patients with RA and control group (P>0.05). Moreover in disagreement with this study, Martin et al. [34] observed that there was no statistically significant difference in IL-6 promoter genotype or allele distribution between patients and control group (P>0.05).

In this study, regarding clinical and laboratory parameters, there was no statistically significant difference in mean age between the studied groups (P=0.402). This disagree with Bing et al. [35] who reported that there was a statistically significant difference in mean age between control group and patients with RA (P<0.05).

There was a statistically significant difference in sex distribution between patients with RA and control group (P=0.043), and according to IL-6 genotypes, there was a statistically significant difference in sex distribution in comparing GG with GC genotypes (P2=0.012). Contrary to this study, Bing et al. [35] found no statistically significant difference for the percentage of females between patients with RA and control group (P>0.05).

Regarding disease duration, there was a statistically significant difference in disease duration in comparing GG with GC genotypes (P2=0.040). Regarding the number of tender joints and swollen joints, there was a highly statistically significant difference on comparing the three groups together (P<0.001). This agree with Jancic et al. [26] who found that statistically significantly higher number of swollen joints were observed in patients with the IL-6 (−174CC) genotype compared with GG or GC genotypes (P<0.05).

Regarding CRP, DAS28, and platelet (PLT), there was a highly statistically significant difference on comparing the three groups together (P<0.001). Regarding ESR, there was a highly statistical significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2<0.001).

Regarding RF, there was a highly statistically significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2<0.001). This agrees with Pavkova et al. [36] who found that patients of the GC genotype had higher levels of RF without significance. Regarding ACPA, there was a highly statistical significant difference on comparing GG with CC and GG with GC genotypes (P1 and P2<0.001).

In accordance with this study, Gaber et al. [20] reported that the higher the frequency of promoter polymorphism GC and CC, the more the disease activity of the patients as assessed by DAS28 score, the more increased in platelet count, ESR, CRP and number of swollen and tender joints; all were statistically significantly increased (P≤0.001). In contrary to our results, some studies [26],[33],[37] reported that no statistically significant differences were found according to sex distribution, disease duration, DAS28, RF. and ACPA statuses on comparing the different IL-6 (−174) genotypes (P>0.05).


  Conclusion Top


The frequencies of GC and CC genotypes and C allele of IL-6 (−174G/C) were statistically significantly increased in patients with RA than control participants (P<0.001), which supports the relationship between IL-6 (−174G/C) polymorphism and the development of RA.

Recommendations

We recommend to study other polymorphisms of IL-6 gene and their relations to RA. Further studies on polymorphism of IL-6 gene will be helpful in indicating the association of this polymorphism with other health problems like diabetes and cancer.

Acknowledgements

The authors thank members of Al-Zahra University Hospital (Rheumatology Outpatient Clinic and Internal Medicine Department) and Medical biochemistry Department, Faculty of Medicine for Girls, Al Azhar University, for help with the preparation of this manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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