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Study of interleukin-17 gene polymorphism and susceptibility to vitiligo in a sample of the Egyptian population

Mohammed, Faisal N.; Sayed Amr, Khalda; Abdel Raheem, Heba M.; Shehata, Hany; ElRasheedy, Maged; Ramzy Elias, Thany; Agaiby, Mervat

Journal of the Egyptian Women's Dermatologic Society: January 2017 - Volume 14 - Issue 1 - p 45–48
doi: 10.1097/01.EWX.0000503399.60863.9f
Original articles
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Background Vitiligo is a common, acquired disorder resulting in depigmentation due to loss of functional melanocytes. The role of the immune system in the pathogenesis of the disease has been studied and hypothesized shifting toward Th17 response with the production of various inflammatory cytokines including interleukin (IL)-17. IL-17 gene polymorphism has been associated with various autoimmune disorders but, to our knowledge, has not been studied before in vitiligo.

Objective To investigate the association of IL-17 gene polymorphism and susceptibility to vitiligo.

Patients and methods IL-17 genotyping was performed for 75 patients with vitiligo and 93 healthy controls using the restriction fragment length polymorphism PCR technique. Serum level of IL-17 was also estimated in 30 of these patients and 40 controls using enzyme-linked immunosorbent assay technique.

Results No significant difference was found between the different genotypes of IL-17 gene (GG, AG, and AA) in vitiligo patients and controls (P=0.250, 0.102, and 0.128, respectively). Moreover, we did not find any significant relation between IL-17 gene polymorphism and any of the clinical aspects of the disease. However, a significant difference was found between serum levels of IL-17 in patients and controls (P=0.003) with a significant negative correlation with age (r=−0.446, P=0.013) and duration of disease (r=−0.439, P=0.015).

Conclusion IL-17 gene polymorphism is not associated with an increased susceptibility to vitiligo, although IL-17 plays an important role in the pathogenesis of the disease.

aDepartment of Dermatology, National Research Center

bDepartment of Molecular Genetics, National Research Center

cDepartment of Dermatology, Cairo University

dDepartment of Medical Biochemistry, National Research Center, Cairo, Egypt

Correspondence to Heba M. Abdel Raheem, MD, 52, Mohamed Mandour Street, Nasr City, PO 11759, Cairo, Egypt Tel: +20 100 241 4104; e-mail: heba.abdelraheem@kasralainy.edu.eg

Received April 16, 2016

Accepted September 23, 2016

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Introduction

Vitiligo is an idiopathic skin disease characterized by selective destruction of melanocytes leading to depigmentation 1 in the form of milky white macules or patches 2. It is frequently associated with autoimmune disorders 3. Research hypotheses for its pathogenesis suggest an inherent defect in the melanocyte, altered development of the peripheral nervous system, or a dysregulation of the immune response 4.

Previous studies have postulated that shifting of the immune system toward Th1 and Th17 responses might be responsible for the development of autoimmune diseases 5. Th17 cells produce interleukin (IL)-17 and IL-6 6. Serum and tissue levels of IL-17 have been shown to be increased in vitiligo patients as compared with controls 7,8, postulating that it might play a role in the pathogenesis of the disease. IL-17 synergizes with other local inflammatory cytokines to inhibit melanocyte proliferation 8.

IL-17 gene polymorphism has been shown to be associated with a number of autoimmune diseases, including asthma, rheumatoid arthritis, and inflammatory bowel disease 9–11. To our knowledge, no previous studies have investigated IL-17 gene polymorphism in vitiligo.

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Patients and methods

Seventy-five patients with vitiligo (and no history of any other autoimmune disease) were included in this case–control study. They were selected from the Dermatology Outpatient Clinic at the Faculty of Medicine, Cairo University, during the period from December 2014 to March 2015. Ninety-three age and sex matched healthy volunteers were included as a control group. Informed written consent from every participant and the approval of the dermatology Research Ethics Committee of the Faculty of Medicine, Cairo University was obtained before enrollment in this study.

All patients were subjected to complete history taking including onset, course, and duration of the disease, activity (as determined by appearance of new lesions in the preceding 6 months 12), relation to stress, and personal and family history. Clinical examination was carried out to determine the skin type of the patient, as well as the type and extent of vitiligo.

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DNA extraction

Two ml of venous blood samples was collected in the morning into EDTA vacutainer tubes for genomic DNA extraction. Extraction of genomic DNA from whole blood using purification of spin column QIA Gene Extraction Kit (Qiagen, Hilden, Germany) was carried out.

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Genotyping of interleukin-17 (A197G) polymorphism

Restriction fragment length polymorphism PCR analysis was used to amplify the IL-17 polymorphism (A197G). The following primers were used to amplify the target fragment containing polymorphism: IL-17 (−197A/G) rs2275913 forward primer: 5′-CAGAAGACCTACATGTTACT-3′, reverse primer: 5′-GTAGCGCTATCGTCTCTCT-3′. PCR was performed in a total volume of 25 µl using 10 pmol of each primer, 1.5 mmol/l HgCl2, 200 µmol/l dNTPs, and 2 U of Taq DNA polymerase. The conditions were as follows: 35 cycles, each consisting of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. The reaction cycles were preceded by 5 min denaturation at 94°C and were followed by 7 min extension at 72°C. The PCR product of 344 bp was digested with 10 U of Xmn1 restriction enzymes (Fermentas, Vilnius, Lithuanias) at 37°C and yielded 213 and 131 bp digested products. The PCR fragments’ sizes were analyzed on a 2% agarose gel stained with ethidium bromide.

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Measurement of serum interleukin-17 level

Five ml of venous blood samples was collected in vacuum tubes under sterile conditions from both patients and controls. Serum was rapidly separated by means of centrifugation and stored at −70°C until processed. Serum IL-17 concentration was estimated using enzyme-linked immunosorbent assay with a kit obtained from R&D Systems Inc. (Minneapolis, Minnesota, USA) according to Cua and Tato 13. Standards and samples were placed into the wells, and any IL-17 present in the sample was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman IL-17 antibody was added, followed by horseradish-conjugated streptavidin. The wells were again washed, tetramethylbenzidine substrate solution was added, and color developed in proportion to the amount of IL-17 bound. The intensity of the color was measured at 450 nm.

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Statistical analysis

Quantitative data were statistically represented in terms of minimum, maximum, and median. Comparison between different groups in the present study was made using the Mann–Whitney test for comparing two nonparametric groups and the Kruskall–Wallis test was used when comparing between more than two nonparametric groups. Qualitative data were statistically represented in terms of number and percent. Comparison between different groups was made using the χ2-test. Correlation between various variables was made using Spearman rank correlation coefficient (R) with graphic representations using linear regression. A P value less than or equal to 0.05 was considered significant. All statistical analyses were performed using statistical software SPSS statistical program (SPSS Inc., Illinois, Chicago, USA). Graphs were obtained using SPSS statistical program, version (16.0).

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Results

This study was conducted on 75 patients with vitiligo, 28 (37.3%) male and 47 (62.7%) female, whose ages ranged from 15 to 65 years with a median of 26 years. Ninety-three age and sex matched healthy individuals (P=0.704 and 0.086, respectively) were included as controls, 23 (24.7%) male and 70 (75.3%) female, whose ages ranged from 13 to 57 years with a median of 34 years. Sixty-three (84%) patients had vitiligo vulgaris, six (8%) patients had acrofacial vitiligo, and six (8%) patients had segmental vitiligo. Twenty-nine patients were of skin type III (38.7%), 45 (60%) patients were of skin type IV, and one (1.3%) patient was of skin type V. The duration of the disease ranged from 0.5 to 35 years with a median of 3.5 years. The extent of body involvement ranged from 1 to 80% with a median of 30%. The disease was active in 36 (48%) patients and stable in 39 (52%) patients. Fifteen (20%) patients had positive family history and 60 (80%) patients had negative family history. Forty-one (54.7%) patients reported relation of the disease activity to stress, whereas 34 (45.3%) patients reported no relation to stress.

Serum level of IL-17 was measured in 30 patients and 40 healthy controls. In patients it ranged from 9.9 to 112.5 pg/ml with a median of 14.18 pg/ml, whereas in controls it ranged from 2.1 to 21.73 pg/ml with a median of 12.02 pg/ml with a statistically significant difference (P=0.003). A significant negative correlation was found between serum IL-17 and both the age of patients and the duration of the disease (r=−0.446, P=0.013, and r=−0.439, P=0.015, respectively) (Figs 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

As regards IL-17 gene polymorphism, no statistically significant difference was found between patients and controls in the different genotypes. GG genotype was found in 22 (29.30%) patients and 15 controls (16.10%) (P=0.250), AG genotype was found in 40 (53.30%) patients and 56 controls (60.20%) (P=0.102), and AA genotype was found in 13 (17.30%) patients and 22 controls (23.70%) (P=0.128) (Table 1). No relation was found between IL-17 gene polymorphism and any of the clinical parameters of the disease, including type of vitiligo (P=0.543), disease activity (P=0.861), relation to stress (P=0.22), presence of family history (P=0.126), or the patient’s skin type (P=0.446).

Table 1

Table 1

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Discussion

Vitiligo is an acquired hypopigmentary disorder characterized by progressive loss of melanocytes. Vitiligo occurs worldwide with an estimated prevalence of 0.5–1%. In almost half of the patients, vitiligo starts before the age of 20 years with no significant sex difference 14.

Several theories have been proposed trying to explain the pathogenesis of vitiligo, including genetic, autoimmune, oxidant–antioxidant, autocytotoxic, neural, viral, and ultraviolet radiation theory for destruction of epidermal melanocytes 15. The autoimmune hypothesis is supported by the association of vitiligo with other autoimmune disorders and that it responds to immunosuppressive treatment 16. Autoantibodies directed against several melanocyte antigens such as tyrosinase and tyrosinase-related protein 1 and tyrosinase-related protein 2 have been found in the sera of patients with vitiligo 17.

Various cytokines have been found to be significantly increased in the sera of vitiligo patients, including tumor necrosis factor-α (TNF-α) and interferon-γ, suggesting that vitiligo is mediated by the Th1 response 18. The role of IL-17, a cytokine produced by the Th17 subset, in the pathogenesis of vitiligo has been investigated in several studies. A study by Bassiouny and Shaker 7 found elevated levels of IL-17 in both serum and tissue of vitiligo patients. IL-17 is a family of cytokines including six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F 19. Th17 cells produce IL-17A and IL-17F under the influence of transforming growth factor-β and IL-6. IL-17 is overexpressed in several autoinflammatory disorders including rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. IL-17 synergizes with other local inflammatory mediators such as IL-1b, IL-6, and TNF-α to inhibit melanocyte proliferation 6,20.

On the basis of the growing evidence of the involvement of IL-17 in the pathogenesis of vitiligo, we attempted to investigate whether there is an association between IL-17 gene polymorphism and susceptibility to vitiligo in Egyptian patients. A previous study was conducted on patients with Vogt–Koyanagi–Harada syndrome and found a significantly higher frequency of the GG genotype in patients than in controls (P=0.005) 21. However, to our knowledge, IL-17 gene polymorphism was not studied before in vitiligo patients.

We assessed the three different genotypes (GG, AG, and AA) for the IL-17 gene in 75 Egyptian vitiligo patients and 93 healthy controls using the restriction fragment length polymorphism PCR technique. We did not find any significant difference between any of the genotypes in patients and controls. We also did not find any relation between the IL-17 genotype and any of the clinical parameters of the disease. This might be explained by the fact that vitiligo is a multifactorial disease influenced by factors other than genetics. All factors work together synergistically to produce the disease, and no disease is like the other. IL-17 polymorphism might be associated with diseases in which the genetic component takes the upper hand. However, further studies are needed to confirm our findings.

We also measured the serum level of IL-17 in 30 patients and 40 controls. We found a significantly higher level in patients than in controls (P=0.003). These results are in agreement with those of Bassiouny and Shaker 7 and Basak et al. 8. We found a significant negative correlation between serum IL-17 and the age of patients (r=−0.446, P=0.013), which was also similar to their results. However, unlike their findings, a significant negative correlation was found between serum IL-17 and the duration of the disease (r=−0.439, P=0.015). Osman et al. 22 also did not find a significant correlation between IL-17 level and disease duration in vitiligo. Both negative correlations imply that IL-17 might play a role in the early onset and early stages of the disease, which highlights its importance as a possible triggering factor in an already predisposed individual, and this supports the significance of the Th17 immune response shift in vitiligo.

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Conclusion

The results of this study negate the association of IL-17 gene polymorphism with susceptibility to vitiligo. However, they support the importance of IL-17 in the pathogenesis of the disease and that autoimmune disorders might result from shifting of the immune system toward Th17 response. Studies on a larger number of patients from different ethnicities are recommended to further investigate this association.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

interleukin-17; polymorphism; vitiligo

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