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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 1  |  Page : 23-28

Interleukin 17A gene polymorphism in alopecia areata


1 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Dermatology, Andrology and STDs, Ministry of Health, Giza, Egypt

Date of Submission20-Apr-2019
Date of Decision30-May-2019
Date of Acceptance01-Jun-2019
Date of Web Publication27-Mar-2021

Correspondence Address:
Samara M Azooz
Abas Riad Street, El Haram, Giza 12574
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_150_19

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  Abstract 


Objective
The aim of the current work was to investigate if interleukin 17A (IL-17A) G197A gene polymorphism confers susceptibility to alopecia areata (AA) in Egyptian patients.
Background
There is strong evidence for an autoimmune etiology of AA. IL-17A is a Th17 proinflammatory cytokine that has been linked to the pathogenesis of diverse autoimmune and inflammatory diseases.
Patients and methods
A total of 60 patients with AA were recruited, along with 40 healthy individuals who were matched for age and sex as a control group. IL-17A (G197A) gene polymorphism was investigated using restriction fragment length polymorphism PCR.
Results
Presence of A allele and AA genotype was significantly associated with AA cases compared with control group (P = 0.00 for both). A significant association was found between AA genotype and A allele and clinical data of the studied cases regarding disease severity, pattern of hair loss, nail changes, and course and response of disease to treatment (P = 0.00 for all).
Conclusion
IL-17A (G197A) gene polymorphism is associated with AA. Further large-scale studies are recommended to underscore and validate the current findings.

Keywords: alopecia areata, interleukin 17, polymorphism


How to cite this article:
Seleit IA, Mohamed OA, El-Hefnawy SM, Azooz SM. Interleukin 17A gene polymorphism in alopecia areata. Menoufia Med J 2021;34:23-8

How to cite this URL:
Seleit IA, Mohamed OA, El-Hefnawy SM, Azooz SM. Interleukin 17A gene polymorphism in alopecia areata. Menoufia Med J [serial online] 2021 [cited 2021 Aug 4];34:23-8. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/23/311994




  Introduction Top


Alopecia areata (AA) is an autoimmune, nonscarring, inflammatory disease of the hair follicle that causes loss of hair from the scalp and/or body. If the patient loses all of the hair on the scalp, the disease is called alopecia totalis. If all body hair is lost, the diagnosis becomes alopecia universalis [1]. The etiology of AA is not exactly known; however, genetic predisposition, autoimmunity, and environmental factors have been suggested to play roles [2]. There is evidence for a T cell-mediated autoimmune process directed against an unknown autoantigen of the hair follicle. Activated CD4+ perifollicular infiltrate, along with CD8+ intrafollicular infiltrate, coincides with hair loss during the active phase of the disease [3].

Several studies in the late 1990s have implicated interleukin 17 (IL-17) in the pathogenesis of autoimmunity. Elevated IL-17 levels were found in patients with rheumatoid arthritis, systemic lupus erythematosus, and psoriasis, although it was not clear from these studies how important a role it may play in disease pathogenesis [4]. IL-17 is a potent proinflammatory cytokine that is produced by activated T lymphocytes. Th17 cells are a subset of CD4+ T lymphocytes named after their hallmark cytokine IL-17 [5], which represents the prototype of a cytokine family comprising six members (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F) and five receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE) [6]. Among IL-17 family, IL-17A and IL-17F are the most researched cytokines responsible for the pathogenic activity of Th17 cells [7].

The most important role of IL-17 is induction and mediation of proinflammatory responses [8]. IL-17 promotes inflammation by inducing local chemokine and cytokine secretion [9] and is essential for the clearance of certain pathogens such as bacteria and fungi. Moreover, IL-17 has been linked to the instigation of immune-mediated organ damage in the context of several autoimmune diseases [6].

Recently, much data have accumulated on the possible role of IL-17 in the pathogenesis of AA. IL-17 levels are consistently reported to be elevated in the serum and lesional skin of patients with AA, and there is no clear connection between IL-17 levels and disease severity or duration. Some evidence has suggested an association between IL-17 and early-onset disease, although this awaits further confirmation [10].

Single nucleotide polymorphism (SNP) is an important type of gene mutation that affects gene regulation by altering transcription and translation by inducing abnormal expression of protein [11].

Some SNPs of Th17 genes have been associated with several diseases and conditions. Of these, IL-17A G197A is the most studied, being located in the promoter region of the IL-17A gene and described as a functional polymorphism, or capable of altering the function of the gene [12].

The purpose of this study is to investigate the association between a IL-17 gene polymorphism and AA to explore if it increases disease risk or influence its clinical pattern.


  Patients and methods Top


Studied population

This case-control study was carried out on 60 patients with AA and 40 healthy individuals matched for age and sex, who have no present, past, or family history of AA, as a control group. Selected control individuals had no blood relationship with the studied patients.

Patients were selected during the period between March 2015 and October 2015. Written consent forms approved by the local ethical research committee were obtained from the studied cases and control subjects before the study initiation. All studied patients were subjected to complete history taking and general and dermatological examination.

Grading of AA was done according to Kavack et al. [13]: mild = the presence of three or less patches of alopecia with a widest diameter of 3 cm or less, or the disease is limited to eye lashes and/or eyebrows, moderate = existence of more than three patches of alopecia or a patch greater than 3 cm at the widest diameter without AT or AU, and severe = AT or AU, with snake-shaped plaques extending on the scalp border or hair loss in a wave form at the circumference of the head known as ophiasis.

Exclusion criteria

Any case or control subject who had dermatologic diseases other than AA or presented with a history of systemic or autoimmune diseases was excluded.

DNA extraction and amplification

Every case and control subject underwent detection of IL-17A gene polymorphism (G197A). DNA was extracted from the whole blood using Qiagen (QIAGEN Inc., Germantown, MD, USA)Genomic DNA purification kit (Qiagen, USA). DNA was eluted and stored at −20°C for PCR procedure. PCR for the IL-17A (G197A) gene was carried out to a total volume of 25 μl, containing 10 μl of genomic DNA; 1 μl of each primer (forward and reverse); 12.5 μl of Master Mix using Thermo Scientific Dream Taq Green PCR Master Mix (Thermo Scientific, USA); and 0.5 μl of distilled water.

The G197A polymorphism in IL-17A gene was analyzed using the following designed primers (Midland, Texas, USA):

Forward: 5′AACAAGTAAGAAT

GAAAAGAGGACATGGT -3′.

Reverse: 5′- CCCCCAATGAGGTC

ATAGAAGAATC -3′.

PCR amplification for IL-17A gene was performed separately using Applied Bio systems 2720 thermal cycler (Applied Bio System, Singapore, Singapore,). PCR condition consisted of the following: one cycle of amplification at 94°C for 5 min followed by 38 cycles at 94°C for 45 s, 58°C for 45 s, 72°C for 1 min, and one final cycle of extension at 72°C for 10 min.

The amplification products were separated by electrophoresis through 3% agarose gel stained with ethidium bromide and visualized on transilluminator with positive band at 102 bp.

IL-17A (G197A) gene polymorphism was detected using restriction fragment length polymorphism technique, where 10 μl of the PCR products for the IL-17A was mixed with 1 μl (1 U) of FastDigest XagI (Fermentas, Lithuania) restriction enzyme with 6.5-μl nuclease-free water and 2.5 of 10× FastDigest Buffer. The mixture was mixed well and incubated for at 37°C for 2 h then 10 μl of the products was loaded onto a 3% agarose gel containing ethidium bromide for electrophoresis. The uncut fragment was 102 bp and digestion products were 68 bp and 34 bp [Figure 1].
Figure 1: (a) IL-17A (G197A) gene. Lanes from 2 to 11 show the length of the PCR amplicon which is 102 bp. Ladder 50 bp was used. (b) For IL-17A (G197A) gene polymorphism, the uncut fragment was 102 bp and digestion products were 68 and 34 bp. Ladder 50 bp was used. Lanes 2, 3, 4, 6, 8, 10, and 12 show AA homozygote genotype at 102 bp. Lane 11 shows AG heterozygote genotype at 102, 68, and 34 bp. Lanes 5, 7, and 9 show GG homozygote genotype at 68 and 34 bp. AA, alopecia areata; IL-17A, interleukin 17A.

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Statistical analysis

Data were collected, tabulated, and statistically analyzed using a personal computer with SPSS IBM. SPSS statistics for Windows. Version 21.0. Armonk, NY: IBM; 2012, version 11 program. Fisher's exact test was used for comparison of qualitative variables in 2 × 2 tables when expected cell count of more than 25% of cases was less than 5. χ2 test was used to study the association between two qualitative normally distributed variables. Mann–Whitney U test was used for comparison between two groups not normally distributed having quantitative variables. Odds ratio (OR) was used to describe the probability that people who are exposed to a certain factor will have a disease compared with people who are not exposed to the factor. Differences were considered statistically significant at P value less than 0.05.


  Results Top


Clinical characteristics of studied groups were as follows: age of the selected cases ranged from 6 to 55 years, with a mean ± SD of 30.22 ± 9.9 years. Age of onset ranged from 3 to 49 years, with a median age of 26 years and a mean ± SD of 26.97 ± 9.45 years. The disease course was progressive in 34 (56.7%) cases, regressive in 17 (28.3%) cases, and stationary in nine (15%) cases. Disease duration varied in the studied cases from 2 to 144 months, with mean ± SD of 35.13 ± 38.45 months. Family history of AA was positive in 11 (18.3%) cases. AA and severity index varied between mild in 20 (33.3%) cases, moderate in 19 (31.7%) cases, and severe in 21 (35%) cases. AA types were patchy in 39 (65%) cases, ophiasis in seven (11.7%) cases, totalis in seven (11.7%) cases, and universalis in seven (11.7%) cases. The site of AA was scalp in 48 (80%) cases and scalp and other body sits in 12 (20%) cases. Nail changes were found in 21 (35%) cases. Response to ttt was present in 43 (71.7%) cases.

Prevalence of alleles and genotypes in cases and controls was as follows: GG genotype was more prevalent among control group than patients with AA (60 vs. 20%). Polymorphism AA genotype was significantly associated with patients with AA (50 vs. 25%). Mosaic AG genotype was significantly associated with patients with AA (30 vs. 15%). AA and AG genotypes were associated with increase in the risk of occurrence of AA [OR 6.00, 95% confidence interval (CI) 2.21–16.24, P = 0.000, and OR 6.00, 95% CI 1.89–19.04, P = 0.00, respectively]. Regarding alleles, G allele was more prevalent among control than patient group (66.25 vs. 35%). Allele A was significantly associated with patient group than control (65 vs. 33.75%). Allele A increased the risk of occurrence of AA (OR 3.65, 95% CI 2.01–6.62, P = 0.00) [Table 1].
Table 1: Comparison between cases and control subjects regarding interleukin 17A (G197A) alleles and genotypes

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Relationship between genotypes and alleles and clinical data of the cases was as follows: no significant association was found between both genotypes and alleles and clinical data of the studied cases regarding site of affection, recurrence, criteria of recurrence, and family history.

GG genotype and G allele were significantly associated with type of AA (100% patchy for both), severity of the disease (100% mild for GG genotype and 66.7% mild for G allele), course of the disease (75% regressive for GG genotype and 50% regressive for G allele), and response to treatment (100% responded to treatment for GG genotype and 86.7% for G allele).

AA genotype and A allele were significantly associated with severity of the disease (70% severe for AA genotype and 43.8% severe for A allele), course of the disease (83.3% progressive for AA genotype and 70% progressive for A allele), nail changes (60% had nail changes for AA genotype and 43.8% for A allele), and response to ttt (56.7% responded to treatment for AA genotype and 64.6% responded to treatment for A allele). A allele was significantly associated with type of AA (56.3% [Table 2] and [Table 3].
Table 2: Relation between genotypes and clinical data of cases

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Table 3: Comparison between alleles of patients with alopecia areata and their clinical data

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  Discussion Top


A polymorphism is a genetic variant that appears in at least 1% of the population. These genetic changes can occur in noncoding parts of the gene, so they would not be seen in the protein product. Changes in these regulatory parts of the gene would then affect the degree of expression of the gene, and thus the levels, functions, or stability of the protein. The association of a certain polymorphism with a phenotype or disease does not necessarily mean that the polymorphism is causing it [14]. To the best of our knowledge, this is the first case–control study to report significantly association between IL-17A (G197A) gene polymorphism and AA. This further substantiates preliminary observations pointing to the possible role of IL-17 in this disease [15].

IL-17 is the key product of Th17 cells; thus, our findings confirm an upregulated Th17 profile in patients with AA. Th17 cells are considered to be pathogenic forms of CD4+ T cells driven by IL-23. In addition to IL-17, Th17 cells produce IL-6 and tumor necrosis factor upon antigen-specific stimulation. These cytokines are essential for the establishment of organ-specific inflammation associated with autoimmunity [16], mainly through the pleiotropic activity of IL-17 on fibroblasts, keratinocytes, endothelial cells, neutrophils, and memory T cells [17], which is mediated by the release of various cytokines and chemokines [18].

Owing to the lack of reports concerning the role of IL-17 allelic variant in AA, we aimed to investigate IL-17 gene polymorphism (G197A) in a sample of 60 Egyptian cases and 40 matched healthy control subjects.

By evaluating the distribution of the rs2275913 SNP in cases and controls, we found significant differences in both allelic and genotypic frequencies. The AA and GA genotypes and A allele were significantly more common among the patients with AA compared with the control subjects, whereas the GG genotype and G allele were significantly more common among the control subjects.

These results suggest that AA and GA genotypes and A allele of IL-17 (G197A) may be involved in the etiopathogenesis of AA and may be associated with an increased predisposition to the disease (OR 6.00, 95% CI 2.21–16.24, P = 0.00; OR 6.00, 95% CI 1.89–19.04, P = 0.00; and OR 3.65, 95% CI 2.01–6.62, P = 0.00, respectively).

This role was suggested to be through the effect of IL-17A polymorphism on IL-17 secretion, as SNP of IL-17A, the rs2275913 G>A polymorphism (G197A), has been identified in the upstream variant 2KB region of IL-17A [19]. It is located within a binding motif for the nuclear factor of activated T cell, a critical regulator of IL-17 expression. Therefore, it is conceivable that this SNP might influence the transcriptional regulation of IL-17 [20]. It has been reported that upon stimulation peripheral blood mononuclear cells with variant genotypes (197AG or 197AA) secreted significantly more IL-17 than the wild-type (197GG) cells [21]. Kubo et al. [22] showed that allele 197A correlates with more efficient IL-17 secretion, and that this resulted from its higher affinity for nuclear factor of activated T cell.

Contrary to our results, Lew et al. [4] reported no significant association between IL-17A gene polymorphism and any of the disease parameters studied. This controversy can be explained by different sample size, different clinical data of selected cases, and geographic differences.

When patients with AA in the present study were compared according to clinical data, the IL-17 (G197A) AA genotype and A allele were significantly associated with type of AA (patchy AA), severity, progressive course of the disease, nail changes, and the response to treatment (P = 0.00 for all).

Tojo et al. [23] reported a positive correlation between the severity and progression of alopecia and the increase in the number of interferon-g-producing Th1 cells or IL-17-producing Th17 cells. A recently published study by Ferreira et al. [24] reported that the percentage of IL-17-producing cells in acute, diffuse, and total alopecia was significantly lower than that in multiple patchy AA. Association with nail changes may be explained by the fact that nail changes are more frequently seen in the severe forms of AA [25] and IL-17 (G197A) AA genotype and A allele in our study were associated with severe cases.


  Conclusion Top


From the aforementioned findings, we can conclude that IL-17 (G197A) polymorphism is associated with increases in the risk of AA. Furthermore, larger prospective studies to underscore and validate current findings are needed. It is necessary also for future studies to extend the research to other populations and ethnicities. Investigating other IL-17 gene polymorphisms in AA is also warranted. As AA is a multifactorial disease, gene–gene and gene–environment interactions should be considered in future geneticepidemiologic researches to provide more comprehensive insight into the role of IL-17 gene in its pathogenesis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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