Investigation of interleukin-12,interleukin-17 and interleukin-23 receptor gene polymorphisms in alopecia areata

Abstract

Objective

To investigate the distribution of interleukin (IL)-12 (IL12; 1188A/C), IL17 (A7488G) and IL-23 receptor (IL23R; +2199A/C) gene polymorphisms in patients with alopecia areata.

Methods

Patients with alopecia areata and healthy controls were enrolled in this case–control study. Genotyping of the IL12 (1188A/C), IL17 (A7488G) and IL23R (+2199A/C) polymorphisms was undertaken. Genotype frequencies were compared between the two groups.

Results

The study enrolled 100 patients with alopecia areata and 71 control subjects. No significant differences were found in the frequencies for the IL12 and IL23R gene polymorphisms between the patient and control groups. The IL17 GG genotype was significantly more common and the IL17 GA genotype was significantly less common in patients with alopecia areata compared with controls, but only 10% of patients had the GG genotype.

Conclusion

The IL17 GG genotype was associated with susceptibility for alopecia areata, but this genotype was only present in a small number of patients. The IL12 and IL23R gene polymorphisms were not found to have a significant association with alopecia areata.

Keywords

Alopecia areata polymorphism interleukin-12 interleukin-17 interleukin-23 receptor

Introduction

Alopecia areata is an autoimmune, nonscarring, inflammatory disease of the hair follicle that causes loss of hair from the scalp and/or body.¹ If the patient loses all of the hair on the scalp, the disease is called alopecia totalis.¹ If all body hair is lost, the diagnosis becomes alopecia universalis.¹ The aetiopathogenesis of alopecia areata is unclear. Alopecia areata is considered to be an organ-specific autoimmune disorder that is associated with a genetic predisposition and environmental triggers, and is mediated by T lymphocytes.^1–3 Genetic factors play important roles in the development of alopecia areata, and the incidence of a positive family history ranges between 10 and 42% among patients with alopecia areata.^4,5

In alopecia areata, an inappropriate immune response is regarded as possibly resulting from an event that promotes the inflammatory process.¹ Defects in cytokine production or signalling may lead to autoimmune disorders and may occur in parallel with the cascade of events involved in the development of alopecia areata.³ Interleukin (IL)-1 is known to be a major trigger for hair loss.^2,3 An aberrant expression of IL-1 in the early stages of alopecia areata suggests a potential negative effect of this cytokine on hair growth.³ Research has demonstrated an association between interferon-γ, IL-5, IL-6 and IL-16 and the pathogenesis of alopecia areata.¹

Interleukin-12 is a cytokine that plays major roles in both natural and acquired immunity. It is produced by several types of cells including primarily B cells, dendritic cells and macrophages, and is involved in the stimulation of cytokine production and the differentiation of various inflammatory cells.^3,6

Interleukin-17 is secreted mainly by T lymphocytes and several other inflammatory cells. IL-17 is involved in the production of proinflammatory cytokines, chemokines, cell adhesion molecules and growth factors.^2,3 IL-17 expression has been shown to be increased in certain autoimmune disorders including asthma, systemic lupus erythematosus and inflammatory bowel disease.^7–9

The IL-23 receptor complex is composed of two different chains – IL-12R1 and IL-23R.^10,11 IL-23 mediates inflammation and autoimmunity by stimulating the proliferation of helper 17 T cells that produce proinflammatory cytokines.¹¹

The present case–control study investigated the relationship between single nucleotide polymorphisms (SNPs) of the IL12 (1188A/C), IL17 (A7488G) and IL23R (+2199A/C) genes and clinical parameters in patients with alopecia areata.

Patients and methods

Study population

This case–control study recruited consecutive patients clinically diagnosed with alopecia areata, admitted to the Department of Dermatology, Medical School, Gaziantep University, Gaziantep, Turkey between April 2012 and March 2013. The study also recruited healthy control subjects without a personal or family history of alopecia areata or any inflammatory disorder. Healthy control subjects were recruited from a clinic in the Department of Dermatology, Medical School, Gaziantep University that they were attending for routine examinations between April 2012 and March 2013. There were no exclusion criteria for this study.

The diagnosis of alopecia areata was based on patient history and clinical examination. During history taking, age, sex, disease duration, autoimmune disorders coexisting with alopecia areata, family history of alopecia areata and atopic state (atopic dermatitis, allergic rhinitis, asthma) were recorded. In addition to loss of scalp hair, loss of eyelashes, eyebrows, beard or body hair, nail changes and nevus flammeus were also evaluated. The percentage scalp hair loss was estimated using the Severity of Alopecia Tool (SALT) score among patients with scalp involvement.¹² The severity of scalp disease was classified as mild (S1, S2) and severe (S3, S4, S5).

Approval was obtained from the Ethics Committee of the Medical School, Gaziantep University before initiation of the study (approval dated: 13.03.2012; no. 13.03.2012/93). Oral and written information about the study was provided to all patients and healthy controls, and their written informed consent was obtained.

DNA isolation and genotyping

All study participants provided 2 ml of venous blood from the antecubital vein, which was added to 1% ethylenediaminetetra-acetic acid tubes in preparation for DNA isolation. Blood samples were stored at −20℃ until DNA extraction. DNA was extracted from peripheral blood samples using a salting-out procedure.¹³ The polymorphisms of IL12 +1188A/C, IL17 +7488A/G and IL23R +2199A/C were determined using a polymerase chain reaction–restriction fragment length polymorphism method, as described previously.^14–16 Primer sequences used in the present study are shown in Table 1 (Bio Basic, Ontario, Canada).^14–16 Each PCR reaction mix comprised 55 ng genomic DNA, 200 µM dNTP, 1U Taq DNA polymerase (Fermentas AB, Vilnius, Lithuania) and 100 µM primers, in a total volume of 25 µl. The cycling programme involved preliminary denaturation at 95℃ for 5 min, followed by 32 cycles of denaturation at 94℃ for 45 s, annealing at 60℃ for 45 s, and elongation at 72℃ for 45 s, followed by a final elongation step at 72℃ for 7 min. PCR products were analysed by 2% agarose gel electrophoresis and visualized using ethidium bromide staining and ultraviolet light. PCR products of IL12 +1188A/C, IL17 +7488A/G and IL23R +2199A/C were 200 (138/62) base pairs (bp), 143 (80/63) bp and 215 (154/61) bp, respectively. To verify reproducibility, 20% of samples were analysed twice, to serve as an internal quality control to avoid sampling or reading errors.

Table 1.

Primer sequences for the polymerase chain reaction amplification of polymorphic sites of the IL12, IL17 and IL23R genes.

Gene polymorphism	Primer sequence	Restriction enzymes and conditions
IL12	F: 5′-TTTGGAGGAAAAGTGGAAGA-3′	Taq1 and 37℃ incubation
1188 A/C	R: 5′-AACATTCCATACATCCTGGC-3′	Taq1 and 37℃ incubation
IL17	F: 5′-ACCAAGGCTGCTCTGTTTCT-3′	NlaIII and 37℃ incubation
7488 T/C	R: 5′-GGTAAGGAGTGGCATTTCTA-3′	NlaIII and 37℃ incubation
IL23R	F: 5′-AGGGGATTGCTGGGCCATAT-3′	Mnl1 and 37℃ incubation
+2199 A/C	R: 5′-TGTGCCTGTATGTGTGACCA-3′	Mnl1 and 37℃ incubation

F, forward; R, reverse.

Statistical analyses

All statistical analyses were performed using the SPSS® statistical package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Differences between patients and controls were estimated using a logistic regression analysis. Odds ratios (OR) in the logistic regression model were corrected for age and sex, and their associated 95% confidence intervals were estimated. Differences between patients and controls in frequencies of IL12, IL17 and IL23R alleles were compared using χ²-test or Fisher’s exact test as appropriate. Additionally, expected and observed genotype frequencies were estimated using the Hardy–Weinberg equilibrium. A P-value ≤0.05 was considered statistically significant.

Results

The study enrolled 100 patients (75 male; 75.0%) with clinically diagnosed alopecia areata with a mean ± SD age of 24.2 ± 4.3 years (range 6–60 years). The mean ± SD age at disease onset was 22.2 ± 6.4 years (range 1–60 years). Those patients with an age at disease onset at <30 years of age accounted for 73.0% (n = 73) of the patient population. The healthy control group was similar in terms of age and sex distribution; with 71 healthy individuals (43 male; 60.5%) who did not have a personal or family history of alopecia areata or any inflammatory disorder. The mean ± SD age of healthy control subjects was 25.3 ± 3.8 years (range 10–63 years). Clinical characteristics of the study groups are shown in Table 2.

Table 2.

Clinical and demographic characteristics of patients with alopecia areata and healthy control subjects included in a study investigating the association between single nucleotide polymorphisms of IL12, IL17 and IL23R genes and risk of alopecia areata.

Characteristic	Patients with alopecia areata n = 100	Control subjects n = 71
Age, years	24.2 ± 4.3	25.3 ± 3.8
Age range, years	6–60	10–63
Sex, male/female	75/25	43/28
Age <30 years at disease onset	73 (73)
Age at disease onset, years	22.2 ± 6.4
Age range at disease onset, years	1–60
Family history of alopecia	16 (16)
Autoimmune disorder	17 (17)
Atopy	11 (11)
Alopecia areata type
Patchy	84 (84)
Alopecia totalis	2 (2)
Alopecia universalis	14 (14)
Nail involvement	15 (15)
Nevus flammeus	3 (3)
Ophiasis	2 (2)

Data presented as mean ± SD or n (%) of study participants.

Patients were divided into three groups based on the clinical type of the disease: 84 patients (84.0%) had patchy alopecia areata, two (2.0%) had alopecia totalis and 14 (14.0%) had alopecia universalis. 77 patients with scalp involvement were grouped into the following classes based on their SALT scores: 49 S1 patients (63.6%), 16 S2 patients (20.8%), six S3 patients (7.8%), one S4 patient (1.3%) and five S5 patients (6.5%). As for the severity of the scalp disease, 65 patients (84.4%) were found to have mild (S1, S2) and 12 (15.6%) had severe (S3, S4, S5) scalp involvement.

Seventeen patients (17.0%) had one or more of coexisting autoimmune disorders including psoriasis in one patient, thyroid disease in six patients, type 1 diabetes mellitus in two patients, rheumatoid arthritis in one patient, urticaria in three patients, urticaria + vitiligo + goitre in one patient, urticaria + hypothyroidism in two patients and type 1 diabetes mellitus + hypothyroidism in one patient.

There was no significant difference in genotypes of the IL12 (1188A/C) gene polymorphism between the patients with alopecia areata and the control subjects (Table 3). In terms of the IL17 (A7488G) gene polymorphism, the majority of patients with alopecia areata and control subjects carried the AA genotype. The GG genotype of the IL17 (A7488G) gene polymorphism was significantly more common in patients with alopecia areata (OR 7.788; P = 0.021), and the GA genotype was significantly less common (OR 0.055; P = 0.014) in these patients compared with control subjects. However, the GG genotype was only expressed in a small subset of patients (10.0%) with alopecia areata. There was no significant difference in the genotypes of the IL23R (+2199A/C) gene polymorphism between patients with alopecia areata and control subjects. As for the Hardy–Weinberg equilibrium P-values, while a deviation from the Hardy–Weinberg equilibrium was observed in patients with alopecia areata for IL17, it was absent for IL12 and IL23R. In control subjects, there was a deviation from the Hardy–Weinberg equilibrium for IL12 only.

Table 3.

Comparison of genotype frequencies of single nucleotide polymorphisms of IL12, IL17 and IL23R genes in patients with alopecia areata and healthy control subjects.

Gene polymorphism	Genotype	Patients with alopecia areata n = 100	Control subjects n = 71	OR	95% CI	Statistical significance
IL12 1188A/C	AA	69 (69.0)	38 (53.5)	0.997*	0.169, 5.873*	NS*
	AC	27 (27.0)	31 (43.7)	0.471*	0.078, 2.866*	NS*
	CC	4 (4.0)	2 (2.8)	1.438†	0.256, 8.071†	NS†
HWE P-value		NS	P < 0.001
IL17 A7488G	AA	84 (84.0)	60 (84.5)	0.148*	0.018, 1.220*	NS*
	GA	6 (6.0)	10 (14.1)	0.055*	0.005, 0.562*	P = 0.014*
	GG	10 (10.0)	1 (1.4)	7.778†	0.972, 62.209†	P = 0.021†
HWE P-value		P < 0.001	NS
IL23R +2199A/C	AA	30 (30.0)	18 (25.4)	1.747*	0.740, 4.125*	NS*
	AC	50 (50.0)	31 (43.7)	1.853*	0.858, 4.003*	NS*
	CC	20 (20.0)	22 (31.0)	0.557†	0.276, 1.124†	NS†
HWE P-value		NS	NS

Data presented as n (%) of study participants.

Age- and sex-adjusted OR (95% CI); †Fisher’s exact test.

OR, odds ratio; CI, confidence interval; HWE, Hardy–Weinberg equilibrium; NS, no statistically significant difference (P > 0.05).

When patients with alopecia areata were stratified according to the extent of scalp involvement based on their SALT scores into two groups, mild (S1, S2) and severe (S3, S4, S5), there were no significant differences in the genotypes of the IL12 (1188A/C) and IL23R (+2199A/C) gene polymorphisms (Table 4). Comparison of the genotypes of the IL17 (A7488G) gene polymorphism with respect to the severity of scalp involvement showed that the AA genotype was significantly more common in the group with mild scalp involvement (S1, S2) (OR 6.867; P = 0.024), whereas the GG genotype was significantly less common (OR 0.167; P = 0.029) compared with the group with severe scalp involvement (S3, S4, S5).

Table 4.

Comparison of genotype frequencies of single nucleotide polymorphisms of the IL12, IL17 and IL23R genes in patients with alopecia areata according to the extent of scalp involvement.

Gene polymorphism	Genotype	Scalp involvement		OR	95% CI	Statistical significance
Gene polymorphism	Genotype	Mild S1, S2 n = 65	Severe S3, S4, S5 n = 12	OR	95% CI	Statistical significance
IL12 1188A/C	AA	43 (66.2)	9 (75.0)	1.535†	0.377, 6.249†	NS†
	AC	18 (27.7)	3 (25.0)	1.149†	0.279, 4.730†	NS†
	CC	4 (6.2)	0 (0.0)	1.197†	1.081, 1.325†	NS†
IL17 A7488G	AA	55 (84.6)	8 (66.7)	6.867*	1.290, 35.551*	P = 0.024*
	GA	5 (7.7)	0 (0.0)	1.200†	1.082, 1.331†	NS†
	GG	5 (7.7)	4 (33.3)	0.167†	0.037, 0.753†	P = 0.029†
IL23R +2199A/C	AA	17 (26.2)	4 (33.3)	1.607*	0.247, 10.436*	NS*
	AC	34 (52.3)	5 (41.7)	1.662*	0.312, 8.849*	NS*
	CC	14 (21.5)	3 (25.0)	0.824†	0.196, 3.456†	NS†

Data presented as n (%) of study participants.

Age- and sex-adjusted OR (95% CI); †Fisher’s exact test.

OR, odds ratio; CI, confidence interval; NS, no statistically significant difference (P > 0.05).

When patients with alopecia areata were stratified according to the coexistence of other autoimmune disorders, there were no significant differences in the genotypes of the IL12 (1188A/C) and IL23R (+2199A/C) gene polymorphisms (Table 5). Comparison of the genotypes of the IL17 (A7488G) polymorphism in patients with alopecia areata with or without coexisting autoimmune disorders showed that the AA genotype was significantly more common among patients who did not have any other autoimmune disorders, compared with those who had a coexisting autoimmune disorder (OR 5.868; P = 0.027). Although it did not reach statistical significance, the GG genotype of the IL17 (A7488G) polymorphism was more common in patients with alopecia areata with coexisting autoimmune disorders: two patients with the GG genotype also had goitre, one had urticaria + vitiligo + goitre, and one had type 1 diabetes mellitus + hypothyroidism.

Table 5.

Comparison of genotype frequencies of single nucleotide polymorphisms of IL12, IL17 and IL23R genes in patients with alopecia areata according to coexistence of any other autoimmune disorder.

Gene polymorphism	Genotype	Autoimmune disorder		OR	95% CI	Statistical significance
Gene polymorphism	Genotype	Negative n = 83	Positive n = 17	OR	95% CI	Statistical significance
IL12 1188A/C	AA	56 (67.5)	13 (76.5)	1.482†	0.441,4.986†	NS†
	AC	23 (27.7)	4 (23.5)	1.172†	0.345, 3.978†	NS†
	CC	4 (4.8)	0 (0.0)	1.215†	1.108, 1.333†	NS†
IL17 A7488G	AA	71 (85.5)	13 (76.5)	5.868*	1.220, 28.225*	P = 0.027*
	GA	6 (7.2)	0 (0.0)	1.221†	1.110, 1.342†	NS†
	GG	6 (7.2)	4 (23.5)	0.253†	0.063, 1.022†	NS†
IL23R +2199A/C	AA	26 (31.3)	4 (23.5)	1.971*	0.375, 10.361*	NS*
	AC	41 (49.4)	9 (52.9)	1.335*	0.323, 5.523*	NS*
	CC	16 (19.3)	4 (23.5)	0.776†	0.223, 2.699†	NS†

Data presented as n (%) of study participants.

Age- and sex-adjusted OR (95% CI); †Fisher’s exact test.

OR, odds ratio; CI, confidence interval; NS, no statistically significant difference (P > 0.05).

Discussion

Alopecia areata is an autoimmune disease with an unknown aetiopathogenesis. Defects in cytokine production or signalling are considered to be involved in the development of autoimmune conditions such as alopecia areata.¹

Several studies have reported SNPs in patients with alopecia areata. For example, a study conducted in 165 patients and a large control group found that polymorphisms within the IL-1 receptor antagonist (IL1RN + 2018) gene and its homologue IL-1L1 (IL1L1 −4734) were associated with the severity of alopecia areata.¹⁷ A study in patients with severe alopecia areata demonstrated that increased frequency of the IL1β 1,2 genotype and the IL1β + 3953 polymorphism was strongly correlated with increased production of IL-1β.¹⁸ A study on a Korean population investigated SNPs in the IL-17 receptor A (IL17RA) gene and reported a significant difference between the patient group with alopecia areata and the control group, suggesting that the IL17RA gene polymorphism may be associated with an early age of alopecia onset.¹⁹ In one study, a tumour necrosis factor-α gene polymorphism (–308 G/A) confered increased susceptibility for the development of patchy alopecia areata.²⁰ The present study investigated polymorphisms in the genes for the cytokines IL-12, IL-17 and IL-23. To the best of our knowledge, this is the first study to investigate IL12 and IL23R gene SNPs in alopecia areata.

The IL12B 1188A/C gene polymorphism located in the 3′ untranslated region appears to have an effect on IL-12 production or protein expression: a report shows its association with type 1 helper T cell-mediated autoimmune disorders including inflammatory and malignant conditions.²¹ In an Iranian population, the frequency of the IL12B −1188C allele and CC genotype was found to be greater in patients with Graves’ ophthalmopathy.²² IL12B and IL23R gene polymorphisms were investigated in patients with psoriatic arthritis and were found to be associated with the disease.²³ In the current study, there was no significant difference between patients with alopecia areata and control subjects in the genotypes of the IL12 (1188A/C) gene polymorphism. Patients with alopecia areata did not differ significantly in the IL12 (1188A/C) gene polymorphism when stratified according to severity of scalp involvement and presence of coexisting autoimmune disorders.

Interleukin-17 is a cytokine involved in the production of proinflammatory cytokines, chemokines, cell adhesion molecules and growth factors.^7,8 IL-17 expression is increased in patients with chronic immune thrombocytopenia.⁸ In a separate study, IL17F gene polymorphism was suggested to confer a greater risk for the development of Graves’ disease.²⁴ In another study in a Chinese population, IL17F SNPs were associated with allergic rhinitis and coexisting asthma.²⁵ In the present study, the genotype distribution for the IL17 (A7488G) gene polymorphism showed that the GG genotype was significantly more common, whereas the GA genotype was significantly less common, in patients with alopecia areata compared with the control subjects. These results suggest that these polymorphisms may be involved in the aetiopathogenesis of alopecia areata, and that the GG genotype may be associated with an increased predisposition to the disease. However, it must be taken into consideration that most patients with alopecia areata (84.0%) carried the IL17 AA genotype, while the GG genotype was only expressed in a small subset of patients (10.0%). Thus its relevance in the aetiopathogenesis of the disease remains questionable. It is interesting to note that a study in patients with inflammatory bowel disease demonstrated that the IL17 (A7488G) GG genotype was associated with a slight decrease in the risk of ulcerative colitis compared with other genotypes.²⁶

When patients with alopecia areata in the present study were compared according to the extent of scalp involvement, the IL17 (A7488G) AA genotype was significantly more common and the GG genotype was significantly less common in the group with mild disease compared with the severe-disease group. These findings suggest that the IL17 (A7488G) AA genotype may have a diminishing effect on disease severity. In contrast, the IL17 (A7488G) GG genotype may increase disease severity in alopecia areata. Only a minority of patients (10.0%) with alopecia areata expressed the IL17 (A7488G) GG genotype in the present study, with the majority (84.0%) expressing the IL17 (A7488G) AA genotype. In terms of coexisting autoimmune disorders in patients with alopecia areata, the IL17 (A7488G) AA genotype was significantly more common in patients who did not have any coexisting autoimmune disorders compared with those who had coexisting autoimmune disorders.

Several studies have shown an association between IL23R gene SNPs and immune-mediated disorders including inflammatory bowel disease, Crohn’s disease, psoriasis and ankylosing spondylitis.^27–29 However, a study in a Chinese population did not find an association between IL23R gene polymorphisms and Vogt–Kayanagi–Harada Syndrome.³⁰ In the present study, the genotype distribution of the IL23R (+2199A/C) gene polymorphism demonstrated no significant difference between patients with alopecia areata and control subjects. There were also no significant differences in genotype distribution of the IL23R (+2199A/C) gene polymorphism when patients with alopecia areata were stratified according to extent of scalp involvement and presence of coexisting autoimmune disorders.

In conclusion, IL12 (1188A/C) and IL23R (+2199A/C) gene polymorphisms were not found to have a significant association with alopecia areata. However, the present findings suggest that the IL17 (A7488G) gene polymorphism may be involved in the aetiopathogenesis and disease severity of alopecia areata. This polymorphism may also be associated with a predisposition to other autoimmune disorders. However, it must be taken into consideration that the IL17 (A7488G) GG genotype was only present in a small subset of patients with alopecia areata in this present study. It is clear that more research is needed in larger patient groups in order to be able to determine the role that polymorphisms in the genes for cytokines play in the aetiopathogenesis of alopecia areata.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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