TLR2 and TLR4 gene polymorphisms in Turkish vitiligo patients

Abstract

Background  It has been shown that toll like receptors (TLR) may be involved in some inflammatory skin diseases such as psoriasis, atopic dermatitis. Vitiligo is an acquired pigmentation disorder of unknown aetiology. A number of genes playing a role in inflammatory response may be associated with development of vitiligo.

Objectives  To investigate whether there is an association between TLR 2 and TLR4 gene polymorphisms in Turkish patients with vitiligo.

Methods  A total of 100 patients (59 women and 41 men) with vitiligo and 100 controls (58 women and 42 men) were included in the study. The TLR2 gene Arg753Gln and TLR4 gene Asp299Gly and Thr399Ile polymorphisms were genotyped by using polymerase chain reaction and restriction fragment length polymorphism method. The data were analysed by Mann–Whitney U-test, chi-squared test and logistic regression analysis.

Results  Significant difference was found in the distribution of TLR2 Arg753Gln genotype and in the allele frequencies TLR2 753Gln between vitiligo patients and healthy subjects (P < 0.05). The distribution of TLR4 Asp299Gly genotype was significantly higher in the patient group (10%) than in the control group (%2) (P < 0.05). The TLR4 Thr399Ile distribution did not show any difference in both vitiligo and healthy groups.

Conclusions  Our findings suggest that Toll-like receptor 2 gene Arg753Gln and Toll-like receptor 4 Asp299Gly gene polymorphisms are associated with vitiligo susceptibility in Turkish patients.

Introduction

Vitiligo is an acquired, chronic disease characterized by depigmented patches with an unknown aetiology displaying a polygenic inheritance pattern. Viral infection, autoimmunity and increased activation of superoxide radicals are also considered in the aetiopathogenesis.^1,2 Melanocyte damage in vitiligo, which is stimulated by viruses such as HIV, HCV and Epstein-Barr virus, has been also mentioned by some authors.^3–6

Toll-like receptors (TLRs) are pattern recognition transmembrane receptors. They recognize some conserved components of endogenous molecules such as heat-shock proteins, apoptotic cells, increased oxidative stress and exogenous molecules such as bacteria or virus. Thus, they initiate the innate immune system by triggering the intracellular signalling pathways and lead to the development of adaptive immune system.^7–10 As a result, stimulated B, T lymphocytes and antigen-presenting cells trigger inflammatory, auto-inflammatory, or autoimmune pathways.^11–14 Activation of TLR increases synthesis and secretion of cytokines and arachidonic acid molecules.^15,16

Among TLRs, TLR2 is critical in the immune response to mycobacterial infections, in sensing oxidative stresses and cellular necrosis and in induction of apoptosis. In addition, TLR4 is critical in the immune response to viruses, gram-negative microorganisms, and also induces apoptosis as TLR 2.¹⁷

Single nucleotide gene polymorphisms in TLR 2 have been reported to be associated with a predisposition to septic shock or gram-positive microorganisms, unresponsive immune system to Borrelia, Treponema,¹⁸ Mycoplasma species,^19,20 urinary tract infection²¹ and acute rheumatic fever,²² and atherosclerosis.²³ On the other hand, single gene polymorphisms in TLR4 have been reported to be associated with a reduced response to endotoxins,²⁴ gram-negative infections,^25,26 and altered risk to atherosclerosis,^23,27 Crohn’s disease, ulcerative colitis²⁸ and Behcet disease²⁹ and familial Mediterranean fever.³⁰

However, the association of TLR 2 and 4 in the aetiopathogenesis of vitiligo remains unknown. Considering the influence of infection and increased oxidative stress, which may be sensed by TLR in development of vitiligo, we aimed to investigate the frequencies of single nucleotide gen polymorphisms including TLR 2 Arg 753 Gln, TLR 4 Asp299Gly and TLR4 Thr399Ile in vitiligo patients.

Materials and methods

The study was approved by the ethical committee of Ege Universiy Hospital and carried out in compliance with the principles of the Declaration of Helsinki. A total of 100 Turkish vitiligo patients who were admitted to our department between January 2006–May 2008 and 100 healthy controls without vitiligo or any other skin disease, and without any family history of vitiligo were included in the study. The individuals with a chronic or acute infection at the time of study were excluded. Informed consent was obtained from all subjects. The vitiligo diagnosis was based on the depigmented lesions detected by clinical examination together with Wood’s lamb examination. The participants were examined for the type of vitiligo and the body surface area of vitiligo was calculated. The coexistence of another autoimmune disease such as Type I diabetes mellitus or Hashimoto’s thyroiditis, the family history of vitiligo, duration of the disease and the time of vitiligo onset were all noted.

Genomic DNA preparation

Two millilitres of whole blood samples was collected into ethylenediaminetetraacetic acid – anticoagulated tubes by the standard venipuncture method. Genomic DNA was extracted from whole blood samples using the QIAmp blood DNA mini-kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.

Polymerase chain reaction (PCR) and enzyme digest

TLR2 Arg753Gln gene polymorphisms were analysed by the method of Schröder NW et al.³¹ Designed primers spanned a region of 340 bp, using the following primers: forward 5′-GCCTACTGGGTGGAGAACCT-3′ and reverse 5′-GGCCACTCCAGGTAGGTCTT-3′. Amplification was carried out on a GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA) in a 25-mL reaction mixture in 0.2 mL thin-wall PCR strip tubes (Axygen Scientific, Inc., CA, USA) containing 1.0 μL genomic DNA solution, GeneAmp Gold Buffer (15 mmol/L Tris-HCl, pH 8.0, 50 mmol/L KCl; PE Applied Biosystems), 2.5 mm MgCl2, 50 mmol/L each of the dGTP, dATp, dTTP and dCTP (Promega Inc., Madison, WI, USA); 25 pmol each forward and reverse primers and 1.0 U AmpliTaq Gold polymerase (PE Applied Biosystems). Seven microlitre of the PCR product was incubated for 2 h with 0.5 U AciI enzyme (NewEngland Biolabs,Beverly, MA, USA), in a total volume of 10 μL at 37 °C.

Determination of the TLR4 gene polymorphisms was accomplished with PCR and restriction fragment length polymorphism by the method of Lorenz E. et al.²⁶ The primers for TLR4 Asp299Gly were forward 5′-GATTAGCATACTTAGACTACTACCTCGA-3′ and reverse 5′-GATCAACTTCTGAAAAAGCATTCCCACC-3′. The primers for TLR4 Thr399Ile were forward 5′-GGTTGCTGTTCTCAAAGTGATTTTGGGACAA-3′ and reverse 5′-CCTGAAGACTGGAGAGTGAGTTAAATGCT-3′. Amplification conditions for TLR4 gene polymorphisms were described above for TLR2 polymorphisms, except for MgCl2 concentrations (4.0 mm MgCl2 for Asp299Gly and 3 mm for Thr399Ile). The cycling conditions comprised a hot start at 95 °C for 10 min., followed by 35 amplification cycles at 95 °C for 30 s, 62 °C for 30 s (Asp299Gly) or 60 °C (Thr399Ile) and 72 °C for 25 s, followed by one elongation step at 72 °C for 5 min. The digest reaction was set up using 4 μL PCR product, appropriate restriction enzyme NcoI (TLR4 Asp299Gly) and HinfI (TLR4 Thr399Ile), 1.0 μL 10 X enzyme buffer (New England Biolabs). It was incubated overnight at 371 °C and electrophoresed in a 3% NuSieve (FMC Bioproducts, Rockland, ME, USA) gel to identify the TLR4 alleles on the basis of the respective allele size. After digestion, the wild-type TLR4 allele sizes of 249 bp for the 299 residue and 406 bp for the 399 residue will not change; fragment sizes for carriers of the polymorphic allele will decrease to 23 bp for the 299 residue and 29 bp for the 399 residue. To confirm our PCR-RFLP results, PCR products for all genotypes were sequenced on an automated DNA sequencer (ABI 3130xl Genetic Analyzer; PE Applied Biosystems).

Statistical analysis

All data analysis was performed using a statistical package (version 15.0, SPSS Inc., Chicago, IL, USA). We compared categorical data and proportions using chi-squared test. Chi-squared analysis was used to test for deviation of genotype frequencies from Hardy–Weinberg equilibrium. Numeric values were compared by the Mann–Whitney U-test. P-values of <.05 were considered significant. Logistic regression analysis was used to test the associated risk of developing vitiligo with the tested TLR 2 and TLR4 polymorphisms. Odds ratios (ORs) and 95% confidence intervals (95% CI) were also assessed.

Results

A total of 100 vitiligo patients [59 women and 41 men, mean age 36.00 ± 1.70 years (range 8–76)] were genotyped. The control group included 53 men and 47 women. The mean age was 22.00 ± 1.82; Vitiligo duration was median 5 years (range 0.6–30.0 years). The types of vitiligo were generalized (51 patients, 51%), generalized acrofacial type (18 patients, 18%), localized focal type (14 patients, 14%), localized segmental type (15 patients, 15%), universal type (2 patients, 2%). The coexistence of autoimmune diseases including Hashimoto’s thyroiditis (4 patients, 4%), type I diabetes mellitus (7 patients, 7%), Myasthenia gravis (1 patients, 1%) were observed in 12 patients (12%).

We found the distribution of TLR2 Arg753Arg, Arg753Gln, Gln753Gln genotypes in vitiligo patients as 70%, 28%, 2% respectively and in the control group as 93%, 7%, 0% respectively. The frequency of TLR2 753Gln allele in vitiligo patients (32 alleles in 30 patients, 30%) was significantly higher than in the healthy controls (7 alleles in 7 patients, 7%) (OR = 5.252, 95% CI 2.259–12.208, P < 0.05). TLR2 Arg753Gln genotype had 5.7 times greater risk of vitiligo compared with TLR2 Arg753Arg genotype (OR = 5.694; 95% CI: 2.364–13.717, P < 0.05) (Table 1a).

Table 1.    (a) The genotypic distribution of TLR2 Arg753Gln polymorphism and allele frequency of patients with vitiligo and healthy controls. (b) The genotypic distribution of TLR4 Asp299Gly polymorphisms and allele frequency of patients with vitiligo and healthy controls

TLR2	Vitiligo n = 100	Control n = 100	P-values	OR (95% CI)
(a)
Genotype
Arg753Arg	70	93
Arg753Gln	28	7	<0.001*	5.694 (2.364–13.717)
Gln753Gln	2	0
Allel frequency
Arg	168	193
Gln	32	7	<0.001*	5.252 (2.259–12.208)

TLR4	Vitiligo n = 100	Control n = 100	P-values	OR (95% CI)
CI, confidence interval; OR, odds ratio. *Pearson Chi-squared test, P < 0.05). (for d.f. = 1).
(b)
Genotype
Asp299Asp	90	97
Asp299Gly	10	2	<0.033*	5.389 (1.149–25.264)
Gly299Gly	0	1
Allel Frequency
Asp	190	196
Gln	10	4	0.115	2.579 (0.795–8.364)

The distribution of TLR 4 Asp299Asp, Asp299Gly, Gly299Gly genotypes in the vitiligo group was as 90%, 10%, 0 and in the control group was as 97%, 2%, 1% respectively. The frequency of TLR4 299Gly alleles in patients with vitiligo (10 alleles in 10 patients, 10%) and healthy controls (4 alleles in 3 patients, 3%) did not show a significant difference (OR = 2.579, 95% CI: 0.795–8.364, P > 0.05). The increased risk of vitiligo was found 5.4 greater in TLR4 Asp299Gly genotype compared with TLR 4 Asp299Asp genotype (OR = 5.389; 95% CI: 1.149–25.264, P < 0.05) (Table 1b). The Hardy-Weinberg equilibrium data were provided for both the vitiligo and control groups (Table 2).

Table 2.   Hardy–Weinberg equilibrium data for both the vitiligo and control groups

	TLR2 Vitiligo	TLR2 Control	TLR4-299 Vitiligo	TLR4-299 Control	TLR4-399 Vitiligo	TLR4-399 Control
N	70	93	90	97	90	95
Het	28	7	10	2	10	4
Mut	2	0	0	1	0	1
Chi-squared	0.1736	0.1315	0.2770		0.2770
P-value (for d.f. = 1)	0.676	0.71	0.598		0.598

The distribution of TLR4 Thr 399Thr, Thr399Ile, Ile399Ile genotypes was detected as 90%, 10, % 0% in the vitiligo group and as 95%, 4%, 1% in the control group respectively (Table 1b). The frequency of TLR4 Ile399 allele in patients with vitiligo (10 alleles in 10 patients) and healthy controls (6 alleles in 5 patients) showed no statistically significant difference (OR = 1.702; 95% CI: 0.607–4.775, P > 0.05). There was no increased risk of vitiligo in TLR4 Thr399Ile genotype compared with TLR4 Thr399Thr genotypes (OR = 2.639; 95% CI: 0.799–8.716, P > 0.05) (Table 3).

Table 3.   The genotypic distribution of TLR4 Thr399Ile polymorphisms and allele frequency of patients with vitiligo and healthy controls

TLR4	Vitiligo n = 100	Control n = 100	P-values	OR* (95% CI)
CI, confidence interval; OR; odds ratio. Pearson Chi-squared test, P < 0.05) (d.f. = 1). *Fisher’s exact test: 0.283 (There is existing problem in expected values of Chi-squared analysis, because of low frequency for MUT. P-value is given according to Fisher’s exact test data in 2 × 2 tables which joined in the form of HET-MUT).
Genotype
Thr399Thr	90	95
Thr399Ile	10	4	0.111	2.639 (0.799–8.716)
Ile399Ile	0	1
Allele frequency
Thr	190	194
Ile	10	6	0.312	1.702 (0.607–4.775)

Multiple logistic regression analysis of TLR2 753Gln, TLR4 299Gly and TLR4 399Ile allele presence in vitiligo patients also confirmed the increased susceptibility of vitiligo in TLR2 753Gln (+) patients compared with others (P < 0.001) (Table 4).

Table 4.   Susceptibility of TLR2 753Gln, TLR4 299Gly and TLR4 399Ile positive genotypes to develop vitiligo

	OR	95% CI	P-values
CI, Confidence interval; OR, odds ratio. *Logistic regression analysis, P < 0.05 (for d.f. = 1).
TLR2 Arg753Gln genotype
753Gln negative genotype
753Gln positive genotype	5.694	2.364–13.717	<0.001*
TLR4 Asp299Gly genotype
299Gly negative genotype
299Gly positive genotype	3.593	0.958–13.472	0.058
TLR4 Thr399Ile genotype
399Ile negative genotype
399Ile positive genotype	2.111	0.695–6.416	0.188

Clinical features of vitiligo patients with at least one of the TLR2 753Gln, TLR4 299Gly ve TLR4 399IIe allele were compared with TLR2 753Gln, TLR4 299Gly ve TLR4 399IIe allele negative patients. The compared clinical features included the age, the duration of the disease, the age of disease onset, family history, association of an autoimmune disease, the vitiligo body surface area. TLR2 753Gln (+) and 753Gln (−) patients’ median age showed a significant difference of 44 (range 8–76) and 32.50 (range 8–67) (P < 0.05). The other features of TLR2 753Gln positive and negative patients were similar (Table 5). The comparison of clinical features of TLR4 299Gly (+) and 299Gly (−) patients revealed a significant difference in median age of 53.00 (range 14–64) and 34.00 (range 8–76) respectively (P < 0.05). The other clinical features were similar in both groups. The median age of TLR4 399IIe (+) patients (53.00, range 43–64) was statistically higher than that of TLR4 399IIe (−) patients (33.50, range 8–76, P < 0.05). Additionally, TLR4 399IIe (+) patients had an older median age of vitiligo onset than TLR4 399IIe (−) patients 46.00 (range 13–54) vs. 24.50 (range 3–64) respectively (P < 0.05) (Table 6).

Table 5.   Clinical features of TLR2 753Gln positive and TLR2 753Gln negative vitiligo patients

TLR2 753Gln	753Gln (+) n = 30	753Gln (−) n = 70	P-values
*Mann–Whitney U-test, P < 0.05.
Median age	44.00 (range 8–76)	32.50 (range 8–67)	0.013*
Vitiligo surface area	10% (range 5–40%)	10% (range 5–100%)	0.988
Family history	11	22	0.647
Associated autoimmune disease	4	8	0.632
Duration of vitiligo (median year)	8.00 (range0–38)	4.00 (range 0–39)	0.085
Onset of vitiligo (median age)	35.00 (range 4–61)	22.50 (range 3–64)	0.179

Table 6.   Clinical features of TLR4 399 Ile positive and TLR4 399 Ile negative vitiligo patients

TLR4 399Ile	399Ile (+) n = 10	399Ile (−) n = 90	P-values
*Mann–Whitney U-test, P < 0.05.
Median age (years)	53.00 (range 43–64)	33.50 (range 8–76)	*0.001
Vitiligo surface area (%)	20 (range 10–40)	10 (range 5–100)	0.174
Family history	7	26	*0.014
Associated autoimmune disease	4	8	0.324
Duration of vitiligo (median year)	6.50 (range 0–35)	4.50 (range 0–39)	0.211
Onset of vitiligo (median age)	46.00 (range 13–54)	24.50 (range 3–64)	*0.005

Discussion

In this study, TLR2 Arg753Gln, TLR4 Asp299Gly and TLR4 Thr399Ile gene polymorphisms were examined in vitiligo patients. TLR2 Arg753Gln genotype distribution and allele frequency in patients with vitiligo in comparison with the control group were observed more evidently, and a strong association between them was determined. TLR4 Asp299Gly genotype was observed in the vitiligo group significantly more than in the control group. However, no significant difference in TLR4 299Gly allele frequency was detected between the two groups.

Different aetiopathological mechanisms underlying the different clinical phenotypes of vitiligo remain to be elucidated. To unravel the aetiological and genetic background, it is very important to distinguish between these different clinical phenotypes to estimate correctly the value of different scientific genetic results.²

Recent studies about the pathogenesis of vitiligo support the role of cellular immunity and autoimmunity.^32–36 However, the antigens, which are supposed to trigger these pathways, are still unknown. According to the data obtained from this study, the altered TLR2 and TLR4 expression and signalling by the polymorphic TLR2 753Gln and TLR4 299Gly may be the reason for the defective immune response to some antigens such as viruses in vitiligo. On the other hand, TLR 4399Ile polymorphism was found to be non-significant for vitiligo.

In humans, TLRs recognize constituents of microbial cell walls or pathogen-specific nucleic acids, trigger NF-κB (nuclear factor kappa light chain enhancer of activated B cells) and/or MAPK (mitogen-activated protein kinase) signalling pathway, and might be involved in the induction of several inflammatory cytokines and chemokines.⁷ Furthermore, TLR-mediated activation is very similar to that of another essential cytokine receptor, inteleukin-1 receptor (IL-1R). Both TLRs and IL-1R share the MyD88 adaptor molecule and promote the production of proinflammatory cytokines.⁸ In the process of viral infection, dsRNA stimulates cellular antiviral activities, and occasionally, cell death, which is another way of protecting the host against virus spreading. Yu N et al. found that in vitro treatment with poly (I : C), a synthetic analogue dsRNA, resulted in massive death and detachment of cultured human melanocytes. Another potential NF-kB stimulator, the TLR4 ligand LPS, failed to induce significant apoptosis in melanocytes. One could speculate that NF-kB in the downstream of Toll-like receptor (TLR)-mediated signalling pathways may not contribute significantly to the apoptosis.³⁷ Polymorphisms of TLR2 and TLR4 genes may lead to activation of NF-κB signalling pathway and this mechanism may result in melanocytes apoptosis.

To our knowledge, this is the first study about the association of TLR2 Arg753Gln, TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms with vitiligo. However, TLR2 and TLR4 gene polymorphisms were previously evaluated in some chronic inflammatory skin diseases. Ahmad-Nejad et al. detected TLR2 Arg753Gln polymorphism in %11.5 of atopic dermatitis. This polymorphism has been found to be associated with severe-to-moderate atopic dermatitis together with the higher levels of total serum IgE and superantigen-specific IgE than the non-polymorphic atopic dermatitis.³⁸ TLR2 has been implicated in the inflammatory process of acne vulgaris. In addition, TLR2 Arg753Gln polymorphism carriers were found to have an increased risk for acne vulgaris in Chinese Han patients.³⁹ TLR2 Arg753Gln polymorphism was found to be irrelevant to Behçet’s disease.⁴⁰ However, TLR4 gene polymorphisms were found to be associated with Behçet’s disease.²⁹

This study also revealed that patients with positive TLR4 399IIe allele have evidently higher onset age for vitiligo in comparison with patients with negative TLR4 399IIe allele. It was thought that individuals, who have this kind of allele, might be prone to late onset of vitiligo. All these lines of evidence indicate that genetic factors play a significant role in vitiligo.

In conclusion, TLR2 Arg 753 Gln and TLR4 Asp299Gly gene polymorphisms are associated with an increased risk for the development of vitiligo.

Acknowledgement

We are indebted to Timur Kose (Biostatistics and Medical Biology Department, Ege University, İzmir) for helping us with the statistical analysis.