Investigating the link between toll-like receptor 9 gene variants and vitiligo susceptibility - A cross-sectional comparative study

Introduction

Vitiligo is an acquired, chronic disorder characterised by depigmented patches with an unknown aetiology and a polygenic inheritance pattern.¹ It is a complex disease influenced by genetic and environmental factors.² Some researchers suggest it is a multifactorial disorder with numerous genetic causes.³

Various pathophysiological mechanisms have been proposed for vitiligo, including autoimmune, neural, autocytotoxic, biochemical, oxidative stress, melanocytorrhagy, and decreased melanocyte survival.⁴

The autoimmune theory is particularly relevant in generalized vitiligo, which is considered a complex disorder involving the combined pathogenic effects of multiple susceptibility genes and unidentified environmental factors, resulting in the autoimmune destruction of melanocytes.⁴ Vitiligo is a polygenic disorder, with many genes related to autoimmunity implicated in its pathogenesis, each genetic factor making a small individual contribution.^5,6 However, the polygenicity of vitiligo is low, and its heritability is high when compared to most other complex traits, which might make its genetic architecture easier to discover.²

Vitiligo serves as a model for investigating the genetic architecture of complex diseases and could offer significant insights for predictive and personalized medicine in complex diseases.⁷ The role of innate immunity in vitiligo is evident by the infiltration of natural killer (NK) cells, which are five times more prevalent in the lesional skin of vitiligo patients when compared to healthy controls, and almost two-fold in unaffected skin.⁸ TLRs, identified in humans in 1998, are components of innate immunity that recognise pathogen-associated molecular patterns (PAMPs) present in microbes.⁹ TLRs function by binding to lipoproteins and peptidoglycans (TLR2, 4, 5, 6, and 11), or nucleic acid ligands associated with viral and bacterial single-strand RNA or double-strand RNA (TLR3, 7, 8, and 9). Upon ligand binding, TLRs participate in pathogen eradication through inflammatory cytokines and co-stimulatory gene expression.¹⁰

TLRs are the most well-characterised pattern recognition receptors (PRRs) and are transmembrane proteins encoded by the toll gene family.⁴ They are expressed in various cell types, including immune and nonimmune cells, and play a crucial role in detecting PAMPs and damage-associated molecular patterns (DAMPs). They also activate the adaptive immune system through the upregulation of costimulatory molecules on antigen-presenting cells (APCs).⁹

The signalling mediated by endogenous TLR ligands is crucial in autoimmune disorders.¹¹ Activation of certain TLRs through DAMP interactions can facilitate tissue repair and the elimination of cell debris; however, this same interaction is linked to the chronic inflammatory processes involved in rheumatologic diseases.¹² Therefore, TLR activation by DAMPs appears to play a role in the self-sustained inflammatory cycles and progression of these chronic diseases.¹³

Single-nucleotide polymorphism (SNP) genotyping technologies investigate the effects of amino acid variations on protein function. SNPs in promoter regions can alter transcription factor binding motifs, suppress wild-type transcript production, modify enhancer or repressor element efficiency, or introduce alternative translation initiation codons.¹⁴

There is a paucity of research on TLR gene polymorphisms in Egyptian vitiligo patients. This study aims to determine whether there is an association between TLR9 polymorphisms, specifically rs187084, in Egyptian vitiligo patients and their clinical characteristics including family history, disease onset, activity, duration, and hair involvement.

Methods

The sample size was determined to provide 80% power for chi-square (X²) goodness-of-fit tests. The type of power analysis used was an a priori computation to determine the required sample size, based on a significance level of 0.05 and an effect size of 0.5. This calculation was performed using G*Power 3.1.9.7 software. The present study entailed a cross-sectional comparison comprising 50 participants in two groups:

Group 1: Consisted of 30 individuals clinically diagnosed with vitiligo, ascertained both through clinical evaluation by dermatologist and Wood’s light examination. These patients were recruited from our outpatient Department of Dermatology, STDs, and Andrology. Detailed medical histories encompassing personal information, disease progression, psychological trauma, other concurrent skin or systemic disorders, and familial predispositions to vitiligo or other autoimmune conditions were documented for each patient.

Group 2: Included twenty healthy subjects selected as a control cohort.

Ethical approval for the study was obtained from the local scientific research ethics committee at the Faculty of Medicine (Approval No. 652:2023). Written consent was taken from all patients and control prior to the study.

Skin assessments in Group 1 involved clinical inspection for vitiligo manifestations, with attention to lesion characteristics, distribution patterns, and associated comorbidities, validated by Wood’s light examination.

Vitiligo activity (appearance of new depigmented lesions or the enlargement of existing ones) was quantified using the Vitiligo Disease Activity Score (VIDA), employing a six-point scale reflective of disease progression over time. VIDA score is based on patients’ opinions and divided into six grades as follows ¹⁵

+4: Activity of 6 weeks or less preceding the assessment

+3: Activity of 6 weeks to 3 months

+2: Activity of 3 to 6 months

+1: Activity of 6 to 12 months

0: Stable for at least 1 year and

−1: Stable for at least 1 year with spontaneous regimentation

Genotyping

Blood specimens were collected in ethylene diamine tetra acetic acid (EDTA) tubes and stored at -20°C until processing. Genomic DNA was isolated from whole blood using the QIAamp DNA Mini Kit (cat no.51104, QIAGEN, Germany) following standard protocols.

Genotyping of SNPs at the TLR9 rs187084 locus was performed via polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP assay. Oligonucleotide primers specific to rs187084 were utilised:

F: 5’-TAT CGT CTT ATT CCC CTG CTGGAATGT -3’ (sense) and

R: 5-TGCCCAGAGCTGACTGCTGG-3’ (anti- sense).¹³

PCR conditions entailed initial denaturation at 95°C, followed by 35 cycles of denaturation for 30 seconds, annealing at 60°C for 25 seconds, and extension at 72°C for 40 seconds using a thermocycler (UNOII). Amplicons were then subjected to Afl II restriction endonuclease digestion at 37°C for 1 hour (Enzynomics, Cat. R042S, Korea), and the resulting fragments were visualised on an agarose gel stained with ethidium bromide. A DNA ladder with 50 bp (Sizer tm DNA Markers from QIAGEN, USA) was used as a marker.

Statistical analysis

Statistical analyses were conducted using IBM SPSS version 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp). Data normality was assessed via the Kolmogov-Smirnov test and Brown-Frosythe test for median (0.232 for duration of disease and 0.342 for VIDA score), and it was noted that the duration of disease and VIDA score had homogenetic variance. Hence, the Kruskal Wallis test was used for these non-parametric quantitative data between three gene groups. The Chi-square test was used to compare cases and controls regarding genotype and phenotype, while the Fisher Exact test was used to compare family history, VIDA score, hair involvement, type of vitiligo and onset of disease between different genotype groups. Multivariable Logistic Regression analysis was used to analyse the effect of phenotype characteristics including gender, family history, age of onset, disease duration, type of vitiligo and disease activity on the dependent variable (allele frequency T and C) in cases. P-value less than 0.05 was considered statistically significant.

Results

Study design and population demographics

This was a cross-sectional comparative study involving 50 subjects sub-divided into two groups:

• Group 1 comprised 30 vitiligo patients, 19 females (63.3%) and 11 males (36.7%). Their ages ranged from 8 to 80 years (mean ± SD, 35.9 ± 23.1 years). A positive family history was reported in 5 (16.7 %) patients. Disease activity using the VIDA score was distributed as in Figure 1, with a mean ± SD of 0.97 ± 1.6. The duration of the disease ranged from 1 to 37 years (mean ± SD, 6.9 ± 8.4 years). The patients included in the study had non-segmental vitiligo in 22 (73.3%) cases and segmental vitiligo in 8 (26.7%) cases. Early onset disease (before 12 years of age) was reported in 16 (53.3%) patients, while 14 cases (46.7%) showed late onset disease. Hair involvement was reported in only 5 (16.7%) cases, while the remaining 25 (83.3%) patients showed no hair involvement.

• Group 2 consisted of 20 healthy controls, with 7 males (35%) and 13 females (65%). Their ages ranged from 8-70 years.

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Figure 1:

Distribution of disease activity in vitiligo patients. (VIDA score: Vitiligo disease activity score)

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Agarose gel electrophoresis analysis of TLR9 rs187084

• CC genotype produced only one 145 bp band, the TT genotype produced 2 small fragments at 111 and 67 bp, and the CT genotype produced 3 bands:145, 111, and 67 bp [Figures 2 and 3].

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Figure 2:

Agarose gel electrophoresis analysis of TLR9 rs18708 in the patient group; lanes 1 (CC genotype produced only one 145 bp band); lane: 2, 5, 6 (CT genotype produced 3 bands: 145, 111 and 67 bp); lane:3 (TT genotype 111, and 67 bp bands); L: ladder 50 bp.

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Figure 3:

Agarose gel electrophoresis analysis of TLR9 rs187084 in control subjects; lanes 1, 2, 3 (CC genotype produced only one 145 bp band), L: ladder 50 bp.

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Discussion

The precise aetiology of vitiligo remains subject to debate, with multiple hypotheses proposed to explain its pathogenesis. Nonetheless, autoimmunity is strongly implicated in the disease’s development.¹⁶

In vitiligo, innate immunity operates via two primary signalling pathways, mediated by TLRs and nod-like receptors (NLRs), which recognize PAMPs on various microbial surfaces.⁴ Notably, TLR9 plays a critical role in autoimmune diseases, and current research is investigating synthetic TLR9 agonists and antagonists to regulate autoimmune inflammation.¹⁷

This study is the first to evaluate TLR9 gene polymorphisms in the context of vitiligo immunopathogenesis. Prior research has assessed TLR9 gene polymorphisms in other autoimmune and chronic inflammatory skin diseases. In our study, the CT genotype of TLR9 rs187084 was observed in 43.3% of vitiligo cases compared to 20% of controls, while the TT genotype was found in 33.3% of vitiligo cases compared to 5% of controls. These results suggest a potential genetic risk factor for vitiligo. Similarly, Bashir et al. linked the TLR9 CT and TT genotypes, particularly the T-allele, with increased susceptibility to systemic lupus erythematosus (SLE).⁵ This finding aligns with observations from other studies on autoimmune diseases.

Oliveira et al. found that TLR9 polymorphisms rs187084, rs5743836, and rs5743708 were associated with the development of spondyloarthritis (SpA),¹³ suggesting these mutations significantly impact immune response profiles and immunological regulation Zhang et al. also reported associations between TLR9 rs187084, rs5743836, and rs352162 polymorphisms and increased risk of multiple sclerosis.¹⁸ Svitich et al. suggested that TLR9 rs352140 polymorphism might increase the risk of atopic dermatitis, with the GG genotype of SNP rs352140 in TLR9 serving as a predictor for moderate AD.¹⁹Additionally, Yu et al. indicated that upregulated TLR7 and TLR9 expression might be related to melanocyte dysfunction and vitiligo pathogenesis.²⁰

Various studies support the role of TLR gene polymorphisms in vitiligo. Traks et al. identified genetic associations between TLR gene SNPs and vitiligo susceptibility, notably TLR7 SNP 179020 and TLR3, with specific associations for TLR10 and TLR4 in subgroups.³ Karaca et al. associated TLR2 Arg753Gln and TLR4 Asp299Gly polymorphisms with vitiligo susceptibility in Turkish patients.¹ Abdelsalam et al. found the protective CT genotype of TLR4 rs1927914 to be more prevalent in controls. ⁴ However, Rajendiran et al. did not find TLR4 rs4986790 to confer risk in the South Indian population,²¹ suggesting possible ethnic differences in genetic predisposition.

Our study did not find significant differences in gene polymorphism distribution with regard to family history of vitiligo, aligning with Abdelsalam et al., who found no association with TLR4 SNP rs10759932 in familial cases.⁴ Conversely, Traks et al. reported significant associations for TLR4 SNP rs10759932 in familial but not sporadic cases³ possibly due to differences in study size, TLRs examined, and ethnic backgrounds. Regarding disease onset, our study did not find a significant association with TLR9 gene polymorphism, unlike Karaca et al., who reported that patients with the TLR4 399IIe allele had a later onset of vitiligo.¹

Our study utilised the VIDA score to assess vitiligo activity, finding no significant associations between SNPs and VIDA scores. This finding is consistent with Karaca et al., who reported no significant association between TLR4 expression and vitiligo surface area,¹ and with Abdelsalam et al., who found no association between TLR4 SNP rs10759932 and disease activity.⁴

Limitation

The study included a smaller control group (20 individuals) compared to the case group (30 participants) due to financial constraints. To optimise resources, we prioritised including more cases to focus on the costly gene expression analysis. Due to the limited sample size, along with the methodological constraints of PCR-RFLP, there is a need for further research to validate our findings and to better understand the mechanisms through which different SNPs influence TLR9 gene function.

Conclusion

Our findings of the significant associations between CT, TT genotypes and vitiligo susceptibility underscore the crucial role of innate immunity in vitiligo pathogenesis.