Vitamin D Receptor Gene Polymorphisms in Vitiligo Patients

Research Ethical Statement

Approval for this research was granted by the Research Ethical Committee of the Faculty of Medicine for Girls, Al-Azhar University. Informed consent was obtained from all study participants.

Patient Selection and Clinical Samples

This prospective cross-sectional study took place between January 2013 and March 2014. Blood samples were collected from individuals attending the outpatient Dermatology Clinic at Al-Zahraa University Hospital in Cairo, Egypt. All laboratory work was conducted at the Biochemistry Department Research Lab, Faculty of Medicine, Ain Shams University, Cairo, Egypt.

Thirty patients who had previously been diagnosed with non-segmental vitiligo (NSV) and 30 apparently healthy individuals, matched for age and sex, were recruited for this study. Eligible participants were required to be over 16 years of age and have normal liver and kidney test results. Controls were not related to the patients and had no family history of vitiligo. Comprehensive demographic data, medical histories, and physical examinations were conducted for all participants. This included assessing Fitzpatrick's skin phototype for everyone, determining vitiligo type [8], and calculating the Vitiligo Area Scoring Index (VASI)[9] for the patients.

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Individuals with segmented vitiligo, other depigmenting disorders, or autoimmune diseases were excluded from the study. Additionally, participants taking vitamin D, calcium, or steroid medications were not eligible for inclusion.

Ten milliliters of venous blood were withdrawn from each participant using sterile labeled vacutainers. Part of the sample was collected in gel vacutainers, then centrifuged at 12,000 rpm for 10 minutes, and the serum was stored at -20°C for measuring 25-OH-D3 levels. The other part of the sample was placed in vacutainers containing EDTA and stored at -80°C for DNA extraction.

Measurement of Serum 25-OH-D3

Serum samples were examined for 25-OH-D3 levels using an enzyme-linked immunosorbent assay (ELISA) kit supplied by Immunodiagnostic USA. A serum 25-OH-D3 level of 30 ng/mL or higher is considered sufficient [10].

Apa-I & Taq-I Gene Polymorphisms Analysis

Whole genomic DNA was isolated from whole blood samples using the Thermo Scientific Gene JET Genomic DNA purification kit from Finland. The samples were stored at -20°C. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) was employed to study Apa-I (rs7975232) and TaqI (rs731236) genotype variations using the Thermo Scientific DreamTaq Green PCR Master Mix, Finland. The PCR mixture in a final volume of 50 μl contained 25 μl of PCR master mix, 13 μl of nuclease-free water, 10 μl of DNA template, and 1 μl of each primer. The primers used were provided by Sigma Genosys, UK.

For the amplification of the Apa-I segment, the forward primer was: ^{5’- CAACCAAGACTACAAGTACCGCGTCAGTGA-3’} [11], and the reverse primer was: ^{5’-CACTTCGAGCACAAGGGGCGTTAG-3’} [12]. The PCR protocol involved an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 67°C for 30 seconds, and extension at 72°C for 2 minutes. The final extension step was carried out at 72°C for 5 minutes. Samples were then digested with the restriction enzyme Apa-I, supplied by Thermo Scientific Fast Digest.

For the amplification of the Taq-I segment, the forward primer was: ^{5’- CAGAGCATCGACAGGGAGCAA-3’}, and the reverse primer was: ^{5’-CACTTCGAGCACAAGGGGCGTTAG-3’} [11]. The PCR protocol for this segment also involved an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 64°C for 30 seconds, and extension at 72°C for 30 seconds. The final extension step was carried out at 72°C for 5 minutes. Samples were then digested with the restriction enzyme Taq-I, supplied by Thermo Scientific Fast Digest. The digested samples were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized using a UV-transilluminator.

Statistical Analysis

The statistical analysis of the data was conducted using SPSS program version 20. Qualitative data were presented as frequency and percentage. Chi-square and Fisher’s exact tests were employed to compare groups. Quantitative data were presented as mean and standard deviation or median and range. For comparisons between two groups with parametric data, the Student t-test and ANOVA test were used. The Mann-Whitney test and Kruskal-Wallis were used for non-parametric data comparisons. The distribution of alleles and genotypes in the studied groups was assessed for conformity with the Hardy-Weinberg equilibrium by comparing the observed and expected frequencies of genetic variants using the chi-square test. Odds ratios and 95% confidence intervals were calculated. A P-value is considered significant if it is less than 0.05 at a 95% confidence interval.

Results

Patients' Data and Criteria

Out of the thirty patients, nineteen were females, and eleven were males, with a mean age ± SD of 43.5 ± 14.35 years. Fourteen females and sixteen males of age-matched controls (mean age ± SD: 40 ± 10.67 years) were also included (Table 1). A positive family history of vitiligo was found in four patients (13.3%). The age of onset of the disease ranged from 10 to 62 years (mean ± SD: 31.8 ± 14.6 years). The duration of the disease ranged from 1 to 32 years, with a median of 8 years. The Vitiligo Area Scoring Index (VASI) score had a median (range) of 2.75 (0.25-100).

Table 1: Demographic Data of Patients and Controls

Table 2: Allele and Genotype Frequencies for VDR Apa-I & Taq-I Polymorphisms in Patients and Controls

Measurement of Serum 25‐OH Vitamin D3

Measuring serum vitamin D levels in patients revealed a median (range) of 12.9 ng/ml (5.8-22.2 ng/ml), while in controls, it was 17.6 ng/ml (1.8-150 ng/ml) with no statistically significant difference (P=0.121) between both groups (Figure 1).

VDR Apa-I & Taq-I Gene Polymorphisms

Apa I (rs7975232) and TaqI (rs731236) genotypes were identified as digested PCR fragments, where:

• AA = the homozygous genotype with the absence of a restriction site and separation of a single band of 2000 bp.
• Aa = the heterozygous genotype with the absence of a restriction site in one allele and the presence of the restriction site in the other allele, separated as three bands of 2000, 1600, and 400 bp.
• aa = the wild genotype with the presence of a restriction site in both alleles and separation of two bands of 1600 & 400 bp (Figure 2).

For Taq-I gene polymorphism:

• TT genotype = homozygous with the absence of a restriction site in both alleles; 500 bp (wild type).
• Tt genotype = heterozygous; absence of a restriction site in one allele and presence of a restriction site in the other allele; 500, 300 & 200 bp.
• tt genotype = homozygous with the presence of a restriction site in both alleles; 300 & 200 bp.

Studying the SNPs of Apa-I & Taq-I in patients to test its correlation with vitiligo revealed the following allele frequency among patients:

• A (83.3%), a (16.7%), T (63.3%), and t (36.7%).

Among control subjects, it was:

• A (76.7%), a (23.3%), T (73.3%), t (26.7%).

The most common VDR genotypes among patients were: AA (73.3%) and TT (50%). In the control group: AA (66.7%), and TT (50%) with no statistically significant difference detected between both groups (Table 2). The frequency distribution of combined VDR genotypes in patients showed that the AATT genotype is the most frequent (36.7%), followed by the AAtt (23.3%). As for the control group, AATt (33.3%) is the most frequent followed by the AATT (30%).

It is noteworthy that the tt genotype showed a marginally significant increase in vitiligo cases compared to control subjects (P=0.053), (Table 2).

Discussion

Non-segmental vitiligo is a depigmentation skin lesion with its pathogenesis extensively studied, and multiple hypotheses have been proposed. The most widely accepted theory is the autoimmune theory. In this study, we investigated 30 NSV patients for VDR gene polymorphism and compared them with 30 age- and sex-matched controls. Vitamin D polymorphisms have been linked to an increased risk of diseases with autoimmune etiology [13].

The association between vitiligo and VDR polymorphisms and serum 25-OH-D3 levels has gained attention in recent years. However, reported results have been inconsistent [8]. Vitamin D plays a crucial role in supporting both the innate and adaptive immune systems. Studies have shown the expression of nuclear VDR and vitamin D-activating enzymes in T- and B-cells. Upon activation, VDR expression significantly increases, allowing the regulation of numerous vitamin D-responsive genes that influence the differentiation and proliferation of these cells [14].

Vitamin D is also believed to regulate melanocyte activity, as evidenced by the presence of vitamin D receptors in these cells. Topical vitamin D has even been prescribed as a treatment for vitiligo due to its potential to protect melanocytes and prevent their destruction [15]. It has been suggested that vitiligo occurs by triggering apoptosis of epidermal and mucosal melanocytes, and vitamin D reduces the apoptotic activity induced by UVB in keratinocytes and melanocytes [16]. Additionally, vitamin D has been shown to induce the synthesis of sphingosine-1-phosphate, which has an anti-apoptotic effect, further protecting melanocytes from destruction [17]. Vitamin D also plays a role in preventing oxidative DNA damage [18]. Thus, vitamin D may be considered as a potential component of the vitiligo treatment protocol due to its protective effects on melanocytes, preventing their destruction and the loss of skin pigment [15].

In our study, measuring serum vitamin D revealed no significant difference between patients and controls. However, it is worth noting that both groups were vitamin D deficient. This finding is consistent with some previous studies that found no significant difference in vitamin D levels between vitiligo patients and controls [19, 20], while others reported significantly lower levels in vitiligo patients [21, 22].

We conducted SNP analysis for Apa I and TaqI, and we found no significant differences between both groups regarding age or sex. This aligns with a few previous studies conducted in the Romanian population [11], the Chinese population [23], and Croatia [24].

Studying the allele frequency revealed the most common alleles among patients: A (83.3%), a (16.7%), T (63.3%), and t (36.7%). Among control subjects, it was: A (76.7%), a (23.3%), T (73.3%), and t (26.7%). The most common VDR genotypes among patients were: AA (73.3%) and TT (50%). In the control group: AA (66.7%), and TT (50%) with no statistically significant difference detected between both groups.

The frequency distribution of combinations of VDR genotypes in patients showed that the AATT genotype is the most frequent (36.7%), followed by the AAtt (23.3%). As for the control group, AATt (33.3%) is the most frequent followed by the AATT (30%).

Regarding the Apa-I polymorphism, we found no significant differences in allele, genotype, or combined genotype frequency. This result is consistent with a previous study in Turkey [25] that also found no significant difference in allele or genotype frequency between vitiligo and control subjects. However, a study in China [23] reported a significantly decreased frequency of AA, Aa, and A alleles associated with a decreased risk of developing vitiligo.

In our study, there was no significant difference in allele, genotype, or combined genotype frequency for Taq-I. However, we observed an increase in the tt genotype, which showed a marginally significant difference (P=0.053) compared to controls. This finding aligns with a Turkish study [25], which reported that the tt genotype and t allele are more frequent in vitiligo cases and are associated with an increased risk for the disease. A study in India [16] revealed that the genotype Tt is higher among vitiligo cases but not significantly higher than controls. In contrast, a Chinese study [23] showed that the t allele frequency is significantly lower among vitiligo patients, and the tt genotype is associated with a decreased risk of vitiligo in China.

Comparing clinical data between vitiligo patients regarding VDR Apa-I and Taq-I SNPs and their combined gene polymorphisms, no significant difference was noted regarding age, sex, family history, age of onset, duration, VASI score, or clinical variants of the disease.

Conclusion

Despite Egypt being a sunny country, vitamin D deficiency is considered prevalent among the population. Although vitiligo patients manifested lower serum levels of vitamin D, there was no detectable significant difference when compared to controls. VDR Apa-I (rs7975232) and TaqI (rs731236) SNPs were not correlated with the risk of developing vitiligo. However, it is noteworthy that the tt genotype frequency was increased among vitiligo patients with a marginally significant difference when compared to the control group.