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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 1  |  Page : 47-52

A study of vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D levels in vitiligo patients and controls


1 Department of Dermatology, Venereology and Leprosy, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
2 Department of Biochemistry, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
3 Molecular Genetic Lab, Central Research Laboratory, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
4 Medgenome Labs Ltd, Bangalore, India

Date of Submission21-Nov-2020
Date of Decision29-Jan-2021
Date of Acceptance01-Feb-2021
Date of Web Publication25-Feb-2022

Correspondence Address:
Banavasi Shanmukha Girisha
Department of Dermatology, Venereology and Leprosy, K. S. Hegde Medical Academy, Deralakatte, Mangalore 575 018, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cdr.cdr_129_20

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  Abstract 


Background: Vitiligo is a common pigmentary disorder affecting 0.1%–2% of the global population. Recently, interest has evoked in the role of Vitamin D in decreasing the risk of several chronic diseases. Little is known about the genetics of vitiligo. This study was conducted to investigate whether Vitamin D receptor (VDR) polymorphisms could be the susceptibility markers for vitiligo. Objectives: (i) To evaluate the potential association between VDR polymorphisms and vitiligo susceptibility and (ii) To estimate the serum levels of 25–hydroxyvitamin D in case and control groups. Materials and Methods: This study included 34 participants (17 with vitiligo and 17 age-and gender-matched healthy controls). After a written informed consent, a detailed history and examination was done. Serum 25(OH)D levels were measured using the enzyme-linked immunosorbent assay kit. Genomic DNA was extracted from the peripheral blood. The VDR polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Fok1, Bsm1, Apa1, and Taq1 restriction enzymes were used to determine the genotypes of the respective polymorphisms. Results: Nonsegmental vitiligo was the most common type of vitiligo seen in 14 (82.4%) cases. The mean serum Vitamin D level in cases was 35.64 ± 15.68 ng/ml and in controls was 37.00 ± 16.45 (P = 0.81). There was no significant difference in the distribution of VDR polymorphisms between the case and control groups. Conclusion: In our study, no significant difference was observed in the distribution of Fok1, Bsm1, Apa1, and Taq1 VDR polymorphisms between the case and control groups. However, we observed a significant association between the Fok1 polymorphism and serum 25(OH) Vitamin D levels in vitiligo patients studied.

Keywords: Polymorphism, Vitamin D, Vitamin D receptor, vitiligo


How to cite this article:
Noronha TM, Girisha BS, Kumari S, Shetty SS, Sampath M. A study of vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D levels in vitiligo patients and controls. Clin Dermatol Rev 2022;6:47-52

How to cite this URL:
Noronha TM, Girisha BS, Kumari S, Shetty SS, Sampath M. A study of vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D levels in vitiligo patients and controls. Clin Dermatol Rev [serial online] 2022 [cited 2022 Aug 19];6:47-52. Available from: https://www.cdriadvlkn.org/text.asp?2022/6/1/47/338586




  Introduction Top


Vitiligo is the most common pigmentary disorder, with a reported frequency of 0.1%–2% in various populations.[1] Autoimmunity plays an important role in the pathogenesis of vitiligo. The discovery of Vitamin D receptors (VDRs) in most cells of the body and the presence of enzymes that synthesize the active form of Vitamin D, namely 1,25-dihydroxyvitamin D in non-renal sites like skin have led to a renewed interest in its functions, particularly its role in decreasing the risk of several chronic, highly morbid conditions including autoimmune diseases.[2]

Little is known about the genetics of vitiligo, despite its long history and high frequency. Vitiligo does not follow a simple Mendelian pattern of inheritance, and most investigators have considered that it most likely has a multifactorial, polygenic basis.[1] The family aggregation, described in 6%–38% of patients, strongly supports the involvement of genetic factors in vitiligo.[3] The VDR gene is located on chromosome 12q12-14.[4]

A polymorphism is a genetic variant that appears in at least 1% of the population. The existence of several restriction fragment length polymorphisms (RFLPs) in the VDR gene has been described using different restriction enzymes.[5] The BAT nomenclature indicating the presence of the restriction site with small letters and its absence with capitals is commonly used in the literature.[6]

Due to the lack of reports concerning the role of Vitamin D and VDR allelic variants in vitiligo in the Indian population, this case–control study was conducted to investigate whether VDR polymorphisms could be the susceptibility markers for vitiligo.

The objectives of this study were to evaluate the potential association between VDR polymorphisms and vitiligo susceptibility and to estimate the serum levels of 25(OH)D in case and control groups.


  Materials and Methods Top


This was a hospital-based case–control study involving 34 participants conducted in the department of Dermatology, Venereology and Leprosy, of a tertiary care teaching hospital in India from May 2016 to May 2018. Seventeen patients with a clinical diagnosis of vitiligo were enrolled in the study along with 17 age-matched (±3 years) and sex-matched controls. Ethical clearance for conducting this study was obtained from the Institutional Ethical Committee before the start of the study. All clinically diagnosed vitiligo patients of 18 years and above of either gender and not treated topically in the previous 1 month or systemically in the previous 3 months were recruited as cases. The controls included age- and gender-matched individuals without vitiligo. Patients <18 years of age, patients not consenting to participate in the study, patients currently on treatment or Vitamin D supplementation or phototherapy, patients with other known autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, autoimmune diabetes, thyroid disease and pernicious anemia or other depigmentation disorders such as piebaldism and albinism, and patients who had received blood transfusions during the previous 6 months were excluded from the study. After fulfilling the selection criteria, all patients were counselled about the study, and informed written consent was obtained. A detailed history and complete physical examination, including age, sex, age of onset, type of vitiligo, vitiligo disease activity score (VIDA),[4] skin surface area affected, and family history of the patients along with local examination of the lesions was done. The percentage of vitiligo involvement was calculated in terms of hand units. One hand unit (which encompasses the palm plus the volar surface of all digits) is approximately equivalent to 1% of the total body surface area.[7]

Laboratory methodology

Serum 25(OH) Vitamin D levels were measured in both cases and controls using 25(OH) Vitamin D enzyme-linked immunosorbent assay kit (Bio-Detect, Laguna Hills, CA, USA). Genomic DNA was extracted from peripheral blood (3 ml in ethylenediaminetetraacetic acid vacutainer). The VDR polymorphisms were genotyped using the polymerase chain reaction (PCR)-RFLP method. Fok1, Bsm1, Apa1, and Taq1 restriction enzymes were used to determine the genotypes of the respective polymorphisms. Amplification of the gene of interest was done by PCR. PCR reaction mixture was prepared for five samples along with a negative control and only primer control. Components of a PCR mixture for Fok1, Bsm1, Taq1+Apa1 polymorphism for 50 μL reaction volume were 25 μL of master mix, 0.8 μL of GC enhancer, 1.0 μL of forward primer (10pmol), 1.0 μL of reverse primer (10pmol), 22.0 μL distilled water, and 1 μL DNA template. The PCR cycle conditions for Fok1 were initial digestion at 93°C for 10 min, denaturation at 93°C for 45s, annealing at 70°C for 30s, extension at 72°C for 45s, and final extension at 72°C for 5 min. The cycle was repeated 35 times. The PCR products were verified using 2% agarose gel containing ethidium bromide [Figure 1]a. PCR products were digested with Fok1 restriction enzyme at 37°C for 3 h. The size of the digested fragments was identified by agarose gel electrophoresis, and the results were visualized under ultraviolet light and photographed [Figure 1]b. The PCR cycle conditions for Bsm1 and Taq1+Apa1 were initial digestion at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 58°C for 30s (for Bsm1), annealing at 65–70°C for 30s (for Taq1+Apa1), extension at 72°C for 30s, and final extension at 72°C for 5 min. The PCR products were verified using 2% agarose gel containing ethidium bromide [Figure 1]c and [Figure 1]d. PCR products were digested with Bsm1 restriction enzyme. The size of the digested fragments was identified by agarose gel electrophoresis, and the results were visualized under ultraviolet light and photographed [Figure 1]e. PCR products were digested with Apa1 and Taq1 restriction enzyme at 65°C for 4 h. The size of the digested fragments was identified by agarose gel electrophoresis, and the results were visualized under ultraviolet light and photographed [Figure 1]f and [Figure 1]g.
Figure 1: (a) Amplified polymerase chain reaction products of Fok1; M: 100 bp DNA ladder. (b) Fok1 restriction enzyme digestion results: FF (lanes V3, V8, V9, V10), Ff (lanes V5, V7), ff (lanes V4, V6). (c) Amplified polymerase chain reaction product of Bsm1. (d) Amplified polymerase chain reaction product of Taq1 and Apa1. (e) Bsm1 restriction enzyme digestion results: Lanes V8, V10, V12, V13, V14, V15 showing BB; lane V9 showing genotype Bb; lane V11 showing genotype bb. (f) Apa1 restriction enzyme digestion results: Lanes V7, V8, V10, V12 and V13 showing AA; lane V9 showing genotype Aa; lane V11 showing genotype aa. (g) Taq1 restriction enzyme digestion results: Lane V26 showing TT; lanes V28, V30 and V31 showing genotype Tt; lanes V27 and V29 showing genotype tt

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Statistical methodology

Collected data were summarized by the frequency and percentage of categorical type of data, and quantitative data were expressed by mean, standard deviation (SD), and median. The comparison between the two groups was performed by the Chi-square test and Fisher's exact test of all categorical data. Quantitative data were compared between the groups by the Mann–Whitney test. The level of significance in the present study was 5%. Analysis was performed using the SPSS Statistics version 17 software (SPSS Inc., Chicago, Illinois, U.S.A.).


  Results Top


A total of 34 participants including 17 patients with vitiligo and 17 age- and gender-matched controls were included in the study. The mean ± SD age of cases was 45.82 ± 13.62 years (range 19–65 years) and controls was 46.00 ± 13.52 years (P = 0.97). Among the 34 participants studied, 18 (52.9%) were female and 16 (47.1%) were male (P = 1.00). The male:female ratio was 0.89:1.

Of the 17 patients with vitiligo, 3 (17.6%) gave a family history of vitiligo in one or two first-degree relatives. Of the 17 patients studied, 14 (82.3%) had nonsegmental vitiligo (NSV). Oral mucosal involvement was present in 11 (64.7%) of vitiligo cases studied. Leukotrichia was present in 10 (58.8%) of vitiligo cases studied. Koebner phenomenon was present in 7 (41.2%) of vitiligo patients in our study. The duration of vitiligo ranged from 6 months to 53 years. Among the 17 patients studied, majority 11 (64.7%) had vitiligo of <10 years duration. The mean duration of vitiligo was 11.2 ± 13.7 years. The mean age of onset of vitiligo was 33.94 ± 14.70 years. Of the 17 patients studied, 12 (70.6%) had active vitiligo and 5 (29.4%) had stable vitiligo. A VIDA score of 0 and below was considered stable vitiligo and a VIDA score greater than 0 was considered active vitiligo. Of the 17 vitiligo patients studied, 10 (58.8%) had <10% skin surface affected by vitiligo.

The genotypic distribution of the Fok1, Bsm1, Apa1, and Taq1 polymorphisms between case and control groups is shown in [Table 1]. There was no significant difference in the distribution of Fok1, Bsm1, Apa1, and Taq1 VDR polymorphisms between the case and control groups.
Table 1: Genotype frequencies of the Vitamin D receptor polymorphisms among cases and controls and their association with vitiligo

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The mean serum 25(OH) Vitamin D level in vitiligo patients was 35.64 ± 15.68 ng/ml and in controls was 37.00 ± 16.45 ng/ml with no statistically significant difference between the two groups (P: 0.667). None of the cases and controls was deficient in Vitamin D [Table 2]. There was no significant association between the area of involvement by vitiligo and serum 25-hydroxyvitamin D levels. No significant association was observed between the stability of vitiligo and serum 25-hydroxyvitamin D levels.
Table 2: Comparison of serum 25-hydroxy Vitamin D levels between case and control groups

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We evaluated any association between the genotype variants assessed and serum 25 hydroxy Vitamin D levels in vitiligo patients. We observed a significant association between the Fok 1 VDR gene polymorphisms and serum 25-hydroxyvitamin D levels in vitiligo patients studied [Table 3]. Among the vitiligo patients in our study, carriers of two Fok1 f alleles had sufficient serum 25-hydroxy Vitamin D levels. Seven (77.8%) of vitiligo patients bearing the FF genotype in our study had insufficient serum Vitamin D levels. No significant association was observed between the Bsm1, Apa1, Taq1 polymorphisms, and serum 25-hydroxyvitamin D levels among vitiligo patients in our study [Table 4], [Table 5], [Table 6].
Table 3: Association between the Fok 1 Vitamin D receptor gene polymorphism and serum 25-hydroxy Vitamin D level in vitiligo patients

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Table 4: Association between the Bsm 1 Vitamin D receptor gene polymorphism and serum 25-hydroxy Vitamin D levels in vitiligo patients

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Table 5: Association between the Apa 1 Vitamin D receptor gene polymorphism and serum 25-hydroxy Vitamin D levels in vitiligo patients

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Table 6: Association between the Taq 1 Vitamin D receptor gene polymorphism and serum 25-hydroxy Vitamin D levels in vitiligo patients

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  Discussion Top


In our study, there was no significant difference in the distribution of Fok1, Bsm1, Apa1, and Taq1 VDR polymorphisms between case and control groups. Li et al. in China found that the Bsm1 variant B allele frequency was significantly lower among vitiligo cases than among controls (P = 0.023).[4] They also observed that the Apa1 variant A allele frequency was significantly lower among cases than among controls (P < 0.001). They also observed that the Taq1 variant t allele frequency was significantly lower among vitiligo cases than among controls (P = 0.004).

Aydingoz et al. in Turkey found that the homozygous variant genotype (CC) for Taq1 was more frequent in vitiligo cases (21.4%) than in controls (12.0%).[6] Those who carried allele C had a 1.41-fold increased risk of developing vitiligo compared to those who carried allele T (P = 0.046). Sobeih et al. in Egypt found that the Apa1 variant “a” allele frequency was significantly higher among vitiligo cases than controls (P = 0.004).[8]

In a study by Hassan et al. in Jammu and Kashmir in India, the differences between the vitiligo and control groups regarding the frequency of the genotype Aa were statistically significant (P = 0.0001).[9]

Serum 25(OH)D levels were categorized into the following clinically acceptable cutoffs: Deficient: <10 ng/ml; insufficient: 10–30 ng/ml; sufficient: ≥30 ng/ml.[10] In our study, no statistically significant difference was observed between the mean serum 25(OH) Vitamin D levels in vitiligo patients and controls (35.64 ± 15.68 ng/ml vs. 37.00 ± 16.45 ng/ml, respectively). Sobeih et al. in Egypt found lower mean serum 25(OH) Vitamin D levels in vitiligo patients compared to controls (17.5 ± 8.1 vs. 28.8 ± 10.5 ng/ml, P < 0.05).[8] In a similar Indian study by Hassan et al.,[9] mean serum Vitamin D levels were lower in vitiligo patients than in the control group, being 16.170 ± 8.629 ng/ml and 25.49 ± 1.02 ng/ml, respectively (P = 0.0001). Our findings are similar to those of Khurrum et al. in Saudi Arabia who found no significant difference in the median of serum 25(OH)D levels between cases and controls (P = 0.25).[10]

None of our cases and controls was deficient in Vitamin D. In a study by Khurrum et al.,[10] 91% of subjects studied had deficient Vitamin D levels (<10 ng/ml). The differences in mean serum 25(OH) Vitamin D levels in different geographic locations may be attributed to differences in food and dressing habits.

We observed a significant association between the Fok 1 VDR gene polymorphisms and serum 25-hydroxyvitamin D levels among vitiligo patients studied. Our findings were similar to the study by Li et al. in China who found that carriers of two Fok1 F alleles had lower serum 25(OH)D levels than did carriers of two Fok1 f alleles.[4] Hassan et al. in Jammu and Kashmir in India observed no significant difference in serum 25(OH)D levels among the different Apa1, Bsm1, Taq1, Cdx2 and Fok1 genotypes among vitiligo cases studied.[9]

Smolders et al. observed an association of the F allele with low serum 25(OH)D levels in patients of multiple sclerosis (MS) and controls in the Netherlands.[11] Monticielo et al. observed that serum 25(OH)D concentrations were significantly higher in Brazilian patients with SLE carrying the FokI f/f genotype compared with patients carrying the F/F genotype (31.6 ± 14.1 ng/ml vs. 23.0 ± 9.2 ng/ml, P = 0.004).[12] Fok1 polymorphisms may have important consequences for Vitamin D metabolism in patients with autoimmune diseases like vitiligo, MS and SLE.[4],[11],[12]

Limitations of our study were small sample size. As the cases and controls were recruited from March to November 2017, we did not consider seasonal variation in serum Vitamin D levels. Further Indian studies with larger sample size are needed to evaluate the association of VDR gene polymorphisms and serum 25-hydroxyvitamin D levels in patients with vitiligo.

It has been reported that the increased expression of proinflammatory and proapoptotic cytokines such as interleukin-6 (IL-6), IL-8, IL-10, IL-12, Interferon alpha and tumor necrosis factor-α (TNF-α) cause vitiligo. Vitamin D might exert immunomodulatory effects by inhibiting the expression of IL-6, IL-8, TNF-α and TNF-γ. The active form of Vitamin D reduces the apoptotic activity induced by ultraviolet B (UVB) in keratinocytes and melanocytes by the production of IL-6.[13]

A meta-analysis by Zhang et al. revealed that VDR Apa1 polymorphism increased the susceptibility risk of vitiligo and that there is a positive correlation between serum 25(OH)D deficiency and the incidence of vitiligo.[14]

Many studies have shown Vitamin D3 analogues like calcipotriol to be effective in combination with psoralen plus ultraviolet A, narrowband UVB, or an excimer laser. As the mechanism of Vitamin D action is slow, Vitamin D analogues will be effective in patients with stable disease or slow-spreading disease.[13]

Finamor et al. treated 16 patients of vitiligo with 35,000 IU of Vitamin D3 once daily for 6 months in association with a low-calcium diet (avoiding dairy products and calcium-enriched foods like oat, rice or soya “milk”) and hydration (minimum 2.5L daily). Fourteen of the 16 patients had 25%–75% repigmentation with no significant change in metabolic parameters. High-dose Vitamin D3 therapy may be effective and safe for vitiligo patients.[15]


  Conclusion Top


In our study, no significant difference was observed in the distribution of Fok1, Bsm1, Apa1 and Taq1 VDR polymorphisms between case and control groups. However we observed a significant association between the Fok1 polymorphism and serum 25(OH) vitamin D levels in vitiligo patients studied. In our study no statistically significant difference was observed between the mean serum 25(OH) vitamin D levels in vitiligo patients and controls.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initial s will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgement

The authors acknowledge Nitte (Deemed to be University) and Molecular Genetic Lab, Central Research Laboratory, K.S.Hegde Medical Academy, Mangalore.

Financial support and sponsorship

The study was financially supported by Nitte (Deemed to be University) Faculty Research Grant 2016.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Alkhateeb A, Fain PR, Thody A, Bennett DC, Spritz RA. Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Res 2003;16:208-214.  Back to cited text no. 1
    
2.
Wadhwa B, Relhan V, Goel K, Kochhar AM, Garg VK. Vitamin D and skin diseases: A review. Indian J Dermatol Venereol Leprol 2015;81:344-55.  Back to cited text no. 2
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3.
Birlea S, Birlea M, Cimponeriu D, Apostol P, Cosgarea R, Gavrila L, et al. Autoimmune diseases and Vitamin D receptor Apa-I polymorphism are associated with vitiligo in a small inbred Romanian community. Acta Derm Venereol 2006;86:209-14.  Back to cited text no. 3
    
4.
Li K, Shi Q, Yang L, Li X, Liu L, Wang L, et al. The association of Vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D levels with generalized vitiligo. Br J Dermatol 2012;167:815–21.0.  Back to cited text no. 4
    
5.
Valdivielso JM. Fernandez E. Vitamin D receptor polymorphisms and diseases. Clin Chim Acta 2006;371:1-12.  Back to cited text no. 5
    
6.
Aydingoz IE, Bingul I, Dogru-Abbasoglu S, Vural P, Uysal M. Analysis of Vitamin D receptor gene polymorphisms in vitiligo. Dermatology 2012;224:361-368.  Back to cited text no. 6
    
7.
Bhor U, Pande S. Scoring systems in dermatology. Indian J Dermatol Venereol Leprol 2006;72:315-21.  Back to cited text no. 7
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8.
Sobeih S, Mashaly HM, Gawdat H, Amr K, Hamid MF, Shaalan E. Evaluation of the correlation between serum levels of Vitamin D and Vitamin D receptor gene polymorphisms in an Egyptian population. Int J Dermatol 2016;55:1329-35.  Back to cited text no. 8
    
9.
Hassan I, Bhat YJ, Majid S, Sajad P, Rasool F, Malik RA, et al. Association of vitamin D receptor gene polymorphisms and serum 25-hydroxy Vitamin D levels in vitiligo- A case-control study. Indian Dermatol Online J 2019;10:131-8.  Back to cited text no. 9
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10.
Khurrum H, AlGhamdi KM. The relationship between the serum level of Vitamin D and vitiligo: A controlled study on 300 subjects. J Cutan Med Surg 2016;20:139-45.  Back to cited text no. 10
    
11.
Smolders J, Damoiseaux J, Menheere P, Tervaert JWC, Hupperts R. Fok-I Vitamin D receptor gene polymorphism (rs10735810) and Vitamin D metabolism in multiple sclerosis. J Neuroimmunol 2009;207:117-21.  Back to cited text no. 11
    
12.
Monticielo OA, Brenol JC, Chies JA, Longo MG, Rucatti GG, Scalco R, et al. The role of BsmI and FokI Vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D in Brazilian patients with systemic lupus erythematosus. Lupus 2012;21:43-52.  Back to cited text no. 12
    
13.
AlGhamdi K, Kumar A, Moussa N. The role of Vitamin D in melanogenesis with an emphasis on vitiligo. Indian J Dermatol Venereol Leprol 2013;79:750-8.  Back to cited text no. 13
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14.
Zhang JZ, Wang M, Ding Y, Gao F, Feng YY, Yakeya B, et al. Vitamin D receptor gene polymorphism, serum 25-hydroxyvitamin D levels, and risk of vitiligo a meta-analysis. Medicine 2018;97:29.  Back to cited text no. 14
    
15.
Finamor DC, Sinigaglia-Coimbra R, Neves LC, Gutierrez M, Silva JJ, Torres LD, et al. A pilot study assessing the effect of prolonged administration of high daily doses of vitamin D on the clinical course of vitiligo and psoriasis. Dermato-Endocrinology 2013;5:222-34.  Back to cited text no. 15
    


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