Lack of association between Vitamin D receptor FokI polymorphism and alopecia areata

ARTICLE

Auteur(s) : Ahmet AKAR¹, Funda Elif ORKUNOGLU², Metin OZATA³, Ali SENGUL², Ali Riza GUR¹

¹ Department of Dermatology 
² Department of Immunology 
³ Department of Endocrinology, Gulhane Military Medical Academy, School of Medicine, 06018 Ankara, Turkey

Article accepted on 18/3/2004

Numerous studies have detected the expression of VDR in epidermis and hair follicle [1-3]. The VDR is expressed in the two major cell populations that comprise the hair follicle: the epidermal keratinocytes and the mesenchymal dermal papilla cells [3]. Expression of the VDR in keratinocytes is necessary for maintenance of the normal hair cycle [4]. Lack of the VDR is associated with reduced epidermal differentiation and hair follicle growth [5]. In addition, patients with hereditary 1,25-dihydroxyvitamin D3-resistant rickets type II (HVDRR) and VDR knockout mice exhibit a phenotype that includes alopecia totalis [6-8]. 

There is strong evidence indicating that AA is a tissue-specific, autoimmune disease [9]. VDR gene polymorphisms influence susceptibility to autoimmune diseases such as Graves’ disease [10] and psoriasis [11-12]. But the role of the VDR gene has not been investigated in patients with AA. 

VDR is expressed in the hair follicle and the lack of it leads to alopecia, that is VDR has great importance for the physiology of the hair follicle. Therefore we hypothesized that the VDR gene could play a role in AA. For this reason, we examined the role of one of the VDR polymorphisms, which is the FokI polymorphism in the translation initiation codon of VDR gene.

Patients and methods

Subjects

The patients were characterized according to AA investigational assessment guidelines [13]. Patients with patchy AA were excluded from this study and only patients with extensive forms of AA (AT, AU and AT/AU) were included. The study was performed on 25 unrelated patients with AT, AU and AT/AU (24 males and 1 female). The mean age of the patients was 23.8 (SD = 4.9) years ranging from 19 to 36 years. The age onset of the first episode of AA ranges from 3 to 17 years. Duration of the current episode of the disease is 2 months to 9 years. Sixteen patients had a history of AT, AU and AT/AU longer than 2 years of duration and nine patients had a history of AT, AU and AT/AU shorter than 2 years of duration at any time. One patient had vitiligo, one patient had vitiligo and thyroiditis. Thyroid antimicrosomal antibody determinations were performed in 20 patients. The levels of these antibodies were increased in 9 patients. Among the 25 patients, 3 had a family history of AA, 1 had a family history of psoriasis vulgaris. The control group consisted of 27 healthy subjects (24 males and 3 females). The mean age of the control subjects was 25.5 (SD = 5.7) years ranging from 20 to 40 years. Subjects with a history of any episode of AA or androgenetic alopecia were excluded from the control group. All subjects were Caucasian. This study was approved by a local ethical committee.

DNA extraction and genotyping of the FokI polymorphism

DNA was extracted from fresh peripheral blood using a commercial kit (Spincolumn DNA extraction kits, Gentra, USA). The PCR by use of primers designed to amplify the FokI site in exon 2 of the VDR gene and digestion of its 265 bp-product with FokI (New England Biolabs Inc., Beverly, MA, USA) enzyme were carried out as described by Chiu and coworkers [14]. The genotypes were classified as FF, homozygotes (absence of the FokI site resulted in one fragment of 265 bp); Ff, heterozygotes, fragments of 265 bp, 196 bp, and 69 bp; and ff homozygotes (presence of the sites resulted in two fragments of 196 bp and 69 bp), (Fig. 1).

Statistical analysis

The frequencies of genotypes from patients and controls was compared using chi square analysis of 2 × 2 tables and Yates’ correction, and strength of associations was estimated by odd ratio (OR) of chi square determination with use of the EPI-INFO 6 statistical program. If five or fewer persons were present per group, Fisher’s exact two-tailed test was used.

Results

FokI genotyping was performed in the group of 25 unrelated patients with AT, AU and AT/AU and 27 healthy control subjects. Homozygous cleavage by FokI generated two fragments, 69 and 196 bp, respectively, whereas the heterozygotes displayed all three bands, thus, the genotypes FF, Ff and ff were identified. The frequencies of F and f alleles were 76.0% and 24.0% in the patient group, and 72.2% and 27.7% in the control group. When patients were compared to controls; no significant differences were found in F and f alleles frequencies. The frequencies of the FF, Ff and ff genotypes were 56.0%, 40.0% and 4.0%, respectively in the patient group. In the control group, the frequencies of FF, Ff and ff genotypes were 48.1%, 48.1% and 3.7%, respectively. The distribution of FokI genotypes in the patient group showed no statically significant differences compared with the control group (Table I).

Discussion

To our knowledge, this study is the first in which the relationship between the VDR gene polymorphism and AA has been investigated. We found no relationship between AA and the FokI restriction fragment length polymorphism VDR genotype in a Turkish population.
It has been reported that the VDR shows considerable polymorphism. A number of polymorphisms have been described in the VDR locus: an exon 2 initiation codon polymorphism, which is detected with FokI restriction enzyme, the BsmI, Tru9I, and ApaI restriction fragment length polymorphism located between exons 8 and 9, the TaqI located in exon 9, and a PolyA polymorphism downstream of the 3’-untranslated region [12]. We investigated only one site including FokI site.
Since there is no study investigating the relationship between VDR polymorphism and AA, we have not compared our data with them. However, we would like to compare our finding for the control group to some previous studies. Saeki et al. [11] found that the frequencies of FF, Ff and ff genotypes were 42%, 45% and 13% in Japanese control subjects. There is only one previous report of VDR FokI polymorphism in a Turkish population. In this study, Kaya et al. [12] investigated the association between VDR gene polymorphism and psoriasis. They found that the frequency of FF, Ff and ff genotypes were 53.7%, 40.7% and 5.6%, respectively in the control group. The genotype frequencies in our control group for FokI were similar to the frequencies in the control group of both the above-mentioned studies, confirming the accuracy of our results. Kanan et al. [15] investigated the VDR gene start codon (FokI) and bone mineral density in healthy male subjects. Most of our patients and control subjects were also males. The frequencies F and f alleles were 64% and 36% in their study. They found the frequencies of FF, Ff and ff genotypes were 44%, 41% and 16%, respectively. Supporting this study, we have also found the alleles frequency for F and f were 72.2% and 27.7% in healthy subjects. However, it has already been revealed that there is a marked ethnic difference in the VDR genotypes [16].
In summary, our findings do not identify the role of VDR gene in pathogenesis of AA, but we investigated only one site among various sites of the VDR gene. More extensive surveys on polymorphisms of the VDR gene, such as BsmI, Tru9I, ApaI, TaqI and polyA, are required. Furthermore, to conclude that there is no relationship between VDR gene polymorphism and AA, the VDR FokI should be further studied in other populations and larger groups. However, this preliminary study may provide insights into the VDR gene in the pathogenesis of AA. n

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