Vitamin D receptor gene BsmI, FokI, ApaI and TaqI polymorphisms and the risk of systemic lupus erythematosus

Recently, several studies have demonstrated the role of vitamin D receptor (VDR) polymorphisms in the development of systemic lupus erythematosus (SLE); however, these results are inconsistent between different cohorts. Therefore, we studied the prevalence of the VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) genotypes and alleles in SLE patients (n = 258) and healthy individuals (n = 545) in a Polish population. We did not observe significant differences for either the VDR FokI, BsmI, ApaI and TaqI genotype and allele frequencies in patients with SLE and healthy individuals. However, the frequency of the VDR F/F and F/f genotypes of FokI was statistically different between patients with renal disease and patients without this symptom OR = 3.228 (1.534–6.792, p = 0.0014), p corr = 0.0476)]. There was no association of the studied VDR BsmI, ApaI and TaqI polymorphisms with clinical manifestations and laboratory profiles in patients with SLE. Our study indicates that the studied VDR FokI variant might increase the risk of some clinical presentations in patients with SLE. Electronic supplementary material The online version of this article (doi:10.1007/s11033-012-2118-6) contains supplementary material, which is available to authorized users.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease for which the underlying cause remains unclear [1]. The putative causations include interactions between environmental factors, disease-prone genetic background, and various pathogen eliciting innate and adaptive immune responses [2][3][4]. Hyperactivation of the immune system results in the overproduction of autoantibodies and in the formation of immune complexes [1]. These immune complexes are deposited in various organs and tissues, causing the symptoms seen in the clinical manifestations of SLE [1]. Some studies have suggested the possible role of vitamin D in the development of rheumatoid arthritis (RA), type 1 diabetes (T1D), Crohn's disease, multiple sclerosis (MS) and SLE [5][6][7]. The primary function of the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH) 2 D 3 ], is calcium homeostasis and bone metabolism [7,8]. In recent years, the function of vitamin D has been further studied and determined to include this molecule as a pleiotropic regulator of human physiology, as having a role in cancer chemoprevention, and as playing a role in cardioprotection and immune system modulation [7,8].
Vitamin D transmits signals to target cells using the vitamin D receptor (VDR), which is composed of both ligand binding and conserved DNA binding domains. The conserved DNA binding domains function primarily as regulators of gene transcription [9]. VDR forms heterodimers with the related retinoid X receptors and binds to DNA to initiate histone modifications, chromatin rebuilding, and RNA polymerase II binding, which is essential for initiation of transcription [10]. The VDR gene is located on chromosome 12q and contains more than 470 single nucleotide polymorphisms (SNPs), some of which modulate 1,25(OH) 2 D 3 uptake. Therefore, these SNPs can lead to this gene's polymorphisms to being considered as candidate disease risk variants [11]. The most commonly studied VDR SNPs include rs10735810/rs2228570 (FokI) situated in exon 2, and three SNPs in linkage disequilibrium, namely rs1544410 (BsmI) located in intron 8, rs731236 (TaqI), and rs7975232 (ApaI), the last being an SNP situated in exon 9 and intron 9 [8,12].
Recently, several studies have demonstrated the role of VDR SNPs in the development of SLE and its clinical manifestations; however, these results are inconsistent between different cohorts [13][14][15][16][17][18][19]. Therefore, we aimed to study whether the FokI, BsmI, ApaI and TaqI SNPs can be a genetic risk factor of SLE in the Polish population. Because SLE is a heterogeneous disease, we also evaluated the association of these SNPs with different SLE symptoms.

Patients and controls
Two hundred and fifty-eight patients (women only) fulfilling the American College of Rheumatology Classification criteria for SLE [20,21] were selected randomly at Institute of Rheumatology in Warsaw, Poland (Table 1). The five hundred and forty-five controls consisted of randomly selected unrelated healthy female blood donors and healthy women who had an examination at the Institute of Mother and Child in Warsaw, Poland. Both patients and control groups were of Polish Caucasian origin. The protocol of the study was approved by the Local Ethical Committee of Poznań University of Medical Sciences. Written agreement was obtained from all participating individuals. The mean age of SLE patients at diagnosis was 40 ± 11 years, and of controls 39 ± 10 years.
Genotyping DNA was isolated from peripheral leucocytes using a standard salting out procedure. The presence of the VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) SNPs was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) according to the manufacturer's instructions New England BioLabs (Ipswich, USA). DNA fragments were separated in 3 % agarose gels and visualized by ethidium bromide staining. Primer sequences and conditions for PCR-RFLP analyses are presented in Supplemental Table 1S. The presence of the FokI, BsmI, ApaI and TaqI polymorphisms was also confirmed by repeated PCR-RFLP analysis and commercial sequencing.

Statistical analysis
The distribution of genotypes in patients and controls was tested for deviation from Hardy-Weinberg equilibrium. Differences in genotypic and allelic distribution between patients and controls and associations between clinical manifestations, production of antibodies (Abs), and polymorphism distribution in patients were determined by Chisquare (v 2 ) or Fisher exact test. Bonferroni correction for multiple comparisons was used and both p values, before (p) and after correction (p corr ), were determined. The FokI and BsmI polymorphisms were tested for association with SLE using the Chi-square test for trend (p trend ). The odds ratio (OR) and 95 % confidence intervals (95 % CI) were also determined. Statistical significance was interpreted as p value \0.05.

Results
Genotype analysis of the VDR FokI, BsmI, ApaI and TaqI polymorphisms did not show a significant deviation form Hardy-Weinberg equilibrium in the SLE and control groups.

Distribution of the VDR FokI (rs2228570) and BsmI (rs1544410) genotypes and alleles in SLE patients and healthy individuals
We observed an increased frequency of the F/F genotype in patients as compared to controls, but these differences were not significant, with OR of the F/F versus f/fgenotypes 1.300 (95 % CI = 0.840-2.010, p = 0.238) ( Table 2). The frequency of the F/f genotype in patients and controls was similar at 0.44 and 0.45, respectively, and the OR for the F/f versus f/f genotypes was 1.163 (95 % CI = 0.757-1.786, Renal 131 Neurologic 54 Hematologic 88 Immunologic 120 Antinuclear antibody 258 Table 2). There was an increased frequency of the F/F and F/f genotypes in patients compared to controls, but these differences were also not significant OR = 1.225 (95 % CI = 0.820-1.829, p = 0.321) ( Table 2).We also observed an increased frequency of the F allele in patients with SLE as compared to the control group; however, these differences were not significant, OR = 1.146 (95 % CI = 0.924-1.421, p = 0.215). The p value of the Chi-square test for the trend observed for the FokI polymorphism was also not statistically significant (p trend = 0.232). There was also no contribution of the VDR BsmI polymorphism to SLE. The frequency of the BB genotype was lower in patients with SLE compared to the control group, but not significantly so. OR of the B/B versus b/b genotypes was 0.683 (95 % CI = 0.419-1.111, p = 0.123). We found an increase in B/b heterozygote frequency in patients than in controls, but these differences were also not significant, OR of B/b versus b/b genotype was 0.988 (95 % CI = 0.719-1.356, p = 0.939) ( Table 2). The frequency of the B/B or B/b genotypes was slightly lower in SLE patients, OR of B/B or B/b versus b/b was 0.9113 (95 % CI = 0.675-1.231, p = 0.545) ( Table 2). We also did not find a significant difference in the prevalence of B alleles between patients and controls. OR for the VDRB allele frequency was 0.869 (95 % CI = 0.698-1.083, p = 0.211). The p value of the Chi-square test for the trend observed for the BsmI polymorphism was also not statistically significant (p trend = 0.271).

Distribution of the VDR ApaI (rs7975232) and TaqI (rs731236) genotypes and alleles in SLE patients and healthy individuals
We did not observe an association of the VDR ApaI and TaqI SNPs with SLE. OR of the A/A versus a/a genotypes was 1.126 (95 % CI = 0.750-1.689, p = 0.568). There was a slight decrease in A/a heterozygote frequency in patients than in controls, OR of A/a versus a/a genotype was 1.007 (95 % CI = 0.695-1.460, p = 0.970) ( Table 2). The frequency of the A/A or A/a genotypes was slightly increased in SLE patients, OR of A/A or A/a versus a/a was 1.051 (95 % CI = 0.744-1.485, p = 0.777) ( Table 2). There was no significant difference in the prevalence of A alleles between patients and controls. OR for the VDR A allele frequency was 1.068 (95 % CI = 0.866-1.317, p = 0.541). The p value of the Chi-square test for the trend observed for the ApaI polymorphism was also not statistically significant (p trend = 0.5533) ( Table 2).
There was also no contribution of the VDR TaqI polymorphism to SLE. The frequency of the TT genotype was slightly increased in SLE patients than in the control group. OR of the T/T versus t/t genotype was 1.440 (95 % CI = 0.884-2.345, p = 0.142). We also observed a slight increase in T/t heterozygote frequency in patients than in controls, OR of T/t versus t/t genotype was 1.429 (95 % CI = 0.883-2.312, p = 0.145) ( Table 2). The frequency of the T/T or T/t genotypes was also slightly increased in SLE  Table 2). We also did not find a significant difference in the prevalence of T alleles between patients and controls. OR for the VDR T allele frequency was 1.140 (95 % CI = 0.916-1.420, p = 0.240). The p value of the Chi-square test for the trend observed for the TaqI polymorphism was also not statistically significant (p trend = 0.2417).
Association of the VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) polymorphisms with clinical symptoms of SLE Since previous studies indicated the association of VDR polymorphisms to some clinical SLE manifestations [13][14][15][16][17][18][19], we evaluated their contribution to clinical manifestations present in the patient group we studied. We found a significant association between the F/F and F/f allele with renal manifestations of SLE OR = 3.228 (1.534-6.792, p = 0.0014), p corr = 0.0476) ( Fig. 1; Table 2S, on line supplementary data). However, we did not observe a significant association of the BsmI, ApaI and TaqI polymorphisms with clinical manifestations in patients (Figs. 2, 3, 4; Tables 3S-5S, online supplementary data). There was also no association of either of the studied polymorphisms with the presence of anti-dsDNA, anti-Smith, anti-snRNP, anti-Ro, anti-Scl-70 or anti-phospholipid Abs (not shown).
Recently, Birmingham et al. [35] suggested that a seasonal decrease in vitamin D levels may trigger an SLE flare in non-African Americans. The role of vitamin D in the development of SLE has also been studied in the murine model. Administration of 1,25(OH) 2 D 3 to mice with SLE resulted in a protective role in the development of this autoimmune disease [36].
Studies performed in vitro have demonstrated that vitamin D inhibits T cell proliferation and leads to a reduced production of interferon-gamma(IFN-c),interleukin-2 (IL-2), IL-6, IL-12, IL-23 and IL-17 [37][38][39][40]. Moreover, the immunosuppressive activity of 1,25(OH) 2 D 3 is demonstrated by an inhibition in the maturation, differentiation, Fig. 1 Odds ratio plot for comparison of genotypes VDR F/F ? F/f versus f/f and F/F versus F/f ? f/f between patients with and patients without a particular manifestation. Each OR value is represented by the corresponding black square or circle with arms representing 95 % confidence intervals (95 % CI). The analysis was performed by v 2 test. The black circle indicates a significant association activation, and survival of dendritic cells, leading to T cell hyporesponsiveness [41]. VDR have been found in monocytes, macrophages, dendritic cells, and effector/memory T cells [25]. This suggests that functional polymorphisms in the VDR gene may modulate the immune system and an individual's susceptibility to developing SLE.
We did not find that the FokI, BsmI, ApaI and TaqI polymorphisms are associated with an individual's susceptibility to SLE in a Polish population. Our observations are consistent with the findings of Monticielo et al. [18], who demonstrated no association of the BsmI and FokI polymorphisms with the development of SLE in a Brazilian-European cohort. There was also no association of the FokI VDR polymorphism with Chinese SLE patients in Taiwan, or of the BsmI VDR polymorphism with SLE in Iranian and Thai populations [15][16][17]. However, the BB genotype of the BsmI VDR SNP was found to be a risk factor of SLE in Taiwanese and Japanese populations [13,14]. In addition to these findings, Luo et al. [19]  In our study, SLE patients with the F/F and F/f genotypes of the FokI VDR polymorphism exhibited a significantly increased risk of developing renal disease. However, we did not find an association of the BsmI VDR polymorphism with clinical manifestations of SLE. There was no association between the BsmI polymorphism and clinical manifestations, laboratory profiles, or lupus nephritisin Chinese SLE patients in Taiwan [14]. However, the VDR B allele was associated with the development of nephritis in a Han Chinese population, and the bb genotype was associated with lupus nephritis in a Japanese population [13,19].
The differences observed between the BsmI and FokI VDR polymorphisms and the susceptibility to SLE development and the occurrence of some clinical manifestations may be due to exposure of the analysed groups to different environmental factors, group size and genetic heterogeneity.
The roles of the BsmI and FokI polymorphisms on the function of the VDR receptor have been already determined in several studies [12,18,19]. Arai et al. [12] demonstrated that the FokI polymorphism located in exon 2 is linked to a second methionine start site, leading to the formation of a shorter protein receptor that has greater transcriptional activity than the wild type receptor. Recently, Monticielo et al. [18] reinforced the functional role of the FokI polymorphism. They demonstrated that vitamin D concentration was significantly increased in individuals with the f/f genotype versus patients having the F/F genotype [18]. The BsmI polymorphism can be linked to a variable-length polyadenylate sequence within the 3 0 -untranslated region [8]. Recently, Luo et al. [19] demonstrated that the level of VDR mRNA was significantly decreased in patients with the VDR B allele versus those not bearing the B allele.
Our study did not demonstrate that the FokI, BsmI, ApaI and TaqI VDR SNPs are risk factors of SLE in the Polish population, but we found an association of the FokI polymorphism with renal manifestations in SLE patients. However, to study the detailed role of these SNPs in SLE, this evaluation should be replicated in other independent cohorts. Fig. 4 Odds ratio plot for comparison of genotypes VDR T/T ? T/t versus t/t and T/T versus t/T ? t/t between patients with and patients without a particular manifestation. Each OR value is represented by the corresponding black square with arms representing 95 % confidence intervals (95 % CI). The analysis was performed by v 2 test