The FokI Polymorphism of the VDR Gene is a Protective Factor for Psoriasis Vulgaris

Ricardo E Vega-Hernandez1, Marina MJ Romero-Prado1, Lucila Sandoval-Ramirez2, Maria G. Moreno-Trevino3, Oscar RFajardo-Ramirez3 and Julio C Salas-Alanis3* 1Departament of Physiology, University Center of Health Sciences (CUCS), Guadalajara, Mexico 2Mexican Social Security Institute, Genetics Division, Biomedical Research Centre of the West (Cibo), Guadalajara, Mexico 3Department of Basic Sciences and Dermatology, Monterrey University, School of Medicine, Nuevo Leon, Mexico


Introduction
Psoriasis vulgaris (PsV) is a multifactorial disease characterized by well-circumscribed erythematous plaques, constant skin flaking and intense pruritus, that affects specific skin areas. PsV worldwide prevalence ranges from 0.4 to 4.8% and in Mexico 2% of the population presents this disease. It is well know that Vitamin D Receptor (VDR) plays a key role in both the metabolism and differentiation of keratinocytes [1]. The gene encoding the VDR is located on chromosome 12cen-q12, contains 11 exons and spans approximately 75 kb of genomic DNA. Several polymorphisms have been identified in the gene including the Fok I polymorphism located in exon 2 at the 5' coding region of the gene [2]. For this reason, researchers have attempted to establish an association between VDR gene polymorphisms and the onset, presence and response of treatment of psoriasis vulgaris. The aim of this study was to determine the association between four polymorphisms on the VDR gen (FokI, BsmI, ApaI and TaqI) and the clinical manifestations of PsV in a sample of Mexican patients.

Samples
Peripheral blood from a total of 52 patients (aged 5-68 years) and 100 healthy donors was taken after obtaining informed consent. The diagnosis was based on the clinical history, classic features, and a positive Auspitz´s sign. The Psoriasis Area Severity Index (PASI) was also assessed. At least one first-degree relative diagnosed with PsV was reported by 48% of the patients.

Analysis of polymorphisms
Total DNA was obtained from blood by the Gustincich method [3]. Three sets of primers previously described were used to amplify exons 4 and 11, and intron 10 of the VDR gen [4]. Amplified fragments were analyzed by RFLP using the enzymes FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232 ) and BsmI (rs1544410); the latter SNP was confirmed with the enzyme HhaI and by direct sequencing (sequencer model ABIPRISM310, Applied Biosystems, Foster City, CA). All PCRs were performed according to the manufacturer's protocol (New England Biolab, Ipswich, MA) and the products were visualized on1% agarose gels stained with bromide after electrophoresis.
recessive binary association were used to estimate the OR with a 95% CI analyzed by binary logistic regression [5]. The estimation of haplotype frequencies was performed using the expectation-maximization algorithm. A p value <0.05 was considered significant. The platform used for statistical analysis was SNPSstats [6].

Results
Of the total of patients (n=52) with PsV, 78% (n=38) corresponded to early onset PsV; 65% (n=39) did not have nail involvement and 48% of patients (n=25) reported at least one first-degree relative diagnosed with psoriasis.
The PASI score obtained by the study universe was mild in 81%, moderate in 13%, and severe in 6% with a mean of 5.04, range 0.3 to 32. The polymorphisms BsmI (p=0.042) and ApaI (p=0.011) did not show HWE in the reference population. By analyzing the distribution of allelic and genotypic frequencies regarding the clinical variables mentioned above, it is observed that under the dominant model, the TT(ff) genotype was significantly more frequent in patients with late onset PsV (40 or older) (p=0.033) and in patients without nail involvement (p=0.015). The TT (aa) genotype was significantly more frequent, under the recessive model, in patients with a negative family history (p=0.049) ( Table 1). According to the haplotype analysis in the SNPstats platform, we observed significant differences in the most common haplotype in the reference population HpVDR1 (FbaT, 21.1%) compared to patients with PsV HpVDR2 (FbAT, 24.1%) (p<0.001). Considering only 3 of the polymorphisms located just before the 3' region of the VDR gene (BsmI, ApaI and TaqI), we found that haplotypes significantly different between healthy donors and PsV patients were HpBAT1 (baT 32.4%) vs. HpBAT2 (bAT, 49.7%), respectively (p<0.001). Although we found no association of any haplotype in particular with the presence of PsV, we did find a significant difference (p<0.001) between the distribution of the haplotypes composed of the recessive alleles FokI and ApaI (f and a) in patients with PsV vs. the reference population (Table 2). This is the first report of a possible association of the ff genotype in VDR gen (rs2228570) with the clinical variables, late age of onset, and no nail involvement, and also the first time a protective effect of the "f " allele is suggested, which contradicts with that demonstrated in Egyptian population, since this same allele, both homozygous (ff) and heterozygous (Ff), proved to be associated with Type 1 Diabetes Mellitus in Egyptian children [7]. In addition, contrary to the protective effect found in this study, Swapna et al. reported that the VDR gene FokI polymorphism is associated with the risk of developing essential hypertension [8].
Similarly, there is no previous history that addresses a possible association between genotype "aa" (rs7975232) and a family history for the disease according to the recessive model. On the other hand, there are reports that the "aa" allele lessens the risk of tuberculosis (p=0.006), according to a meta-analysis published in 2014, which also suggests that it may be a protector allele [5]. In this study, the genotype frequencies obtained in Mexican population (AA 22%, Aa 35%, aa 43%) show that this polymorphism is in HW disequilibrium with BsmI (P=0.011), which is consistent with previous reports in African population (D´0.974, P<0.00001) [9]. Furthermore, we found that the bAT haplotype was present in 49.7% of our patients vs. 17.2% as reported by Rucevic in Croatia [10]. In contrast with Acikbas et al. [11] we found no haplotype associated with susceptibility to PsV in patients vs. healthy donors [11].
Finally, vitamin-D is a member of the steroid receptor family and mediates the effects of the active metabolite 1, 25(OH) 2 vit D3 by regulating transcription of a number of different cellular genes. The action of vitamin D is mediated through its binding to nuclear receptor (VDR) [12]. The FokI Vitamin D Receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids [13]. This study found an association in FokI polymorphism of VDR gene and two major clinical variables, suggesting that the genotype [T/T]/ff may be a protective factor for nail involvement and age of onset in Mexican patients with PsV.