Vitamin D receptor genetic polymorphisms were associated with oral lichen planus susceptibility in Chinese Han population

Vitamin D receptor (VDR) is involved in multiple immune-mediated disorders including Oral lichen planus (OLP). This study was aimed to investigate the association between VDR gene polymorphisms and the risk of OLP. 177 OLP patients and 207 healthy participants were recruited from Affiliated Hospital of Stomatology, Nanjing Medical University. Eight single nucleotide polymorphisms (SNPs: rs731236, rs739837, rs757343, rs2107301, rs2239185, rs7975232, rs11574129 and rs11568820) on the VDR gene were selected and genotyped. The results showed that the OLP risk was increased in subjects with the rs2239185 TT genotype (Recessive model: adjusted OR = 2.68, 95% CI = 1.28-5.62, P = 0.009) and rs7975232 CC genotype (Recessive model: adjusted OR = 2.25, 95% CI = 1.10-4.58, P = 0.026). And the significant cumulative effects on OLP risk were found in rs2239185 and rs7975232 (P < 0.01). The haplotype analysis showed that haplotype CC (rs2239185-rs7975232) was associated with increased OLP risk (OR =3.11, 95% CI = 1.42-6.83, P = 0.005), compared with haplotype AC. In conclusion, the variants of VDR rs2239185 and rs7975232 may influence the OLP susceptibility and VDR gene polymorphisms may be the candidate susceptibility region of OLP in Chinese Han population.

The oral mucosa of all participants was assessed by two experienced oral clinicians. If there was a disagreement between two examiners, a third clinically experienced mucosal dentist would make the judgment. Main clinical features, including clinical subtype, affected sites, the number of sites, the presence of cutaneous lesions, the type of mouth lesions and symptoms, were collected for further analyses. All subjects were informed of the purpose of the study and signed the informed consent. Information, such as demographic data, alcohol consumption habits and oral hygiene, were collected by one-toone survey using a questionnaire designed according to our research content. Prior to OLP diagnosis, Participants who drank more than 20 alcohol drinks per week were classified as heavy drinkers [11]. The periodontal status of all subjects including gingival index (GI), periodontal index (PI) and bleeding on probing (BOP) were evaluated in both groups. Oral hygiene of subjects was defined as poor when GI and PI both ≥2, and BOP score was 1. In addition, 10 ml venous blood was collected from each subject for biochemical test and SNPs determination.

DNA isolation and genotyping
Genomic DNA was extracted from peripheral blood samples using protease K digestion and phenol/chloroform purification according to standard protocol. The TaqMan allelic discrimination technology through ABI 7900HT Sequence Detection system (Applied Biosystems, San Diego, California, USA) was used to explore polymorphisms at the chosen SNPs. Polymerase chain reaction (PCR) was executed by the following thermal profile: 50 twarer 2 min to preheat, 95 °C for 10 min to preincubate, then 40 cycles at 95 e following thermal profile: 50 twarer 2 mito anneal.

Statistical analysis
All analyses were operated in Stata/SE (V.12.0 for Windows). The difference of individual demographic characteristics was analyzed by Student t test or the chi-square (χ 2 ) test (for categorical variables). The relationship between candidate SNP and OLP risk was estimated by multivariate logistic regression analysis, and the results were expressed as odds ratio (ORs) and its 95% confidence intervals (CIs). The heterogeneity between the corresponding subgroups was examined by Q test. The Cochran-Armitage test was used for trend analysis. And haplotype analysis was performed to explore the relationship between two significant SNPS and OLP risks. PHASE software (v2.1) was used to estimate haplotype frequency based on observed genotypes. Single-fold view software (version 4.2) was used to analyze linkage disequilibrium (LD) parameters (i.e., D and r2) [12], and Thesias software (version 3.1) was used to analyze associations of identified haplotypes in the VDR gene with OLP [13].

Results
The demographic information of 177 OLP patients and 207 healthy subjects were shown in Table 1. There was similar distribution of age and gender between the two groups (P=0.155 and 0.091, respectively). However, compared with the control group, OLP patients had more alcohol consumption and better oral hygiene (P <0.05).
The genotype distribution of these eight SNPs in two groups is described using dominant, recessive and additive genetic models in Table 2. The recessive genetic model computed by logistic regression analyses showed that rs2239185 and rs7975232 were significantly associated with OLP susceptibility. Patients carrying the rs2239185-TT genotype (adjusted OR=2.39, 95%CI=1.10 -5.18, P = 0.027) and those with rs7975232-CC genotype (adjusted OR=2.65, 95%CI=1.24 -5.66, P = 0.012) tend to have a higher risk of OLP.
Cumulative effects of the two SNPs on OLP were evaluated by comparing the effects among genotypes with different degrees of mutation. The results showed that as the number of mutation increased, the risk of OLP increased ( Table 3). The Cochran-Armitage trend test also showed that the OLP susceptibility increased in subjects carrying one or two alleles of rs2239185 and rs7975232 (OR= 2.33, 95% CI=1.22-4.43). In stratified analyses on the combined variant alleles (rs2239185 and rs7975232) and OLP risk susceptibility, no heterogeneity was observed (Table 4). Table 5. We performed haplotype analysis to assess the effect of the haplotype rs2239185 and rs7975232 variant alleles on OLP risk (Table 5). When compared with the most frequent AC haplotype, CC haplotype was significantly associated with OLP susceptibility (OR= 3.11, 95%CI=1.42-6.83), which was consistent with the single SNP analysis.

LD information of the two SNPs is shown in
To further explore the biological significance of VDR rs2239185 and rs7975232, we also searched for expression quantitative trait loci (eQTL) evidence based on the public GTEx database (https://gtexportal.org/). It is found that VDR rs2239185 and rs7975232 genotype were significantly associated with the expression of VDR in whole blood.
Mutations in VDR rs7975232 and rs2239185 would down-regulate of VDR gene expression in whole blood (P = 0.002 and 0.006, respectively, Fig. 1).

Discussion
Though the etiology of OLP is unknown, immunodeficiency, heritable variation, stress, trauma, virus, diabetes, hypertension and Vitamin D deficiency can be considered as one of etiological factors, and meanwhile which may interact with each other. A family study further emphasized the importance of genetic tendency to OLP susceptibility [14].0.5% to 2.0% of OLP patients can develop a frequent malignant transformation [15].Finding new molecular biomarkers could effectively contribute in the identification of OLP patients with a higher tendency to frequent malignant transformation. This retrospective study aimed to discuss the relationship of environment, clinical features, genetic variation and susceptibility of OLP.
In our study, adult females could increase the susceptibility of OLP, which was the same as other studies [16,17]. Through the recessive model, VDR rs2239185 and rs7975232 polymorphism of significant differences in allele and genotype distribution between OLP patients and control group suggested the possible importance in OLP susceptibility. The cumulative effects of VDR rs2239185 and rs7975232 in OLP indicated that carrying two unfavorable alleles offered the highest risk effect (P<0.05). One finding in African-American (AA) men showed that, nine VDR SNPs were analyzed in a case-control study .The tendency for the number of risk alleles to increase in the haplotype was more associated with prostate cancer risk, which was consisted with our study [23].The heterogeneity test suggested no interaction between different genotypes in different population (P>0.05), which was consistent with previous studies [24,25]. Cumulative analysis showed the importance of considering the SNPs-SNPs combined with VDR gene analysis for OLP susceptibility.
In the current study, TT genotype of VDR gene rs2239185 might be one of the potential genetic risk factors for community-acquired pneumonia(CAP), to increase the susceptibility and severity [24]. In our study, LD was found between VDR rs7975232 and rs2239185 polymorphisms in both OLP and control groups. Compared with the most frequent haplotype AC, carrying the haplotype CC showed an increased risk of OLP, which was consistent with the single SNP effects. However, the haplotype carrying rs2239185 C expressed a significant risk effect, although the single SNP analysis did not show obvious associations. Thus, effects of rs2239185 and rs7975232 may not be independent, and further fine mapping studies are needed. Recent study found that the distribution of AT and CC haplotypes were significantly different between patients and controls, indicating that VDR haplotype had an effect on OLP susceptibility [11]. However, the effect of AT on OLP risk reduction was not found in our study, which might be attributed to the racial difference. But it also underscores again the necessity of further research. Moreover, our findings highlighted the importance of haplotype blocks analysis over individual SNPs approach for complex diseases.

Conclusion
This study was the first time to discover that genetic mutations at VDR rs2239185 and rs7975232 were associated with OLP susceptibility, which might be the candidate

Conflicts of Interest
The authors declare no conflict of interest.

Informed consent:
All subjects were provided the voluntary informed consent to participate in the study.     Logistic regression was used in the implicit model to determine the adjusted P a value according to age, gender, alcohol consumption and oral hygiene, heterogeneity was used to test P b-value.