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

Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa mediated by T cells, whose etiology remains unknown. OLP is characterized as dense lymphocyte infiltration and basal keratinocyte degeneration observable under a microscope [1]. OLP, the typical clinical feature of which include white stripes, can manifest as reticular, papular, plaque-like, erosive, atrophic and bullous [2, 3]. Erosive-like lesions are considered to be the most threatening condition and are characterized by pain, ranging from mild discomfort to severe onset [4].The pain seriously affects the patient’s eating experience and food digestion, reducing the patients quality of life.

Previous studies have suggested that vitamin D (VD) deficiency may be associated with an increased risk of some inflammatory diseases, such as OLP and inflammatory bowel disease [5, 6]. OLP patients presented a nearly 50% reduction in mucosal VD levels, which may be caused by immunoreactions [6]. As a ligand-induced transcription factor, vitamin D receptors (VDRs) encoded by the VDR gene (chromosomal location 12q12–14) play an important role in regulating the role of vitamin D [7.8]. Increasing evidence suggests that single nucleotide polymorphisms (SNPs) in vitamin D-related genes could affect the properties of vitamin D, such as its anti-carcinogenic effects [7]. Thus, we speculated that VDR gene polymorphisms may be related to OLP. Since OLP is considered as a potential precancerous lesion, specific SNPs in VDR or vitamin D pathway genes may also play an important role in oral cancer.

Demographic information on 177 OLP patients and 207 healthy subjects is shown in Table 1. There were similar age and gender distributions 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 the eight SNPs in the two groups was 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 rs7975232-CC genotype (adjusted OR = 2.65, 95%CI = 1.24–5.66, P = 0.012) tended to have a higher risk of OLP.

The cumulative effects of the two SNPs on OLP were evaluated by the Cochran-Armitage trend test. The results showed that the risk of OLP increased as the number of mutations increased (Table 3). The OLP risk increased in subjects carrying one or both of the rs2239185 and rs7975232 alleles (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).

LD information for the two SNPs was shown in Table 5. We performed haplotype analysis to assess the effects of the rs2239185 and rs7975232 haplotype variant alleles on the OLP risk (Table 5). When compared with the most frequent AC haplotype, the 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.

To further explore the biological significance of rs2239185 and rs7975232 in VDR, we also searched for expression quantitative trait loci (eQTL) evidence based on the public Genotype-Tissue Expression(GTEx) database (https://​gtexportal.​org/​). We found that the VDR rs2239185 and rs7975232 genotypes were significantly associated with the expression of VDR in whole blood. Mutations associated with VDR rs7975232 and rs2239185 down-regulated of VDR gene expression in whole blood (P = 0.002 and 0.006, respectively, Fig. 1).

OLP is one of the main causes of oral cancer and approximately 0.5 to 2.0% of patients with OLP can progress to malignant transformation [12]. Finding new markers that can effectively identify people at a high risk for OLP can help prevent and control OLP, thereby reducing the progression of malignant transformation. Previous studies have confirmed the role of multiple factors including genetic polymorphisms in OLP susceptibility [13]. The current study aimed to further explore the effects of genetic variation in the VDR gene and related environmental factors on OLP susceptibility in Chinese Han population and the results showed that VDR rs2239185-TT and rs7975232-CC increased the susceptibility to OLP, after adjusting for factors such as drinking and oral hygiene [14, 15].

Vitamin D and its receptors are mainly involved in immune regulation and calcium and phosphorus metabolism regulation. Evidence from population studies suggests that vitamin D deficiency is highly prevalent among the general population in China, which may be related to genetic variation in VDR [16]. Previous studies have shown that VDR genetic variants (such as rs1544410, rs7975232 and rs731236) can participate in the development of various tumors (such as prostate cancer and hepatocellular carcinoma) by affecting the biological effects of vitamin D [17, 18]. Shortly thereafter, vitamin D deficiency was found to be associated to immune-related diseases (systemic lupus erythematosus) and infectious diseases (tuberculosis) [19]. On this basis, our study found the association between the genetic variation of VDR and OLP susceptibility, which further confirmed the findings of Zhao Bin et al. And their study found that the VD/VDR signaling pathway could prevent OLP development by mediating the NF-κB pathway [20]. The current results revealed that mutations in VDR rs2239185 and rs7975232 increased the risk of OLP, and OLP risk increased with increasing adverse alleles. The heterogeneity test suggested that our final results were robust and unaffected by other factors. Additionally, due to the strong LD between VDR rs7975232 and rs2239185, haplotype analysis was further conducted with the results suggested that SNP-SNP interactions in the VDR gene increased susceptibility to OLP, which further validated the single-gene analysis results [21‐23].

The gene encoding VDR, which is located on chromosome 12q13, consists of 9 exons and 8 introns, and is approximately 75 kb in length [24]. Both rs2239185 and rs7975232 are located in a VDR gene intron, and their polymorphism can affect messenger RNA transcription and protein translation by regulating gene transcription to change the expression and function of VDR [25]. Moreover, the GTEx database showed that VDR expression in the blood was indeed affected by genetic variation associated with rs2239185 and rs7975232. Therefore, we hypothesized that the rs2239185 and rs7975232 variants, as well as the CC haplotypes between the two, may affect VDR expression in whole blood by interfering with transcription. The binding of VD to VDR in whole blood and oral keratinocytes was reduced, preventing the activation of the vitamin D/VDR signaling pathway [20], thereby inhibiting functions related to the VDR signaling pathway, such as anti-inflammatory effects, and eventually led to the occurrence of OLP.

It is worth noting that our findings differ from those of Bojan Kujundzic et al., which found that the genetic polymorphism associated with OLP risk was not VDR rs7975232 but VDR rs2228570 (rs2239185 was not included in the study) [9]. And the haplotype analysis involved rs7975232 and rs731236 suggested that the AT haplotype decreased OLP risk (compared with the most common haplotype CT), further confirming that OLP susceptibility was not caused by a single SNP. Many previous epidemiological studies have shown that the incidence of OLP, clinical symptoms and the mutation rate of VDR genes are different in different ethnic populations, so we speculate that this may also be the main reason for the difference between the two studies [26, 27]. The sample size for this study was smaller than our study (only 65 patients with oral lichen planus and 100 healthy blood donors), and all of study subjects were Caucasian and Serbian. Nonetheless, the results needs to be further confirmed in larger sample cohort studies and functional experimental studies.

We acknowledge that there are limitations in this study. Although various analyses were used to reveal the potential impact of VDR gene polymorphisms on OLP susceptibility, the existing epidemiological evidence presented in this study was not sufficient to speculate on the role of the VDR gene, indicating that our findings require confirmation by functional studies. However, our study also has advantages. First, this is the first study to find genetic evidence that the VDR gene is associated with OLP susceptibility in a Chinese Han population. Second, the sample size of this study is larger than the existing related research [9].