| Abstract|| |
Background: The influence of environmental factors on dental caries is a well-established fact; however, little is known about the impact of genetics on the caries process. Aims: This study was aimed to analyse the association between the vitamin D receptor (VDR) gene polymorphism and dental caries. It also compared the risk of dental caries between individuals with homozygous (tt and TT) genotype and heterozygous (Tt) genotype. Further, this study also aimed to compare VDR gene polymorphism by gender. Methodology: A total of 196 subjects were selected based on their decayed, missing, filled teeth (DMFT) index according to the WHO guidelines (1997), and were divided into two groups based on caries experience. The case group was further classified into low and high caries groups. The saliva samples were collected, DNA was extracted, and VDR Taq1 (T/C) gene polymorphism was analysed by polymerase chain reaction–based restriction fragment length polymorphism (PCR-RFLP) and visualized under 2% agarose gel. Results: There was no significant difference in the VDR Taq1 polymorphism and dental caries between the case and control group. However, further stratification showed a statistically significant difference based on caries experience in the study group. The heterozygous mutant genotype (Tt) was associated with a higher caries experience (62.1%) (P = 0.008) with the frequency of 't' allele being 77% as compared to 'T' allele (63.6%) in the high caries group. Conclusion: VDR Taq1 gene polymorphism does not seem to have a role in the incidence of dental caries in the studied population; however, it may have a role in the severity of the caries process.
Keywords: Dental caries, gene polymorphism, RFL-PCR, vitamin D receptor
|How to cite this article:|
Protyusha G B, Sundharam B S. Analysis of the association between polymorphisms in Vitamin D receptor gene and dental caries. Indian J Dent Res 2021;32:3-7
|How to cite this URL:|
Protyusha G B, Sundharam B S. Analysis of the association between polymorphisms in Vitamin D receptor gene and dental caries. Indian J Dent Res [serial online] 2021 [cited 2021 Sep 17];32:3-7. Available from: https://www.ijdr.in/text.asp?2021/32/1/3/321367
| Introduction|| |
Dental caries is one of the most prevalent oral diseases worldwide, affecting people from all age groups and segments of society. It is a complex, chronic and dynamic multifactorial disease affecting 60%–90% of the population both in industrialized and developing countries alike. The influence of host and environmental factors in causing dental caries is well established. However, it is interesting to note that when exposed to the same environmental challenges such as oral hygiene, tooth brushing frequency, or dietary habits, the degree of caries susceptibility varies among individuals. These discrepancies indicate a genetic component regulating one's susceptibility to dental caries in addition to the other factors. An important host factor affecting the incidence of dental caries is the diet and nutrition of an individual.
Vitamin D is a hormone that is of paramount importance in the control of calcium and phosphorus metabolism. This hormone is responsible for the absorption of calcium from a diet rich in fish, egg, and dairy products. This is responsible for maintaining the normal levels of serum vitamin D, in turn harmonizing the acts of bone formation and resorption. Deficiency of this vitamin, thus, leads to various problems including impaired composition and mineralization of teeth and bones. An established role of vitamin D when exposed in early life is the prevention of dental caries. The possible mechanisms by which vitamin D decreases dental caries include better immune function and improved mineralization of teeth and their surrounding bone in response to caries. It helps in the formation of enamel and dentin by helping in odontoblast differentiation and matrix calcification., The ameloblasts and odontoblasts are the target cells for 1,25-dihydroxyvitamin D, the most active form of vitamin D. The circulating levels of this active form is strictly monitored and acts through a specific intracellular vitamin D receptor (VDR) to mediate its genomics actions on almost every aspect of calcium homeostasis.,
The gene encoding the VDR, located on chromosome 12q13.11, contains several polymorphic regions. Four common polymorphisms of this gene are TaqI, ApaI, BsmI, and FokI.
The Taq I polymorphism that is characterized by a single base transition (T > C) leading to a synonymous change at codon 352 in exon 9, is the most studied variant. It creates a TaqI restriction site, and the resulting alleles are called ''T'' (Taq I site absent) and ''t'' (Taq I site present). The presence of the ''t'' allele correlates with increased transcriptional activity, mRNA stability, and a high serum level of 1,25 (OH)2D3. Therefore, nutritional vitamin D deficiency or VDR gene mutation may lead to improper functioning of the vitamin D pathway leading to crucial defects in gene activation, affecting calcium metabolism, cell proliferation, and immune function.,
Several studies have evaluated the potential association between the VDR gene polymorphism and increased susceptibility to periodontitis, diabetes, tuberculosis, and osteoporosis. Limited studies have been done to analyse the allele and genotype frequencies of the VDR TaqI polymorphism in patients with dental caries. However, no study has been conducted on the Indian population yet. Hence, this study was carried out to analyse the association between this VDR gene variant and dental caries in the Chennai population.
| Materials and Methods|| |
Selection of subject
A total of 196 individuals between 18 and 60 years of age were included in this study from the southern part of India, belonging to the city of Chennai. The recruited subjects were diagnosed with dental caries based on the decayed, missing, and filled teeth (DMFT) index according to the WHO guidelines (1997). The subjects were then divided into two groups depending on caries experience. Group 1 (Case group) consisted of 96 subjects with dental caries experience (DMFT ≥1). They were further sub-classified into: low caries group (DMFT ≤3) and high caries group (DMFT >3). Group 2 (control group) consisted of 100 subjects with no previous caries experience (DMFT=0) [Table 1].
Method of examination
A detailed history was obtained from the subjects which included their past medical and dental treatments, drug history as well as other general health concerns. Except for the presence of caries, the subjects included in this study were apparently healthy. The subjects with periodontitis or other systemic conditions that could influence the occurrence of dental caries were excluded. A thorough intraoral examination was carried out and the DMFT index scoring was done thereafter. The clearance for conducting the study was obtained from the Institutional Review Board, and an informed consent was obtained from the subjects.
Sample collection and DNA extraction
Ten mL of unstimulated saliva was collected in a sterile container from the subjects in both groups in a sitting position, after a saline rinse. All the samples were collected between 10:00 am and 1:00 pm to prevent diurnal variations. The collected samples were labeled with the group name and case number and stored at -20°C until processing.
The DNA was extracted from these saliva samples using a DNA extraction kit. The VDR receptor/TaqI polymorphic regions were amplified using PCR in the isolated genomic DNA.
The VDR Taq1 (T/C) gene polymorphisms were genotyped by polymerase chain reaction–based restriction fragment length polymorphism (PCR-RFLP) both in the case as well as in the control group [Figure 1]. The PCR amplification reagents consisted of PCR Master mix (2X) [Takara, Japan], Primers: [Stock- 100 pmol/μL; working stock- 5 pmol/μL, Forward :5′-ATGGAAGGACCTAGGTCTGGAT-3′, Reverse: 5′-TTCAGGATCATCTTGGCATA-3′ and Template DNA: [100 ng/μl].
|Figure 1: Genotyping using restriction fragment length polymorphism (RFLP) method|
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| Methodology|| |
A cocktail containing PCR master mix, primers (forward and reverse), genomic DNA, and water were made to a volume of 20 μL, and PCR was performed as per the conditions: 35 cycles of initial denaturation: 94°C – 4 min, denaturation: 94°C – 35 sec, annealing: 59°C – 35 sec, extension: 72°C – 35 sec, final extension: 72°C – 5 min. The PCR products were digested with the Taq1 enzyme using the optimal buffer for the specific enzyme. The restriction digested samples were run on agarose gel of appropriate concentration (1.5% – 2% w/v). The DNA bands were visualized by trans-illumination with UV light and documented. The genotypes were recorded [Figure 1], and the data were analyzed.
The Hardy-Weinberg equilibrium was assessed for the VDR Taq1 genotypes. Genotypic and allelic frequencies of VDR Taq1 gene variants were calculated in two groups. Qualitative data such as age, gender, genotypic and allelic frequencies were calculated between the case and the control group using the Pearson Chi-square test. The odds ratio (OR) with a 95% confidence interval (95% CI) was also calculated. Data analysis was performed on the SPSS 16.0 software platform (SPSS, IBM Chicago, IL, USA). A P value of less than 0.05 (P < 0.05) was considered to be statistically significant.
| Results|| |
All the genotype frequencies of the VDR TaqI polymorphism were consistent with the Hardy-Weinberg equilibrium (Cases - P = 0.330; Control P = 0.373). In this study, there were no significant differences in the VDR Taq I genotype distributions and allele frequencies between the case group and the control group [Table 2]. However, on further stratification, there was a statistically significant difference based on caries experience among the patients in the studied population. It was observed that the heterozygous mutant genotype (Tt) was associated with a higher caries experience (62.1%) (P value = 0.008). The frequency of the 't' allele was 77% as compared to 'T' allele (63.6%) in the high caries group [Table 3].
|Table 2: Frequency Of VDR TaqI SNP Genotypes (Rs731236) and alleles in the case and control groups|
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|Table 3: Frequency of VDR TaqI snp genotypes and alleles by caries experience|
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Binary regression analysis was done for the significant variables. The odds's ratio with 95% confidence intervals was calculated for the genotypes using 'TT' genotype as the reference [Table 4] and [Table 5].
The individuals with the 'Tt' genotype were predisposed to developing higher caries than the individuals with the 'TT' genotype by 4.5 times (95% CI 1.6–12.2). Additionally, the individuals with the 'tt' genotype showed a 2 times greater risk of developing higher caries than the individuals with 'TT' genotype (95% CI 0.50–7.9) although the P value was not statistically significant [Table 4]. The frequency of the 't' allele was found to be 1.7 times more in individuals with high caries than in individuals with low caries [Table 5].
The frequency of the genotypes was analysed with gender both in the case and in the control group. No significant differences were observed between the male and female individuals either in the case (P = 0.515) or in the control group (P = 0.548).
| Discussion|| |
The multifaceted nature of dental caries involving the interplay between several factors accounts for its complex etiology. Over the years, studies have associated dental caries not only with microorganisms but also with socio-economic, demographic, and behavioral aspects. The implication of the role of genetics in its etiology has further added a newer dimension.
The role of nutrients, especially that of vitamin D in the etiology of dental caries has been widely accepted., Published research has reported that 1,25 (OH) 2D3, the active form of vitamin D, functions through its widely distributed vitamin D receptors (VDR) in the body and regulates the expression of a myriad of genes. This calciotropic hormone, after binding to the Vitamin D receptor, plays a pivotal role in regulating calcium metabolism and enhances its deposition on the teeth and bones rendering them more mineralized thereby shielding them against destruction.
It has been previously reported that polymorphisms in the functionally critical areas on the VDR gene can alter the expression of the receptor protein or modulate its affinity for vitamin D, in turn influencing the calcium metabolism and homeostasis. Studies in the past have reported a possible association of Taq1polymorphism in the VDR gene with vitamin D3 levels. The TaqI RFLP being a “synonymous” polymorphism, has been found useful in association studies as they can be used as markers for several diseases. Recent studies have reported the association of VDR Taq1 gene polymorphism with dental caries., However, all published studies do not share similar views, and there have been conflicts in their results.
The present study analysed the association of the VDR gene polymorphism and dental caries in the population of Chennai using the Taq1 (rs731236) polymorphism. The saliva was used to isolate genomic DNA in the present study, unlike other studies, where blood or buccal epithelial cells have been used.,, Though blood is the conventional source of genomic DNA, the procedure of drawing blood is invasive and requires trained personnel. Thus, the present study used saliva which is non-invasive, convenient and an equally effective alternative.,
The results of this study did not provide convincing evidence of a causal relationship between the overall risk of dental caries and VDR Taq1 gene polymorphism. However, the results do seem to indicate a significant association between the incidence of this polymorphism and the severity of dental caries. Stratification of the patients according to their caries experiences showed a significant correlation between the VDR Taq1 gene polymorphism and caries risk with the “t” allele being a potential caries susceptibility marker. It was seen that the frequency of “Tt” genotype in the high caries group (62.1%) was significantly higher than the low caries group (30%). The frequency of 't' allele was 77% as compared to 'T' allele (63.6%) in the high caries group. The individuals with 'Tt' and 'tt' genotypes had 4.5 times and 2 times higher odds of experiencing high caries, respectively. The frequency of the “t” allele was also found to be 1.7 times more in individuals with high caries than in individuals with low caries.
This suggests that despite having no significant association with the overall risk of the disease, VDR Taq1 gene polymorphism may be suggested to have a strong association with a higher caries risk among the studied population. While the influence of environmental factors, oral hygiene awareness, and habitual aspects of an individual in the initiation of caries cannot be denied, a statistically significant difference was noted between this polymorphism and a higher caries rate in people already affected with caries.
Interestingly, the 'tt' genotype which has been previously associated with caries, was found to be slightly more in the control group than in the caries group in this study. This suggests that though the individuals with 'tt' genotype in the control group may be more susceptible to caries, they may not necessarily express the disease clinically because the other confounding factors such as regulating this multifactorial disease, may compensate for the genetic impact.
A study done on Czech children showed the absence of a significant association between VDR gene polymorphism and overall risk of dental caries which was similar to the result of the present study, but contradicted the studies carried out on the Chinese and Turkish population., However, the association of this polymorphism with higher caries susceptibility seen in this study was in accordance with the previously published studies., The difference in the results may be accounted for by the methods applied, sample size and variation in racial, ethnic, geographic, genetic and environmental background.
VDR Taq1 (rs731236) is one of the polymorphisms of the VDR gene that has been evaluated in the present study. However, the other polymorphisms of the gene may have some association with dental caries and require to be evaluated in the future. Previous researches have shown an association of dental caries with other polymorphisms of the VDR gene, apart from Taq1., However, it is to be noted that both these studies were carried out on children with primary and mixed dentitions unlike the present study, which only dealt with permanent dentitions. Furthermore, dental caries is influenced by a number of genes, one or more of which may cause silencing of the VDR gene via imprinting. This may also be responsible for the lack of clinical expression of the polymorphism in the studied population.
In addition, information on the other potential regulators of dental caries such as socio-economic status, dietary habits, fluoride exposure, the status of individual's serum vitamin D levels and oral hygiene habits was not obtained from the studied population, which may have further contributed to the difference of result in the present study.
In summary, it may be stated that though the incidence of VDR Taq1 gene polymorphism does not seem to have a role in the incidence of dental caries in the studied population, it may, however, have a role in the severity of the caries process. Despite the limitations discussed, the present study may prove useful in providing a baseline and starting point for the genotyping of caries susceptibility genes in the Indian population. Prospective studies utilising these values, evaluating genetic linkages while using all polymorphisms in a larger population could provide the exact role of the VDR gene in caries incidence, its interactions with the environment, and other susceptibility genes while allowing important associations to be derived for this complex multifaceted disease.
The authors would like to thank Dr. J. Vijayashree Priyadharshini (Research Associate, Meenakshi Ammal Dental College and Hospital) for her guidance, support, and for providing the facilities in the technical aspects of this study.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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G B Protyusha
Department of Oral and Maxillofacial Pathology, Meenakshi Ammal Dental College, Alapakkam Main Road, Maduravoyal, Chennai - 600 095, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]