INTRODUCTION:

Dental caries is a multifactorial disease with no single causation pathway and is recognized to be a biofilm-mediated disease that occurs when acidogenic oral flora disrupts the homeostatic balance of the plaque biofilm and initiate the disease process¹.Caries progression occurs as a result of an imbalance in the processes of demineralization caused by acid produced by cariogenic oral bacteria and remineralization mediated by buffering action of saliva eventually leading to dental cavitations².Multiple factors contribute to a person’s risk for caries, including: environmental factors such as diet, oral hygiene, fluoride exposure and the level of colonization of cariogenic bacteria and host factors, such as salivary flow, salivary buffering capacity and surface characteristics of tooth enamel³.

Genetic variation of the host factors and altered immune response to the cariogenic bacteria also contribute to the increased risk and incidence of dental caries⁴. In a large cohort study of twin pairs between 18 months to 8 years of age, at least 70% of the variation in frequency and severity of dental caries was explained by genetic contribution to dental caries traits⁵.Host genetics lead to inter-individual variation in susceptibility to caries suggesting dental caries could be heritable².Several genes likely influence individual susceptibility to caries and these include genes involved in enamel development, salivary factors which influence bacterial adhesion or acidic buffer capacity and immune response⁶.

Antimicrobial peptides (AMPs) form the first line of defense and are involved in the host innate immune responses which implies their potential role in protecting tooth structure from bacterially-induced caries, either by direct killing or by prevention of biofilm formation on the tooth surface⁷.β-Defensins are a group of antimicrobial peptides produced by epithelial cells of many organs including skin, kidney, pancreas, eyes and nasal and oral mucosa⁸.They exhibit broad-spectrum activity against Gram-positive and-negative bacteria, viruses and fungi and antimicrobial activity was observed against Streptococcus mutans, Candida albicans and also against periodontal pathogens including Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum⁹.β-Defensins bind to bacterial membrane and aggregate to form pores that kill bacteria¹⁰ and also show potent chemotactic activity to immune cells, activate immature dendritic cells and memory T cells and induce the immune cells to release chemokines¹¹.

β-Defensin 1(DEFB1) is expressed in oral cavity and plays an important role against cariogenic bacteria¹².It acts synergistically with other antimicrobials and has coexpression with salivary peptides such as histatin, proline-rich proteins and calprotectin and provides a natural antibiotic barrier¹³.Studies have shown association between risk of dental caries and salivary protein polymorphisms in different populations worldwide¹⁴. So we have specifically chosen to study how single nucleotide polymorphisms (SNPs) in
β-defensin 1 region affect susceptibility to dental caries. Polymorphisms in DEFB1 have been associated with several diseases^15-18but there is little information available regarding the distribution of DEFB1 polymorphism in adult dental caries. SNPs located in 5’ UTRs are associated with deregulation in gene expression at the transcriptional and post-transcriptional levels^19.Alterations of these regulatory mechanisms are known to modify molecular pathways leading to disease processes. Here, we analyse genetic polymorphisms in the regulatory elements of DEFB1- 5’ UTR (-20G>A (rs11362), -44C>G (rs1800972), -52G>A (rs1799946)) in individuals having caries experience (Decayed, Missing teeth due to caries, Filled Teeth- DMFT>5) in tamil ethnic population. Our work aims to ascertain whether polymorphism in β-defensin 1 gene is associated with caries susceptibility in adults.

MATERIALS AND METHODS:

Study subjects:

The participants were recruited from the outpatient Department of Conservative Dentistry and Endodontics, Sree Balaji Dental College and Hospital (SBDCH), Chennai, Tamil Nadu, India. This study was approved by the Ethical Committee of SBDCH (Reference no: SBDCH/IEC/12/2019) and the participants gave written informed consent. Molecular genetic studies have been carried out previously with saliva for dental caries^14,20 and the Decayed–Missing–Filled (DMF) method proposed by WHO is the most common method in oral health epidemiology for assessing and measuring dental caries among populations²¹. So, we collected unstimulated saliva from the participants for genetic analysis and used DMFT index as a criteria for grouping the participants. The recruited individuals comprising 30-60 years were diagnosed according to DMFT index and divided into two groups based on caries experience: case group (n=60) with caries (DMFT >5) and control group (n=60) with no previous caries experience (DMFT = 0). For the age group between 35 and 44 years, the mean DMFT score is reported as 5-8.9 by the WHO Global Review of Oral Health in India²². So, we considered DMFT>5 as our criteria. Most individuals studied reported that they brushed their teeth at least once a day. The individuals were refrained from eating for at least 1 hour and saliva collection was done between 9am – 12pm. Patients with chronic diseases such as diabetes, carcinoma, tuberculosis and patients undergoing steroid therapy for more than one month were excluded from the study.

Extraction of genomic DNA:

Unstimulated saliva was collected from the participants with caries and healthy controls without caries and genomic DNA was extracted by a standard salting-out method. Briefly, salivary samples were centrifuged at 10000rpm for 10 mins. To the pellet 500µl of lysis buffer (50mM TrisHCl at pH 8, 10mM ethylenediaminetetraacetic acid and 0.2% sodium dodecyl sulfate) was added and incubated at 65 ̊C for 2 hrs. The samples were then cooled to room temperature and 5M NaCl was added to each tube and vortexed. The samples were kept at 4 ̊C for 10 mins and then centrifuged at 10000rpm for 5 mins. To the supernatant, an equal volume of isopropanol was added, mixed well and centrifuged at high speed for 5 mins. To the pellet, 300µl of 70% ethanol was added and centrifuged at high speed for 5 mins. The DNA pellet was air-dried and dissolved in nuclease-free water and stored in the freezer (-20^ᵒC) (Synergy Scientific, Chennai) for further use. The quality and quantity of DNA were checked using 1% Agarose gel electrophoresis apparatus (EPS Biosolutions, Chennai) and Quantus Fluorometer (Promega, India), respectively.

Genotyping of β-defensin 1

Genotyping of rs11362, rs1800972, rs1799946 polymorphisms of 5’ UTR of DEFB1 was carried out by PCR for amplification using primer sequences and thermal cycling parameters as described previously in the literature²³and followed by Sanger method of sequencing of DEFB1 gene, for cases and controls. Since all three SNPs lie in the 5’UTR region of DEFB1 (-20G>A (rs11362), -44C>G (rs1800972), -52G>A (rs1799946)), same primer sequences were designed for amplification of the sequence region covering three SNPs. The variations in the genetic region of DEFB1 corresponding to rs11362, rs1800972, rs1799946 were then determined using Sanger sequencing.

An amplicon of 261bp was generated by PCR using the DEFB1 forward primer
5'-GTGGCACCTCCCTTCAGTTCCG and the reverse primer 5'-CAGCCCTGGGGATGGGAAACTC. PCR was performed with 50-100ng of DNA under the following conditions. After the initial denaturation at 95°C for 10 min, the reaction mixture was subjected to 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 67°C and extension for 30 s at 72°C followed by the final extension at 72°C for 5 min. Amplification was performed in a 20µl reaction and was confirmed for their specificity by running a 5µl aliquot of the reaction in 1% agarose gel and samples migrated at the expected size of 261 bp and PCR amplified products were given for DNA sequencing.

Statistical analysis:

The statistical analysis was performed using EpiInfo software v.7.0. The risk associated with the genotypes was calculated as odds ratio (OR) with 95% confidence intervals. Statistical significance in the test was set at p<0.05. Hardy–Weinberg equilibrium (HWE) was determined for both cases and controls for all the SNPs. The distribution of genotypes and allele frequencies in dental caries and control groups were compared using the Chi-square (χ2-test) goodness-of-fit test.

RESULTS:

Polymorphisms of DEFB1 (rs11362, rs1800972, rs1799946) were analyzed in dental caries patients and healthy controls to find any association between these genotypes and susceptibility to dental caries. Demographic details of the study groups are shown in Table 1. The genotype and allele frequencies for the DEFB1 polymorphisms -20G>A (rs11362), -44C>G (rs1800972), -52G>A (rs1799946) are shown in Tables 2 and 3, respectively. Sequence results of three polymorphisms of DEFB1 in dental caries patients are shown in Figure 1. The case and control groups for all the three SNPs were found to be in HWE. There was a significant association between DEFB1 rs11362 (p=.025, odds ratio=3.72, 95% CI=1.289-10.742), rs1799946 (p=.023, odds ratio=4.32, 95% CI=1.33-14.028) polymorphisms and the risk of dental caries. For rs11362 (-20G>A), the frequency of AA polymorphism was 68.2% in caries group and 31.8 % in control group. For rs1799946 (-52G>A), the frequency of AA polymorphism was 70.6% in caries group and 29.4% in control group. However, for rs1800972 (-44C>G), the variant genotype GG of DEFB1 polymorphism was found to be high in cases (69.2%) than controls (30.8%) but the odds ratio did not show statistical significance (p=0.136).

Table 1: Demographic characteristics of the study groups

Category	Case Group (DMFT>5) (%) n=60	Control Group (DMFT=0) (%) n=60
Male	31 (51.7)	22 (36.7)
Female	29 (48.3)	38 (63.3)
Age Range	30-60	30-60

Table 2: Genotype frequency distribution for DEFB1 polymorphisms

Genotypes

Number of Cases (DMFT

>5) %

Number of Controls (DMFT

=0)%

Odds Ratio (95% Confidence Interval)

p-Value

rs11362

(-20G>A)

19 (36.5)

26 (56.5)

15 (68.2)

33 (63.5)

20 (43.5)

7 (31.8)

3.72 (1.289-10.742)

0.025*

rs1800972

(-44C>G)

30 (42.3)

21 (58.3)

9 (69.2)

41 (57.7)

15 (41.7)

4 (30.8)

3.07 (0.864-10.932)

0.136

rs1799946

(-52G>A)

20 (35.7)

28 (59.6)

12 (70.6)

36 (64.3)

19 (40.4)

5 (29.4)

4.32 (1.33-14.028)

0.023*

* Indicates significance at P < 0.05.

Table 3: Allele frequency distribution for DEFB1 polymorphisms

Allele Distribution

Cases (DMFT>5)

Controls (DMFT=0)

rs11362 (-20G>A)

0.53

0.47

0.72

0.28

rs1800972(-44C>G)

0.67

0.33

0.81

0.19

rs1799946(-52G>A)

0.57

0.43

0.76

0.24

Allelic distribution in cases and controls were consistent with Hardy–Weinberg Equilibrium.

Figure 1: Chromatogram showing single nucleotide polymorphisms of DEFB1 of dental caries subjects.

A. Heterozygous genotype (GA) (peak colours: G-Black, A-Green) in rs11362 polymorphism, B. Homozygous wild type genotype (CC) (peak colour: C-Blue) in rs1800972 polymorphism, C. Homozygous variant genotype (AA) (peak colour: A-Green) in rs1799946 polymorphism.

DISCUSSION:

Caries is still a major oral health problem in most countries affecting a vast majority of children and adults. The wide prevalence of caries among certain groups and also the evidence that environmental factors such as fluoride exposures do not protect all individuals have particularly encouraged scientific communities to embark on research towards identifying genetic contributors to caries.⁶Polymorphism in genes has been found to play a significant role in disease susceptibility or in how a person responds to different treatments. Genetic association studies between polymorphisms in a particular gene and disease susceptibility are carried throughout the world and many genes are studied for their role. Some of the genes studied with different diseases are IL-4^24-26, IL-10^27-29, TNF alpha^30,31, TLR³², CYP (cytochrome P450)^33-35 and Vitamin D receptor^36,37. With respect to caries risk, the role of genetic factors is still largely unknown but has been investigated.^38-40 This study is a case-control study investigating genetic variations in β-defensin 1 and whether the polymorphisms affect dental caries status. We investigated the genetic background related to caries experience in tamil ethnic adults.

Our study conducted on individuals from the tamil ethnicity revealed that there was a significant association between DEFB1 rs11362 and rs1799946 polymorphisms with the susceptibility to develop caries.Ozturk A
et al., 2010 showed that two polymorphisms in the DEFB1 gene were associated with caries experience: rs11362 increased DMFT up to 5 times and rs1799946 was associated with lower caries experience²⁰.In a study conducted by Yildiz G et al., 2016, for rs11362 polymorphism, the frequency of AA polymorphism was 12.8% in the low caries risk group (DMFT≤5) and 87.2% in the high caries risk group (DMFT≥14)⁴¹. In an Italian population, for rs11362 polymorphism, the study performed by Navarra CO et al., 2016 showed that GG homozygous individuals had a higher DMFT index (mean DMFT=15.5±7.0) compared to both GA heterozygous and AA homozygous individuals⁴².A lack of association between DEFB1 polymorphism rs1800972 and susceptibility to develop caries was reported in a group of Turkish children⁴³.Our study demonstrated that DEFB1 polymorphisms (rs11362, rs1799946) could influence disease susceptibility and progression in dental caries. The identified genetic variants are located in 5’ UTRs of DEFB1 therefore the susceptibility to caries experience might be due to the deregulation of gene expression through modulation of the transcriptional or post-transcriptional process as reported⁴⁴^,⁴⁵.

As AMPs such as β-defensin 1 play an important role in innate immune defense, identifying polymorphisms in AMPs could help in determining an individual's susceptibility to caries in adults. This could aid in finding novel methods using peptides such as β-defensin to treat people who are particularly susceptible to tooth decay and develop new tools for caries risk assessment. Finding people who are more prone to caries might help in early detection, prevention and also can alter the prognosis of the disease. Planned teaching programmes are being conducted throughout India which are found to be effective in imparting knowledge about oral health to school children^46,47 and such initiatives are required to bring awareness to adults also.

CONCLUSION:

Polymorphism in β-defensin 1 plays an important role in the susceptibility to develop dental caries. A detailed genetic variant analysis in β-defensin 1 transcriptional regulatory region in a larger population size is necessary to understand the role of innate defense mechanism at the gene expression level. β-defensin 1 could be used as a prognostic marker in caries associated inflammatory disease prevention regimen. Identification of such genetic risk factors will help to screen and identify susceptible patients and to better understand the contribution of genes involved in caries aetiopathogenesis.

Financial support:

We wish to thank DST-FIST (Ref. No.SR/FST/College-23/2017), Government of India, New Delhi, for providing the funded research equipment facilities to Sree Balaji Dental College and Hospital, Chennai, Tamil Nadu, India.

This study was supported by Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India.

ACKNOWLEDGEMENT:

We thank Dr. Subbiya Arunajatesan, Department of Conservative Dentistry and Endodontics, Sree Balaji Dental College and Hospital, for providing the clinical samples.

We wish to thank Dr. Vettriselvi Venkatesan, Dr. Priyanka Iyer and Ms. Deepika Ramu of the Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research for their research guidance.

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Received on 10.07.2020 Modified on 25.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4731-4735.

DOI: 10.52711/0974-360X.2021.00823

Association of β-defensin 1 gene Polymorphism and dental caries susceptibility in Tamil Ethnicity