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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 4  |  Page : 381-386
 

Evaluation of the association between tuftelin gene polymorphism, Streptococcus mutans, and dental caries susceptibility


1 Department of Pediatric Dentistry, Nair Hospital Dental College, Mumbai, Maharashtra, India
2 Department of Pediatric Dentistry, Vice Chancellors Office, Maharishi Markandeshwar University, Ambala, Haryana, India

Date of Submission12-Mar-2020
Date of Decision28-Aug-2020
Date of Acceptance06-Dec-2020
Date of Web Publication5-Jan-2021

Correspondence Address:
Dr. Priti Sushil Jain
Department of Pediatric Dentistry, Nair Hospital Dental College, Dr. A. L. Nair Road, Mumbai - 400 008, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_98_20

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   Abstract 


Context: Dental caries can be conceptualized as an interaction between genetic and environmental factors. Aims: The purpose of this study was to identify any polymorphism in tuftelin gene and its association with dental caries susceptibility, either singly or in combination with the microbial causing agent: Streptococcus mutans. Settings and Design: The presented study included a total of 30 children of age group 12–16 years categorized into two groups: 15 children with no detectable caries in Group I and 15 children with high caries (DMFS ≥10) in group II. Materials and Methods: The stimulated salivary samples were inoculated in mitis salivarius bacitracin agar plates and growth of S. mutans was estimated. DNA extraction was done from whole blood and amplification was done with the help of real-time polymerase chain reaction technique. Oligonucleotide primers were designed to flank single nucleotide polymorphism in the gene. Statistical Analysis Used: The collected data was statistically analyzed by unpaired t-test, paired t-test, Chi-square test, Pearson correlation, and regression analysis. Results: The difference in mean salivary S. mutans counts between the two groups was highly significant. Correlation between tuftelin gene polymorphism and dental caries susceptibility was not significant in both Group I and Group II. Only 4.1% of the variability in dental caries risk can be explained by interaction between tuftelin gene and S. mutans. Conclusions: Future research studies including parents and siblings should be carried out to focus on further investigation into the mechanism of this gene-environment interaction.


Keywords: Dental caries, polymorphism, Streptococcus mutans, tuftelin gene


How to cite this article:
Jain PS, Damle SG, Dedhia SP, Jetpurwala AM, Gupte TS. Evaluation of the association between tuftelin gene polymorphism, Streptococcus mutans, and dental caries susceptibility. J Indian Soc Pedod Prev Dent 2020;38:381-6

How to cite this URL:
Jain PS, Damle SG, Dedhia SP, Jetpurwala AM, Gupte TS. Evaluation of the association between tuftelin gene polymorphism, Streptococcus mutans, and dental caries susceptibility. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2021 Jan 25];38:381-6. Available from: https://www.jisppd.com/text.asp?2020/38/4/381/306229





   Introduction Top


Dental caries being a multifactorial, infectious disease has many contributing host and environmental factors. These factors can influence caries susceptibility by their direct or indirect effects on enamel structure and enamel mineralization. During the recent years, the genetic influence over enamel development has been accentuated.[1],[2] Several studies provide evidence of influence of enamel development genes on the susceptibility of dental caries in humans.[3],[4],[5],[6]

Tuftelin is one of the nonamelogenin, enamel matrix protein implied to play an eminent role in mineralization of enamel.[6],[7],[8] It has been seen that tuftelin gene overexpression leads to gross deficiencies in enamel which is evident at the lower levels of enamel hierarchy (at the mesoscale and nanoscale).[9] The part of tuftelin in enamel development may be diminutive, however, studies show that sequence variations in the tuftelin gene could be indirectly affecting caries susceptibility by interfering with other gene or protein interactions.[2] Tuftelin genotypes have additionally been identified to interrelate with Streptococcus mutans levels and early childhood caries in children.[2],[3]

The present study was conducted to identify any polymorphism in tuftelin gene and its association with dental caries susceptibility, either individually or in combination with the microoraganism responsible for initiation of dental caries; S. mutans.


   Materials and Methods Top


The present study was carried out, in collaboration with Microbiology department, Molecular Laboratory, and Roche Diagnostics Ltd. Necessary permissions were obtained from the Institutional Ethical Committee.

A total of 30 children from a municipal school of age group 12–16 years with good general health were included. Informed consent from their parents were taken and the children were categorized into two groups according to their caries status using Modified WHO index (1997);[10] 15 children with no clinically detectable caries in Group I and 15 children with high caries (DMFS ≥10) in Group II.

The paraffin stimulated salivary samples were collected from each child in duly labeled sterile containers containing 4 ml of Thioglycollate transport media[11],[12] which were transported to the department of microbiology, for microbial culturing.

Mitis salivarius-bacitracin (MSB) agar was prepared by adding 0.2 units of bacitracin per ml and sucrose to a concentration of 20% to mitis salivarius agar.[13] The MSB agar plates were prepared and an inoculated platinum loop was used to streak saliva from the sample onto MSB Agar plates. The plates were placed in an anaerobic jar containing a H2 and CO2 mixture (Gas Pak BBL System, São Paulo, SP, Brazil) and then incubated at 37°C for 48 h in a 5% CO2 incubator at the department of microbiology and examined for the growth of S. mutans.

Following incubation the colonies of S. mutans were identified as round or spherical, highly convex, raised, dark blue to black in color ranging from pinpoint to pinhead size with rough surface [Figure 1] as described by Emilson (1983).[14]The identification was confirmed using biochemical tests such as mannitol fermentation and catalase test[15] (the absence of effervescence with hydrogen peroxide) along with gram stained smears showing Gram-positive cocci in chains. Representative colonies with typical morphology of S. mutans were counted using a magnifying glass (10X).
Figure 1: A MSB agar plate with growth of Mutans streptococci

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Five milliliter of intravenous blood were collected using disposable syringe and needle by venipuncture of the median cubital vein. DNA extraction was done from whole blood according to the manual of high pure polymerase chain reaction (PCR) template preparation kit. Amplification was done with the help of real-time PCR technique using Light Cycler FastStart DNA Master SYBR Green protocol, as it allows direct evaluation of single nucleotide polymorphism in the particular gene [Figure 2]. Oligonucleotide primers were designed to flank polymorphism of single nucleotide in the gene. Specificity of the amplified product was assessed by performing a melting curve analysis. When melting curve analysis is used to monitor the melting of the primers using the LightCycler 480 System, the assay identifies even single base variations in the amplicon. Thus, this is an ideal tool for the detection of single-nucleotide polymorphism (SNPs) or genotyping.
Figure 2: Amplification curves of all the DNA samples

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The data were collected and statistically analyzed by unpaired t-test, paired t-test, Chi-square test, Pearson correlation, and regression analysis using SPSS 21.0 software (IBM Corp., Armonk, New York, USA).


   Results Top


The mean DMFS in children in group I was 0 and group II was found to be 11.73 ± 1.75. 14 samples out of the 15 children in Group I and all the samples of the 15 children in Group II showed growth of S. mutans. In Group II, the highest salivary S. mutans count was found to be 106 CFU/ml. The mean salivary S. mutans counts in Group I was 9 ± 5.75 × 104 and in Group II was 52 ± 22.66 × 104 CFU/ml [Table 1]. The difference in S. mutans colony counts between both the groups was highly significant with P < 0.01 [Table 1].
Table 1: Comparison of salivary Streptococcus mutans counts in children with no detectable caries (Group I) and children with high caries (Group II)

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A positive correlation was found between salivary S. mutans counts and dental caries susceptibility in Group II (correlation coefficient of 0.202), however, it was not statistically significant (P = 0.471, P > 0.05).

The melting curve analysis of the PCR products of the DNA samples of both the groups revealed that the first peak was at a temperature range of 81.38°C–82.13°C [Table 2]. The second peak was at a temperature range of 84.83°C to 85.96°C. However, there was only one DNA sample (6.66%) in Group II which had the third melting peak at 89.45°C signifying presence of SNPs in tuftelin gene in that sample.
Table 2: Melting curve analysis of the polymerase chain reaction products of all the DNA samples (Group I and Group II)

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In Group I, none of the children showed tuftelin gene polymorphism. Only 6.66% of children in Group II showed tuftelin gene polymorphism. Comparison of the presence of tuftelin gene polymorphism in Group I and Group II showed no statistical significant difference (P = 0.309, P > 0.05).

There was a positive correlation between tuftelin gene polymorphism and dental caries in Group II (Pearson r being 0.177), however, this correlation was not statistically significant (P = 0.681, P > 0.05).

When all the three variables, dental caries susceptibility, tuftelin gene polymorphism, and Salivary S. mutans counts in Group II were correlated using regression analysis to assess gene-environment interaction on dental caries susceptibility, an insignificant interaction between tuftelin and S. mutans was noted, with about 4.1% of the variability accounted for by the model (R2 = 0.041%). This implies that only 4.1% of the variability in dental caries risk can be explained by interaction between tuftelin gene and S. mutans.


   Discussion Top


Dental caries is a disease process that depends on the conjunction and interaction of cariogenic microorganisms, the dietary pattern and the susceptible tooth for maximal expression. The dilemma of this multifactorial disease can be solved if the causative factors are combated at an initial stage. Therefore, it is imperative to identify and evaluate the extent of the factors that challenge the tooth or protect it.

The role of S. mutans in the initiation of dental caries has been suggested by many researchers.[16],[17] The relationship of S. mutans with the prevalence of caries has been clearly established in several epidemiological studies.[18],[19] In the present study, the difference in mean salivary S. mutans counts in children with no detectable caries and in children with high caries was highly significant. This was in accordance with the studies conducted by Campus G et al., 2000[20]and Hegde PP et al., 2005.[21]

Although the effects of environmental factors such as variation in diet, the oral microbial flora, and oral hygiene status on dental caries have been extensively studied, the importance of genetic factors has so far been downplayed.[22] The possibility of genetic involvement in dental caries has been reported in the literature by a number of investigators who have evaluated the pattern of dental caries among family members and twins.[23],[24] The twin and family studies provided strong evidence of a genetic contribution to dental caries risk, however none provided any evidence of linkage to specific genes.

The advances in the field of molecular biology have made it possible to comprehend a multifactorial disease like dental caries on a “molecular level.” The understanding of the genes that produce the enamel matrix proteins has greatly expanded in the past few years with the discovery of genome information.[25] Several investigators have contributed to the cloning of enamel matrix genes, chromosomal localization of the genes, and association of genes with altered tooth development as seen in various syndromes.[26],[27] In each of these analyses, a highly defined clinical phenotype is correlated with the altered mineralization matrix protein and followed by the identification of the genome for linkage to a precise mutation or polymorphism in DNA sequence.

Some studies have proven that susceptibility or resistance to caries would be the result of one or more gene-environment interactions.[28] The cumulative evidence from the studies of Slayton et al., 2005;[3]Patir et al., 2008;[4]Saha et al., 2015[5]and Gereth et al., 2016[6] strongly suggests a role of the genes involved in the enamel formation in caries susceptibility in humans. The current evidences support that genetic variations in these genes contribute to structural alterations of the enamel that may cause higher levels of mineral loss under acidic conditions and/or facilitate bacterial attachment and biofilm deposition.[29]

Tuftelin is one of the enamel matrix protein proposed to play a vital role during the development of mineralized tissues. There is also an evidence that tuftelin is broadly expressed in nonmineralizing tissues too, and it is highly preserved among various species as was demonstrated in a study by Mao et al., 2001.[8] This suggests a more widespread function of this gene than was previously understood. There also continues to be substantial data for the involvement of tuftelin in enamel development. It was observed by Deutsch et al., 1995[7] and Paine CT et al., 2000[30] that tuftelin protein is secreted into the enamel matrix (E19 and 1 day postnatal) and can be identified at the dentinoenamel junction (3–11 days' postnatal). Mao et al., 2001 suggested that tuftelin is a good candidate for contribution in the initial stages of enamel mineralization due to its expression just before the commencement of enamel mineralization and its acidic nature.[8] In addition, in a study by Luo et al., 2004, carried out in a transgenic mouse model, the overexpression of tuftelin in the extracellular enamel matrix led to deficiencies in both crystallite structure and enamel prisms.[9] These alterations in dental enamel development can lead to reduced mineral content, increased enamel porosity, and the presence of enamel crystal inhibitory proteins which are directly linked to dental caries predisposition.[31]

Slayton et al., 2005 showed that the presence of an relationship between genetic deviation of tuftelin and caries was improved when the interaction with S. mutans levels was included in the model, hence reporting that 26.8% of the variation in DMFS can be explained by the interaction between tuftelin gene and S. mutans.[3] In the present study, tuftelin gene and dental caries susceptibility did show a positive association; however, the association was not significant and only 4.1% of the variability in dental caries risk can be explained by interaction between tuftelin gene and S. mutans. Similarly, Patir et al., in 2008 found that the inclusion of S. mutans in the model did not improve the positive association between caries experience and the tuftelin rs3790506 marker which was evident in their study.[4]

There are some variations among the studies regarding the genetic associations with dental caries. Some previous studies showed that polymorphisms of the amelogenin, ENAM and TUFT1 genes were closely associated with the susceptibility for dental caries, while other studies did not find the same association.[4],[5],[6],[32],[33],[34],[35] Results in some studies were inconsistent, especially in people from different geographical locations and ethnic minorities.[36],[37],[38]

These differences can be the result of several factors such as heterozygosity of the genetic markers in the studied populations and dissimilarities in the study designs; reports on primary versus permanent dentitions, utilization of caries experience scores by tooth versus by surface, availability of S. mutans data, etc.

Even though, the association between tuftelin gene, S. mutans, and dental caries susceptibility could not be demonstrated significantly in this study, further investigations should be carried out on larger sample to arrive at conclusive results. This was a preliminary study, and subjects were limited to children. Future research studies including parents and siblings should be carried out to focus on further investigation into the mechanism of this gene-environment interaction with dental caries.


   Conclusions Top


The association of Streptococcus mutans with the prevalence of caries has been clearly established in the present study. Recognizing the genetic factors contributing to caries risk and resistance will equip clinicians with new tools for targeting individuals and/or populations for more efficient and effective preventive therapies especially in children. It may also facilitate new, more targeted approaches to the prevention and treatment of a disease that is pandemic among certain subsets of the population. If risk could be identified prior to the occurrence of the disease, or prior to the eruption of teeth, limited resources could be used most efficiently to prevent the pain and suffering that many young children currently endure.

Acknowledgment

I express my sincere gratitude to Dr. Adesh Kakade, Professor and Head of Department, Department of Pediatric and Preventive Dentistry, Nair Hospital Dental College, Mumbai for his constant encouragement which has helped me complete this dissertation successfully. I am grateful to the authorities of Gilderlane Municipal school and the children who willingly participated in the study. I sincerely thank Mr. Kaizad Wadia and Mrs. Ravindra Kaur of Roche Diagnostics Ltd., Mumbai for their wholehearted support and cooperation during the study. My special thanks to Dr. Umesh Aigle and Dr. Nilima, Kasturba Hospital, for providing the Molecular Laboratory and other facilities for the study at my disposal. I would like to acknowledge Dr. (Mrs.) Jayanti Shashtri (Head), and Dr. Anjali (Associate Professor) and Mrs. Mangal (Lab. technician) Department of Microbiology, Topiwala National Medical College, Mumbai for their help.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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