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Tuftelin, Mutans Streptococci, and Dental Caries Susceptibility
1 Department of Pediatric Dentistry, University of Washington School of Dentistry, Box 357136, 1959 NE Pacific St., Seattle, WA 98195, USA; Correspondence: * corresponding author, rslayton{at}u.washington.edu
The purpose of this study was to identify genetic factors that contribute to dental caries susceptibility, either alone or in combination with environmental factors. Dental examinations were performed and buccal swab samples collected from 3- to 5-year-old children with at least 4 surfaces of decay, or with no evidence of decay. SNP assays for each of 6 candidate genes were performed for all cases and controls. Chi-square analysis and regression analysis were used for the evaluation of individual gene effects, environmental effects, and gene-environment interactions. There were no significant associations between single candidate genes and caries susceptibility. Levels of S. mutans were positively and Lactobacilli were negatively associated with caries. Regression analysis revealed a significant interaction between tuftelin and S. mutans, with 26.8% of the variation in dmfs explained by the interaction. Future research will focus on the identification of these additional factors and the development of functional assays so that these interactions can be better understood.
Key Words: caries • genetics • S. mutans • polymorphism • tuftelin
Dental caries is one of the most common diseases of childhood. In the United States, disparities in oral health have led to substantially higher average disease prevalence among children in poverty and in people of color. Efforts to identify those individuals at risk for dental caries prior to the occurrence of the disease have had limited success. Currently, the most accurate predictor of a child’s risk for caries is past or current caries experience. Although there is clear evidence that dental caries is a multifactorial, infectious disease, with many contributory environmental factors, there is also strong evidence for a genetic component in the etiology of this disease. This has been supported by studies in both humans and animals (Boraas et al., 1988; Conry et al., 1993; Liu et al., 1998; Shuler, 2001; Nariyama et al., 2004). The etiology of dental caries has been studied for many years. Multiple factors contribute to a person’s risk for caries, including: (1) environmental factors, such as diet, oral hygiene, fluoride exposure, and the level of colonization of cariogenic bacteria; and (2) host factors, such as salivary flow, salivary buffering capacity, position of teeth relative to each other, surface characteristics of tooth enamel, and depth of occlusal fissures on posterior teeth. In spite of all that is known about this disease, there are still individuals who appear to be more susceptible to caries and those who are extremely resistant, regardless of the environmental risk factors to which they are exposed. Disorders with both genetic and environmental influences do not follow simple Mendelian inheritance patterns and are referred to as ‘complex’ disorders. Knowledge gained through the human genome project has made it feasible for investigators to study the genetics underlying complex disorders such as cleft lip and palate, diabetes, periodontal disease, and multiple sclerosis (Wright and Hart, 2002). The susceptibility or resistance to dental caries is similar in its complexity and must be dissected in a manner similar to that established for other complex disorders. The most compelling evidence for the existence of a genetic component in caries susceptibility comes from studies of twins reared apart. In two related studies, investigators found significant resemblance within monozygotic (MZ) but not dizygotic (DZ) twin pairs for number of teeth present, percentage of teeth and surfaces restored or carious, tooth size, and malalignment (p < 0.001) (Boraas et al., 1988; Conry et al., 1993). The second of these studies estimated the genetic contribution to caries as 40% (Conry et al., 1993). There has been a substantial effort, in the recent past, to establish a caries risk profile for children that could be used to predict those children who are most likely to have serious dental decay (Bohannan et al., 1985; Disney et al., 1992; Featherstone et al., 2003). In general, efforts to develop a single, inclusive, and accurate model for caries risk assessment have been unsuccessful, particularly for young children. The identification of genetic risk factors has the potential to lead to the development of a caries risk assessment tool with a high level of predictive ability. The purpose of this study was to identify genetic markers that are associated with dental caries susceptibility, either singly or in combination with other genes or environmental factors. Understanding the genetics of dental caries susceptibility or resistance will provide new insights into the caries process in individuals and will facilitate the development of targeted preventive strategies. In addition, identification of genetic susceptibility factors can be used for the development of a valid, early screening test for caries risk. This may allow early preventive intervention to be directed at those who would benefit the most and would facilitate more cost-effective strategies for oral health promotion.
Subject Screening Subjects were recruited for this study according to The University of Iowa Institutional Review Board (IRB) guidelines, and informed consent was obtained. Eligible children were from 3 to 5 years of age and were enrolled in Head Start, a Federally funded child development program for low-income children. Cases were defined as children with 4 or more decayed or filled tooth surfaces, while controls were defined as children with no evidence of caries (including white-spot lesions) and no history of caries. Examinations were done with the use of a flashlight and mouth mirror. Cavitated lesions were classified according to the combined d2-d3 criteria (Pitts and Fyffe, 1988). We determined the extent of caries experience by calculating the dmfs (decayed, missing, and filled surfaces of primary teeth) for each subject. Subjects with dmfs > 0 but < 4 were considered intermediate in phenotype and were not included in the current study. All subjects lived in a community with optimally fluoridated water, and had two meals per day provided at the Head Start center. Dietary habits during the subjects’ time outside the center were not evaluated.
Sample Collection
DNA Extraction, Amplification, Visualization, and Analysis
Candidate Genes
Statistical Analysis In general, cases and controls were matched by race, age, and gender. In situations where there were more controls than cases, the ‘optimal’ algorithm described by Rosenbaum was used (Rosenbaum, 1989). In this study, since race and gender are categorical variables, and age can be used as a ratio variable, optimal matching was done with age. The association of individual genes with caries susceptibility or resistance was assessed by Chi-square analysis to compare allele frequencies for cases and controls. Association of caries with levels of S. mutans and Lactobacillus was performed with the Chi-square test. In addition, we used regression analysis to investigate main-effects models, after which interactions (Gene-Environment, i.e., SNP-bacteria) with covariates/confounders (race, gender) were tested. A power analysis demonstrated that a statistically significant correlation of 0.3—with a single-sided test with alpha equal to 0.05 and a power of 80%—can be found with a sample size of 78 or more.
Screening examinations were performed on 588 children according to the criteria developed for primary tooth exams in the Iowa Fluoride Study (Warren et al., 2002). Of those who were screened, 562 (95.4%) agreed to participate in the proposed study. Informed consent was obtained from at least one parent, following the University of Iowa IRB-approved guidelines. Microbiological tests were performed on 419 of the 562 subjects. Ninety-two (16.4%) of the subjects were classified as ‘cases’, based on a dmfs  4. Three hundred and forty-three children (61.1%) were classified as ‘controls’, while the remainder (127 children) had an intermediate phenotype and were not included in the genetic analysis. For the current study, 92 control subjects matched for race, age, and gender were selected for genetic analysis. Average decayed-missing-filled surfaces (dmfs) and decayed-missing-filled teeth (dmft) scores were calculated for all subjects (Table 2 ).
Chi-square analysis of S. mutans levels in children with and those without dental caries demonstrated a statistically significant positive association with caries (P < 0.0001), with more individuals with caries in each of the ‘low, medium, and high’ categories for S. mutans. As noted in previous studies, the level of S. mutans was not necessarily indicative of the presence of disease (Table 3 ). There was also a statistically significant negative association between the presence of Lactobacillus and caries (P = 0.0003) (Table 3), with more individuals with caries than expected in the ‘none and low’ Lactobacillus categories and fewer in the ‘medium and high’ categories.
Screening of Candidate Genes At least one SNP was analyzed for each of the candidate genes described in "MATERIALS & METHODS". Allele frequencies for cases and controls and the results of the Chi-square analysis are summarized in Table 4 . There was no significant association between a single gene and caries susceptibility. Two of the genes (KLK4 and AMBN) showed no heterozygosity among the subjects for the selected SNPs and were not analyzed further.
Gene-Environment Interaction We used regression analysis to assess gene-environment interactions and the effects of confounders such as race or gender. This analysis demonstrated a significant interaction between tuftelin and S. mutans, with about 27% of the variability accounted for by the model (R-square equal to 0.268). The best-fitting model incorporating this interaction was:
When we modeled severity of caries using dmfs as a continuous variable within the 50 cases with complete data, then race (Caucasian vs. non-Caucasian) became a significant confounder (R2 = 0.262). In this sample, the majority of children with severe caries were Caucasian (mean dmfs = 10.4), while the mean dmfs for non-Caucasians was 7.46 (t test p-value = 0.13).
In this study, the finding that dental caries susceptibility was not associated with a single gene was not surprising. Based on the complex, multifactorial nature of the disease, it was anticipated that susceptibility or resistance to caries would be the result of one or more gene-environment interactions. The finding that tuftelin, a gene involved in enamel development and mineralization, combined with high levels of S. mutans results in increased susceptibility to dental caries is a significant, exciting, and potentially perplexing result. Recent studies have demonstrated that tuftelin is widely expressed in non-mineralizing tissues, and that it is highly conserved among species (Mao et al., 2001). This suggests a more universal function for this gene than was previously understood. There also continues to be evidence for the involvement of tuftelin in enamel development. The tuftelin protein is secreted into the enamel matrix and can be detected at the dentin-enamel junction (Deutsch et al., 1998; Paine et al., 2000). Its expression (just before the beginning of enamel mineralization) and its acidic nature make it a good candidate for involvement in the initial stages of enamel mineralization (Mao et al., 2001). In addition, in a recently published study, the over-expression of tuftelin in the extracellular enamel matrix led to imperfections in both enamel prisms and crystallite structure in a transgenic mouse model (Luo et al., 2004). Based on our understanding of the biology of caries, it is conceivable that alterations in enamel development could influence both bacterial adherence and/or the resistance of enamel to acid pH. Although the role of tuftelin in enamel development is only a part of what this protein does, it is still a viable candidate gene for disorders that affect enamel. It should be noted that sequence changes in the tuftelin gene could also be indirectly affecting caries susceptibility by interfering with other gene or protein interactions. While the selection of candidate genes is based on the biology of caries, it is necessarily limited to genes that have already been identified and characterized. There are potentially many other genes that contribute to this process. An alternate approach, and one that would remove selection bias on the part of the authors, is a genome-wide scan. The goal of a genome-wide scan would be to identify regions of the genome in which there are loci that contain potential candidate genes. This approach would identify genes that may not have been obvious choices for involvement in the caries process. Because this was a preliminary study, and subjects were limited to children, this approach was not feasible. Future studies will include parents and siblings so that a genome-wide scan can be performed. Of the children who participated in this study, the greatest severity of disease was found among the Caucasian children. The reason for this disparity is not clear, and further investigation of this finding is warranted. This is one of the first studies to use a candidate gene approach to investigate host susceptibility to dental caries. An understanding of caries at the molecular level has the potential to provide the caries research community with novel insights into the complexity of this disease. 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. Currently, the most accurate prediction of caries risk in children is the presence of cavitated caries lesions. 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. Findings such as these, although interesting, require testing and validation in additional subjects. Future research will focus on repeating these studies and on further investigation into the mechanism for this gene-environment interaction.
I am extremely grateful to Dr. Jeffrey C. Murray, for his constant support, encouragement, and helpful conversations. I thank my research assistants, Sally Santiago and Elizabeth Palmer, for their dedication and skill in the laboratory. This study was supported by NIH grants R03 DE014445-03 (R. Slayton, PI), R01 DE014899 (M.L. Marazita, PI), P30 DE10126 (J. Wefel, PI), and P60-DE-13076 (J.C. Murray, PI).
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication September 13, 2004. Revision received March 21, 2005. Accepted for publication May 6, 2005.
Journal of Dental Research, Vol. 84, No. 8,
711-714 (2005)
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