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Genome-wide Scan Finds Suggestive Caries Loci
1 Departments of Oral Biology, Correspondence: * corresponding author, arv11{at}pitt.edu
Here we report the first genome-wide scan performed for caries. Evidence from twin studies suggests a genetic component to caries. We aimed to identify genetic factors contributing to caries in a population similarly influenced by confounding factors, such as diet, oral hygiene habits, fluoride exposure, and access to dental care. Forty-six families with similar cultural and behavioral habits, and living in the Philippines, were studied, and genome-wide genotype data and DMFT (Decayed, Missing due to caries, Filled Teeth) scores were evaluated. Suggestive loci logarithmic odds (LOD) scores above 2.0 or non-parametric p-values below 0.0009) were found for low caries susceptibility (5q13.3, 14q11.2, and Xq27.1) and high caries susceptibility (13q31.1 and 14q24.3). Genes that may be related to saliva flow and diet preferences are proposed as possible candidates. A protective locus for caries in the X chromosome may explain the gender differences seen in caries frequency.
Key Words: caries • genetics • linkage
Caries is a major public health concern worldwide, affecting more than 80% of the population alive in the world today. Its impact on individuals and communities—as a result of pain and suffering, impairment of function, and reduced quality of life—is considerable (World Health Organization, 2003). Moreover, traditional treatment of caries is extremely costly, representing the fourth most expensive disease to treat in most of the industrialized world. Access to appropriate treatment is difficult in many low-income countries; if treatment were available in such countries, the costs of caries alone in children would exceed the total current health care budgets for children (Yee and Sheiham, 2002). Although caries is largely preventable, it remains the most common chronic disease for children ages 5 to 17 yrs in the US, as well as in the rest of the world (US Department of Health and Human Services, 2005). In the US, caries is 5 times more common than asthma (59% vs. 11%). Caries is an infectious localized disease that results in loss of mineral from the affected teeth, caused by organic acids that originate from the microbial fermentation of carbohydrates. Caries is related to 3 essential interactive factors: the host, represented by teeth and saliva; the oral microbial flora; and type of diet (Keyes, 1960, 1962). The factors related to the host are under strong genetic control, but external factors—such as fluoride exposure, quality of dental hygiene, microbiota, and type of diet—may overcome an individual’s a priori genetic susceptibility. Therefore, hereditary aspects of caries have been seldom studied and thus are poorly understood. Further complicating family studies of caries are shared behavior, practices, and habits within families. These factors can be expected to contribute to covariance between relatives, and to mimic genetic correlation if not controlled by the experimental design (Potter, 1990). This is particularly true for caries, which can be profoundly affected by dietary sugar intake and/or oral hygiene practices within families. Studies of twins (Boraas et al., 1988; Conry et al., 1993), families (Klein and Palmer, 1940; Klein, 1946), and animal models (Hunt et al., 1944) have all indicated that caries has a genetic component. Some of the most compelling evidence for a genetic component to caries comes from studies of twins reared apart. In two related studies, investigators found significant resemblance within monozygotic, but not dizygotic, twin pairs reared apart for percentage of teeth and surfaces restored or carious (Boraas et al., 1988; Conry et al., 1993), and estimated the genetic contribution to caries to be 40% (Conry et al., 1993). Other recent studies of twins reared together estimated the heritability for caries, adjusted for age and gender, as ranging from 45 to 64% (Bretz et al., 2005). Despite the strong evidence for a genetic component in caries, conclusions have usually not gone beyond the observation of familial aggregation. Animal models have provided the first interesting connections between loci and caries susceptibility. The HLA-DR antigen, which corresponds to the human major histocompatibility complex (MHC), has been associated with helper T-cell activity in the control of dental caries (Lehner et al., 1981). However, this finding was not replicated in a second independent study (de Vries et al., 1985). Much later, the H-2 region on chromosome 17 was linked to susceptibility to caries in mice (Suzuki et al., 1998). Chromosome 2 was also reported as linked to susceptibility to caries in mice (Uematsu et al., 2003). A genome-wide scan performed in genetic crosses of C3H/HeJ (caries-resistant) and C57BL/6J (caries-susceptible) mice inoculated with Streptococcus mutans serotype c detected 3 suggestive quantitative trait loci (QTL) on chromosomes 1, 2, and 7, one significant QTL on chromosome 2, and one highly significant QTL on chromosome 8 (Nariyama et al., 2004). Only recently has a genetics study in human caries been reported. Tuftelin genotypes appeared to interact with levels of Streptococcus mutans infection in children with early childhood caries (with 4 or more affected tooth surfaces) compared with caries-free children (Slayton et al., 2005). In the current study, we compared individuals living in the same area in the Philippines, and therefore with similar cultural backgrounds and access to dental care, in an attempt to reduce the influence of environmental confounders. The families studied all come from the central part of the country, mostly Cebu Island, and the surrounding islands. All families are small-scale fishermen or landless rural dwellers. They all appear to be descendents from a proto-Malay stock. Caries was measured by the caries experience score DMFT (Decayed, Missing due to caries, Filled Teeth) (Klein et al., 1938). Here we report the first genome-wide search performed to identify caries susceptibility and protective loci in humans.
The study sample consisted of 46 families with genome scan genotypes available from our previous work with oral clefts (Riley et al., 2007). The genotyping was done in the Center for Inherited Disease Research (CIDR) for 392 markers from the Marshfield Genetics screening set 8. These families are part of a project where complete dental descriptions were obtained in an attempt to correlate dental characteristics with oral clefts. This protocol has both the University of Pittsburgh and local Filipino IRB approval. Appropriate informed consent was obtained from all participants. The DMFT cut-offs are modified from the descriptions used by the WHO (World Health Organization, 2003), to allow for the creation of more discrete age groups and risk classifications (Table 1  ).
We first accessed each genome scan marker with PedCheck (O’Connell and Weeks, 1998), to test for inconsistencies due to non-paternity or other errors. For the parametric linkage analyses, we estimated allele frequencies for unaffected founders from a panel of 458 Filipino cleft families with available genotypes. The genetic model parameters were taken from segregation analysis results in a sample of the Filipino families (unpublished results). The dominant model was a disease allele frequency of 0.002 with a penetrance of 0.60. The recessive model was a disease allele frequency of 0.04 with a penetrance of 0.90. We calculated two-point LOD scores in the families using the LINKAGE program with updates to speed calculations (VITESSE and FASTLINK) (Cottingham et al., 1993; Terwilliger and Ott, 1994; O’Connell and Weeks, 1995). For multipoint LOD and heterogeneity LOD (HLOD) (Smith, 1963) calculations, we used the descent graph method (Sobel and Lange, 1996; Sobel et al., 1996; Lange, 2002) implemented in the computer program SIMWALK2. Given the probable complexities in the genetic model for caries (e.g., heterogeneity), we also calculated model-free linkage statistics (both two-point and multipoint), using MERLIN (Abecasis et al., 2002).
The 46 families have 624 people recorded in the pedigrees. DMFT data were available for 279 people. We performed 2 genome scan analyses. In the first pass, individuals classified with very low and low caries experiences were assigned as ‘affected’. In the second pass, individuals classified with moderate-to-high caries experiences were assigned as ‘affected’. Three regions yielded suggestive positive results (LOD scores above 2.0 and or non-parametric LOD p-values below 0.0009) for low caries susceptibility: 5q13.3 (dominant multipoint LOD score = 2.30), 14q11.2 (recessive single-point LOD score = 2.29), and Xq27.1 (non-parametric LOD p-value = 0.00005). Another 2 regions yielded suggestive positive results for high caries susceptibility: 13q31.1 (recessive single-point LOD score = 2.33 and recessive multipoint LOD score = 2.20) and 14q24.3 (recessive single-point LOD score = 2.06). Several other regions yielded LOD scores above 1.0 or non-parametric LOD p-values below 0.05 (Table 2 and Figs. 1, 2).
Among the 5 regions with the most significant linkage results, only 1 overlaps with the regions mapped by the genome-wide scan performed in genetic crosses of C3H/HeJ (caries-resistant) and C57BL/6J (caries-susceptible) mice inoculated with Streptococcus mutans serotype c (Nariyama et al., 2004). Human 14q11.2 corresponds to the region at 45.5cM at mouse chromosome 8, which yielded the most significant QTL results in the mouse genome-wide scan. The C57BL/6J allele for the QTL was associated with caries-resistant properties, while the C3H/HeJ allele was associated with caries susceptibility. About 1100 genes map on the mouse chromosome 8 in which the QTL were detected, including genes for which functions are known and unknown. The only possible candidate gene highlighted by Nariyama et al.(2004) on the mouse genome-wide scan that maps to human 14q11.2 is NFATC4 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4). According to the authors, this gene is involved in intra- and extracellular transport of calcium and potassium ions relating to the flow rate of saliva. Chromosome 1q21.3, where tuftelin is located, did not yield any suggestive linkage results. The previous report that suggested tuftelin involvement in caries (Slayton et al., 2005) showed significant regression analysis data, suggesting the existence of an interaction between tuftelin genotypes and infection by Streptococcus mutans. In our study, we did not have microbiologic data available, and this possible interaction could not be investigated. Candidate genes for caries could range from genes contributing to enamel formation to those for saliva composition and immune responses. There are no obvious candidate genes in the 5 regions with the most significant results. The marker that yielded the suggestive LOD score in 14q11.2 (D14S742) is very close to OR4E2 (olfactory receptor, family 4, subfamily E, member 2), which interacts with odorant molecules in the nose, to initiate a neuronal response that triggers the perception of a smell. Taste and smell are subsumed under the term ‘flavor’. Many flavors are recognized mainly through the sense of smell (e.g., anyone will have trouble identifying the chocolate flavor if one holds one’s nose while eating chocolate, even though one can distinguish the food’s sweetness or bitterness). Genetic variation in genes regulating olfactory and taste sensations may predispose someone to be more or less inclined to eat certain foods, and therefore to have a less or more cariogenic diet. The other 2 regions with the most significant results for lower caries experience are 5q13.3 and Xq27.1. CARTPT (cocaine- and amphetamine-regulated transcript) is located in between the markers that yielded suggestive LOD scores in 5q13.3 (D5S2500 and D5S424). This gene appears to have a role in reward, feeding, and stress, and it has the functional properties of an endogenous psycho-stimulant (Kuhar et al., 2002). A behavior that includes decreased ingestion of sweet foods would contribute to a lower caries experience. A protective locus for caries in the human X chromosome raises interesting possibilities. There is a nearly universal pattern of sex differences in caries. An extensive literature review of more than 50 epidemiological and clinical reports and 50 paleopathological studies of caries demonstrated a clear female gender bias in caries prevalence (Lukacs and Largaespada, 2006). Higher caries prevalence among females is often explained by one of 3 factors: (1) early eruption of teeth in girls, and longer exposure to a cariogenic oral environment; (2) proximity of women to food supplies and snacking during food preparation; and (3) pregnancy and hormonal influences. The evidence of a contribution of a locus in the X chromosome to low caries susceptibility may shed some light on the poorly understood gender differences in caries prevalence. In our data, the group of fathers of the studied families had a lower average DMFT score (10.96), compared with that in the mothers (14.45). Therefore, X-linked genetic variation could partly explain why men tend to have fewer cavities than women. The 2 regions with the most significant results for the ‘higher caries experience’ scan were 13q31.1 and 14q24.3. The marker that yielded the most significant LOD score in 13q31.1 (D13S317) is near SPRY2 (Sprouty2). Sprouty2 has been shown to inhibit the Ras/MAP kinase pathway (Yusoff et al., 2002). The MAP kinase cascades constitute highly conserved signaling systems that have been deemed to play various roles in physiological responses, including immune responses. In addition, SPRY2 is an antagonist of FGF signaling, which is implicated in controlling the integrity of oral mucosa and as having mitogenic effects in the salivary glands as well (Kagami et al., 2000). The other region, 14q24.3, had a marker (D14S53) close to ESRRB (estrogen-related receptor beta). This gene encodes a protein with similarity to the estrogen receptor. Its function is unknown; however, this gene is likely to have diverse biological functions (Zhou et al., 2006). One can argue that a gene with estrogen-related function could also contribute to the observed gender differences in caries frequency. Estrogens have been known to have a depressing effect on the secretion of growth hormone from the anterior pituitary. Growth hormone is known to be closely related to the development and maintenance of normal histologic structure of salivary glands, the function of which, in turn, might influence caries formation (Liu, 1967). This is the first time a genome-wide search has been performed for caries in humans, and the investigation of these chromosomal regions can unveil genes that contribute to caries susceptibility and provide a new framework for caries prevention strategies. If future studies can identify predisposing and/or protective genetic factors, we will potentially be able to design more effective treatments aimed at preventing dental caries in children worldwide.
The authors are indebted to the families who participated in this project. We are thankful to Jeffrey C. Murray for many helpful discussions. Sandra Daack-Hirsch provided family and genotyping information. Phenomics Group of the Philippines, Inc. provided technical support. Support was provided by NIH Grant R21-DE16718 and R01-DE14899. Genotyping services were provided by the Center for Inherited Disease Research (CIDR). The CIDR is fully funded through a Federal contract from the National Institutes of Health to The Johns Hopkins University (contract number N01-HG-65403). Received for publication August 14, 2007. Revision received February 5, 2008. Accepted for publication February 26, 2008.
Journal of Dental Research, Vol. 87, No. 5,
435-439 (2008)
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