Human genes influence the interaction between Streptococcus mutans and host caries susceptibility: a genome-wide association study in children with primary dentition

Streptococcus mutans is a well-known cause of dental caries, due to its acidogenicity, aciduricity, and ability to synthesize exopolysaccharides in dental plaques. Intriguingly, not all children who carry S. mutans manifest caries, even with similar characteristics in oral hygiene, diet, and other environmental factors. This phenomenon suggests that host susceptibility potentially plays a role in the development of dental caries; however, the association between host genetics, S. mutans, and dental caries remains unclear. Therefore, this study examined the influence of host gene-by-S. mutans interaction on dental caries. Genome-wide association analyses were conducted in 709 US children (<13 years old), using the dbGap database acquired from the center for oral health research in appalachia (COHRA) and the Iowa Head Start programmes (GEIRS). A generalized estimating equation was used to examine the gene-by-S. mutans interaction effects on the outcomes (decayed and missing/filled primary teeth due to caries). Sequentially, the COHRA and GEIRS data were used to identify potential interactions and replicate the findings. Three loci at the genes interleukin 32 (IL32), galactokinase 2 (GALK2), and CUGBP, Elav-like family member 4 (CELF4) were linked to S. mutans carriage, and there was a severity of caries at a suggestive significance level among COHRA children (P < 9 × 10−5), and at a nominal significance level among GEIRS children (P = 0.047–0.001). The genetic risk score that combined the three loci also significantly interacted with S. mutans (P < 0.000 1). Functional analyses indicated that the identified genes are involved in the host immune response, galactose carbohydrate metabolism, and food-rewarding system, which could potentially be used to identify children at high risk for caries and to develop personalized caries prevention strategies.


INTRODUCTION
Although largely preventable, dental caries remains the single most common chronic childhood disease, with nearly 1.8 billion new cases per year globally. [1][2][3] Dental caries is a chronic infectious disease initiated from the virulent dental biofilms/plaque formed on tooth surfaces. 4 Within the dental biofilms/plaque, oral cariogenic bacteria metabolize dietary carbohydrates, produce acid, and initiate demineralization of the tooth enamel. 5 Although an enamel remineralization process takes place when the enamel is exposed to salivary calcium, phosphate, and fluoride ions, when the demineralization exceeds the remineralization process, dental caries occur. 6 Traditional microbial risk markers for caries include Streptococcus mutans, a well-known culprit for dental caries, due to its acidogenicity, aciduricity, and capability of synthesizing dental plaque extracellular matrix. 7-11 In theory, higher S. mutans carriage indicates higher caries risk. Interestingly, studies have identified a group of children who are colonized by S. mutans but do not manifest the disease, even with similar social-demographic-hygiene behavior characteristics compared with those of their counterparts. 12 This phenomenon suggests that host susceptibility potentially plays a role in modulating the risk of S. mutans on the development of dental caries. Therefore, elucidating the interaction between host susceptibility, S. mutans carriage and their impact on caries onset could help identify high-risk child populations, and develop personalized precision dental caries prevention strategies.
It is speculated that host susceptibility to dental caries is influenced by each individual's genetic constitution, including the tooth anatomy (deep groove and fissure), tooth hard tissue (enamel and dentin) quality, salivary properties, host immunity, oral microbiome composition, and taste preference, 13 and the interactions between host gene and environmental factors, e.g., fluoride exposure. 14 Recent genome-wide studies have reported novel associations of common genetic variants and gene-byenvironment interactions with dental caries, [15][16][17][18] such as the genes TUFT1, NAMPT, and BMP7, which are involved in enamel matrix and tooth development. The NAMPT gene (rs190395159) and the BMP7 gene (rs72626594) were found to be associated with dental caries in a Hispanic/Latino community. 17 In addition, an intergenic locus on chromosome 4q32 (rs4690994) was found to be strongly associated with early childhood caries. 15 Zeng et al. 19 reported that BCOR, BCORL1, INHBA, CXCR1, and CXCR2 were associated with caries in permanent dentition adjusted for the presence of S. mutans.
However, given the bacterial infectious nature of dental caries, previous researchers have not examined the association between host genetics, oral microorganisms, and the onset of dental caries. Therefore, in this study, we hypothesized that certain host genes modulate host susceptibility to S. mutans in developing dental caries. To test this hypothesis, we investigated the influence of host gene-by-S. mutans interaction on dental caries in 709 children younger than 13 years of age in the United States.

RESULTS
Participants' characteristics Genome-wide association analyses were conducted in 709 US children (<13 years old) using a dbGap database acquired from the center for oral health research in appalachia (COHRA) and the Iowa Head Start programmes (GEIRS). The participants' characteristics included in this study are shown in Table 1. The majority of the participants were white (84%) and non-Hispanic (97%). There were slightly more male participants than females (52% vs. 48%). The prevalence of dental caries (dmft and dmftw) was 48% and 50%, respectively. S. mutans was detected in 53% of children. The mean age of children without S. mutans was lower than that of children with S. mutans (5.7 ± 3.0 vs. 7.1 ± 2.9 years of age, P < 0.000 1). S. mutans carriage was strongly associated with dental caries analyzed using the generalized estimating equation (GEE) and adjusting for covariates (P < 0.000 1). The prevalence of dental caries (dmft and dmftw) in children with S. mutans was nearly twice that in children without S. mutans (61% vs. 33%, P < 0.000 1) (Fig. 1).
Single-nucleotide polymorphisms associated with host caries susceptibility modified by S. mutans carriage Three loci (rs4786370, rs11635005, and rs1539849) interacted with S. mutans carriage and caries severity at a suggestive significance level among COHRA children (P < 9 × 10 −5 ), and at a nominal significant level among GEIRS children (P = 0.047-0.001). At the discovery stage, genome-wide gene-by-S. mutans interaction analyses were performed. Manhattan plots are shown in Fig. 2. No interactions met the genome-wide significance level after Bonferroni correction (P < 9 × 10 −8 ). The interactions with the lowest significance levels were between rs1831292 and S. mutans for both dmft and dmftw (P = 5.2 × 10 −6 and 5.05 × 10 −6 , respectively). A relatively lenient significance value (P < 9 × 10 −5 ) was then used to select the potential suggestive loci. A total of 53 single-nucleotide polymorphisms (SNPs) for dmft and dmftw were selected for replication.
At the replication stage, the conservative significance level was determined by Bonferroni correction for 53 assessed loci (P < 9.4 × 10 −4 ). For dmft, the interaction between rs4786370 and S. mutans was close to the significance level, which had a P-value of 1 × 10 −3 ( Table 2). Three additional SNPs, rs11635005, rs6004787, and rs1539849 were significant at the nominal level (P < 0.05). For dmftw, one SNP, rs1539849 was significant at the nominal level. These four SNPs are located on chromosomes 15, 16, 18, and 22, respectively. The minor allele frequency ranged from 0.2 to 0.42. Allele frequencies of the four SNPs in the COHRA and GEIRS sites are shown in Appendix Table 1. The interactive effects between the four SNPs and S. mutans were consistent with those at the discovery stage ( Table 2). The marginal effects of these SNPs, except for rs6004787 modified by S. mutans carriage status, were also concordant with those at the discovery stage (Appendix Fig. 1).
Caries severity (dmft) was dependent on individual genotypes and S. mutans carriage. The effect of the three gene-by-S. mutans interactions on dmft is presented in Fig. 3. The effect of rs1539849by-S. mutans on dmftw was similar to its effect on dmft (data not shown). For rs4786370 (Fig. 3a), S. mutans was significantly associated with dental caries severity when children carried the TC and CC alleles (P < 0.05). For rs11635005 (Fig. 3b), S. mutans was significantly associated with dental caries severity when children carried the CC alleles (P < 0.05). For rs1539849 (Fig. 3c), regardless of alleles, S. mutans was significantly associated with caries susceptibility (P < 0.05). The difference in caries severity affected by S. mutans carriage was more prominent among children, who carried the AA and CA alleles compared with children with CC alleles. When the marginal effects of the three SNPs in the whole sample were compared with those in the COHRA and GEIRS sites separately, the effects were consistent between the two sites and the whole sample ( Fig. 3 and Appendix Fig. 1).
When the caries severity was compared in children with or without S. mutans, children with varied genotypes of rs11635005 had significant differences in caries severity when S. mutans was present (pairwise comparison P < 0.05). Children with varied genotypes of rs4786370 or rs1539849 had significant differences in caries severity when S. mutans was absent (pairwise comparison P < 0.05).  Fig. 1 The relationship between dental caries and S. mutans carriage. *Indicates P < 0.05. S. mutans+ means positive for S. mutans carriage. S. mutans-means negative for S. mutans carriage The joint effect of the three SNPs, rs4786370, rs11635005, and rs1539849 was calculated as the genetic risk score (GRS), which ranged from −2 to 4. The interaction between the GRS and S. mutans was significant in the whole sample and the two sites (P < 0.000 1). The marginal effect of the GRS is presented in Fig. 3d. For children without S. mutans carriage, the risk for dental caries increased when the GRS increased, whereas for children with S. mutans carriage, the risk of dental caries decreased with increasing GRS.
The results of sensitivity analyses in white children and the subsample with the assessment of home water source are presented in Table 3. The effect sizes of the three SNPs in sensitivity analyses were similar to those in the whole sample. All interactions between SNPs and S. mutans were significant (P < 0.05).
Functional analysis of the identified SNPs Functional analysis of the three SNPs and SNPs in linkage disequilibrium (LD; R 2 ≥ 0.8) was conducted using HaploReg. Two SNPs, rs11635005 and rs1539849, are located within intron regions of the galactokinase 2 (GALK2) and CUGBP, Elav-like family member 4 (CELF4) genes, and rs4786370 is located within 2 kb upstream of the interleukin 32 (IL32) gene. Although no SNPs are located within exons (protein-coding regions), all identified SNPs and SNPs in LD are located at the promoter/enhancer histone markers and/or DNAse hypersensitive regions (Appendix Table 2), which means that these SNPs or SNPs in LD may affect the expression of relevant genes. Two SNPs, rs11635005 and rs4786370, have been identified as the expression quantitative trait loci that affect the expression levels of mRNAs. Moreover, analysis of the genes listed in Table 2 that harbor these SNPs using GTEx showed that GALK2 is ubiquitously expressed (Appendix Fig. 2). IL32 is highly expressed in the blood and gastrointestinal organs. CELF4 is mainly expressed in the brain. The impact of the effective alleles on annotated genes was assessed in expression-specific tissues and the minor salivary gland (Fig. 4). The effective alleles of rs11635005 and rs4786370 are significantly associated with increased expression of GALK2 in the EBV-transformed lymphocytes, and IL32 in the whole blood, respectively. These alleles have similar but nonsignificant effects on their annotated genes in the minor salivary glands. The effective alleles of rs1539849 are not associated with expression changes in their annotated genes.
Ontological analysis of the three annotated genes using Enrichr and ToppFun indicated that (1) GALK2 is involved in the galactose metabolic process and carbohydrate phosphorylation (Appendix Fig. 3); (2) IL32 participates in the cytokine-mediated signaling pathway and is associated with multiple inflammatory diseases (Appendix Table 3). CELF4 is related to the modulation of excitatory postsynaptic potential and regulation of mRNA splicing. CELF4 is significantly expressed in the nucleus accumbens, both in humans and mice, which is the center of the food reward circuitry. In addition, CELF4 is linked to an increased susceptibility to weight gain (P = 0.001 5) (Appendix Fig. 4).

DISCUSSION
Our results indicate that dental caries severity in the primary dentition was potentially related to individual genotypes and  S. mutans carriage. Although no gene-by-S. mutans interactions met the strict genome-wide significance level, we identified three potential SNPs, rs4786370, rs11635005, and rs1539849, that interacted with S. mutans at the suggestive significance level (P < 9 × 10 −5 ). These interactions were replicated at the nominal level in an independent cohort. We adopted the approach used in previous GWAS with a relatively lenient p-value to select suggestive genetic loci for replication. 15,18 In the replication step, we used the conservative significance level and nominal significance level, which was used in a previous genome-wide study. 18 These SNPs were not identified by previous genome-wide studies, because we assessed gene-by-environment interactions instead of direct genetic associations, and previous studies have shown that novel genes could be identified by examining the modification effect of environmental factors on genetic associations. 14,20 The severity of dental caries was associated with   Fig. 4 The impact of effective alleles on gene expression. The labels were organized as SNP_effective allele_reference allele_tissue. The slope values were obtained from SNP-gene association tests conducted by GTEx. A positive slope value indicates that the effect allele is associated with increased gene expression. *P < 0.05 Human genes influence caries susceptibility Meng et al.
individual host genotypes and S. mutans carriage. For instance, dental caries severity was significantly influenced by S. mutans carriage in children who carried CC alleles but not TC or TT alleles at rs11635005. In addition to the individual SNP, the GRS that combined the effect of these three SNPs significantly interacted with S. mutans carriage. Further ontological analysis of the annotated genes for the identified SNPs suggests that IL32, GALK2, and CELF4 are potentially plausible genes that may play a role in the development of dental caries and interact with S. mutans through their involvement in galactose and carbohydrate metabolism, host immune response, and food consumption.

IL32
The SNP rs4786370 is mapped to IL32, which encodes a member of the proinflammatory cytokine family. 21 IL32 has nine isoforms and regulates the expression of numerous inflammatory cytokines, such as TNF-α, IL1β, IL6, and IL10. Some of these cytokines are elevated in saliva, in children with dental caries. 22,23 Furthermore, IL32 has been associated with various infectious/ inflammatory diseases, [24][25][26][27] including periodontitis. 28 In this study, we found that children with the C allele of rs4786370 were associated with increased caries severity in the presence of S. mutans. When S. mutans was absent, dental caries severity was significantly different among children with varied genotypes of rs4786370. Children who carried the C allele presented with lower caries severity when S. mutans was absent. This phenomenon is possibly because the C allele at rs4786370 is associated with higher expression of IL32. When S. mutans is absent, higher IL32 expression may indicate an enhanced immune response and better host protection from dental caries.

GALK2
The SNP rs11635005 is located within GALK2. GALK2 encodes an Nacetylgalactosamine (GalNAc) kinase. This enzyme can efficiently phosphorylate GalNAc at low concentrations and phosphorylate galactose at high concentrations. 29 The activity of GalNAc kinase is pH-dependent, with the highest activity at pH 7-8. 30 GalNAc and galactose are monosaccharides (simple sugar). GalNAc is found in a variety of human glycoproteins that participate in numerous biological processes, including immune response 31 and tumor cell surface expression. 32 Galactose is often found in dairy products, which form lactose with glucose. 33 In the oral cavity, GalNAc and galactose could be metabolized through two paths, including phosphorylation by human enzymes, such as GalNAc kinase encoded by GALK2, and metabolism by acidogenic oral bacteria, including S. mutans, S. salivarius, and Lactobacillus. 34 The bacteria-involved galactose metabolism is associated with more acid production and lower plaque pH. Our study results showed that for children who have CC alleles at rs11635005, the presence of S. mutans significantly increased caries severity. This phenomenon can be explained as follows: the C allele of rs11635005 is associated with lower expression of GALK2, which manifested as attenuated GalNAc kinase-involved galactose metabolism; without S. mutans carriage, the caries severity among children with different GALK2 genotypes was not significantly different. However, with S. mutans carriage, while children who carry CC alleles may have lower host enzyme activity to metabolize galactose, the bacteria-involved galactose metabolism may be enhanced. Therefore, more acid production and lower plaque pH increase caries risk among children with CC alleles.
In contrast, for children with the T allele of rs11635005, the carriage of oral S. mutans was not significantly associated with caries severity. The T allele is associated with higher expression of GALK2 and higher production of GalNAc kinase. It is speculated that with sufficient GalNAc kinase-involved galactose metabolism, bacteria-involved galactose metabolism is not significantly increased with the presence of S. mutans. Therefore, children with the T allele are not susceptible to S. mutans carriage in relation to caries risk.

CELF4
The SNP rs1539849 is located at an intron region of CELF4. CELF4 is predominantly expressed in the brain. It plays a role in maintaining the stability and availability of mRNA for numerous proteins in excitatory neurons. 35 Consequently, CELF4 regulates synaptic plasticity and transmission. Other than epilepsy, CELF4 mutant mice also presented hyperactivity and late-onset obesity. 36 This obesity trait was possibly due to increased food intake rather than reduced energy expenditure, as CELF4 mutant mice were hyperactive. Furthermore, CELF4 is highly expressed in the nucleus accumbens, the center of food reward circuitry that is linked to overeating and palatable food intake. 37,38 Therefore, CELF4 may play a role in the regulation of dietary intake.
In this study, CELF4 was found to be associated with S. mutans carriage and dental caries risk in primary dentition. The dental caries severity was significantly different among children with varied genotypes at rs1539849, when S. mutans was absent. CELF4 might not have a direct relationship with S. mutans carriage or its pathogenicity. However, children with certain genotypes at variants within CELF4, such as rs1539849, might be at higher risk for overeating, particularly sweetened food consumption, and consequently have an increased risk for dental caries.
The findings from this study need to be cautiously interpreted with the following considerations: (1) the limited sample size, which is potentially the reason that no genome-wide significant interactions were identified; (2) the limited microbiological data. Only binary (yes/no) data of S. mutans assessments were available, and we were not able to assess the impact of the oral carriage scale of S. mutans. In addition, the detection of oral S. mutans depends on clinical sample types (swab, saliva, or plaque samples; carious or non-carious lesions) and S. mutans identification methods (culture-dependent or culture-independent). This critical information was not provided in the original dataset. Furthermore, there are no microbiological data on other cariogenic or beneficial microorganisms. For instance, Candida species and Lactobacillus species have been shown to be associated with caries in children; 39,40 Streptococcus salivarius, Streptococcus sobrinus, Streptococcus parasanguinis, Streptococcus wiggsiae, Streptococcus exigua, Parascardovia denticolens, Porphyromonas, Actinomyces, and Veillonella have been identified in the oral microbiota of caries-active children in addition to S. mutans; 41-46 the role of these cariogenic or beneficial microorganisms in this hypothesized host gene-by-S. mutans interaction on dental caries could not be tested using the current dataset; (3) the lack of data on other environmental factors associated with dental caries and S. mutans, such as water fluoridation (only 48% of the participants had data on home fluoride exposure level), oral hygiene, salivary flow, socioeconomic status, and dietary intake; and (4) the limited portion of racially and ethnically diverse participants, which precludes the generalization of our findings to nonwhite and Hispanic children. Therefore, future studies with a larger, more diverse population and additional caries risk factors are desirable to validate the current findings.
To summarize, we identified three human genes that are likely associated with the interaction between the host, S. mutans, and dental caries in children. These human genes are involved in the host immune response, galactose carbohydrate metabolism, and potential food-rewarding system. Further validation of these candidate genes in the development of dental caries in children is warranted. Given that additional oral cariogenic microorganisms have been identified in addition to S. mutans, further research should consider investigating the relationship of these genes with the oral microbiota and the development of dental caries. The integration of human genome and oral microbiome findings in dental caries risk assessment in children could be used to develop more effective, personalized, predictive, and preventive strategies for dental caries.

Study population
This study was approved by the University of Rochester Research Subject Review Board. The assessed dataset, Dental Caries: Whole Genome Association and Gene×Environment Studies, was obtained from the NIH Database of Genotypes and Phenotypes (https://www.ncbi.nlm.nih.gov/gap). The study subjects in this dataset were recruited from four sites at the University of Pittsburgh and the University of Iowa: (1) the COHRA, (2) the Dental Registry and DNA Repository (DRDR), (3) the Iowa Fluoride Study (IFS), and (4) the GEIRS. 18,47 Study subject recruitment and data collection were detailed previously. 18,47 The dataset obtained from dbGaP included 5 418 participants with dental phenotype data. In this study, participants who met the following inclusion criteria were selected: (a) younger than 13 years of age; (b) having S. mutans data; (c) having records of dental caries in primary dentition; and (d) having genotyping data. In total, 709 children from 526 families (COHRA and GEIRS sites) were eligible for the gene-by-S. mutans interaction analysis. Twostage analysis (discovery and replication) was performed to identify potential gene-by-S. mutans interactions. The data from the COHRA site (n = 547) were used to conduct the genome-wide analysis at the discovery stage. The data from the GEIRS site (n = 162) were used at the replication stage to assess the findings identified at the discovery stage.
Phenotypes and covariates Dental caries diagnosis was detailed by Shaffer et al. 18 Upon intraoral examination, each primary tooth was scored as sound (no white spot or cavitated lesion), white spots (non-cavitated lesion), decayed (cavitated lesion), or missing/filled due to caries. Based on these scorings, two indices were calculated: dmft including decayed (d) missing due to decay (m), filled (f), and teeth (t) and dmftw (including both dmft and white spots). S. mutans detection status was coded as "yes/no" in the original study. Demographic characteristics that were marginally associated with primary dental caries (P < 0.2) were included as covariates, which were age, gender, and race (white/nonwhite).
Genotyping methods DNA samples were extracted from blood, saliva, buccal mucosal swabs, or mouthwash. A custom panel of 580 000 SNPs was genotyped using the Illumina 610 platform (Illumina, Inc., San Diego, CA, USA). In this study, SNPs were filtered based on the following exclusion criteria: (a) located on autosomal chromosomes; (b) genotyping call rate <95%; (c) minor allele frequency <1%; and (d) Hardy-Weinberg equilibrium P value < 5 × 10 −7 . In total, 556 839 SNPs passed the quality assurance criteria and were used for the genome-wide analyses.

Statistical analysis
The population substructure was assessed using principal component analysis in PLINK1.9 (https://www.cog-genomics.org/ plink2). 48,49 The first five eigenvectors were generated and adjusted as covariates. At the discovery stage, the genome-wide gene-by-S. mutans interaction analyses were conducted using the GWAF package in R, which utilizes GEE to adjust for family clusters. 50 Binomial distribution of the dichotomized dental caries outcomes (dmft and dmftw) was applied. An additive genetic model with the number of effective alleles in a SNP treated as a numeric variable was used in the analyses. The interactions between SNPs and S. mutans were assessed by including a product term (x additive x S:mutans ) in the regression models. 50,51 To maximize sample size and power, stratified analyses were not conducted at the genome-wide association analysis stage. 52 logitðpÞ ¼ β 0 þ β 1 x additive þ β 2 x S:mutans þ β 3 x additive x S:mutans þ β 4 x covariates þ ε The genome-wide P value was set at 9 × 10 −8 after Bonferroni correction for the total number of SNPs tested in the analyses. The Bonferroni correction is a commonly used method to control multiple testing in GWAS. 53 Age, gender, and race were controlled in the statistical models, although the ages were different among the dmft = 0 and dmft > = 1 groups.
At the replication stage, we assessed the potential suggestive SNPs selected at the discovery stage. Dental caries outcomes used as count data were analyzed using GEE, with a negative binomial distribution in STATA 15.0. Two significance levels were used. The conservative significance level was set based on Bonferroni correction, adjusting for the number of SNPs assessed. The nominal significance level was also used. Finally, the effects of gene-by-S. mutans interactions were estimated using GEE with a negative binomial distribution in the whole sample (n = 709).
The joint effect of the replicated SNPs represented as a GRS was calculated based on the effective alleles' impact on dmft when S. mutans carriage was negative. If the effective allele of an SNP was associated with an increased risk of dmft, the GRS score of this SNP was coded as "0, + 1, + 2" based on the number of the effective allele. If the effective allele of a SNP was associated with a decreased risk of dmft, the GRS score of the SNP was coded as "0, −1, −2" based on the number of the effective allele. The interactive effect of GRS with S. mutans was estimated using GEE with a negative binomial distribution.
We also conducted sensitivity analyses to evaluate the robustness of our identified SNPs using the GEE model with a negative binomial distribution in STATA 15.0. The top SNPs were assessed in white children (n = 587). Nonwhite children were not assessed due to limited sample size. We also assessed the top SNPs in a subsample containing an assessment of home water source (n = 509). Home water source was classified as water from the city/public, well, or others.
The functional annotation of the identified SNPs was conducted using HaploReg v4.1 54 and GTEx Portal v7. Functional inference of the genes harboring the identified SNPs was conducted using Enrichr 55 and ToppFun. 56 This study was conducted following the STREGA reporting guidelines. 57

CONCLUSIONS
This study identified three gene-by-S. mutans interactions that were potentially associated with the severity of dental caries in the primary dentition with plausible biological roles. These genetic markers were linked to varied caries risk modified by S. mutans carriage status. Our study findings could help to elucidate the relationship between host susceptibility, S. mutans carriage, and caries development. The identified human genes could be used to identify children at high risk of caries and develop personalized dental caries prevention strategies. The identified genes could also serve as potential targets for caries prevention.

DATA AVAILABILITY
The datasets used for the analyses described in this paper were obtained from the NIH Database of Genotypes and Phenotypes through dbGaP accession number phs000095.v3.p1. The study, Dental Caries: Whole Genome Association and Gene × Environment Studies, was funded by the NIDCR, grant number U01-DE018903. https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000095. v3.p1.