AMBN gene polymorphism Alters the Caries Susceptibility of Adolescents in South China

The aim of this study was to identify genetic factors under additive and dominance models that contribute to caries susceptibility, and to investigate into the interactions between these genes. A cross-sectional study was conducted among 1055 adolescents in Foshan, South China. The International Caries Detection and Assessment System (ICDAS) was used to identify caries. Demographic and environmental variables were analyzed. Twenty-three single nucleotide polymorphisms (SNPs) in 14 genes were identified from saliva samples. Regression analysis was used for the evaluation of direct and epistasis of genes under the hypothesis of an additive model and using the minor allele as the reference allele. After the adjustment by environmental factors, the G allele in AMBN (rs13115627) was a protective factor for caries under both additive model (P=0.007; OR=0.728; 95% CI, 0.579-0.916) and dominance model (P=0.021; OR=0.728; 95% CI, 0.556-0.953). No interactions between selected genes met the Bonferroni correction significance cutoff for multiple testing. Our results suggests that gender, one-child family, Cariostat score and Plaque Index have independent protective effects on dental caries. The polymorphisms in AMBN (rs13115627), under both additive and dominance models alters caries susceptibility of Adolescents in South China. No epistatic effect has been found between selected genes.


Introduction
Dental caries is one of the most common chronic diseases among children and adults worldwide. The cost of direct treatment was estimated to account for an average of 4.6% of global health expenditures (1). Dental caries is a major public health problem due to the increasing prevalence in developing countries and the heavy disease burden (2). According to the 4th National Oral Health Survey in China, the prevalence of caries in 12-year-old children increased from 28.9% to 38.5% in the last decade, which has attracted the attention of the government (3).
Dental caries is a multifactorial disease, and environmental risk factors such as addiction to sugary snacks and drinks, poor oral hygiene, high levels of cariogenic bacteria, salivary dysfunction, and insufficient fluoride exposure are critical to its development (2). However, even when exposed to the same level of environmental risks, some people are more susceptible to caries than others. The high prevalence of caries among certain groups has motivated research towards identifying genetic contributors to caries(4) (5).
Genes involved in enamel formation, the immune response, saliva proteins and food preferences have been considered to be involved in the etiology of dental caries(6) (7). Evidences found in studies regarding twins estimated that 40-60% of caries susceptibility is genetically determined (8)(9). Some studies have identified large numbers of single nucleotide polymorphisms (SNPs) associated with caries (6).
However, few studies have taken different genetic models into consideration, particularly in Chinese population. The present study investigated environmental factors and genetic factors related with dental caries, focusing on the genes involved in enamel formation, immune response, saliva proteins, and taste preferences under different genetic models.

Subject
The study was approved by the Ethical Review Committee of Guanghua School of Stomatology, Sun Yat-Sen University (ERC-[2018]01). A written informed consent was obtained from each student's guardian before the study.
The oral examination and saliva tests were performed in March 2018 in Foshan.
Foshan is a medium-sized city in Guangdong Province in southern China with a population of 7.6 million. The GDP was CNY 124,324 (USD 18,018) per capita in 2017 (10). The water fluoride concentration is 0.16 mg/L (11).
According to Peduzzi, for logistic regression analysis, the number of events (death or illness) should be 5-10 times the number of independent variables (12). In total, 43 independent variables were investigated via logistic regression in this study, and the caries prevalence in 12-year-old adolescents was 38.5%; thus, the total sample size should be between 559 and 1117 (12)(3).
A random cluster sampling technique was employed in 6 middle schools according to the number of children that we needed to recruit. All Grade 7 students living in Foshan for the previous 2 years who reported no systematic illness and no antibiotic use for at least the preceding 2 weeks were invited to participate in the study.

Questionnaire
The questionnaire was mainly designed with reference to the Fisher-Owens conceptual model of influence on children's oral health (2). The structured questionnaire recorded demographic information, socioeconomic status, diet, selfreported oral health behaviors and past dental experiences. The questionnaire was self-administered and completed in schools by students with the guidance of guardians. The reliability of the questionnaire was assessed by an internal consistency test, and the Cronbach's alpha was 0.75.

Oral examination
The oral examination was conducted in the classrooms. The examinee was lying in a portable dental chair. The teeth were examined visually with the help of a plain mouth mirror, a CPI probe and compressed air. The International Caries Detection and Assessment System (ICDAS) criteria were used (13). All tooth surfaces were first examined with a wet surface and then reexamined after the teeth were dried with compressed air. Filled surfaces and missing teeth due to caries (the reasons for missing teeth were obtained from the questionnaire) were also recorded. The DMFT was calculated both at the early clinical stage of decay, with enamel and dentine caries (at the D 1 level, as in the ICDAS method), and at the later stages of decay, with dentine-only caries (at the D 3 level, as in the ICDAS method), and these values were recorded as D 1 MFT and D 3 MFT, respectively. Individuals with lesions scoring from 3-6 were classified into the caries group, and the others were classified into the caries-free group in this study (14). The Plaque Index (PlI) was recorded according to the Silness and Löe scale (15).
The three examiners had taken a 12-hour e-learning course on ICDAS before the study. Ten percent of the subjects were randomly selected to obtain a duplicate examination in order to monitor the reliability of the diagnoses and calculate the interexaminer consistency. The Cohen's kappa values were 0.85, 0.83 and 0.91 for caries examination.

Microbiological examination
The Microbiological assessment was performed by the Cariostat method, which is a colorimetric test for measuring caries activity based on the presence of acidogenic microorganisms (16) (Ramesh et al. 2013). Plaque was collected using the sterile swab included in the Cariostat kit (GangDa Medical Technology Co. LTD., Beijing, China) package. According to the Cariostat kit instructions, the examiners scrubbed the buccal surfaces of the maxillary molars and mandibular incisors 3-5 times and immersed the swab into the culture medium ampule. The samples were incubated at 37°C for 48 hours. The color of the culture medium was compared with the reference color on the color chart supplied with the Cariostat kit. The reference color was scored from 0 to 3 with every 0.5 grade as an interval. The color turns from blue to green and, ultimately, to yellow, indicating an increased acid production ability of the plaque in the sample.

Saliva tests
All saliva was collected after the students had rinsed their mouths with tap water.
The unstimulated saliva was collected for 15 minutes. Students were asked to spit the saliva through a funnel into a scaled tube every 3 minutes. Then, the unstimulated saliva flow rate (ml/min) was calculated. The saliva buffering capability was measured according to the Ericsson method (17). One milliliter of saliva was added to 3 ml of 3.3 mmol HCl within 5 minutes after collection and then allowed to stand for 20 minutes to remove CO 2 . The final pH of the saliva was evaluated by an electrical pH meter. The buffering capability of unstimulated saliva was recorded as 'low', 'normal' and 'high' when the pH value fell into the ranges of <4.25, 4.25-4.75 and >4.75, respectively.

Candidate Genes Selection
The major candidate gene categories identified to date include enamel formation genes, immune response genes, genes related to saliva, and genes related to taste and dietary habits (6). The candidate genes selected for this study are listed in Table 1. The Haploview 4.2 software package (http://www.broad.mit.edu/mpg/haploview/) was used to select tag SNPs in each candidate gene. SNPs reported having associations with dental caries in previous studies or located in a gene fragment that could have functional effects were selected.

DNA analysis
Genomic DNA was extracted from 1-ml saliva samples according to the manufacturer's instructions (Saliva DNA Sample Collection Kit, ZEESAN, Xiamen, China). The DNA concentration and purity of each sample were determined by spectrophotometry. Twenty-three SNPs in 14 genes were identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Statistical analysis
The data were analyzed by SPSS version 22 for Windows (IBM Inc., Chicago, IL, USA). The differences in the characteristics between the caries and caries-free groups were compared by binary regression analysis and an independent samples T test. A binary regression model was also applied to analyze the environmental risk factors. For both the characteristics and environmental risk factors, each risk indicator with a P value of less than 0.2 in the univariate analysis was applied to stepwise multivariate analysis. Risk factors with a final P value in the multivariate analysis of less than 0.05 were used as covariates in further analysis.
The Hardy-Weinberg equilibrium was estimated by the chi-square test. Genotypes of the candidate genes were analyzed under the hypothesis of an additive model and using the major allele as the reference. Each SNP was treated as a continuous exposure under additive model, coded 0,1, or 2(minor alleles). For direct associations, a binary regression model was applied. Each SNP with a P value of less than 0.2 in the univariate analysis would be applied to stepwise multivariate analysis. All possible pairwise SNP-SNP interactions between the selected SNPs were assessed by a bivariate logistic regression model. Significant characteristics and environmental risk indicators were used as covariables. The interaction between two SNPs was defined as the additional effect of their concomitant carriage on caries beyond the addition of their independent effects (departure from additivity):

Characteristics
In total, 1055 students participated in the survey. The mean D 1-6 MFT was 4.42±0.11, and the mean D 3-6 MFT was 0.72±0.40. A total of 382 (36.2%) students had caries scored from 3-6 (categorized into the caries group in this study), and 673 (63.8%) students had caries scored from 0-2 (categorized into the caries-free group in this study), among which 129 (12.2%) scored 0. The characteristics are summarized in Table 2. Girls, and students from families with more than one child had a higher risk of having caries experience. As a result, gender and family children number were taken as covariables and used in further analysis.

Environmental factors
According to multivariate analysis, Plaque Index and the Cariostat score were associated with caries in this population (Table 3). Students with a Plaque Index score of 2-3 or 1-2 had a higher risk of having caries experience than those with a score of 0-1. Students with a Cariostat score of 2-3 or 1-2 had a higher risk of having caries experience than those with a score of 0-1.

Direct associations
The results of univariate analyses of the association between genotypes and caries under additive model and dominance model are presented in Table 4&5. Only one Pvalue for SNPs was less than 0.2 in both models, so the multivariable analysis was omitted. For additive model, SNP rs13115627 in AMBN were observed significantly associated to caries in both the unadjusted and adjusted analyses (Table 4).
Individuals with the G allele in rs13115627 (AMBN) had a lower risk of having caries experience than those without the G allele. For dominance model, SNP rs13115627 in AMBN were observed significantly associated to caries after adjusted by gender, family children number, Cariostat score and Plaque Index (Table 5). Individuals carries GG or AG in rs13115627 (AMBN) had a lower risk of having caries experience than those carries AA.

Interactions
After pairwise SNP-SNP interactions analyzed by binary regression adjusted for gender, the number of children in the family, the Plaque Index and the Cariostat score, 11 interactions were significant under addition model, and 5 interactions were significant under dominance model (Table 6&7). None of them met the significance cut-off P value (0.0001) after the Bonferroni correction for multiple testing. No epistaic effect was found between these genes to be related to caries risk in this population.

Discussion
The present study gives a profile of dental caries of adolescents in South China using the scales of ICDAS. To our knowledge, this work is the first to show the caries situation of Chinese adolescent by ICDAS, and is the first to examine the contribution of caries related genes under different genetic models.
For demographic and environmental factors, girls, families with more than one child, a high Plaque Index and a high Cariostat score were associated with caries experience which was consistent with the previous studies (19) (20). Therefore, these factors were included as covariates in the multivariate analysis to elucidate the independent contributions of genes.
For genetic factors, we found significant associations between rs13115627 in AMBN and caries under additive model and dominance model. Previous studies examined the relationship between other SNPs in AMBN and caries. Shimizu el al. found that the frequency of the C allele of rs4694075 in AMBN was significantly higher in individuals with high caries experience than in those with low caries experience in five different populations(4). Ergöz el al. found that a variation in AMBN (rs4694075) was related to lower caries experience among asthmatic children in Turkey (21).
AMBN encodes ameloblastin, which is believed to control the elongation of enamel crystals and generally directs enamel mineralization during tooth development (22) (23)(24). Our previous study found that the GG genotype of AMBN (rs13115627) was significantly associated with an increased calcium-phosphorus ratio in adolescents in southern China(25). Das et al. found that a lower calcium-phosphorus ratio may lead to higher caries risk (26). Thus, AMBN (rs13115627) was assumed to affect caries susceptibility through altering enamel composition.
In the present study, AMBN(rs13115627) was significantly associated with dental caries under both additive and dominance model, so we attempted to check the epistasis between AMBN and other genes related with dental caries. Our results show that the P values of interaction between AMBN(rs13115627) and Understanding the contribution of a network of genes to variations in dental caries risk may lead to new approaches to prevention, early identification of high-risk patients, and therapeutic targets.

Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions
Ketian Wang and Liangyue Pang substantially contributed to conception and design