To explore whether variations in odontogenesis-related genes are associated with tooth-size discrepancies.
Measurements of the width of permanent teeth were obtained from dental casts of 62 orthodontic patients (age 15.65 ± 6.82 years; 29 males and 33 females). Participants were classified according to the anterior and overall Bolton ratios as without tooth-size discrepancy or with maxillary or mandibular tooth-size excess. Genomic DNA extracted from buccal cells was used, and 13 single nucleotide polymorphisms (SNPs) across nine genes were genotyped by polymerase chain reaction using TaqMan chemistry. χ2 or Fisher exact tests were applied to determine the overrepresentation of genotypes/alleles depending on the type of tooth-size discrepancy (α = .05; corrected P value: P < 5.556 × 10−3). Odds ratios (ORs) and their correspondent 95% confidence intervals (CIs) were also calculated to investigate the risk of this phenotype for the SNPs having significant association.
Individuals carrying the FGF10 rs900379 T allele were more likely to have larger mandibular teeth (OR = 3.74; 95% CI: 1.65–8.47; P = .002). This effect appeared to be stronger when two copies of the risk allele (TT) were found (recessive model, OR = 6.16; 95% CI: 1.71–22.16; P = .006). On the other hand, FGF13 rs5931572 rare homozygotes (AA, or male A hemizygotes) had increased risk of displaying tooth-size discrepancies when compared with the common homozygotes (GG, or male G hemizygotes; OR = 10.32; 95% CI: 2.20–48.26; P = .003).
The results suggest that FGF10 and FGF13 may contribute to the presence of tooth-size discrepancies.
The lack of size correspondence between the upper and lower teeth, also known as tooth-size discrepancy, is a condition that contributes to the development of malocclusions.1 It has been suggested that tooth-size discrepancy could be an inheritable trait.2 In fact, tooth-size variability is largely caused by genetic factors, with or without a contribution from the environment.3–6 Hundreds of genes participate in odontogenesis, being involved not only in growth, differentiation, and cell function but also in the patterning and morphogenesis of teeth.7,8 Genetic variants in some of these genes have been associated with specific dental phenotypes.9–12
Tooth alterations are not isolated conditions. It was proposed that variation in tooth number and size were linked.13 Various investigations reported the co-occurrence of hypodontia and supernumerary teeth, with size disparities in isolated teeth, as well as with consistent discrepancies in the size of the entire dentition, suggesting that these conditions could have a common etiologic origin.14–20 Interestingly, previous studies showed that relatives of patients with hypodontia had decreased dental sizes, which could indicate a genetic link between both traits.21,22 Based on this aggregate evidence, it was hypothesized that genetic variants suggested to contribute to the development of dental number anomalies could also be involved with the presence of altered tooth-size patterns. Thus, the objective of this study was to test for associations between odontogenesis-related genes and tooth-size discrepancies.
MATERIALS AND METHODS
The protocol of this study was reviewed and approved by the Research Ethics Committee at the School of Dentistry of Ribeirão Preto, University of São Paulo (01451418.3.0000.5419/3.150.551). The sample consisted of 62 healthy, unrelated individuals (age: 15.65 ± 6.82 years; 33 females, 29 males), self-reported as white, who were undergoing treatment at the orthodontic graduate clinic of the School of Dentistry of Ribeirão Preto. The main eligibility criterion was to have pretreatment dental casts with fully erupted permanent dentition (up to the first molar). Individuals with the presence of craniofacial congenital anomalies or syndromes (including tooth anomalies of number, size, or shape); preceding Class I, II, or III restorations; severe occlusal dental wear; interproximal caries; preceding orthodontic treatment including interproximal enamel reduction; extreme tooth misalignment hindering correct measurements; or poor-quality or fractured dental casts were not included. Signed written informed consent documents were obtained from all participants and their legal guardians.
Tooth width was measured on all teeth for each of the upper and lower dental casts to the nearest 0.01 mm using an electronic handheld digital caliper (Digimatic CD-15DCX; Mitutoyo, Kawasaki, Japan) by the same individual. Tooth width was defined as the largest crown distance between the lateral contact points parallel to the occlusal plane. Measurements were performed three times consecutively and remeasured when they differed by more than 0.2 mm. Ten randomly chosen dental casts were measured twice at least 2 weeks apart to test intraexaminer reproducibility and agreement, using the intraclass correlation coefficient (ICC) for rater consistency in a two-way mixed model (95% confidence interval [CI]) and the estimation of the limits of agreement (LoA) using the Bland-Altman method. Intraexaminer reproducibility was high for all tooth measurements (ICC ranging from 0.888 to 0.996, all P < .001). Similarly, Bland-Altman tests showed small LoA.
Individuals were separately classified by their Bor and Bar, according to the original values reported by Bolton,1 as without tooth-size discrepancy (89.39 ≤ Bor ≤ 93.21; 75.55 ≤ Bar ≤ 78.85), with maxillary tooth-size excess (Bor < 89.39; Bar < 75.55), or with mandibular tooth-size excess (Bor > 93.21, Bar > 78.85).
Genomic DNA was extracted from buccal cells as previously described.23 Genetic variants studied were single nucleotide polymorphisms (SNPs) selected based on the evidence of a previously reported association with tooth-related phenotypes and/or their known role in odontogenesis. A total of 13 SNPs across nine genes (Table 1) were blindly genotyped using TaqMan chemistry and end-point analysis in a real-time polymerase chain reaction (PCR) system (Prism QuantStudio 6 Flex PCR System, Applied Biosystems, Thermo Fisher Scientific Inc, Foster City, Calif) following a standardized protocol.24 Because of the exploratory nature of the study, only SNPs with a genotyping failure rate of up to 20% were included for further analyses. In addition, SNPs in Hardy-Weinberg disequilibrium at P < 10−3 were not assessed. All retained SNPs had a minimum minor allele frequency >15%.
The Pearson χ2 (3 × 2 contingency tables; with continuity correction, when necessary) and Fisher exact (2 × 2 contingency tables) tests were performed to determine associations between genotype/allele frequencies on each SNP and the presence of tooth-size discrepancy. The absence of tooth-size discrepancy was considered as the reference phenotype for the analyses. For FGF13 rs5931572 (located on the chromosome X), a multivariate logistic regression was performed to test associations, adjusting the analyses for the covariate sex. The threshold for statistical significance after Bonferroni correction for multiple testing was P < 5.556 × 10−3 (0.05/9 SNPs). The odds ratios (ORs) and their 95% CIs were also calculated to estimate the risk of tooth-size discrepancy when carrying variant alleles. Additional analyses were also performed for the dominant and recessive models for the significantly associated SNPs. All analyses were performed using two-tailed tests with a significance level of 5% using SPSS Statistics 23 (IBM, Armonk, NY).
Nine SNPs were included for genotype/phenotype analyses. FGF10 rs900379, GHR rs1509460, GLI3 rs929387, and FGF3 rs1893047 showed association with the presence of tooth-size discrepancy at the nominal level (P < .05; Tables 2 and 3). FGF10 rs900379 allele frequency was associated with the overall mandibular tooth-size excess, even after Bonferroni correction (P < 5.556 × 10−3; Table 2). Individuals carrying the rs900379 T allele were more likely to have larger mandibular teeth (OR = 3.74; 95% CI: 1.65–8.47; P = .002). This effect appeared to be stronger when two risk alleles (TT) were considered (recessive model, OR = 6.16; 95% CI: 1.71–22.16; P = .006).
Sex-adjusted analysis showed significant association of FGF13 rs5931572 with the presence of tooth-size discrepancy (P < 5.556 × 10−3; Table 4). FGF13 rs5931572 rare homozygotes (AA, or male A hemizygotes) had an increased risk of presenting this phenotype when compared with the common homozygotes (GG, or male G hemizygotes; OR = 10.32; 95% CI: 2.20–48.26; P = .003). Although only at the nominal level, subsequent analysis showed that rare homozygotes were more likely to have larger mandibular anterior teeth (OR = 4.25; 95% CI: 1.00–17.99; P = .050).
The importance of phenotype-genotype correlation data in determining the role of genes in dentofacial morphology variations associated with malocclusions has been suggested.25 Although relevant phenotypes such as the Class II and Class III craniofacial patterns26–30 have been widely investigated, studies regarding the underlying genetic component of variation in tooth-size patterns are still lacking. This is the first study to report genetic variants associated with the presence of tooth-size discrepancy.
The results indicated that FGF10 rs900379 and FGF13 rs5931572 were associated with the presence of tooth-size discrepancy, specifically with a larger size of mandibular teeth. FGF10 rs900379 was previously reported as not associated with hypodontia, although another SNP in this gene was.9 Mutations in FGF10 cause the lacrimo-auriculo-dento-digital syndrome, an autosomal dominant multiple congenital anomaly disorder, which displays, among many other features, alterations in tooth size and structural defects.31 The current findings, together with the information mentioned above, reinforce the idea that there would be a genetic link between tooth number anomalies and tooth-size variations and that FGF10 and FGF13 could be involved in this.
FGF10 and FGF13 are part of the family of fibroblast growth factors (FGFs) that participate in one of the most important signaling pathways during odontogenesis.8,32–37 Fgf10-null mice have hypoplastic teeth38 but no other significant dental defects.39,40 For this reason, the functional redundancy of FGF10 in the dental formation process has been proposed.33,41 However, on the other hand, it has been shown that the dental epithelium of these animals showed limited growth. Dental epithelium lacking a cervical loop exhibited a decreased growth rate.35 Based on this, it may be assumed that genetic variation in FGF10 could alter the rate of dental growth, which could explain variations in the size of the teeth.
FGF3 rs1893047 showed a trend for association with tooth-size discrepancy. Previous studies already demonstrated the possible involvement of markers of this gene (rs1893047, rs12574452, rs7932320) in the presence of hypodontia.9,10 Fgf3 is expressed in mesenchymal cells from E13.5 (bud stage) in regions adjacent to the inner enamel epithelium.33,35 Unlike FGF10, FGF3 could stimulate cell proliferation in isolated dental mesenchyme.33 Although it is not responsible for cervical loop formation, since Fgf3-/- mice present smaller molars with structural alterations compared with wild-type and Fgf3 +/− molars,42 FGF3 may be involved in tooth-size–related alterations.
Further research is necessary to replicate the current findings in larger samples of different ethnic origin. The absence of positive associations for tested variations in the other genes could be due to a type II error due to an insufficient sample size (explorative study). In addition, future studies could use digital methods to possibly increase the accuracy of the acquisition processes and/or the measurement of the studied phenotype, as well as consider the study of individual tooth sizes expressed as continuous data or different approaches for tooth size analysis.
FGF10 and FGF13 may contribute to the presence of tooth-size discrepancy.
PhD candidate, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Professor, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Undergraduate student, School of Dentistry of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.
PhD candidate, Department of Orthodontics, State University of Rio de Janeiro, Rio de Janeiro, Brazil.
PhD candidate, Department of Oral and Maxillofacial Surgery, Positivo University, Curitiba, Brazil.
Associate Professor, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Full Professor, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Professor, Department of Oral and Maxillofacial Surgery, Positivo University, Curitiba, Brazil.
Associate Professor, Department of Pediatric Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.