Background

Tooth agenesis (TA) is the congenital absence of one or more teeth. This condition results from disturbances at early stages of odontogenesis. Several studies have demonstrated that mutations and genetic polymorphisms within specific genes may contribute to the presence of TA; among them are MSX1 [1], PAX9 [1], TGF-α [2] and genes from the FGF family [3, 4].

MSX1 (muscle segment homeobox 1) is expressed during epithelial-mesenchymal interactions that occur at the beginning of tooth formation [5]. Mutations on this gene are related to failures in the development of multiple teeth, preferentially premolars [6]. Msx1 deficient mice showed cleft palate, alveolar bone defects, anomalies in various facial bones, and failure of tooth development [7], suggesting that TA could be genetically related to the development of cranium, maxillary, and mandibular complex [8]. On the other hand, PAX9 (paired box gene 9) is expressed in the neural-crest-derived mesenchyme of the maxillary/mandibular arches and also contributes to tooth and palate formation [9]. Mutation in PAX9 gene was associated with autosomal dominant oligodontia, usually affecting the majority of permanent molars [10]. Pax9 deficient mice presented agenesis of all teeth, cleft palate, and other craniofacial anomalies [5].

TGF-α (transforming growth factor-alpha) is a gene expressed during craniofacial development [11]. Although, mice with Tgf-α deficiency did not show tooth anomalies [12], human studies evidenced that genetic polymorphisms in TGF-α contributes to the presence of TA [2]. Regarding the FGF (fibroblast growth factor) signaling, its role in craniofacial development has extensively investigated [13, 26]. Quantification of the concentration and purity of the DNA was determined using a spectrophotometer (Nanodrop 1000; Thermo Scientific, Wilmington, DE, USA).

Eight genetic polymorphisms, located in intronic regions, were assessed: MSX1 (rs1042484), PAX9 (rs8004560), TGF-α (rs2902345), FGF3 (rs1893047), FGF10 (rs900379), and FGF13 (rs12838463, rs5931572, and rs5974804) (Table 1). The polymorphisms were blindly genotyped by polymerase chain reactions (PCR) using the TaqMan method (ABI Prism 7900HT, Applied Biosystems, Foster City, CA, USA) [27] and end-point analysis. The interpretation of the data was performed using software provided by Applied Biosystems (Foster City, CA, USA) for allelic discrimination.

Table 1 Studied genetic polymorphisms
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Statistical analysis

Statistical analyses were performed on Epi Info 3.5.2 (www.cdc.gov/epiinfo) and Plink (http://zzz.bwh.harvard.edu/plink/), using an established α of 0.05. Chi-square test (with Yates’ correction for continuity, when necessary) or Fisher’s exact test were performed to determine association between allele/genotype frequencies and the craniofacial phenotypes assessed. For the polymorphisms located in the chromosome X (polymorphisms in FGF13), an analysis adjusted by the gender was performed. Due to the multiple comparisons made, a Bonferroni correction was applied for each evaluated outcome (corrected p value = 0.00625; 0.05/8 genetic polymorphisms assessed). Genotype/phenotype associations were also tested in the dominant and recessive models. Chi-square test was also used to evaluate the Hardy-Weinberg equilibrium.

Results

The distribution of genotypes followed Hardy-Weinberg equilibrium (data not shown). Information regarding the association between skeletal malocclusion and TA was reported in a previous published paper [24]; individuals presenting class II skeletal malocclusion showed lower frequency of TA. There was no significant association between genotype/allele distributions and the presence of TA for any polymorphism assessed in the present study (p > 0.05) (Table 2).

Table 2 Genotype and allele distribution between TA and non-TA participants
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Genotype (p = 0.038) and allele (p = 0.037) distributions for the FGF3 rs1893047 were significantly different between class III and class I individuals (Table 3). Analysis in the dominant model (AG + GG vs. AA) demonstrated that carrying at least one G allele increased in more than two times the chance of presenting skeletal class III malocclusion (OR = 2.21, 95% CI = 1.14–4.32; p = 0.017). There was no association between the facial growth pattern and any polymorphism assessed in the present study (p > 0.05) (Table 4). No reported associations remained significant after the Bonferroni correction.

Table 3 Genotype and allele distribution among class I, class II and class III skeletal malocclusions
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Table 4 Genotype and allele distribution among mesofacial, dolichofacial, and brachyfacial growth patterns
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Discussion

Many human and animal studies support that dental anomalies, mainly TA, and craniofacial alterations could share a common genetic background [7, 9, 12,13,7]. Also, Pax9 deficient mice present agenesis of all teeth, cleft palate, and other craniofacial anomalies [5]. TGF-α is a gene expressed during craniofacial development [11]; mice with Tgf-α deficiency presented eye and hair anomalies [12]. Despite the above-described roles of these genes, the genetic polymorphisms studied within MSX1, PAX9, and TGF-α were not associated with none of the craniofacial morphological patterns assessed (skeletal malocclusions and facial type). Considering that previous studies support that genetic polymorphisms and mutations in these genes did show association with craniofacial patterns [2, 6, 10, 31,32,33], it is possible that other genetic variants within these genes are involved in the etiology of the craniofacial phenotypes in this population.

Fgf signaling is involved in various regulatory processes during embryogenesis as well as in the adult organism [34, 35]. This signal pathway has key roles in suture and synchondrosis regulation; mutations in FGF receptors cause craniosynostosis, which is the premature suture fusion [36, 37]. On the other hand, Fgf signal participates in multiple stages of palatogenesis, from palatal shelf elevation to the completion of fusion [38]. Therefore, disturbances in Fgf-related pathways are possible mechanisms of palatal cleft. Based on the above, it is clear that FGF is considered a candidate gene for study in relation to variations in the morphology of the craniofacial skeleton. In our study, carrying at least one G allele in the polymorphism rs1893047 within FGF3 increased the chance of presenting skeletal class III malocclusion. Considering that multiple members of this gene family are expressed mainly during midfacial region development [39], among them FGF3, we hypothesize that variations on this gene could contribute to the development of class III due to maxillary growth deficiency. A stratified analysis according to the maxilla contribution on class III was not possible due to the low prevalence of class III individuals in our sample, and the consequent significant reduction that would have existed in the power of the analyses.

It has been shown that Fgf3 is expressed during development and outgrowth of the facial primordia and branchial arches [39,40,41], and during odontogenesis [42, 43]. Additionally, this gene has been associated with oral cleft [31] and TA [2], suggesting that FGF3 plays a role in both craniofacial phenotypes. Our data must be carefully interpreted since significant results could be due to chance. Statistical significance did not persist for the reported associations after the Bonferroni correction.

Briefly, the knowledge regarding the role of genetic polymorphisms on craniofacial development offers the possibility of establishing new strategies to prevent these disorders. Further investigations with other genetic polymorphisms in these genes are necessary to confirm our results.

Conclusion

Our result suggests that genetic polymorphism rs1893047 in FGF3 might contribute to variations in the craniofacial sagittal pattern, specifically to the establishment of the Class III skeletal malocclusion.