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Single nucleotide polymorphisms in odontogenesis-related genes associated with tooth-size discrepancy

Single nucleotide polymorphisms in odontogenesis-related genes associated with tooth-size... IntroductionA tooth-size discrepancy (TSD) is characterised by a dimensional disproportion in the mesiodistal size between the maxillary and mandibular teeth1,2. A TSD contributes to an altered relationship between the dental arches because accepted overbite, overjet and interdigitation require a certain proportional size relationship between the teeth.3,4 Successful orthodontic treatment with regard to a functional and aesthetic occlusion also, in part, depends on an accurate TSD diagnosis and intervention.1,2Tooth size is determined at the commencement of the dental development stage.5 Odontogenesis involves a complex mechanism of interaction between signalling networks and growth factors.6–8 Bone morphogenetic protein 4 (BMP4) is an important growth factor inducing tooth development as it stimulates the differentiation of mesenchymal-derived dental-specific cells from the beginning of tooth formation.6,9 BMP4 also regulates Runt-related transcription factor 2 (RUNX2), which is a critical transcriptional regulator of tooth formation.7,10 RUNX2 controls the morpho-differentiation and growth of the embryonic epithelium of the enamel organ which precedes the formation of tooth enamel.8 In addition, RUNX2 stimulates BMP2 production which is a key protein involved in odontogenic differentiation and the control of enamel mineralisation.11 Additionally, SMAD proteins are important mediators affecting this network of signaling pathways.8,12Single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide in the genome differs between members or paired chromosomes of an individual. Recent studies have investigated SNPs in odontogenesis-related genes and identified some as possible relevant factors related to tooth-size variability in humans.13–15 Therefore, SNPs associated with growth factors that play an important role in odontogenesis could be a risk factor for TSD. Therefore, the current study aimed to investigate the association between SNPs in BMP4, BMP2, RUNX2 and SMAD6 genes and TSD.MethodsThe present cross-sectional study was approved by the Local Research Ethics Committee (protocol: 01451418.3.0000.5419/3.150.551) and all included patients and their legal guardians signed written informed consent according to the Declaration of Helsinki. The STREGA (STrengthening the REporting of Genetic Association Studies) checklist was used to design and report this study.16The study included pretreatment dental casts and genomic DNA samples from self-reported white and biologically unrelated patients undergoing orthodontic treatment at School of Dentistry of Ribeirão Preto, University of São Paulo from 2016 to 2018. Patients with a previous history of orthodontic treatment, congenital syndromes, tooth anomalies, interproximal caries, restorations or enamel reduction, occlusal dental wear, fractured or poor-quality dental casts, and extreme tooth misalignment, were excluded. The sample was previously described by Marañón-Vásquez et al.13 Sixty-two patients were included in the current study.A single dentist evaluated ten random dental casts twice within a two-week period to conduct an intra-examiner reliability test. The intraclass correlation coefficient (ICC) estimated a high reproducibility for all teeth (ICC ranging from 0.888 to 0.996). Mesiodistal tooth width was obtained by the largest crown distance between the lateral contact point parallel to the occlusal plane (maximum side-distal distance). A digital calliper (Digimatic CD-15DCX; Mitutoyo, Kawasaki, Japan) was used for measurements. All teeth of each maxillary and mandibular cast were consecutively measured three times and, when the difference between the measurements was more than 0.2 mm, the tooth was remeasured.TSD was assessed using the Bolton analysis.17 Bolton ratios were calculated according to the preexisting formula for the establishment of an anterior discrepancy ratio and overall discrepancy ratio, as follows:AnteriorTSDBar=Sumofmesiodistalwidthof6anteriormandibularteethSumofmesiodistalwidthof6anteriormaxillaryteeth×100OverallTSDBor=Sumofmesiodistalwidthof12mandibularteethSumofmesiodistalwidthof12maxillaryteeth×100\[\begin{array}{c}Anterior\,TSD\,{B_{ar}} = \frac{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}6{\rm{ }}anterior{\rm{ }}mandibular{\rm{ }}teeth{\rm{ }}}}{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}6{\rm{ }}anterior{\rm{ }}\max illary{\rm{ }}teeth{\rm{ }}}} \times 100\\ Overall\,TSD\,{B_{or}} = \frac{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}12{\rm{ }}mandibular{\rm{ }}teeth{\rm{ }}}}{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}12{\rm{ }}\max illary{\rm{ }}teeth{\rm{ }}}} \times 100\end{array}\]TSD was classified according to Bolton17 as Anterior TSD: Bar < 75.55 and Bar > 78.85 were considered as maxillary and mandibular tooth-size anterior excess, respectively. Bar ranging from 75.55 to 78.85 was classified as an absence of anterior TSD.Overall TSD: Bor < 89.39 and Bor > 93.21 were considered as maxillary or mandibular overall tooth-size excess, respectively. Bor ranging from 89.39 to 93.21 was classified as an absence of overall TSD.DNA was extracted from saliva according to the protocol previously published by Küchler et al.18 and was used for the molecular analysis. SNPs in RUNX2, SMAD6, BMP2, and BMP4 genes that were previously associated with permanent tooth size14 were selected for genotyping analysis. The SNP characteristics are shown in Table I. Probes (Applied Biosystems, Foster City, CA, USA) were used for genotyping in a real-time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA) according to the Taqman method.19 All reactions were performed blindly and 10% of the sample was genotyped twice to test for reproducibility, which was 100%.Table I.Characteristics of SNPs.GenesSNPsType of AlterationBase ChangeGlobal MAF*Genotyping success rateRUNX2rs59983488Upstream VariantG > T0.1588.7%Rs1200425Intron VariantG > A0.4487.0%SMAD6rs3934908Intron VariantC > T0.4191.9%Rs211261Intron VariantC > T0.1990.3%BMP2rs1005464Intron VariantG > A0.1991.9%rs235768Missense VariantT > A0.3290.3%BMP4Rs17563Missense VariantA > G0.4288.7%A, adenine; C, cytosine; G, guanine; MAF, Minor Allele Frequency; T, thymine.Data are available in https://www.ncbi.nlm.nih.gov/snp.Statistical analysisThe Hardy–Weinberg equilibrium of each SNP was evaluated by the chi-square test. Genotype and allele distributions were compared using Fisher’s exact test. Odds Ratios (OR) and 95% Confidence Intervals (95% CI) were also calculated. A Bonferroni adjustment was applied for the total number of SNPs (0.05/7 = 0.007). IBM SPSS Version 25.0 (IBM Corp, Armonk, USA) software was used for all analyses.ResultsThe number of excluded patients from the study and associated reasons are shown in Figure 1. Sixty-two patients participated, 33 females (53.2%) and 29 (46.8%) males. The mean age of the patients was 15.65 years (Standard Deviation = 6.82 years).Figure 1.Patient flow of the study.The genotyping success rate of each SNP is presented in Table I. The Hardy–Weinberg equilibrium (HWE) was assessed by a chi-square test (clinicalc.com), and all SNPs were within the HWE (p>0.05). This indicated that allele frequencies in this population did not change from generation to generation.Table II demonstrates the allele and genotype distribution between the groups for the overall TSD analysis. The rs59983488 SNP in the RUNX2 gene was significantly associated with the presence of an anterior mandibular tooth-size excess in allele (T allele: p<0.001; OR = 11.74; 95% CI =2.61–55.80), and genotype models (GT genotype: p = 0.002; OR = 12.69; 95% CI = 2.47–64.83).Table II.Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size anterior excessControl vs. Mandibular tooth-size anterior excessGeneSNPComparison TypeAlleles or GenotypesControl (%)Maxillary (%)p-valueOR95% CIMandibular (%)p-valueOdds Ratio95% Confidence IntervalRUNX2rs59983488AllelicG60 (96.8)14 (87.5)Ref.23 (71.9)Ref.T2 (3.2)2 (12.5)0.1844.280.61–28.449 (28.1)<0.001**' 11.742.61–55.80GenotypicGG29 (93.5)6 (75.0)Ref.8 (50)Ref.GT2 (6.5)2 (25.0)0.1804.830.62–33.707 (43.8)0.002**12.692.47–64.83TT0 (0.0)0 (0.0)>0.999--1 (6.3)0.236--rs1200425AllelicG37 (63.8)13 (81.3)Ref.21 (61.8)Ref.A21 (36.2)3 (18.7)0.2370.400.11–1.5613 (38.2)>0.9991.090.47–2.64GenotypicGG14 (48.3)6 (75.0)Ref.6 (35.3)Ref.GA9 (31.0)1 (12.5)0.3710.250.02–2.479 (52.9)0.3202.330.61–7.90AA6 (20.7)1 (12.5)0.6330.380.02–2.872 (11.8)0.2684.660.44–71.52SMAD6rs3934908AllelicC33 (53.2)7 (43.7)Ref.19 (52.8)Ref.T29 (46.8)9 (56.3)0.5801.460.47–4.1217 (47.2)>0.9991.010.45–2.25GenotypicCC9 (29.0)2 (25.0)Ref.4 (22.2)Ref.CT15 (48.4)3 (37.5)>0.9990.900.15–5.8411 (61.1)0.7281.650.38–5.76TT7 (22.6)3 (37.5)0.6351.920.31–12.803 (16.7)0.1340.250.06–1.12rs211261AllelicC29 (55.8)10 (62.5)Ref.19 (55.9)Ref.T23 (44.2)6 (37.5)0.7740.750.22–2.4215 (44.1)>0.9990.990.39–2.41GenotypicCC10 (32.3)2 (25.0)Ref.3 (17.6)Ref.CT19 (61.3)6 (75.0)>0.9991.570.27–8.7113 (76.5)0.3222.280.53–8.75TT2 (6.5)0 (0.0)>0.999--1 (5.9)>0.9991.660.08–18.07BMP2rs1005464AllelicG5213Ref.29Ref.A1030.7231.200.31–4.595>0.9990.890.31–2.82GenotypicGG23 (74.2)6 (75.0)Ref.12 (70.6)Ref.GA6 (19.4)1 (12.5)>0.9990.630.04–4.545 (29.4)0.7211.590.41–5.53*p < 0.05. **p < 0.007.Table II.Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.GeneSNPComparison TypeAlleles or GenotypesControl (%)Control vs. Maxillary tooth-size anterior excessControl vs. Mandibular tooth-size anterior excessMaxillary (%)p-valueOR95% CIMandibular (%)p-valueOdds Ratio95% Confidence IntervalAA2 (6.5)1 (12.5)0.5361.910.11–18.280 (0.0)>0.999--rs235768AllelicT47 (75.8)11 (68.7)Ref.26 (72.2)Ref.A15 (24.2)5 (31.3)0.5391.420.46–4.8810 (27.8)0.8101.200.44–2.92GenotypicTT16 (51.6)4 (50.0)Ref.9 (50.0)Ref.TA15 (48.4)3 (37.5)>0.9990.800.17–3.418 (44.4)>0.9990.940.30–2.84AA0 (0.0)1 (12.5)0.23--1 (5.6)0.384--BMP4rs17563AllelicA39 (62.9)9 (64.3)Ref.21 (61.8)Ref.G23 (37.1)5 (35.7)>0.9990.940.31–3.1113 (38.2)>0.9991.050.46–2.47GenotypicAA13 (41.9)2 (28.6)Ref.7(41.2)Ref.AG13 (41.9)5 (71.4)0.4132.500.37–13.977(41.2)>0.9991.000.25–3.96GG5 (16.1)0 (0.0)>0.999--3 (17.6)>0.9991.110.23–5.39*p < 0.05. **p < 0.007.Table III demonstrates the allele and genotype distribution between the groups for the anterior Bolton analysis. The rs3934908 SNP in the SMAD6 gene was significantly associated with the presence of overall maxillary tooth-size excess in allele (T allele: p < 0.001) and genotype models (TT genotype: p = 0.010).Table III.Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size overall excessControl vs. Mandibular tooth-size overall excessGeneSNPModelAlleles or GenotypesControl (%)Maxillary (%)p-valueOdds ratio95% Confidence IntervalMandibular (%)p-valueOdds Ratio95% Confidence IntervalRUNX2rs59983488AllelicG56 (93.3)8 (80.0)Ref.33 (82.5)Ref.T4 (6.7)2 (20.0)0.2013.500.57–17.987 (17.5)0.1102.970.83–9.51GenotypicGG26 (86.7)3 (60.0)Ref.14 (70.0)Ref.GT4 (13.3)2 (40.0)0.1954.330.58–25.795 (25.0)0.2812.320.53–8.40TT0 (0.0)0 (0.0)>0.999--1 (5.0)0.365--rs1200425AllelicG35 (60.3)8 (80.0)Ref.28 (70.0)Ref.A23 (39.7)2 (20.0)0.3030.380.07–1.8812 (30.0)0.3930.650.28–1.49GenotypicGG14 (48.3)3 (60.0)Ref.9 (45.0)Ref.GA7 (24.1)2 (40.0)>0.9991.330.19 - 7.7310 (50.0)0.3372.220.64–7.51AA8 (27.6)0 (0.0)0.527--1 (5.0)0.2100.190.01–1.58SMAD6rs33408AllelicC38 (59.4)0 (0.0)Ref.21 (52.5)Ref.T26 (40.6)10 (100)<0.001**∞3.75 - ∞19 (47.5)0.5441.320.57–2.85GenotypicCC10 (31.3)0 (0.0)Ref.5 (25.0)Ref.CT18 (56.3)0 (0.0)>0.999--11 (55.0)>0.9991.220.34–4.33TT4 (12.4)5 (100)0.010*∞1.96-∞4 (20.0)0.6572.000.39–10.88rs211261AllelicC38 (61.3)6 (60.0)Ref.24 (60.0)Ref.T24 (38.7)4 (40.0)>0.9991.050.31–3.7216 (40.0)>0.9991.050.47–2.28GenotypicCC9 (29.0)1 (20.0)Ref.5 (25.0)Ref.CT20 (64.5)4 (80.0)>0.9991.800.22–24.2114 (70.0)>0.9991.260.36–4.31TT2 (6.5)0 (0.0)>0.999--1 (5.0)>0.9990.900.05–9.37BMP2rs1005464AllelicG50 (80.6)8 (80.0)Ref.36 (90.0)Ref.A12 (19.4)2 (20.0)>0.9991.040.20–5.724 (10.0)0.2690.460.15–1.44GenotypicGG22 (71.0)3 (60.0)Ref.16 (80.0)Ref.GA6 (19.3)2 (40.0)0.5732.440.35–13.914 (20.0)>0.9990.910.25–3.29Notes: *p < 0.05. **p < 0.007.Table III.Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size overall excessControl vs. Mandibular tooth-size overall excessGeneSNPModelAlleles or GenotypesControl (%)Maxillary (%)p-valueOdds ratio95% Confidence IntervalMandibular (%)p-valueOdds Ratio95% Confidence IntervalAA3 (9.7)0 (0.0)>0.999--0 (0.0)0.268--rs235768AllelicT48 (75.0)8 (80.0)Ref.28 (70.0)Ref.A16 (25.0)2 (20.0)>0.9990.750.14–3.8412 (30.0)0.6511.280.55–3.18GenotypicTT17 (53.1)3 (60.0)Ref.9 (45.0)Ref.TA14 (43.8)2 (40.0)>0.9990.800.13–4.4410 (50.0)0.7711.340.40–3.89AA1 (3.1)0 (0.0)>0.999--1 (5.0)>0.9991.880.09–37.71BMP4rs17563AllelicA34 (55.7)6 (60.0)Ref.29 (72.5)Ref.G26 (43.3)4 (40.0)>0.99911 (27.5)0.1390.490.21–1.13GenotypicAA10 (33.3)2 (40.0)Ref.10 (50.0)Ref.AG14 (46.7)2 (40.0)>0.9990.710.10–5.219 (45.0)0.5470.640.19–1.99GG6 (20.0)1 (20.0)>0.9990.830.05–8.471 (5.0)0.1830.160.01–1.64Notes: *p < 0.05. **p < 0.007.DiscussionPredictions of skeletal and tooth patterns is a challenging aspect of orthodontic practice.20 Knowledge regarding the genes involved in tooth morphology that could be used to predict tooth-size excess in each patient will assist an individual orthodontic treatment plan and prognosis as well as in the screening of patients. In the present study, the association between SNPs in RUNX2 and SMAD6 genes and TSD, is identified for the first time which may, in the future, be used in orthodontic finishing strategies to optimise TSD-dependent treatment outcomes.21RUNX2 was selected in the study due to its important role in tooth development and, potentially, in final tooth morphology.8 Besides regulating odontoblast differentiation, RUNX2 also plays a role in the later stages of odontogenesis, mainly before crown formation. RUNX2 mRNA is strongly expressed in immature and mature ameloblasts and is also known to regulate the transcription of the Amelobastin (Ambn) gene, one of the major proteins of enamel matrix.8 SNPs in RUNX2 have been previously associated with tooth development and size.14 In the present study, the recessive allele (T) of rs59983488 was associated with the presence of anterior mandibular tooth-size excess. This SNP is located in a binding site of an intronic region of the RUNX2 gene and has already been associated with cleidocranial dysplasia,22 and also with tooth-size variability.14 The exact functional effect of this SNP on Runx2 is still unknown; however, Napierala et al.22 showed that the recessive allele prevents binding between the RUNX2 gene and the Myeloid Zinc-Finger transcription factor 1 (MZF1). MZF1 is a factor that acts as a transcriptional repressor in stem cells, which may subsequently modify RUNX2 expression and affect tooth development.SMAD6 is characterised by an inhibitory SMAD protein that restricts the cellular response to BMPs and transforming growth factor b (TGFb ).6,23 This mediator is present in all tooth-formation stages, but mainly at the initiation stage of tooth development.12 Therefore, it is hypothesised that SNPs in SMAD6 are involved in tooth morphogenesis. In addition, SNPs in SMAD6 have already been associated with other craniofacial traits, as craniosynostosis,24 skeletal patterns,25 palatal rugae,26 and tooth agenesis.27 Of note, some of these factors have been associated with tooth size variation,14 suggesting that these different phenotypes share a common genetic background. The present results suggest an association between rs3934908 in SMAD6 and overall maxillary tooth-size excess. Although Gerber et al.14 did not find an association between this SNP and mesiodistal and buccal-lingual tooth sizes, it is important to consider that previous studies focused on the evaluation of TSD/tooth excess and therefore differed from the present study and possibly explains this divergence of results. Further research is indicated to clarify the exact role of SMAD6 in odontogenesis and tooth-size determination.The findings of the present study indicated that rs59983488 in RUNX2 was associated with anterior tooth-size excess, but not with overall tooth-size excess. Similarly, the rs3934908 in SMAD6 was associated with overall tooth-size excess, but not with anterior tooth-size excess. Furthermore, the SNPs were associated with tooth-size excess in only one of the arches, and not in both arches. Previous studies have indicated that the morphogenesis of each type of tooth is regulated by different growth factors and different sets of genes.6,8,12 Therefore, it is reasonable to suggest that SNPs in growth factor decoder genes can influence the tooth size of only one type, one arch or one group of teeth, and impact the occlusal relationship,13–15 depending on the group of genotypes and alleles that the patient carries. The investigated SNPs in BMP2 and BMP4 were not associated with tooth-size excess in the present study. Gerber et al.14 evaluated tooth size in a Brazilian sample and showed that the same SNPs in BMP2 and BMP4 were associated with size variability of some groups of permanent teeth and so these genes were selected for examination. However, the present results did not demonstrate that the SNPs in BMP2 and BMP4 were involved in TSD. More studies are indicated with larger sample sizes in other populations to analyse these and other SNPs to confirm the findings.The method of assessing tooth-size discrepancies proposed by Bolton17 was adopted for the current study due to its accuracy and long-standing acceptance by the dental profession.4 It is possible that conventional plaster casts may not accurately reproduce exact tooth size, which is a likely limitation of this study. Nevertheless, carefully prepared impressions and plaster casts should have negligible size errors. The absence of positive associations for other analysed SNPs may also be due to the small sample size. However, the present study contributes to the identification of some genes and SNPs that may affect tooth development and impact directly on the occlusal relationship of the individual, and therefore provides valuable information for future studies.ConclusionsThe present results suggest that SNPs in RUNX2 and SMAD6 may be associated with the presence of tooth-size excess. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Australasian Orthodontic Journal de Gruyter

Single nucleotide polymorphisms in odontogenesis-related genes associated with tooth-size discrepancy

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References (23)

Publisher
de Gruyter
Copyright
© 2023 Caio Luiz Bitencourt Reis et al., published by Sciendo
eISSN
2207-7480
DOI
10.2478/aoj-2023-0008
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Abstract

IntroductionA tooth-size discrepancy (TSD) is characterised by a dimensional disproportion in the mesiodistal size between the maxillary and mandibular teeth1,2. A TSD contributes to an altered relationship between the dental arches because accepted overbite, overjet and interdigitation require a certain proportional size relationship between the teeth.3,4 Successful orthodontic treatment with regard to a functional and aesthetic occlusion also, in part, depends on an accurate TSD diagnosis and intervention.1,2Tooth size is determined at the commencement of the dental development stage.5 Odontogenesis involves a complex mechanism of interaction between signalling networks and growth factors.6–8 Bone morphogenetic protein 4 (BMP4) is an important growth factor inducing tooth development as it stimulates the differentiation of mesenchymal-derived dental-specific cells from the beginning of tooth formation.6,9 BMP4 also regulates Runt-related transcription factor 2 (RUNX2), which is a critical transcriptional regulator of tooth formation.7,10 RUNX2 controls the morpho-differentiation and growth of the embryonic epithelium of the enamel organ which precedes the formation of tooth enamel.8 In addition, RUNX2 stimulates BMP2 production which is a key protein involved in odontogenic differentiation and the control of enamel mineralisation.11 Additionally, SMAD proteins are important mediators affecting this network of signaling pathways.8,12Single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide in the genome differs between members or paired chromosomes of an individual. Recent studies have investigated SNPs in odontogenesis-related genes and identified some as possible relevant factors related to tooth-size variability in humans.13–15 Therefore, SNPs associated with growth factors that play an important role in odontogenesis could be a risk factor for TSD. Therefore, the current study aimed to investigate the association between SNPs in BMP4, BMP2, RUNX2 and SMAD6 genes and TSD.MethodsThe present cross-sectional study was approved by the Local Research Ethics Committee (protocol: 01451418.3.0000.5419/3.150.551) and all included patients and their legal guardians signed written informed consent according to the Declaration of Helsinki. The STREGA (STrengthening the REporting of Genetic Association Studies) checklist was used to design and report this study.16The study included pretreatment dental casts and genomic DNA samples from self-reported white and biologically unrelated patients undergoing orthodontic treatment at School of Dentistry of Ribeirão Preto, University of São Paulo from 2016 to 2018. Patients with a previous history of orthodontic treatment, congenital syndromes, tooth anomalies, interproximal caries, restorations or enamel reduction, occlusal dental wear, fractured or poor-quality dental casts, and extreme tooth misalignment, were excluded. The sample was previously described by Marañón-Vásquez et al.13 Sixty-two patients were included in the current study.A single dentist evaluated ten random dental casts twice within a two-week period to conduct an intra-examiner reliability test. The intraclass correlation coefficient (ICC) estimated a high reproducibility for all teeth (ICC ranging from 0.888 to 0.996). Mesiodistal tooth width was obtained by the largest crown distance between the lateral contact point parallel to the occlusal plane (maximum side-distal distance). A digital calliper (Digimatic CD-15DCX; Mitutoyo, Kawasaki, Japan) was used for measurements. All teeth of each maxillary and mandibular cast were consecutively measured three times and, when the difference between the measurements was more than 0.2 mm, the tooth was remeasured.TSD was assessed using the Bolton analysis.17 Bolton ratios were calculated according to the preexisting formula for the establishment of an anterior discrepancy ratio and overall discrepancy ratio, as follows:AnteriorTSDBar=Sumofmesiodistalwidthof6anteriormandibularteethSumofmesiodistalwidthof6anteriormaxillaryteeth×100OverallTSDBor=Sumofmesiodistalwidthof12mandibularteethSumofmesiodistalwidthof12maxillaryteeth×100\[\begin{array}{c}Anterior\,TSD\,{B_{ar}} = \frac{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}6{\rm{ }}anterior{\rm{ }}mandibular{\rm{ }}teeth{\rm{ }}}}{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}6{\rm{ }}anterior{\rm{ }}\max illary{\rm{ }}teeth{\rm{ }}}} \times 100\\ Overall\,TSD\,{B_{or}} = \frac{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}12{\rm{ }}mandibular{\rm{ }}teeth{\rm{ }}}}{{{\rm{ }}Sum{\rm{ }}of{\rm{ }}mesiodistal{\rm{ }}width{\rm{ }}of{\rm{ }}12{\rm{ }}\max illary{\rm{ }}teeth{\rm{ }}}} \times 100\end{array}\]TSD was classified according to Bolton17 as Anterior TSD: Bar < 75.55 and Bar > 78.85 were considered as maxillary and mandibular tooth-size anterior excess, respectively. Bar ranging from 75.55 to 78.85 was classified as an absence of anterior TSD.Overall TSD: Bor < 89.39 and Bor > 93.21 were considered as maxillary or mandibular overall tooth-size excess, respectively. Bor ranging from 89.39 to 93.21 was classified as an absence of overall TSD.DNA was extracted from saliva according to the protocol previously published by Küchler et al.18 and was used for the molecular analysis. SNPs in RUNX2, SMAD6, BMP2, and BMP4 genes that were previously associated with permanent tooth size14 were selected for genotyping analysis. The SNP characteristics are shown in Table I. Probes (Applied Biosystems, Foster City, CA, USA) were used for genotyping in a real-time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA) according to the Taqman method.19 All reactions were performed blindly and 10% of the sample was genotyped twice to test for reproducibility, which was 100%.Table I.Characteristics of SNPs.GenesSNPsType of AlterationBase ChangeGlobal MAF*Genotyping success rateRUNX2rs59983488Upstream VariantG > T0.1588.7%Rs1200425Intron VariantG > A0.4487.0%SMAD6rs3934908Intron VariantC > T0.4191.9%Rs211261Intron VariantC > T0.1990.3%BMP2rs1005464Intron VariantG > A0.1991.9%rs235768Missense VariantT > A0.3290.3%BMP4Rs17563Missense VariantA > G0.4288.7%A, adenine; C, cytosine; G, guanine; MAF, Minor Allele Frequency; T, thymine.Data are available in https://www.ncbi.nlm.nih.gov/snp.Statistical analysisThe Hardy–Weinberg equilibrium of each SNP was evaluated by the chi-square test. Genotype and allele distributions were compared using Fisher’s exact test. Odds Ratios (OR) and 95% Confidence Intervals (95% CI) were also calculated. A Bonferroni adjustment was applied for the total number of SNPs (0.05/7 = 0.007). IBM SPSS Version 25.0 (IBM Corp, Armonk, USA) software was used for all analyses.ResultsThe number of excluded patients from the study and associated reasons are shown in Figure 1. Sixty-two patients participated, 33 females (53.2%) and 29 (46.8%) males. The mean age of the patients was 15.65 years (Standard Deviation = 6.82 years).Figure 1.Patient flow of the study.The genotyping success rate of each SNP is presented in Table I. The Hardy–Weinberg equilibrium (HWE) was assessed by a chi-square test (clinicalc.com), and all SNPs were within the HWE (p>0.05). This indicated that allele frequencies in this population did not change from generation to generation.Table II demonstrates the allele and genotype distribution between the groups for the overall TSD analysis. The rs59983488 SNP in the RUNX2 gene was significantly associated with the presence of an anterior mandibular tooth-size excess in allele (T allele: p<0.001; OR = 11.74; 95% CI =2.61–55.80), and genotype models (GT genotype: p = 0.002; OR = 12.69; 95% CI = 2.47–64.83).Table II.Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size anterior excessControl vs. Mandibular tooth-size anterior excessGeneSNPComparison TypeAlleles or GenotypesControl (%)Maxillary (%)p-valueOR95% CIMandibular (%)p-valueOdds Ratio95% Confidence IntervalRUNX2rs59983488AllelicG60 (96.8)14 (87.5)Ref.23 (71.9)Ref.T2 (3.2)2 (12.5)0.1844.280.61–28.449 (28.1)<0.001**' 11.742.61–55.80GenotypicGG29 (93.5)6 (75.0)Ref.8 (50)Ref.GT2 (6.5)2 (25.0)0.1804.830.62–33.707 (43.8)0.002**12.692.47–64.83TT0 (0.0)0 (0.0)>0.999--1 (6.3)0.236--rs1200425AllelicG37 (63.8)13 (81.3)Ref.21 (61.8)Ref.A21 (36.2)3 (18.7)0.2370.400.11–1.5613 (38.2)>0.9991.090.47–2.64GenotypicGG14 (48.3)6 (75.0)Ref.6 (35.3)Ref.GA9 (31.0)1 (12.5)0.3710.250.02–2.479 (52.9)0.3202.330.61–7.90AA6 (20.7)1 (12.5)0.6330.380.02–2.872 (11.8)0.2684.660.44–71.52SMAD6rs3934908AllelicC33 (53.2)7 (43.7)Ref.19 (52.8)Ref.T29 (46.8)9 (56.3)0.5801.460.47–4.1217 (47.2)>0.9991.010.45–2.25GenotypicCC9 (29.0)2 (25.0)Ref.4 (22.2)Ref.CT15 (48.4)3 (37.5)>0.9990.900.15–5.8411 (61.1)0.7281.650.38–5.76TT7 (22.6)3 (37.5)0.6351.920.31–12.803 (16.7)0.1340.250.06–1.12rs211261AllelicC29 (55.8)10 (62.5)Ref.19 (55.9)Ref.T23 (44.2)6 (37.5)0.7740.750.22–2.4215 (44.1)>0.9990.990.39–2.41GenotypicCC10 (32.3)2 (25.0)Ref.3 (17.6)Ref.CT19 (61.3)6 (75.0)>0.9991.570.27–8.7113 (76.5)0.3222.280.53–8.75TT2 (6.5)0 (0.0)>0.999--1 (5.9)>0.9991.660.08–18.07BMP2rs1005464AllelicG5213Ref.29Ref.A1030.7231.200.31–4.595>0.9990.890.31–2.82GenotypicGG23 (74.2)6 (75.0)Ref.12 (70.6)Ref.GA6 (19.4)1 (12.5)>0.9990.630.04–4.545 (29.4)0.7211.590.41–5.53*p < 0.05. **p < 0.007.Table II.Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.GeneSNPComparison TypeAlleles or GenotypesControl (%)Control vs. Maxillary tooth-size anterior excessControl vs. Mandibular tooth-size anterior excessMaxillary (%)p-valueOR95% CIMandibular (%)p-valueOdds Ratio95% Confidence IntervalAA2 (6.5)1 (12.5)0.5361.910.11–18.280 (0.0)>0.999--rs235768AllelicT47 (75.8)11 (68.7)Ref.26 (72.2)Ref.A15 (24.2)5 (31.3)0.5391.420.46–4.8810 (27.8)0.8101.200.44–2.92GenotypicTT16 (51.6)4 (50.0)Ref.9 (50.0)Ref.TA15 (48.4)3 (37.5)>0.9990.800.17–3.418 (44.4)>0.9990.940.30–2.84AA0 (0.0)1 (12.5)0.23--1 (5.6)0.384--BMP4rs17563AllelicA39 (62.9)9 (64.3)Ref.21 (61.8)Ref.G23 (37.1)5 (35.7)>0.9990.940.31–3.1113 (38.2)>0.9991.050.46–2.47GenotypicAA13 (41.9)2 (28.6)Ref.7(41.2)Ref.AG13 (41.9)5 (71.4)0.4132.500.37–13.977(41.2)>0.9991.000.25–3.96GG5 (16.1)0 (0.0)>0.999--3 (17.6)>0.9991.110.23–5.39*p < 0.05. **p < 0.007.Table III demonstrates the allele and genotype distribution between the groups for the anterior Bolton analysis. The rs3934908 SNP in the SMAD6 gene was significantly associated with the presence of overall maxillary tooth-size excess in allele (T allele: p < 0.001) and genotype models (TT genotype: p = 0.010).Table III.Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size overall excessControl vs. Mandibular tooth-size overall excessGeneSNPModelAlleles or GenotypesControl (%)Maxillary (%)p-valueOdds ratio95% Confidence IntervalMandibular (%)p-valueOdds Ratio95% Confidence IntervalRUNX2rs59983488AllelicG56 (93.3)8 (80.0)Ref.33 (82.5)Ref.T4 (6.7)2 (20.0)0.2013.500.57–17.987 (17.5)0.1102.970.83–9.51GenotypicGG26 (86.7)3 (60.0)Ref.14 (70.0)Ref.GT4 (13.3)2 (40.0)0.1954.330.58–25.795 (25.0)0.2812.320.53–8.40TT0 (0.0)0 (0.0)>0.999--1 (5.0)0.365--rs1200425AllelicG35 (60.3)8 (80.0)Ref.28 (70.0)Ref.A23 (39.7)2 (20.0)0.3030.380.07–1.8812 (30.0)0.3930.650.28–1.49GenotypicGG14 (48.3)3 (60.0)Ref.9 (45.0)Ref.GA7 (24.1)2 (40.0)>0.9991.330.19 - 7.7310 (50.0)0.3372.220.64–7.51AA8 (27.6)0 (0.0)0.527--1 (5.0)0.2100.190.01–1.58SMAD6rs33408AllelicC38 (59.4)0 (0.0)Ref.21 (52.5)Ref.T26 (40.6)10 (100)<0.001**∞3.75 - ∞19 (47.5)0.5441.320.57–2.85GenotypicCC10 (31.3)0 (0.0)Ref.5 (25.0)Ref.CT18 (56.3)0 (0.0)>0.999--11 (55.0)>0.9991.220.34–4.33TT4 (12.4)5 (100)0.010*∞1.96-∞4 (20.0)0.6572.000.39–10.88rs211261AllelicC38 (61.3)6 (60.0)Ref.24 (60.0)Ref.T24 (38.7)4 (40.0)>0.9991.050.31–3.7216 (40.0)>0.9991.050.47–2.28GenotypicCC9 (29.0)1 (20.0)Ref.5 (25.0)Ref.CT20 (64.5)4 (80.0)>0.9991.800.22–24.2114 (70.0)>0.9991.260.36–4.31TT2 (6.5)0 (0.0)>0.999--1 (5.0)>0.9990.900.05–9.37BMP2rs1005464AllelicG50 (80.6)8 (80.0)Ref.36 (90.0)Ref.A12 (19.4)2 (20.0)>0.9991.040.20–5.724 (10.0)0.2690.460.15–1.44GenotypicGG22 (71.0)3 (60.0)Ref.16 (80.0)Ref.GA6 (19.3)2 (40.0)0.5732.440.35–13.914 (20.0)>0.9990.910.25–3.29Notes: *p < 0.05. **p < 0.007.Table III.Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.Control vs. Maxillary tooth-size overall excessControl vs. Mandibular tooth-size overall excessGeneSNPModelAlleles or GenotypesControl (%)Maxillary (%)p-valueOdds ratio95% Confidence IntervalMandibular (%)p-valueOdds Ratio95% Confidence IntervalAA3 (9.7)0 (0.0)>0.999--0 (0.0)0.268--rs235768AllelicT48 (75.0)8 (80.0)Ref.28 (70.0)Ref.A16 (25.0)2 (20.0)>0.9990.750.14–3.8412 (30.0)0.6511.280.55–3.18GenotypicTT17 (53.1)3 (60.0)Ref.9 (45.0)Ref.TA14 (43.8)2 (40.0)>0.9990.800.13–4.4410 (50.0)0.7711.340.40–3.89AA1 (3.1)0 (0.0)>0.999--1 (5.0)>0.9991.880.09–37.71BMP4rs17563AllelicA34 (55.7)6 (60.0)Ref.29 (72.5)Ref.G26 (43.3)4 (40.0)>0.99911 (27.5)0.1390.490.21–1.13GenotypicAA10 (33.3)2 (40.0)Ref.10 (50.0)Ref.AG14 (46.7)2 (40.0)>0.9990.710.10–5.219 (45.0)0.5470.640.19–1.99GG6 (20.0)1 (20.0)>0.9990.830.05–8.471 (5.0)0.1830.160.01–1.64Notes: *p < 0.05. **p < 0.007.DiscussionPredictions of skeletal and tooth patterns is a challenging aspect of orthodontic practice.20 Knowledge regarding the genes involved in tooth morphology that could be used to predict tooth-size excess in each patient will assist an individual orthodontic treatment plan and prognosis as well as in the screening of patients. In the present study, the association between SNPs in RUNX2 and SMAD6 genes and TSD, is identified for the first time which may, in the future, be used in orthodontic finishing strategies to optimise TSD-dependent treatment outcomes.21RUNX2 was selected in the study due to its important role in tooth development and, potentially, in final tooth morphology.8 Besides regulating odontoblast differentiation, RUNX2 also plays a role in the later stages of odontogenesis, mainly before crown formation. RUNX2 mRNA is strongly expressed in immature and mature ameloblasts and is also known to regulate the transcription of the Amelobastin (Ambn) gene, one of the major proteins of enamel matrix.8 SNPs in RUNX2 have been previously associated with tooth development and size.14 In the present study, the recessive allele (T) of rs59983488 was associated with the presence of anterior mandibular tooth-size excess. This SNP is located in a binding site of an intronic region of the RUNX2 gene and has already been associated with cleidocranial dysplasia,22 and also with tooth-size variability.14 The exact functional effect of this SNP on Runx2 is still unknown; however, Napierala et al.22 showed that the recessive allele prevents binding between the RUNX2 gene and the Myeloid Zinc-Finger transcription factor 1 (MZF1). MZF1 is a factor that acts as a transcriptional repressor in stem cells, which may subsequently modify RUNX2 expression and affect tooth development.SMAD6 is characterised by an inhibitory SMAD protein that restricts the cellular response to BMPs and transforming growth factor b (TGFb ).6,23 This mediator is present in all tooth-formation stages, but mainly at the initiation stage of tooth development.12 Therefore, it is hypothesised that SNPs in SMAD6 are involved in tooth morphogenesis. In addition, SNPs in SMAD6 have already been associated with other craniofacial traits, as craniosynostosis,24 skeletal patterns,25 palatal rugae,26 and tooth agenesis.27 Of note, some of these factors have been associated with tooth size variation,14 suggesting that these different phenotypes share a common genetic background. The present results suggest an association between rs3934908 in SMAD6 and overall maxillary tooth-size excess. Although Gerber et al.14 did not find an association between this SNP and mesiodistal and buccal-lingual tooth sizes, it is important to consider that previous studies focused on the evaluation of TSD/tooth excess and therefore differed from the present study and possibly explains this divergence of results. Further research is indicated to clarify the exact role of SMAD6 in odontogenesis and tooth-size determination.The findings of the present study indicated that rs59983488 in RUNX2 was associated with anterior tooth-size excess, but not with overall tooth-size excess. Similarly, the rs3934908 in SMAD6 was associated with overall tooth-size excess, but not with anterior tooth-size excess. Furthermore, the SNPs were associated with tooth-size excess in only one of the arches, and not in both arches. Previous studies have indicated that the morphogenesis of each type of tooth is regulated by different growth factors and different sets of genes.6,8,12 Therefore, it is reasonable to suggest that SNPs in growth factor decoder genes can influence the tooth size of only one type, one arch or one group of teeth, and impact the occlusal relationship,13–15 depending on the group of genotypes and alleles that the patient carries. The investigated SNPs in BMP2 and BMP4 were not associated with tooth-size excess in the present study. Gerber et al.14 evaluated tooth size in a Brazilian sample and showed that the same SNPs in BMP2 and BMP4 were associated with size variability of some groups of permanent teeth and so these genes were selected for examination. However, the present results did not demonstrate that the SNPs in BMP2 and BMP4 were involved in TSD. More studies are indicated with larger sample sizes in other populations to analyse these and other SNPs to confirm the findings.The method of assessing tooth-size discrepancies proposed by Bolton17 was adopted for the current study due to its accuracy and long-standing acceptance by the dental profession.4 It is possible that conventional plaster casts may not accurately reproduce exact tooth size, which is a likely limitation of this study. Nevertheless, carefully prepared impressions and plaster casts should have negligible size errors. The absence of positive associations for other analysed SNPs may also be due to the small sample size. However, the present study contributes to the identification of some genes and SNPs that may affect tooth development and impact directly on the occlusal relationship of the individual, and therefore provides valuable information for future studies.ConclusionsThe present results suggest that SNPs in RUNX2 and SMAD6 may be associated with the presence of tooth-size excess.

Journal

Australasian Orthodontic Journal – de Gruyter

Published: Jan 1, 2023

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