A 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.1,2
Tooth 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.8,12
Single 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
The 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.16
The 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:
TSD was classified according to Bolton17 as Anterior TSD:
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
Characteristics of SNPs.
Genes | SNPs | Type of Alteration | Base Change | Global MAF* | Genotyping success rate |
---|---|---|---|---|---|
RUNX2 | rs59983488 | Upstream Variant | G > T | 0.15 | 88.7% |
Rs1200425 | Intron Variant | G > A | 0.44 | 87.0% | |
SMAD6 | rs3934908 | Intron Variant | C > T | 0.41 | 91.9% |
Rs211261 | Intron Variant | C > T | 0.19 | 90.3% | |
BMP2 | rs1005464 | Intron Variant | G > A | 0.19 | 91.9% |
rs235768 | Missense Variant | T > A | 0.32 | 90.3% | |
BMP4 | Rs17563 | Missense Variant | A > G | 0.42 | 88.7% |
A, adenine; C, cytosine; G, guanine; MAF, Minor Allele Frequency; T, thymine.
Data are available in
The 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.
The 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).
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 (
Table II demonstrates the allele and genotype distribution between the groups for the overall TSD analysis. The rs59983488 SNP in the
Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.
Control vs. Maxillary tooth-size anterior excess | Control vs. Mandibular tooth-size anterior excess | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SNP | Comparison Type | Alleles or Genotypes | Control (%) | Maxillary (%) | OR | 95% CI | Mandibular (%) | Odds Ratio | 95% Confidence Interval | ||
rs59983488 | Allelic | G | 60 (96.8) | 14 (87.5) | Ref. | 23 (71.9) | Ref. | |||||
T | 2 (3.2) | 2 (12.5) | 0.184 | 4.28 | 0.61–28.44 | 9 (28.1) | <0.001** | ' 11.74 | 2.61–55.80 | |||
Genotypic | GG | 29 (93.5) | 6 (75.0) | Ref. | 8 (50) | Ref. | ||||||
GT | 2 (6.5) | 2 (25.0) | 0.180 | 4.83 | 0.62–33.70 | 7 (43.8) | 0.002** | 12.69 | 2.47–64.83 | |||
TT | 0 (0.0) | 0 (0.0) | >0.999 | - | - | 1 (6.3) | 0.236 | - | - | |||
rs1200425 | Allelic | G | 37 (63.8) | 13 (81.3) | Ref. | 21 (61.8) | Ref. | |||||
A | 21 (36.2) | 3 (18.7) | 0.237 | 0.40 | 0.11–1.56 | 13 (38.2) | >0.999 | 1.09 | 0.47–2.64 | |||
Genotypic | GG | 14 (48.3) | 6 (75.0) | Ref. | 6 (35.3) | Ref. | ||||||
GA | 9 (31.0) | 1 (12.5) | 0.371 | 0.25 | 0.02–2.47 | 9 (52.9) | 0.320 | 2.33 | 0.61–7.90 | |||
AA | 6 (20.7) | 1 (12.5) | 0.633 | 0.38 | 0.02–2.87 | 2 (11.8) | 0.268 | 4.66 | 0.44–71.52 | |||
rs3934908 | Allelic | C | 33 (53.2) | 7 (43.7) | Ref. | 19 (52.8) | Ref. | |||||
T | 29 (46.8) | 9 (56.3) | 0.580 | 1.46 | 0.47–4.12 | 17 (47.2) | >0.999 | 1.01 | 0.45–2.25 | |||
Genotypic | CC | 9 (29.0) | 2 (25.0) | Ref. | 4 (22.2) | Ref. | ||||||
CT | 15 (48.4) | 3 (37.5) | >0.999 | 0.90 | 0.15–5.84 | 11 (61.1) | 0.728 | 1.65 | 0.38–5.76 | |||
TT | 7 (22.6) | 3 (37.5) | 0.635 | 1.92 | 0.31–12.80 | 3 (16.7) | 0.134 | 0.25 | 0.06–1.12 | |||
rs211261 | Allelic | C | 29 (55.8) | 10 (62.5) | Ref. | 19 (55.9) | Ref. | |||||
T | 23 (44.2) | 6 (37.5) | 0.774 | 0.75 | 0.22–2.42 | 15 (44.1) | >0.999 | 0.99 | 0.39–2.41 | |||
Genotypic | CC | 10 (32.3) | 2 (25.0) | Ref. | 3 (17.6) | Ref. | ||||||
CT | 19 (61.3) | 6 (75.0) | >0.999 | 1.57 | 0.27–8.71 | 13 (76.5) | 0.322 | 2.28 | 0.53–8.75 | |||
TT | 2 (6.5) | 0 (0.0) | >0.999 | - | - | 1 (5.9) | >0.999 | 1.66 | 0.08–18.07 | |||
rs1005464 | Allelic | G | 52 | 13 | Ref. | 29 | Ref. | |||||
A | 10 | 3 | 0.723 | 1.20 | 0.31–4.59 | 5 | >0.999 | 0.89 | 0.31–2.82 | |||
Genotypic | GG | 23 (74.2) | 6 (75.0) | Ref. | 12 (70.6) | Ref. | ||||||
GA | 6 (19.4) | 1 (12.5) | >0.999 | 0.63 | 0.04–4.54 | 5 (29.4) | 0.721 | 1.59 | 0.41–5.53 |
Allelic and Genotypic distribution between anterior TSD groups and comparison by Fisher Test.
Gene | SNP | Comparison Type | Alleles or Genotypes | Control (%) | Control vs. Maxillary tooth-size anterior excess | Control vs. Mandibular tooth-size anterior excess | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Maxillary (%) | OR | 95% CI | Mandibular (%) | Odds Ratio | 95% Confidence Interval | |||||||
AA | 2 (6.5) | 1 (12.5) | 0.536 | 1.91 | 0.11–18.28 | 0 (0.0) | >0.999 | - | - | |||
rs235768 | Allelic | T | 47 (75.8) | 11 (68.7) | Ref. | 26 (72.2) | Ref. | |||||
A | 15 (24.2) | 5 (31.3) | 0.539 | 1.42 | 0.46–4.88 | 10 (27.8) | 0.810 | 1.20 | 0.44–2.92 | |||
Genotypic | TT | 16 (51.6) | 4 (50.0) | Ref. | 9 (50.0) | Ref. | ||||||
TA | 15 (48.4) | 3 (37.5) | >0.999 | 0.80 | 0.17–3.41 | 8 (44.4) | >0.999 | 0.94 | 0.30–2.84 | |||
AA | 0 (0.0) | 1 (12.5) | 0.23 | - | - | 1 (5.6) | 0.384 | - | - | |||
rs17563 | Allelic | A | 39 (62.9) | 9 (64.3) | Ref. | 21 (61.8) | Ref. | |||||
G | 23 (37.1) | 5 (35.7) | >0.999 | 0.94 | 0.31–3.11 | 13 (38.2) | >0.999 | 1.05 | 0.46–2.47 | |||
Genotypic | AA | 13 (41.9) | 2 (28.6) | Ref. | 7(41.2) | Ref. | ||||||
AG | 13 (41.9) | 5 (71.4) | 0.413 | 2.50 | 0.37–13.97 | 7(41.2) | >0.999 | 1.00 | 0.25–3.96 | |||
GG | 5 (16.1) | 0 (0.0) | >0.999 | - | - | 3 (17.6) | >0.999 | 1.11 | 0.23–5.39 |
Table III demonstrates the allele and genotype distribution between the groups for the anterior Bolton analysis. The rs3934908 SNP in the
Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.
Control vs. Maxillary tooth-size overall excess | Control vs. Mandibular tooth-size overall excess | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SNP | Model | Alleles or Genotypes | Control (%) | Maxillary (%) | Odds ratio | 95% Confidence Interval | Mandibular (%) | Odds Ratio | 95% Confidence Interval | ||
rs59983488 | Allelic | G | 56 (93.3) | 8 (80.0) | Ref. | 33 (82.5) | Ref. | |||||
T | 4 (6.7) | 2 (20.0) | 0.201 | 3.50 | 0.57–17.98 | 7 (17.5) | 0.110 | 2.97 | 0.83–9.51 | |||
Genotypic | GG | 26 (86.7) | 3 (60.0) | Ref. | 14 (70.0) | Ref. | ||||||
GT | 4 (13.3) | 2 (40.0) | 0.195 | 4.33 | 0.58–25.79 | 5 (25.0) | 0.281 | 2.32 | 0.53–8.40 | |||
TT | 0 (0.0) | 0 (0.0) | >0.999 | - | - | 1 (5.0) | 0.365 | - | - | |||
rs1200425 | Allelic | G | 35 (60.3) | 8 (80.0) | Ref. | 28 (70.0) | Ref. | |||||
A | 23 (39.7) | 2 (20.0) | 0.303 | 0.38 | 0.07–1.88 | 12 (30.0) | 0.393 | 0.65 | 0.28–1.49 | |||
Genotypic | GG | 14 (48.3) | 3 (60.0) | Ref. | 9 (45.0) | Ref. | ||||||
GA | 7 (24.1) | 2 (40.0) | >0.999 | 1.33 | 0.19 - 7.73 | 10 (50.0) | 0.337 | 2.22 | 0.64–7.51 | |||
AA | 8 (27.6) | 0 (0.0) | 0.527 | - | - | 1 (5.0) | 0.210 | 0.19 | 0.01–1.58 | |||
rs33408 | Allelic | C | 38 (59.4) | 0 (0.0) | Ref. | 21 (52.5) | Ref. | |||||
T | 26 (40.6) | 10 (100) | <0.001** | ∞ | 3.75 - ∞ | 19 (47.5) | 0.544 | 1.32 | 0.57–2.85 | |||
Genotypic | CC | 10 (31.3) | 0 (0.0) | Ref. | 5 (25.0) | Ref. | ||||||
CT | 18 (56.3) | 0 (0.0) | >0.999 | - | - | 11 (55.0) | >0.999 | 1.22 | 0.34–4.33 | |||
TT | 4 (12.4) | 5 (100) | 0.010* | ∞ | 1.96-∞ | 4 (20.0) | 0.657 | 2.00 | 0.39–10.88 | |||
rs211261 | Allelic | C | 38 (61.3) | 6 (60.0) | Ref. | 24 (60.0) | Ref. | |||||
T | 24 (38.7) | 4 (40.0) | >0.999 | 1.05 | 0.31–3.72 | 16 (40.0) | >0.999 | 1.05 | 0.47–2.28 | |||
Genotypic | CC | 9 (29.0) | 1 (20.0) | Ref. | 5 (25.0) | Ref. | ||||||
CT | 20 (64.5) | 4 (80.0) | >0.999 | 1.80 | 0.22–24.21 | 14 (70.0) | >0.999 | 1.26 | 0.36–4.31 | |||
TT | 2 (6.5) | 0 (0.0) | >0.999 | - | - | 1 (5.0) | >0.999 | 0.90 | 0.05–9.37 | |||
rs1005464 | Allelic | G | 50 (80.6) | 8 (80.0) | Ref. | 36 (90.0) | Ref. | |||||
A | 12 (19.4) | 2 (20.0) | >0.999 | 1.04 | 0.20–5.72 | 4 (10.0) | 0.269 | 0.46 | 0.15–1.44 | |||
Genotypic | GG | 22 (71.0) | 3 (60.0) | Ref. | 16 (80.0) | Ref. | ||||||
GA | 6 (19.3) | 2 (40.0) | 0.573 | 2.44 | 0.35–13.91 | 4 (20.0) | >0.999 | 0.91 | 0.25–3.29 |
Notes: *p < 0.05.
Allelic and Genotypic distribution between overall TSD groups and comparison by Fisher Test.
Control vs. Maxillary tooth-size overall excess | Control vs. Mandibular tooth-size overall excess | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SNP | Model | Alleles or Genotypes | Control (%) | Maxillary (%) | Odds ratio | 95% Confidence Interval | Mandibular (%) | Odds Ratio | 95% Confidence Interval | ||
AA | 3 (9.7) | 0 (0.0) | >0.999 | - | - | 0 (0.0) | 0.268 | - | - | |||
rs235768 | Allelic | T | 48 (75.0) | 8 (80.0) | Ref. | 28 (70.0) | Ref. | |||||
A | 16 (25.0) | 2 (20.0) | >0.999 | 0.75 | 0.14–3.84 | 12 (30.0) | 0.651 | 1.28 | 0.55–3.18 | |||
Genotypic | TT | 17 (53.1) | 3 (60.0) | Ref. | 9 (45.0) | Ref. | ||||||
TA | 14 (43.8) | 2 (40.0) | >0.999 | 0.80 | 0.13–4.44 | 10 (50.0) | 0.771 | 1.34 | 0.40–3.89 | |||
AA | 1 (3.1) | 0 (0.0) | >0.999 | - | - | 1 (5.0) | >0.999 | 1.88 | 0.09–37.71 | |||
rs17563 | Allelic | A | 34 (55.7) | 6 (60.0) | Ref. | 29 (72.5) | Ref. | |||||
G | 26 (43.3) | 4 (40.0) | >0.999 | 11 (27.5) | 0.139 | 0.49 | 0.21–1.13 | |||||
Genotypic | AA | 10 (33.3) | 2 (40.0) | Ref. | 10 (50.0) | Ref. | ||||||
AG | 14 (46.7) | 2 (40.0) | >0.999 | 0.71 | 0.10–5.21 | 9 (45.0) | 0.547 | 0.64 | 0.19–1.99 | |||
GG | 6 (20.0) | 1 (20.0) | >0.999 | 0.83 | 0.05–8.47 | 1 (5.0) | 0.183 | 0.16 | 0.01–1.64 |
Notes: *p < 0.05.
Predictions 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
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
The findings of the present study indicated that rs59983488 in
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.
The present results suggest that SNPs in