ABSTRACT

Objectives

To explore whether variations in odontogenesis-related genes are associated with tooth-size discrepancies.

Materials and Methods

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.

Results

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).

Conclusions

The results suggest that FGF10 and FGF13 may contribute to the presence of tooth-size discrepancies.

INTRODUCTION

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.36  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.912 

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.1420  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.

Phenotyping

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.

Overall (Bor) and anterior (Bar) Bolton ratios were calculated according to the following formulas1 :
formula
formula

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).

Genotyping

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%.

Table 1.

Studied Single Nucleotide Polymorphismsa

Studied Single Nucleotide Polymorphismsa
Studied Single Nucleotide Polymorphismsa

Statistical Analysis

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).

RESULTS

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).

Table 2.

Genotype and Allele Frequencies According to the Presence of TSD (Bor)a

Genotype and Allele Frequencies According to the Presence of TSD (Bor)a
Genotype and Allele Frequencies According to the Presence of TSD (Bor)a
Table 3.

Genotype and Allele Frequencies According to the Presence of TSD (Bar)a

Genotype and Allele Frequencies According to the Presence of TSD (Bar)a
Genotype and Allele Frequencies According to the Presence of TSD (Bar)a

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).

Table 4.

Genotype Frequencies for the FGF13rs5931572 According to the Presence of TSD (Bor and Bar)a

Genotype Frequencies for the FGF13rs5931572 According to the Presence of TSD (Bor and Bar)a
Genotype Frequencies for the FGF13rs5931572 According to the Presence of TSD (Bor and Bar)a

DISCUSSION

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 patterns2630  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.8,3237 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.

CONCLUSION

  • FGF10 and FGF13 may contribute to the presence of tooth-size discrepancy.

REFERENCES

1. 
Bolton
WA.
Disharmony in tooth size and its relation to the analysis and treatment of malocclusion
.
Angle Orthod
.
1958
;
28
:
113
130
.
2. 
Baydas
B,
Oktay
H,
Dagsuyu
IM.
The effect of heritability on Bolton tooth-size discrepancy
.
Eur J Orthod
.
2005
;
27
:
98
102
.
3. 
Osborne
RH,
Horowitz
SL,
De George
FV.
Genetic variation in tooth dimensions: a twin study of the permanent anterior teeth
.
Am J Hum Genet
.
1958
;
10
:
350
356
.
4. 
Townsend
GC,
Brown
T.
Heritability of permanent tooth size
.
Am J Phys Anthropol
.
1978
;
49
:
497
504
.
5. 
Dempsey
PJ,
Townend
GC.
Genetic and environmental contributions to variation in human tooth size
.
Heredity (Edinb)
.
2001
;
86
:
685
693
.
6. 
Kabban
M,
Fearne
J,
Jovanovski
V,
Zou
L.
Tooth size and morphology in twins
.
Int J Paediatr Dent
.
2001
;
11
:
333
339
.
7. 
Thesleff
I.
The genetic basis of tooth development and dental defects
.
Am J Med Genet A
.
2006
;
140
:
2530
2535
.
8. 
Bei
M.
Molecular genetics of tooth development
.
Curr Opin Genet Dev
.
2009
;
19
:
504
510
.
9. 
Küchler
EC,
Lips
A,
Tannure
PN,
et al.
Tooth agenesis association with self-reported family history of cancer
.
J Dent Res
.
2013
;
92
:
149
155
.
10. 
Vieira
AR,
D'Souza
RN,
Mues
G,
et al.
Candidate gene studies in hypodontia suggest role for FGF3
.
Eur Arch Paediatr Dent
.
2013
;
14
:
405
410
.
11. 
Vieira
AR,
Meira
R,
Modesto
A,
Murray
JC.
MSX1, PAX9, and TGFA contribute to tooth agenesis in humans
.
J Dent Res
.
2004
;
83
:
723
727
.
12. 
Liu
H,
Han
D,
Wong
S,
Nan
X,
Zhao
H,
Feng H. rs929387 of GLI3 is involved in tooth agenesis in Chinese Han population
.
PLoS One
.
2013
;
8
:
e80860
.
13. 
Brook
AH.
A unifying aetiological explanation for anomalies of human tooth number and size
.
Arch Oral Biol
.
1984
;
19
:
373
378
.
14. 
Schalk-van der Weide
Y,
Steen
WH,
Beemer
FA,
Bosman
F.
Reductions in size and left-right asymmetry of teeth in human oligodontia
.
Arch Oral Biol
.
1994
;
39
:
935
939
.
15. 
Brook
AH,
Elcock
C,
al-Sharood
MH,
McKeown
HF,
Khalaf
K,
Smith
RN.
Further studies of a model for the etiology of anomalies of tooth number and size in humans
.
Connect Tissue Res
.
2002
;
43
:
289
295
.
16. 
Khalaf
K,
Robinson
DL,
Elcock
C,
Smith
RN,
Brook
AH.
Tooth size in patients with supernumerary teeth and a control group measured by image analysis system
.
Arch Oral Biol
.
2005
;
50
:
243
248
.
17. 
Khalaf
K,
Smith
RN,
Elcock
C,
Brook
AH.
Multiple crown size variables of the upper incisors in patients with supernumerary teeth compared with controls
.
Arch Oral Biol
.
2009
;
54
:
S71
S78
.
18. 
Brook
AH,
Griffin
RC,
Smith
RN,
et al.
Tooth size patterns in patients with hypodontia and supernumerary teeth
.
Arch Oral Biol
.
2009
;
54
:
S63
S70
.
19. 
Gungor
AY,
Turkkahraman
H.
Tooth sizes in nonsyndromic hypodontia patients
.
Angle Orthod
.
2013
;
83
:
16
21
.
20. 
Higashihori
N,
Takada
JI,
Minami
K,
Takahashi
Y,
Moriyama
K.
Frequency of missing teeth and reduction of mesiodistal tooth width in Japanese patients with tooth agenesis
.
Prog Orthod
.
2018
;
19
:
30
.
21. 
Schalk-van der Weide
Y,
Bosman
F.
Tooth size in relatives of individuals with oligodontia
.
Arch Oral Biol
.
1996
;
41
:
469
472
.
22. 
McKeown
HF,
Robinson
DL,
Elcock
C,
al-Sharood
M,
Brook
AH.
Tooth dimensions in hypodontia patients, their unaffected relatives and a control group measured by a new image analysis system
.
Eur J Orthod
.
2002
;
24
:
131
141
.
23. 
Küchler
EC,
Tannure
PN,
Falagan-Lotsch
P,
Lopes
TS,
Granjeiro
JM,
Amorim
LMF.
Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR
.
J Appl Oral Sci.
2012
;
20
:
467
471
.
24. 
Ranade
K,
Chang
MS,
Ting
CT,
et al.
High-throughput genotyping with single nucleotide polymorphisms
.
Genome Res
.
2001
;
11
:
1262
1268
.
25. 
Moreno Uribe
LM,
Miller
SF.
Genetics of the dentofacial variation in human malocclusion
.
Ortho Craniofac Res
.
2015
;
18
:
91
99
.
26. 
Cruz
CV,
Mattos
CT,
Maia
JC,
et al.
Genetic polymorphisms underlying the skeletal Class III phenotype
.
Am J Orthod Dentofacial Orthop
.
2017
;
151
:
700
707
.
27. 
Da Fontoura
CSG,
Miller
SF,
Wehby
GL,
et al.
Candidate gene analyses of skeletal variation in malocclusion
.
J Dent Res
.
2015
;
94
:
913
920
.
28. 
Frazier-Bowers
S,
Rincon-Rodriguez
R,
Zhou
J,
Alexander
K,
Lange
E.
Evidence of linkage in a Hispanic cohort with a Class III dentofacial phenotype
.
J Dent Res
.
2009
;
88
:
56
60
.
29. 
Tobón-Arroyave
SI,
Jiménez-Arbeláez
GA,
Alvarado-Gómez
VA,
Isaza-Guzmán
DM,
Flórez-Moreno
GA,
Pérez-Cano
MI.
Association analysis between rs6184 and rs6180 polymorphisms of growth hormone receptor gene regarding skeletal-facial profile in a Colombian population
.
Eur J Orthod
.
2018
;
40
:
378
386
.
30. 
Zebrick
B,
Teeramongkolgul
T,
Nicot
R,
et al.
ACTN3 R577X genotypes associate with Class I and deepbite malocclusions
.
Am J Orthod Dentofacial Orthop
.
2014
;
146
:
603
611
.
31. 
Milunsky
JM,
Zhao
G,
Maher
TA,
Colby
R,
Everman
DB.
LADD syndrome is caused by FGF10 mutations
.
Clin Genet
.
2006
;
69
:
349
354
.
32. 
Porntaveetus
T,
Otsuka-Tanaka
Y,
Basson
MA,
Moon
AM,
Sharpe
PT,
Ohazama
A.
Expression of fibroblast growth factors (Fgfs) in murine tooth development
.
J Anat
.
2011
;
218
:
534
543
.
33. 
Kettunen
P,
Laurikkala
J,
Itäranta
P,
Vainio
S,
Itoh
N,
Thesleff
I.
Association of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis
.
Dev Dyn
.
2000
;
219
:
322
332
.
34. 
Harada
H,
Kettunen
P,
Jung
H,
Mustonen
T,
Wang
YA,
Thesleff
I.
Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling
.
J Cell Biol
.
1999
;
147
:
105
120
.
35. 
Harada
H,
Toyono
T,
Toyoshima
K,
et al.
FGF10 maintains stem cell compartment in developing mouse incisors
.
Development
.
2002
;
129
:
1533
1541
.
36. 
Klein
OD,
Lyons
DB,
Balooch
G,
et al.
An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors
.
Development
.
2008
;
135
:
377
385
.
37. 
Kettunen
P,
Furmanek
T,
Chaulagain
R,
Kvinnsland
IH,
Luukko
K.
Developmentally regulated expression of intracellular Fgf11-13, hormone-like Fgf15 and canonical Fgf16, -17 and -20 mRNAs in the developing mouse molar tooth
.
Acta Odontol Scand
.
2011
;
69
:
360
366
.
38. 
Ohuchi
H,
Hori
Y,
Yamasaki
M,
et al.
FGF10 acts as a major ligand for FGF receptor 2IIIb in mouse multi-organ development
.
Biochem Biophys Res Commun
.
2000
;
277
:
643
649
.
39. 
Min
H,
Danilenko
DM,
Scully
SA,
et al.
Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless
.
Genes Dev
.
1998
;
12
:
3156
3161
.
40. 
Sekine
K,
Ohuchi
H,
Fujiwara
M,
et al.
Fgf10 is essential for limb and lung formation
.
Nat Genet
.
1999
;
21
:
138
141
.
41. 
Kratochwil
K,
Galceran
J,
Tontsch
S,
Roth
W,
Grosschedl
R.
FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1-/- mice
.
Genes Dev
.
2002
;
16
:
3173
3185
.
42. 
Charles
C,
Lazzari
V,
Tafforeau
P,
et al.
Modulation of Fgf3 dosage in mouse and men mirrors evolution of mammalian dentition
.
Proc Natl Acad Sci USA
.
2009
;
106
:
22364
22368
.

Author notes

a

PhD candidate, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

b

Professor, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

c

Undergraduate student, School of Dentistry of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.

d

PhD candidate, Department of Orthodontics, State University of Rio de Janeiro, Rio de Janeiro, Brazil.

e

PhD candidate, Department of Oral and Maxillofacial Surgery, Positivo University, Curitiba, Brazil.

f

Associate Professor, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

g

Full Professor, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

h

Professor, Department of Oral and Maxillofacial Surgery, Positivo University, Curitiba, Brazil.

i

Associate Professor, Department of Pediatric Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.