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Polymorphisms in genes involved in enamel development are associated with dental fluorosis
Archives of Oral Biology 76 (2017) 66–69
Contents lists available at ScienceDirect
Archives of Oral Biology journal homepage: www.elsevier.com/locate/aob
Original Article
Polymorphisms in genes involved in enamel development are associated with dental fluorosis Erika Calvano Küchlera,1, Patricia Nivoloni Tannureb,f,1, Daniela Silva Barroso de Oliveiraa , Senda Charonec, Paulo Nelson-Filhoa , Raquel Assed Bezerra da Silvaa , Marcelo de Castro Costad, Leonardo Santos Antunese,f , Mônica Diuana Calasans Maiaf , Lívia Azeredo Alves Antunese,f,* a
Department of Pediatric Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil School of Dentistry, Veiga de Almeida University, Rio de Janeiro-RJ, Brazil c Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil d Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro-RJ, Brazil e Department of Specific Formation, School of Dentistry, Fluminense Federal University, Nova Friburgo, Rio de Janeiro, Brazil f Postgraduate Program in Dentistry, Fluminense Federal University, Niterói, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 May 2016 Accepted 15 January 2017
Objective: To evaluate the association between polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 genes with dental fluorosis (DF) phenotype. Design: Four hundred and eighty one subjects (108 with DF and 373 DF free) from 6 to 18 years of age were recruited. This population lived in Rio de Janeiro, a city with fluoridation of public water supplies. DF was assessed using the Dean’s index modified. Only erupted permanent teeth were assessed. Genetic polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 were analyzed by real-time PCR from genomic DNA. Association between DF, genotype, and allele distribution were evaluated using chi-square and logistic regression analyses with an alpha level of 5%. Results: DF was more prevalent in Afro-descendants than in Caucasians (p = 0.08; OR = 1.83; CI 95% = 1.18– 2.82). Logistic regression analysis adjusted by the ethnicity demonstrated a statistical difference for TIMP1 genotype (p = 0.033; OR = 2.93, 95%CI, 1.09–7.90). When only the severer cases of DF were analyzed, polymorphisms in DLX1 and DLX2 were associated with DF (p < 0.05). Conclusion: Our results provided evidence that polymorphisms in TIMP1, DLX1 and DLX2 genes may be associated with DF phenotypes. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Dental fluorosis Gene Polymorphism
1. Introduction Dental fluorosis (DF) occurs as a result of excess fluoride ingestion during enamel formation (Bronckers, Lyaruu, & DenBesten, 2009; DenBesten & Li, 2011). Many of the changes caused by excess fluoride are related to cell, matrix, and mineral interactions while enamel is being formed (Aoba & Fejerskov, 2002; Bronckers
* Corresponding author at: Fluminense Federal University, Rua Doutor Silvio Henrique Braune, 22 – Centro, 28625-650, Nova Friburgo, Rio de Janeiro, Brazil. E-mail addresses: [email protected] (E.C. Küchler), [email protected] (P.N. Tannure), [email protected] (D.S.B.d. Oliveira), [email protected] (S. Charone), [email protected] (P. Nelson-Filho), [email protected] (M.d.C. Costa), [email protected] (L.S. Antunes), [email protected] (M.D. Calasans Maia), [email protected] (L.A.A. Antunes). 1 These authors contributed equal to this work. http://dx.doi.org/10.1016/j.archoralbio.2017.01.009 0003-9969/© 2017 Elsevier Ltd. All rights reserved.
et al., 2009). During the early maturation stage, the relative quantity of protein is increased in fluorosed enamel in a doserelated manner (DenBesten, 1986). This appears to result from a delay in the removal of protein during enamel maturation (DenBesten et al., 2002; DenBesten, 1986). Genetic factors influence DF susceptibility in mice and humans. Two mice strains have been used to identify loci associated with DF susceptibility (Everett et al., 2009; Everett, Yin, Yan, & Zou, 2011). The “susceptible” strain has a severe development of DF, while the “resistant” strain has a minimum development of DF (Everett et al., 2002). Studies in humans with endemic DF confirm the genetic influence in DF etiology (Ba et al., 2009; Ba et al., 2011; Huang et al., 2008; Jiao, Mu, Wang, An, & Jiang, 2013; Jiang, Mu, Wang, Yan, & Jiao, 2015; Wen et al., 2012; Zhang et al., 2013). Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved with tissue remodeling
E.C. Küchler et al. / Archives of Oral Biology 76 (2017) 66–69
and extracellular matrix degradation. MMPs and TIMPs participate in the enamel formation, and regulate biomineralization by controlling the proteoglycan turnover (Hannas, Pereira, Granjeiro, & Tjäderhane, 2007). MMP13 has been previously associated with caries risk (Tannure et al., 2012). Distal-less homologs, the DLX genes, are expressed during initial stages of the odontogenesis, and enamel formation. DLX have a participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression (Lézot et al., 2008; Zhao, Stock, Buchanan, & Weiss, 2000). Based on these evidences, polymorphisms in genes expressed during enamel formation may be involved in the DF etiology. Therefore, the aim of this study was to investigate the association between polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 genes with DF phenotype. 2. Materials and methods The local Human Ethics Committee (113/09) approved this study. Informed consent was obtained from all participating individuals or parents/legal guardians. 2.1. Type of study and sampling This cross-sectional eligible unrelated healthy subjects from 6 to 18 years of age recruited at the Pediatric Dental Clinics, Federal University of Rio de Janeiro. This population lived in Rio de Janeiro, a city with fluoridation of public water supplies. The subjects were divided in groups according presence or absence of DF. The current analysis used data from early-erupting permanent teeth. For the mixed dentition, only erupted permanent teeth were assessed. 2.2. Sample characterization The following data were obtained: age, gender, dentition, ethicinity. The ethnicity definition was ascertained based on selfreported information. The institution where the subjects were recruited is located in the Southeast of Brazil, the most densely heterogeneous populated and industrialized region of the country. 2.3. Determination of dental fluorosis phenotype Trained examiners conducted dental examinations. The kappa scores on intra- and inter-examiner were between good to excellent; weighted kappa scores were 1.00 for intra-examiner reliability and 0.89 for inter-examiner reliability. Subjects were seated in a dental chair, and the examiner used a probe and dental mirror according to the criteria recommended by the World Health Organization guidelines (WHO, 2013). DF was assessed using the Dean’s index modified (Rozier, 1994) with the examination and a score was registered. This index allows the classification of DF into three degrees: mild (very mild and mild), moderate, and severe. The questionable degree was excluded.
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2.4. DNA samples and genotyping Genomic DNA for molecular analysis was extracted from buccal cells based on the reported method (Küchler et al., 2012). Genetic polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 were genotyped by real time polymerase chain reactions (Real Time PCR) using the Taqman assay (Agilent Technologies, Stratgene Mx3005P) (Shen, Abdullah, & Wang, 2009). The selected genes are expressed in at least one stage of the enamel development (http:// bite-it.helsinki.fi/). The characteristics of the studied polymorphisms are presented in Table 1. 2.5. Statistical analysis The data were analyzed using the Epi Info 3.5.7. The t test, chisquare test and odds ratio calculations were used to compare age, ethnicity, gender, between DF group and DF free group. Chi-square or Fisher’s exact tests and odds ratio, at a level of significance of 0.05, were used to compare allele and genotype distributions between ‘All DF’ and ‘DF free’ groups and between ‘Moderate plus severe DF’ and ‘DF free’. For TIMP1, gender was used as a covariant in the model because is a gene in a sex chromosome. Logistic regression analysis was also implemented for all polymorphisms analysed using ethnicity as covariate in order to test the possibility of ethnicity background influence. A level of significance of 0.10 was used for multivariate analysis. Hardy-Weinberg equilibrium was evaluated using the chisquare test within each polymorphism. 3. Results From 618 subjects evaluated, 481 subjects were included in this study (108 subjects with DF and 373 DF free subjects). The dropout due to questionable degree of DF was 8. Table 2 summarizes the characteristics of the studied population. There were no significant differences in age and gender between the groups (p > 0.05). DF was more prevalent in Afro-descendants than in Caucasians (p = 0.008; OR = 1.83; CI 95% = 1.18–2.82). Mild DF affected 85 subjects (78.7%), moderate DF affected 18 subjects (16.6%) and the severe DF affected 5 subjects (4.7%). Table 3 presents the genotypes frequency distribution of the genes among the groups. We were not able to identify any statistical difference of genotype and allele distribution between ‘All DF’ and ‘DF free’ groups (p > 0.05); however, a borderline association was observed between TIMP1 and genotype distribution (p = 0.073). When only the severer cases of DF (Mild DF plus Severe DF) were analyzed, genotype distribution was different in DLX1 gene, in which GG genotype was less frequent in the DF free group (p = 0.048). In DLX2 gene, genotype and allele distributions were different among the groups (p = 0.022 and p = 0.013; respectively). Table 4 demonstrated the results of the logistic regression analysis adjusted by the ethnicity, statistical difference was
Table 1 Candidate studied genes and polymorphisms. Gene
Polymorphism
Locus
Location in the gene
Base Change
Alteration
MAF
DLX1 DLX2 MMP13 TIMP1 TIMP2
rs788173 rs743605 rs2252070 rs4898 rs7501477
2q31.1 17q24.1 11q22.3 Xp11.23 17q25
UTR UTR promoter Intron promoter
A/G A/G A/G C/T G/T
unknown upstream variant up regulationa unknown down regulationa
0.403 0.316 0.364 0.469 0.153
Note: Bold form indicates ancestral allele.MAF means Minor Allele Frequency obtained from databases: http://www.ncbi.nlm.nih.gov. a Change in transcription.
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Table 2 Characteristics of the studied subjects. Variables
Total of subjects (n = 481)
All DF (n = 108)
DF free (n = 373)
p-valuea
Mean Age (SD)
9.82 (2.78)
9.53 (2.51)
9.90 (2.85)
0.22a
Gender (%) Female Male
233 (48.4) 248 (51.6)
54 (50.0) 54 (50.0)
179 (48.0) 194 (52.0)
0.74b
Ethnicity (%) Caucasian Afro-descendants
274 (57.0) 207 (43.0)
49 (45.4) 59 (54.6)
225 (60.3) 148 (39.7)
0.008b
Dentition (%) mixed permanent
359 (74.3) 122 (25.4)
83 (76.8) 25 (23.2)
276 (73.9) 97 (26.1)
0.616b
Fluorosis (%) mild moderate severe
85 (17.7) 18 (3.74) 5 (1.03)
85 (78.7) 18 (16.6) 5 (4.7)
– – –
–
Note: bold forms indicated statistical significance. a t-test. b Chi-square test.
Table 3 Results of genotype and allele comparisons between fluorosis and fluorosis free groups. Gene and base change rs#
All DF DF free dd/dD/DD dd/dD/DD
p-value Genotype Allele
DLX1 (A/G) DLX2 (A/G) MMP13 (A/G) TIMP1 (C/T) TIMP2 (G/T)
19/37/21 17/25/31 7/23/37 6/27/35 2/27/38
0.608 0.408 0.715 0.073 0.114
788173 743605 2252070 4898 7501477
74/147/106 77/139/116 47/131/180 43/65/117 15/98/241
0.648 0.672 0.769 0.286 0.170
y
Upper case letters denote the more frequent allele in controls; dd is the less frequent homozygotyc genotype, DD is the more frequent homozygotyc genotype and dD is the heterozygotyc.
Table 4 Genotype results of the multivariate logistic regression analysis. Gene
rs#
Reference
Genotype
p value
OR (95% CI)
DLX1
788173
AA
AG GG
0.909 0.697
1.03 (0.55–1.93) 0.87 (0.43–1.75)
DLX2
743605
CC
CT TT
0.155 0.621
0.65 (0.36–1.17) 0.84 (0.43–1.64)
MMP13
2252070
CC
CT TT
0.915 0.587
1.05 (0.41–2.63) 1.27 (0.53–3.0)
TIMP1
4898
CC
CT TT
0.033 0.130
2.93 (1.09–7.90) 2.06 (0.80–5.27)
TIMP2
7501477
GG
GT TT
0.789 0.834
1.64 (0.94–2.85) 0.85 (0.18–3.89)
Note: p 0.05 (95% CI) = Odds ratios; 95% confidence intervals; bold form indicated statistical association. Covariated included age, dietary factors, ethnicity, use of dental floss and the DLXs, MMPs and TIMPs polymorphism. For TIMP1 gender was also used as covariate.
observed for TIMP1 genotype (p = 0.033; OR = 2.93, 95%CI, 1.09– 7.90). 4. Discussion Although DF occurs as a result of excess fluoride ingestion during different stages of enamel formation, evidence from studies with animal models (Everett et al., 2011) and humans (Ferreira
et al., 2010; Suzuki, Shin, Simmer, & Bartlett, 2014; Zhang et al., 2015) clearly support the genetic background in DF etiology. Some previous studies evaluated the association between genetic polymorphisms and DF (Ba et al., 2009; Ba et al., 2011; Huang et al., 2008; Jiang et al., 2015; Jiao et al., 2013; Wen et al., 2012; Zhang et al., 2013) and some genes, such as estrogen receptor (Ba et al., 2009; Ba et al., 2011), calcitonin receptor (Jiang et al., 2015), ameloblastin (Jiao et al., 2013) and myeloperoxidase (Zhang et al., 2013) have already been associated with this condition in the population who live in the endemic fluorosis area. Our study evaluated subjects raised and living in Rio de Janeiro Country with water fluoride supplementation. This region is not considered an endemic area like the regions studied above. The ethnic background of the population from Rio de Janeiro State is basically composed of Europeans descendants and African descendants. Some diseases are more common in particular ethnicity, based on differences in genetic variants frequencies. In our population, DF was almost twice more common in Afrodescendant than in Caucasian. For this reason, we decided to perform multivariate analyses using the ethnicity as co-variant. In our logistic regression analyses TIMP1 was associated with DF. It is well know that MMPs and TIMPs genes participate in enamel formation, in histomorphogenesis and cytodifferentiation (Yoshiba et al., 2003) and some evidences also indicate that MMPs and TIMPs could be involved in the etiology of DF (Hannas et al., 2007). The excessive fluoride can disturb the balance MMP/TIMP, which lead to the delay of amelogenin removal and enamel demineralization (Zhang, Xi, Guo, Cheng, & Zhang, 2011). Fluoride-induced delay in the hydrolysis and removal of amelogenin matrix during enamel maturation may lead to DF. The underlying mechanisms involved in the severity of the DF phenotype are still not fully understood. The dose-dependent effects on the severity of the DF phenotype are well-known. This is related to the concentration of fluoride in the plasma, considered to be in equilibrium with the tissue fluid that bathes the enamel organ (Angmar-Mansson & Whitford, 1984). Furthermore, dose level alone is not the only factor affecting DF phenotype; the role of genetic factors has been emphasized in studies with animal models (Carvalho et al., 2009; Everett et al., 2009; Everett et al., 2011) and in epidemiological and clinical studies (Dequeker & Declerck, 1993). DLX1 and DLX2 were associated with the most severe form of DF in our population. Our study suggests that the
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phenotype severity of DF in humans has a genetic background, in which some genes are associated with the most severe forms of DF. Lézot et al. (2008) demonstrated a role for DLX homeoproteins in the morphological control of the enamel formation. During the secretory phase of amelogenesis, when DLX2 is transitorily switched off, DLX1 expression is upregulated. The authors also suggested that the regulation of amelogenin expression in ameloblasts may be one process by which DLX homeoproteins control enamel formation. Briefly, our study demonstrated that polymorphisms in genes involved in the enamel development might be involved in the DF susceptibility. Further studies should evaluate other genes according to the DF phenotype. 5. Conclusion Our study suggests that the polymorphisms in TIMP1, DLX1 and DLX2 genes are associated with DF phenotypes. Conflicts of interest Authors declare that there is no conflicts of interest. Acknowledgements The authors are grateful to the volunteers. Funding resources from Brazilian funding agencies FAPERJ (LSA and LAA) and individual fellowships from CAPES (DSBO), CNPq (ECK) and FAPESP (SC) supported this work. References Angmar-Mansson, B., & Whitford, G. M. (1984). Enamel fluorosis related to plasma F levels in the rat. Caries Research, 18, 25–32. Aoba, T., & Fejerskov, O. (2002). Dental fluorosis: Chemistry and biology. Critical Reviews in Oral Biology and Medicine, 13, 155–170. Ba, Y., Li, H. X., Yin, G. J., Wu, W. H., Yu, B., Cheng, X. M., et al. (2009). Study on the relationship between ER Rsa I gene polymorphism and children's dental fluorosis. Sichuan Da Xue Xue Bao Yi Xue Ban, 40, 869–872. Ba, Y., Zhang, H., Wang, G., Wen, S., Yang, Y., Zhu, J., et al. (2011). Association of dental fluorosis with polymorphisms of estrogen receptor gene in Chinese children. Biological Trace Element Research, 143, 87–96. Bronckers, A. L. J. J., Lyaruu, D. M., & DenBesten, P. K. (2009). The impact of fluoride on ameloblasts and the mecanisms of enamel fluorosis. Journal of Dental Research, 88, 877–893. Carvalho, J. G., Leite, A. L., Yan, D., Everett, E. T., Whitford, G. M., & Buzalaf, M. A. (2009). Influence of genetic background on fluoride metabolism in mice. Journal of Dental Research, 88, 1054–1058. DenBesten, P. K., & Li, W. (2011). Chronic fluoride toxicity: Dental fluorosis. Monographs in Oral Science, 22, 81–96. DenBesten, P. K., Yan, Y., Featherstone, J. D., Hilton, J. F., Smith, C. E., & Li, W. (2002). Effects of fluoride on rat dental enamel matrix proteinases. Archives of Oral Biology, 47, 763–770. DenBesten, P. K. (1986). Effects of fluoride on protein secretion and removal during enamel development in the rat. Journal of Dentistry Research, 65, 1272–1277. Dequeker, J., & Declerck, K. (1993). Fluor in the treatment of osteoporosis: An overview of thirty years clinical research. Schweizerische Medizinische Wochenschrift, 123, 2228–2234.
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Everett, E. T., McHenry, M. A., Reynolds, N., Eggertsson, H., Sullivan, J., Kantmann, C., et al. (2002). Dental fluorosis: Variability among different inbred mouse strains. Journal of Dental Research, 81, 794–798. Everett, E. T., Yan, D., Weaver, M., Liu, L., Foroud, T., & Martinez-Mier, E. A. (2009). Detection of dental fluorosis-associated quantitative trait Loci on mouse chromosomes 2 and 11. Cells Tissues Organs, 189, 212–218. Everett, E. T., Yin, Z., Yan, D., & Zou, F. (2011). Fine mapping of dental fluorosis quantitative trait loci in mice. European Journal of Oral Sciences, 119(Suppl. 1), 8– 12. Ferreira, E. F., Vargas, A. M. D., Castilho, L. S., Velásquez, L. N. M., Fantinel, L. M., & Abreu, M. H. N. G. (2010). Factors associated to endemic dental fluorosis in brazilian rural communities. International Journal of Environmental Research and Public Health, 7, 3115–3128. Hannas, A. R., Pereira, J. C., Granjeiro, J. M., & Tjäderhane, L. (2007). The role of matrix metalloproteinases in the oral environment. Acta Odontologica Scandinavica, 65, 1–13. Huang, H., Ba, Y., Cui, L., Cheng, X., Zhu, J., Zhang, Y., et al. (2008). COL1A2 gene polymorphisms (Pvu II and Rsa I), serum calciotropic hormone levels, and dental fluorosis. Community Dentistry and Oral Epidemiology, 36, 517–522. Jiang, M., Mu, L., Wang, Y., Yan, W., & Jiao, Y. (2015). The relationship between Alu I polymorphisms in the calcitonin receptor gene and fluorosis endemic to Chongqing, China. Medical Principles and Practice, 24, 80–83. Jiao, Y. Z., Mu, L. H., Wang, Y. X., An, W., & Jiang, M. (2013). Association between ameloblastin gene polymorphisms and the susceptibility to dental fluorosis. Zhonghua Liu Xing Bing Xue Za Zhi, 34, 28–32. Küchler, E. C., Tannure, P. N., Falagan-Lotsch, P., Lopes, T. S., Granjeiro, J. M., & Amorim, L. M. F. (2012). Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and real-time PCR. Journal of Applied Oral Science, 20, 467–471. Lézot, F., Thomas, B., Greene, S. R., Hotton, D., Yuan, Z.-A., Castaneda, B., et al. (2008). Physiological implications of DLX homeoproteins in enamel formation. Journal of Cellular Physiology, 216, 688–697. Rozier, R. G. (1994). Epidemiologic indices for measuring the clinical manifestations of dental fluorosis: Overview and critique. Advances in Dental Research, 8, 39–55. Shen, G. Q., Abdullah, K. G., & Wang, Q. K. (2009). The TaqMan method for SNP genotyping. Methods in Molecular Biology, 578, 293–306. Suzuki, M., Shin, M., Simmer, J. P., & Bartlett, J. D. (2014). Fluoride affects enamel protein content via TGF-b1-mediated KLK4 inhibition. Journal of Dental Research, 93, 1022–1027. Tannure, P. N., Küchler, E. C., Falagan-Lotsch, P., Amorim, L. M., Raggio Luiz, R., Costa, M. C., et al. (2012). MMP13 polymorphism decreases risk for dental caries. Caries Research, 46, 401–407. WHO World Health Organization (2013). Oral health surveys: Basics methods, 5th ed. Geneva: Word Health Organization. Wen, S., Li, A., Cui, L., Huang, Q., Chen, H., & Guo, X. (2012). The relationship of PTH Bst BI polymorphism, calciotropic hormone levels, and dental fluorosis of children in China. Biological Trace Element Research, 147, 84–90. Yoshiba, N., Yoshiba, K., Stoetzel, C., Perrin-Schmitt, F., Cam, Y., Ruch, J. V., et al. (2003). Temporospatial gene expression and protein localization of matrix metalloproteinases and their inhibitors during mouse molar tooth development. Developmental Dynamics, 228, 105–112. Zhang, X. L., Xi, S. H., Guo, X. Y., Cheng, G. Y., & Zhang, Y. (2011). Effect of fluoride on the expression of MMP-20/TIMP-2 in ameloblast of rat incisor and the antagonistic effect of melatonin against fluorosis. Shanghai Kou Qiang Yi Xue, 20, 10–15. Zhang, T., Shang, K. R., Tu, X., He, Y., Pei, J. J., & Guan, Z. Z. (2013). Myeloperoxidase activity and its corresponding mRNA expression as well as gene polymorphism in the population living in the coal-burning endemic fluorosis area in Guizhou of China. Biological Trace Element Research, 152, 379–386. Zhang, S., Zhang, X., Liu, H., Qu, W., Guan, Z., Zeng, Q., et al. (2015). Modifying effect of COMT gene polymorphism and a predictive role for proteomics analysis in children’s intelligence in endemic fluorosis area in Tianjin, China. Toxicological Sciences, 144, 238–245. Zhao, Z., Stock, D., Buchanan, A., & Weiss, K. (2000). Expression of Dlx genes during the development of the murine dentition. Development Genes and Evolution, 210, 270–275.
Contents lists available at ScienceDirect
Archives of Oral Biology journal homepage: www.elsevier.com/locate/aob
Original Article
Polymorphisms in genes involved in enamel development are associated with dental fluorosis Erika Calvano Küchlera,1, Patricia Nivoloni Tannureb,f,1, Daniela Silva Barroso de Oliveiraa , Senda Charonec, Paulo Nelson-Filhoa , Raquel Assed Bezerra da Silvaa , Marcelo de Castro Costad, Leonardo Santos Antunese,f , Mônica Diuana Calasans Maiaf , Lívia Azeredo Alves Antunese,f,* a
Department of Pediatric Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil School of Dentistry, Veiga de Almeida University, Rio de Janeiro-RJ, Brazil c Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil d Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro-RJ, Brazil e Department of Specific Formation, School of Dentistry, Fluminense Federal University, Nova Friburgo, Rio de Janeiro, Brazil f Postgraduate Program in Dentistry, Fluminense Federal University, Niterói, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 May 2016 Accepted 15 January 2017
Objective: To evaluate the association between polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 genes with dental fluorosis (DF) phenotype. Design: Four hundred and eighty one subjects (108 with DF and 373 DF free) from 6 to 18 years of age were recruited. This population lived in Rio de Janeiro, a city with fluoridation of public water supplies. DF was assessed using the Dean’s index modified. Only erupted permanent teeth were assessed. Genetic polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 were analyzed by real-time PCR from genomic DNA. Association between DF, genotype, and allele distribution were evaluated using chi-square and logistic regression analyses with an alpha level of 5%. Results: DF was more prevalent in Afro-descendants than in Caucasians (p = 0.08; OR = 1.83; CI 95% = 1.18– 2.82). Logistic regression analysis adjusted by the ethnicity demonstrated a statistical difference for TIMP1 genotype (p = 0.033; OR = 2.93, 95%CI, 1.09–7.90). When only the severer cases of DF were analyzed, polymorphisms in DLX1 and DLX2 were associated with DF (p < 0.05). Conclusion: Our results provided evidence that polymorphisms in TIMP1, DLX1 and DLX2 genes may be associated with DF phenotypes. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Dental fluorosis Gene Polymorphism
1. Introduction Dental fluorosis (DF) occurs as a result of excess fluoride ingestion during enamel formation (Bronckers, Lyaruu, & DenBesten, 2009; DenBesten & Li, 2011). Many of the changes caused by excess fluoride are related to cell, matrix, and mineral interactions while enamel is being formed (Aoba & Fejerskov, 2002; Bronckers
* Corresponding author at: Fluminense Federal University, Rua Doutor Silvio Henrique Braune, 22 – Centro, 28625-650, Nova Friburgo, Rio de Janeiro, Brazil. E-mail addresses: [email protected] (E.C. Küchler), [email protected] (P.N. Tannure), [email protected] (D.S.B.d. Oliveira), [email protected] (S. Charone), [email protected] (P. Nelson-Filho), [email protected] (M.d.C. Costa), [email protected] (L.S. Antunes), [email protected] (M.D. Calasans Maia), [email protected] (L.A.A. Antunes). 1 These authors contributed equal to this work. http://dx.doi.org/10.1016/j.archoralbio.2017.01.009 0003-9969/© 2017 Elsevier Ltd. All rights reserved.
et al., 2009). During the early maturation stage, the relative quantity of protein is increased in fluorosed enamel in a doserelated manner (DenBesten, 1986). This appears to result from a delay in the removal of protein during enamel maturation (DenBesten et al., 2002; DenBesten, 1986). Genetic factors influence DF susceptibility in mice and humans. Two mice strains have been used to identify loci associated with DF susceptibility (Everett et al., 2009; Everett, Yin, Yan, & Zou, 2011). The “susceptible” strain has a severe development of DF, while the “resistant” strain has a minimum development of DF (Everett et al., 2002). Studies in humans with endemic DF confirm the genetic influence in DF etiology (Ba et al., 2009; Ba et al., 2011; Huang et al., 2008; Jiao, Mu, Wang, An, & Jiang, 2013; Jiang, Mu, Wang, Yan, & Jiao, 2015; Wen et al., 2012; Zhang et al., 2013). Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved with tissue remodeling
E.C. Küchler et al. / Archives of Oral Biology 76 (2017) 66–69
and extracellular matrix degradation. MMPs and TIMPs participate in the enamel formation, and regulate biomineralization by controlling the proteoglycan turnover (Hannas, Pereira, Granjeiro, & Tjäderhane, 2007). MMP13 has been previously associated with caries risk (Tannure et al., 2012). Distal-less homologs, the DLX genes, are expressed during initial stages of the odontogenesis, and enamel formation. DLX have a participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression (Lézot et al., 2008; Zhao, Stock, Buchanan, & Weiss, 2000). Based on these evidences, polymorphisms in genes expressed during enamel formation may be involved in the DF etiology. Therefore, the aim of this study was to investigate the association between polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 genes with DF phenotype. 2. Materials and methods The local Human Ethics Committee (113/09) approved this study. Informed consent was obtained from all participating individuals or parents/legal guardians. 2.1. Type of study and sampling This cross-sectional eligible unrelated healthy subjects from 6 to 18 years of age recruited at the Pediatric Dental Clinics, Federal University of Rio de Janeiro. This population lived in Rio de Janeiro, a city with fluoridation of public water supplies. The subjects were divided in groups according presence or absence of DF. The current analysis used data from early-erupting permanent teeth. For the mixed dentition, only erupted permanent teeth were assessed. 2.2. Sample characterization The following data were obtained: age, gender, dentition, ethicinity. The ethnicity definition was ascertained based on selfreported information. The institution where the subjects were recruited is located in the Southeast of Brazil, the most densely heterogeneous populated and industrialized region of the country. 2.3. Determination of dental fluorosis phenotype Trained examiners conducted dental examinations. The kappa scores on intra- and inter-examiner were between good to excellent; weighted kappa scores were 1.00 for intra-examiner reliability and 0.89 for inter-examiner reliability. Subjects were seated in a dental chair, and the examiner used a probe and dental mirror according to the criteria recommended by the World Health Organization guidelines (WHO, 2013). DF was assessed using the Dean’s index modified (Rozier, 1994) with the examination and a score was registered. This index allows the classification of DF into three degrees: mild (very mild and mild), moderate, and severe. The questionable degree was excluded.
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2.4. DNA samples and genotyping Genomic DNA for molecular analysis was extracted from buccal cells based on the reported method (Küchler et al., 2012). Genetic polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 were genotyped by real time polymerase chain reactions (Real Time PCR) using the Taqman assay (Agilent Technologies, Stratgene Mx3005P) (Shen, Abdullah, & Wang, 2009). The selected genes are expressed in at least one stage of the enamel development (http:// bite-it.helsinki.fi/). The characteristics of the studied polymorphisms are presented in Table 1. 2.5. Statistical analysis The data were analyzed using the Epi Info 3.5.7. The t test, chisquare test and odds ratio calculations were used to compare age, ethnicity, gender, between DF group and DF free group. Chi-square or Fisher’s exact tests and odds ratio, at a level of significance of 0.05, were used to compare allele and genotype distributions between ‘All DF’ and ‘DF free’ groups and between ‘Moderate plus severe DF’ and ‘DF free’. For TIMP1, gender was used as a covariant in the model because is a gene in a sex chromosome. Logistic regression analysis was also implemented for all polymorphisms analysed using ethnicity as covariate in order to test the possibility of ethnicity background influence. A level of significance of 0.10 was used for multivariate analysis. Hardy-Weinberg equilibrium was evaluated using the chisquare test within each polymorphism. 3. Results From 618 subjects evaluated, 481 subjects were included in this study (108 subjects with DF and 373 DF free subjects). The dropout due to questionable degree of DF was 8. Table 2 summarizes the characteristics of the studied population. There were no significant differences in age and gender between the groups (p > 0.05). DF was more prevalent in Afro-descendants than in Caucasians (p = 0.008; OR = 1.83; CI 95% = 1.18–2.82). Mild DF affected 85 subjects (78.7%), moderate DF affected 18 subjects (16.6%) and the severe DF affected 5 subjects (4.7%). Table 3 presents the genotypes frequency distribution of the genes among the groups. We were not able to identify any statistical difference of genotype and allele distribution between ‘All DF’ and ‘DF free’ groups (p > 0.05); however, a borderline association was observed between TIMP1 and genotype distribution (p = 0.073). When only the severer cases of DF (Mild DF plus Severe DF) were analyzed, genotype distribution was different in DLX1 gene, in which GG genotype was less frequent in the DF free group (p = 0.048). In DLX2 gene, genotype and allele distributions were different among the groups (p = 0.022 and p = 0.013; respectively). Table 4 demonstrated the results of the logistic regression analysis adjusted by the ethnicity, statistical difference was
Table 1 Candidate studied genes and polymorphisms. Gene
Polymorphism
Locus
Location in the gene
Base Change
Alteration
MAF
DLX1 DLX2 MMP13 TIMP1 TIMP2
rs788173 rs743605 rs2252070 rs4898 rs7501477
2q31.1 17q24.1 11q22.3 Xp11.23 17q25
UTR UTR promoter Intron promoter
A/G A/G A/G C/T G/T
unknown upstream variant up regulationa unknown down regulationa
0.403 0.316 0.364 0.469 0.153
Note: Bold form indicates ancestral allele.MAF means Minor Allele Frequency obtained from databases: http://www.ncbi.nlm.nih.gov. a Change in transcription.
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Table 2 Characteristics of the studied subjects. Variables
Total of subjects (n = 481)
All DF (n = 108)
DF free (n = 373)
p-valuea
Mean Age (SD)
9.82 (2.78)
9.53 (2.51)
9.90 (2.85)
0.22a
Gender (%) Female Male
233 (48.4) 248 (51.6)
54 (50.0) 54 (50.0)
179 (48.0) 194 (52.0)
0.74b
Ethnicity (%) Caucasian Afro-descendants
274 (57.0) 207 (43.0)
49 (45.4) 59 (54.6)
225 (60.3) 148 (39.7)
0.008b
Dentition (%) mixed permanent
359 (74.3) 122 (25.4)
83 (76.8) 25 (23.2)
276 (73.9) 97 (26.1)
0.616b
Fluorosis (%) mild moderate severe
85 (17.7) 18 (3.74) 5 (1.03)
85 (78.7) 18 (16.6) 5 (4.7)
– – –
–
Note: bold forms indicated statistical significance. a t-test. b Chi-square test.
Table 3 Results of genotype and allele comparisons between fluorosis and fluorosis free groups. Gene and base change rs#
All DF DF free dd/dD/DD dd/dD/DD
p-value Genotype Allele
DLX1 (A/G) DLX2 (A/G) MMP13 (A/G) TIMP1 (C/T) TIMP2 (G/T)
19/37/21 17/25/31 7/23/37 6/27/35 2/27/38
0.608 0.408 0.715 0.073 0.114
788173 743605 2252070 4898 7501477
74/147/106 77/139/116 47/131/180 43/65/117 15/98/241
0.648 0.672 0.769 0.286 0.170
y
Upper case letters denote the more frequent allele in controls; dd is the less frequent homozygotyc genotype, DD is the more frequent homozygotyc genotype and dD is the heterozygotyc.
Table 4 Genotype results of the multivariate logistic regression analysis. Gene
rs#
Reference
Genotype
p value
OR (95% CI)
DLX1
788173
AA
AG GG
0.909 0.697
1.03 (0.55–1.93) 0.87 (0.43–1.75)
DLX2
743605
CC
CT TT
0.155 0.621
0.65 (0.36–1.17) 0.84 (0.43–1.64)
MMP13
2252070
CC
CT TT
0.915 0.587
1.05 (0.41–2.63) 1.27 (0.53–3.0)
TIMP1
4898
CC
CT TT
0.033 0.130
2.93 (1.09–7.90) 2.06 (0.80–5.27)
TIMP2
7501477
GG
GT TT
0.789 0.834
1.64 (0.94–2.85) 0.85 (0.18–3.89)
Note: p 0.05 (95% CI) = Odds ratios; 95% confidence intervals; bold form indicated statistical association. Covariated included age, dietary factors, ethnicity, use of dental floss and the DLXs, MMPs and TIMPs polymorphism. For TIMP1 gender was also used as covariate.
observed for TIMP1 genotype (p = 0.033; OR = 2.93, 95%CI, 1.09– 7.90). 4. Discussion Although DF occurs as a result of excess fluoride ingestion during different stages of enamel formation, evidence from studies with animal models (Everett et al., 2011) and humans (Ferreira
et al., 2010; Suzuki, Shin, Simmer, & Bartlett, 2014; Zhang et al., 2015) clearly support the genetic background in DF etiology. Some previous studies evaluated the association between genetic polymorphisms and DF (Ba et al., 2009; Ba et al., 2011; Huang et al., 2008; Jiang et al., 2015; Jiao et al., 2013; Wen et al., 2012; Zhang et al., 2013) and some genes, such as estrogen receptor (Ba et al., 2009; Ba et al., 2011), calcitonin receptor (Jiang et al., 2015), ameloblastin (Jiao et al., 2013) and myeloperoxidase (Zhang et al., 2013) have already been associated with this condition in the population who live in the endemic fluorosis area. Our study evaluated subjects raised and living in Rio de Janeiro Country with water fluoride supplementation. This region is not considered an endemic area like the regions studied above. The ethnic background of the population from Rio de Janeiro State is basically composed of Europeans descendants and African descendants. Some diseases are more common in particular ethnicity, based on differences in genetic variants frequencies. In our population, DF was almost twice more common in Afrodescendant than in Caucasian. For this reason, we decided to perform multivariate analyses using the ethnicity as co-variant. In our logistic regression analyses TIMP1 was associated with DF. It is well know that MMPs and TIMPs genes participate in enamel formation, in histomorphogenesis and cytodifferentiation (Yoshiba et al., 2003) and some evidences also indicate that MMPs and TIMPs could be involved in the etiology of DF (Hannas et al., 2007). The excessive fluoride can disturb the balance MMP/TIMP, which lead to the delay of amelogenin removal and enamel demineralization (Zhang, Xi, Guo, Cheng, & Zhang, 2011). Fluoride-induced delay in the hydrolysis and removal of amelogenin matrix during enamel maturation may lead to DF. The underlying mechanisms involved in the severity of the DF phenotype are still not fully understood. The dose-dependent effects on the severity of the DF phenotype are well-known. This is related to the concentration of fluoride in the plasma, considered to be in equilibrium with the tissue fluid that bathes the enamel organ (Angmar-Mansson & Whitford, 1984). Furthermore, dose level alone is not the only factor affecting DF phenotype; the role of genetic factors has been emphasized in studies with animal models (Carvalho et al., 2009; Everett et al., 2009; Everett et al., 2011) and in epidemiological and clinical studies (Dequeker & Declerck, 1993). DLX1 and DLX2 were associated with the most severe form of DF in our population. Our study suggests that the
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phenotype severity of DF in humans has a genetic background, in which some genes are associated with the most severe forms of DF. Lézot et al. (2008) demonstrated a role for DLX homeoproteins in the morphological control of the enamel formation. During the secretory phase of amelogenesis, when DLX2 is transitorily switched off, DLX1 expression is upregulated. The authors also suggested that the regulation of amelogenin expression in ameloblasts may be one process by which DLX homeoproteins control enamel formation. Briefly, our study demonstrated that polymorphisms in genes involved in the enamel development might be involved in the DF susceptibility. Further studies should evaluate other genes according to the DF phenotype. 5. Conclusion Our study suggests that the polymorphisms in TIMP1, DLX1 and DLX2 genes are associated with DF phenotypes. Conflicts of interest Authors declare that there is no conflicts of interest. Acknowledgements The authors are grateful to the volunteers. Funding resources from Brazilian funding agencies FAPERJ (LSA and LAA) and individual fellowships from CAPES (DSBO), CNPq (ECK) and FAPESP (SC) supported this work. References Angmar-Mansson, B., & Whitford, G. M. (1984). Enamel fluorosis related to plasma F levels in the rat. Caries Research, 18, 25–32. Aoba, T., & Fejerskov, O. (2002). Dental fluorosis: Chemistry and biology. Critical Reviews in Oral Biology and Medicine, 13, 155–170. Ba, Y., Li, H. X., Yin, G. J., Wu, W. H., Yu, B., Cheng, X. M., et al. (2009). Study on the relationship between ER Rsa I gene polymorphism and children's dental fluorosis. Sichuan Da Xue Xue Bao Yi Xue Ban, 40, 869–872. Ba, Y., Zhang, H., Wang, G., Wen, S., Yang, Y., Zhu, J., et al. (2011). Association of dental fluorosis with polymorphisms of estrogen receptor gene in Chinese children. Biological Trace Element Research, 143, 87–96. Bronckers, A. L. J. J., Lyaruu, D. M., & DenBesten, P. K. (2009). The impact of fluoride on ameloblasts and the mecanisms of enamel fluorosis. Journal of Dental Research, 88, 877–893. Carvalho, J. G., Leite, A. L., Yan, D., Everett, E. T., Whitford, G. M., & Buzalaf, M. A. (2009). Influence of genetic background on fluoride metabolism in mice. Journal of Dental Research, 88, 1054–1058. DenBesten, P. K., & Li, W. (2011). Chronic fluoride toxicity: Dental fluorosis. Monographs in Oral Science, 22, 81–96. DenBesten, P. K., Yan, Y., Featherstone, J. D., Hilton, J. F., Smith, C. E., & Li, W. (2002). Effects of fluoride on rat dental enamel matrix proteinases. Archives of Oral Biology, 47, 763–770. DenBesten, P. K. (1986). Effects of fluoride on protein secretion and removal during enamel development in the rat. Journal of Dentistry Research, 65, 1272–1277. Dequeker, J., & Declerck, K. (1993). Fluor in the treatment of osteoporosis: An overview of thirty years clinical research. Schweizerische Medizinische Wochenschrift, 123, 2228–2234.
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