Journal of Dental Research http://jdr.sagepub.com/
Identification of Genetic Risk Factors for Maxillary Lateral Incisor Agenesis M. Alves-Ferreira, T. Pinho, A. Sousa, J. Sequeiros, C. Lemos and I. Alonso J DENT RES 2014 93: 452 originally published online 19 February 2014 DOI: 10.1177/0022034514523986 The online version of this article can be found at: http://jdr.sagepub.com/content/93/5/452
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Apr 15, 2014 OnlineFirst Version of Record - Feb 19, 2014 What is This?
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research-article2014
JDR
93510.1177/0022034514523986
RESEARCH REPORTS Clinical
M. Alves-Ferreira1, T. Pinho1,2, A. Sousa1,3, J. Sequeiros1,3,4, C. Lemos1,3, and I. Alonso1,3* 1
UnIGENe, Instituto Biologia Molecular Celular, Universidade do Porto, Porto, Portugal; 2Centro de Investigação Ciências da Saúde, Instituto Superior de Ciências Saúde–Norte / CESPU, Gandra-PRD, Portugal; 3Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; and 4CGPP, Instituto Biologia Molecular Celular, Universidade do Porto, Porto, Portugal; *corresponding author, [email protected]
Identification of Genetic Risk Factors for Maxillary Lateral Incisor Agenesis
J Dent Res 93(5):452-458, 2014
Abstract
Tooth agenesis affects 20% of the world population, and maxillary lateral incisors agenesis (MLIA) is one of the most frequent subtypes, characterized by the absence of formation of deciduous or permanent lateral incisors. Odontogenesis is a complex mechanism regulated by sequential and reciprocal epithelialmesenchymal interactions, controlled by activators and inhibitors involved in several pathways. Disturbances in these signaling cascades can lead to abnormalities in odontogenesis, resulting in alterations in the formation of the normal teeth number. Our aim was to study a large number of genes encoding either transcription factors or key components in signaling pathways shown to be involved in tooth odontogenesis. We selected 8 genes—MSX1, PAX9, AXIN2, EDA, SPRY2, TGFA, SPRY4, and WNT10A—and performed one of the largest case-control studies taking into account the number of genes and variants assessed, aiming at the identification of MLIA susceptibility factors. We show the involvement of PAX9, EDA, SPRY2, SPRY4, and WNT10A as risk factors for MLIA. Additionally, we uncovered 3 strong synergistic interactions between MLIA liability and MSX1-TGFA, AXIN2-TGFA, and SPRY2-SPRY4 gene pairs. We report the first evidence of the involvement of sprouty genes in MLIA susceptibility. This large study results in a better understanding of the genetic components and mechanisms underlying this trait.
Introduction
M
axillary lateral incisors agenesis (MLIA) is characterized by the absence of formation of deciduous or permanent upper lateral incisors. Tooth agenesis affects approximately 20% of the world population (Vastardis, 2000), and MLIA is one of the most frequent forms of hypodontia (agenesis of fewer than 6 teeth, excluding the third molars) (Arte et al., 2001), with a prevalence ranging from 1% to 4%. In Portugal, the prevalence of MLIA was estimated at 1.3% (Pinho et al., 2005). We found evidence of familial aggregation of MLIA in a sample of Portuguese families, suggesting the presence of a strong genetic component (relative risk =15) for this trait (Pinho et al., 2010a). Odontogenesis is a complex mechanism regulated by sequential and reciprocal epithelial-mesenchymal interactions, controlled by genetic factors, and is responsible for the determination of the position, number, shape, and size of teeth (Thesleff, 2003). Several genes have been identified as being expressed during odontogenesis, and it is known that mutations in MSX1, PAX9, AXIN2, EDA, SPRY2, TGFA, SPRY4, and WNT10A genes are involved in several forms of tooth agenesis, including syndromes in which tooth agenesis is a regular feature (Nieminen, 2009). These genes are related with all major signaling pathways and with the transcription factors mediating these signal transduction cascades. New findings on the function of these genes that are mutated in particular human syndromes further strengthen the important role of these genes in tooth agenesis (Nieminen, 2009). Here, we present a case-control study with the largest number of genes and single-nucleotide polymorphisms (SNPs) assessed in the same population, aiming at identifying MLIA susceptibility factors focusing on these genes involved in tooth odontogenesis.
KEY WORDS: hypodontia, odontogenesis, association studies, candidate genes, polymorphism, gene-gene interaction.
Material & Methods
DOI: 10.1177/0022034514523986
A case-control sample of 306 unrelated Portuguese individuals was ascertained: 102 individuals with MLIA and 204 controls (without any kind of hypodontia) (Table 1). We had a special concern in obtaining a high case:control ratio, to increase the statistical power of the study. In all cases, MLIA was confirmed radiographically, and both groups were observed by experienced clinicians. Informed consent was obtained in all cases, and this study followed the “Strengthening the Reporting of Observational Studies in Epidemiology” guidelines (von Elm et al., 2008).
Received July 31, 2013; Last revision January 21, 2014; Accepted January 24, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Subjects
452 Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(5) 2014 453 Maxillary Lateral Incisor Agenesis Candidate Gene and SNP Selection Details on SNP selection and genotyping are presented as supplementary material.
Statistical Analysis The statistical power of our sample was calculated with the Genetic Power Calculator (http://pngu.mgh.harvard.edu/ ~purcell/gpc). We performed these estimates assuming a frequency of 0.1 for the high-risk allele, a relative risk of 2.5 for the homozygous genotype and 2.2 for the heterozygous genotype, and a prevalence of 1.3% for MLIA in the Portuguese population, based on α = 0.05 (Pinho et al., 2005). Hardy-Weinberg (H-W) equilibrium was tested with SNPator software (Morcillo-Suarez et al., 2008). To compare allele frequencies between cases and controls, a chi-square test was used, and odds ratios (ORs) were estimated with 95% confidence intervals (CI), also per SNPator. A logistic regression was performed (with the commonest allele as the reference category) to evaluate the genotypic associations. The significance level was set to α = 0.007 (considering 7 logistic regressions), based on the Bonferroni test, to correct for multiple comparisons. As EDA gene is located in the X chromosome, the analyses were performed taking sex into account. These analyses were performed with SPSS 18. Haplotype frequencies in cases and controls were compared with Haploview 4.1 (Barrett et al., 2005); haplotype sequences were analyzed from 5′ to 3′. To correct for multiple testing, 10,000 permutations were used when allelic and haplotype frequencies were estimated. To explore possible gene-gene interactions, we used Multifactor Dimensionality Reduction software (version 2.0), which is a nonparametric and genetic model-free approach that can identify combinations of SNPs involved in disease susceptibility (Ritchie et al., 2001; Moore, 2004). Subsequently, we used the Permutation Testing Module (version 1.0) of Multifactor Dimensionality Reduction to correct our results for multiple testing, based on a 1000-fold permutation test.
Results Here we present a large case-control study composing the analysis of common variation in 8 genes involved in tooth odontogenesis to identify MLIA susceptibility factors. Our sample had a power of 72% to detect a significant association. Genotypic frequencies in the control group were in H-W equilibrium (p > .05). In the cases group, H-W equilibrium deviations were found for some SNPs, although in all these cases, an association with MLIA susceptibility was confirmed. We were able to show the involvement of PAX9, EDA, SPRY2, SPRY4, and WNT10A as risk factors for MLIA. No significant allelic, genotypic, and haplotypic associations were found regarding AXIN2, TGFA, and MSX1 genes (data not shown). All the analyses, were repeated, excluding patients with MLIA and other agenesis (always fewer than 6 teeth), and the results were similar (data not shown). We assessed gene-gene interactions and uncovered 3 strong synergistic interactions between MLIA liability and the following gene pairs: MSX1-TGFA, AXIN2-TGFA, and SPRY2-SPRY4.
Table 1. Clinical and Demographic Characteristics of the Study Sample Study Sample Cases MLIA only Bilateral (12 and 22) Unilateral (12) + microdontia (22) Unilateral (22) + microdontia (12) Unilateral (12) Unilateral (22) MLIA + other agenesis 12; 22; 18; 28; 38; 48 12; 22; 18 12; 22; 45 12; 22; 35 12; 22; 45; 35 12; 35; 45 22; 35; 45 12; 22; 31; 41 12; 22;41 12; 22; 15; 25 12; 32; 42 12; 41; 45; and microdontia (22) 22; 31; 41; and microdontia (13) Males Females Controls Males Females
No. 102 86 50 11 1 12 12 16 1 1 1 1 3 1 1 2 1 1 1 1 1 29 73 204 85 119
MLIA, maxillary lateral incisors agenesis
PAX9 Genotype as a MLIA Risk Factor The A allele of rs8004560 was associated with an increased MLIA susceptibility (p = .02); however, this result did not remain significant after permutation-based correction (Table 2). Importantly, 2 highly significant genotypic associations were found. The AA genotype of rs7149262 was correlated with higher trait susceptibility (p < .001). In agreement with the allelic results, the AA genotype of rs8004560 was associated with an increased MLIA susceptibility (p = .003). These associations remained significant after Bonferroni correction (Table 3). The haplotypic analysis did not reveal any significant association between PAX9 and MLIA susceptibility (data not shown).
Nominal Significant Associations of EDA SNPs With MLIA Susceptibility The A allele of rs1160315, the C allele of rs2428151, the T allele of rs2520378, and the A allele of rs2853659 were associated with an increase in MLIA susceptibility. None of the results remained significant after multiple-testing correction by permutation tests (Table 2). Regarding the genotypic analysis, the GA genotype of rs12853659 presented a higher risk for MLIA (p = .021), while the
Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
454
Alves-Ferreira et al.
J Dent Res 93(5) 2014
Table 2. Allelic Association Results for PAX9, EDA, SPRY2, and WNT10A Genes Alleles (%) PAX9 SNP rs8004560 A G EDA SNP rs1160315 A G rs2428151 C T rs2520378 T C rs12853659 A G SPRY2 SNP rs504122 A G WNT10A SNP rs11680244 A G rs2385199 G A rs7349332 T C
Cases
Controls
81 (39.7) 123 (60.3)
123 (30.1) 285 (69.9)
101 (57.7) 74 (42.3)
155 (48.0) 168 (52.0)
117 (66.9) 58 (33.1)
178 (55.1) 145 (44.9)
153 (87.4) 22 (12.6)
256 (79.3) 67 (20.7)
103 (58.9) 72 (41.1)
152 (47.1) 171 (52.9)
89 (43.6) 115 (56.4)
146 (35.8) 262 (64.2)
4 (2.0) 200 (98.0)
9 (2.2) 391 (97.8)
51 (25.0) 153 (75.0)
58 (14.4) 344( 85.6)
36 (17.6) 168 (82.4)
49 (12.2) 353 (87.8)
χ2
Odds Ratio (95% CI)
5.59
1.53 (1.07, 2.17)
4.30
1.48 (1.02, 2.14)
6.49
1.64 (1.12, 2.41)
5.16
1.82 (1.08, 3.07)
6.32
1.61 (1.11, 2.33)
3.54
1.39 (0.99, 1.96)
0.05
1.15 (0.35, 3.78)
10.25
1.98 (1.30, 3.01)
3.34
1.54 (0.97, 2.46)
p .02* .04* .01* .02* .01* .06 .82 .0014* .068
CI, confidence interval; EDA, ectodysplasin A; PAX9, paired box gene 9; SNP, single-nucleotide polymorphism; SPRY2, sprouty homolog 2 gene; WNT10A, wingless-type MMTV integration site family, member 10A. *p < .05.
GA genotype of rs1160315 (p = .015) and the CT of rs5936523 (p = .038) showed a protective effect. However, the results did not remain significant after Bonferroni correction (Table 3). In the haplotypic analysis, a specific EDA haplotype (T-G-TA-A-C-T-C-A-A-A-G-G-T-G-T) revealed a significant association (p = .032) with increased MLIA susceptibility, although it did not retain its significance after permutation correction.
SPRY2 SNP and Increased Risk for MLIA No significant differences were found regarding allelic frequencies of SPRY2 SNPs. However, we observed a trend toward an increased risk for MLIA conferred by the A allele of rs504122 (p = .06) (Table 2). Regarding the genotypic analysis, we found that the GA genotype of rs504122 presented a higher risk for individuals with MLIA (p = .005; OR = 2.49; 95% CI: 1.31, 4.73), which remained significant after Bonferroni correction (Table 3). No
significant association was found between SPRY2 haplotypes and MLIA.
SPRY4 Gene Haplotypes and MLIA Susceptibility The allelic and genotypic analyses did not reveal any significant association between this gene and MLIA susceptibility (data not shown). Importantly, the T-G-G-A-T-C haplotype revealed a nominal significant association (p = .016) with an increased MLIA susceptibility (OR = 3.46; 95% CI: 1.19, 10.08); however, this did not remain significant after permutation-based correction (p = .137).
WNT10A Variants Increase the Risk for MLIA The G allele of rs2385199 was associated with an increased MLIA susceptibility (p = .0014), a result that remained significant after permutation-based correction (Table 2).
Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(5) 2014 455 Maxillary Lateral Incisor Agenesis Table 3. Results from the PAX9, EDA, SPRY2, and WNT10A Multivariable Logistic Regression Analysis Genotype (%) PAX9 SNP rs8004560 GG (ref) GA AA rs7149262 CC (ref) AC AA EDA SNP rs1160315 GG(ref) GA AA rs5936523 CC(ref) CT TT rs12853659 GG(ref) GA AA SPRY2 SNP rs504122 GG (ref) AA GA WNT10A SNP rs2385199 GG (ref) GA AA
Cases
Controls
Odds Ratio (95% CI)
45 (44.1) 33 (32.4) 24 (23.5)
104 (51.0) 77 (37.7) 23 (11.3)
1.00 (—) 1.27 (0.72, 2.24) 3.03 (1.47, 6.28)
66 (64.7) 23 (22.6) 13 (12.7)
132 (64.7) 68 (33.3) 4 (2.0)
1.00 (—) 0.88 (0.49, 1.58) 9.39 (2.79, 30.97)
15 (44.1) 33 (32.4) 25 (23.5)
30 (51.0) 62 (37.7) 27 (11.3)
1.00 (—) 0.05 (0.01, 0.57) 0.33 (0.02, 5.72)
14 (64.7) 23 (22.6) 36 (12.7)
11 (64.7) 67 (33.3) 41 (2.0)
1.00 (—) 0.46 (0.22, 0.96) 2.59 (0.82, 8.21)
12 (44.1) 38 (32.4) 23(23.5)
31 (51.0) 61 (37.7) 27 (11.3)
25 (24.5) 12 (11.8) 65 (63.7)
81 (39.7) 23 (11.3) 100 (49.0)
1.00 (—) 1.97 (0.73, 5.28) 2.49 (1.31, 4.73)
56 (54.9) 41(40.2) 5 (4.9)
147 (73.1) 50 (24.9) 4 (2.0)
1.00 (—) 2.14 (1.28, 3.58) 3.26 (0.84, 12.58)
1.00 (—) 14.43 (1.51, 138.12) 3.24 (0.21, 49.00)
p .011 .412 .003* .001 .662
Identification of Genetic Risk Factors for Maxillary Lateral Incisor Agenesis M. Alves-Ferreira, T. Pinho, A. Sousa, J. Sequeiros, C. Lemos and I. Alonso J DENT RES 2014 93: 452 originally published online 19 February 2014 DOI: 10.1177/0022034514523986 The online version of this article can be found at: http://jdr.sagepub.com/content/93/5/452
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Apr 15, 2014 OnlineFirst Version of Record - Feb 19, 2014 What is This?
Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
research-article2014
JDR
93510.1177/0022034514523986
RESEARCH REPORTS Clinical
M. Alves-Ferreira1, T. Pinho1,2, A. Sousa1,3, J. Sequeiros1,3,4, C. Lemos1,3, and I. Alonso1,3* 1
UnIGENe, Instituto Biologia Molecular Celular, Universidade do Porto, Porto, Portugal; 2Centro de Investigação Ciências da Saúde, Instituto Superior de Ciências Saúde–Norte / CESPU, Gandra-PRD, Portugal; 3Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; and 4CGPP, Instituto Biologia Molecular Celular, Universidade do Porto, Porto, Portugal; *corresponding author, [email protected]
Identification of Genetic Risk Factors for Maxillary Lateral Incisor Agenesis
J Dent Res 93(5):452-458, 2014
Abstract
Tooth agenesis affects 20% of the world population, and maxillary lateral incisors agenesis (MLIA) is one of the most frequent subtypes, characterized by the absence of formation of deciduous or permanent lateral incisors. Odontogenesis is a complex mechanism regulated by sequential and reciprocal epithelialmesenchymal interactions, controlled by activators and inhibitors involved in several pathways. Disturbances in these signaling cascades can lead to abnormalities in odontogenesis, resulting in alterations in the formation of the normal teeth number. Our aim was to study a large number of genes encoding either transcription factors or key components in signaling pathways shown to be involved in tooth odontogenesis. We selected 8 genes—MSX1, PAX9, AXIN2, EDA, SPRY2, TGFA, SPRY4, and WNT10A—and performed one of the largest case-control studies taking into account the number of genes and variants assessed, aiming at the identification of MLIA susceptibility factors. We show the involvement of PAX9, EDA, SPRY2, SPRY4, and WNT10A as risk factors for MLIA. Additionally, we uncovered 3 strong synergistic interactions between MLIA liability and MSX1-TGFA, AXIN2-TGFA, and SPRY2-SPRY4 gene pairs. We report the first evidence of the involvement of sprouty genes in MLIA susceptibility. This large study results in a better understanding of the genetic components and mechanisms underlying this trait.
Introduction
M
axillary lateral incisors agenesis (MLIA) is characterized by the absence of formation of deciduous or permanent upper lateral incisors. Tooth agenesis affects approximately 20% of the world population (Vastardis, 2000), and MLIA is one of the most frequent forms of hypodontia (agenesis of fewer than 6 teeth, excluding the third molars) (Arte et al., 2001), with a prevalence ranging from 1% to 4%. In Portugal, the prevalence of MLIA was estimated at 1.3% (Pinho et al., 2005). We found evidence of familial aggregation of MLIA in a sample of Portuguese families, suggesting the presence of a strong genetic component (relative risk =15) for this trait (Pinho et al., 2010a). Odontogenesis is a complex mechanism regulated by sequential and reciprocal epithelial-mesenchymal interactions, controlled by genetic factors, and is responsible for the determination of the position, number, shape, and size of teeth (Thesleff, 2003). Several genes have been identified as being expressed during odontogenesis, and it is known that mutations in MSX1, PAX9, AXIN2, EDA, SPRY2, TGFA, SPRY4, and WNT10A genes are involved in several forms of tooth agenesis, including syndromes in which tooth agenesis is a regular feature (Nieminen, 2009). These genes are related with all major signaling pathways and with the transcription factors mediating these signal transduction cascades. New findings on the function of these genes that are mutated in particular human syndromes further strengthen the important role of these genes in tooth agenesis (Nieminen, 2009). Here, we present a case-control study with the largest number of genes and single-nucleotide polymorphisms (SNPs) assessed in the same population, aiming at identifying MLIA susceptibility factors focusing on these genes involved in tooth odontogenesis.
KEY WORDS: hypodontia, odontogenesis, association studies, candidate genes, polymorphism, gene-gene interaction.
Material & Methods
DOI: 10.1177/0022034514523986
A case-control sample of 306 unrelated Portuguese individuals was ascertained: 102 individuals with MLIA and 204 controls (without any kind of hypodontia) (Table 1). We had a special concern in obtaining a high case:control ratio, to increase the statistical power of the study. In all cases, MLIA was confirmed radiographically, and both groups were observed by experienced clinicians. Informed consent was obtained in all cases, and this study followed the “Strengthening the Reporting of Observational Studies in Epidemiology” guidelines (von Elm et al., 2008).
Received July 31, 2013; Last revision January 21, 2014; Accepted January 24, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Subjects
452 Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(5) 2014 453 Maxillary Lateral Incisor Agenesis Candidate Gene and SNP Selection Details on SNP selection and genotyping are presented as supplementary material.
Statistical Analysis The statistical power of our sample was calculated with the Genetic Power Calculator (http://pngu.mgh.harvard.edu/ ~purcell/gpc). We performed these estimates assuming a frequency of 0.1 for the high-risk allele, a relative risk of 2.5 for the homozygous genotype and 2.2 for the heterozygous genotype, and a prevalence of 1.3% for MLIA in the Portuguese population, based on α = 0.05 (Pinho et al., 2005). Hardy-Weinberg (H-W) equilibrium was tested with SNPator software (Morcillo-Suarez et al., 2008). To compare allele frequencies between cases and controls, a chi-square test was used, and odds ratios (ORs) were estimated with 95% confidence intervals (CI), also per SNPator. A logistic regression was performed (with the commonest allele as the reference category) to evaluate the genotypic associations. The significance level was set to α = 0.007 (considering 7 logistic regressions), based on the Bonferroni test, to correct for multiple comparisons. As EDA gene is located in the X chromosome, the analyses were performed taking sex into account. These analyses were performed with SPSS 18. Haplotype frequencies in cases and controls were compared with Haploview 4.1 (Barrett et al., 2005); haplotype sequences were analyzed from 5′ to 3′. To correct for multiple testing, 10,000 permutations were used when allelic and haplotype frequencies were estimated. To explore possible gene-gene interactions, we used Multifactor Dimensionality Reduction software (version 2.0), which is a nonparametric and genetic model-free approach that can identify combinations of SNPs involved in disease susceptibility (Ritchie et al., 2001; Moore, 2004). Subsequently, we used the Permutation Testing Module (version 1.0) of Multifactor Dimensionality Reduction to correct our results for multiple testing, based on a 1000-fold permutation test.
Results Here we present a large case-control study composing the analysis of common variation in 8 genes involved in tooth odontogenesis to identify MLIA susceptibility factors. Our sample had a power of 72% to detect a significant association. Genotypic frequencies in the control group were in H-W equilibrium (p > .05). In the cases group, H-W equilibrium deviations were found for some SNPs, although in all these cases, an association with MLIA susceptibility was confirmed. We were able to show the involvement of PAX9, EDA, SPRY2, SPRY4, and WNT10A as risk factors for MLIA. No significant allelic, genotypic, and haplotypic associations were found regarding AXIN2, TGFA, and MSX1 genes (data not shown). All the analyses, were repeated, excluding patients with MLIA and other agenesis (always fewer than 6 teeth), and the results were similar (data not shown). We assessed gene-gene interactions and uncovered 3 strong synergistic interactions between MLIA liability and the following gene pairs: MSX1-TGFA, AXIN2-TGFA, and SPRY2-SPRY4.
Table 1. Clinical and Demographic Characteristics of the Study Sample Study Sample Cases MLIA only Bilateral (12 and 22) Unilateral (12) + microdontia (22) Unilateral (22) + microdontia (12) Unilateral (12) Unilateral (22) MLIA + other agenesis 12; 22; 18; 28; 38; 48 12; 22; 18 12; 22; 45 12; 22; 35 12; 22; 45; 35 12; 35; 45 22; 35; 45 12; 22; 31; 41 12; 22;41 12; 22; 15; 25 12; 32; 42 12; 41; 45; and microdontia (22) 22; 31; 41; and microdontia (13) Males Females Controls Males Females
No. 102 86 50 11 1 12 12 16 1 1 1 1 3 1 1 2 1 1 1 1 1 29 73 204 85 119
MLIA, maxillary lateral incisors agenesis
PAX9 Genotype as a MLIA Risk Factor The A allele of rs8004560 was associated with an increased MLIA susceptibility (p = .02); however, this result did not remain significant after permutation-based correction (Table 2). Importantly, 2 highly significant genotypic associations were found. The AA genotype of rs7149262 was correlated with higher trait susceptibility (p < .001). In agreement with the allelic results, the AA genotype of rs8004560 was associated with an increased MLIA susceptibility (p = .003). These associations remained significant after Bonferroni correction (Table 3). The haplotypic analysis did not reveal any significant association between PAX9 and MLIA susceptibility (data not shown).
Nominal Significant Associations of EDA SNPs With MLIA Susceptibility The A allele of rs1160315, the C allele of rs2428151, the T allele of rs2520378, and the A allele of rs2853659 were associated with an increase in MLIA susceptibility. None of the results remained significant after multiple-testing correction by permutation tests (Table 2). Regarding the genotypic analysis, the GA genotype of rs12853659 presented a higher risk for MLIA (p = .021), while the
Downloaded from jdr.sagepub.com at UNIV OF SOUTH DAKOTA on June 10, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
454
Alves-Ferreira et al.
J Dent Res 93(5) 2014
Table 2. Allelic Association Results for PAX9, EDA, SPRY2, and WNT10A Genes Alleles (%) PAX9 SNP rs8004560 A G EDA SNP rs1160315 A G rs2428151 C T rs2520378 T C rs12853659 A G SPRY2 SNP rs504122 A G WNT10A SNP rs11680244 A G rs2385199 G A rs7349332 T C
Cases
Controls
81 (39.7) 123 (60.3)
123 (30.1) 285 (69.9)
101 (57.7) 74 (42.3)
155 (48.0) 168 (52.0)
117 (66.9) 58 (33.1)
178 (55.1) 145 (44.9)
153 (87.4) 22 (12.6)
256 (79.3) 67 (20.7)
103 (58.9) 72 (41.1)
152 (47.1) 171 (52.9)
89 (43.6) 115 (56.4)
146 (35.8) 262 (64.2)
4 (2.0) 200 (98.0)
9 (2.2) 391 (97.8)
51 (25.0) 153 (75.0)
58 (14.4) 344( 85.6)
36 (17.6) 168 (82.4)
49 (12.2) 353 (87.8)
χ2
Odds Ratio (95% CI)
5.59
1.53 (1.07, 2.17)
4.30
1.48 (1.02, 2.14)
6.49
1.64 (1.12, 2.41)
5.16
1.82 (1.08, 3.07)
6.32
1.61 (1.11, 2.33)
3.54
1.39 (0.99, 1.96)
0.05
1.15 (0.35, 3.78)
10.25
1.98 (1.30, 3.01)
3.34
1.54 (0.97, 2.46)
p .02* .04* .01* .02* .01* .06 .82 .0014* .068
CI, confidence interval; EDA, ectodysplasin A; PAX9, paired box gene 9; SNP, single-nucleotide polymorphism; SPRY2, sprouty homolog 2 gene; WNT10A, wingless-type MMTV integration site family, member 10A. *p < .05.
GA genotype of rs1160315 (p = .015) and the CT of rs5936523 (p = .038) showed a protective effect. However, the results did not remain significant after Bonferroni correction (Table 3). In the haplotypic analysis, a specific EDA haplotype (T-G-TA-A-C-T-C-A-A-A-G-G-T-G-T) revealed a significant association (p = .032) with increased MLIA susceptibility, although it did not retain its significance after permutation correction.
SPRY2 SNP and Increased Risk for MLIA No significant differences were found regarding allelic frequencies of SPRY2 SNPs. However, we observed a trend toward an increased risk for MLIA conferred by the A allele of rs504122 (p = .06) (Table 2). Regarding the genotypic analysis, we found that the GA genotype of rs504122 presented a higher risk for individuals with MLIA (p = .005; OR = 2.49; 95% CI: 1.31, 4.73), which remained significant after Bonferroni correction (Table 3). No
significant association was found between SPRY2 haplotypes and MLIA.
SPRY4 Gene Haplotypes and MLIA Susceptibility The allelic and genotypic analyses did not reveal any significant association between this gene and MLIA susceptibility (data not shown). Importantly, the T-G-G-A-T-C haplotype revealed a nominal significant association (p = .016) with an increased MLIA susceptibility (OR = 3.46; 95% CI: 1.19, 10.08); however, this did not remain significant after permutation-based correction (p = .137).
WNT10A Variants Increase the Risk for MLIA The G allele of rs2385199 was associated with an increased MLIA susceptibility (p = .0014), a result that remained significant after permutation-based correction (Table 2).
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J Dent Res 93(5) 2014 455 Maxillary Lateral Incisor Agenesis Table 3. Results from the PAX9, EDA, SPRY2, and WNT10A Multivariable Logistic Regression Analysis Genotype (%) PAX9 SNP rs8004560 GG (ref) GA AA rs7149262 CC (ref) AC AA EDA SNP rs1160315 GG(ref) GA AA rs5936523 CC(ref) CT TT rs12853659 GG(ref) GA AA SPRY2 SNP rs504122 GG (ref) AA GA WNT10A SNP rs2385199 GG (ref) GA AA
Cases
Controls
Odds Ratio (95% CI)
45 (44.1) 33 (32.4) 24 (23.5)
104 (51.0) 77 (37.7) 23 (11.3)
1.00 (—) 1.27 (0.72, 2.24) 3.03 (1.47, 6.28)
66 (64.7) 23 (22.6) 13 (12.7)
132 (64.7) 68 (33.3) 4 (2.0)
1.00 (—) 0.88 (0.49, 1.58) 9.39 (2.79, 30.97)
15 (44.1) 33 (32.4) 25 (23.5)
30 (51.0) 62 (37.7) 27 (11.3)
1.00 (—) 0.05 (0.01, 0.57) 0.33 (0.02, 5.72)
14 (64.7) 23 (22.6) 36 (12.7)
11 (64.7) 67 (33.3) 41 (2.0)
1.00 (—) 0.46 (0.22, 0.96) 2.59 (0.82, 8.21)
12 (44.1) 38 (32.4) 23(23.5)
31 (51.0) 61 (37.7) 27 (11.3)
25 (24.5) 12 (11.8) 65 (63.7)
81 (39.7) 23 (11.3) 100 (49.0)
1.00 (—) 1.97 (0.73, 5.28) 2.49 (1.31, 4.73)
56 (54.9) 41(40.2) 5 (4.9)
147 (73.1) 50 (24.9) 4 (2.0)
1.00 (—) 2.14 (1.28, 3.58) 3.26 (0.84, 12.58)
1.00 (—) 14.43 (1.51, 138.12) 3.24 (0.21, 49.00)
p .011 .412 .003* .001 .662
