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Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients
ORIGINAL ARTICLE
Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients Fabio Bernardino,b Felix Hoette,c Maria Luiza S. Simas Netta Fontana,a Cleber Machado de Souza,a Jose Maura Levi Hoette,c Lotario Thum,d Terumi O. Ozawa,e Leopoldino Capelozza Filho,e Marcia Olandoski,f and Paula Cristina Trevilattof Curitiba, Parana, Joinville, Santa Catarina, and Bauru, S~ ao Paulo, Brazil
Introduction: Vitamin D is responsible for the regulation of certain genes at the transcription level, via interaction with the vitamin D receptor, and influences host immune responses and aspects of bone development, growth, and homeostasis. Our aim was to investigate the association of TaqI vitamin D receptor gene polymorphism with external apical root resorption during orthodontic treatment. Methods: Our subjects were 377 patients with Class II Division 1 malocclusion, divided into 3 groups: (1) 160 with external apical root resorption #1.43 mm, (2) 179 with external apical root resorption .1.43 mm), and (3) 38 untreated subjects. External apical root resorption of the maxillary incisors was evaluated on periapical radiographs taken before and after 6 months of treatment. After DNA collection and purification, vitamin D receptor TaqI polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate analyses were performed to verify the association of clinical and genetic variables with external apical root resorption (P \0.05). Results: There was a higher proportion of external apical root resorption in orthodontically treated patients compared with the untreated subjects. In patients orthodontically treated, age higher than 14 years old, initial size of the maxillary incisor root superior to 30 mm, and premolar extraction were associated with increased external apical root resorption. Genotypes containing the C allele were weakly associated with protection against external apical root resorption (CC 1 CT 3 TT [odds ratio, 0.29; 95% confidence interval, 0.07-1.23; P 5 0.091]) when treated orthodontic patients were compared to untreated individuals. Conclusions: Clinical factors and vitamin D receptor TaqI polymorphism were associated with external apical root resorption in orthodontic patients. (Am J Orthod Dentofacial Orthop 2012;142:339-47)
Postgraduate student, School of Health and Biosciences, Pontifıcia Universidade Cat olica do Parana, Curitiba-PR, Brazil. b Graduate student, School of Health and Biosciences, Pontifıcia Universidade Cat olica do Parana, Curitiba-PR, Brazil. c Private practice, Curitiba-PR, Brazil. d Chair, Dental Clinics of the Graduation Course in Orthodontics, Thum Institute of Research, Joinville-SC, Brazil. e Professor, Dental Clinics of the Graduate Course in Orthodontics, S~ao Paulo University, Bauru-SP, Brazil. f Professor, School of Health and Biosciences, Pontifıcia Universidade Catolica do Parana, Curitiba-PR, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. The first author was supported by a scholarship from the Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior. Reprint requests to: Paula Cristina Trevilatto, Center for Health and Biological Sciences (CCBS), Pontifıcia Universidade Cat olica do Parana (PUCPR), Rua Imaculada Conceic¸~ao, 1155, 80215-901, Curitiba, PR, Brazil; e-mail, paula. [email protected]. Submitted, October 2011; revised and accepted, April 2012. 0889-5406/$36.00 Copyright Ó 2012 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2012.04.013 a
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xternal apical root resorption (EARR) is a common consequence of orthodontic tooth movement.1-6 Many studies have aimed to elucidate the causal relationship between orthodontic tooth movement and root resorption, but to date this issue is poorly understood, and it is not possible to predict who will develop EARR.4,7,8 The clinical manifestation of EARR in orthodontic patients is highly variable.2 Most orthodontic patients have moderate resorption that does not harm the dentition; in others, it is severe, with an unfavorable prognosis.9 Root resorption during orthodontic movement is considered to be the result of an inflammatory reaction; therefore, some researchers have called this process orthodontically induced inflammatory root resorption.10 Because the inflammatory extent of root resorption is mainly manifested across the root contour, it can be more specifically defined as EARR. 339
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It is believed that EARR is not caused only by orthodontic movement, but, in patients under treatment, it can result from many variables, such as host (genetic aspects)11,12 and environmental (mechanical forces, trauma)6 factors. A difficulty of evaluating the causes of EARR is to separate the contributions made by genetics from environmental factors such as treatment.13 It was reported that genetic factors account for at least 50% of the variation in EARR.11 It has been suggested that there is a strong genetic component for EARR,7,14 estimated in 70% of patients,13 especially when dizygotic are compared to monozygotic twins. In spite of efforts to identify the genetic component for EARR, how many and which genes contribute to the phenotype of EARR are yet largely unknown.13 Vitamin D is responsible for both positive and negative control of certain genes at the level of transcription, via interaction with the vitamin D receptor15 and is important for skeletal growth and bone homeostasis.16 The human vitamin D receptor is a product of 1 gene, which is located on chromosome 12q13-14.17 The gene comprises 9 exons that, together with intervening introns, span approximately 63 kilobases (kb).18 Genome-wide analyses have identified over 100 polymorphisms in the vitamin D receptor gene.19 Polymorphisms refer to the existence of 2 or more alleles at a given locus; if such alleles occur at a frequency of more than 1% in a population, the locus is said to be polymorphic.20 Single nucleotide polymorphisms are the most common form of DNA variation in the human genome. Patterns of linkage disequilibrium in the vitamin D receptor gene were proposed for a Canadian population, and 2 linkage disequilibrium blocks are believed to represent the whole gene.18 Block 1 is located toward the 50 end and spans roughly 8.4 kb, and block 2 is located toward the 30 end of the vitamin D receptor and spans approximately 5.8 kb. Near the 30 untranslated region, there is a single nucleotide polymorphism identified by a restriction site for TaqI enzyme in vitamin D receptor exon 9 (rs731236). This single nucleotide polymorphism might represent the second linkage disequilibrium block.18 Alleles of this polymorphism are in linkage disequilibrium with other polymorphisms in the same block and are linked and inherited together. Allelic variations in this region could be responsible for messenger RNA stability and differences in translational efficiency, resulting in changes in cellular expression of vitamin D receptor.21 This polymorphism has been associated with bone mass, turnover, and mineral loss22 and diseases such as osteoarthritis23 and periodontal disease.24 However, no study has investigated vitamin D receptor gene polymorphisms and their association with EARR.
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Table I. Baseline clinical characteristics of the study
groups Group 1 Group 2 n 5 160 (%) n 5 179 (%)
Group 3 n 5 38 (%)
P value Sex (n)* 86 (53.7%) 99 (55.3%) 23 (60.5%) 0.695 Female Male 74 (46.3%) 80 (44.7%) 15 (39.5%) Age (y)y Mean 6 SD 14.50 6 3.01 15.33 6 2.64 16.46 6 1.93 \0.001 (range) (8-21) (9.9-20) (11-19) Group 1, treated patients with EARR #1.43 mm; group 2, treated patients with EARR .1.43 mm; group 3, untreated subjects. *Chi-square test; ymean 6 standard deviation.
The aim of this study was to investigate the association of TaqI vitamin D receptor enzyme polymorphism (rs731236, exon 9) and clinical variables with EARR in orthodontic patients. MATERIAL AND METHODS
A sample of 377 unrelated white patients of both sexes (mean age, 14.9 years; range, 8-21 years) was selected between 2005 and 2009. Of these, 339 patients had a Class II Division 1 malocclusion and were orthodontically treated with edgewise or straight-wire techniques, and 38 patients had a Class II Division 1 malocclusion but were untreated. The reason for Class II Division 1 subjects was because this type of malocclusion is the most frequent and requires more treatment25; in addition, it can lead to higher levels of EARR.1,26,27 The patients were selected from the Dental Clinics of the Graduate Course in Orthodontics (Bauru, S~ao Paulo, Brazil), Dental Clinics of the Graduate Course in Orthodontics of Thum Institute of Research (Joinville, Santa Catarina, Brazil), and 2 private orthodontic clinics (Curitiba, Parana, Brazil) (Table I). Although all subjects were white, the Brazilian white population is heterogeneous. Recent articles have not recommended grouping Brazilians into ethnic groups based on color, race, and geographic origin because Brazilians classified as white or black have significantly overlapping genotypes, probably due to miscegenation.28 The subjects completed personal, medical, and dental history questionnaires and, with the protocol approved by the institutional review board of Pontifical Catholic University of Parana, signed a consent form after being advised of the nature of the study (approved by the ethical committee in research at Pontificia Unieversidade Cat olica do Parana, protocol number 546/05). The patients could not have chronic usage of antiinflammatory drugs, human immunodeficiency virus
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infection, and immunosuppressive chemotherapy history of any disease known to severely compromise immune function, current pregnancy or lactation, oral trauma, parafunctional behavior, endodontic treatment, or extensive caries lesions. The periapical x-rays of the maxillary central incisors with the longer roots (reference teeth) were taken before treatment and 6 months after the beginning of treatment. The evaluation method included measuring the root and crown lengths directly from the radiographs. The root apex, incisal edge, and cementoenamel junction of each tooth were marked on the x-rays on a light table. The longitudinal axis of each tooth was constructed from the root apex to the incisal edge following the root canal as accurately as possible. A perpendicular axis was then projected to the longitudinal axis from the mesial to the distal cementoenamel junction sides. The crown length was measured from the incisal edge to the projected cementoenamel junction, and the root length from the projected cementoenamel junction to the root apex (Fig 1). The differences between the 2 measurements indicate the EARR. A correction factor was calculated by using the following formula: correction factor 5 C1/C2 (C1, crown length before treatment; C2, crown length 6 months after starting treatment). Then EARR was calculated with the following formula: EARR 5 R1 – (R2 3 CF) (R1, root length before treatment; R2, root length 6 months after treatment start; CF, correction factor). EARR was also expressed as a percentage of the original root length: EARR 3 100/R1. Only teeth with complete root formation were considered for investigation. Any distortions between the pretreatment and follow-up radiographic images were corrected by using the crown length registration, assuming crown length to be unchanging over the observation period.29,30 The EARR was evaluated by 1 examiner (M.L.S.S.N.F). The radiographs were examined over a light table, and the measurements were made with a fine-tip digital caliper with accuracy up to 0.02 mm (electronic digital vernier caliper; Utustools Professional, Santiago, Chile). A receiver operating characteristic curve was constructed to verify the cutoff point based on the sample data distribution for EARR, and the value of 1.43 mm was obtained. Then, the sample was divided into 3 groups: (1) 160 treated patients with EARR 1.43 mm or less, (2) 179 treated patients with EARR greater than 1.43 mm, and (3) 38 untreated subjects. The following parameters were evaluated in the treated patients: age, sex, initial size of the root of the reference tooth, premolar extraction, use of pendulum appliance, rapid palatal expansion, and use of elastics (Table II).
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Fig 1. A, Anatomic references to measure EARR: cementoenamel junction (CEJ); B, references of measurements in the x-rays.
Cells were obtained by a mouthwash with 3% glucose solution for 1 minute and by scraping the oral mucosa with a sterile spatula.31 DNA was extracted from epithelial oral cells with ammonium acetate (10 mol/L) and EDTA (1 mmol/L).32 The following primer pair was used for polymerase chain reaction amplification of genomic DNA samples: (F – 50 CAG AGC ATG GAC AGG GAG CAA G 30 and R – 50 GGA TGT ACG TCT GCA GTG TG 30 ). Reaction conditions and cycling parameters were as follows: 1 mL of the genomic DNA was used for polymerase chain reaction amplification in a reaction mixture containing 22.5 mL GoTaq Green Master Mix (Promega, Madison, Wis) and 0.7 mL of each 25 mmol/L primer. The reactions were performed in a thermal cycler (T-512; Techne, Chelmsford, UK) and consisted of an initial denaturation step at 95 C for 5 minutes, followed by 37 cycles at 95 C for 1 minute, 55 C for 1 minute, 72 C for 1 minute, and a final extension at 72 C for 7 minutes. The restriction fragment length polymorphism technique was performed in a final reaction volume of 20 mL, by using 1 unit of TaqI (TYCGA) (Invitrogen Life Technologies, Carlsbad, Calif) and a 10-mL aliquot of polymerase chain reaction products, digested at 65 C overnight. The digested products were separated in 10% polyacrylamide gel electrophoresis stained by silver. The genotypes were determined by comparing the restriction fragment length polymorphism band patterns with a 1-kb-plus DNA ladder (Invitrogen Life Technologies). The restriction fragment length polymorphism is formed by a single base transition (TaqI enzyme) at codon 352 in exon 9 of the vitamin D receptor gene that creates a TaqI
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Table II. Clinical variables influencing EARR in treated patients EARR Variable Age (y)z #14 .14 Sex Female Male Initial root (mm)§ \30 $30 Premolar extraction No Yes Elastics No Yes Rapid palatal expansion No Yes Pendulum No Yes Genotype TT TC 1 CC
P value* (univariate) P valuey (multivariate) #1.43 mm .1.43 mm 95% Confidence n n 5 160 (47.2%) n 5 179 (52.8%) n 5 339 n 5 339 Odds ratio interval 147 192
80 (54.4) 80 (41.7)
67 (45,6) 112 (58.3)
0.021
185 154
86 (46.5) 74 (48.1)
99 (53.5) 80 (51.9)
0.827
258 81
133 (51.6) 27 (33.3)
125 (48.4) 54 (66.7)
291 48
143 (49.1) 17 (35.4)
276 63
0.022
1.69
1.08-2.66
0.005
0.002
2.34
1.36-4.03
148 (50.9) 31 (64.6)
0.087
0.052
1.94
0.99-3.78
133 (48.2) 42 (42.9)
143 (51.8) 36 (57.1)
0.486
285 54
130 (45.6) 30 (55.6)
155 (54.4) 24 (44.4)
0.185
252 87
113 (44.8) 47 (54)
139 (55.2) 40 (46)
0.171
58 274
33 (56.9) 124 (45.3)
25 (43.1) 150 (54.7)
0.114
0.104
1.64
0.90-2.97
y
*Fisher exact test, P \0.05; regression logistic model, Wald test, P \0.05 (variables were included when P \0.20 in univariate analysis); zcutoff point (14 years) suggested by receiver operating characteristic curve (0.574, P 5 0.017); §cutoff point (30 mm) suggested by receiver operating characteristic curve (0.620, P \0.001).
restriction site. The alleles that result from the cleavage of TaqI are designated “C” (TaqI site present, with 2 fragments: 293 base pairs and 47 base pairs) or “T” (TaqI site absent, with a fragment: 340 base pairs).
Mean; mean + se; mean + sd
32 p<0.001 p<0.001
p=0.067
30
The results observed in the study were expressed in means and standard deviations (quantitative variable) or frequencies and percentages (qualitative variable). To evaluate the association between 2 qualitative variables, the chi-square test or the Fisher exact test was used. Comparisons between the groups in relation to the quantitative variables were made with analysis of variance (ANOVA) with 1 factor and the least significant difference test for multiple comparisons. Adjustments of the receiver operating characteristic curve were made for EARR, age, and initial root length to determine cutoff points associated with EARR. The unpaired t test was used to compare EARR, age, and initial root length between the groups. For the multivariate analysis, the logistic regression model and the Wald test were used. Values of P \0.05 indicated statistical significance. Data were organized in an Excel spread
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EARR (mm)
Statistical analysis 28
26
24
Group 1
Group 2
Group 3
Initial Final
Fig 2. Comparison of the EARR values (initial and final root length) among the groups (paired t test): group 1, treated patients with EARR #1.43 mm; group 2, treated patients with EARR .1.43 mm; group 3, untreated subjects.
sheet (Microsoft, Redmond, Wash) and analyzed with the computational program Statistica (version 8.0; StatSoft, Tulsa, Okla).
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Table III. Genotype and allele distribution of the vitamin D receptor TaqI polymorphism between the groups Genotypes TT TC CC Alleles T C
Group 1, n 5 157 (%) 33 (21.02) 117 (74.52) 7 (4.46) Group 1, n 5 314 (%) 183 (58.28) 131 (50.00)
Group 2, n 5 175 (%) 25 (14.29) 139 (79.43) 11 (6.29) Group 2, n 5 350 (%) 164 (46.85) 138 (39.42)
Group 3, n 5 35 (%) 2 (5.71) 33 (94.29) 0 (0.00) Group 3, n 5 70 (%) 35 (50.00) 33 (47.14)
P value* 0.051 P value* 0.455
Group 1, EARR #1.43 mm; group 2, EARR .1.43 mm; group 3, untreated subjects. *Chi-square test, P \0.05.
RESULTS
We observed a higher proportion of EARR in the treated patients (EARR #1.43 mm, 0.81 mm; EARR .1.43 mm, 2.24 mm) compared with the untreated subjects (EARR, 0.05 mm). The clinical impact of orthodontic appliances on root resorption is shown in Figure 2. Regarding the treated patients, no statistically significant differences were observed between the groups in relation to sex, pendulum appliance, rapid palatal expansion, or elastics. A statistically significant difference was found between the groups regarding age (P 5 0.021) and initial size of the root of the reference tooth (P 5 0.005) in the univariate analysis. After the multivariate analysis, age (P 5 0.022), initial size of the root of the reference tooth (P 5 0.002), and premolar extractions (P 5 0.052) were associated with EARR (Table II). Considering the study's single nucleotide polymorphism, the genotype distribution was not consistent with the assumption of Hardy-Weinberg equilibrium either in the control group or for the whole sample. Differences were observed in vitamin D receptor TaqI polymorphism genotype frequency (P 5 0.051), but not in allele distribution (P 5 0.455) between the groups (Table III). However, when the treated patients were analyzed vs the untreated subjects, we observed a weak protection effect of allele C against EARR (CC 1 CT 3 TT [odds ratio, 0.29; 95% confidence interval, 0.071.23; P 5 0.091]). DISCUSSION
Most clinicians are highly concerned about EARR as an undesirable side effect of orthodontic treatment. The etiology of EARR has been studied for the past few decades but remains unclear.33 It is believed that no orthodontic tooth movement is possible without some EARR,34 but, in most cases, it will be minor and therefore has no clinical significance.35 However, moderate to severe root resorption has been reported to occur with a frequency of 10% to 20%.1,36
The cause of EARR is considered to be multifactorial.37 Several factors have been mentioned to influence EARR,5 both mechanical and biological.38 Several studies have suggested that excessive orthodontic force,39,40 tooth intrusion,41 rapid palatal expansion,10 and tooth movement against hard and highly mineralized tissues are critical factors for EARR.10 Regarding the biologic aspects, associations between EARR and age, sex,42 tooth morphology, periodontal condition,5 immune response,43,44 bone metabolism,45,46 and systemic and genetic factors2 have been suggested. In this study, impacts of age, initial length of the root of the maxillary central incisor (reference tooth), and premolar extraction were found on EARR in the treated subjects. It was recently reported that older patients have more EARR than do younger ones.47 However, to our knowledge, no other studies in the literature have reported that root length influences EARR. It was reported that incisors' root resorption increased from 15% before treatment to 73% after treatment,48,49 and the number of teeth with moderate and severe root resorption increased from 1% before treatment to 25% after treatment.50 We also identified an association of EARR with orthodontic treatment with extractions, in accordance to other studies.1,26,27,30 However, other authors failed to find a relationship between EARR and premolar extraction.6,47 Regarding sex, there was no statistical difference between orthodontically treated patients with and without EARR; this has been reported by several authors.13,30,47 However, Baumrind et al51 found a higher prevalence of EARR in men than in women, and Kjaer52 observed more EARR among female than male subjects. No statistical difference was found in relation to the pendulum appliance in our study. All patients who used the pendulum were treated with the straightwire technique, and all patients who did not use the pendulum were treated with the edgewise technique. No influence was observed of the type of orthodontic appliance on EARR in other studies,30,37 but Mavragani et al53 found greater EARR in patients
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Table IV. Summary of studies investigating the genetic aspects of EARR Authors (year) Newman (1975)14
Type of study Descriptive
Harris et al (1997)7 Twin study
AL-Qawasmi et al (2003)2
Sample (n) 47 subjects (case: 41; control: 6)
103 twin pairs
Family-based 35 families association study (118 persons: 73 siblings and 45 parents)
Mean age (y) 14.5
Population X-ray Malocclusion Results North American Pe 50% Class I Familial aggregation suggested
boys: 14.5 North American
P
girls: 13.3
C
12.1
North American
P
C AL-Qawasmi et al (2003)3
Linkage analysis
38 families (124 persons; 79 siblings and 45 parents)
12.3
North American
P
C Ngan et al (2004)13 Twin study
26 twin pairs (16 MZ and 10 DZ)
13.5 MZ Australian
P
12.9 DZ Austrian Lages et al (2009)63 Population-based 61 persons association study (case: 23; control: 38)
?
Brazilian
Pe
27% Class II D1 6.8% Class II D2 9.1% Class III 30% Class I 70% heritability to EARR of MCI and LM 63% Class II 9% Class III Class I IL1A (-889) polymorphism not associated. IL1B (13954) genotype CC (OR, 5.6; 95% CI, 1.9-21.2; P 5 0.0003) associated with EARR.2mm in MCI, MdCI, LM Class II Class III Class I Evidence for linkage of D18S64 microsatellite marker [lightly linked to TNFRSF11A (RANK)] with EARR .2 mm (LOD 5 2.5; P 5 0.02) in MCI Class II Class III Class I 49% heritability to EARR in MCI and 66% in LM Class II Class III 54% Class I
Allele C of IL1B (13954) is associated with EARR (OR, 4.0; 95% CI, 1.23-12.9; P \0.05) in MCI and MLI
39% Class II 6% Class III Gulden et al (2009)8 Population-based 94 persons association study (case: 45; control: 49)
$12
North American
P
IL1B (13954) polymorphism not associated with EARR. Genotype TT of IL1A (-889) polymorphism is associated with EARR (P \0.032)
Pe, Periapical x-ray; P, panoramic x-ray; C, cephalometric x-ray; MZ, monozygotic; DZ, dizygotic; MCI, maxillary central incisor; LM, lower molar; MdCI, mandibular central incisor; MLI, maxillary lateral incisor; D, Division; OR, odds ratio; CI, confidence interval; LOD, logarithm of the odds (to the base 10).
treated with the edgewise technique than the straight wire. Data from this study suggest that the maxillary incisors have no susceptibility to EARR during rapid palatal expansion; however, other authors have found such an association.54 All patients who used the Haas appliance in this study also used the pendulum
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appliance. Finally, in our study, no significant difference was found when Class II elastics were used, in contrast with other authors.53 The x-ray is the commonly used method to diagnose EARR and is considered to be effective and predictive.8,55 However, it has limitations, such as standardization,
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radiation exposure, and a restricted view.9 Moreover, the radiographic method is static and cannot indicate whether root resorption has finished or is ongoing; it just shows the result of the EARR process.8,56,57 So far, EARR has been measured on lateral cephalometric and panoramic radiographs.2,7 When diagnosis with those types of x-rays is thought to be imprecise and questionable, standardized intraoral radiographs should be used instead because they provide more detailed information.29,58 In this study, periapical xrays were used to measure EARR. Even in the periapical x-rays, the image interpretations of the resorption have limitations, but they are the most indicated method of analysis among the accessible ones.59 Although EARR can even be found in untreated subjects,37 the results of this study are consistent with the literature showing that teeth without forces have less resorption than teeth that have undergone orthodontic treatment.30 Our findings of a clinically significant association between the degree of EARR and orthodontic treatment suggest that patients susceptible to EARR can be identified early in treatment.60,61 It has been shown that EARR of the maxillary incisors, observed during the initial months of treatment, could be a predictor of a higher risk for continuing resorption during treatment.50,60 Consequently, it has been recommended that periapical x-rays should be obtained after the first 6 months of treatment.60-62 Susceptibility to EARR is believed to have a genetic basis.7,13,14 The genetic component controlling the occurrence and extent of EARR is from 50% to 70% and was especially identified in the maxillary and mandibular central incisors.7,13 This might be because those teeth are more subject to anterior retraction forces in patients with Class II Division 1 malocclusion during orthodontic treatment. Efforts to investigate host factors influencing EARR are scarce and have focused on linkage and association analysis (Table IV). Concerning linkage analysis, only 1 study found evidence for linkage of TNFRSF11A (RANK) with EARR.3 In the association studies, only 2 candidate genes of the immune-inflammatory response have been investigated: IL1A and IL1B, coding for IL1a and IL-1b proinflammatory mediators, respectively.2,9,63 Evidence for an association was identified between EARR and alleles from IL1A and IL1B gene polymorphisms. In the case of IL1A (–889) polymorphism, a significant difference in the genotype distribution was found between EARR patients and the control group, with an augmentation of genotype TT in the group with EARR in a North American population.9 Concerning IL1B (13954), allele C63 and
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genotype CC2 were significantly associated with the EARR in Brazilian and North American populations, respectively. Vitamin D is the major regulator of calcium homeostasis and protects the organism from calcium deficiency via effects on the intestines, kidneys, and bones. Vitamin D is well known as a hormone involved in mineral metabolism and bone formation, and its effect is to facilitate intestinal absorption of calcium and phosphate.64 Many effects of vitamin D on bones have been demonstrated, as a synthesis of bone matrix protein such as type I collagen, alkaline phosphatase, osteocalcin, and osteopontin.65,66 Studies have reported positive associations between osteoporosis or low bone density and alveolar bone and tooth loss.67 Vitamin D also inhibits lymphocyte proliferation and stimulates monocyte differentiation.17 Thus, there is considerable scientific evidence that vitamin D has a variety of effects on immune system functions that might enhance innate immunity and inhibit the development of autoimmunity.68 The effects of vitamin D on cells are thought to be mediated through the vitamin D receptor that belongs to the steroid receptor superfamily,69 a transcription factor regulating gene expression in several cell types, including osteoblasts.70 Polymorphisms in the vitamin D receptor gene have been associated with bone mineral density, bone turnover, and diseases in which mineral loss is a cardinal sign.71 However, to our knowledge, this is the first study to investigate the association of vitamin D receptor polymorphisms and susceptibility to EARR. We observed an enrichment of heterozygotes in the subjects genotyped for polymorphism vitamin D receptor TaqI (rs731236) in this study. The inclusion of subjects with Class II Division 1 malocclusion might have favored the selection of such a genotype. It can be suggested that the craniofacial grow pattern could be related to the heterozygosity. Also, genotypes containing the C allele were observed to be weakly associated with protection against root resorption in the treated when compared to untreated patients. Although the literature shows some controversy, it seems that C allele of polymorphism TaqI increases mRNA stability and vitamin D receptor expression.72 The protection effect of allele C might only be observed in orthodontic patients. The mechanical forces might regulate gene expression during orthodontic treatment though a mechanism involving transducing molecules and mechanosensitive ion channels.73 CONCLUSIONS
We observed that clinical aspects such as age, initial root length, premolar extractions, and vitamin D
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receptor TaqI polymorphism (rs731236) were associated with EARR in orthodontic patients. REFERENCES 1. Brin I, Tulloch JFC, Koroluk L, Philips C. External apical root resorption in Class II malocclusion: a retrospective review of 1- versus 2phase treatment. Am J Orthod Dentofacial Orthop 2003;124:151-6. 2. AL-Qawasmi RA, Hartsfield JK, Everett ET, Flury L, Liu L, Foroud TM, et al. Genetic predisposition to external apical root resorption. Am J Orthod Dentofacial Orthop 2003;123: 242-52. 3. AL-Qawasmi RA, Hartsfield JK, Everett ET, Flury L, Liu L, Foroud TM, et al. Genetic predisposition to external apical root resorption in orthodontic patients: linkage of chromosome-18 marker. J Dent Res 2003;82:356-60. 4. Licata ME, Guiglia R, Giuliana G. Root resorption and orthodontic treatment. Review of the literature. Minerva Stomatol 2007;56: 31-44. 5. Huang Y, Wang X, Zhang J, Liu C. Root shortening in patients treated with two-step and en masse space closure procedures with sliding mechanics. Angle Orthod 2010;80:492-7. 6. Zhuang L, Bai Y, Meng X. Three-dimensional morphology of root and alveolar trabecular bone during tooth movement using microcomputed tomography. Angle Orthod 2011;81:420-5. 7. Harris EF, Kineret SE, Tolley EA. A heritable component for external apical root resorption in patients treated orthodontically. Am J Orthod Dentofacial Orthop 1997;111:301-9. 8. G€ ulden N, Eggermann T, Zerres K, Beer M, Meinett A, Deidrich P. Interleukin-1 polymorphisms in relation to external apical root resorption (EARR). J Orofac Orthop 2009;3:20-38. 9. Mah J, Prasad N. Dentine phosphoproteins in gingival crevicular fluid during root resorption. Eur J Orthod 2004;26:25-30. 10. Liu Z, Xu J, Lingling E, Wang D. Ultrasound enhances the healing of orthodontically induced root resorption in rats. Angle Orthod 2012;82:48-55. 11. Hartsfield JK, Everett ET, Al-Qawasmi RA. Genetic factors in external apical root resorption and orthodontic treatment. Crit Rev Oral Biol Med 2004;15:115-22. 12. Hartsfield JK. Pathways in external apical root resorption associated with orthodontia. Orthod Craniofac Res 2009;12: 236-42. 13. Ngan DC, Kharbanda OP, Byloff FK, Darendeliler MA. The genetic contribution to orthodontic root resorption: a retrospective twin study. Aust Orthod J 2004;20:1-9. 14. Newman WG. Possible etiologic factors in external root resorption. Am J Orthod 1975;67:522-39. 15. Sutton AL, MacDonald PN. Vitamin D: more than a “bone-a-fide” hormone. Mol Endocrinol 2003;17:777-91. 16. Davideau JL, Lezot F, Kato S, Bailleul-Forestier I, Berdal A. Dental alveolar bone defects related to vitamin D and calcium status. J Steroid Biochem Mol Biol 2004;89-90:615-8. 17. Labuda M, Fujiwara TM, Ross MV, Morgan K, Garcia-Heras J, Ledbetter DH, et al. Two hereditary defects related to vitamin D metabolism map to the same region of human chromosome 12q13-14. J Bone Miner Res 1992;7:1447-53. 18. Poon AH, Laprise C, Lemire M, Montpetit A, Sinnett D, Schurr E, et al. Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy. Am J Respir Crit Care Med 2004; 170:967-73. 19. Uitterlinden AG, Fang Y, Bergink AP, van Meurs JB, van Leeuwen HP, Pols HA. The role of vitamin D receptor gene polymorphisms in bone biology. Mol Cell Endocrinol 2002;197:15-21.
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20. Chiba-Falek O, Nussbaum RL. Effect of allelic variation at the NACP-Rep1 repeat upstream of the alpha-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system. Hum Mol Genetics 2001;10:3101-9. 21. Durrin LK, Haile RW, Ingles SA, Coetzee GA. Vitamin D receptor 3'untranslated region polymorphisms: lack of effect on mRNA stability. Biochim Biophys Acta 1999;1453:311-20. 22. Karkoszka H, Chudek J, Strzelczyk P, Wiecek A, Schmidt-Gayk H, Ritz E, et al. Does the vitamin D receptor genotype predict bone mineral loss in haemodialysed patients? Nephrol Dial Transplant 1998;13:2077-80. 23. Horst-Sikorska W, Wawrzyniak A, Celczynska-Bajew L, Marcinkowska M, Dabrowski S, Kalak R, et al. Polymorphism of VDR gene—the most effective molecular marker of osteoporotic bone fractures risk within postmenopausal women from Wielkopolska region of Poland. Endokrynol Pol 2005;56:233-9. 24. Brito Junior RB, Scarel-Caminaga RM, Trevilatto PC, de Souza AP, Barros SP. Polymorphisms in the vitamin D receptor gene are associated with periodontal disease. J Periodontol 2004;75:1090-5. 25. Silva Filho OG, Ferrari Junior FM, Ozawa TO. Dimens~oes dos arcos dentarios na ma oclus~ao de Classe II divis~ao 1 com defici^encia mandibular. Rev Dent Press Ortodon Ortop Facial 2009;14: 120-30. 26. Tanner T, Ciger S, Sencift Y. Evaluation of apical root resorption following extraction therapy in subjects with Class II and Class I malocclusions. Eur J Orthod 1999;21:491-6. 27. Liou EJ, Chang PM. Apical root resorption in orthodontic patients with en-masse maxillary anterior retraction and intrusion with miniscrews. Am J Orthod Dentofacial Orthop 2010;137: 207-12. 28. Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM, Pena SD. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci USA 2003;7:177-82. 29. Linge L, Linge BO. Patient characteristics and treatment variables associated with apical root resorption during orthodontic treatment. Am J Orthod Dentofacial Orthop 1991;99:35-43. 30. Mohandesan H, Ravanmehr H, Valaei N. A radiographic analysis of external apical root resorption of maxillary incisors during active orthodontic treatment. Eur J Orthod 2007;29:134-9. 31. Trevilatto PC, Line SR. Use of buccal epithelial cells for PCR amplification of large DNA fragments. J Forensic Odontostomatol 2000; 18:6-9. 32. Aidar M, Line SR. A simple and cost-effective protocol for DNA isolation from buccal epithelial cells. Braz Dent J 2007;18: 148-52. 33. Gonzales C, Hotokeka H, Yoshimatsu M, Yozgation JH, Darendeliler MA, Yoshida N. Force magnitude and duration effects on amount of tooth movement and root resorption in the rat molar. Angle Orthod 2008;78:502-9. 34. Reitan K, Rygh P. Biomechanical principles and reactions. In: Graber TM, Vanarsdall RL, editors. Orthodontics, current principles and techniques. 2nd ed. St Louis: Mosby; 1994. p. 168-9. 35. Loenen M, Dermaut LR, Degrieck J, De Pauw GA. Apical root resorption of upper incisors during the torquing stage of the tipedge technique. Eur J Orthod 2007;29:583-8. 36. Levander E, Malmgren O. Evaluation of the risk of root resorption during orthodontic treatment: a study of upper incisors. Eur J Orthod 1988;10:30-8. 37. Lopatiene K, Dumbravaite A. Risk factors of root resorption after orthodontic treatment. Stomalogija 2008;10:89-95. 38. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part I: the basic science aspects. Angle Orthod 2002;72:175-9.
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39. Chan E, Darendeliler MA. Physical properties of root cementum: part 5. Volumetric analysis of root resorption craters after application of light and heavy orthodontic forces. Am J Orthod Dentofacial Orthop 2005;127:186-95. 40. Harris DA, Jones AS, Darendeliler MA. Physical properties of root cementum: part 8. Volumetric analysis of root resorption craters after application of controlled intrusive light and heavy orthodontic forces: a microcomputed tomography scan study. Am J Orthod Dentofacial Orthop 2006;130:639-47. 41. Han G, Huang S, Von den Hoff JW, Zeng X, Kuijpers-Jagtman AM. Root resorption after orthodontic intrusion and extrusion: an intraindividual study. Angle Orthod 2005;75:912-8. 42. George A, Evans CA. Detection of root resorption using dentin and bone markers. Orthod Craniofac Res 2009;12:229-35. 43. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. CD40-CD40L expression during orthodontic tooth movement in rats. Angle Orthod 2004;74:100-5. 44. Nishioka M, Ioi H, Nakata S, Nakasima A, Counts A. Root resorption and immune system factors in the Japanese. Angle Orthod 2006;76:103-8. 45. Verna C, Melsen B. Tissue reaction to orthodontic tooth movement in different bone turnover conditions. Orthod Craniofac Res 2003; 6:155-63. 46. Takada K, Kajiya H, Fukushima H, Okamoto F, Motokawa W, Okabe K. Calcitonin in human odontoclasts regulates root resorption activity via protein kinase A. J Bone Miner Metab 2004;22: 12-8. 47. Pandis N, Nasika M, Polychronopoulou A, Eliades T. External apical root resorption in patients treated with conventional and selfligating brackets. Am J Orthod Dentofacial Orthop 2008;134: 646-51. 48. Harris RJ. Successful root coverage: a human histologic evaluation of a case. Int J Periodontics Restorative Dent 1999;19: 439-47. 49. Sameshima GT, Sinclair PM. Characteristics of patients with severe root resorption. Orthod Craniofac Res 2004;7:108-14. 50. Wierzbicki T, El-Bialy T, Aldaghreer S, Li G, Doschak M. Analysis of orthodontically induced root resorption using micro-computed tomography (micro-CT). Angle Orthod 2009;79:91-6. 51. Baumrind S, Korn EL, Boyd RL. Apical root resorption in orthodontically treated adults. Am J Orthod Dentofacial Orthop 1996;110: 311-20. 52. Kjaer I. Morphological characteristics of dentitions developing excessive root resorption during orthodontic treatment. Eur J Orthod 1995;17:25-34. 53. Mavragani M, Vergari A, Selliseth NJ, Boe OE, Wisth PJ. A radiographic comparison, of apical root resorption after orthodontic treatment with a standard edgewise and a straight-wire edgewise technique. Eur J Orthod 2000;22:665-74. 54. Vardimon AD, Levy T, Weinreb M. Maxillary incisor root resorption after rapid palatal expansion in Feliscatus. Eur J Oral Sci 2005;113: 41-6. 55. Sameshima GT, Asgarifar KO. Assessment of root resorption and root shape: periapical vs panoramic films. Angle Orthod 2001; 71:185-9. 56. Jiang RP, Zhang D, Fu MK. A factors study of root resorption after orthodontic treatment. Zhonhua Kou Qiang Yi Xue Za Zhi 2003; 38:455-7.
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57. Makedonas D, Odman A, Hansen K. Management of root resorption in a large orthodontic clinic. Swed Dent J 2009;33:173-80. 58. Levander E, Malmgren O, Stenback K. Apical root resorption during orthodontic treatment of patients with multiple aplasia: a study of maxillary incisors. Eur J Orthod 1998;20:427-34. 59. Consolaro A. Sobre alguns aspectos metodol ogicos de pesquisas ~es dentarias: em movimentac¸~ao dentaria induzida e das reabsorc¸o uma proposta de guia e cuidados para analise de trabalhos. R Dent Press Ortodon Ortop Facial 2004;9:104-9. 60. Levander E, Bajka R, Malmgren O. Early radiographic diagnosis of apical root resorption during orthodontic treatment: a study of maxillary incisors. Eur J Orthod 1998;20:57-63. 61. Levander E, Malmgren O. Long-term follow-up of maxillary incisors with severe apical root resorption. Eur J Orthod 2000;22: 85-92. 62. Levander E, Malmgren O, Eliasson S. Evaluation of root resorption in relation to two orthodontic treatment regimes. A clinical experimental study. Eur J Orthod 1994;16:223-8. 63. Lages EMB, Drummond AF, Pretti H, Costa FO, Lages EJ, Gontijo AI, et al. Association of functional gene polymorphism IL-1beta in patients with external apical root resorption. Am J Orthod Dentofacial Orthop 2009;136:542-6. 64. Fleet JC. Molecular regulation of calcium and bone metabolism through the vitamin D receptor. J Musculoskelet Neuronal Interact 2006;6:336-7. 65. Gallagher JC, Riggs BJ. Action of 1,25-dihydroxyvitamin D3 on calcium balance and bone turnover and its effect on vertebral fracture rate. Metabolism 1990;39:30-4. 66. Jones G, Hogan DB, Yendt E, Hanley DA. Prevention and management of osteoporosis: consensus statements from the Scientific Advisory Board of the Osteoporosis Society of Canada. 8. Vitamin D metabolites and analogs in the treatment of osteoporosis. CMAJ 1996;155:955-61. 67. Naito M, Miyaki K, Naito T, Zhang L, Hoshi K, Hara A, et al. Association between vitamin D receptor gene haplotypes and chronic periodontitis among Japanese men. Int J Med Sci 2007;22: 216-22. 68. Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation. Ann Rev Nutr 2003;23:117-45. 69. Sinotte M, Rousseau F, Ayotte P, Dewailly E, Berube S, Brisson J. Vitamin D receptor polymorphisms (FokI, BsmI) and breast cancer risk: association replication in two case-control studies within French Canadian population. Endocr Relat Cancer 2008;15: 975-83. 70. Masuyama R, Stockmans I, Torrekens S, Van Looveren R, Maes C, Carmeliet P, et al. Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. J Clin Invest 2006;116:3150-9. 71. Gunes S, Sumer AP, Keles GC, Kara N, Koprulu H, Bagci H, et al. Analysis of vitamin D receptor gene polymorphisms in patients with chronic periodontitis. Indian J Med Res 2008; 127:58-64. 72. Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene 2004;1(338):143-56. 73. Kung C. A possible unifying principle for mechanosensation. Nature 2005;436:647-54.
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Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients Fabio Bernardino,b Felix Hoette,c Maria Luiza S. Simas Netta Fontana,a Cleber Machado de Souza,a Jose Maura Levi Hoette,c Lotario Thum,d Terumi O. Ozawa,e Leopoldino Capelozza Filho,e Marcia Olandoski,f and Paula Cristina Trevilattof Curitiba, Parana, Joinville, Santa Catarina, and Bauru, S~ ao Paulo, Brazil
Introduction: Vitamin D is responsible for the regulation of certain genes at the transcription level, via interaction with the vitamin D receptor, and influences host immune responses and aspects of bone development, growth, and homeostasis. Our aim was to investigate the association of TaqI vitamin D receptor gene polymorphism with external apical root resorption during orthodontic treatment. Methods: Our subjects were 377 patients with Class II Division 1 malocclusion, divided into 3 groups: (1) 160 with external apical root resorption #1.43 mm, (2) 179 with external apical root resorption .1.43 mm), and (3) 38 untreated subjects. External apical root resorption of the maxillary incisors was evaluated on periapical radiographs taken before and after 6 months of treatment. After DNA collection and purification, vitamin D receptor TaqI polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate analyses were performed to verify the association of clinical and genetic variables with external apical root resorption (P \0.05). Results: There was a higher proportion of external apical root resorption in orthodontically treated patients compared with the untreated subjects. In patients orthodontically treated, age higher than 14 years old, initial size of the maxillary incisor root superior to 30 mm, and premolar extraction were associated with increased external apical root resorption. Genotypes containing the C allele were weakly associated with protection against external apical root resorption (CC 1 CT 3 TT [odds ratio, 0.29; 95% confidence interval, 0.07-1.23; P 5 0.091]) when treated orthodontic patients were compared to untreated individuals. Conclusions: Clinical factors and vitamin D receptor TaqI polymorphism were associated with external apical root resorption in orthodontic patients. (Am J Orthod Dentofacial Orthop 2012;142:339-47)
Postgraduate student, School of Health and Biosciences, Pontifıcia Universidade Cat olica do Parana, Curitiba-PR, Brazil. b Graduate student, School of Health and Biosciences, Pontifıcia Universidade Cat olica do Parana, Curitiba-PR, Brazil. c Private practice, Curitiba-PR, Brazil. d Chair, Dental Clinics of the Graduation Course in Orthodontics, Thum Institute of Research, Joinville-SC, Brazil. e Professor, Dental Clinics of the Graduate Course in Orthodontics, S~ao Paulo University, Bauru-SP, Brazil. f Professor, School of Health and Biosciences, Pontifıcia Universidade Catolica do Parana, Curitiba-PR, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. The first author was supported by a scholarship from the Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior. Reprint requests to: Paula Cristina Trevilatto, Center for Health and Biological Sciences (CCBS), Pontifıcia Universidade Cat olica do Parana (PUCPR), Rua Imaculada Conceic¸~ao, 1155, 80215-901, Curitiba, PR, Brazil; e-mail, paula. [email protected]. Submitted, October 2011; revised and accepted, April 2012. 0889-5406/$36.00 Copyright Ó 2012 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2012.04.013 a
E
xternal apical root resorption (EARR) is a common consequence of orthodontic tooth movement.1-6 Many studies have aimed to elucidate the causal relationship between orthodontic tooth movement and root resorption, but to date this issue is poorly understood, and it is not possible to predict who will develop EARR.4,7,8 The clinical manifestation of EARR in orthodontic patients is highly variable.2 Most orthodontic patients have moderate resorption that does not harm the dentition; in others, it is severe, with an unfavorable prognosis.9 Root resorption during orthodontic movement is considered to be the result of an inflammatory reaction; therefore, some researchers have called this process orthodontically induced inflammatory root resorption.10 Because the inflammatory extent of root resorption is mainly manifested across the root contour, it can be more specifically defined as EARR. 339
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It is believed that EARR is not caused only by orthodontic movement, but, in patients under treatment, it can result from many variables, such as host (genetic aspects)11,12 and environmental (mechanical forces, trauma)6 factors. A difficulty of evaluating the causes of EARR is to separate the contributions made by genetics from environmental factors such as treatment.13 It was reported that genetic factors account for at least 50% of the variation in EARR.11 It has been suggested that there is a strong genetic component for EARR,7,14 estimated in 70% of patients,13 especially when dizygotic are compared to monozygotic twins. In spite of efforts to identify the genetic component for EARR, how many and which genes contribute to the phenotype of EARR are yet largely unknown.13 Vitamin D is responsible for both positive and negative control of certain genes at the level of transcription, via interaction with the vitamin D receptor15 and is important for skeletal growth and bone homeostasis.16 The human vitamin D receptor is a product of 1 gene, which is located on chromosome 12q13-14.17 The gene comprises 9 exons that, together with intervening introns, span approximately 63 kilobases (kb).18 Genome-wide analyses have identified over 100 polymorphisms in the vitamin D receptor gene.19 Polymorphisms refer to the existence of 2 or more alleles at a given locus; if such alleles occur at a frequency of more than 1% in a population, the locus is said to be polymorphic.20 Single nucleotide polymorphisms are the most common form of DNA variation in the human genome. Patterns of linkage disequilibrium in the vitamin D receptor gene were proposed for a Canadian population, and 2 linkage disequilibrium blocks are believed to represent the whole gene.18 Block 1 is located toward the 50 end and spans roughly 8.4 kb, and block 2 is located toward the 30 end of the vitamin D receptor and spans approximately 5.8 kb. Near the 30 untranslated region, there is a single nucleotide polymorphism identified by a restriction site for TaqI enzyme in vitamin D receptor exon 9 (rs731236). This single nucleotide polymorphism might represent the second linkage disequilibrium block.18 Alleles of this polymorphism are in linkage disequilibrium with other polymorphisms in the same block and are linked and inherited together. Allelic variations in this region could be responsible for messenger RNA stability and differences in translational efficiency, resulting in changes in cellular expression of vitamin D receptor.21 This polymorphism has been associated with bone mass, turnover, and mineral loss22 and diseases such as osteoarthritis23 and periodontal disease.24 However, no study has investigated vitamin D receptor gene polymorphisms and their association with EARR.
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Table I. Baseline clinical characteristics of the study
groups Group 1 Group 2 n 5 160 (%) n 5 179 (%)
Group 3 n 5 38 (%)
P value Sex (n)* 86 (53.7%) 99 (55.3%) 23 (60.5%) 0.695 Female Male 74 (46.3%) 80 (44.7%) 15 (39.5%) Age (y)y Mean 6 SD 14.50 6 3.01 15.33 6 2.64 16.46 6 1.93 \0.001 (range) (8-21) (9.9-20) (11-19) Group 1, treated patients with EARR #1.43 mm; group 2, treated patients with EARR .1.43 mm; group 3, untreated subjects. *Chi-square test; ymean 6 standard deviation.
The aim of this study was to investigate the association of TaqI vitamin D receptor enzyme polymorphism (rs731236, exon 9) and clinical variables with EARR in orthodontic patients. MATERIAL AND METHODS
A sample of 377 unrelated white patients of both sexes (mean age, 14.9 years; range, 8-21 years) was selected between 2005 and 2009. Of these, 339 patients had a Class II Division 1 malocclusion and were orthodontically treated with edgewise or straight-wire techniques, and 38 patients had a Class II Division 1 malocclusion but were untreated. The reason for Class II Division 1 subjects was because this type of malocclusion is the most frequent and requires more treatment25; in addition, it can lead to higher levels of EARR.1,26,27 The patients were selected from the Dental Clinics of the Graduate Course in Orthodontics (Bauru, S~ao Paulo, Brazil), Dental Clinics of the Graduate Course in Orthodontics of Thum Institute of Research (Joinville, Santa Catarina, Brazil), and 2 private orthodontic clinics (Curitiba, Parana, Brazil) (Table I). Although all subjects were white, the Brazilian white population is heterogeneous. Recent articles have not recommended grouping Brazilians into ethnic groups based on color, race, and geographic origin because Brazilians classified as white or black have significantly overlapping genotypes, probably due to miscegenation.28 The subjects completed personal, medical, and dental history questionnaires and, with the protocol approved by the institutional review board of Pontifical Catholic University of Parana, signed a consent form after being advised of the nature of the study (approved by the ethical committee in research at Pontificia Unieversidade Cat olica do Parana, protocol number 546/05). The patients could not have chronic usage of antiinflammatory drugs, human immunodeficiency virus
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infection, and immunosuppressive chemotherapy history of any disease known to severely compromise immune function, current pregnancy or lactation, oral trauma, parafunctional behavior, endodontic treatment, or extensive caries lesions. The periapical x-rays of the maxillary central incisors with the longer roots (reference teeth) were taken before treatment and 6 months after the beginning of treatment. The evaluation method included measuring the root and crown lengths directly from the radiographs. The root apex, incisal edge, and cementoenamel junction of each tooth were marked on the x-rays on a light table. The longitudinal axis of each tooth was constructed from the root apex to the incisal edge following the root canal as accurately as possible. A perpendicular axis was then projected to the longitudinal axis from the mesial to the distal cementoenamel junction sides. The crown length was measured from the incisal edge to the projected cementoenamel junction, and the root length from the projected cementoenamel junction to the root apex (Fig 1). The differences between the 2 measurements indicate the EARR. A correction factor was calculated by using the following formula: correction factor 5 C1/C2 (C1, crown length before treatment; C2, crown length 6 months after starting treatment). Then EARR was calculated with the following formula: EARR 5 R1 – (R2 3 CF) (R1, root length before treatment; R2, root length 6 months after treatment start; CF, correction factor). EARR was also expressed as a percentage of the original root length: EARR 3 100/R1. Only teeth with complete root formation were considered for investigation. Any distortions between the pretreatment and follow-up radiographic images were corrected by using the crown length registration, assuming crown length to be unchanging over the observation period.29,30 The EARR was evaluated by 1 examiner (M.L.S.S.N.F). The radiographs were examined over a light table, and the measurements were made with a fine-tip digital caliper with accuracy up to 0.02 mm (electronic digital vernier caliper; Utustools Professional, Santiago, Chile). A receiver operating characteristic curve was constructed to verify the cutoff point based on the sample data distribution for EARR, and the value of 1.43 mm was obtained. Then, the sample was divided into 3 groups: (1) 160 treated patients with EARR 1.43 mm or less, (2) 179 treated patients with EARR greater than 1.43 mm, and (3) 38 untreated subjects. The following parameters were evaluated in the treated patients: age, sex, initial size of the root of the reference tooth, premolar extraction, use of pendulum appliance, rapid palatal expansion, and use of elastics (Table II).
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Fig 1. A, Anatomic references to measure EARR: cementoenamel junction (CEJ); B, references of measurements in the x-rays.
Cells were obtained by a mouthwash with 3% glucose solution for 1 minute and by scraping the oral mucosa with a sterile spatula.31 DNA was extracted from epithelial oral cells with ammonium acetate (10 mol/L) and EDTA (1 mmol/L).32 The following primer pair was used for polymerase chain reaction amplification of genomic DNA samples: (F – 50 CAG AGC ATG GAC AGG GAG CAA G 30 and R – 50 GGA TGT ACG TCT GCA GTG TG 30 ). Reaction conditions and cycling parameters were as follows: 1 mL of the genomic DNA was used for polymerase chain reaction amplification in a reaction mixture containing 22.5 mL GoTaq Green Master Mix (Promega, Madison, Wis) and 0.7 mL of each 25 mmol/L primer. The reactions were performed in a thermal cycler (T-512; Techne, Chelmsford, UK) and consisted of an initial denaturation step at 95 C for 5 minutes, followed by 37 cycles at 95 C for 1 minute, 55 C for 1 minute, 72 C for 1 minute, and a final extension at 72 C for 7 minutes. The restriction fragment length polymorphism technique was performed in a final reaction volume of 20 mL, by using 1 unit of TaqI (TYCGA) (Invitrogen Life Technologies, Carlsbad, Calif) and a 10-mL aliquot of polymerase chain reaction products, digested at 65 C overnight. The digested products were separated in 10% polyacrylamide gel electrophoresis stained by silver. The genotypes were determined by comparing the restriction fragment length polymorphism band patterns with a 1-kb-plus DNA ladder (Invitrogen Life Technologies). The restriction fragment length polymorphism is formed by a single base transition (TaqI enzyme) at codon 352 in exon 9 of the vitamin D receptor gene that creates a TaqI
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Table II. Clinical variables influencing EARR in treated patients EARR Variable Age (y)z #14 .14 Sex Female Male Initial root (mm)§ \30 $30 Premolar extraction No Yes Elastics No Yes Rapid palatal expansion No Yes Pendulum No Yes Genotype TT TC 1 CC
P value* (univariate) P valuey (multivariate) #1.43 mm .1.43 mm 95% Confidence n n 5 160 (47.2%) n 5 179 (52.8%) n 5 339 n 5 339 Odds ratio interval 147 192
80 (54.4) 80 (41.7)
67 (45,6) 112 (58.3)
0.021
185 154
86 (46.5) 74 (48.1)
99 (53.5) 80 (51.9)
0.827
258 81
133 (51.6) 27 (33.3)
125 (48.4) 54 (66.7)
291 48
143 (49.1) 17 (35.4)
276 63
0.022
1.69
1.08-2.66
0.005
0.002
2.34
1.36-4.03
148 (50.9) 31 (64.6)
0.087
0.052
1.94
0.99-3.78
133 (48.2) 42 (42.9)
143 (51.8) 36 (57.1)
0.486
285 54
130 (45.6) 30 (55.6)
155 (54.4) 24 (44.4)
0.185
252 87
113 (44.8) 47 (54)
139 (55.2) 40 (46)
0.171
58 274
33 (56.9) 124 (45.3)
25 (43.1) 150 (54.7)
0.114
0.104
1.64
0.90-2.97
y
*Fisher exact test, P \0.05; regression logistic model, Wald test, P \0.05 (variables were included when P \0.20 in univariate analysis); zcutoff point (14 years) suggested by receiver operating characteristic curve (0.574, P 5 0.017); §cutoff point (30 mm) suggested by receiver operating characteristic curve (0.620, P \0.001).
restriction site. The alleles that result from the cleavage of TaqI are designated “C” (TaqI site present, with 2 fragments: 293 base pairs and 47 base pairs) or “T” (TaqI site absent, with a fragment: 340 base pairs).
Mean; mean + se; mean + sd
32 p<0.001 p<0.001
p=0.067
30
The results observed in the study were expressed in means and standard deviations (quantitative variable) or frequencies and percentages (qualitative variable). To evaluate the association between 2 qualitative variables, the chi-square test or the Fisher exact test was used. Comparisons between the groups in relation to the quantitative variables were made with analysis of variance (ANOVA) with 1 factor and the least significant difference test for multiple comparisons. Adjustments of the receiver operating characteristic curve were made for EARR, age, and initial root length to determine cutoff points associated with EARR. The unpaired t test was used to compare EARR, age, and initial root length between the groups. For the multivariate analysis, the logistic regression model and the Wald test were used. Values of P \0.05 indicated statistical significance. Data were organized in an Excel spread
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EARR (mm)
Statistical analysis 28
26
24
Group 1
Group 2
Group 3
Initial Final
Fig 2. Comparison of the EARR values (initial and final root length) among the groups (paired t test): group 1, treated patients with EARR #1.43 mm; group 2, treated patients with EARR .1.43 mm; group 3, untreated subjects.
sheet (Microsoft, Redmond, Wash) and analyzed with the computational program Statistica (version 8.0; StatSoft, Tulsa, Okla).
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Table III. Genotype and allele distribution of the vitamin D receptor TaqI polymorphism between the groups Genotypes TT TC CC Alleles T C
Group 1, n 5 157 (%) 33 (21.02) 117 (74.52) 7 (4.46) Group 1, n 5 314 (%) 183 (58.28) 131 (50.00)
Group 2, n 5 175 (%) 25 (14.29) 139 (79.43) 11 (6.29) Group 2, n 5 350 (%) 164 (46.85) 138 (39.42)
Group 3, n 5 35 (%) 2 (5.71) 33 (94.29) 0 (0.00) Group 3, n 5 70 (%) 35 (50.00) 33 (47.14)
P value* 0.051 P value* 0.455
Group 1, EARR #1.43 mm; group 2, EARR .1.43 mm; group 3, untreated subjects. *Chi-square test, P \0.05.
RESULTS
We observed a higher proportion of EARR in the treated patients (EARR #1.43 mm, 0.81 mm; EARR .1.43 mm, 2.24 mm) compared with the untreated subjects (EARR, 0.05 mm). The clinical impact of orthodontic appliances on root resorption is shown in Figure 2. Regarding the treated patients, no statistically significant differences were observed between the groups in relation to sex, pendulum appliance, rapid palatal expansion, or elastics. A statistically significant difference was found between the groups regarding age (P 5 0.021) and initial size of the root of the reference tooth (P 5 0.005) in the univariate analysis. After the multivariate analysis, age (P 5 0.022), initial size of the root of the reference tooth (P 5 0.002), and premolar extractions (P 5 0.052) were associated with EARR (Table II). Considering the study's single nucleotide polymorphism, the genotype distribution was not consistent with the assumption of Hardy-Weinberg equilibrium either in the control group or for the whole sample. Differences were observed in vitamin D receptor TaqI polymorphism genotype frequency (P 5 0.051), but not in allele distribution (P 5 0.455) between the groups (Table III). However, when the treated patients were analyzed vs the untreated subjects, we observed a weak protection effect of allele C against EARR (CC 1 CT 3 TT [odds ratio, 0.29; 95% confidence interval, 0.071.23; P 5 0.091]). DISCUSSION
Most clinicians are highly concerned about EARR as an undesirable side effect of orthodontic treatment. The etiology of EARR has been studied for the past few decades but remains unclear.33 It is believed that no orthodontic tooth movement is possible without some EARR,34 but, in most cases, it will be minor and therefore has no clinical significance.35 However, moderate to severe root resorption has been reported to occur with a frequency of 10% to 20%.1,36
The cause of EARR is considered to be multifactorial.37 Several factors have been mentioned to influence EARR,5 both mechanical and biological.38 Several studies have suggested that excessive orthodontic force,39,40 tooth intrusion,41 rapid palatal expansion,10 and tooth movement against hard and highly mineralized tissues are critical factors for EARR.10 Regarding the biologic aspects, associations between EARR and age, sex,42 tooth morphology, periodontal condition,5 immune response,43,44 bone metabolism,45,46 and systemic and genetic factors2 have been suggested. In this study, impacts of age, initial length of the root of the maxillary central incisor (reference tooth), and premolar extraction were found on EARR in the treated subjects. It was recently reported that older patients have more EARR than do younger ones.47 However, to our knowledge, no other studies in the literature have reported that root length influences EARR. It was reported that incisors' root resorption increased from 15% before treatment to 73% after treatment,48,49 and the number of teeth with moderate and severe root resorption increased from 1% before treatment to 25% after treatment.50 We also identified an association of EARR with orthodontic treatment with extractions, in accordance to other studies.1,26,27,30 However, other authors failed to find a relationship between EARR and premolar extraction.6,47 Regarding sex, there was no statistical difference between orthodontically treated patients with and without EARR; this has been reported by several authors.13,30,47 However, Baumrind et al51 found a higher prevalence of EARR in men than in women, and Kjaer52 observed more EARR among female than male subjects. No statistical difference was found in relation to the pendulum appliance in our study. All patients who used the pendulum were treated with the straightwire technique, and all patients who did not use the pendulum were treated with the edgewise technique. No influence was observed of the type of orthodontic appliance on EARR in other studies,30,37 but Mavragani et al53 found greater EARR in patients
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Table IV. Summary of studies investigating the genetic aspects of EARR Authors (year) Newman (1975)14
Type of study Descriptive
Harris et al (1997)7 Twin study
AL-Qawasmi et al (2003)2
Sample (n) 47 subjects (case: 41; control: 6)
103 twin pairs
Family-based 35 families association study (118 persons: 73 siblings and 45 parents)
Mean age (y) 14.5
Population X-ray Malocclusion Results North American Pe 50% Class I Familial aggregation suggested
boys: 14.5 North American
P
girls: 13.3
C
12.1
North American
P
C AL-Qawasmi et al (2003)3
Linkage analysis
38 families (124 persons; 79 siblings and 45 parents)
12.3
North American
P
C Ngan et al (2004)13 Twin study
26 twin pairs (16 MZ and 10 DZ)
13.5 MZ Australian
P
12.9 DZ Austrian Lages et al (2009)63 Population-based 61 persons association study (case: 23; control: 38)
?
Brazilian
Pe
27% Class II D1 6.8% Class II D2 9.1% Class III 30% Class I 70% heritability to EARR of MCI and LM 63% Class II 9% Class III Class I IL1A (-889) polymorphism not associated. IL1B (13954) genotype CC (OR, 5.6; 95% CI, 1.9-21.2; P 5 0.0003) associated with EARR.2mm in MCI, MdCI, LM Class II Class III Class I Evidence for linkage of D18S64 microsatellite marker [lightly linked to TNFRSF11A (RANK)] with EARR .2 mm (LOD 5 2.5; P 5 0.02) in MCI Class II Class III Class I 49% heritability to EARR in MCI and 66% in LM Class II Class III 54% Class I
Allele C of IL1B (13954) is associated with EARR (OR, 4.0; 95% CI, 1.23-12.9; P \0.05) in MCI and MLI
39% Class II 6% Class III Gulden et al (2009)8 Population-based 94 persons association study (case: 45; control: 49)
$12
North American
P
IL1B (13954) polymorphism not associated with EARR. Genotype TT of IL1A (-889) polymorphism is associated with EARR (P \0.032)
Pe, Periapical x-ray; P, panoramic x-ray; C, cephalometric x-ray; MZ, monozygotic; DZ, dizygotic; MCI, maxillary central incisor; LM, lower molar; MdCI, mandibular central incisor; MLI, maxillary lateral incisor; D, Division; OR, odds ratio; CI, confidence interval; LOD, logarithm of the odds (to the base 10).
treated with the edgewise technique than the straight wire. Data from this study suggest that the maxillary incisors have no susceptibility to EARR during rapid palatal expansion; however, other authors have found such an association.54 All patients who used the Haas appliance in this study also used the pendulum
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appliance. Finally, in our study, no significant difference was found when Class II elastics were used, in contrast with other authors.53 The x-ray is the commonly used method to diagnose EARR and is considered to be effective and predictive.8,55 However, it has limitations, such as standardization,
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radiation exposure, and a restricted view.9 Moreover, the radiographic method is static and cannot indicate whether root resorption has finished or is ongoing; it just shows the result of the EARR process.8,56,57 So far, EARR has been measured on lateral cephalometric and panoramic radiographs.2,7 When diagnosis with those types of x-rays is thought to be imprecise and questionable, standardized intraoral radiographs should be used instead because they provide more detailed information.29,58 In this study, periapical xrays were used to measure EARR. Even in the periapical x-rays, the image interpretations of the resorption have limitations, but they are the most indicated method of analysis among the accessible ones.59 Although EARR can even be found in untreated subjects,37 the results of this study are consistent with the literature showing that teeth without forces have less resorption than teeth that have undergone orthodontic treatment.30 Our findings of a clinically significant association between the degree of EARR and orthodontic treatment suggest that patients susceptible to EARR can be identified early in treatment.60,61 It has been shown that EARR of the maxillary incisors, observed during the initial months of treatment, could be a predictor of a higher risk for continuing resorption during treatment.50,60 Consequently, it has been recommended that periapical x-rays should be obtained after the first 6 months of treatment.60-62 Susceptibility to EARR is believed to have a genetic basis.7,13,14 The genetic component controlling the occurrence and extent of EARR is from 50% to 70% and was especially identified in the maxillary and mandibular central incisors.7,13 This might be because those teeth are more subject to anterior retraction forces in patients with Class II Division 1 malocclusion during orthodontic treatment. Efforts to investigate host factors influencing EARR are scarce and have focused on linkage and association analysis (Table IV). Concerning linkage analysis, only 1 study found evidence for linkage of TNFRSF11A (RANK) with EARR.3 In the association studies, only 2 candidate genes of the immune-inflammatory response have been investigated: IL1A and IL1B, coding for IL1a and IL-1b proinflammatory mediators, respectively.2,9,63 Evidence for an association was identified between EARR and alleles from IL1A and IL1B gene polymorphisms. In the case of IL1A (–889) polymorphism, a significant difference in the genotype distribution was found between EARR patients and the control group, with an augmentation of genotype TT in the group with EARR in a North American population.9 Concerning IL1B (13954), allele C63 and
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genotype CC2 were significantly associated with the EARR in Brazilian and North American populations, respectively. Vitamin D is the major regulator of calcium homeostasis and protects the organism from calcium deficiency via effects on the intestines, kidneys, and bones. Vitamin D is well known as a hormone involved in mineral metabolism and bone formation, and its effect is to facilitate intestinal absorption of calcium and phosphate.64 Many effects of vitamin D on bones have been demonstrated, as a synthesis of bone matrix protein such as type I collagen, alkaline phosphatase, osteocalcin, and osteopontin.65,66 Studies have reported positive associations between osteoporosis or low bone density and alveolar bone and tooth loss.67 Vitamin D also inhibits lymphocyte proliferation and stimulates monocyte differentiation.17 Thus, there is considerable scientific evidence that vitamin D has a variety of effects on immune system functions that might enhance innate immunity and inhibit the development of autoimmunity.68 The effects of vitamin D on cells are thought to be mediated through the vitamin D receptor that belongs to the steroid receptor superfamily,69 a transcription factor regulating gene expression in several cell types, including osteoblasts.70 Polymorphisms in the vitamin D receptor gene have been associated with bone mineral density, bone turnover, and diseases in which mineral loss is a cardinal sign.71 However, to our knowledge, this is the first study to investigate the association of vitamin D receptor polymorphisms and susceptibility to EARR. We observed an enrichment of heterozygotes in the subjects genotyped for polymorphism vitamin D receptor TaqI (rs731236) in this study. The inclusion of subjects with Class II Division 1 malocclusion might have favored the selection of such a genotype. It can be suggested that the craniofacial grow pattern could be related to the heterozygosity. Also, genotypes containing the C allele were observed to be weakly associated with protection against root resorption in the treated when compared to untreated patients. Although the literature shows some controversy, it seems that C allele of polymorphism TaqI increases mRNA stability and vitamin D receptor expression.72 The protection effect of allele C might only be observed in orthodontic patients. The mechanical forces might regulate gene expression during orthodontic treatment though a mechanism involving transducing molecules and mechanosensitive ion channels.73 CONCLUSIONS
We observed that clinical aspects such as age, initial root length, premolar extractions, and vitamin D
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