ORIGINAL ARTICLE
Association of functional gene polymorphism IL-1b in patients with external apical root resorption Elizabeth Maria Bastos Lages,a Alexandre Fortes Drummond,a Henrique Pretti,a Fernando Oliveira Costa,b Eugeˆnio Jose´ Pereira Lages,c Amanda Ianotta Gontijo,c Luı´s Ota´vio Miranda Cota,c and Rui Barbosa Brito, Jrd Belo Horizonte, Minas Gerais, and Campinas, Sa˜o Paulo, Brazil Introduction: External apical root resorption (EARR) is a frequent complication of orthodontic treatment. Interleukin 1ß (IL-1ß) is a potent stimulus for bone resorption and osteoclastic cell recruitment during orthodontic tooth movement. The purpose of this study was to assess the association between a polymorphism in the IL-1b genes and EARR during orthodontic treatment. Methods: The sample included 61 Brazilian orthodontic patients, divided into 2 groups according to the presence (affected group, n 5 23) or absence (control group, n 5 38) of EARR in the central and lateral maxillary incisors in the posttreatment period. DNA was obtained from buccal swab cells. The polymorphism was analyzed by the polymerase chain reaction followed by digestion with restriction enzyme. The polymerase chain reaction products were analyzed in 10% polyacrylamide gel and stained with silver. Results: There were significant statistical differences (P\0.05) among the frequencies of the alleles and genotypes of the IL-1b gene polymorphism between the affected and unaffected groups, suggesting that allele 1 predisposed the subjects to EARR (odds ratio 5 4.0). Conclusions: The polymorphism of the IL-1b gene is associated with root resorption in the studied population. (Am J Orthod Dentofacial Orthop 2009;136:542-6)
E
xternal apical root resorption (EARR) is an undesirable sequel of orthodontic treatment that results in permanent loss of the dental structure of the root apex.1 According to Brezniak and Wasserstein2 and Hartsfield et al,3 this is a frequent iatrogenic result associated with orthodontic treatment, especially reported for the maxillary incisors. The prevalence of EARR associated with orthodontic treatment greatly varies in the literature, depending on the methods used to determine it in the studies. Histologic studies show high prevalence, whereas clinical studies show variable prevalence rates.4 More than a Professor, Department of Orthodontics, Dental School, Federal University of Minas Gerais, Belo Horizonte, Brazil. b Professor, Department of Periodontology, Dental School, Federal University of Minas Gerais, Belo Horizonte, Brazil. c Postgraduate student, Dental School, Federal University of Minas Gerais, Belo Horizonte, Brazil. d Professor, Department of Molecular Biology, Dental School, Sa˜o Leopoldo Mandic, Campinas, Sa˜o Paulo, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Reprint requests to: Elizabeth Maria Bastos Lages, Federal University of Minas Gerais, Department of Orthodontics, Antonio Carlos Ave, 6627, Pampulha, PO Box 359,31270-901, Belo Horizonte, Minas Gerais, Brazil; e-mail, bethlages@ uai.com.br. Submitted, May 2007; revised and accepted, October 2007. 0889-5406/$36.00 Copyright Ó 2009 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2007.10.051
542
one third of patients have root resorptions greater than 3 mm, and severe root resorption (.5 mm) can occur in 2% to 5% of the population.3,5,6 Several factors might be involved in the etiopathology of EARR. Graber and Vanarsdall7 suggested that the etiology of root resorption during orthodontic treatment is complex, and several factors, isolated or associated, might contribute to its development, such as dental vulnerability, patient’s age, type of orthodontic appliance, magnitude and duration of the force, and direction of tooth movement. In addition, Harris et al8 studied the hypothesis of the genetic influence on the EARR and found a great possibility of inheritance. The first description of a genetic marker that identifies subjects who are more likely to have EARR was established by Al-Qawasmi et al.1 The association of EARR during orthodontic treatment and the polymorphism of the interleukin 1ß (IL-1ß) (13954) gene mentioned by these authors suggest a role for this cytokine in the pathogenesis of EARR and for the protective mechanism of the cementum against root resorption. However, in the study by Al-Qawasmi et al,1 lateral cephalograms and panoramic radiographs were used to measure EARR. McFadden et al9 indicated that errors in measurement using electronic calipers on lateral cephalometric films were approximately 2.5 times the errors with periapical radiographs. Sameshima and
American Journal of Orthodontics and Dentofacial Orthopedics Volume 136, Number 4
Lages et al
543
Asgarifar10 suggested that the use of panoramic radiographs to measure root resoption might overestimate the amount of root loss by 20% or more, and that they are not as precise or reliable as intraoral radiographs. We assessed, in Brazilian subjects, the association of the polymorphisms in the IL-1b genes with EARR and orthodontic treatment. MATERIAL AND METHODS
Sixty-one white patients of a private orthodontic practice in Belo Horizonte and Itabirito, Brazil, were invited to participate in this research study. All received comprehensive orthodontic treatment (straight-wire technique) by the same orthodontist (E.M.B.L.). They were divided into 2 groups: the affected group (23 subjects), with at least 1 maxillary incisor with EARR $2 mm, and the control group (38 subjects), with EARR \2 mm in the central and lateral maxillary incisors. This cutoff point of 2 mm to determine the 2 groups was methodologically chosen to prevent potential superestimation of the occurrence of EARR because of limitations of radiographic images. They were genotyped for the candidate loci. All subjects gave written informed consent. This study was approved by the Commission on Research Ethics of Sa˜o Leopoldo Mandic Dental School, Campinas, Sa˜o Paulo, Brazil. To verify the presence or absence of EARR, periapical radiographs of the maxillary incisors were digitalized by a scanner (scanjet model 3570c, Hewlett-Packard, Palo Alto, Calif), with 300 dpi resolution. The images were analyzed directly in the computer with the ARARA program (an environment for image segmentation) to better standardize and perform digital measurements of root length.11 The central and lateral maxillary incisors were measured according to the method of Linge and Linge,12 modified by Brezniak et al.13 Changes in dental and root length were determined in 2 radiographs through the initial and final root (r1 and r2, respectively) and crown (c1 and c2, respectively) lengths (Fig 1). The rule of 3 of Brezniak et al13 was used to calculate the change in root length after treatment (if during treatment the root was shortened, the amount of EARR is r1 r2 [c1/c2]). The sampling of buccal epithelial cells was performed as described previously.14 Briefly, 61 subjecfts used a mouthwash for 1 minute, containing 5 mL of 3% glucose. After the mouthwash, a sterile wooden spatula was used to scrape the oral mucosa. Buccal epithelial cells were pelleted by centrifugation at 2000 rpm for 10 minutes. The supernatant was discarded, and the cell pellet resuspended in 500 mL of extraction buffer (10 mmol/L tris-HCl [pH 7.8], 5 mmol/L
Fig 1. Reference measurements to determine EARR, modified and reprinted with permission from Brezniak et al.13
EDTA, 0.5% SDS). The samples were frozen at –20 C until used for DNA extraction. After defrosting, the samples were incubated overnight with 100 ng per milliliter proteinase K (Sigma Chemical, St Louis, Mo) at 37 C under agitation. Then, the DNA was purified by sequential phenol/chloroform extraction and salt/ethanol precipitation. The DNA was dissolved in a 70 mL TE buffer (10 mmol/L Tris [pH 7.8], 1 mmol/L EDTA). The concentration was estimated by measurements of OD 260. The following primers were used (Al-Qawasmi et al1): 50 -CTCAGGTGTCCTCGAAGAAATCAA-30 (foward) and 50 -GCTTTTTTGCTGTGAGTCCCG-30 (reverse). Amplification reactions were carried out with 500 ng genomic DNA in a total volume of 50 mL, containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1 mmol/ L of each primer, 200 mmol/L each dATP, dCTP, dGTP, and dTTP, 2.5 mmol/L MgCl2, and 2.5 units Taq DNA polymerase (Amersham Pharmacia Biotech, Uppsala, Sweden). Cycling was 1 cycle of 2 minutes at 95 C followed by 38 cycles of 1 minute at 95 C, 45 seconds at 67 C, and 1 minute at 72 C. The products were digested with 3 U per 25 mL reaction of TaqI at 37 C overnight to detect allele 1 (85 bp 1 97 bp) and allele 2 (182 bp). Restriction products were visualized by electrophoresis on vertical 10% polyacrylamide gels in 1X TBE (89 mmol/L tris-borate, 89 mmol/L boric acid, 2 mmol/L EDTA), followed by silver staining (Bio-rad Silver Stain Kit, Bio-Rad Laboratories, Hercules, Calif). The reproducibility of the measurements on periapical radiographs was assessed by statistically analyzing
544
Lages et al
Table I.
American Journal of Orthodontics and Dentofacial Orthopedics October 2009
Baseline clinical parameters of the subjects
Variable Sex Female Male Angle classification Class I Class II Class III Extraction pattern Extraction Nonextraction
Treated subjects (% for variable)
Treated subjects with EARR $2 mm* (% for subject)
35 (57.4) 26 (42.6)
11 (31.4) 12 (46.2)
33 (54.1) 24 (39.3) 4 (6.6)
15 (45.5) 7 (29.2) 1 (25)
9 (14.8) 52 (85.2)
6 (66.7) 17 (32.7)
*At least 1 maxillary incisor.
RESULTS
The mean age of the subjects was 18.9 years (SD 6 5.2). The data describing the baseline clinical parameters of the subjects are summarized in Table I. According to the genotype, 27.9% of the subjects had the 1/1 genotype, 31.1% the 1/2 genotype, and 41% the 2/2 genotype. Regarding the genotype, the subjects are described according to the occurrence of EARR in Figure 2. EARR occurred in 64.7% of the subjects with the 1/1 genotype, 36.8% with the 1/2 genotype, and 20% with the 2/2 genotype. These differences were statistically significant (P \0.014). In addition, EARR was greater in the 1/1 genotype than in the 2/2 genotype. The allele and genotype frequencies of the gene IL1b are shown in Table II. Allele 2 was present in 72.1% of the subjects. There was a statistically significant difference (5%) between allele 1 and allele 2 (P 5 0.0013). The distribution of the genotypes shows that 19 subjects (31.1%) were heterozygous for this gene. Table II shows that subjects with allele 1 had 4 times more chances (OR 5 4.00) of EARR than those with the 2/2 genotype (P \0.0349). It was also observed that subjects with the 1/1 genotype had 7.3 more chances (OR 5 7.3) of EARR compared with those with the 2/2 genotype (P \0.0095). DISCUSSION
Fig 2. Description of the subjects according to EARR by genotype. The significance probability refers to the chi-square test.
the difference between 2 measurements 7 days apart on 20 periapical radiographs. The measurements were made by 1 examiner (E.M.B.L.). The answers were classified according to differences of less than 0.5 mm. Subsequently, to assess the reproducibility of the radiographic measurements, the kappa index was used.15 A value of kappa 5 0.93 was obtained; this is considered a highly positive result. Statistical analysis was performed by using the chisquare test to investigate the polymorphism of the IL-1b gene, the distribution of the genotype, and the allelic frequency in the subjects of the 2 groups. The risk of EARR, associated with the alleles or the genotypes, was calculated with the odds ratio (OR). A significance level of 5% was used, and all statistical tests were performed with SPSS software (version 12.0, Statisical Package for the Social Sciences, Chicago, Ill).
EARR is a frequent iatrogenic result of orthodontic treatment, especially in maxillary incisors, because of compression of the periodontal ligament. This compression causes a decrease or an interruption of the microcirculation; this can cause sterile necrosis. During the removal of this necrotic tissue by macrophages and clastic cells, the root integrity can be damaged.3,4,16,17 Nevertheless, the pathogenesis of EARR is still obscure, complex, and influenced by several risk variables. Despite all the investigations, no isolated or associated factor has been identified as responsible for EARR during orthodontic treatment.18,19 Newman20 and Harris et al8 suggested a genetic influence for EARR, but only recently was it demonstrated that the polymorphism of the IL-1 genes and the TNFRSF11A are associated with increased risk of EARR during orthodontic treatment.1,21 Since molecular investigations were necessary to elucidate the etiopathogeny of EARR, we described a genetic marker to identify subjects who are susceptible to EARR before orthodontic treatment. The association between EARR and the polymorphism of IL-1b verified in this study suggests the participation of this cytokine in the etiopathology of EARR. A statistical difference
Lages et al
American Journal of Orthodontics and Dentofacial Orthopedics Volume 136, Number 4
Allele and genotype frequencies (n [%*]) of the gene IL-1b
Table II.
Genotypes (n 5 61)
Control EARR Total
1/1
1/2
2/2
Chi-square
6 (9.8) 11 (18.0) 17 (27.9)
12 (19.6) 7 (11.5) 19 (31.1)
20 (32.8) 5 (8.3) 25 (41)
P 5 0.014†
2/2 vs 1/2 1 1/1, OR 5 4.00; IC 95% 5 1.23 # m #12.9; P 5 0.0349. 2/2 vs 1/1, OR 5 7.33; IC 95% 5 1.81 # m #29.6; P 5 0.0095. 1
Alleles (n 5 122) 2
Control 24 (19.7) EARR 29 (23.7) Total 53 (43.4)
52 (42.7) 17 (13.9) 69 (56.6)
P 5 0.0013†
*Percentage in relation to the total sample: genotypes (61) and alleles (122); †P value for goodness of fit for all cells in each test.
between the affected and unaffected groups for the genotype and allele frequencies was verified. Subjects with allele 1 are more predisposed to EARR, and subjects homozygous for allele 2 are more protected against EARR. This evidence of predisposition is of utmost importance for the determination and implementation of measures to prevent and control EARR. The association of the IL-1b with the severity of EARR clears the path for many lines of research. The magnitude and duration of the force do not totally explain the differences in EARR severity. Furthermore, individual variations obscure the effect of the magnitude and duration of the force when root resorption is observed.1,19 We used periapical radiographs to determine the presence and severity of EARR to reduce the bias related to the diagnosis of EARR from other types of radiographs. Lateral cephalograms and panoramic radiographs have been used in recent studies, and some errors have been reported. Al-Qawasmi et al1 used lateral cephalograms to determine EARR in the incisors and panoramic radiographs for EARR in the mandibular molars. McFadden et al9 indicated that errors in measurement using electronic calipers on lateral cephalometric films were approximately 2.5 times those of measurements on periapical radiographs. Moreover, according to Sameshima and Asgarifar,10 panoramic radiographs show greater means of root resoprtion than do periapical radiographs, and the use of panoramic radiographs to measure root resorption before and after treatment might overestimate the quantity of root loss by 20% or more. Although Katona22 ques-
545
tioned the accuracy of periapical x-rays for EARR measurements, it is unlikely that any inconsistencies in evaluating root resorption by this method in our study seriously biased the estimates of EARR. Errors certainly will continue until an accurate 3-dimensional imaging system is available. IL-1 has been frequently associated with inflammatory events in connective and bone tissues. 23 Moreover, IL-1b has been characterized as a potent bone-resorptive cytokine and implicated as a key component of the complex pathways leading to root resorption.3 Our results support the clinical perception of the existence of complex mechanisms in the etiopathology of root resorption. Al-Qawasmi et al1 suggested that IL-1b stimulates clastic cells during orthodontic movement, and that a low production of IL-1b might result in less resorption of the cortical bone at the periodontal ligament interface. This lower rate of bone resorption would result in prolonged stress concentrated in the root, thus leading to necrotic areas in the periodontal ligament and causing root resorption. Although our findings are similar to those of Al-Qawasmi et al,1 we found higher ORs. This could be in part explained by differences in the population of the sample (Indiana vs Brazil), the type of teeth that were measured (permanent maxillary central incisors vs permanent maxillary central and lateral incisors), and the type of radiograph used to evaluate EARR (lateral cephalometric vs periapical). Additional studies with standardized radiographs and measurements of EARR, as well as different populations, are required to better clarify this issue. At this time, there is no safe mechanism to determine, before orthodontic treatment, which patients will be affected by EARR. Therefore, confirmation of the influence of genetic polymorphisms might be an important tool in preventing it. In the future, before orthodontic treatment, patients might be analyzed according to the genes IL-1 (and others) by DNA analysis from buccal swab cells. Likewise, the presence of high-risk alleles could also be verified. Thus, it would be possible to advise patients about their predisposition to EARR before treatment. This would prevent the interaction of other risk variables and make it possible to control higher-risk variables. Furthermore, these data are of great value in orthodontic planning that includes large tooth movements, so that further damage can be prevented, and treatment success can be ensured. CONCLUSIONS
The polymorphism of the IL-1b gene is associated with root resorption in the studied population.
546
Lages et al
REFERENCES 1. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, Flury L, Foroud TM, Macri JV, et al. Genetic predisposition to external apical root resorption. Am J Orthod Dentofacial Orthop 2003; 123:242-52. 2. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part II: the clinical aspects. Angle Orthod 2002;72:180-4. 3. Hartsfield JK Jr, Everett ET, Al-Qawasmi RA. Genetic factors in external apical root resorption and orthodontic treatment. Crit Rev Oral Biol Me´d 2004;15:115-22. 4. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part I: the basic science aspects. Angle Orthod 2002;72:175-9. 5. Taithongchai R, Sookkorn K, Killiany DM. Facial and dentoalveolar structure and the prediction of apical root shortening. Am J Orthod Dentofacial Orthop 1996;110:296-302. 6. Killiany DM. Root resorption caused by orthodontic treatment: an evidence-based review of literature. Semin Orthod 1999;5:128-33. 7. Graber TM, Vanarsdall RL Jr. Ortodontia: princı´pios e te´cnicas atuais. Rio de Janeiro, Brazil: Guanabara Koogan; 2002. 8. 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. 9. McFadden WM, Engstrom C, Engstrom H, Anholm JM. A study of the relationship between incisor intrusion and root shortening. Am J Orthod Dentofacial Orthop 1989;96:390-6. 10. Sameshima GT, Asgarifar KO. Assessment of root resorption and root shape: periapical vs panoramic films. Angle Orthod 2001;71: 185-9. 11. Andrade MC. An interactive algorithm for image smoothing and segmentation. Electronic Letters on Computer Vision and Image Analysis 2004;4:32-48. Available at: http://elcvia.cvc.uab.es/ journal/publish.php?art5a2002011-2. Accessed October 3, 2005.
American Journal of Orthodontics and Dentofacial Orthopedics October 2009
12. 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. 13. Brezniak N, Goren S, Zoizner R, Dinbar A, Arad A, Wasserstein A, et al. A comparison of three methods to accurately measure root length. Angle Orthod 2004;74:786-91. 14. Trevilatto PC, Line SRP. Use of buccal epithelial cells for PCR amplification of large DNA fragments. J Forensic Odontostomatol 2000;18:6-9. 15. Siegel S, Castellan NJ. Nonparametric statistics for the behavioral sciences. 2nd ed. New York: McGraw-Hill; 1988. 16. Brudvik P, Rygh P. The initial phase of orthodontic root resorption incident to local compression of the periodontal ligament. Eur J Orthod 1993;15:249-63. 17. Brudvik P, Rygh P. Root resorption beneath the main hyalinized zone. Eur J Orthod 1994;16:249-63. 18. Fritz U, Rudzki-Janson I, Paschos E, Diedrich P. Light microscopic and SEM findings after orthodontic treatment-analysis of a human specimen. J Orofac Orthop 2005;66:39-53. 19. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, Weaver MR, Foroud TM, Faust DM, et al. Root resorption associated with orthodontic force in inbred mice: genetic contributions. Eur J Orthod 2006;28:13-9. 20. Newman WG. Possible etiologic factors in external root resorption. Am J Orthod 1975;67:522-39. 21. Al-Qawasmi RA, Hartsfield JK Jr, 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. 22. Katona TR. Flaws in root resorption assessment algorithms: role of tooth shape. Am J Orthod Dentofacial Orthop 2006;130:19-27. 23. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop 2001;119:307-12.