J Periodontol September 2010

Association of Interleukin-1 Receptor Antagonist Gene Polymorphisms With Generalized Aggressive Periodontitis in an Iranian Population
Hesam Baradaran-Rahimi,* Mehrdad Radvar, Hamid R. Arab,* Jalil Tavakol-Afshari, and Ahmad R. Ebadian
Background: Periodontitis is a multifactorial disease that occurs in the presence of bacteria, environmental factors, and genetic predispositions. It is suggested that polymorphisms in the interleukin 1 (IL1) receptor antagonist gene have an important role in the susceptibility of the host to periodontitis. This study investigates the association of a variable number of tandem repeat polymorphism in the IL1RN gene with generalized aggressive periodontitis (GAgP). Methods: Sixty-six subjects with GAgP and 56 periodontally healthy subjects took part in the study. All subjects were of Iranian Khorasanian (north-east province of Iran) descent. Subjects were identied through clinical examinations and radiographs at the Mashad Dental School and Dental Research Center. DNA was extracted from peripheral blood cells, and different genotypes were detected using polymerase chain reaction amplication and fragment-size analysis. Data were analyzed using the x2 test. Results: The frequencies of the IL1RN genotype A1A2 (x2 test; P = 0.001) and allele A2 (x2 test; P = 0.006) were found to be signicantly increased in patients with GAgP compared to normal subjects. Conclusion: These ndings suggest that the polymorphic IL1RN gene is a risk determinant for generalized aggressive periodontitis in the Iranian Khorasanian population. J Periodontol 2010;81:1343-1347. KEY WORDS Aggressive periodontitis; interleukin-1 receptor antagonist protein; polymerase chain reaction; polymorphism, genetic.
* Department of Periodontology, School of Dentistry and Dental Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Currently, Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA; previously, Department of Periodontology, School of Dentistry and Dental Research Center, Mashhad University of Medical Sciences. Department of Immunogenetic, Bu-Ali Research Institute, Immunology Research Center, Mashhad University of Medical Sciences.

lthough bacteria are necessary to initiate periodontitis, there is not an identied mechanism for predicting the clinical status and progression of the disease for individual patients (i.e., differentiating those patients who respond well to simple professional care while having a mild to moderate form of disease from those similar patients who are susceptible to develop more severe periodontitis that demands more extensive therapy and nally leads to tooth morbidity).1,2 One study3 in twins illustrated that genetic factors played a signicant role in the clinical measure variances of periodontitis. Genetic polymorphisms affect qualitative and quantitative aspects of the host response. Because cytokines are the most important signals in immune regulation, it is of interest to study the genetic polymorphisms attributable to cytokine function and their association with periodontitis.1 The gene encoding IL1 family plays a redundant role in periodontitis. There are three known IL1 genes in a cluster on human 2q13.4 Two of the genes encode proinammatory proteins (IL1A and IL1B, which produce IL-1a and IL-1b, respectively), whereas the third gene (IL1RN) encodes a related protein that binds to IL-1 receptors but does not stimulate intracellular signaling and acts as a receptor antagonist (IL-1ra).5
doi: 10.1902/jop.2010.100073

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Association of IL-1RN Polymorphisms With GAgP in Iranians

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Because periodontal diseases are long-term chronic inammatory conditions, any changes in the function and quantity of IL-1RN could inuence the susceptibility of the host to periodontal disease.6 The individual ability to produce IL-1RN is inuenced by genetic factors. The level of IL-1ra mRNA was shown to be higher in aficted tissues than in healthy tissues.7 Subjects with allele 2 of IL-1RN have lower salivary levels and higher serum levels of IL-1ra compared to patients without this allele.8 In vitro, the presence of IL-1RN allele 2 is associated with an increase in IL-1b production,9 and the carriage of IL-1RN allele 2 was shown to have an association with a reduction in total IL-1RN protein production in cultured mononuclear leukocytes.10 Tai et al.11 indicated that the frequency of IL-1RN variable number of tandem repeat (VNTR) polymorphic alleles was found to have a considerable increase in generalized aggressive periodontitis (GAgP) in Japanese patients. In another study, Parkhill et al.12 found no evidence for a relation between IL-1RN genotypes and aggressive periodontitis (AgP). However, the combination of IL-1b allele 1 and IL-1RN allele 1 (corresponding to four repeats) was associated with AgP.12 Zhong et al.13 pointed out that allele 2 of IL-1RN was considerably higher in severe chronic periodontitis subjects, and it was suggested to be a risk factor for severe periodontitis in individuals of Uighur descent. Laine et al.14 reported that the higher prevalence of IL-1RN allele 2 in non-smoking periodontitis patients indicated that polymorphisms in the IL-1RN gene, in the absence of other risk factors, can be assumed as a risk factor for periodontitis. Studies in non-white populations, based on evaluation of the impact of IL-1RN polymorphism upon development of periodontitis are few.11 The present study explores the possible genetic association of this VNTR polymorphism with GAgP in the Iranian Khorasanian (north-east province of Iran) population. MATERIALS AND METHODS Selection of Subjects This study was approved by the Ethics Committee of Mashhad University of Medical Sciences, Mashhad, Iran (no. 8401) and was performed at the Mashhad Dental School and Dental Research Center, Mashhad University of Medical Sciences by collaborating with the immunology group at the Bu-Ali Research Institute, Mashhad University of Medical Sciences, over a period of 3 years (2005 to 2008). In this project, 66 Iranian Khorasanian subjects (25 males and 41 females) with GAgP, which was detected in the Periodontology Department of Mashhad Dental School, and 56 race-matched periodontally healthy subjects (19 males and 37 females) as a control group were included. The mean age of the GAgP group was
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30.5 years (age range: 20 to 39 years), and the mean age of the control group was 30.2 years (age range: 18 to 36 years). All subjects signed a consent form after being advised of the aims of the study. Diagnoses were based on past dental histories, clinical parameters, and radiographic patterns of bone loss. Clinical evaluations consisted of probing depth (PD), clinical attachment level, plaque, and bleeding on probing. Attachment loss was measured by identifying the cemento-enamel junction and measuring the distance to the base of the pocket. Sets of intraoral radiographs were taken using a standardized parallel technique. Patients with GAgP showed radiographic evidence of attachment loss at multiple permanent teeth, three of which were not incisors or rst molars. The healthy control subjects did not show attachment loss or PDs >3 mm at more than one site. Full medical histories were obtained to clear conditions that might have predisposed subjects to periodontal disease (e.g., diabetes, pregnancy, lactation, human immunodeciency virus, or hepatitis infection). None of the subjects were smokers, and they did not have a familial relationship with each other. The patientselection methods were similar to those of other studies.11,15-18 Genotype Identication Three milliliters of arm-vein blood from each subject was taken by venipuncture and mixed with EDTA. DNA was isolated by means of a salting-out method using a whole-blood kit and stored at -70C until further processing. The intron 2 of the IL-1RN gene contains a sequence with an 86-base pair (bp) VNTR, which gives rise to ve different alleles. IL-1RN genotyping was performed by polymerase chain reaction (PCR), and length differences were shown by electrophoresis.18 Amplication was performed by a thermocycleri on a 20 ml mixture of the following components: 100-150 ng DNA, 500 mm ol of specic primers, 0.5 unit Taq DNA polymerase, 10x PCR reaction buffer (10mM/L Tris-HCl, 50 mM/L KCl, 1.5 mM/L MgCl2), 0.2 mM each dNTP . The primer sequences used in our study were 59CTCAGCAACACTCCTAT 39 (+2,879/+2,895) and 59-TCC.TGG.TCT.GCA.GGT.AA-39 (+3,274/+3,290). The PCR protocol was as follows: primary denaturation was conducted at 94C for 3 minutes and was followed by 35 cycles at 94C for 1 minute (secondary denaturation), 62C for 1 minute (annealing), 72C for 2 minutes (extension), and completed with a nal extension at 72C for 5 minutes. PCR products were identied by electrophoresis on a 2% agarose gel

BioGene whole blood kit, BioGene, Mashhad, Iran. i Biometra T3 thermocycler, Biometra, Gottingen, Germany. Cinna Gene, Tehran, Iran.

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Baradaran-Rahimi, Radvar, Arab, Tavakol-Afshari, Ebadian

stained with ethidium bromide (0.5 mg/ml) (Figs. 1 and 2). Statistical Analyses Associations of allele frequencies and genotypes between patients with GAgP and healthy controls were established by x2 analysis. The analysis showed that the x2 validity criteria held true, and no other test was required. Statistical software** was used. P <0.05 was considered statistically signicant. Chi-square analysis was used to test for the deviation of genotype frequencies from Hardy-Weinberg expectations, and it showed that the population was in Hardy-Weinberg equilibrium. RESULTS Because the frequency of genotypes other than A1A1 and A1A2 among test and control groups was quite low, all of these genotypes were categorized and analyzed under the name of other genotypes. The A1A2 genotype was signicantly less frequent among control subjects (n = 9 [16.1%]) than among patients (n = 30 [45.5%]), whereas the A1A1 genotype was more prevalent among control subjects (P = 0.001) (Table 1). The frequency of allele A1 was signicantly greater among controls (n = 94 [83.9%]) than among patients (n = 89 [67.4%]) (P = 0.006). Moreover, the A2 allele frequencies in patients and normal subjects were also quite different (Table 2). DISCUSSION In this study, the VNTR polymorphism of the IL-1RN gene was investigated among subjects with GAgP and normal individuals. A signicant association was found between the IL-1RN genotype and the periodontal status of individuals (P = 0.001). Similarly, a signicant association was found between IL-1RN allele frequencies and periodontal status (P = 0.006). From the literature, it appears that the carriage rate of IL-1RN allele 2 is higher among white subjects12 than among Japanese11 and African American subjects.19 Our data show that the carriage rate for the IL-1RN allele A2 was 10.7% among healthy Iranian Khorasanians. The corresponding reported rates were 48.6% in white subjects,12 20.4% in African American subjects,19 and 8.2% in Japanese subjects.11 In particular, the rate of the IL-1RN allele A2 tended to be lower in Iranian Khorasanians than in white subjects, whereas the rate in African American subjects was nearly double the rate in the present study. The Japanese rate was lower than the rate in the present study. From these ndings, there appears to be a great variability in the frequency of IL-1RN (VNTR) polymorphism among different populations. Moreover, ethnic and racial differences in disease-susceptibility

Figure 1.
PCR products of eight patients (1 to 8) with GAgP after electrophoresis on an agarose gel stained with ethidium bromide. Allele 1 (four repeats) was 412 bp in size, allele 2 (two repeats) was 240 bp in size, allele 3 (ve repeats) was 498 bp in size, allele 4 (three repeats) was 326 bp in size, and allele 5 (six repeats) was 584 bp in size. Genotypes of patients were as follows: A1A2 = patients 1, 2, 5, 7, and 8; A2A3 = patient 3; and A1A1 = patients 4 and 6. c+ = positive control; L50 = 50-bp ladder.#

Figure 2.
PCR products of eight patients (1 to 8) with GAgP after electrophoresis on an agarose gel stained with ethidium bromide. Allele 1 (four repeats) was 412 bp in size, allele 2 (two repeats) was 240 bp in size, allele 3 (ve repeats) was 498 bp in size, allele 4 (three repeats) was 326 bp in size, and allele 5 (six repeats) was 584 bp in size. Genotypes of patients were as follows: A1A1 = patients 4 and 5; A1A2 = patients 1, 3, and 6; A2A3 = patient 2; A1A3 = patient 7. c+ = positive control; c- = negative control; L50 = 50-bp ladder.#

# Fermentas, Burlington, ON. ** SPSS, version 10, SPSS, Chicago, IL.

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Association of IL-1RN Polymorphisms With GAgP in Iranians

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Table 1.

Distribution (n [%]) of IL-1RN Genotypes Among GAgP and Control Subjects

Genotype A1A1 A1A2 Others
2

Controls (n = 56) 41 (73.2) 9 (16.1) 6 (10.7)

Patients (n = 66) 26 (39.4) 30 (45.5) 10 (15.2)

Total (N = 122) 67 (54.9) 39 (32) 16 (13.1)

x = 14.947; P = 0.001.

Table 2.

Distribution (n [%]) of IL-1RN Alleles Among GAgP and Control Subjects

Allele A1 A2 Others Control (n = 112) 94 (83.9) 12 (10.7) 6 (5.4) Patients (n = 132) 89 (67.4) 35 (26.5) 8 (6.1) Total (N = 244) 183 (75) 47 (19.3) 14 (5.7)

x2 = 10.106; P = 0.006.

gene polymorphisms were reported by others.20,21 For example, the IL-1RN allele A2 was associated with a susceptibility in white subjects to systemic lupus erythematosus (SLE), where this gene was present in 24.1% of controls and 32.7% of patients with SLE (P <0.05).20 In contrast, only 4.1% of Japanese controls carried this polymorphism compared to 9.7% of SLE patients (P <0.05).21 The prevalence of the marker gene was clearly lower among Japanese subjects compared to white subjects.21 In a study by Kornman et al.,1 the carriage rate of the IL-1RN allele A2 was not associated with the severity of periodontitis (59.2% in the mild group compared to 48.8% in the severe group).1 Moreover, in another study, no signicant difference was found in the distribution of IL-1RN (VNTR) genotypes between AgP patients and healthy controls (the allele A2 carriage rate was 31.4% in GAgP patients compared to 48.6% in healthy subjects).12 We did not investigate the relationship between the severity of disease and the allele frequency in the present study; the sample primarily included individuals with moderate to severe GAgP. Like our study, Tai et al.11 reported that the frequency of IL-1RN (VNTR) polymorphic alleles was signicantly increased in GAgP patients. In addition, the carriage rate of IL-1RN (VNTR) polymorphic alleles was signicantly higher in GAgP patients than in healthy controls; consequently, Tai et al.11 sug1346

gested that IL-1RN (VNTR) polymorphisms were associated with GAgP in Japanese subjects. Interestingly, the A2A2 genotype was absent in their study subjects.11 In the present study, we found one A2A2 genotype in the test (GAgP) group and one in the control group. Although the results of this study indicate that the presence of A1A2 genotype might be associated with an increased frequency of periodontitis among subjects, the presence of other environmental risk determinants, such as specic bacteria, oral hygiene, and smoking status, and their interaction with genetic risk determinants play an important role in the manifestation of disease. Patients with the A1A2 genotype and a healthy periodontal status in the present study might have been lacking the environmental or other genetic determinants necessary for the initiation and progression of periodontal disease. Because of known interactions between periodontal disease and smoking, we only selected non-smoking individuals in the present study. Another study11 did not mention the smoking status of the subjects, whereas in a study by Meisel et al.,22 smoking and non-smoking subjects were analyzed separately. Moreover, they evaluated the effects of the coincidence of IL-1 gene cluster (IL-1a, IL-1b, and IL-1RN) polymorphism and smoking as an environmental factor on the periodontal status of 154 white subjects. Although their smoking group consisted of 19 subjects and was not quite statistically powerful, Meisel et al.22 concluded that the composite genotypes showed an interaction with smoking, the main environmental risk factor of periodontal disease. In contrast, they found that non-smoking subjects were not at increased risk even if they were genotype positive. CONCLUSIONS In the present study, there is strong evidence of an association between the IL-1RN genotype and the periodontal status of subjects. Furthermore, there appears to be a signicant association between the alleles of this particular polymorphism and periodontal status. It seems that the IL-1RN (VNTR) gene polymorphism could serve as a useful marker of GAgP among the Iranian Khorasanian population. ACKNOWLEDGMENTS This study was supported by a grant from the ofce of the research vice chancellor, Mashhad University of Medical Sciences. The authors report no conicts of interest related to this study. REFERENCES
1. Kornman KS, Crane A, Wang HY, et al. The interleukin1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 1997;24:72-77.

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2. Loe H, Anerud A, Boysen H, Morrison E. Natural history of periodontal disease in man. Rapid, moderate and no loss of attachment in Sri Lankan laborers 14 to 46 years of age. J Clin Periodontol 1986;13:431-445. 3. Michalowicz BS. Genetic and heritable risk factors in periodontal disease. J Periodontol 1994;65(Suppl. 5)479-488. 4. Nicklin MJ, Weith A, Duff GW. A physical map of the region encompassing the human interleukin-1 alpha, interleukin-1 beta, and the interleukin-1 receptor antagonist genes. Genomics 1994;19:382-384. 5. Symons JA, Young PR, Duff GW. Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of IL-1b precursor and loses afnity for IL-1 receptor antagonist. Proc Natl Acad Sci U S A 1995; 92:1714-1718. 6. Kinane DF, Hodge P, Eskdale J, Ellis R, Gallagher G. Analysis of genetic polymorphisms at the interleukin10 and tumour necrosis factor loci in early-onset periodontitis. J Periodontal Res 1999;34:379-386. 7. Roberts FA, Hockett RD, Jr, Bucy RP, Michalek SM. Quantitative assessment of inammatory cytokine gene expression in chronic adult periodontitis. Oral Microbiol Immunol 1997;12:336-344. 8. Perrier S, Coussediere C, Dubost JJ, Albuisson E, Sauvezie B. IL-1 receptor antagonist (IL-1RA) gene polymorphism in Sjogrens syndrome and rheumatoid arthritis. Clin Immunol Immunopathol 1998;87:309-313. 9. Santtila S, Savinainen K, Hurme M. Presence of the IL-1RA allele 2 (IL1RN*2) is associated with enhanced IL-1b production in vitro. Scand J Immunol 1998;47: 195-198. 10. Tountas NA, Casini-Raggi V, Yang H, et al. Functional and ethnic association of allele 2 of the interleukin-1 receptor antagonist gene in ulcerative colitis. Gastroenterology 1999;117:806-813. 11. Tai H, Endo M, Shimada Y, et al. Association of interleukin-1 receptor antagonist gene polymorphisms with early onset periodontitis in Japanese. J Clin Periodontol 2002;29:882-888. 12. Parkhill JM, Hennig BJ, Chapple ILC, Heasman PA, Taylor JJ. Association of interleukin-1 gene polymorphisms with early-onset periodontitis. J Clin Periodontol 2000;27:682-689. 13. Zhong LJ, Zhang YH, Zhang JC, Feng JH, Yang AL. The association between interleukin-1 receptor antagonist genotype and chronic periodontitis of Uighur patients (in Chinese). Zhonghua Kou Qiang Yi Xue Za Zhi 2003;38:370-373.

14. Laine ML, Farre MA, Garca-Gonzalez MA, et al. Risk factors in adult periodontitis: polymorphism in the interleukin-1 gene family (in Dutch). Ned Tijdschr Tandheelkd 2002;109:303-306. 15. Armitage GC. Development of a classication system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6. 16. Bajestan MN, Radvar M, Afshari JT, Naseh MR, Arab HR. Interleukin-6 production by cultured peripheral blood monocytes before and after stimulation by E. coli lipopolysaccharide in Iranian patients with aggressive periodontitis. Med Sci Monit 2006;12: CR393-396. 17. Mellati E, Arab HR, Afshari JT, Ebadian AR, Radvar M. Analysis of -1082 IL-10 gene polymorphism in Iranian patients with generalized aggressive periodontitis. Med Sci Monit 2007;13:CR510-514. 18. Radvar M, Tavakkol-Afshari J, Bajestan MN, Naseh MR, Arab HR. The effect of periodontal treatment on IL-6 production of peripheral blood monocytes in aggressive periodontitis and chronic periodontitis patients. Iran J Immunol 2008;5:100-106. 19. Rider LG, Artlett CM, Foster CB, et al. Polymorphisms in the IL-1 receptor antagonist gene VNTR are possible risk factors for juvenile idiopathic inammatory myopathies. Clin Exp Immunol 2000; 121:47-52. 20. Blakemore AI, Tarlow JK, Cork MJ, Gordon C, Emery P, Duff GW. Interleukin-1 receptor antagonist gene polymorphism as a disease severity factor in systemic lupus erythematosus. Arthritis Rheum 1994;37:13801385. 21. Suzuki H, Matsui Y, Kashiwagi H. Interleukin-1 receptor antagonist gene polymorphism in Japanese patients with systemic lupus erythematosus. Arthritis Rheum 1997;40:389-390. 22. Meisel P, Siegemund A, Dombrowa S, Sawaf H, Fanghaenel J, Kocher Th. Smoking and polymorphisms of the interleukin-1 gene cluster (IL-1a, IL-1b, and IL-1RN) in patients with periodontal disease. J Periodontol 2002;73:27-32. Correspondence: Dr. Mehrdad Radvar, Department of Periodontics, School of Dental Medicine, University of Pennsylvania, 240 (Suite W1), S. 40th St., Philadelphia, PA 19104. E-mail: radvar@dental.upenn.edu. Submitted February 6, 2010; accepted for publication April 17, 2010.

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