ORIGINAL ARTICLE

Iran J Allergy Asthma Immunol June 2009; 8(2): 95-98

Polymorphism of IL-1(-889) Gene and Its Association with Aggressive Periodontitis

Zahra Kiani1, Jalil Tavakkol-Afshari2, Hamed Hojjat2, Hamid Reza Arab3, Mehrdad Radvar3, Mehrdad Sadeghizadeh4, and Ahmad Reza Ebadian2

ABSTRACT

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Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. The pro-inflammatory and bone resorption properties of Interleukine-1 (IL-1) strongly suggest a role for this cytokine in the pathogenesis of periodontal disease. Eighty Iranian adult patients with periodontitis and 80 Iranian controls were investigated in this study. In this study we report that the frequency of IL-1 genotypes including allele 2 of the IL1(-889) restriction fragment length bi-allele polymorphism were significantly increased in patients with advanced aggressive adult periodontitis compared to those with early and moderate disease. Furthermore, allele 2 was associated with increased production of IL-1 by activated peripheral blood polymorphonuclear cells of patients with advanced disease, although this increase failed to reach statistical significance. Finally, the data obtained revealed significant linkage disequilibrium between allele 2of the IL-1 (-889) polymorphism and allele 2 of the bi-allelic IL-1 (+3953) polymorphism in both patients and orally normal controls. These findings provide new insight into the possible rate of IL-1 and genes polymorphism in the susceptibility to adult periodontitis. Key words: IL-1; Periodontitis; Polymorphism; RFLP

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INTRODUCTION The etiopathogenesis of adult periodontitis is poorly understood, therefore, predictive diagnostic test for this
Corresponding Author: Zahra Kiani, MS;
Chabahar International University of Medical Sciences, Chabahar, Iran. Tel: (+98 915158 2481), Fax: (+98 545) 222 3017, Email: zkiani604@yahoo.com

Copyright 2009, IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY. All rights reserved.

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Received: 28 May 2008; Received in revised form: 24 February 2009; Accepted: 14 May 2009

disease remains an elusive. Adult periodontitis is a complex multifactorial disease whose etiology is not well defined1. The result from bacterial plaque and influenced by environmental and genetic factors. Proinflammatory cytokines such as IL-1 have an important role in the pathogenesis of periodontal disorders. It has been proposed that IL-1 gene polymorphism plays an important role in the

Department of Immunology, Chabahar International University of Medical Sciences, Chabahar, Iran Department of Immunogenetic, Bu-Ali Research Center, Mashhad University of Medical Sciences, Mashhad, Iran 3 Department of Periodontology, Mashhad University of Medical Sciences, Mashhad, Iran 4 Department of Molecular Genetic, Tariat Modarres University, Tehran, Iran

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susceptibility to periodontal disease. The aim of this study was to investigate the relationship between IL-1 (-889) gene polymorphism and generalized aggressive periodontitis (GAP). Implicated in the onset and pathogenesis of the adult periodontitis are risk factors such as age, sex, race and smoking 2,3. Cytokines are of the special interest in the contest of periodontitis due to their effective bone metabolizing and inflammatory properties4. IL-1 in particular has been studied as it is the more potent inducer of bone resorption5 and therefore may play a more significant role in the pathogenesis of periodontal disease. Increased levels of IL-1 have been found in both gingival crecicular fluid6 and gingival tissues7 of patients with adult periodontitis addition oral polymorphonuclear cells of patients have been shown to produce substantial levels of IL18.Variation in cytokine levels among individual is well-documented and may contribute to disease susceptibility. Such differences may be attributed in part to the alleles of polymorphic cytokine genes since particular alleles have been associated with increased cytokine levels .Scientists showed that allele 2 of the IL-1(+3953)polymorphism is associated with increased IL-1 production. The bi-allelic restriction fragment length polymorphism (RFLP) of the IL-1 gene at position (-511) and (+3953) and of the IL-1 gene at position (-889) have been characterized previously9 .In recent study10, found an association of composite genotype including both IL-1(-889)allele2 and IL-1(+3953)allele 2, with advanced adult periodontitisis in non-smokers. In the present study, RFLP analysis was performed to determine the distribution of IL-1 and genotype in patients with aggressive periodontal disease and their matched controls. In addition, possible association of genotype with cytokine production by oral and peripheral blood polymorphonuclear cells (PMN) was examined. subject patients diagnosis and classification of disease severity were determined using the American Academy of Periodotology Current Procedural Terminology (http://www.perio.org) following physical examination and a complete radiograph series. We did not separate smokers from non smokers. All subjects were in good health overall and were taking no medications. This study was confirmed by the ethic committee of Mashhad University of Medical Sciences. Laboratory Procedures Blood samples were sent to Mashhad Bu-Ali Research Institute for laboratory tests. Genomic DNA was extracted by commercially DNA extraction kit (Biogene, Mashhad, Iran), using salting- out method from 10ml of whole blood, which was used genotyping assays with the Polymerase Chain Reaction Restriction Fragment Lengh Polymorphism (PCR-RFLP) technique. We used following primers for amplifying IL-1(-889)gene: 5'AAG CTT GTT CTA CCA CCT GAA CTA GGC 3'and 5'TTA CAT ATG AGC CTT CCATG 3'.The 99 bp region of IL-1gene was amplified for 35 cycles of incubation at 95 C for 5 min, 95 C for 30 sec,60 C for 30 sec and 72 C for 30 sec and 72 C for 5 min (final extension)11. The 6microlitre product was digested with 0.3 unit of Nco1 restriction enzyme at 37C for 1.20 hour, yielding 2 fragments of 83bp and 16 bp in subjects homozygous for allele 1 and one fragment of 99bp in subjects homozygous for allele 2.All 3 fragments were present in heterozygous subject, but fragment of 16bp was washed out and we could not saw that. Statistical Analysis Statistical Analysis was performed using Fishers exact and chi-Square tests. The P-value and df for statistical analysis have given in result section.

Study population Subjects recruited for this study included 80 Iranian adult periodontitis patients individually matched for sex and age with 80 Iranian controls found to exhibit no signs of periodontal disease upon clinical examination. All patients have gone to Dentology Department of Mashhad University and examined by dentist. Peripheral blood samples from all subjects were obtained upon informed consent using a protocol approved by the Institutional Review Board for Human

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PATIENTS AND METHODS

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Figure 1. Polyacrylamide gel electrophoresis. IL-1 (-889) gene was amplified by PCR and digested with Nco-I restriction enzyme and electerophoretically separated on 17% polyacrylamide gel. All lanes related to patients. Lanes 1,2,3,6 and 8 are heterozygote (1.2), samples 4, 7 are homozygote (2.2) and sample 5 is homozygote (1.1).

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Polymorphism of IL-1(-889) Gene and Aggressive Periodontitis

RESULTS At first, we investigated the relationship between 3 kind of genotype of IL-1 (-889) gene and periodontitisis (Figure1). In control group: 17.5% had 1/1 genotype, 43.75% had genotype and 38.75% had 2/2 genotype (Table1 and Figure 2). In patients group: 15%had 1/1 genotype, 53.75% had genotype and 31.25%had 2/2 genotype in Table2 and Figure2. Then with Chi-Square Test we realized that there is not any relationship between several kind of genotype of IL-1 (-889) gene and periodontitis. At second, in control group: 39.37%had allele1 and 60.62% had allele 2.In patients group: 41.87% had allele 1 and 58.12% had allele 2 .With Chi-square Test and Fishers Exact Test we realized that there is not any relationship between 2 kind of allele of IL-1(-889) gene and periodontitis (pvalue=0.867 and df=1).At third ,we compared people without allele 1 and people with at least one allele 1 in control and patient groups. Then we did Chi-square Test and Fishers Exact Test. The result revealed that there is not any significant relationship between presence or absence of allele1 and periodontitis. Finally we compared people without allele 2 with people who had at least one allele 2 in control and patient groups. With Chi-square Test and Fishers Exact Test we realized that presence or absence of allele2 isnt important in periodontitis (p-value=0.528 and df=1). DISCUSSION IL-1 has long been considered to play a role in the pathogenesis of periodontal disease due to its proinflammatory and bone resorption properties, Although IL-1 has been studied as a potential effector of periodontitis, more attention has been given to IL-1 as it has been found in higher concentrations at periodontal disease sites. Due to the complex multifactorial etiology of periodontitis, a definite role for IL-1 has been difficult to establish. However, there are several reports of increased levels of IL-1 in the gingival crevicular fluid and gingival tissues of periodontitis patients.3 Furthermore, it has been shown that oral polymorphonuclear cells, the first line of defense against microbial insult, produce substantial levels of both IL-1 and TNF-, which increase with severity of periodontal disease.8 The alleles of the IL1 gene polymorphisms, which may be involved, in the regulation of IL-1 production ,could contribute greatly to the development of periodontal disease. The results of the present study showed that there is not any significant increase in frequency of the IL-1(-889) genotypes containing the allele 2(IL-1(-889)1/2 and 2/2) and allele1 (IL-1(-889)1/1 and ) in patients with severe periodontitis compared to control group. Smoking has been established as a major risk factor in the development of periodontal disease10-13 and should be considered when conducting genetic studies of adult periodontitisis.

Healthy

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14 17.5% 1/1 12 15%

Table 1. Genotype information for normal group. Genotype information 1/1 1/2 2/2 35 43.75% 31 38.75%

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2/2 25 31.25%

Total 80 100%

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Periodontitis

Count %within group

As this table illustrates, mostly had heterozygote genotype in this group by 35 in number and 43.75% in percentage. Of course the number of heterozygotes and hemozygote2/2 were close to each other and had significant interval with hemozygote1/1,by14 in number and 17.5% in percentage.

Table 2. Genotype information for periodontitis group. Genotype information Total 80 100%

Count %within group

As this table illustrates, mostly had heterozygote genotype in this group by 43 in number and 53.75% in percentage. Subsequently, 25 patients had 2/2 genotype with 31.25% and 12patients had 1/1genotype with 15%.

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REFERENCES
1. Gore EA, Sanders JJ, Pandey JP, Palesch Y, Galbraith GM.

Interleukin-1beta+3953 allele 2: association with disease status in adult periodontitis. J Clin Periodontol 1998; 25(10):781-5.
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GENOTYPE
1.1

Salvi GE, Brown CE, Fujihashi K, Kiyono H, Smith FW,

Beck JD, et al. Inflammatory mediators of the terminal dentition in adult and early onset periodontitis. J Periodont Res1998;33(4):21225. 3. 4. Offenbacher S, Periodontal diseases. Pathogenesis. Ann Eftimiadi C, Stashenko P, Tonetti M, Mangiante PE, Massara Periodontol 1996; 1(1):821-78. R, Zupo S, et al. Divergent effect of the anaerobic bacteria byproduct butyric acid on the immune response: suppression of Tproduction. Oral Microbiol Immunol 1991; 6(1):17-23. 5. 6. Irwin CR, Myrillas TT. The role of IL-6 in the pathogenesis Page RC, Altman LC, Ebersole JL, Vandesteen GE, Dahlberg

Count

1.2 0 healthy periodontitis 2.2

GROUP

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The patient population studied was therefore not entirely comparable with that examined by Kornman et al.9 After the data obtained in the present were analyzed to determine the frequency of composite genotype in the different subject groups, it was found to occur in 50% of patients with severe periodontitis and only 25% of those with early or moderate disease. Interestingly, the increase in frequency of genotype in severe disease did not reach significance in the present study. Several studies have shown an increase of IL-1 production by circulating monocytes of patients with periodontal disease. However, contradictory data have been published. These conflicting findings may be due to in part to methods of detection and inter individual variation in cytokine production. In the present study, a strong but not significant trend towards increased IL-1 production by peripheral blood polymorphonuclear cells was found in patients with advanced periodontitis. we suggest that another study in this field in the future, other risk factors in periodontitis should be investigated.

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ACKNOLEDGMENTS

We thank Miss Brook for her valuable technical assistance and great helps during the project. This research was supported by a grant from vice chanceller for research at MUMS.

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of periodontal disease. Oral Dis 1998; 4(1):43-47.

WH, Williams BL, et al. Rapidly progressive periodontitis. A distinct clinical condition. Dent Health(London) 1984; 23(5):3-4,6Honig J, Rordorf-Adam C, Siegmund C, Wiedemann W,

Erard F. Increased interleukin-1 beta (IL-1)concentration in gingival tissue from periodontitis patients. J Periodont Res 1989; Galbraith GM, Hendley TM, Sanders JJ, Palesch Y, Pandey

JP. Polymorphic cytokine genotypes as markers of disease severity in adult periodontitis. J Clin Periodontol 1999; 26(11):705-9. Kornman KS, Crane A, Wang HY, di Giovine FS, Newman MG, Pirk FW, et al. The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 1997; 24(1):72-7. 10. Torres de Hees GL, Van der Valden U, Loos BG. Cigarette smoking enhances T cell activation and a Th2 immune response; an aspect of the pathophysiology in periodontal disease. Cytokine 11. Agnihotri R, Pandurang P, Kamath SU, Goyal R, Ballal S, et al. Association of cigarette smoking with superoxide dismutase enzyme levels in subjects with chronic periodontitis.J Periodontal 12. Adler L, Modin C, Friskopp J, Jansson L. Relationship between smoking and periodontal probing pocket depth profile. Swed Dent J 2008; 32(4):157-63 13. Laxman VK, Annaji S:Tobacco use and its effects on the periodontium and periodontal therapy.J Contemp Dent Pract 2008;

Figure 2. Histogram of healthy and periodontitiss genotypes. It shows in both group genotype1.2 has the highest level and after that are 2.2 and 1.1, respectively.on the other hand heterozygotes are the majority in both group.

lymphocyte prolifration and stimulation of interleukin-1 beta

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