Association study of IL-β (rs ) and IL- 1RN (rs ) with susceptibility to chronic periodontitis in Northern Indian population

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1 Gene Therapy and Molecular Biology Vol: 17, page 1 Gene Ther Mol Biol Vol 17, 1-12, 2015 Association study of IL-β (rs ) and IL- 1RN (rs ) with susceptibility to chronic periodontitis in Northern Indian population Research Article Garima Prakash 1, Sadhana Ajay 2, Krishna Kumar Gupta 3, Deepika Bali 4, Jaya Dixit 5, Balraj Mittal 1 * 1Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Department of Biochemistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow Department of Periondontics and Implantology, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow Department of Periodontology and Implantology, D.A.V (c) Dental College, Yamuna Nagar, Haryana Department of Periodontics, King George s Medical University, Lucknow *Correspondence: Prof. Balraj Mittal, Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow , India, Phone: , 004 8, Ext: 4322, Fax: , , bml_pgi@yahoo.com Keywords: IL-1β; IL-1RN; periodontitis; susceptibility Key Message: IL-β (rs ) and IL-1RN (rs ) are not associated with the susceptibility to chronic periodontitis in Northern Indian population. Received: 10 December 2014; Revised: 16 December 2014 Accepted: 7 January 2015; electronically published: 13 March 2015 Summary Background: Chronic Periodontitis (CP) is an inflammatory disease and Interleukin1 is believed to play key role in its pathogeneses. Previous studies have elucidated the role of IL-1 polymorphisms in periodontitis; however findings are contradictory. Aims: So, we aimed to study the association of IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphism in susceptibility to chronic periodontitis in northern Indian population. Settings and Design: Case-control study was carried out in individuals recruited from North Indian population. Materials and methods: The genotyping of IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphisms was carried out by PCR-RFLP and PCR respectively in 200 age and sex matched clinically diagnosed CP cases and 200 healthy controls. Statistical Analysis: SPSS ver.15.0 software was used to calculate risk estimation via binary logistic regression. Results: None of the IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphisms conferred risk of CP in northern Indian population. Gender stratification or tobacco usage also did 1

2 Prakash et al: IL-1β and IL-1RN polymorphism and periodontitis not modulate risk in CP. Results shows that IL-1β (-511C>T) and IL-1RN (VNTR) are not involved in CP susceptibility in northern Indian population. Conclusion: In conclusion IL-1β (-511C>T) and IL-1RN (VNTR) are not implied in risk modification. However, the present study should be replicated in larger samples for more consistent results. I. Introduction: Periodontitis is a chronic inflammatory disease which results in the devastation of tissue supporting tooth (periodontium), leading to loosening and loss of teeth. Around 10% to 15% of adults population are affected worldwide (Petersen and Ogawa, 2005). In northern Indian population, prevalence of chronic periodontitis (CP) is 30 to 35.4% in year age group as per reported by World Health Organization in one of its survey in India (Shah et al., 2007). Periodontitis is believed to be multi-factorial disease: infection from bacteria, tobacco consumption, smoking and diabetes are well established risk factors. However, inter-individual variation in genetic factors also plays key role in susceptibility to periodontitis and its pathogenesis (Van Dyke and Sheilesh, 2005). Host genetic factors are supposed to modulate immune response by production of pro-inflammatory cytokines and affect the risk of periodontitis (Noguchi and Ishikawa, 2007). During last several years, inflammatory factors has been studied profoundly and IL-1 was found to play vital role in various chronic inflammatory diseases like rheumatoid arthritis, COPD, Crohn s disease and periodontitis (Shete et al., 2010; Botelho et al., 2011; Ferraccioli et al., 2012; Szczeklik et al., 2012). IL-1 gene family is located on long arm of chromosome 2 at position 2q13-21 in the form of cluster. This family consists of two proinflammatory proteins (IL-1α and IL-1β) and an antagonist protein (IL-1Ra) encoded by IL- 1α, IL-1β and IL-1RNgene respectively (Upadhyay et al. 2008).IL-1gene family is highly polymorphic and various polymorphisms are known in IL-1β and IL- 1RNgenes (Bidwell et al., 1999). IL-1β is pro-inflammatory cytokine and expressed in high levels in many inflammatory diseases (Botelho et al., 2011, Ferraccioli et al., 2012 and Szczeklik et al., 2012).Due to its bone resorption and proinflammatory features, higher expression of IL-1β in response to pathogenic infection is involved in tissue destruction. Increased levels ofil-1β have also been found in gingival crevicular fluid which in turn is related to the severity of periodontitis (Ferreira et al., 2008). IL1-β (+3954) polymorphism present in 5th exon is extensively studied and shows increased risk with the severity of CP (Brett et al. 2005).IL1- β (-511C>T) polymorphism is present in promoter region and shows-t base transitions, at positions -511 from transcriptional site. This SNP is found to be in linkage disequilibrium with IL1-RNVNTR polymorphism (Hartland et al. 2004). IL-1RN gene codes for IL-1RA (receptor antagonist) an endogenous inhibitor which modulates the activity of IL1-β (Dinarello CA, 1996).A VNTR polymorphism is present in intron 2 of IL-1 RN. TheIL-1 RNVNTR polymorphism generally found in six allelic variants (one to six copies of the 86-bp 2

3 Gene Therapy and Molecular Biology Vol: 17, page 3 sequence repeat) where IL-1RN*1(4 repeats) and IL-1RN*2 (2 repeats) allele are observed more frequently in normal individuals (Upadhyay et al., 2008). Genotype 2/2 of IL- 1RNhas been observed as putative risk for chronic periodontitis among Afro-American and mulattos (Trevilatto et al. 2011).In periodontic patients low salivary levels and high serum levels of IL-1Ra were observed in the subjects with allele 2 (Perrier et al. 1998).Studies from Caucasian; African- American, Chinese and Asians show inconsistent differences in distribution of IL- 1RN*2 (Laine et al. 2010).Therefore, in the present study, we investigated the role of IL- 1β-511C>T (rs ) and IL-1RN VNTR (rs )polymorphisms with the susceptibility to CP in northern Indian population. II. Materials and Methods: II.A. Ethical Statement From each subject written inform consent was taken and study protocol was approved by local ethical committee of the Institute, namely Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow and Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow. All research work was conducted in accordance with ethical principles as per the declaration of Helsinki. II.B. Study subjects Patients and controls were recruited from same geographical region from January 2011 to December A total of 200 patients were selected from the outpatient clinic of Department of Periodontics and Implantology in Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, for the clinical diagnosis, criteria declared by IWC 1999 International Workshop for Classification of Periodontal Diseases and Conditions were followed (Armitage, 1999). To validate the diagnosis bone resorption level was measured by radiographic analysis of full mouth. Cases were grouped in CP if there is clinical attachment loss > 3mm in 30% site and in control this attachment loss is 3mm. Sex, age and ethnicity matched 200 controls were enrolled who visited for regular dental checkup in same hospital and were free from any dental disease. Clinical attachment loss, bleeding on probing, pocket depth, gingival index and calculus index were noted as clinical parameters shown in Table.1 and all these parameters were examined by junior examiner and confirmed by a senior consultant (Loe and Silness, 1963; Ramfjord 1967; Harold and George 1974). Cases and control who showed up presence of any systemic disease like diabetes, hepatitis, bleeding disorder, immunosuppressive disease or taking any immunosuppressive drugs, pregnant women/or lactating mothers were excluded. The case and control were from same ethos i.e. North Indian. At the time of diagnosis, all data on clinical, demographic and environmental exposure from medical records and personal interview were noted. Patients using tobacco in any form (smoking, chewing, sniffing or any combinations) were grouped as tobacco users. 3

4 Prakash et al: IL-1β and IL-1RN polymorphism and periodontitis Table 1. Characteristics of study subjects Variables Controls Cases Mean Age±SD (years) 40.25± ±10.46 Gender Males N (%) 111 (55.5) 111 (55.5) Females N (%) 89 (44.5) 89 (44.5) Tobacco users N (%) - 25 (12.5) Non-tobacco users N (%) 175 (87.5) Smoker N (%) - 6 (3) Non-smokers N (%) 194 (97) Clinical measurements Mean clinical attachment loss (mm) Mean pocket depth (mm) Bleeding on Probing Yes No II.C. DNA extraction and Genotyping Five ml of peripheral blood was collected in EDTA (ethylenediaminetetraacetic acid) vial and kept frozen. Genomic DNA was isolated by salting out method (Miller et al. 1988) and stored at -70 C. The isolated DNA was then checked on NanoDropAnalyzer (ND-1000) spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) for its quality and quantity assurance. The genotyping was done by PCR/PCR-RFLF (polymerase chain reaction- restriction fragment length polymorphism) method (Figure 1). Figure 1. Graphic representation of PCR method showing DNA extraction from blood used for gene amplification, digested and checked on Agarose Gel 4

5 Gene Therapy and Molecular Biology Vol: 17, page 5 II.D. IL-1 polymorphisms PCR-RFLP and PCR respectively were used to genotype IL-1β (-511C>T) and IL-1RN (VNTR) polymorphisms. The PCR was carried out in total volume of 50 µl with 25 pmol of each primer. Primer sequences and restriction enzyme used as described previously (Upadhyay et al., 2008; di Giovine et al., 1992; Tarlow et al., 1993). PCR master-mix without DNA was used as negative control and to make sure that PCR product is contamination free. Representative genotypes are shown in Figure 2 and 3 respectively. 10% of cases and control of each genotype was sequenced to ensure the quality and quantity of genotype and to avoid genotypic error which shows 100% accuracy. Figure 2. Representative gel picture showing IL-1 β(-511 C>T). Lane 1 shows TT genotype, Lane 2 shows CT genotype and Lane 4 shows CC genotype. Lane 3 is 100 bp ladder. 5

6 Prakash et al: IL-1β and IL-1RN polymorphism and periodontitis Figure 3. Representative gel picture showing IL-1 RN (VNTR). Lane 2 shows 1/1 genotype, Lane 3 shows 1/2 genotype, Lane 4 shows 2/2 genotype, Lane 6 shows 1/3 genotype, Lane 7 shows 2/4, Lane 8 shows 1/4 genotype. Lane 1 is 50 bp ladder and Lane 5 is 100 bp ladder. II.E. Statistical analysis Quanto ver.1.2 ( software was used to calculate the sample size for 80% power of the study. The IL-1β (-511C>T) genotype frequency in controls followed Hardy- Weinberg Equilibrium (HWE), confirming that controls were representatives of general population. Pair of 200 cases-and controls was adequate sample size for this study to give 80% power in log additive model (to avoid possible Type II error).for gene environment interaction, analysis was carried out in cases only due to low response rate of tobacco usage in controls. Age and gender adjusted odd ratio (OR) was estimated by binary logistic regression with 95% confidence interval. P-values less than 0.05 were considered significant for all statistical tests. Statistical Package for Social Sciences version 15.0 (SPSS, Chicago, IL, USA) was used to carry out all statistical analyses. III. Results: III.A. Characteristics of Participants The baseline characteristics of CP cases and age-gender matched controls are shown in Table 1. The age was expressed in Mean age ± Standard Deviation (SD). The mean age and SD among cases and controls (37.8±10.46 and 39.5±8.63 years respectively) and gender distribution in cases (males: 55.5%, female: 45.5%) and in controls (male: 57.5%, female: 43.5%) were comparable. 12.5% of patients reported tobacco habit (tobacco chewers, smokers or both) and 3% of cases were smokers alone(table 1). In controls, tobacco usage was not recorded and case only analysis was performed for the risk estimation. III.B. Association of IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphism with risk of CP Genotypic and allelic frequency distributions ofil-1β (-511C>T) and IL-1RN (VNTR) are 6

7 Gene Therapy and Molecular Biology Vol: 17, page 7 shown in Table.2. The genotypes of IL-1β (-511C>T) were according to Hardy-Weinberg Equilibrium in controls. Frequencies at genotypic and allelic levels for IL-1β (-511C>T) and IL-1RN (VNTR) were not significantly different between cases and controls. On application of binary logistic regression also IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphism did not yield any significant association with risk of periodontitis (Table 2). Table 2. Association of IL-1β and IL-1RN gene polymorphisms with risk of chronic periodontitis Genotypes/Alleles Patients N (%) Controls N (%) Odds Ratio*(95% CI) P-value IL-1β (-511C>T) polymorphism CC 30(15.0%) 21(10.5%) Reference CT 84(42.0%) 105(52.5%) 0.59( )0.107 TT 86(43.0%) 74(37.0%) 0.90( )0.750 C allele 144(36.0%) 147(36.8%) Reference T allele 256(64.0%) 253(63.3%) 1.03( )0.826 IL-1RN (86 bp VNTR) polymorphism 1/1 103(51.5%) 104(52.0%) Reference 1/2 72(36.0%) 78(39.0%) 0.93( ) /2 18(9.0%) 13(6.5%) 1.39( ) /4 3(1.5%) 0(0%) - 1/3 3(1.5%) 0(0%) - 1/4 1(0.5%) 5(2.5%) 0.20( ) allele 282(70.5%) 291(72.8%) Reference 2 allele 111(27.8%) 104(26.0%) 1.10( ) allele 3(0.8%) 0(0%) - 4 allele 4(1.0%) 5(1.3%) 0.82( )

8 Prakash et al: IL-1β and IL-1RN polymorphism and periodontitis III.C. Gender specific association of IL- 1β (-511C>T) and IL-1RN (VNTR) gene polymorphisms with risk of CP When IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphism were stratified on the base of gender, none of the genotypes showed sex specific risk of CP. AlthoughIL- 1β-511CT genotype frequency was lower in cases (40.5%) as compared to controls (51.4%) but there was no significant difference (P > 0.05) in cases and controls (Table.3). Table 3. Gender specific association of IL-1β and IL-1RN gene polymorphisms with risk of chronic periodontitis Genotypes Male N (%) Odds Ratio*(95% Female N (%) Odds Ratio* (95% Cases Controls CI) P value Cases Controls CI) P value IL-1β (-511C>T) polymorphism CC 17(15.3) 11(9.9) Reference 13(14.6) 10(11.2) Reference CT 45(40.5) 57(51.4) 0.51( ) (43.8) 48(53.9) 0.64( )0.358 TT 49(44.1) 43(38.7) 0.74( ) (41.6) 31(34.8) 1.01( )0.972 IL-1RN (86 bp VNTR) polymorphism 1/1 56(50.5) 61(54.5) Reference 47(52.8) 43(48.9) Reference 1/2 42(37.8) 41(36.6) 1.11( ) (33.7) 37(42.0) 0.74( ) /2 10(9) 7(6.3) 1.57( ) (9.0) 6(6.8) 1.22( ) / (3.4) - - 1/3 3(2.7) /4 0(0) 3(2.7) - 1(1.1) 2(2.3) 0.45( )0.529 III. D. Association of IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphism with tobacco usage Case-only analysis was carried out to study the interaction of IL-1β (-511C>T) and IL-1RN (VNTR) gene polymorphisms with environmental risk factors. Among 200 cases 12.5 % were tobacco users. Frequencies of IL- 1β (-511C>T) and IL-1RN (VNTR) genotypes did not differ in tobacco users and non-users and thus did not modulate significant risk of CP with tobacco usage (P > 0.05) (Table.4). 8

9 Gene Therapy and Molecular Biology Vol: 17, page 9 Table 4. Association of IL-1β and IL-1RN gene polymorphism with risk of chronic periodontitis in tobacco users Genotypes Users N (%) Non-Users N (%) Odds Ratio* (95% CI) P-value IL-1β (-511C>T) polymorphism CC 1(4.0) 29(16.6) Reference CT 14(56.0) 70(40.0) 6.64( )0.079 TT 10(40.0) 76(43.4) 4.00( )0.204 IL-1RN (86 bp VNTR) polymorphism 1/1 12(48%) 91(52%) Reference 1/2 10(40%) 62(35.48%) 1.223( ) /2 3(12%) 15(8.6%) 1.517( ) /4 0 3(1.7%) - 1/3 0 3(1.7%) - 1/4 0 1(0.6%) - IV. Discussion IL-1 is one of the major cytokines produced at inflamed sites and is involved in the initiation and progression of connective tissue destruction. Large numbers of genes expressed during inflammation are directly affected by IL-1 and these include genes for other cytokines, cytokine receptors and extracellular matrix components (Dinarello, 1996).Various functional polymorphisms have been reported in the IL-1 gene family (Bidwell et al. 1999) and the variants affect the function by altering either the amount of protein or the activity of the protein produced. In the present study we looked into the role of IL-1β (-511C>T) and IL-1RN (VNTR) with the risk of CP in North Indian Population. Findings from our study suggest that IL-1β (-511C>T) and IL-1RN (VNTR) polymorphism do not affect the risk of CP in North Indian Population. Till date majority of studies on association of IL-1β (- 511C>T) polymorphism with CP have been carried out in Caucasian and Japanese populations and most of them do not show any association (Shete et al., 2010, Brett et al., 2005, Soga et al., 2003 and Geismar et al., 2008).However, Ferreira et al. reported higher serum level of IL-1βin periodontitis patients (Ferreira et al. 2008). The IL-1β (-511C>T) polymorphism is present in promoter region may cause alteration of gene expression. A 9

10 Prakash et al: IL-1β and IL-1RN polymorphism and periodontitis possible reason could be that there may be other variants in IL-1β gene which may be involved in CP. Alternatively, this polymorphism may have role in severity of disease but not with susceptibility. IL1RN codes for a natural inhibitor for IL- 1β. A VNTR in intron 2 of IL1RN influences its expression. However, its association with CP is controversial. Cheng et al. in his metaanalysis of IL-1RN (VNTR) showed increased risk of CP in overall population which included Caucasians, Asians and Mixed (Brazil and Chile) populations (Ding et al., 2012). However, no association was observed in Caucasians and Asians population when subgroups analysis was done (Ding et al., 2012).Geismar and Sankellari et al. also reported no association in Caucasian population (Geismar et al., 2008; Sankellari et al., 2006 ). Similarly, in present study also we did not find significant association with the risk of CP. The inconsistent reporting may be results of small number of subject in the previous studies and also mixed populations. Stratification of subjects based on gender showed that both polymorphism IL-1β (- 511C>T) and IL-1RN (VNTR) did not confer gender specific risk in CP cases. Tobacco in any form whether smokeless or non-smokeless tobacco is the well-known and one of the causative agent for the risk exposure of CP (Burgan 1997 ; Johnson 2001). Since there was low response regarding this information among controls, so case-only analysis was done to look into the role of tobacco usage with the risk of CP. In the present study no such difference in the frequency IL-1βand IL1RN VNTR polymorphisms was seen between tobacco users and non-users. It is also possible that negative association may be because of lower number of tobacco users in the present study. Strength of present study include well defined set of cases and controls while collecting samples we followed stringent criteria, sufficient sample size for 80% power of study, consistencies of genotypic frequency with HWE in controls and comparative MAF in present study to that of other reported studies from same population. Limited number of subjects in the subgroup analysis could be the limitation of present study. In conclusion, polymorphism IL-1β (- 511C>T) and IL-1RN (VNTR) do not modulate the risk of CP independently in North Indian Population. Analysis based on tobacco consumption also didn t predict any significant risk although it is one the major risk factors. This is the first study from North Indian Population on the study of IL-1β (- 511C>T) and IL-1RN (VNTR) gene polymorphism with the susceptibility to CP. However, the study requires replication in larger sample size. Acknowledgements Financial assistance for the study was provided by Indian Council of Medical Research (ICMR), New Delhi (Grant No. 5/4/2-5/09- NCD-II). 10

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