Abstract
Objective: The objectives of this study were to determine the association between single nucleotide polymorphisms (SNPs) in IL1B (−511, +3954), IL1A (−889, +4845), and the variable number of tandem repeats (VNTRs) polymorphism in the IL-1RN gene with chronic periodontitis susceptibility and to analyze gene–gene interactions in a hospital-based sample population from South India. Subjects and Methods: A total of 400 individuals were recruited for this study; 200 individuals with healthy gingiva and 200 chronic periodontitis patients. Genomic DNA was isolated from peripheral blood samples and genotyping was performed for the above-mentioned single nucleotide and VNTR polymorphisms by polymerase chain reaction, DNA sequencing, and agarose gel electrophoresis. Results: A higher proportion of the variant alleles were observed in the chronic periodontitis group for all the SNPs examined. The SNP at +3954 (C>T) in the IL1B gene was found to be significantly associated with chronic periodontitis (p=0.007). VNTR genotypes (χ2 value: 5.163, df=1, p=0.023) and alleles (χ2 value: 6.818, df=1, p=0.009) were found to have a significant association with chronic periodontitis susceptibility. Conclusion: In the study population examined, the SNP in the IL1B gene (+3954) and VNTR polymorphisms in the IL1RN gene were found to have a significant association with chronic periodontitis susceptibility.
Introduction
Chronic periodontitis is an inflammatory disease of the supporting structures of the tooth with a microbial etiology (Haffajee and Socransky, 1994). Its progression and severity have been determined to be influenced by several factors: environmental factors (smoking) (Haber et al., 1993), genetic factors (candidate gene polymorphisms) (Loos et al., 2005), and systemic disease (diabetes mellitus type II) (Chapple and Genco, 2013). An important cytokine that plays a role in the pathogenesis of chronic periodontitis is interleukin-1 (Birkedal-Hansen, 1993). The interleukin-1 gene cluster is found in chromosome 2 and encodes for the cytokines; interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), and interleukin-1 receptor antagonist (IL-1 ra) (Deng et al., 2013). IL-1α and IL-1β have a proinflammatory role in periodontitis pathogenesis, whereas IL-1ra is antagonistic to these cytokines (Rawlinson et al., 2003).
The usefulness of IL-1β, IL-1α levels in gingival crevicular fluid (GCF) as a biomarker for chronic periodontitis has been evaluated in several studies. Masada et al. (1990) evaluated IL-1β m-RNA (gingival tissue) and protein levels in GCF samples from chronic periodontitis patients. An elevated level of the IL-1β was observed and it was concluded that IL-1β was produced locally and contributed to bone loss observed in chronic periodontitis. A positive correlation of GCF IL-1β levels with probing pocket depth (Perozini et al., 2010) and IL-1α, IL-1β, and IL-1 activity (IL-1/IL-1ra ratio) with alveolar bone loss (Ishihara et al., 1997) has been reported among patients with chronic periodontitis.
A landmark study by Kornman et al. (1997), evaluated the association of single nucleotide polymorphisms (SNPs) in genes coding for interleukin-1 cytokines, tumor necrosis factor alpha (TNFα), and susceptibility to chronic periodontitis. The authors demonstrated a positive association between the composite genotype (IL1A −889 and IL1B +3954) and severe chronic periodontitis. Ever since, several studies have evaluated the role of these polymorphisms in periodontitis susceptibility with varied reports in different ethnic populations (Trevilatto et al., 2011; Tawil et al., 2012; Yücel et al., 2013). Previous studies in an Indian population have given conflicting results with a few studies showing a positive association (Shete et al., 2010; Archana et al., 2012), and one study showing no association between SNP at +3954 in the IL1B gene and chronic periodontitis (Kaarthikeyan et al., 2009).
India is inhabited by 5000 anthropologically well-defined caste and tribal populations; they are linguistically classified into Indo-European (northern part of the country), Austro-Asiatic (central and north eastern states), Dravidian (southern states), and Tibeto-Burman (northeastern states and the foothills of the Himalayas). In this study, we have analyzed the association between SNPs in IL1B (−511, +3954), IL1A (−889, +4845), and variable number of tandem repeat (VNTR) polymorphism in IL1RN gene with chronic periodontitis susceptibility and gene–gene interactions in a hospital-based sample population from South India.
Materials and Methods
Study population
The study population consisted, a total of 400 individuals (>18 years) recruited from the Out Patient Department of Periodontology, Faculty of Dental Sciences, Sri Ramachandra University, Chennai. Sri Ramachandra University is a tertiary care teaching hospital located on the outskirts of the city of Chennai and patients visiting the institute represent a fair cross section of urban, semiurban, and rural population. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University and has followed the Helsinki guidelines on Ethics for Human Research (IEC-NI/10/APR/15/05).
Sample size calculation
The sample size was calculated based on the allele frequencies reported by Masamatti et al. (2012). The variant allele frequency among cases and controls was 44.2% and 33.3% respectively. Based on the true probability of exposure, inclusion of 200 cases and 200 controls will be able to reject the null hypothesis with type I error of 5% and power of 80%.
All study participants were recruited after obtaining informed consent. Out of the 400, 200 were control individuals (healthy gingiva) and 200 patients were diagnosed to have generalized chronic periodontitis. The inclusion criteria for individuals with healthy gingiva were as follows: probing pocket depth of ≤3 mm, no attachment loss, absence of gingival bleeding on probing, absence of any clinical signs of gingival inflammation, and no previous history of periodontal disease. Individuals were diagnosed to have generalized chronic periodontitis based on attachment loss of >1 mm in at least 30% of the sites examined (Armitage, 1999) and radiographic evidence of bone loss, and presence of at least 10 natural teeth. Current and former smokers, pregnant women, individuals who were on any medication, and individuals with any known systemic disease were excluded from the study.
Clinical examination
A full mouth periodontal examination was performed for the following clinical parameters: Gingival Index (Silness and Loe, 1964), Plaque Index (Loe, 1967), Oral Hygiene Index-Simplified (OHI-S) (Greene and Vermillion, 1964), Clinical Attachment Level (CAL), and probing pocket depth at six sites per tooth excluding third molars. All the clinical parameters were assessed by the same examiner (Vamsi Lavu) using a UNC 15 manual periodontal probe.
Sample collection
Peripheral blood (3 mL) was collected with aseptic precautions from the ante-cubital vein in a vacutainer containing 0.5 mL of 0.129 mM sodium citrate (BD Biosciences, San Jose, CA). DNA extraction was done in 200 μL of the blood using commercially available extraction kits (Nucleo-pore DNA Sure Blood Mini Kit; Shivaji Marg, New Delhi, India) as per manufacturer advised protocols. The quality of extracted DNA was checked using nanodrop (260/280 nm ratio).
Genotyping
VNTR polymorphism in the IL1RN gene was determined by polymerase chain reaction (PCR) and agarose gel (2%) electrophoresis. The other polymorphisms in the IL-1 gene cluster, IL1A (+4845), IL1A (−889), IL1B (−511), and IL1B (+3954) were determined by PCR and DNA sequencing. PCR was performed on 50–100 μg of DNA in a 10 μL reaction containing 5 μL PCR Master Mix (Merck PCR Master Mix Kit; Merck, Rahway, NJ), 2.8 μL sterile water, and 30 pM of forward and reverse primers using an Eppendorf thermocycler (Eppendorf, Hamburg, Germany). The primers used, PCR conditions, and the different number of repeats for the VNTR polymorphism were referenced from a previous study (Meisel et al., 2002). The quality of the PCR products was assessed by electrophoresis using a 2% agarose gel.
DNA sequencing
Cycle sequencing, for the PCR products, to determine the polymorphisms was performed using an ABI PRISM Big Dye Terminator Ready Reaction Cycle Sequencing Kit (Life Technologies, Carlsbad, CA). Sequencing was done in a final volume of 10 μL containing 0.5 μL of ready reaction mix, 1.5 μL of sequencing dilution buffer, 1 μL of primer (forward/reverse), 6 μL of sterile water, and 1 μL of PCR product. The cycle sequenced products were then subjected to purification before the sequencing. Analysis of sequencing results was done using Applied Biosystems Seqscape v2.7 software (Life Technologies) to determine the base transition in the sites of interest.
Statistical analysis
The mean, frequency, and standard deviation for all the continuous variables were determined. The allele and genotype frequencies for all the SNPs were calculated and their association with chronic periodontitis was assessed by chi-square test. The association of the VNTR genotypes and alleles with chronic periodontitis was assessed by the CLUMP software package (Sham and Curtis, 1995). The association of the genotypes of SNPs with clinical parameters and indices of chronic periodontitis was done by one-way ANOVA. Hardy–Weinberg equilibrium (HWE) was determined for both cases and controls for all the SNPs and VNTR and significance of any deviation was assessed by chi-square test. Linkage disequilibrium (LD) was assessed between the loci of the SNPs of the IL-1 gene cluster in cases and controls and the standardized disequilibrium coefficient (D′) and squared correlation coefficient (r2) were calculated by Haploview software. Multifactorial dimensionality reduction (MDR) analysis was performed to analyze the gene–gene interactions among the SNPs analyzed. The results were assumed to be statistically significant at p<0.05.
Results
The demographic data pertaining to study population are summarized in Table 1. The genotype frequencies for the IL1A −889 C>T (rs1800587), IL1A +4845G>T (rs17561), IL1B −511C>T (rs16944) and IL1B +3954C>T (rs1143634) polymorphisms are shown in Table 2. The genotype distribution of three SNPs was found to be in HWE in both cases and controls. A deviation of the HWE was observed in the case group for the SNP in IL1B +3954C>T. The IL1A +4845G>T, and IL1B −511C>T genotypes were not associated with chronic periodontitis (Table 2). Both codominant and dominant models of IL1B −511C>T showed an increased risk for periodontitis, but the association was not found to be statistically significant (Table 2). An association of the CT genotype and dominant genotype model (CT+TT) in IL1B +3954C>T polymorphism with chronic periodontitis was observed (p=0.007, p=0.006). The IL1B +3954C>T locus also showed significant association with chronic periodontitis in the allelic model (Table 3). The distribution of IL1RN VNTR genotypes and alleles for both chronic periodontitis and control individuals are shown in Table 4. The allele A1 was the most frequent allele followed by A2 in this study population. The allele A5 was observed only in the periodontitis group with low frequency (Table 4). The IL1RN VNTR genotypes significantly deviated from HWE in both periodontitis (p<0.001) and control groups (p<0.001). CLUMP analysis revealed a statistically significant difference between periodontitis and control group for both genotype and alleles of IL1RN VNTR using chi-square clumped 2×2 table (genotype T4 χ2=5.163, df=1, p=0.023 and allele T3 χ2=6.818, df=1, p=0.009). One-way ANOVA revealed no significant association between the clinical parameters and genotype frequencies of any of the SNPs evaluated in the chronic periodontitis group (Table 5). The pairwise LD was not strong and significant between the SNPs examined (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/gtmb). Haplotypes constructed using IL1A −889C>T, IL-1A 4845G>T, IL1B −511C>T and 3954C>T revealed the presence of a total of 16 haplotypes. Of the total 16 haplotypes formed, only the CTCC and TGTT haplotypes were found to be associated with periodontitis group (Supplementary Table S2). MDR analysis between the SNPs of the IL-1 gene revealed two-, and three-factor genotype–genotype interaction models and none of the model/s had an average cross-validation consistency (CVC) of 9 out of 10 and p-value of<0.05 (Table 6).
Table 1.
Descriptive Data of the Study Population
| Clinical indices | Control (n=200) | Chronic periodontitis (n=200) | p-Value |
|---|---|---|---|
| Age range (years) | 20–55 | 22–60 | |
| Mean age (mean±SD) | 29.64±5.5 | 38.16±8.4 | <0.001 |
| Gender | |||
| Males | 95 (47.5%) | 112 (56.0%) | |
| Females | 105 (52.4%) | 88 (44.0%) | |
| OHI Score (mean±SD) | 0.415±0.251 | 2.810±0.857 | <0.001 |
| Gingival Index (mean±SD) | 0.227±0.190 | 1.837±0.290 | <0.001 |
| Plaque Index (mean±SD) | 0.250±0.228 | 1.902±0.392 | <0.001 |
| Probing pocket depth (mm) (mean±SD) | 1.099±0.199 | 4.124±0.445 | <0.001 |
| CAL (mm) (mean±SD) | 0.00±0.00 | 3.414±1.238 | |
p-Value<0.05, statistically significant.
CAL, Clinical Attachment Level; OHI, Oral Hygiene Index.
Table 2.
Association Analysis of Genotypes of SNP of IL-1 Gene with Chronic Periodontitis
| SNP in IL-1 gene | Controls (n=200) | Chronic periodontitis (n=200) | OR (95% CI) | p-Value |
|---|---|---|---|---|
| (rs1800587) IL1A −889 C>T | ||||
| CC | 107 (53.5) | 104 (52.0) | Ref | |
| CT | 79 (39.5) | 79 (39.5) | 1.029 (0.68–1.55) | 0.892 |
| TT | 14 (7.0) | 17 (8.5) | 1.25 (0.59–2.66) | 0.564 |
| CT+TT | 93 (46.5) | 96 (48.0) | 1.062 (0.72–1.57) | 0.764 |
| HWE (p-value) | 0.911 | 0.717 | ||
| (rs17561) IL1A +4845 G>T | ||||
| GG | 101 (50.5) | 96 (48.0) | Ref | |
| GT | 75 (37.5) | 81 (40.5) | 1.14 (0.75–1.73) | 0.551 |
| TT | 24 (12.0) | 23 (11.5) | 1.01 (0.53–1.91) | 0.980 |
| GT+TT | 99 (49.5) | 104 (52.0) | 1.11 (0.75–1.64) | 0.617 |
| HWE (p-value) | 0.091 | 0.354 | ||
| (rs16944), IL1B −511C>T | ||||
| CC | 97 (48.5) | 83 (41.5) | Ref | |
| CT | 82 (41.0) | 86 (43.0) | 1.23 (0.80–1.87) | 0.344 |
| TT | 21 (10.5) | 31 (15.5) | 1.73 (0.92–3.23) | 0.088 |
| CT+TT | 103 (51.5) | 117 (58.5) | 1.33 (0.89–1.97) | 0.160 |
| HWE (p-value) | 0.556 | 0.272 | ||
| rs1143634, IL1B +3954C>T | ||||
| CC | 130 (65.0) | 103 (51.5) | Ref | |
| CT | 66 (33.0) | 92 (46.0) | 1.76 (1.17–2.65) | 0.007* |
| TT | 4 (2.0) | 5 (2.5) | 1.58 (0.41–6.03) | 0.505 |
| CT+TT | 70 (35.0) | 97 (48.5) | 1.75 (1.17–2.61) | 0.006* |
| HWE (p-value) | 0.182 | 0.003 | ||
| IL1RN VNTR | ||||
| a1a1 | 127 (63.5) | 137 (68.5) | Ref | |
| Rest all | 73 (36.5) | 63 (31.5) | 0.80 (0.524–1.21) | 0.292 |
| HWE (p-value) | <0.001 | <0.001 | ||
Bold indicates p-value<0.05, statistically significant.
95% CI, 95% confidence interval; HWE, Hardy–Weinberg equilibrium; OR, odds ratio; SNP, single nucleotide polymorphism; VNTR, variable number of tandem repeat.
Table 3.
Allele Frequencies of SNPs in IL1A and IL1B Genes in Periodontitis Patients and Controls
| SNP in IL-1 gene | Controls | Cases | OR (unadjusted) 95% CI | p-Value |
|---|---|---|---|---|
| (rs1800587) IL1A −889 C>T | ||||
| C | 293 | 287 | Ref | — |
| T | 107 | 113 | 1.08 (0.79–1.47) | 0.635 |
| (rs17561) IL1A +4845 G>T | ||||
| G | 277 | 273 | Ref | — |
| T | 123 | 127 | 1.05 (0.78–1.41) | 0.760 |
| (rs16944), IL1B −511 C>T | ||||
| C | 276 | 252 | Ref | — |
| T | 124 | 148 | 1.31 (0.97–1.75) | 0.073 |
| (rs1143634), IL1B +3954C>T | ||||
| C | 326 | 298 | Ref | — |
| T | 74 | 102 | 1.51 (1.07–2.11) | 0.017* |
Bold indicates p-value <0.05, statistically significant.
Table 4.
Association Analysis (CLUMP) of ILRN VNTR Polymorphism with Chronic Periodontitis
| Case (periodontitis) | Control | T1 | T2 | T3 | T4 | |
|---|---|---|---|---|---|---|
| Genotype | ||||||
| a1a1 | 137 (68.5) | 127 (63.5) | ||||
| a1a2 | 21 (10.5) | 23 (11.5) | ||||
| a2a2 | 7 (3.5) | 18 (9) | ||||
| a1a3 | 5 (2.5) | 4 (2) | ||||
| a3a3 | 7 (3.5) | 7 (3.5) | χ2 value: 7.841 | χ2 value: 5.567 | χ2value: 5.163 | χ2 value:5.163 |
| a1a4 | 13 (6.5) | 11 (5.5) | df=9 | df=4 | df=1 | df=1 |
| a2a4 | 1 (0.5) | 1 (0.5) | p=0.550 | p=0.234 | p=0.023 | p=0.468 |
| a3a4 | 3 (1.5) | 4 (2) | ||||
| a4a4 | 4 (2) | 5 (2.5) | ||||
| a1a5 | 2 (1) | 0 (0) | ||||
| HW p-value | <0.001 | <0.001 | ||||
| Alleles | ||||||
| a1 | 315 (78.8) | 292 (73.0) | ||||
| a2 | 36 (9.0) | 60 (15.0) | ||||
| a3 | 22 (5.5) | 22 (5.5) | χ2 value: 8.891 | χ2 value: 6.978 | χ2 value: 6.818 | χ2 value: 6.818 |
| a4 | 25 (6.3) | 26 (6.5) | df=4 | df=3 | df=1 | df=1 |
| a5 | 2 (0.5) | 0 (0.0) | p=0.064 | p=0.072 | p=0.009 | p=0.041 |
T1, T2, T3, and T4 are Pearson's χ2 respectively for raw, rare alleles grouped together, 2×2 tables that compare one column against the rest grouped, and 2×2 tables comparing any combination of columns against the rest. p-Value<0.05, statistically significant.
Table 5.
Comparison of Clinical Indices with IL-1 Genotypes for rs1800587, rs17561, rs16944, and rs1143634
| TT (n=104) | CT (n=79) | CC (n=17) | F-value | p-Value | |
|---|---|---|---|---|---|
| IL1A −899 | |||||
| OHI Score | 2.78+0.90 | 2.92+0.82 | 2.50+0.69 | 1.826 | 0.164 |
| Gingival Index | 1.84+0.28 | 1.84+0.30 | 1.81+0.31 | 0.091 | 0.913 |
| Plaque Index | 1.87+0.41 | 1.93+0.37 | 1.92+0.36 | 0.569 | 0.567 |
| PPD | 4.12+0.43 | 4.14+0.49 | 4.10+0.32 | 0.071 | 0.931 |
| CAL | 3.32+1.28 | 3.58+1.13 | 3.22+1.42 | 1.196 | 0.305 |
| GG (n=96) | GT (n=81) | TT (n=23) | F-value | p-Value | |
|---|---|---|---|---|---|
| IL1A +4845 | |||||
| OHI Score | 2.80+0.89 | 2.82+0.82 | 2.83+0.84 | 0.025 | 0.975 |
| Gingival Index | 1.87+0.31 | 1.80+0.26 | 1.86+0.30 | 1.272 | 0.283 |
| Plaque Index | 1.92+0.38 | 1.86+0.40 | 1.97+0.41 | 0.892 | 0.409 |
| PPD | 4.14+0.43 | 4.14+0.47 | 4.00+0.39 | 1.053 | 0.351 |
| CAL | 3.36+1.27 | 3.47+1.17 | 3.44+1.35 | 0.189 | 0.829 |
| CC (n=83) | CT (n=86) | TT (n=31) | F-value | p-Value | |
|---|---|---|---|---|---|
| IL1B −511 | |||||
| OHI Score | 2.72+0.92 | 2.89+0.82 | 2.85+0.76 | 0.873 | 0.419 |
| Gingival Index | 1.82+0.29 | 1.86+0.29 | 1.83+0.29 | 0.511 | 0.601 |
| Plaque Index | 1.82+0.44 | 1.94+0.36 | 2.01+0.33 | 3.31 | 0.039 |
| PPD | 4.13+0.47 | 4.15+0.47 | 4.04+0.28 | 0.63 | 0.534 |
| CAL | 3.37+1.32 | 3.58+1.21 | 3.09+1.04 | 1.908 | 0.151 |
| CC (n=103) | CT (n=92) | TT (n=5) | F-value | p-Value | |
|---|---|---|---|---|---|
| IL1B +3954 | |||||
| OHI Score | 2.85+0.83 | 2.74+0.90 | 3.28+0.47 | 1.200 | 0.303 |
| Gingival Index | 1.81+0.29 | 1.85+0.29 | 2.08+0.26 | 2.220 | 0.111 |
| Plaque Index | 1.88+0.40 | 1.91+0.38 | 2.19+0.45 | 1.639 | 0.197 |
| PPD | 4.13+0.42 | 4.12+0.48 | 4.21+0.25 | 0.124 | 0.884 |
| CAL | 3.37+1.17 | 3.42+1.31 | 4.12+1.39 | 0.867 | 0.422 |
SNP's among chronic periodontitis subjects. p-Value<0.05, statistically significant.
Table 6.
Interaction Models by MDR Analysis
| Best model | TA | CVC | χ2 | p-Value |
|---|---|---|---|---|
| rs16944, rs1143634 | 0.528 | 8/10 | 0.124 | 0.725 |
| rs17561, rs16944, rs1143634 | 0.48 | 7/10 | 0.067 | 0.796 |
p-Value<0.05, statistically significant.
CVC, cross-validation consistency; MDR, multifactorial dimensionality reduction; TA, testing accuracy.
Discussion
The association of polymorphic regions in the interleukin-1 gene with chronic periodontitis susceptibility was assessed in this study. The SNP at +3954 (C>T) and VNTR in the intron 2 of IL1RN region were found to be associated with chronic periodontitis susceptibility.
Interleukin-1 is a potent proinflammatory cytokine, which plays a key role in periodontitis pathogenesis (Birkedal-Hansen, 1993). SNPs have been identified at various loci of the interleukin-1 gene (Kinane et al., 2005). The potential role of these SNPs in periodontitis susceptibility has been studied in different populations across the world. Trevilatto et al. (2011), reported genotypes 2/2 of IL1RN and T allele of IL1B (−511) were risk indicators for chronic periodontitis in an Afro-American segment of the Brazilian population. Among a diabetic African American population with periodontitis, an over-representation of allele 1 at IL1B (−511) and IL1B (+3954) was observed (Guzman et al., 2003). Cullinan et al. (2001), in a prospective study in Queensland reported IL-1 genotype-positive subjects demonstrated attachment loss and increased mean pocket depth in nonsmokers. SNPs in the IL-1 gene were found not to contribute to chronic periodontitis susceptibility in Greek (Sakellari et al., 2006), Jordanian (Karasneh et al., 2011), Chinese (Armitage et al., 2000), and Thai (Anusaksathien et al., 2003) populations. The wide variation in this association may be due to the ethnicity of the target population. A recent systematic review and meta-analysis by Karimbux et al. (2012), reported a significant association between IL-1 genotype and chronic periodontitis in a white population only.
In this study, a statistically significant association could be established between SNP at +3954 in the IL1B gene and chronic periodontitis susceptibility in the dominant and allelic models. This result gains significance as an important confounding variable; smoking/use of tobacco in any form was excluded from the study during subject selection. The SNP at IL1B +3954 position has been identified as a potential risk marker for chronic periodontitis in a similar ethnic population in recent studies (Shete et al., 2010; Archana et al., 2012; Masamatti et al., 2012). The findings of this study are in agreement with the above-mentioned studies. In contrast, a previous study by Kaarthikeyan et al. (2009), reported a lack of association between the SNP at +3954 in the IL1B gene and chronic periodontitis susceptibility in a similar population. The observed difference in the results may be attributed to the small sample size used in the study by Kaarthikeyan et al. (2009), which may not have been adequate to detect the difference in genotypic or allelic variation between the groups.
A considerable variation in the variant (T) allele frequency has been observed in different studies in a similar demographic population. The variant (T) allele frequency observed in the chronic periodontitis subjects of this study population was 25.5% as compared to 38.5% (Masamatti et al., 2012), 46.7% (among severe periodontitis group) (Archana et al., 2012), 14% (Shete et al., 2010) as reported in studies involving subjects of a similar demographic population.
IL-1ra is negative regulator of the interleukin-1 function and is coded for by the IL1RN gene (Rawlinson et al., 2003). A VNTR polymorphism in the intron 2 of the IL1RN gene was evaluated and showed an association with chronic periodontitis susceptibility in this study. The results of our study are in agreement with previous studies, which have reported a significant over-representation of IL1RN gene among early onset (Tai et al., 2002) and chronic periodontitis patients in an Asian population (Zhong et al., 2003). A recent systematic review and meta-analysis (Ding et al., 2012) also reported; IL1RN VNTR polymorphism could contribute to an increased risk for chronic periodontitis.
In our study, the haplotype analysis was performed for four SNPs of the IL-1 gene. A total of 16 different haplotypes were obtained as no LD was present between the SNPs examined. The CTCC haplotype was found to be significantly over-represented in subjects with healthy gingiva and TGTT, haplotype was over-represented in chronic periodontitis patients as compared to individuals with healthy gingiva. The haplotype analysis revealed CTCC to have a protective effect with respect to periodontitis susceptibility and the TGTT haplotype to be more susceptible for chronic periodontitis. A recent study on IL1B (−31, −511, and +3954) SNP haplotype distribution in a similar population (Shete et al., 2010) reported a significant difference in the distribution of the rare allele haplotype (T-T) among controls and chronic periodontitis groups.
Strong LD between IL1B −31 and −511 in control, aggressive periodontitis and chronic periodontitis groups have been reported in a similar population by Shete et al. (2010). In the same study the authors reported, the T allele of IL1B −31 was in linkage with IL1B +3954 in the control group. In this study the SNP at IL1B −31 was not examined and no LD could be detected between the SNPs (IL1B −511, +3954, IL1A −889, and IL1A +4845) examined in this study.
A positive relationship between the rare alleles of the SNPs in IL-1 gene and severity of periodontitis has been reported in a previous study (Papapanou et al., 2001). However in this study, the variant genotype frequencies of the SNPs in interleukin-1 gene were found to have no association with clinical periodontal parameters, and other clinical indices assessed.
Engebretson et al. (1999) reported a corelation between GCF levels of IL1B and periodontitis-associated genotype (PAG). The authors demonstrated that the IL1β in GCF was 2.5 times higher in the PAG-positive group before treatment and 2.2 times higher in the same group even after periodontal therapy. One of the limitations of this study is that the IL-1 protein levels in GCF were not assessed and corelated with the SNP prevalence in the individuals.
In conclusion, in the population examined, the individuals with SNP (C>T) in IL1B +3954 gene and variant alleles of the VNTR in IL1RN gene demonstrated an increased susceptibility for chronic periodontitis.
Supplementary Material
Acknowledgment
The study has been funded by a grant from the Department of Science and Technology, Government of India (Ref No.: SR/SO/HS/0080/2009).
Author Disclosure Statement
No competing financial interests exist.
References
- Archana PM, Arif Salman A, Kumar TSS, et al. (2012) Association between interleukin-1 gene polymorphism and severity of chronic periodontitis in a south Indian population group. J Indian Soc Periodontol 16:174–178 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Armitage GC. (1999) Development of a classification system for periodontal disease and conditions. Ann Periodontol 4:1–6 [DOI] [PubMed] [Google Scholar]
- Armitage GC, Wu Y, Wang HY, et al. (2000) Low prevalence of a periodontitis-associated interleukin-1 composite genotype in individuals of Chinese heritage. J Periodontol 71:164–171 [DOI] [PubMed] [Google Scholar]
- Anusaksathien O, Sukboon A, Sitthiphong P, et al. (2003) Distribution of interleukin-1beta(+3954) and IL-1alpha(-889) genetic variations in a Thai population group. J Periodontol 74:1796–1802 [DOI] [PubMed] [Google Scholar]
- Birkedal-Hansen H. (1993) Role of cytokines and inflammatory mediators in tissue destruction. J Periodontal Res 28:500–510 [DOI] [PubMed] [Google Scholar]
- Chapple IL, Genco R. (2013) Diabetes and periodontal diseases: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases; working group 2 of the joint EFP/AAP workshop. J Periodontol 84:S106–S112 [DOI] [PubMed] [Google Scholar]
- Cullinan MP, Westerman B, Hamlet SM, et al. (2001) A longitudinal study of interleukin-1 gene polymorphisms and periodontal disease in a general adult population. J Clin Periodontol 28:1137–1144 [DOI] [PubMed] [Google Scholar]
- Deng JS, Qin P, Li XX, et al. (2013) Association between interleukin-1β C (3953/4)T polymorphism and chronic periodontitis: evidence from a meta-analysis. Hum Immunol 74:371–378 [DOI] [PubMed] [Google Scholar]
- Ding C, Zhao L, Sun Y, et al. (2012) Interleukin-1 receptor antagonist polymorphism (rs2234663) and periodontitis susceptibility: a meta-analysis. Arch Oral Biol 57:585–593 [DOI] [PubMed] [Google Scholar]
- Engebretson SP, Lamster IB, Herrera-Abreu M, et al. (1999) The influence of interleukin gene polymorphism on expression of interleukin-1beta and tumor necrosis factor-alpha in periodontal tissue and gingival crevicular fluid. J Periodontol 70:567–573 [DOI] [PubMed] [Google Scholar]
- Greene JC, Vermillion JR. (1964) The simplified oral hygiene index. J Am Dent Assoc 68:7–13 [DOI] [PubMed] [Google Scholar]
- Guzman S, Karima M, Wang HY, et al. (2003) Association between interleukin-1 genotype and periodontal disease in a diabetic population. J Periodontol 74:1183–1190 [DOI] [PubMed] [Google Scholar]
- Haber J, Wattles J, Crowley M, et al. (1993) Evidence for cigarette smoking as a major risk factor for periodontitis. J Periodontol 64:16–23 [DOI] [PubMed] [Google Scholar]
- Haffajee AD, Socransky SS. (1994) Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 5:78–111 [DOI] [PubMed] [Google Scholar]
- Ishihara Y, Nishihara T, Kuroyanagi T, et al. (1997) Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites. J Periodontal Res 32:524–5299 [DOI] [PubMed] [Google Scholar]
- Kaarthikeyan G, Jayakumar ND, Padmalatha O, et al. (2009) Analysis of the association between interleukin-1beta (+3954) gene polymorphism and chronic periodontitis in a sample of the south Indian population. Indian J Dent Res 20:37–40 [DOI] [PubMed] [Google Scholar]
- Karasneh JA, Ababneh KT, Taha AH, et al. (2011) Investigation of the interleukin-1 gene cluster polymorphisms in Jordanian patients with chronic and aggressive periodontitis. Arch Oral Biol 56:269–276 [DOI] [PubMed] [Google Scholar]
- Karimbux NY, Saraiya VM, Elangovan S, et al. (2012) Interleukin-1 gene polymorphisms and chronic periodontitis in adult whites: a systematic review and meta-analysis. J Periodontol 83:1407–1419 [DOI] [PubMed] [Google Scholar]
- Kinane DF, Shiba H, Hart TC. (2005) The genetic basis of periodontitis. Periodontol 2000 39:91–117 [DOI] [PubMed] [Google Scholar]
- Kornman KS, Crane A, Wang HY, et al. (1997) The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 24:72–77 [DOI] [PubMed] [Google Scholar]
- Loe H. (1967) The Gingival index, the Plaque index and the Retention index system. J Periodontol 38:610–616 [DOI] [PubMed] [Google Scholar]
- Loos BG, John RP, Laine ML. (2005) Identification of genetic risk factors for periodontitis and possible mechanisms of action. J Clin Periodontol 32:159–179 [DOI] [PubMed] [Google Scholar]
- Masada MP, Persson R, Kenney JS, et al. (1990) Measurement of interleukin-1 alpha and -1 beta in gingival crevicular fluid: implications for the pathogenesis of periodontal disease. J Periodontal Res 25:156–163 [DOI] [PubMed] [Google Scholar]
- Masamatti SS, Kumar A, Kumar ABT, et al. (2012) Evaluation of interleukin-1B (+3954) gene polymorphism in patients with chronic and aggressive periodontitis: a genetic association study. Contemp Clin Dent 3:144–149 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meisel P, Siegemund A, Dombrowa S, et al. (2002) Smoking and polymorphisms of the interleukin-1 gene cluster (IL-1alpha, IL-1beta, and IL-1RN) in patients with periodontal disease. J Periodontol 73:27–32 [DOI] [PubMed] [Google Scholar]
- Papapanou PN, Neiderud AM, Sandros J, et al. (2001) Interleukin-1 gene polymorphism and periodontal status. A case-control study. J Clin Periodontol 28:389–396 [DOI] [PubMed] [Google Scholar]
- Perozini C, Chibebe PC, Leao MV, et al. (2010) Gingival crevicular fluid biochemical markers in periodontal disease: a cross-sectional study. Quintessence Int 41:877–883 [PubMed] [Google Scholar]
- Rawlinson A, Grummitt JM, Walsh TF, et al. (2003) Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers. J Clin Periodontol 30:42–48 [DOI] [PubMed] [Google Scholar]
- Sakellari D, Katsares V, Georgiadou M, et al. (2006) No correlation of five gene polymorphisms with periodontal conditions in a Greek population. J Clin Periodontol 33:765–770 [DOI] [PubMed] [Google Scholar]
- Sham PC, Curtis D. (1995) Monte Carlo tests for associations between disease and alleles at highly polymorphic loci. Ann Hum Genet 59:97–105 [DOI] [PubMed] [Google Scholar]
- Shete AR, Joseph R, Vijayan NN, et al. (2010) Association of single nucleotide gene polymorphism at interleukin-1beta +3954, −511, and −31 in chronic periodontitis and aggressive periodontitis in Dravidian ethnicity. J Periodontol 81:62–69 [DOI] [PubMed] [Google Scholar]
- Silness J, Loe H. (1964) Periodontal disease in pregnancy. II Corelation between oral hygiene and periodontal conditions. Acta Odontol Scand 22:112–135 [DOI] [PubMed] [Google Scholar]
- Tai H, Endo M, Shimada Y, et al. (2002) Association of interleukin-1 receptor antagonist gene polymorphisms with early onset periodontitis in Japanese. J Clin Periodontol 29:882–888 [DOI] [PubMed] [Google Scholar]
- Tawil G, Akl FA, Dagher MF, et al. (2012) Prevalence of IL-1β +3954 and IL-1α-889 polymorphisms in the Lebanese population and its association with the severity of adult chronic periodontitis. J Biol Regul Homeost Agents 26:597–606 [PubMed] [Google Scholar]
- Trevilatto PC, de Souza Pardo AP, Scarel-Caminaga RM, et al. (2011) Association of IL1 gene polymorphisms with chronic periodontitis in Brazilians. Arch Oral Biol 56:54–62 [DOI] [PubMed] [Google Scholar]
- Yücel OO, Berker E, Mescil L, et al. (2013) Association of interleukin-1 beta (+3954) gene polymorphism and gingival crevicular fluid levels in patients with aggressive and chronic periodontitis. Genet Couns 24:21–35 [PubMed] [Google Scholar]
- Zhong LJ, Zhang YH, Zhang JC, et al. (2003) The association between interleukin-1 receptor antagonist genotype and chronic periodontitis of Uighur patients (in Chinese). Zhonghua Kou Qiang Yi Xue Za Zhi 38:370–373 [PubMed] [Google Scholar]
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