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Analysis of TNF-α (-308) polymorphism and gingival crevicular fluid TNF-α levels in aggressive and chronic periodontitis: A preliminary report
Cytokine 72 (2015) 173–177
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Analysis of TNF-a (-308) polymorphism and gingival crevicular fluid TNF-a levels in aggressive and chronic periodontitis: A preliminary report _ Tezcan e Özlem Özer Yücel a,⇑, Ezel Berker b, Lütfiye Mesci c, Kenan Eratalay b, Eser Tepe d, Ilhan a
Department of Oral Pathology, Faculty of Dentistry, University of Gazi, Ankara, Turkey Department of Periodontology, Faculty of Dentistry, Hacettepe University, Ankara, Turkey _ Turkey Genetic Technologies, Diagnostics and Therapies INC., Istanbul, d T.C. Ministry of Health, Tepebasßı Mouth and Tooth Health Hospital, Ankara, Turkey e Hacettepe University, Medical Faculty, Children’s Hospital, Immunology Unit, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 22 December 2014 Accepted 2 January 2015
Keywords: TNF-a Aggressive periodontitis Chronic periodontitis Gene polymorphism GCF
a b s t r a c t Objectives: The purpose of this study was to determine the differences in the distribution of TNF-a (-308) gene polymorphism among aggressive periodontitis, chronic periodontitis and periodontally healthy individuals and also to investigate whether this polymorphism is associated with gingival crevicular fluid TNF-a levels and periodontal disease severity. Material and methods: A total of 93 individuals were enrolled in the study including 38 aggressive periodontitis, 29 chronic periodontitis patients, and 26 healthy controls. Single nucleotide polymorphism at TNF-a (-308) is analyzed by PCR-RFLP method. Gingival crevicular fluid samples were analyzed for TNF-a, using ELISA. Results: The distribution of genotypes and allele frequencies for TNF-a (-308) were similar among the groups. After stratification of patients with respect to attachment level, aggressive periodontitis patients with clinical attachment level P4 mm was observed to have a higher frequency of TNF-a (-308) allele 2 compared to the chronic periodontitis patients with clinical attachment level P4 mm. No significant differences were found between the TNF-a levels of the different genotypes in spite of an insignificant increase in patient groups carrying TNF-a (-308) allele 2. Conclusion: The results of this study revealed an association between TNF-a (-308) allele 2 frequency and aggressive periodontitis patients with clinical attachment level P4 mm in the population studied. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Periodontal diseases are chronic inflammatory infectious diseases that lead to the destruction of the tooth supporting tissues. While the presence of periodontopathogens is essential for the development of periodontal disease, host-related factors are the determining parameters for the progression and severity of the clinical outcome. Host response to bacteria triggers the secretion of pro-inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-a) leading extracellular matrix catabolism and bone resorption in periodontitis [1–3].
⇑ Corresponding author at: Department of Oral Pathology, Faculty of Dentistry, Gazi University, 06510 Ankara, Turkey. Tel.: +90 3122034383; fax: +90 3122239226. E-mail address: [email protected] (Ö. Özer Yücel). http://dx.doi.org/10.1016/j.cyto.2015.01.001 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
TNF-a is an important mediator in inflammatory reactions and appears to play a central role in the pathogenesis of severe chronic inflammatory diseases, autoimmune diseases and septic shock. Monocytes and macrophages are the potent cell types producing TNF-a. The local cellular effects of TNF-a include the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells, degranulation of PMNs, activation of phagocytosis and intercellular adhesion molecule-1 (ICAM-1) expression. It is known that TNF-a induces bone and cartilage resorption by activating the osteoclasts in a similar way with IL-1 [4]. The amount of TNF-a was demonstrated at high levels in GCF and diseased periodontal tissues [5–7] and experimental studies have shown a central role for TNF-a in alveolar bone resorption [8,9]. Variation in cytokine levels among periodontitis patients is well documented and is associated with disease severity [10,11]. The mechanisms which regulate the production and biological activity of TNF-a are under genetic control and play an important role in
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the initiation and progression of periodontal disease. Single nucleotide polymorphisms (SNPs), particularly the G to A transition at position – 308, have been demonstrated to increase TNF-a production up to fivefold in vitro [12]. Although the functional role of TNF-a (-308) polymorphism is shown in vitro, there are no studies demonstrating significant association between in vivo TNF levels and the assessed polymorphism in patients with periodontal disease. Limited number of studies reporting on the TNF-a (-308) polymorphism and TNF levels evaluated small numbers of subjects with chronic periodontitis and healthy individuals [13–15]. (Galbraith et al. 1999 [20 patients], Engebretson et al. 1999 [24 patients], Soga et. al. 2003 [15 healthy donors were used for monocyte cultures]) All the studies mentioned above, except by Engebretson et. al. [13] evaluated TNF expression in blood cultures [14–16]. In fact, the influence of different TNF-a genotypes on the phenotypic cytokine production is not fully established. The possible effect of TNF-a (-308) polymorphism on the periodontal disease severity and clinical outcome remains to be elucidated. While there is a considerable number of studies investigating the association of TNF-a (-308) gene polymorphisms in different ethnicities [14,16–24], the literature contains no data regarding the association of TNF-a (-308) genotype, GCF TNF-a levels and clinical parameters in periodontal disease, concerning both aggressive and chronic periodontitis. Therefore, the aims of the present study were to analyze TNF-a (-308) genotype and allele frequency in both chronic and aggressive periodontitis patients, and also to investigate whether this polymorphism is associated with GCF TNF-a levels, periodontal disease severity and clinical parameters. 2. Materials and methods 2.1. Selection of subjects Ninety-three adult Turkish subjects referred to the Department of Periodontics at Hacettepe University were included for the study. Informed consent was obtained from the patients and the protocol was approved by the Ethics Committee of Hacettepe University. The Turkish ethnicity belongs to the Caucasian racial group, and the study population was drawn from a homogenous group residing in the same geographic region. The patients were diagnosed according to clinical and radiographic criteria as aggressive periodontitis (AP, n = 38) chronic periodontitis (CP, n = 29), and controls (C, n = 26). All of the subjects were non-smokers, or had a pack year history of less than or equal to 5, and had at least 20 teeth. Both chronic and aggressive periodontitis patients were classified according to the AAP (1999) classification [25]. Patients with CP had moderate-to-advanced periodontitis (probing depth P5 mm at least on six sites one of which is the anterior, bone loss P 25%) and had not received any periodontal treatment before the time of examination. For a diagnosis of AP, subjects had to present with a generalized pattern of severe periodontal destruction (probing depth P5 mm at least on three sites other than first molars and incisors). The patients with AP had a mean age of 29.6 and were drawn from a sample of patients with a history of rapid attachment loss and bone destruction. C group subjects were periodontally healthy and had no proximal bone and attachment loss. None of the subjects had any known systemic disorders or used antibiotics and antiinflammatory medications in the last 3 months. Subjects with active infectious diseases such as hepatitis, HIV infection, and tuberculosis or chronically treated with medications (phenytoin, cyclosporine-A, or calcium channel blockers), as well as females, who were lactating or pregnant, were excluded. Clinical parameters included probing depth (PD), clinical attachment level (CAL), plaque index (PI) [26], gingival index (GI)
[27] and bleeding on probing (BOP). Six sites were examined for each tooth: mesiobuccal, buccal, distobuccal, mesiolingual, lingual and distolingual. All the measurements were made by the same examiner. 2.2. Genotyping methods Peripheral venous blood was obtained by standard venipuncture and genomic DNA was extracted using a DNA isolation kit (QIAGEN DNA mini kit). Polymerase chain reaction (PCR) analysis was performed following DNA extraction as described previously [16]. Details of the PCR-RFLP method is summarized below: Forward primer: 50 -AGGCAATAGG-TTTTGAGGGCCAT. Reverse primer: 50 -TCCT-CCCTGCTCCGATTCCG-30 . Cycling was carried out for 1 cycle at 94 °C for 3 min, 35 cycles for 1 min each at 94 °C, 60 °C and 72 °C; and 1 cycle at 72 °C for 1 min. Digestion of PCR products with NcoI yielded 87 + 20-bp fragments (allele 1: G nucleotide at position -308), and a single 170-bp fragment (allele 2: A nucleotide at position -308). The restriction fragments were determined on 3% agarose gel electrophoresis, stained with ethidium bromide (DNA Grade + Nuisieve agarose, Rockland, ME, USA). The identification number of SNP was rs1800629, typically called -308. 2.3. GCF sampling A total of 45 patients including 18 aggressive periodontitis, 17 chronic periodontitis, and 10 healthy subjects were sampled for the analysis of TNF-a in GCF. The selected sites included anterior maxillary sites with probing depth P5 mm and radiographic evidence of bone loss. The sampling teeth selected were free of restorations. GCF samples were collected from 6 maxillary sites per patient. The strips obtained from each patient were studied as pooled sample. Sampling teeth were isolated with cotton rolls and gently dried. A standard paper strip (Perio-paper, IDE Interstate, Amityville, NY, USA) was inserted into the sulcus to the depth of 1–2 mm for 30 s and the strips were moved immediately to a calibrated Periotron 8000 (Oroflow, Inc, NY, USA) to determine the GCF volume. Strips contaminated by blood were excluded from the sampled group. After collection of the gingival fluid, the strips were immediately placed in sterile Eppendorf tubes containing 10 mm NaH2PO4 and 150 mm NaCl, pH 7.2, followed by mixing and centrifugation at 800 g. The GCF samples were stored at 80 °C until subsequent analysis. 2.4. Assay of TNF-a GCF samples were analyzed for TNF-a using commercially available enzyme-linked immunosorbent assay (ELISA, R&D System Inc., MN, USA). Analyses were performed according to the manufacturer’s protocol. All ELISA determinations were performed in duplicate. Results were calculated using the standard curves created in each assay. GCF TNF-a levels were reported as total amount in picograms (pg), or as concentration (pg/ll) by converting Periotron units to microliters using calibration curves described previously [28]. 2.5. Statistical analysis The associations of allele and genotype frequencies in patient and control groups were analyzed using the v2 (Pearson ki-square, likelihood ratio) and Fisher’s Exact test. The difference between the clinical parameters was determined by ANOVA test. Because the data were not normally distributed, the total amounts of cytokines in GCF were expressed as medians and the significance of differences was assessed using Kruskall–Wallis test. A p value of less than 0.05 was considered to be statistically significant. The Kruskall–Wallis
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test was used to analyze the correlations between TNF-a (-308) genotype, clinical parameters and GCF cytokine levels. SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. 3. Results 3.1. TNF-a (-308) frequency analysis in AP, CP and C groups Demographic data of the individuals involved in the study is presented in Table 1. The clinical parameters such as PD, CAL, PI, G and BOP were significantly more pronounced in the patient groups. The allele frequencies and genotype distributions for TNF-a (-308) gene polymorphism among the study groups are presented in Table 2. The genotype frequencies were in agreement with the Hardy–Weinberg equilibrium (P > 0.01 for all analyses). The TNFa (-308) frequencies of genotypes and alleles were found to be similar on comparing the patient group (CP + AP), and the control group; and also among all the groups (P > 0.05). Homozygous allele 1 was the most common genotype for all the patient groups. The TNF-a (-308) allele 2 was found in 35 (26.3%) of the AP, and in 23 (20.7%) in CP patients as compared to 21 (32.7%) of the healthy
Table 1 Subject data and clinical parameters (mean ± standard error) of patient groups and healthy controls.
Age Female/male PD (mm) CAL (mm) PI GI BOP (%)
AP (n = 38)
CP (n = 29)
C (n = 26)
29.63 ± 8.3 (18–50) 23/15 3.54 ± 0.69a 4.24 ± 1.2a 1.35 ± 0.6a 1.58 ± 0.6a 86.90 ± 21.1a
41.63 ± 7.3 (30–59) 16/13 3.56 ± 0.91a 4.01 ± 0.7a 1.5 ± 0.4a 1.6 ± 0.5a 91.57 ± 17.9a
34.44 ± 8.1 (20–49) 17/9 1.53 ± 0.27 0.1 ± 0.2 0.5 ± 0.4 0.2 ± 0.3 13.0 ± 14.4
control individuals. The homozygous allele 2 was not detected in any of the groups (Table 2). The patient groups (AP, CP, AP + CP) were further divided into two groups according to clinical attachment level (CAL < 4 mm and CAL P 4 mm) in order to investigate the possible link between the polymorphisms and periodontal disease severity. According to the features described by AAP, patients with mean CAL between 4 and 5 mm are not included in any of the sub classifications. (mild: 1–2 mm of attachment loss; moderate: 3–4 mm of attachment loss and severe: P5 mm of attachment loss) Since patients with moderate periodontitis are described to have 3–4 mm of attachment loss, patients with CAL P 4 mm are throwed in the ‘‘severe’’ group [29,30]. In addition, a considerable number of patients presented with a mean value of CAL between 4 and 5 mm. After stratification of subjects according to the clinical attachment level (CAL), AP patients with CAL P 4 mm were observed to have a higher frequency of allele 2 compared to the CP patients with CAL P 4 mm (Table 3). 3.2. TNF-a (-308) genotype versus GCF TNF-a levels and clinical parameters No differences were found between the GCF TNF-a levels of the different genotypes in spite of a statistically insignificant increase in patient groups (AP,CP) with allele 2 when compared to carriers of (1,1) genotype (Table 4). Similar clinical scores of bleeding on probing, gingival index, plaque index, probing depth and attachment levels were observed in all genotype groups (p > 0.05, Kruskall–Wallis). 4. Discussion
AP: aggressive periodontitis, CP: chronic periodontitis, C: control group, PD: probing depth, CAL: clinical attachment level, PI: plaque index, GI: gingival index, BOP: bleeding on probing. a Significantly different in comparison with the control group (P < 0.05, ANOVA).
TNF-a is an important pro-inflammatory cytokine playing a central role in periodontal disease due to its effects on bone and cartilage resorption by activating the osteoclasts in a similar way with IL-1. It is assumed that the individual susceptibility and severity for periodontitis might be related to genetically determined differences in the TNF-a production [16,31,32]. The most studied SNP in TNF-a gene locus is the G to A transition at position -308, which have been demonstrated to increase tumor necrosis
Table 2 Genotype and allele frequencies of patient groups and healthy controls. TNF-a (-308)
AP n = 38 (%)
CP n = 29 (%)
P (AP + CP) n = 67 (%)
C n = 26 (%)
Statistical Analysis
Genotype (1,1) (1,2)
18 (47.4) 20 (52.6)
17 (58.6) 12 (41.4)
35 (52.2) 32 (47.8)
9 (34.6) 17 (65.4)
(x2 = 2.759 p = 0.252)
Alleles
n = 76 (%)
n = 58 (%)
n = 134 (%)
n = 52 (%)
Allele 1 Allele 2
56 (73.7) 20 (26.3)
46 (79.3) 12 (20.7)
102 (76.1) 32 (23.9)
35 (67.3) 17 (32.7)
(x2 = 1.757 p = 0.415)
AP: aggressive periodontitis, CP: chronic periodontitis, P: periodontitis, C: control group. No statistical differences were observed at all comparisons (v2 test, p > 0.05).
Table 3 Allele frequencies after stratification of subjects according to the clinical attachment level (CAL). TNF-a (-308)
AP (n = 38)
Alleles
Allele1 (%) n = 56
Allele 2 (%) n = 20
Allele1 (%) n = 46
Allele 2 (%) n = 12
Allele1 (%) n = 102
Allele 2 (%) n = 32
CAL < 4 mm CAL P 4 mm
15 (26.8) 41 (73.2)
7 (18.4) 13 (71.6)a
16 (34.7) 30 (64.3)
6 (50) 6 (50)
31 (30.4) 71 (69.6)
13 (40.6) 19 (59.4)
CP (n = 29)
P (AP + CP) (n = 67)
AP: aggressive periodontitis, CP: chronic periodontitis, P: periodontitis, CAL: clinical attachment level. a Significantly higher allele 2 (%) values compared to CP patients with CAL P 4 mm (Fisher’s exact, p = 0,04).
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Table 4 GFC TNF-a levels (median) in patient groups and healthy controls regarding their TNF-a (-308) genotype.
Genotype TNF-a (pg) TNF-a (pg/ ll)
AP (n = 18) (min–max)
CP (n = 17) (min–max)
P (n = 35) (min–max)
C (n = 10) (min–max)
(1,1) 70.38 (19.87– 219.50) 41.10 (13.42– 187.61)
(1,1) 51.14 (17.47– 30.27) 37.17 (7.9– 286.92)
(1,1) 53.55 (17.47– 301.27) 40.84 (7.91– 286.92)
(1,1) 207.47 (171.39– 291.65) 373.25 (285.65– 506)
(1,2) 190.64 (39.11– 262.79) 108.9 (39.5– 159.27)
(1,2) 75.19 (17.47– 113.67) 36.68 (8.91– 115.59)
(1,2) 108.86 (17.47– 262.79) 76.61 (8.91– 159.27)
(1,2) 231.52 (10– 363.81) 190.88 (17.24– 641.08)
No statistical differences were observed at all comparisons (p > 0.05, Kruskall–Wallis).
factor-alpha production up to fivefold in vitro [12]. In this study, we investigated the possible association of the TNF-a (-308) genotype with GCF TNF-a levels, periodontal disease severity and clinical parameters in both aggressive and chronic periodontitis patients in a group of Turkish subjects. Our results revealed no significant difference regarding the allele or genotype distribution in controls and patients, in spite of a trend toward a higher frequency of heterozygosity in the control group. The allele 2 frequency, which is thought to be associated with periodontal disease is reported between 5% [14] and 29% [31] in the literature for chronic periodontitis. The literature contains no other data regarding the association of TNF-a (-308) polymorphism and chronic periodontitis in a Turkish population. Majority of the studies in parallel to ours, revealed no significant difference between chronic periodontitis and healthy [17,19– 21,23,31,33–35]. Kornmann et al. [16], reported the frequency of TNF-a (-308) allele 2 20.9%, 38.1%, and 26.5% for severe, moderate and mild periodontitis respectively. In the present study, TNF-a (-308) allele 2 was found in 35 (26.3%) of the AP, and in 23 (20.7%) in CP patients as compared to 21 (32.7%) of the healthy control individuals. The homozygous allele 2 was not detected in any of the groups. The lack of association between TNF-a (-308) genotype and periodontitis reported by Galbraith et al. [36], Craandijk et al. [31], Sakellari et al. [21] and Schulz et al. [22] is consistent with observations made in the present study. However Galbraith et al. [14] reported higher prevalence of TNF-a (-308) allele 1 in periodontitis compared to gingivitis. The controversial results from different studies could partly be explained by the sample size, and different criteria to select diseased and control groups (subjects of unknown periodontal status as a reference group) as well as ethnic differences which could be effective on genetic variations. Another study which have analyzed TNF-a (-308) polymorphism in aggressive periodontitis have shown no differences with regard to the TNF-a (-308) genotype between patients and controls. Similarly, Shapira et al. [32] found no association between LagP (localized aggressive periodontitis) and TNF-a (-308) polymorphism. Guzeldemir et al. [24] studied 31 localized aggressive patients in Turkish subjects and reported a similar distribution for allele and genotype frequency for TNF-a (-308) compared to controls. In the present study, no differences were observed between aggressive periodontitis and controls, in agreement, with findings in populations of Caucasian [18,20–22,32,34]. Data from our study are among the few in the literature referring to both aggressive and chronic periodontitis patients, indicate a similar distribution of TNF-a (-308) genotype between the two [21,22,34]. On the other hand, when the patient groups were stratified with respect to attachment level (CAL < 4 mm and CAL P 4 mm), AP patients with CAL P 4 mm was observed to have a higher frequency of allele 2 compared to the CP patients with CAL P 4 mm. The genotype distribution and allele frequencies were not different regarding the subjects with CAL < 4 mm. These findings suggest that TNF-a (-308) allele 2 frequency might play a significant role for rapidly progressing periodontitis rather than periodontal disease susceptibility. Longitudinal studies are required in order to support and clarify this finding. While there
are few longitudinal studies investigating IL-1 genotype and periodontal disease severity, alveolar bone loss, tooth loss, and response to periodontal therapy [37,38]; there is no longitudinal study concerning the putative role of TNF-a (-308) in periodontal disease outcome. The occurrence of the allele 2 in the TNF-a (-308) was associated with an increased level of TNF-a in vitro in response to different stimuli [12]. The results of our study revealed no significant differences in GCF TNF-a levels of the different genotypes, in spite of a statistically insignificant increase in the median TNF-a levels in GCF from patient groups (AP,CP) with allele 2 when compared to carriers of (1,1) genotype. Similarly Trombone et al. [23]. reported a slight statistically insignificant increase in TNF-a mRNA levels in periodontal tissues from TNF-a (-308) allele 2 carriers, moreover they demonstrated that allele 2 carriers presented significantly higher levels of TNF-a in the absence of red-complex bacteria. The search for a functional role of TNF-a (-308) polymorphism in the TNF-a expression led controversial results [15,18,23]. Single nucleotide polymorphism at position -308, can increase tumor necrosis factor-alpha production up to fivefold in vitro [12], while a lower increase rate was demonstrated in vivo [23]. Taken together, our results do not permit to make a definite conclusion for the effect of TNF-a (-308) polymorphism in the modulation of GCF TNF-a levels. In agreement with the previous reports, we found a wide inter-individual variability in the TNF-a expression in both patient groups and controls. This may be one of the factors avoiding to establish a statistically significant association. Lack of observance of specific bacteria is a limitation of the present study, since different stimuli and cell-type were shown to be modulating factors for the transcriptional activity of the TNF-a [12]. Despite the limitations of clinic association studies, the results of our study revealed an association between TNF-a (-308) allele 2 frequency and aggressive periodontitis patients with CAL P 4 mm. TNF-a expression may aggravate the rapid destruction of periodontal attachment and bone loss in aggressive periodontitis patients by inducing the certain types of matrix metalloproteinases (MMP-1, MMP-3), cytokines (IL-1, interferon-b) and prostaglandins (PGE2) which mediate bone and extracellular matrix resorption. Acknowledgments The authors wish to thank the staff members of Hacettepe University, Faculty of Dentistry Department of Periodontology for their participation. The authors declare that there are no conflicts of interest in this study. Dr. Özlem Özer Yücel was supported by H.U.B.A.B (Hacettepe University Research Foundation) Ph.D. Thesis Grant (07T06T02004). References [1] Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge in periodontitis: assembling the players. Periodontol 2000;1997(14):33–53. [2] Nares S. The genetic relationship to periodontal disease. Periodontol 2000;2003(32):36–49.
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[20] Folwaczny M, Glas J, Torok HP, Mende M, Folwaczny C. Lack of association between the TNF alpha G -308 A promoter polymorphism and periodontal disease. J Clin Periodontol 2004;31:449–53. [21] Sakellari D, Katsares V, Georgiadou M, Kouvatsi A, Arsenakis M, Konstantinidis A. No correlation of five gene polymorphisms with periodontal conditions in a Greek population. J Clin Periodontol 2006;33:765–70. [22] Schulz S, Machulla HK, Altermann W, Klapproth J, Zimmermann U, Glaser C, et al. Genetic markers of tumour necrosis factor alpha in aggressive and chronic periodontitis. J Clin Periodontol 2008;35:493–500. [23] Trombone AP, Cardoso CR, Repeke CE, Ferreira Jr SB, Martins Jr W, Campanelli AP, et al. Tumor necrosis factor-alpha -308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased periodontal tissues. J Periodontal Res 2009;44:598–608. [24] Guzeldemir E, Gunhan M, Ozcelik O, Tastan H. Interleukin-1 and tumor necrosis factor-alpha gene polymorphisms in Turkish patients with localized aggressive periodontitis. J Oral Sci 2008;50:151–9. [25] International workshop for a classification of periodontal diseases and conditions. Annals of periodontology 1999; 4. [26] Loe H. The gingival index, the plaque index and the retention index systems. J Periodontol 1967;38(Suppl):610–6. [27] Silness J, Loe H. Periodontal disease in pregnancy. Ii. correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121–35. [28] Lamster IB, Oshrain RL, Fiorello LA, Celenti RS, Gordon JM. A comparison of 4 methods of data presentation for lysosomal enzyme activity in gingival crevicular fluid. J Clin Periodontol 1988;15:347–52. [29] Engebretson SP, Grbic JT, Singer R, Lamster IB. GCF IL-1beta profiles in periodontal disease. J Clin Periodontol 2002;29:48–53. [30] Meisel P, Siegemund A, Grimm R, Herrmann FH, John U, Schwahn C, et al. The interleukin-1 polymorphism, smoking, and the risk of periodontal disease in the population-based SHIP study. J Dent Res 2003;82:189–93. [31] Craandijk J, van Krugten MV, Verweij CL, van der Velden U, Loos BG. Tumor necrosis factor-alpha gene polymorphisms in relation to periodontitis. J Clin Periodontol 2002;29:28–34. [32] Shapira L, Stabholz A, Rieckmann P, Kruse N. Genetic polymorphism of the tumor necrosis factor (TNF)-alpha promoter region in families with localized early-onset periodontitis. J Periodontal Res 2001;36:183–6. [33] Endo M, Tai H, Tabeta K, Kobayashi T, Yamazaki K, Yoshie H. Analysis of single nucleotide polymorphisms in the 50 -flanking region of tumor necrosis factoralpha gene in Japanese patients with early-onset periodontitis. J Periodontol 2001;72:1554–9. [34] Brett PM, Zygogianni P, Griffiths GS, Tomaz M, Parkar M, D’Aiuto F, et al. Functional gene polymorphisms in aggressive and chronic periodontitis. J Dent Res 2005;84:1149–53. [35] Loos BG, John RP, Laine ML. Identification of genetic risk factors for periodontitis and possible mechanisms of action. J Clin Periodontol 2005;32(Suppl 6):159–79. [36] Galbraith GM, Steed RB, Sanders JJ, Pandey JP. Tumor necrosis factor alpha production by oral leukocytes: influence of tumor necrosis factor genotype. J Periodontol 1998;69:428–33. [37] Cattabriga M, Rotundo R, Muzzi L, Nieri M, Verrocchi G, Cairo F, et al. Retrospective evaluation of the influence of the interleukin-1 genotype on radiographic bone levels in treated periodontal patients over 10 years. J Periodontol 2001;72:767–73. [38] Ehmke B, Kress W, Karch H, Grimm T, Klaiber B, Flemmig TF. Interleukin-1 haplotype and periodontal disease progression following therapy. J Clin Periodontol 1999;26:810–3.
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Analysis of TNF-a (-308) polymorphism and gingival crevicular fluid TNF-a levels in aggressive and chronic periodontitis: A preliminary report _ Tezcan e Özlem Özer Yücel a,⇑, Ezel Berker b, Lütfiye Mesci c, Kenan Eratalay b, Eser Tepe d, Ilhan a
Department of Oral Pathology, Faculty of Dentistry, University of Gazi, Ankara, Turkey Department of Periodontology, Faculty of Dentistry, Hacettepe University, Ankara, Turkey _ Turkey Genetic Technologies, Diagnostics and Therapies INC., Istanbul, d T.C. Ministry of Health, Tepebasßı Mouth and Tooth Health Hospital, Ankara, Turkey e Hacettepe University, Medical Faculty, Children’s Hospital, Immunology Unit, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 22 December 2014 Accepted 2 January 2015
Keywords: TNF-a Aggressive periodontitis Chronic periodontitis Gene polymorphism GCF
a b s t r a c t Objectives: The purpose of this study was to determine the differences in the distribution of TNF-a (-308) gene polymorphism among aggressive periodontitis, chronic periodontitis and periodontally healthy individuals and also to investigate whether this polymorphism is associated with gingival crevicular fluid TNF-a levels and periodontal disease severity. Material and methods: A total of 93 individuals were enrolled in the study including 38 aggressive periodontitis, 29 chronic periodontitis patients, and 26 healthy controls. Single nucleotide polymorphism at TNF-a (-308) is analyzed by PCR-RFLP method. Gingival crevicular fluid samples were analyzed for TNF-a, using ELISA. Results: The distribution of genotypes and allele frequencies for TNF-a (-308) were similar among the groups. After stratification of patients with respect to attachment level, aggressive periodontitis patients with clinical attachment level P4 mm was observed to have a higher frequency of TNF-a (-308) allele 2 compared to the chronic periodontitis patients with clinical attachment level P4 mm. No significant differences were found between the TNF-a levels of the different genotypes in spite of an insignificant increase in patient groups carrying TNF-a (-308) allele 2. Conclusion: The results of this study revealed an association between TNF-a (-308) allele 2 frequency and aggressive periodontitis patients with clinical attachment level P4 mm in the population studied. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Periodontal diseases are chronic inflammatory infectious diseases that lead to the destruction of the tooth supporting tissues. While the presence of periodontopathogens is essential for the development of periodontal disease, host-related factors are the determining parameters for the progression and severity of the clinical outcome. Host response to bacteria triggers the secretion of pro-inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-a) leading extracellular matrix catabolism and bone resorption in periodontitis [1–3].
⇑ Corresponding author at: Department of Oral Pathology, Faculty of Dentistry, Gazi University, 06510 Ankara, Turkey. Tel.: +90 3122034383; fax: +90 3122239226. E-mail address: [email protected] (Ö. Özer Yücel). http://dx.doi.org/10.1016/j.cyto.2015.01.001 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
TNF-a is an important mediator in inflammatory reactions and appears to play a central role in the pathogenesis of severe chronic inflammatory diseases, autoimmune diseases and septic shock. Monocytes and macrophages are the potent cell types producing TNF-a. The local cellular effects of TNF-a include the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells, degranulation of PMNs, activation of phagocytosis and intercellular adhesion molecule-1 (ICAM-1) expression. It is known that TNF-a induces bone and cartilage resorption by activating the osteoclasts in a similar way with IL-1 [4]. The amount of TNF-a was demonstrated at high levels in GCF and diseased periodontal tissues [5–7] and experimental studies have shown a central role for TNF-a in alveolar bone resorption [8,9]. Variation in cytokine levels among periodontitis patients is well documented and is associated with disease severity [10,11]. The mechanisms which regulate the production and biological activity of TNF-a are under genetic control and play an important role in
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the initiation and progression of periodontal disease. Single nucleotide polymorphisms (SNPs), particularly the G to A transition at position – 308, have been demonstrated to increase TNF-a production up to fivefold in vitro [12]. Although the functional role of TNF-a (-308) polymorphism is shown in vitro, there are no studies demonstrating significant association between in vivo TNF levels and the assessed polymorphism in patients with periodontal disease. Limited number of studies reporting on the TNF-a (-308) polymorphism and TNF levels evaluated small numbers of subjects with chronic periodontitis and healthy individuals [13–15]. (Galbraith et al. 1999 [20 patients], Engebretson et al. 1999 [24 patients], Soga et. al. 2003 [15 healthy donors were used for monocyte cultures]) All the studies mentioned above, except by Engebretson et. al. [13] evaluated TNF expression in blood cultures [14–16]. In fact, the influence of different TNF-a genotypes on the phenotypic cytokine production is not fully established. The possible effect of TNF-a (-308) polymorphism on the periodontal disease severity and clinical outcome remains to be elucidated. While there is a considerable number of studies investigating the association of TNF-a (-308) gene polymorphisms in different ethnicities [14,16–24], the literature contains no data regarding the association of TNF-a (-308) genotype, GCF TNF-a levels and clinical parameters in periodontal disease, concerning both aggressive and chronic periodontitis. Therefore, the aims of the present study were to analyze TNF-a (-308) genotype and allele frequency in both chronic and aggressive periodontitis patients, and also to investigate whether this polymorphism is associated with GCF TNF-a levels, periodontal disease severity and clinical parameters. 2. Materials and methods 2.1. Selection of subjects Ninety-three adult Turkish subjects referred to the Department of Periodontics at Hacettepe University were included for the study. Informed consent was obtained from the patients and the protocol was approved by the Ethics Committee of Hacettepe University. The Turkish ethnicity belongs to the Caucasian racial group, and the study population was drawn from a homogenous group residing in the same geographic region. The patients were diagnosed according to clinical and radiographic criteria as aggressive periodontitis (AP, n = 38) chronic periodontitis (CP, n = 29), and controls (C, n = 26). All of the subjects were non-smokers, or had a pack year history of less than or equal to 5, and had at least 20 teeth. Both chronic and aggressive periodontitis patients were classified according to the AAP (1999) classification [25]. Patients with CP had moderate-to-advanced periodontitis (probing depth P5 mm at least on six sites one of which is the anterior, bone loss P 25%) and had not received any periodontal treatment before the time of examination. For a diagnosis of AP, subjects had to present with a generalized pattern of severe periodontal destruction (probing depth P5 mm at least on three sites other than first molars and incisors). The patients with AP had a mean age of 29.6 and were drawn from a sample of patients with a history of rapid attachment loss and bone destruction. C group subjects were periodontally healthy and had no proximal bone and attachment loss. None of the subjects had any known systemic disorders or used antibiotics and antiinflammatory medications in the last 3 months. Subjects with active infectious diseases such as hepatitis, HIV infection, and tuberculosis or chronically treated with medications (phenytoin, cyclosporine-A, or calcium channel blockers), as well as females, who were lactating or pregnant, were excluded. Clinical parameters included probing depth (PD), clinical attachment level (CAL), plaque index (PI) [26], gingival index (GI)
[27] and bleeding on probing (BOP). Six sites were examined for each tooth: mesiobuccal, buccal, distobuccal, mesiolingual, lingual and distolingual. All the measurements were made by the same examiner. 2.2. Genotyping methods Peripheral venous blood was obtained by standard venipuncture and genomic DNA was extracted using a DNA isolation kit (QIAGEN DNA mini kit). Polymerase chain reaction (PCR) analysis was performed following DNA extraction as described previously [16]. Details of the PCR-RFLP method is summarized below: Forward primer: 50 -AGGCAATAGG-TTTTGAGGGCCAT. Reverse primer: 50 -TCCT-CCCTGCTCCGATTCCG-30 . Cycling was carried out for 1 cycle at 94 °C for 3 min, 35 cycles for 1 min each at 94 °C, 60 °C and 72 °C; and 1 cycle at 72 °C for 1 min. Digestion of PCR products with NcoI yielded 87 + 20-bp fragments (allele 1: G nucleotide at position -308), and a single 170-bp fragment (allele 2: A nucleotide at position -308). The restriction fragments were determined on 3% agarose gel electrophoresis, stained with ethidium bromide (DNA Grade + Nuisieve agarose, Rockland, ME, USA). The identification number of SNP was rs1800629, typically called -308. 2.3. GCF sampling A total of 45 patients including 18 aggressive periodontitis, 17 chronic periodontitis, and 10 healthy subjects were sampled for the analysis of TNF-a in GCF. The selected sites included anterior maxillary sites with probing depth P5 mm and radiographic evidence of bone loss. The sampling teeth selected were free of restorations. GCF samples were collected from 6 maxillary sites per patient. The strips obtained from each patient were studied as pooled sample. Sampling teeth were isolated with cotton rolls and gently dried. A standard paper strip (Perio-paper, IDE Interstate, Amityville, NY, USA) was inserted into the sulcus to the depth of 1–2 mm for 30 s and the strips were moved immediately to a calibrated Periotron 8000 (Oroflow, Inc, NY, USA) to determine the GCF volume. Strips contaminated by blood were excluded from the sampled group. After collection of the gingival fluid, the strips were immediately placed in sterile Eppendorf tubes containing 10 mm NaH2PO4 and 150 mm NaCl, pH 7.2, followed by mixing and centrifugation at 800 g. The GCF samples were stored at 80 °C until subsequent analysis. 2.4. Assay of TNF-a GCF samples were analyzed for TNF-a using commercially available enzyme-linked immunosorbent assay (ELISA, R&D System Inc., MN, USA). Analyses were performed according to the manufacturer’s protocol. All ELISA determinations were performed in duplicate. Results were calculated using the standard curves created in each assay. GCF TNF-a levels were reported as total amount in picograms (pg), or as concentration (pg/ll) by converting Periotron units to microliters using calibration curves described previously [28]. 2.5. Statistical analysis The associations of allele and genotype frequencies in patient and control groups were analyzed using the v2 (Pearson ki-square, likelihood ratio) and Fisher’s Exact test. The difference between the clinical parameters was determined by ANOVA test. Because the data were not normally distributed, the total amounts of cytokines in GCF were expressed as medians and the significance of differences was assessed using Kruskall–Wallis test. A p value of less than 0.05 was considered to be statistically significant. The Kruskall–Wallis
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test was used to analyze the correlations between TNF-a (-308) genotype, clinical parameters and GCF cytokine levels. SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. 3. Results 3.1. TNF-a (-308) frequency analysis in AP, CP and C groups Demographic data of the individuals involved in the study is presented in Table 1. The clinical parameters such as PD, CAL, PI, G and BOP were significantly more pronounced in the patient groups. The allele frequencies and genotype distributions for TNF-a (-308) gene polymorphism among the study groups are presented in Table 2. The genotype frequencies were in agreement with the Hardy–Weinberg equilibrium (P > 0.01 for all analyses). The TNFa (-308) frequencies of genotypes and alleles were found to be similar on comparing the patient group (CP + AP), and the control group; and also among all the groups (P > 0.05). Homozygous allele 1 was the most common genotype for all the patient groups. The TNF-a (-308) allele 2 was found in 35 (26.3%) of the AP, and in 23 (20.7%) in CP patients as compared to 21 (32.7%) of the healthy
Table 1 Subject data and clinical parameters (mean ± standard error) of patient groups and healthy controls.
Age Female/male PD (mm) CAL (mm) PI GI BOP (%)
AP (n = 38)
CP (n = 29)
C (n = 26)
29.63 ± 8.3 (18–50) 23/15 3.54 ± 0.69a 4.24 ± 1.2a 1.35 ± 0.6a 1.58 ± 0.6a 86.90 ± 21.1a
41.63 ± 7.3 (30–59) 16/13 3.56 ± 0.91a 4.01 ± 0.7a 1.5 ± 0.4a 1.6 ± 0.5a 91.57 ± 17.9a
34.44 ± 8.1 (20–49) 17/9 1.53 ± 0.27 0.1 ± 0.2 0.5 ± 0.4 0.2 ± 0.3 13.0 ± 14.4
control individuals. The homozygous allele 2 was not detected in any of the groups (Table 2). The patient groups (AP, CP, AP + CP) were further divided into two groups according to clinical attachment level (CAL < 4 mm and CAL P 4 mm) in order to investigate the possible link between the polymorphisms and periodontal disease severity. According to the features described by AAP, patients with mean CAL between 4 and 5 mm are not included in any of the sub classifications. (mild: 1–2 mm of attachment loss; moderate: 3–4 mm of attachment loss and severe: P5 mm of attachment loss) Since patients with moderate periodontitis are described to have 3–4 mm of attachment loss, patients with CAL P 4 mm are throwed in the ‘‘severe’’ group [29,30]. In addition, a considerable number of patients presented with a mean value of CAL between 4 and 5 mm. After stratification of subjects according to the clinical attachment level (CAL), AP patients with CAL P 4 mm were observed to have a higher frequency of allele 2 compared to the CP patients with CAL P 4 mm (Table 3). 3.2. TNF-a (-308) genotype versus GCF TNF-a levels and clinical parameters No differences were found between the GCF TNF-a levels of the different genotypes in spite of a statistically insignificant increase in patient groups (AP,CP) with allele 2 when compared to carriers of (1,1) genotype (Table 4). Similar clinical scores of bleeding on probing, gingival index, plaque index, probing depth and attachment levels were observed in all genotype groups (p > 0.05, Kruskall–Wallis). 4. Discussion
AP: aggressive periodontitis, CP: chronic periodontitis, C: control group, PD: probing depth, CAL: clinical attachment level, PI: plaque index, GI: gingival index, BOP: bleeding on probing. a Significantly different in comparison with the control group (P < 0.05, ANOVA).
TNF-a is an important pro-inflammatory cytokine playing a central role in periodontal disease due to its effects on bone and cartilage resorption by activating the osteoclasts in a similar way with IL-1. It is assumed that the individual susceptibility and severity for periodontitis might be related to genetically determined differences in the TNF-a production [16,31,32]. The most studied SNP in TNF-a gene locus is the G to A transition at position -308, which have been demonstrated to increase tumor necrosis
Table 2 Genotype and allele frequencies of patient groups and healthy controls. TNF-a (-308)
AP n = 38 (%)
CP n = 29 (%)
P (AP + CP) n = 67 (%)
C n = 26 (%)
Statistical Analysis
Genotype (1,1) (1,2)
18 (47.4) 20 (52.6)
17 (58.6) 12 (41.4)
35 (52.2) 32 (47.8)
9 (34.6) 17 (65.4)
(x2 = 2.759 p = 0.252)
Alleles
n = 76 (%)
n = 58 (%)
n = 134 (%)
n = 52 (%)
Allele 1 Allele 2
56 (73.7) 20 (26.3)
46 (79.3) 12 (20.7)
102 (76.1) 32 (23.9)
35 (67.3) 17 (32.7)
(x2 = 1.757 p = 0.415)
AP: aggressive periodontitis, CP: chronic periodontitis, P: periodontitis, C: control group. No statistical differences were observed at all comparisons (v2 test, p > 0.05).
Table 3 Allele frequencies after stratification of subjects according to the clinical attachment level (CAL). TNF-a (-308)
AP (n = 38)
Alleles
Allele1 (%) n = 56
Allele 2 (%) n = 20
Allele1 (%) n = 46
Allele 2 (%) n = 12
Allele1 (%) n = 102
Allele 2 (%) n = 32
CAL < 4 mm CAL P 4 mm
15 (26.8) 41 (73.2)
7 (18.4) 13 (71.6)a
16 (34.7) 30 (64.3)
6 (50) 6 (50)
31 (30.4) 71 (69.6)
13 (40.6) 19 (59.4)
CP (n = 29)
P (AP + CP) (n = 67)
AP: aggressive periodontitis, CP: chronic periodontitis, P: periodontitis, CAL: clinical attachment level. a Significantly higher allele 2 (%) values compared to CP patients with CAL P 4 mm (Fisher’s exact, p = 0,04).
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Table 4 GFC TNF-a levels (median) in patient groups and healthy controls regarding their TNF-a (-308) genotype.
Genotype TNF-a (pg) TNF-a (pg/ ll)
AP (n = 18) (min–max)
CP (n = 17) (min–max)
P (n = 35) (min–max)
C (n = 10) (min–max)
(1,1) 70.38 (19.87– 219.50) 41.10 (13.42– 187.61)
(1,1) 51.14 (17.47– 30.27) 37.17 (7.9– 286.92)
(1,1) 53.55 (17.47– 301.27) 40.84 (7.91– 286.92)
(1,1) 207.47 (171.39– 291.65) 373.25 (285.65– 506)
(1,2) 190.64 (39.11– 262.79) 108.9 (39.5– 159.27)
(1,2) 75.19 (17.47– 113.67) 36.68 (8.91– 115.59)
(1,2) 108.86 (17.47– 262.79) 76.61 (8.91– 159.27)
(1,2) 231.52 (10– 363.81) 190.88 (17.24– 641.08)
No statistical differences were observed at all comparisons (p > 0.05, Kruskall–Wallis).
factor-alpha production up to fivefold in vitro [12]. In this study, we investigated the possible association of the TNF-a (-308) genotype with GCF TNF-a levels, periodontal disease severity and clinical parameters in both aggressive and chronic periodontitis patients in a group of Turkish subjects. Our results revealed no significant difference regarding the allele or genotype distribution in controls and patients, in spite of a trend toward a higher frequency of heterozygosity in the control group. The allele 2 frequency, which is thought to be associated with periodontal disease is reported between 5% [14] and 29% [31] in the literature for chronic periodontitis. The literature contains no other data regarding the association of TNF-a (-308) polymorphism and chronic periodontitis in a Turkish population. Majority of the studies in parallel to ours, revealed no significant difference between chronic periodontitis and healthy [17,19– 21,23,31,33–35]. Kornmann et al. [16], reported the frequency of TNF-a (-308) allele 2 20.9%, 38.1%, and 26.5% for severe, moderate and mild periodontitis respectively. In the present study, TNF-a (-308) allele 2 was found in 35 (26.3%) of the AP, and in 23 (20.7%) in CP patients as compared to 21 (32.7%) of the healthy control individuals. The homozygous allele 2 was not detected in any of the groups. The lack of association between TNF-a (-308) genotype and periodontitis reported by Galbraith et al. [36], Craandijk et al. [31], Sakellari et al. [21] and Schulz et al. [22] is consistent with observations made in the present study. However Galbraith et al. [14] reported higher prevalence of TNF-a (-308) allele 1 in periodontitis compared to gingivitis. The controversial results from different studies could partly be explained by the sample size, and different criteria to select diseased and control groups (subjects of unknown periodontal status as a reference group) as well as ethnic differences which could be effective on genetic variations. Another study which have analyzed TNF-a (-308) polymorphism in aggressive periodontitis have shown no differences with regard to the TNF-a (-308) genotype between patients and controls. Similarly, Shapira et al. [32] found no association between LagP (localized aggressive periodontitis) and TNF-a (-308) polymorphism. Guzeldemir et al. [24] studied 31 localized aggressive patients in Turkish subjects and reported a similar distribution for allele and genotype frequency for TNF-a (-308) compared to controls. In the present study, no differences were observed between aggressive periodontitis and controls, in agreement, with findings in populations of Caucasian [18,20–22,32,34]. Data from our study are among the few in the literature referring to both aggressive and chronic periodontitis patients, indicate a similar distribution of TNF-a (-308) genotype between the two [21,22,34]. On the other hand, when the patient groups were stratified with respect to attachment level (CAL < 4 mm and CAL P 4 mm), AP patients with CAL P 4 mm was observed to have a higher frequency of allele 2 compared to the CP patients with CAL P 4 mm. The genotype distribution and allele frequencies were not different regarding the subjects with CAL < 4 mm. These findings suggest that TNF-a (-308) allele 2 frequency might play a significant role for rapidly progressing periodontitis rather than periodontal disease susceptibility. Longitudinal studies are required in order to support and clarify this finding. While there
are few longitudinal studies investigating IL-1 genotype and periodontal disease severity, alveolar bone loss, tooth loss, and response to periodontal therapy [37,38]; there is no longitudinal study concerning the putative role of TNF-a (-308) in periodontal disease outcome. The occurrence of the allele 2 in the TNF-a (-308) was associated with an increased level of TNF-a in vitro in response to different stimuli [12]. The results of our study revealed no significant differences in GCF TNF-a levels of the different genotypes, in spite of a statistically insignificant increase in the median TNF-a levels in GCF from patient groups (AP,CP) with allele 2 when compared to carriers of (1,1) genotype. Similarly Trombone et al. [23]. reported a slight statistically insignificant increase in TNF-a mRNA levels in periodontal tissues from TNF-a (-308) allele 2 carriers, moreover they demonstrated that allele 2 carriers presented significantly higher levels of TNF-a in the absence of red-complex bacteria. The search for a functional role of TNF-a (-308) polymorphism in the TNF-a expression led controversial results [15,18,23]. Single nucleotide polymorphism at position -308, can increase tumor necrosis factor-alpha production up to fivefold in vitro [12], while a lower increase rate was demonstrated in vivo [23]. Taken together, our results do not permit to make a definite conclusion for the effect of TNF-a (-308) polymorphism in the modulation of GCF TNF-a levels. In agreement with the previous reports, we found a wide inter-individual variability in the TNF-a expression in both patient groups and controls. This may be one of the factors avoiding to establish a statistically significant association. Lack of observance of specific bacteria is a limitation of the present study, since different stimuli and cell-type were shown to be modulating factors for the transcriptional activity of the TNF-a [12]. Despite the limitations of clinic association studies, the results of our study revealed an association between TNF-a (-308) allele 2 frequency and aggressive periodontitis patients with CAL P 4 mm. TNF-a expression may aggravate the rapid destruction of periodontal attachment and bone loss in aggressive periodontitis patients by inducing the certain types of matrix metalloproteinases (MMP-1, MMP-3), cytokines (IL-1, interferon-b) and prostaglandins (PGE2) which mediate bone and extracellular matrix resorption. Acknowledgments The authors wish to thank the staff members of Hacettepe University, Faculty of Dentistry Department of Periodontology for their participation. The authors declare that there are no conflicts of interest in this study. Dr. Özlem Özer Yücel was supported by H.U.B.A.B (Hacettepe University Research Foundation) Ph.D. Thesis Grant (07T06T02004). References [1] Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge in periodontitis: assembling the players. Periodontol 2000;1997(14):33–53. [2] Nares S. The genetic relationship to periodontal disease. Periodontol 2000;2003(32):36–49.
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