SINGLE NUCLEOTIDE POLYMORPHISMS IN IL-1A RS1800587, IL-1B RS1143634 AND VITAMIN D RECEPTOR RS731236 IN STAGE III GRADE B/C PERIODONTITIS
Özturk Özener H.1, Tacal Aslan B.2, Eken B.F.2, Agrali Ö.B.1, Yildrim H.S.1, Altunok E.Ç.3, Ulucan K.2, Kuru L.1
*Corresponding Author: Assist. Prof. Dr. Hafize Öztürk Özener, Address: Marmara University, Faculty of Dentistry, Department of Periodontology Basıbuyuk, Maltepe, Istanbul, Turkey. Fax number: +90 216 421 02 21; E-mail: hafize.ozturk@marmara.edu.tr
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METHODS

Study Population The protocol of this cross-sectional study was approved by the Clinical Studies Ethical Committee of Faculty of Dentistry, Marmara University on 23.10.2017 with the reference number 2017-143, and registered at clinicaltrials.gov (identification number NCT04806971). According to the 2013 revision of the 1975 Helsinki Declaration, each participant signed an informed consent form prior to the study. All individuals provided a written informed consent. The present study recruited 200 individuals from the Department of Periodontology, Faculty of Dentistry, Marmara University between January 2018 and September 2019. The study population was made up of 100 periodontitis patients and 100 periodontally healthy subjects of Turkish origin. Inclusion criteria for entry were as follows: previously untreated periodontitis patients, systemically healthy study population. Exclusion criteria included smoking (current and past); presence of systemic disease such as diabetes, rheumatic fever, kidney or liver diseases, neurologic deficiencies, immunologic diseases; pregnancy; received antibiotics within the last 6 months; use of any medication that may influence periodontium (chronic use of non-steroidal anti-inflammatory drugs, cyclosporine, nifedipine, phenytoin, etc.). Periodontitis group: Stage III, Grade B/C periodontitis patients were diagnosed on the base of the new classification criteria [2,24]. Stage III periodontitis patients enrolled had ≥20 teeth, probing depth (PD) ≥6 mm, clinical attachment loss ≥5 mm, and bone loss reaching to the middle third of the root radiographically. Grade B was assessed by indirect consideration of radiographic bone loss throughout the most affected tooth in the dentition as a function of age (% bone loss/age: 0.25-1.0), and Grade C was evaluated based on the radiographic bone loss in the most affected tooth in whole dentition as a function of age (% bone loss/age: >1.0). Subjects in the periodontitis group were categorized into two subgroups, 51 patients Grade B and 49 patients Grade C. Healthy group: The periodontally healthy subjects with the absence of any sign of clinical inflammation, not having periodontitis history; absence of detectable bone and/or attachment loss; <10% of sites with bleeding on probing (BOP); PD ≤3 mm; presence of 28 permanent teeth without extensive restorations or caries. In addition, all subjects in the healthy group did not show any local or systemic pathology. Periodontal Examination Periodontal examinations were carried out by the same periodontist (HOO) using a periodontal probe (PCP 15 UNC, Hu-Friedy, Chicago, USA). Plaque index (PI) [25] and gingival index (GI) [26] were evaluated at 4 sites, PD, clinical attachment level (CAL) and BOP at 6 sites per tooth, excluding third molars. A calibration session was performed by using 10 subjects (not part of the study groups) to assess the intra-examiner reliability and full mouth PD and CAL scores were measured at two separate sessions, 24 hours apart. The intra-examiner correlation for PD and CAL measurements were calculated as 92.2% and 93% reproducibility, respectively. Genotyping 4–6 ml venous blood samples were collected from the antecubital fossa of each individual and stored in ethylenediaminetetraacetic acid (EDTA) vacationer. DNA was extracted from the blood by using commercially available kits (Invitrogen, USA), following manufacturer’s guidelines. Sections of the IL-1A, IL-1B and VDR genes that contain the SNPs of interest were amplified. All genotyping assays were validated prior to processing the study samples. After the validation step, negative controls (water), positive controls of known genotypes and a subset of study samples were genotyped in duplicate. All samples were genotyped for rs1800587, rs1143634 and rs731236 polymorphisms using Real-Time PCR (Thermofisher Quantstudio 3, USA) by using a Taqman genotyping assay (Catalog no: 4362691 Thermofisher, USA). The 10 μl reaction volume comprised of 5 μl genotyping master mix (Applied Biosystems, Foster City, CA), 0.5 μl genotyping assay (Applied Biosystems), 3.5 μl nucleasefree H2O (Thermofisher, USA), and 1 μl DNA. Reactions were incubated in a 96-well optical plate for denaturation at 92°C for 15 s, followed by annealing and extension at 60°C for 1 min (40 cycles). Statistical Analysis SPSS software version 25 was used for analyses. The descriptive statistics were presented using mean and standard deviation. Frequencies and percentages were used for categorical data. The Kolmogorov Smirnov test was performed to investigate to whether the variables are normally distributed or not. For association analysis between SNPs and periodontitis susceptibility, various genetic models were used by SNPassoc. The Mann-Whitney U test was done to compare the groups, since the variables were not normally distributed. Chi-Square test and Fisher’s exact test, where appropriate, was applied for comparison of the proportions of the groups. The study population obeys the rule of the Hardy-Weinberg equilibrium law, which was carried out with a goodness-of-fit χ2. Odds ratios with a 95% confidence interval were calculated for risk factors. A 5% type-I error level was used to interpret a statistical significance.



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