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This study was designed as a case-controlled study. The study group was composed of 1150 individuals, ranging from 27–62 years of age. It took almost one year (June 2012 to April 2013) to complete sample collection. All of the subjects were unrelated Han Chinese individuals randomly selected from the Shaanxi Province, with no migration history within the previous 3 generations. Additionally, all participants were of a similar socio-economic level, which is important because there is a strong association between low socio-economic status and a higher risk of periodontal disease [31]. All enrolled individuals answered a questionnaire to obtain information regarding dental history, family history of periodontal disease, cigarette smoking habits and general health. All of the subjects were required to have at least 10 teeth, be in good general health and be free of oral soft tissue abnormalities or severe dental caries, except for the presence of chronic periodontitis. Patients who reported the following characteristics were excluded from the study: use of orthodontic appliances, chronic anti-inflammatory drugs or immunosuppressive chemotherapy, antibiotics within the previous 3 months, chronic inflammatory diseases, a history of diabetes mellitus, hepatitis, HIV infection, nephritis, bleeding or autoimmune disorders, diseases with severe commitment of the immune function, current pregnancy or breastfeeding. Other authors have previously used these exclusion criteria [32]–[35]. Because tobacco is associated with increased clinical attachment loss (CAL) and supports alveolar bone loss, it represents an important risk factor for the initiation and progression of periodontal disease [3], [36]–[38]. The individuals enrolled in this study were all nonsmokers (never smoked before). Individuals were categorized into the control and chronic periodontitis groups. The healthy control group did not have signs of any periodontal disease at the time of sample collection and did not have a history of previous periodontal disease as determined by a lack of sites with probing depth (PD) >3 mm and the absence of gingival recession, CAL, and bleeding on probing (BOP). The 750 blood samples from periodontally healthy Han Chinese subjects were obtained randomly, which represented the controlled population (375 males and 375 females, aged 27 to 60 years with a mean age of 50.32±8.27 years; Table 1).
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0104436.t001 Demographic characteristics and clinical parameters of chronic periodontitis and controls. Patients with chronic periodontitis often presented an amount of destruction consistent with the amount of microbial deposits, presence of subgingival calculus, probable association with local predisposing factors and a slow to moderate rate of progression. All of the chronic periodontitis subjects were previously diagnosed with moderate or severe chronic periodontal disease. Diagnosis of chronic periodontitis (CP) was established clinically and by X-ray verification, according to the criteria of the American Academy of Periodontal Disease (AAP, 1999) [39], presence of chronic gingivorrhagia, bleeding on probing, clinical attachment loss, and horizontal or vertical loss of alveolar bone. In all patients, the degree of clinical attachment loss was defined using confirmed periodontal probe. Patients with probing depths greater than 5 mm, CAL greater than 4 mm, and some degree of gingival recession and tooth mobility were chosen. This clinical form is most prevalent in adults, but its occurrence may be present in younger individuals [40]. This patient group was composed of 400 subjects (200 males and 200 females, aged 28 to 62 years with a mean age of 50.46±9.14 years; Table 1) that were recruited from the inpatient and outpatient clinical services at the First Affiliated Hospital of Xi'an Jiaotong University, the second Affiliated Hospital of Xi'an Jiaotong University and the Stomatology Hospital of Xi'an Jiaotong University. This study was approved by the Xi'an Jiaotong University Ethics Committee. All participants completed written informed consent forms. Data related to the participants are described in Table 1.
IL-8 polymorphisms were identified using the National Center for Biotechnology Information single nucleotide polymorphism database, and 209 SNPs were identified. For the first screen of the most common SNPs in Han Chinese chronic periodontitis patients, a MAF≥0.01 was used as the cut-off. Based on these criteria, we selected 14 SNPs in IL-8 (rs2227528, rs7682639, rs2227531, rs2227532, rs4073, rs2227538, rs2227307, rs2227549, rs2227306, rs2227543, rs2227545, rs1126647, rs10938092 and rs13112910). Next, we then searched for all SNPs with minor allele frequencies (MAF)≥0.01 between 20 kb upstream and 20 kb downstream of IL-8 in the HapMap HCB database using Haploview [41], which identified 9 SNPs (rs12506479, rs10805066, rs10031141, rs46946336, rs11730667, rs1951242, rs11730284, rs10938095 and rs2886920). Therefore, we selected 23 SNPs in the 45 kb region containing IL-8 (Fig. 1).
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0104436.g001 Distribution of the 23 SNPs across the IL-8 gene selected for the association analysis and their relationship with gene exons. Patients and controls were mixed on the same plates using a double-blind procedure. Plasma samples were stored at −20°C. Genomic DNA was isolated from peripheral blood leukocytes according to the manufacturer's protocol (Genomic DNA kit, Axygen Scientific Inc., California, USA), and DNA samples were stored at −20°C for SNP analysis. SNP genotyping was performed using the Sequenom MassARRAY platform with iPLEX GOLD chemistry (Sequenom, San Diego, CA, USA), according to the manufacturer's protocol. Polymerase chain reaction (PCR) primers and locus-specific extension primers were designed using MassARRAY Assay Design software package (v. 3.1). 50 nanograms of DNA template were used in each multiplexed PCR well. PCR products were treated with shrimp alkaline phosphatase (USB, Cleveland, OH, USA) prior to use in the iPLEX GOLD primer extension reaction. Single base extension products were desalted with SpectroCLEAN resin (Sequenom), and 10 nL of the desalted product was spotted onto a 384-format SpectroCHIP using the MassARRAY Nanodispenser. Mass determination was performed with a MALDI-TOF mass spectrometer, and MassARRAY Typer 4.0 software was employed for data acquisition. The final genotype call rate of each SNP was greater than 96% and the overall genotyping call rate was 98.1%, confirming the reliability of further statistical analyses.
Statistical analysis and power analysis: Hardy–Weinberg equilibrium (HWE) for each SNP was assessed using GENEPOP v4.0. Allelic and genotypic association tests were performed using CLUMP v2.4 with 10,000 simulations, and this program employed an empirical Monte Carlo test of significance through simulation. To control for possible confounding effects, age and gender were used as independent variables in a multiple logistic regression analysis for adjustment by commercially available software (Statistical Package for Social Sciences, version 16.0 for windows, SPSS Inc., Chicago, IL, USA). The D′ values for each pair of markers were calculated using the software program 2LD [42]. Haplotype frequencies were estimated using GENECOUNTING v2.2, which computes maximum-likelihood estimates of haplotype frequencies from unknown phase data using an expectation–maximization algorithm [43]. The significance of a haplotypic association with chronic periodontitis was evaluated using a likelihood ratio test, followed by permutation testing that compared the estimated haplotype frequencies in patients and controls [43], [44]. Differences were considered significant when the p value was less than 0.05. For haplotype analyses, the global p value was based on a comparison of the frequency distribution of all possible combinations of haplotypes among patients and controls. Furthermore, we performed power calculations for case–control genetic association analyses using PGA v2.0 [45]. Our sample size can detect SNP and haplotype associations with 91% and 85% power, respectively, at a false positive rate of 5%, and a presumed minimum odds ratio (OR) of 1.5.
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