Frequency Of MCP-1 And CCR2 Gene Polymorphisms And Its Effect On Gene Expression In Patients With AgP

Differences in structure of gene or non genetic factors such as nutrition/environmental factors may alter functional gene product: mRNA/protein which play crucial roles in immune system. This can lead developing defective immune responses, exacerbation of current inflammation and increased host susceptibility to inflammatory diseases. Monocyte functions can be critical with regard to getting individuals susceptible to periodontitis. Similarly Garrison and Nichols (1989) reported that hyper-inflammatory monocyte phenotype can be deterministic in periodontal destruction. For this reason, regulation of MCP-1 and CCR2 expression, which have an essential role in defense system, may be a crucial step for managing inflammatory diseases as well as periodontitis.

Because of complex genetic nature of periodontal disease, we hypothesized that gene polymorphisms of MCP-1 and CCR2 could be associated with AgP and could alter the production of functional proteins, as a result might influence the susceptibility. Therefore, the primary aim of this study was to estimate genetic impact of MCP-1 and its receptor CCR2 polymorphisms on AgP patients among Turkish individuals and secondary outcome was whether MCP-1 genotype effects mRNA levels of PBML.

A total of 215 Turkish subjects from inner Anatolia including, 108 Aggressive periodontitis (AgP) and 107 age, gender and ethnic matched periodontally healthy (H) controls were recruited in this cross-sectional case control study. The control group included periodontally healthy volunteers from staff and other subjects referring to the School of Dentistry. The diagnosis of subjects were established on the basis of clinical and radiographic examination. Periodontally H control group (n:107) had <3mm probing Depth (PD), <2 gingival Index (GI) and no signs of interproximal attachment loss and a history of periodontal disease. Patients with AgP (n:108) were diagnosed by the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions. The AgP group included individuals diagnosed with localized AgP (LAgP) or generalized AgP (GAgP) who were otherwise healthy. Periodontal attachment loss ≥4 mm not involving more than two permanent teeth, other than the first molars and incisors were diagnosed with LAgP (n: 43); patients with involvement of at least three teeth, other than the first molars and incisors with an attachment loss ≥4 mm were diagnosed with GAgP (n: 65) Genomic DNA was isolated from a peripheral blood sample obtained from each subject. Gene polymorphisms of MCP-1 -2518 A/G and CCR2 -190 G/A were analyzed by a standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Gene expression levels were quantified in peripheral blood leukocytes from 25 AgP and 15 periodontally H controls by quantitative real-time PCR. Threshold cycles (Ct ) values obtained from the RT-PCR analysis based on SYBR Green detection and data was normalized via ΔC t .

Sample size was determined by power analysis prior to study. According to this; an expected difference 20% in allele frequencies with 80% power, and % 95 confidence interval it was calculated that minimum of 102 patients were necessary in each group with a significance level of 0.05. ( ⃰ G. Power: 3.1.2). When comparing the numeric characteristics between H and AgP groups,non-parametric Mann-Whitney U test; between H, LAgP and GAgP groups then Kruskal Wallis with Bonferroni correction were calculated.

Deviations from Hardy-Weinberg equilibrium were assessed in the H and AgP groups based on genotype distribution for MCP-1 -2518 and CCR2 -190 by using a chi-squared test. The differences in genotype and allele frequencies between groups were also detected by the chi-square test. We used independent samples t-test for comparison of gene expression levels between groups and one way ANOVA was used to determine the effect of MCP-1 -2518 genotype on gene expression, based on log-transformed data. Significance level was P = 0.05.