title: TLR2 and TLR4 gene promoter methylation status during chronic periodontitis.
abstract: OBJECTIVE
  The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis.
  
  
  METHODS
  Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels.
  
  
  RESULTS
  The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05).
  
  
  CONCLUSIONS
  The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.
authors: Department of Morphology, Laboratory of Molecular Biology, Division of Histology, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, São Paulo, SP, Brazil.
authors: Naila Francis Paulo
authors: NF
authors: De Oliveira
authors: Denise Carleto
authors: DC
authors: Andia
authors: Aline Cristiane
authors: AC
authors: Planello
authors: Silvana
authors: S
authors: Pasetto
authors: Marcelo Rocha
authors: MR
authors: Marques
authors: Francisco Humberto
authors: FH
authors: Nociti
authors: Sérgio Roberto Peres
authors: SR
authors: Line
authors: Ana Paula
authors: AP
authors: De Souza
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D016009:Chi-Square Distribution; D055113:Chronic Periodontitis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D005881:Gingiva / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012907:Smoking / Q000235:genetics; D018709:Statistics, Nonparametric; D051195:Toll-Like Receptor 2 / Q000235:genetics* / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism
chemical_list: D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4