DNA Methylation Profile of the SOCS-1 Gene Promoter in Smokers Patients With Chronic Periodontitis.

Completed

Interventions: Genetic: oral epithelial cells obtained by rinsing with 3% sucrose

Brief Summary: Periodontitis is related to host genetics, constitution of the dental biofilm and environmental factors such as smoking. DNA methylation is a mechanism of genetic expression that can inhibit or silence gene expression. In this way several researchers have been dedicated to study the genetic influence on the susceptibility and / or increased risk to periodontal disease. Studies have reported association between several epigenetic biomarkers with periodontal inflammation. Considering the hypothesis that there is an association between smoking and methylation in genes related to periodontal disease, the objective of this study was to verify the DNA methylation pattern in oral epithelial cells of patients with chronic periodontitis (CP) in the promoter of a specific gene involved in the control of inflammation, as suppressor of cytokine signaling (SOCS) 1 in smokers and nonsmokers patients.

Detailed Description: This was an experimental type study with parallel controls, comparing two groups, a group with consumption of 10 minimum cigarettes per day, with a diagnosis of chronic periodontitis. And another control group were non-smokers with chronic periodontitis. For this, genomic DNA was purified from oral epithelial cells obtained by rinsing with 3% sucrose, for a single time of collection, patients received periodontal treatment after collection and data analysis. The DNA was modified by Sodium bisulfite and the methylation patterns of the DNA were analyzed with the MSPCR technique (Polymerase chain reaction). This study was approved by the Institutional Review Board of the School of Dentistry of Ribeirão Preto (CAAE:57171816.2.0000.5419 ), and all patients need to provide written informed consent.

Recruitment & Eligibility

Inclusion Criteria

Chronic Periodontitis carriers - presence of proximal insertion loss ≥5 mm in more than 30% of the teeth present.
Periodontal pocket ≥ 5mm
Smokers who have smoked 10 or more cigarettes per day for the past five years.
No positive history of basic periodontal treatment in the last six months.

Exclusion Criteria

Extensive prosthetic involvement.
Using anti-inflammatories.
Pregnant or nursing.
Presence of systemic alterations that compromise host response or require prophylactic medication for treatment.
History of constant use of oral antiseptics in the last six months.

Arm && Interventions

Group	Intervention	Description
Smokers/Non-smokers	oral epithelial cells obtained by rinsing with 3% sucrose	It was an experimental study with parallel controls, comparing two groups, a group with patients who smoked for more than 10 years, consuming 10 more cigarettes per day and diagnosing chronic periodontitis (case) and another group (control) were non-smokers with chronic periodontitis, according to the standard of World Health Organization (WHO) definition of the smoking population

Primary Outcome Measures

Name	Time	Method
Extraction of genomic DNA from oral epithelial cells	Baseline (at the beginning of the study)	Epithelial cells were collected after mouthwash with 5ml dextrose autoclaved 3% and stored under refrigeration at -20ºC. After collection, the samples were taken to the Epigenetics and Reproduction Laboratory of the Department of Genetics of the Medical School of Ribeirão Preto-USP (FMRP-USP) where nucleic acid extraction and molecular analysis were performed. After thawing the saliva, 2ml of each sample collected were transferred separately to eppendorf tubes, for processing. The material was then diluted in 50 μl of cobalt Mili-Q water, incubated at 37 ° C in a water bath for 1 hour and stored in a freezer at -20 ° C. At the end, DNA quality, purity and integrity were measured using a spectrophotometer (NanoDrop 2000-Thermo Scientific), using OD 260/280 and 260/230. Considering ideal values for both reasons, between 1.8 and 2.2.

Secondary Outcome Measures

Name	Time	Method
Bleeding on probing (BOP)	Baseline (at the beginning of the study)	Clinical parameter was recorded at baseline by one trained periodontist.Bleeding on probing (BOP) was recorded based on the presence or absence of bleeding up to 30 seconds after probing on four sides of each tooth,the evaluation was done with the unit of measure in percentage (%) whole mouth. Probing measurement was performed using a manual University of North Carolina - UNC Periodontal probe (Hu- Friedy, Chicago, IL, USA).
Plaque index (PI)	Baseline (at the beginning of the study)	Clinical parameter was recorded at baseline by one trained periodontist. Plaque index (PI) was used to assess the oral hygiene status of the patients, the evaluation was done with the unit of measure in percentage (%) whole mouth. Probing measurement was performed using a manual University of North Carolina - UNC Periodontal probe (Hu- Friedy, Chicago, IL, USA).
Probing pocket depth (PD)	Baseline (at the beginning of the study)	Clinical parameter was recorded at baseline by one trained periodontist.Probing pocket depth (PD) was measured from the free gingival margin to the bottom of the periodontal pocket and clinical attachment level ( CAL) was measured from the cementum-enamel junction (CEJ) to the bottom of periodontal pocket. Probing measurement was performed using a manual University of North Carolina - UNC Periodontal probe (Hu- Friedy, Chicago, IL, USA).