Blood samples were obtained from 237 cigarette smoker individuals (mean age = 34 ± 12) and 90 healthy nonsmokers as controls (mean age = 36 ± 11), with no history of psychiatric illnesses. Before data collection, all respondents were informed about the aims of the study and data confidentiality and gave written informed consent. All participants took part in the survey voluntarily. Exclusion criteria were as follow: a history of primary neurologic diseases such as Parkinson’s disease or depressive disorders, schizophrenia, suicide disorder, Alzheimer's and Huntington disease, alcohol usage. Smoker individuals were classified into three groups according to the Fagerstrom protocol: sever, moderate and low users.
Bisulfite Modification Of Genomic Dna And Methylation Analysis:
Genomic DNA was extracted from whole blood leukocytes according to the protocol described with the (Pars Tous Company, Iran) kit. Briefly extracted DNA samples were treated with sodium bisulfite and then PCR amplified using primers specific for either the methylated and modified unmethylated promoter region of each growth factor genes. The primers are listed in Table 1. In all MSP reactions, DNA from normal leukocytes and universal human methylated DNA standards from Zymo Research (ZYMO Research, Freiburg, Germany) were used as unmethylated (negative) and methylated (positive) controls, respectively.
Methylation Specific Pcr (ms-pcr):
The results from MS-PCR showed that the percentage of the people that their BDNF gene ( in exon I, in promoter I (was methylated, was significantly higher in heavy smokers than in healthy nonsmokers, Table 4, (P-value = 0.001). Also, the rate of cigarette dependence directly correlates with the methylation status in the subjects. (Fig. 1).
Figure 1. Correlation of nicotine dependence with BDNF gene methylation status. the percentage of the people that their BDNF gene was methylated, was higher in the heavy smokers > medium smokers > low smokers. ** P-value < 0.001
Quantitative Real-time Polymerase Chain Reaction:
The RT-PCR products were quantified using 2−∆∆ct equation. The threshold cycle of BDNF gene was used to calculate the relative expression of the gene in smokers and control groups. The results showed that the relative expression of BDNF gene in heavy smokers is significantly (p-value < 0.001) lower than the healthy nonsmokers (Fig. 2).
Figure 2. Relative expression of BDNF gene in heavy smokers and healthy control. The results from the groups of moderate and low smokers did not show any significant difference, data not shown. ** P-value < 0.001
Enzyme-linked Immunosorbent Assay (elisa):
The serum level of BDNF was significantly (p-value < 0.05) lower in heavy smokers than healthy nonsmokers; these data confirm the results from real-time PCR (Fig. 3).
Figure 3. Comparison between heavy smokers and healthy nonsmokers in the serum level of BDNF. The results from the groups of moderate and low smokers did not show any significant difference, data not shown. ** P-value < 0.05