Studies On The Association Of 5-HT2A Receptor Gene Polymorphism And Methylation With Nicotine Dependence

Objective To investigate the distribution of 5-HT2 AR gene polymorphism and the methylation level of 5-HT2 AR gene promoter in nicotine dependent population.Methods Chapter 1: Chapter 1: From June 2018 to November 2019,130 middle-aged and elderly smokers and non-smokers who met the inclusion criteria of this study were selected from the health care department of Affiliated Hospital of Qingdao University.According to the score of nicotine dependence test(FTND),the patients were divided into two groups: nicotine dependence group and control group.Polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)and DNA direct sequencing were used to detect the rs6311 polymorphism of 5-HT2 AR gene.On line analysis of rs6311 polymorphism of 5-HT2 AR gene by primer3 software.SPSS 26.0 software was used to analyze the data.Shapiro Wilk's was used for normal test,and the counting data was expressed in the number of cases and percentage;the measurement data was expressed in the mean ± standard deviation,the comparison between the two groups was t-test,the comparison between the two groups was chi square test,the correlation analysis was Spearman correlation analysis,P< 0.05,which showed that there was statistical significance.Chapter 2: Using Illumina hiseq platform,high-throughput sequencing was carried out with 2 × 150 bp double terminal sequencing mode to detect the level of genomic methylation of all subjects.If the continuous variable is normal distribution,t-test is used.If it is not normal distribution,Wilcoxon rank combination test is used.The methylation level of promoter region of 5-HT2 AR gene was determined by Wilcoxon rank test.The difference was statistically significant(P < 0.05).Results Chapter 1: 5-HT2 AR gene polymorphism analysis The rs6311 locus of 5-HT2 AR gene was consistent with Hardy Weinberg equilibrium(P > 0.05).The genotype frequency distribution of rs6311 was 31.5% for GG,49.2% for GA and 19.3% for AA.The statistical analysis showed that there was no significant difference in genotype distribution and allele frequency of rs6311 between nicotine dependent group and control group(P > 0.05).Chapter 2: Methylation degree of promoter region of 5-HT2 AR gene in samples 1.There is no complete CpG island in 5-HT2 AR gene,so the methylation level of two fragments in promoter region of 5-HT2 AR gene was detected.The results of methylation showed that there was no significant difference between smoking group and control group(P > 0.05);2.There was no significant difference between the two groups(P > 0.05).The methylation level of CpG 84 and CpG177 in the promoter region of 5-HT2 AR gene in nicotine dependent group was higher than that in the control group,and the methylation level of CpG69 and CpG87 in nicotine dependent group was lower than that in the control group;3.By linear regression analysis of the methylation sites of rs6311 and promoter region of 5-HT2 AR gene,it was found that the polymorphism of rs6311 and CpG69,CpG84,CpG87 and CpG177 in the two CpG fragments of 5-HT2 AR promoter region were correlated,and the difference was statistically significant(P < 0.05).Conclusion 1.The rs6311 polymorphism of 5-HT2 AR gene has no significant correlation with nicotine dependence.2.The methylation levels of CpG69,CpG84,CpG87 and CpG177 in the two CpG fragments of 5-HT2 AR gene were not significantly correlated with nicotine dependence.However,the rs6311 polymorphism of 5-HT2 AR gene is closely related to the methylation degree of CpG69,CpG84,CpG87 and CpG177 of 5-HT2 AR gene.