The Relationship Between CDH23 Gene Methylation And Carotid Atherosclerosis In Male Cigarette-dependent Patients

Objective:To investigate the altered methylation of the CDH23 gene and its relationship with carotid atherosclerosis in male cigarette-dependent patients.Methods:Chapter I:Selected 130 male subjects in the Health Department and Health examination Center of Qingdao University Hospital from September 2019 to January 2021,who were divided into cigarette-dependent group(n=64)and non-smoking control group(n=66).After professional training,health care doctors should collect subject questionnaire data,scale data,medical history.The questionnaire records included the subjects’general condition(name,age,years of education)and the history of cigaertte dependence;mini-mental state examination(MMSE)that assessed the cognitive level of the subjects;fagerstorm test for nicotine dependence(FTND)to assess cigaertte dependence in cigarette-dependent people;Medical history collected included hypertension,diabetes,hyperlipidemia,All were diagnosed according to the newly published diagnostic criteria.Subjects took 5ml of peripheral venous blood in their fasting state and stored it in the EDTA anticoagulant tube.1ml of blood sample from the anticoagulant tube was extracted and put into the EP tube and stored in the-80℃refrigerator.Using target region methylation sequencing(Methyl Target)analysis to detect the methylation level per gene site of the CDH23 gene and compare the average methylation difference per Cp G site of the CDH23 gene between the cigarette-dependent group and control group.Statistical analysis was performed using SPSS26.0 and Shapiro-Wilk’s was applied for the normality test.For normally distributed measurement data,mean±standard deviation is analyzed by t-test;Count data is expressed by frequency,count data is analyzed by c~2test;P<0.05 is statistically significant.Chapter II:The 130 subjects included had a carotid ultrasound examination,carotid artery intimal thickness was recorded;Selected the Cp G sites statistically significant in the cigarette-dependent group and control group,median methylation values were calculated for all subjects at this locus;Using the median number as the standard,subjects methylation levels were divided into hypomethylation and hypermethylation;According to the factorial analysis of variance comparing the difference in carotid intima thickness between cigarette-dependent and methylation at two levels(cigarette-dependent hypomethylation,cigarette-dependent hypermethylation,control hypomethylation and control hypermethylation)(P<0.05 was statistically significant);Further analysis of the relationship between methylation changes in the CDH23 gene and carotid atherosclerosis in cigarette-dependent patients.Results:Chapter 1:Methylation analysis of the CDH23 gene1.No significant difference between age,years of education,and MMSE scores between cigarette-dependent group and control groups(P>0.05).2.No significant difference in medical history(diabetes,hypertension,hyperlipidemia)between cigarette-dependent group and control group(P>0.05).3.Analysis of cigaertte dependence in the cigarette-dependent group:the initial age of smoking was 24.06±7.48 years,duration 31.45±9.36 years,daily smoking 20.73±9.92,FTND score 6.38±1.30.4.The methylation degree of Cp G246 site in CDH23-1 fragment and Cp G48 site in CDH23-2 fragment in cigarette-dependent group was significantly lower than that in control group,and the methylation degree of Cp G110,Cp G112,Cp G127,Cp G131,Cp G135,Cp G139,Cp G147,Cp G156,Cp G162,Cp G170,Cp G191,Cp G195,Cp G200,Cp G222,Cp G224 in cigaertte-dependent group,Cp G36,Cp G38,Cp G43,Cp G57,Cp G60,Cp G62,Cp G64,Cp G88,Cp G101,Cp G140,Cp G152,Cp G185 in CDH23-2 fragment.The methylation degree of Cp G50,Cp G52,Cp G62,Cp G80,Cp G91,Cp G98 in CDH23-3fragment and Cp G33,Cp G55,Cp G89,Cp G138,Cp G166,Cp G192,Cp G208 in CDH23-4fragment were lower than those in control group,but the difference was not statistically significant(P>0.05).5.The average methylation degree of CDH23 gene fragments(CDH23-1,CDH23-2,CDH23-3,CDH23-4)in cigaertte-dependent group was lower than that in control group,but the difference was not significant(P>0.05).Chapter2:Study on the relationship between methylation of CDH23 gene and carotid atherosclerosis incigaertte-dependent patients1.The Cp G248 site:The median methylation of Cp G246 site is 0.010556,carotid intima thickness among different groups:cigaertte-dependent on low methylation group(1.403±0.503 mm),cigaertte-dependent on hypermethylation group(1.138±0.504 mm),control hypomethylation group(1.204±0.537 mm),control hypermethylation group(1.023±0.463 mm);2×2 factorial analysis of variance results:Total correction model F=3.851,P=0.011;There was no significant difference in main effect cigarette-dependent:F=3.113,P=0.080;The difference was statistically significant in main effect Cp G246 hypomethylation:F=6.235,P=0.014;There was no significant difference in interactions between the two main effects:F=0.220,P=0.640.In conclusion,the results showed that carotid intima thickness increased significantly with hypomethylation at Cp G246,and altered hypomethylation at Cp G246 in CDH23 gene correlated with the degree of carotid atherosclerosis.2.The Cp G48 site:The median methylation of Cp G48 site is 0.056351,carotid intima thickness among different groups:cigaertte-dependent on low methylation group(1.477±0.462 mm),cigaertte-dependent on hypermethylation group(1.135±0.524mm),control hypomethylation group(1.114±0.513mm),control hypermethylation group(1.077±0.489mm);2×2 factorial analysis of variance results:Total correction model F=4.266,P=0.007;There was no significant difference in main effect cigarette-dependent:F=5.793,P=0.018;The difference was statistically significant in main effect Cp G48hypomethylation:F=4.692,P=0.032;There was no significant difference in interactions between the two main effects:F=3.041,P=0.084..In conclusion,the carotid intima thickness increased significantly during hypomethylation at Cp G48,and the hypomethylation at Cp G48 in CDH23 gene was correlated with the degree of atherosclerosis;the carotid intima in the cigarette-dependent group was significantly higher than the control group,and the degree of carotid atherosclerosis correlated with cigarette-dependent.Conclusion:1.In this study,we detected the methylation level of 66 Cp G sites of CDH23 gene.It was found that the methylation level of CDH23 gene at Cp G246 site(CDH23-1)and Cp G48site(CDH23-2)was significantly decreased in cigaertte-dependent patients.2.By further analyzing the relationship between the methylation level of Cp G246 site and Cp G48 site of CDH23 gene and carotid atherosclerosis,we found that the changes of hypomethylation at Cp G246 site(CDH23-1)and Cp G48(CDH23-2)site of CDH23 gene were related to the degree of carotid atherosclerosis.