| Objective: OPRK1 and OPRM1 belong to opioid receptor family and spread widely in nervous system,moreover,they show association with AD.In our previous study,it has been mentioned that DNA methylation modification of both genes was associated with AD,we further explored their methylated roles in its prophase,namely MCI.Moreover,high throughput method will be used to identify the risk genes and provide evidence for the future the study.Methods: Gender,age and ethnicity-matched MCI and controls were recruited from epidemiological study in Xinjiang region which included 5398 samples.DNA was extracted from blood samples according to paramagnetic particle method.Pyrosequencing was conducted after the bisulphite conversion.Dual-luciferase assays were used to detect the activities of promoters.450 K methylation array was further used to detect the genome-wide methylation.Results: Part one: case-control comparisons: OPRK1 showed no association with MCI in Uygur and Han group,but analyses stratified by gender indicated that OPRK1 was hypernethylated in Xinjiang Han female MCI(p = 0.015);as for OPRM1,CpG1 hypermethylation was associated with Uygur MCI(total p = 5.16E-05,male p = 0.015,female p = 1.20E-03),CpG2-4 hypomethylation was associated with Han MCI(total p =0.002,male p = 0.018,female p = 4.98E-02).Comparisons between two ethnic groups: logistic regression was used to adjust the different characteristics between two ethnicities,OPRK1 methylation showed no ethnic difference(p > 0.05);as for OPRM1,Cp G1 methylation was associated with ethnicity in MCI group and Cp G2-4 methylation showed association with ethnicity control group(Cp G1 p = 0.002,adjust p = 0.009,Cp G2-4 p = 0.005,adjust p = 0.009).In addition,we compared OPRK1 and OPRM1 methylation between Zhejiang Han controls and Xinjiang Han controls,OPRK1 methylation showed regional difference in total and male groups(total p = 0.006,male p = 2.31E-04);as for OPRM1,CpG1 and CpG2-4 were hypermethylated in Xinjiang Han controls(CpG1: total p = 2.99E-05,male p = 0.006,female p = 0.001;CpG2-4: total p = 1.32E-13,male p = 1.71E-08,female p = 2.74E-05).Partial correlation analyses showed that OPRM1 CpG2-4 methylation was positively correlated with age in Uygur group(r = 0.220,p = 0.039).Dual-luciferase experiments indicated that,compared with negative control,luciferase reporter gene activity was significantly enhanced by OPRK1 and OPRM1 promoter fragments(OPRK1: fold change = 2.248,p = 0.008;OPRM1 fold change = 2.616,p = 0.003).Part two: 450 K method was used to detect the genome-wide methylation profiles of 6 samples,given the different level of triglyceride between AD and controls(p = 0.025),it was corrected using linear models in limma package.Differently methylated CpG site was selected based on |delta beta| > 0.2 and p < 0.05.In present result,41 CpG sites in which 18 were hypermethylated and 23 were hypomethylated were identified.Conclusion: Hypermethylation in OPRK1 served as a risk factor in Han female MCI and its fragment showed promoter activity.Environmental factor was associated with OPRK1 methylation in Han males while not in Han females.Ethnic diversity of DNA methylation at certain CpG site in OPRM1 promoter showed association with MCI.CpG1 methylation was associated MCI in Uygur group and CpG2-4 methylation was associated MCI in Han group.In addition,CpG1 methylation in MCI showed ethnic difference while CpG2-4 in controls showed ethnic difference.Environment factor would influence OPRM1 methylation.Age served as an important risk factor in MCI,in oue present study,it positively correlated with OPRM1 CpG2-4 methylation in Uygur population.Fragments in OPRM1 showed promoter activityIn addition,our genome-wide methylation results provided evidence for subsequent study,thereinto,CpG sites in proximal promoter should be studied in enlarged samples,especially in the progression of MCI. |
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