Genome-wide DNA Methylation Patterns Study Of Alcohol-dependent Patients

Objective In this study, the Illumina Infinium Human Methylation450was performed on peripheral blood mononuclear cells from alcohol-dependent patients and siblings without alcohol dependence as controls. Gene-specific methylation of DNA isolated from peripheral blood mononuclear cells was assessed. By which, the relation between the differential methylation status of these genes and alcohol dependence was discussed. Some genes were selected to validate beadchip results by pyrosequencing to increase the credibility. We analyze differential genes of high degree methylation by case-control of large sample. We can understand preliminarily the function and significance of epigenetics in the pathological mechanism of alcohol use disorder.Methods1. The standard of diagnosis is DSM-Ⅳ with its formulary inspection tools SCID-I/P (Structured Clinical Interview for DSM-Ⅳ). With the standard of diagnosis, who were diagnosed alcohol dependence were recruited as cases and we adopt The Alcohol Use Disorders Identification Test (AUDIT) to assess the severity of alcohol dependence, LES and Wheatley Stress Scale to understand the stress factors related to AD. Controls matched with cases on age, degree of education and region were recruited. We selected one of alcohol dependence’s brothers who had not yet appeared the status of alcohol dependence (sick), and intaked non-sick siblings on the basis of informed consent. This research collected10discordant sib pairs,63cases and65controls, all were male, and all come from Xinxiang city and nearby areas.2. We drawn the peripheral anticoagulant blood of all the research objects8-10ml, and was extracted DNA (Qiagen, Germany) to spare. DNA was made sulfite treatment and PCR amplification. We detected and analyzed genes with the use of genome methylation chip (the Illumina Infinium Human Methylation450).3. With the software of BeadStudio Methylation Module v3.2, we analyzed and compared the status of methylation, and concluded the sites of gene difference and high degree of methylation. The results were screened and analyzed according to AVGJBeta value (reflects the methylation degree of the CpG site, the values were higher and tend to1, the methylation degree of the CpG site was higher). According to the absolute value of DiffScore>20, we screened methylation difference sites.4. Genes GABRP, ALDH1L2, GAD1and DBH were chosen from the Illumina assay repertoire for pyrosequencing to validate beadchip results among discordant sib pairs with alcohol dependence.5. To identify the function of genes, including differential CG sites, the Database for Annotation, Visual-ization and Integrated Discovery6.7(DAVID; http://david.abcc.ncifcrf.gov), Gene Ontology (GO; http://www. geneontology.org/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) were used.6. Genes GABRP and SSTR4were chosen to detect methylation difference sites by pyrosequencing between cases and controls.Results1. Clinical characteristics(1) The mean age of cases and sib pairs were (44.9±5.9) years old and (44.2±7.2) years old, cases and sib pairs had no significant differences on age (P>0.05); The mean age of cases and controls were (39.1±7.3) years old and (39.6±8.1) years old, cases and controls had no significant differences on age (P>0.05).(2) Scale analysis of AUDIT, LES and Wheatley Stress Scale of discordant sib pairs had significant differences. AUDIT (t=8.246, P<0.05), LES (t=5.453, P<0.05), Wheatley Stress Scale (t=7.981, P<0.05).(3) Scale analysis of AUDIT, LES and Wheatley Stress Scale of case-control had significant differences. AUDIT (t=16.301, P<0.05), LES (t=14.440, P<0.05), Wheatley Stress Scale (t=21.779, P<0.05).2. Detection results of the genome methylation chip and verification in10discordant sib pairs with alcohol dependenceThere were, according to the absolute value of the DiffScore,865hypomethylated and716hypermethy-lated CG sites in the peripheral blood mononuclear cell DNA in AD patients. These sites were involved in827known genes. The most hypermethylated CG site is located in GABRP and the most hypomethylated CG site is located in the promoter of SSTR4. Linear regression analysis among10discordant sib pairs revealed the microarray methylation and pyrosequencing were well correlated. The R2of gene GABRP, ALDH1L2, GAD1and DBH were0.998,0.954,0.986, and0.996.3. Analysis of biological pathwaysThe differential methylation genes are part of13biological processes,19molecular functions and20cellular components in GO analysis. Biological regulation accounted for the largest proportion of biological processes. Thirty-three genes of130differential methylation genes used in KEGG analysis belonged to metabolic pathways.4. Validation of analysis of case and controlMethylation difference of gene SSTR4between cases and controls had statistical significance(t=14.723, P<0.05), and the gene SSTR4CG%of cases is lower than that of controls. Methylation difference of gene GABRP between cases and healthy controls had statistical significance(t=32.622, P<0.05), and the gene GABRP CG%of cases is higher than that of controls.Conclusions1. Compared to sib pairs and healthy controls, alcohol-dependent patients suffered from more negative events and higher stress level. Environment played an important part in the formation and maintenance of alcohol dependence.2. Alcohol dependence exist abnormal DNA methylation.865hypomethylated and716hypermethylated CG sites in the peripheral blood mononuclear cell DNA in AD patients, these sites were involved in827known genes. Biological regulation accounted for the largest proportion of biological processes. DNA methylation change may have effect on the pathological mechanism of alcohol use disorder.3. The most hypermethylated CG site is located in GABRP and the most hypomethylated CG site is located in the promoter of SSTR4. Gene SSTR4may be a new gene related to AD.