Genome Wide Methylation Analysis Of Panic Disorder And The Relationship Between DNA Methylation And Expression Of Differential Genes And Early Efficacy Of Antidepressants

Objective:Panic disorder,also known as acute anxiety attack,is a common type of anxiety disorder,with a high incidence and a large disease burden.However,its pathogenesis have not been fully elucidated.Studies have found that PD is related to genetic factors,but there is a problem of poor reproducibility in molecular genetic studies.However,the repeatability is quite low in molecular genetic studies.As more and more studies focused on epigenetic mechanism and its expression regulation,researchers have found that epigenetic,especially DNA methylation,plays an important role in the occurrence of adult psychiatric disorders and the efficacy of drugs influenced by early negative experiences.Therefore,based on the genome-wide methylation 850 K microarray and mRNA sequencing,this study is aimed to investigate differentially methylated DNA and expressed genes between panic disorder patients and healthy controls,and to find whether DNA methylation and expression can be used as the disease biomarkers.Meanwhile,escitalopram was used as an effective antidepressant and anxiety drug to analyze the relationship between DNA methylation and expression level of candidate genes and the early efficacy of escitalopram.This study will help to reveal the epigenetic modification related genes of PD,and provide valuable clues and basis for the discovery of epigenetic markers for disease diagnosis and the prediction of early efficacy of antidepressants in the treatment of PD.Materials and Methods:In this study,the peripheral blood samples of 30 patients with panic disorder and 30 matched healthy controls were selected.The whole genome DNA methylation sites were detected by 850 k methylation chip,and the mRNA expression was sequenced.Then,gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)were used for gene function enrichment analysis,combined with the methylation microarray and expression results to screen out the candidate genes.Secondly,101 PD patients and 107 healthy controls were selected to validate the methylation and expression level of candidate genes in peripheral blood by pyrosequencing and fluorescent quantitative PCR.To analyze whether DNA methylation and its expression of candidate genes can be used as biomarkers for disease diagnosis.Finally,the efficacy of escitalopram was evaluated by PDSS after 4 weeks’ treatment,and the relationship between methylation and expression of candidate genes and early efficacy of escitalopram was analyzed.Results:There were 3613 different methylation sites detected by 850 k methylation chip(Delta β ≥ 0.06,adj.P < 0.05).There were 1938 genes corresponding to different methylation sites,including 1758 hypermethylated genes and 150 hypomethylated genes.The coexistence of both of the hypermethylation and hypomethylation sites were found in 30 genes.523 GO items were found statistically significant(adj.P <0.05),such as signal complex assembly.And FC γ R-mediated phagocytosis,platelet activation,T cell receptor signaling pathway,sphingolipid signaling pathway and endocytosis were enriched in KEGG pathways(adj.P < 0.05).The results of mRNA sequencing showed that there were 212 differentially expressed genes were screened(FC≥2 or FC≤0.5,adj.P < 0.05),including 40up-regulated genes and 172 down regulated genes.Go function enrichment analysis showed that 58 GO items were statistically significant(adj.P < 0.05),such as speroxidase activity.KEGG enrichment analysis showed that 43 pathways were enriched(adj.P < 0.05).The results showed that CBL,MPZL3,NR2C2,RNF24,STK4 and RAB31 genes were overlapped based on the cross-joint analysis for differentially methylated and expressed genes,and the methylation level of the gene locus was correlated with mRNA expression(adj.P < 0.05).The methylation and expression level of SLC23A2,CHPT1,CBL,NR2C2 and RNF24 genes were validated.Pyrosulfate sequencing results showed that the DNA methylation level of SLC23A2 gene loci(cg11294350)in PD patients was lower than that in normal controls(adj.P < 0.05).The results remained significant after controlling for covariates such as age,sex and so on.Also,the results remained significant after FDR correction.ROC curve analysis showed that the sensitivity was64.4%,specificity was 78.5% and AUC value of SLC23A2 gene loci(cg11294350)was 0.729(95% CI: 0.661-0.798;P < 0.001).There was no significant difference in DNA methylation level of other candidate loci between the two groups(P > 0.05).q PCR results showed that the expression levels of CBL,RNF24 and SLC23A2 genes in PD patients were lower than those in normal control group,while the expression levels of CHPT1 and NR2C2 genes in PD patients were higher than those in normal control group(P < 0.05).The results remained significant after controlling for covariates such as age,sex and so on.Also,the results remained significant after FDR correction.ROC curve analysis showed that the AUC value of CHPT1 gene expression was 0.759(95% CI: 0.690-0.828;P < 0.001),the sensitivity was 67.7%,and the specificity was 79.4%.The spearman correlation analysis has shown that the panic disorder severity scale score of PD patients was negatively associated with the expression of SLC23A2.The results remained significant after FDR correction.All PD patients were treated with escitalopram for four weeks.The efficacy was determined by the scale of panic disorder severity.42 patients in the effective group and 59 in the ineffective group were found.The results showed that there was no significant difference in the methylation levels of CBL,CHPT1,NR2C2,RNF24 and SLC23A2 gene loci between the effective group and the ineffective group(P >0.05).However,the CBL gene expression level of the effective group was higher than that of the ineffective group,and the difference was statistically significant(adj.P < 0.05).The results remained significant after controlling for covariates such as age,sex and so on.The sensitivity,specificity and AUC for the CBL gene expression level to predicte the efficacy of escitalopram after four weeks’ treatment was 42.1%,85.7% and 0.656(95% CI: 0.549-0.763;P < 0.01)respectively after the ROC curve analysis.Conclusion:There are differences in genome-wide DNA methylation and mRNA expression between PD patients and healthy controls.Some different DNA methylation sites and mRNA expression may be used as important molecular markers for PD diagnosis or early drug efficacy.The hypomethylation of SLC23A2 gene loci(cg11294350)and the high expression of CHPT1 gene may be used as biomarkers for the diagnosis of PD.The changes in expression level of CBL gene may predict the early therapeutic effect of antidepressant in PD patients but need larger sample to validate the results.