| BackgroundIn recent years,the incidence of chronic insomnia is on the rise and has become a global public health problem.Long-term chronic insomnia can lead to diabetes,hypertension and cardiovascular diseases.The pathogenesis of chronic insomnia is not clear,but epigenetics may play a role through environmental influence on genes.DNA methylation is one of the earliest epigenetic modifications and plays an important role in regulating gene expression and silencing.At present,some genes related to insomnia have been found,but their role in chronic insomnia remains to be explored.In order to provide new insights into the pathogenesis of chronic insomnia from the perspective of epigenetics,this study aims to analyze DNA methylation characteristics of chronic insomnia patients at the genome-wide level through whole blood samples,and to study insomnia related genes by using methylation level sequencing technology in the target region.Objectives1.Methylation EPIC Bead Chip（850K chip）was used to analyze the genome wide differences in DNA methylation levels between chronic insomnia patients and healthy controls,so as to screen out genes with significant changes in DNA methylation levels in chronic insomnia patients and conduct functional enrichment analysis.To elucidate DNA methylation patterns in chronic insomnia.2.Target Methyl Target was used to analyze the differences in DNA methylation levels of insomnia related genes BDNF,PAX8 and genes screened by 850 K chip between chronic insomnia patients and healthy controls,and to explore the methylation changes of related genes between chronic insomnia patients and healthy people.To search for molecular markers of DNA methylation associated with chronic insomnia.Methods1.According to strict inclusion and exclusion criteria,chronic insomnia patients and healthy volunteers hospitalized in the Second Affiliated Hospital of Xinxiang Medical College were recruited.Polysomnography（PSG）was used to analyze the objective sleep situation of patients with chronic insomnia,and the monitoring indexes included: total sleep time,sleep latency,REM sleep time and sleep efficiency.General relevant information was collected for all samples,and sleep quality was assessed with Pittsburgh Sleep Quality Index（PSQI）,Insomnia Severity Index（ISI）,The Hamilton Anxiety Scale（HAMA）and Hamilton Rating Scale for Depression（HAMD）were used to assess Anxiety and depressive symptoms.The 850 K chip group included: 8 patients with chronic insomnia and 8 healthy controls.The Methyl Target group consisted of 30 chronic insomnia patients and 30 healthy controls.2.Whole blood samples from 8 chronic insomnia patients and 8 healthy controls were collected.DNA was extracted and sulfite transformation was performed,and hybridization was performed by 850 K chip analysis.More than 850000 methylation sites were detected and data processing was performed to obtain differential genes.Gene Ontology Analysis（GO）and the Kyoto Encyclopedia of Genes and Genomes（KEGG）functional Analysis of different Genomes.Bumphunter algorithm was used to obtain differential methylation regions and related genes.3.Methyl Target was performed on 30 chronic insomnia patients and 30 healthy controls to analyze the methylation levels of sleep related genes BDNF and PAX8 as well as the selected LHX6 gene promoter region,and to clarify the differences in methylation levels of related genes between chronic insomnia patients and healthy people.4.Statistical software SPSS 22.0 was used for statistical analysis,and P < 0.05 was considered statistically significant.Results1.General data analysis,850 K chip group: There were no significant differences in gender,age,years of education,HAMA and HAMD between chronic insomnia group and healthy control group（P > 0.05）,while there were significant differences in PSQI and ISI（t=10.16,P < 0.05）and ISI（t=22.10,P < 0.05）.Methyl Target group:There were no statistically significant differences in gender,age and years of education between chronic insomnia group and healthy control group（P > 0.05）,but there were statistically significant differences in PSQI,ISI,HAMA and HAMD,PSQI（T =15.78,P < 0.05）,ISI（t=37.28,P < 0.05）,HAMA（t=3.22,P < 0.05）,HAMD（t=2.22,P < 0.05）.2.By comparing the genome-wide DNA methylation levels between the chronic insomnia group and the healthy control group,a total of 369 methylation sites were selected with different methylation levels（P < 0.05 and absolute value of delta Beta >0.1 were the differential methylation sites）,among which 153 sites were up-regulated in the chronic insomnia group compared with the healthy control group.There were216 down-regulated methylation sites,and all differential methylation sites covered175 genes.GO analysis and KEGG pathway analysis revealed that the biological functions of the differential genes were mainly focused on nervous system development,vascular smooth muscle contraction,cell adhesion and calcium channels.Analysis of differential methylation regions revealed 23 differential methylation regions covering 29 related genes,among which LHX6 gene was most associated with insomnia and the difference was statistically significant（P < 0.001）.3.Methyl Target was used to analyze the methylation levels in promoter regions of BDNF,PAX8 and LHX6 genes,and there was no significant difference in methylation levels between chronic insomnia group and healthy control group（P >0.05）.Conclusions1.Genome-wide methylation levels in patients with chronic insomnia were significantly changed compared with healthy controls,which may be one of the important epigenetic mechanisms leading to chronic insomnia.2.No significant changes in methylation levels in promoter regions of relat ed genes BDNF,PAX8 and LHX6 in patients with chronic insomnia. |
PDF Full Text Request