Analysis Of Genome-wide Methylation Differences And Candidate Gene Research In Amyotrophic Lateral Sclerosis

Background and objectives:Amyotrophic lateral sclerosis(ALS)is a progressive and complex neurodegenerative disease,However,the exact mechanism of pathogenesis remains unclear.Both genetic and environmental factors play an important role in the occurrence and development of ALS,but the common mechanism is still unclear,and epigenetics might be the bridge for the interaction between the two,and the genetic research is expected to be a breakthrough in the diagnosis and treatment of ALS.Methods:1.Venous blood was collected from ALS patients and normal controls,and genome-wide DNA was extracted for the detection of genome-wide DNA methylation differences,and the differential methylation sites were analyzed.The differential methylation regions were analyzed according to gene annotation classification and Cp G island annotation classification,and the predicted results were analyzed by GO pathway enrichment.2.PCSK9 was selected for further verification and functional analysis.UCSC search and Li Lab online prediction were performed to corroborate whether there is methylation site in PCSK9 gene promoter region.Methylation of PCSK9 gene was detected by methylation specific PCR(MSP)MSP.PCSK9 m RNA and protein expression levels in peripheral blood samples of ALS patients and normal controls were detected by q RT-PCR and ELISA.3.ALS cell models were established to explore the effect of PCSK9 methylation on ALS cell models in vitro.Methylation level of PCSK9 in model group and control group was detected by MSP.The PCSK9 expression at m RNA and protein levels in model group and control group was detected by q RT-PCR and western blot.The expression of methylated transferases DNMT1 and DNMT3 A in model group and control group was also detected by western blot analysis.ALS cells were administrated with methylated transferase inhibitor 5-Aza-Cd R,and the effects on apoptosis and apoptosis-related protein expression as well as the PCSK9 expression were detected.PCSK9 overexpression vector or PCSK9 si RNA and their controls were transfected into the ALS model group cells to detect the effects on apoptosis and expression of apoptosis-related proteins.Result:1.By the detection and analysis of methylation differences in genome-wide DNA,it was found that a total of 76 genes in the experimental group and the control group showed methylation difference changes(|beta.difference| > 0.8).There were 44 genes with elevated methylation level and 32 genes with down-regulated methylation level.The differential methylation regions were analyzed according to gene annotation classification and Cp G island annotation classification,and the results showed that there were differentiation sites except SSHELF areas.The GO enrichment analysis results showed that there were several DMR-related genes that were significantly correlated with the functional regions in the known GO database,which involved cell components,biological processes and molecular functions.2.According to UCSC search and Li Lab online prediction,methylation site was found ini PCSK9 promoter.MSP method was used to detect the methylation of PCSK9 gene in 20 ALS samples and 10 control samples,and the results showed that the promoter region of PCSK9 gene was partially methylated in ALS samples.q RTPCR and ELISA results revealed that compared with the control group,PCSK9 expression at m RNA and protein levels in ALS group were significantly downregulated.3.PCSK9 methylation level was detected by MSP,and no statistically significant difference was observed in PCSK9 methylation level in the control group,and there was a significantly increased methylation level in ALS cells.The results of q RT-PCR and western blot showed that the PCSK9 expression at m RNA and protein levels in ALS cells were significantly reduced relative to the control group.Western blot results revealed that compared with the control group,the expression levels of DNMT1 and DNMT3 A proteins in ALS cells were significantly increased.5-AzaCd R significantly inhibited ALS cells apoptosis and the expression of apoptosisrelated proteins,and significantly increased of PCSK9 expression at the m RNA and protein level in ALS cells.Conclusion:In this study,genome-wide DNA methylation chip technology was applied to comprehensively explore the genome-wide methylation characteristics of ALS.MSP was used to detect the difference in the PCSK9 methylation level between ALS group and the control group,and it was confirmed that there was abnormal methylation in the promoter region of PCSK9 gene in the serum of ALS patients.PCSK9 methylation promoted apoptosis of ALS cells through ALS cell model experiment.The findings in this study are helpful to provide new ideas and methods for the study of the pathogenesis of ALS.