DNA Methylation Research In Amyotrophic Lateral Sclerosi

Part 1 Characteristics of DNA methylation in amyotrophic lateral sclerosisObjective:This study explored the role of DNA methylation in the pathogenesis of amyotrophic lateral sclerosis(ALS)by comparing DNA methylation profiles in whole-blood samples taken from patients with sporadic ALS with those of healthy controls(HC).Methods:This study included 32 patients with sporadic ALS who received clinically definite,clinically probable,or clinically probable laboratory-supported diagnoses in the Department of Neurology of our hospital from December 2020 to May 2021.Healthy family members accompanying the patients during the same period were enrolled as the control group.Demographic data and clinical data were collected from the two groups.The clinical data included the diagnostic category,age of onset,site of onset,disease duration,Revised ALS Functional Rating Scale(ALSFRS-R)score,King’s stage,andΔALSFRS-R,which is a variable that measures disease progression at an early stage.and Whole-blood samples were collected,genomic DNA was extracted from the samples,subjected to bisulfite conversion,and amplified.The DNA was then fragmented using a random endonuclease,hybridized with Infinium MethylationEPIC BeadChip,and labeled according to the manufacturer’s protocol.The chip was stained for imaging,scanned,and relevant bioinformatics analysis was performed on the resulting data.Results:Thirty-four differentially methylated positions(DMPs)were identified in the ALS group;5 were hypermethylated and 29 were hypomethylated.These DMPs mapped to 13 genes,including 2 hypermethylated genes(ATAD3B and BLK)and 11 hypomethylated genes(DDO,IQCE,ABCB1,DNAH9,FIGN,NRP1,TMEM87B,CCSAP,ST6GALNAC5,MYOM2 and RUSC1-AS1).Twelve differentially methylated regions(DMRs)were identified in the ALS group;these mapped to 12 genes(NWD1,LDHD,C1S,IQCE,TNF,PDE1C,LGALS1,CSNK1E,LRRC23,ENO2,ELOVL2 and ELOVL2-AS1).According to data in the Kyoto Encyclopedia of Genes and Genomes database,DNAH9 and TNF are involved in the ALS pathway.Correlation analysis between clinical features and DNA methylation profiles indicated that the methylation levels of ELOVL2 and ARID1B were positively associated with,respectively,age of onset(r=0.86,adjusted P=0.001)and disease duration(r=0.83,adjusted P=0.01).However,no methylation sites were correlated with the ALSFRS-R score orΔALSFRS-R.There was no significant difference between the two groups with respect to the total DNA methylation level(P>0.05).Conclusions:The aberrant methylation in DMP-and DMR-related genes suggests that epigenetic alterations,such as the hypomethylation of DNAH9 and TNF,play important roles in the etiology of ALS.This provides a new insight and epigenetic perspective to the pathogenesis of ALS.In future studies,more attention should be focused on DMPand DMR-related genes,which should be evaluated in cohorts with larger sample sizes to provide a basis for developing new therapeutic targets.Part 2 Association between transcriptome characteristics and DNA methylation in amyotrophic lateral sclerosisObjective:To identify differentially expressed genes(DEGs)between patients with amyotrophic lateral sclerosis(ALS)and healthy controls(HC),this study measured mRNA expression levels using transcriptome sequencing of peripheral blood samples.Combined with the results of DNA methylation studies(Part 1 of the study),we explored the relationship between DEGs and differentially methylated genes(DMGs)and to establish criteria for identifying genes involved in ALS.Methods:The analysis included 12 patients with ALS and 12 HCs from Part 1 of the study.A PAXgene Blood RNA Tube was used to collect the peripheral blood of the participants,and extract total RNA for library preparation and transcriptome sequencing.The resulting sequence data was then aligned to the human genome for mapping.The mRNA expression levels were quantified,and the differential expression was analyzed using a FoldChange>1.2 and P-value<0.05 as the screening criteria.Enrichment analysis of DEGs and gene set enrichment analysis were performed,and the DEGs detected in this part of the study were compared with the DMGs identified in Part 1 of this study to identify target genes.The quantitative real-time polymerase chain reaction(qRT-PCR)was used to measure the mRNA expression levels of the genes.Results:There were 1001 DEGs identified in the peripheral blood samples of patients with ALS,including 629 upregulated genes and 372 downregulated genes.These genes are enriched in several ALS-related pathways such as the oxidative stress pathway,and in ribosomal and mitochondrial functions.The intersecting genes from the DEG and DMG analyses were LGALS1,TNF,LDHD and RUSC1-AS1.The mRNA expression level of LGALS1 was significantly higher in the ALS group than in the HC group,as measured by qRT-PCR.Conclusions:Transcriptome sequencing identified an association between mRNA expression levels and DNA methylation in peripheral blood samples from patients with ALS.The intersecting gene LGALS is a candidate for further in-depth study.Part 3 Association between DNA methylation of the LGALS1 promoter and amyotrophic lateral sclerosisObjective:To explore the relationship between methylation of the LGALS1 promoter and amyotrophic lateral sclerosis(ALS),this study measured methylation of the LGALS1 promoter in the peripheral blood samples taken from patients with ALS and healthy controls(HC).To explore the effect of LGALS1 promoter methylation on galectin-1 expression in the peripheral blood,we compared the galectin-1 levels between the two groups.Methods:There were 45 patients with ALS and 32 HCs included in this study.Peripheral blood samples were collected at the time of enrollment.Twelve CpG sites in one CpG island in the LGALS1 promoter region were identified using the bisulfite sequencing PCR method,and the methylation level at each CpG site was quantified.The amount of galectin-1 in the peripheral blood was measured using an enzyme-linked immunosorbent assay(ELISA).The methylation levels at the CpG sites were compared between the ALS and HC groups,and the relationship between these methylation levels and the clinical characteristics of patients with ALS were analyzed.The galectin-1 levels in the peripheral blood from the ALS and HC groups were compared,and the relationship between the LGALS1 methylation state and its protein level was analyzed.Results:The CpG island in the LGALS1 promoter had significantly lower methylation levels of CpGl,CpG3,CpG4,CpG5,CpG7,CpG8,CpG9,CpG11 and CpG12 in the ALS group compared with the HC group(P<0.001,P<0.001,P=0.001,P=0.004,P<0.001,P<0.001,P<0.001,P<0.001,P<0.001,respectively).The total methylation level of the 12 CpG sites was significantly lower(P<0.001),and the galectin-1 levels were significantly higher(P=0.007),in the ALS group compared to the HC group.In both groups,there was a significant reverse correlation between the LGALS1 promoter methylation level and the galectin-1 level(r=-0.571,P<0.001).The methylation levels at the CpC3,CpG4,CpG7,CpG11 and 12 total CpG sites were significantly lower in the older-onset age group(>45 years)than in the younger-onset age group(≤ 45 years)(P=0.006,P=0.041,P=0.003,P=0.013,P<0.001,respectively).A significant inverse relationship was observed between the age of onset and the DNA methylation levels at the CpG1,CpG7,CpG11,CpG12 and 12 total CpG sites(P=0.020,P<0.001,P=0.025,P=0.044,P=0.001,respectively).Conclusions:In patients with ALS,the LGALS1 promoter was hypomethylated and its protein level was elevated.DNA methylation levels were inversely associated with the age of onset.This study establishes an association between LGALS1 promoter methylation and ALS and provides a basis for further mechanistic research.