Screening Of Molecular Markers Of DNA Methylation In Cerebral Microbleeds Based On 850K Gene Chip

Objective:Illumina Infinium Methylation EPIC Bead Chip(850K chip)was used to screen for cerebral microbleeds(CMBs)-related candidate genes in cerebral small vessel disease(CSVD).To explore the possible mechanisms of action of genes with methylation differences and their possible relationships with clinical imaging phenotypes,and to find the molecular markers of DNA methylation associated with the pathogenesis of CMBs and affecting their development.Methods:The patients with CMBs in the Small cerebral vascular disease treated in the Affiliated Hospital of Qingdao University from January 2020 to December 2021 and the subjects who underwent physical examination at the same time were selected.(1)the primary screening population was 6 patients(4 males and 2 females)in the CMBs group(72.83 ±11.26 years old)and 6 patients(4 males and 2 females)in the control group(71.50 ±9.77 years old).(2)According to the same criteria,17 patients(10 males and 7 females)in the CMBs group(72.90±9.20 years old)and 21 patients in the control group(72.75 ±8.73 years old).The850 K DNA methylation chip was used to detect genome-wide DNA methylation sites for sites whose absolute value of methylation difference(meth.diff)is greater than 0.1 and P<0.05 significance for bioinformatics analysis(KEGG,GO analysis);Sites located in the 5 ′UTR,first exon,TSS200 or TSS1500 regions and with potential regulatory functions were selected to select candidate genes mechanically related to CMBs and differential in DNA methylation expression;The sample size was expanded to 17 patients in the CMBs group and21 controls,and Methyl Target analysis of the candidate genes was performed for validation;Analysis of the correlation between methylation of differentiated genes and imaging phenotypes in patients with CMBs.Results:(1)According to the results of 850 K DNA methylation chip detection,there were 1700 methylation differential sites,including 616 high methylation sites and 1084 low methylation sites,and 4858 functional items involved in biological processes,730 molecular functional items and 520 cellular functional items by GO analysis.KEGG Pathway analysis showed that there were 362 methylated differential genes involved in 291 pathways.(2)Ten candidate genes with methylation differences were screened out:ESYT3,FUCA1,PALLD,NEDD1,FOXN2,TMEM9 B,HDHD3,COX11,TANC1 and PRSS50.(3)Methyl Target validation analysis showed that the fragment and gene regional methylation levels of COX11,FUCA1 and TANC1 varied between CMBs and control groups and were statistically significant(P <0.05).Among them,COX11 and TANC1 were hypermethylated in the CMBs group relative to the control group,while FUCA1 was hypomethylated in the CMBs group relative to the control group.(4)The correlation analysis results suggest that there is no correlation between the methylation values of COX11,FUCA1 and TANC1 and the number of cerebral microbleeding involvement sites,the number of microbleeding at each site and the total number of microbleeding.(P>0.05)Conclusion:(1)The hypermethylation status of the target genes COX11 and TANC1,and the hypomethylation status of FUCA1,may be associated with the occurrence of CMBs through cell adhesion,intercell junctions,calcium binding,GTP enzyme mediation signals,and signaling pathways such as MAPK,calcium and RAS.(2)FUCA1 may promote the occurrence of CMBs,and COX11 and TANC1 may have protective effects in CMBs.