The Screening For Molecular Markers Of DNA Methylation In Cerebral Amyloid Angiopathy Based On Methylation Chip And Sequencing Technology

Purpose:The cerebral amyloid angiopathy(CAA)is the main cause of spontaneous cerebral hemorrhage and age-related cognitive impairment in the elderly.The CAA is caused by the deposition of beta-amyloid protein(Aβ)in the cerebrovascular.The pathogenesis of CAA has not been fully clarified so far and the neurovascular and genetic factors are important risk factors for CAA.The neurovascular unit(NVU)is an important functional domain in the brain,which plays a crucial role in the accumulation and clearance of Aβ.The genetic factors such as β-amyloid precursor protein(APP)and apolipoprotein E(APOE)motivate the occurrence and development of CAA by influencing the conformation,fiber formation,toxicity and deposition sites of Aβ.Furthermore,the CAA is a complex age-related disease and the epigenetic regulation may play an important role in the pathogenesis of CAA.The epigenetic modification regulates the expression of genes without altering the DNA sequence,affecting and regulating the function and characteristics of genes.However,whether the epigenetic modification plays an important role in the occurrence,development and prognosis of CAA has not been systematically studied.Therefore,this study analyzed the differences in DNA methylation levels in peripheral blood of CAA patients and controls and screened out the differential genes via the Illumina Infinium Methylation EPIC850 K Bead Chip and independent sample verification.This study further defined the role of DNA methylation in the occurrence and development of CAA,explored the possible pathological mechanism of differential methylated genes and providied new ideas and theoretical basis for the early diagnosis and treatment of CAA.Methods:(1)The blood samples of CAA patients were collected according to Boston 1.5diagnostic criteria from September 2019 to December 2021 in the affiliated hospital of Qingdao University.At the same time,we also collected the blood samples of controls according to age and sex of the case group.Then,the DNA was extracted from peripheral blood samples.The genomic DNA samples of 4 CAA patients and 4 controls were hybridized and analyzed by Illumina Infinium Methylation EPIC 850 K Bead Chip,and then the differential genes were obtained via data processing.Furthermore,we performed the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analysis in the differential genes.(2)Seven candidate genes(PRSS50,RUFY1,HTRA4,CIZ1,CCDC3,NFKB2 and LRP1)were screened out for verification according to GO and KEGG enrichment analysis results and related literatures.The annotated Cp G sites or regions of PRSS50,RUFY1,HTRA4,CCDC3,CIZ1,NFKB2 and LRP1 were analyzed by Methyl Target DNA methylation Target sequencing in 32 independent samples(16 CAA patients and 16 controls)to compare the differences in methylation levels between CAA patients and controls.The clinical and imaging characteristics of CAA patients were summarized via reviewing the medical history and imaging examination of 16 patients with CAA carefully.The statistical analysis were peformed on the levels of methylation of differential methylated genes and clinical phenotypes of CAA patients.Results:(1)A total of 7378 methylation differential sites were screened by comparing the genome-wide methylation levels between the CAA group and the control group(P <0.05,the absolute value of meth.diff > 0.1),which including 1169 hypermethylated sites involving 665 genes,and 6029 hypomethylated sites involving 2926 genes.In addition,45 methylation differential regions were identified(P<0.05),involving 42 genes.The results of GO and KEGG enrichment analysis revealed that the biological functions of the differential genes mainly focused on small GTPase-mediated signal transduction,hormone and steroid hormone-mediated signal transduction,central nervous system development,lipid metabolism,calcium signaling pathway and so on.The changes in DNA methylation levels of differential genes of functional pathways may be one of the causes of CAA.(2)The methylation levels of 176 Cp G sites and 17 fragments of seven screened genes(PRSS50,RUFY1,HTRA4,CIZ1,CCDC3,NFKB2 and LRP1)were detected for the independent sample validation.The results showed that there were significant differences in methylation levels of several sites in the target region of HTRA4 gene between CAA group and control group(P<0.05).The methylation level of HTRA4 gene was decreased in CAA patients compared with controls.In addition,4 fragments(HTRA4＿3,HTRA4＿4,HTRA4＿5and HTRA4＿6)and 24 Cp G sites of HTRA4 gene were hypomethylated.(3)It was found that the CAA patients mainly suffered from occipital lobe hemorrhage and accompanied by white matter lesions,enlarged perivascular space,lacunar infarction or subarachnoid hemorrhage via analyzing the clinical data of CAA patients.The statistical analysis was conducted on the relationship between HTRA4 methylation level and clinical phenotype of CAA patients.The results indicated that there was no difference in HTRA4 methylation levels of CAA patients with or without cerebral hemorrhage and different position of cerebral hemorrhage(frontal lobe,temporal lobe,parietal lobe and occipital lobe)(P<0.05).In addition,the levels of HTRA4 methylation has no correlation with the amount of bleeding in CAA patients with hemorrhage(P<0.05).Conclusions:(1)The genome-wide methylation levels of CAA patients were significantly different from that of controls,which may be one of the important epigenetic causes for the occurrence and development of CAA.(2)The methylation levels of HTRA4 were significantly decreased in CAA patients,indicating that HTRA4 may be associated with the pathogenesis of CAA.