Gene Identification Of Alzheimer’s Disease And The Study Of Early Blood Biomarkers

Multiple studies have shown that Alzheimer’s disease(AD)is the result of the combined effects of multiple complex etiologies.Among them,more and more attention has been paid to the study of gene therapy,especially the genome-wide association study(GWAS).In this study,the AD gene pathway,core associated genes,susceptible cell types and blood biomarkers obtained by bioinformatics technology and clinical experimental research were comprehensively used to provide new ideas for the pathogenesis,early diagnosis and clinical drug development of AD.This study was divided into the following three parts:Part 1 Alzheimer’s disease-associated gene pathway and cell type Part 11 expression enrichment analysisObjective:Using bioinformatics methods such as FUMA,MAGMA and DAVID to perform gene annotation analysis on Alzheimer’s disease GWAS data,to find susceptibility genes,and to perform pathway enrichment analysis on associated genes in order to find out the biological pathways that may play a role on AD,and then to explore the brain cell types affected by related genes according to the different expression analysis of cell types.Methods:The study explored AD genes and pathways by using GWAS summary statistics for AD from the Center for Genomics and Cognitive Research.First,the polymorphic single nucleotides were mapped to genes by magma,and the Cell type tool in the FUMA database was used to determine the susceptible cell type and to analyze the data.Then the DAVID method was used to conduct pathway enrichment analysis of Alzheimer’s susceptibility genes,associated susceptibility genes with biological annotation terms,and found the most significantly enriched susceptibility genes.Results:Twenty susceptibility genes were identified by repeated analysis using the GWAS statistics of AD.Combined with comprehensive analysis of pathway enrichment,166 AD-related highly expressed genes and 24 enriched pathways were finally obtained,and the genes were highly enriched in microglia and GABA neurons.Conclusion:A total of 20 susceptibility genes that are highly associated with AD were obtained in this study;166 susceptibility genes that were highly associated with AD were found after extraction and analysis of GWAS data,and 24 pathways were obtained according to the analysis results of DAVID genes;microglia Dysregulated gene expression in cells and GABA neurons is highly associated with AD pathogenesis.Part 2 Identification of Alzheimer’s Disease Hub Gene and Part 211 Characterization of Immune Cell InfiltrationObjective:A large amount of genetic data and clinical information,most of which is openly available,had been accumulated through multiple high-throughput sequencing studies and advances in genomics.This part of our study,combined with the research in the first part,would give us a clearer direction for gene research,and have more theoretical basis to use bioinformatics technology to find core genes that are highly related to AD.In this part of the experimental study,high-throughput sequencing,GO analysis,KEGG analysis,and protein-protein interaction(PPI)network analysis were used to determine differential gene expression,and to determine Hub genes and their roles.Meanwhile,we analyzed the infiltration characteristics of inflammatory cells and the association analysis with core associated genes in all data analysis processes involved,which is a major innovation of this part of the study.Methods:This section was based on the Alzheimer’s transcriptome data downloaded from the GEO database.We performed differential analysis of gene expression in AD and normal human brains.After obtaining the differentially expressed genes,the cluster Profiler package was used to conduct pathway analysis.This analysis covered many common metabolic and biological pathways such as KEGG,biological process(BP),cellular component(CC),and molecular function(MF).We aimed to elucidate the key biological functions and the key pathways of the differentially expressed genes in AD.Furthermore,PPI information was obtained through the STRING database and HUB gene were identified.Finally,the AD immune cell infiltration analysis was performed by preprocess Core,the level of immune cell infiltration in each sample was obtained,and the correlation between gene expression and immune cell fraction was determined.Results:A total of 119 differentially expressed genes(DEGs)were obtained in this part of the study,of which 30 genes were significantly up-regulated and 89 genes were down-regulated.The biological process(BP)of DEG is mainly focused on the positive regulation of chemical synaptic transmission,nervous system development,ion transport and neuronal projection development.DEG cellular structure(CC)is closely related to the Golgi membrane,cell junctions and neuronal cell bodies;DEG molecular function(MF)is related to calmodulin binding,extracellular ligand-gated channel activity and GABA;searching the KEGG database found DEG Mainly matched with retrograde endocannabinoid signaling,morphine addiction,and GABAergic synapses.The top 10 network hub genes obtained by PPI network analysis were SLC32A1,STMN2,GFAP,GABRA1,SST,GABRG2,SYN2,GNG3,PVALB and SH3GL2.Compositional analysis of infiltrating immune cells revealed differences in the overall composition of immune cells between AD and control groups,and the expression of multiple genes correlated with immune cell composition.GFAP gene is associated with immune infiltrating cells.Conclusions:This study identified 10 network hub genes(SLC32A1,STMN2,GFAP,GABRA1,SST,GABRG2,SYN2,GNG3,PVALB and SH3GL2).GFAP is associated with certain immune cells.These genes may be candidate disease diagnostic markers and possible therapeutic targets.Part 3 Study on early blood biomarkers of Alzheimer’s diseaseObjective:Through the first two parts of the study,we found the infiltration characteristics of inflammatory cells.According to the existing literature reports,the GSN,the MMP3,and the Aβ in the plasma of Alzheimer’s disease patients had a complex relationship with inflammatory factors,which had obvious changes between the groups.Mild cognitive impairment(MCI)is a state of cognitive impairment between conventional aging and dementia,and can be understood as a certain state in the pre-dementia stage.According to the very similar pathophysiological processes of AD and MCI,specific biological indicators in the plasma of MCI patients,such as some obtained inflammatory indicators,are used to compare with the enrolled control patients to identify and prevent early cognitive impairment.Methods:To study the outpatient and inpatient population of the Second Affiliated Hospital of Shandong First Medical University from May 2019 to December 2020.According to the results of cognitive function(diagnostic criteria for Alzheimer’s disease and mild cognitive impairment),the patients were divided into NC group,MCI group,AD group.The general information and required examination results of the patients for the research samples were collected.Fasting blood was collected in the standard procedure and separated for testing.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of GSN,Aβ40,Aβ42 and MMP3 in plasma.We uesd SPSS25.0statistical software for statistical analysis.Results:This part of the study collected a total of 180 subjects who met the inclusion and exclusion criteria,including 63 subjects in the NC group,41 subjects in the MCI group,and 76 subjects in the AD group.(1)Comparison of basic data: There were no statistically significant differences in gender,marriage,and place of residence among the NC group,MCI group,and AD group;compared with the NC group,the AD group had a statistically significant difference in age;the MCI group had a statistically significant difference in age.There was a statistically significant difference between the educational levels;the AD group had no statistical difference between the educational levels.(2)There were no significant differences in blood lipids,blood glucose,and renal function among the three groups;(3)Blood routine:red blood cells,hemoglobin,and platelets had no significant differences among the three groups;compared with the NC group: white blood cells The difference of the content in MCI group was statistically significant;in AD group,the difference was not statistically significant and the change of white blood cell count was unstable among the three groups.(4)Plasma protein levels:(1)GSN: there were statistical differences among the three groups;compared with the NC group,there was no significant difference in the GSN content in the MCI group;there was a statistically significant difference in the GSN content in the AD group;compared with the MCI group: the GSN concentration in the AD group The difference was statistically significant and the GSN concentration showed a downward trend in the NC,MCI and AD groups.(2)Aβ40: There was no statistical difference between the three groups;(3)Aβ42: There was a statistical difference between the three groups;compared with the NC group,there was no significant difference in the Aβ42content between the MCI group and AD group;compared with the MCI group:the difference of Aβ42 content in AD group was statistically significant,and the Aβ42 concentration in the three groups showed a trend of first decreasing and then increasing,and the change trend was unstable.(4)MMP3: There was no statistical difference among the three groups.Conclusion:(1)Plasma GSN can be used as a biological marker for AD diagnosis,which might have little significance for the diagnosis of MCI.(2)Plasma MMP3 is still controversial as a biomarker for the diagnosis of AD.(3)Plasma GSN content may be related to cognitive level.The higher the GSN content has,the higher the cognitive level is.(4)The contents of Aβ40 and Aβ42 in plasma have little significance for the diagnosis of MCI and AD.(5)The influence of blood lipids,blood sugar,blood routine and renal function on cognitive function have not been determined.