Research On DNA Methylation And Immune Abnormalities Of Autism Spectrum Disorder

Autism Spectrum Disorder(ASD)is a kind of neurodevelopmental disorder.In recent years,the incidence of ASD has been increasing globally,while still lacking effective treatment which causes a heavy burden on families and society.The research on ASD has been increasing.However,its pathogenesis is still unclear.It is suggested that epigenetic mechanism and abnormal immunity are associated with the pathogenesis of ASD.DNA methylation is a major epigenetic modality that affects the stability of gene structure and gene expression,and is associated with some disease processes.Currently,DNA methylation is believed to be involved in the occurrence and development of ASD,and differentially methylated genes may affect important biological processes and signaling pathways through abnormal gene expression,thereby promoting the occurrence of ASD.Therefore,studying differentially methylated genes of ASD from the perspective of epigenetics and conducting functional analysis will help to better understand the pathogenesis of ASD from the genetic level.Abnormal immune function and inflammatory response are related to the pathogenesis of ASD.Lymphocyte subsets,immunoglobulin,complements,and cytokines are important indicators to reflect the immune function of the body,which jointly maintain the balance of immune and inflammatory processes of the body.Once this balance is disturbed,neurological pathological processes will be caused.Understanding the changes of immune indicators in ASD will provide new ideas for studying the pathogenesis of ASD and seeking new clinical immune detection indicators.This study was divided into three parts.The first part was an integrated analysis of ASD methylation microarray data from the Gene Expression Omnibus database(GEO)to explore the differentially methylated genes associated with ASD.To study the pathogenesis of ASD at the epigenetic level,Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were also conducted to detect whether abnormal DNA methylation was involved in immune and inflammatory processes.In the second part,the m RNA expressions of ASD-related differentially methylated genes were studied.The PPI network of core differentially methylated genes was established to explore whether the abnormal expressions of differentially methylated genes were related to immune and inflammatory processes.In the third part,the immune function-related indicators of ASD were analyzed to understand the systemic immune status and inflammatory factor levels of children with ASD.The combination of clinical phenotypes and epigenetics in this study will provide new ideas for studying the pathogenesis and intervention methods of ASD.Part One Research on DNA methylation of ASDObjective: This part aimed to investigate differentially methylated genes and related signaling pathways in ASD,to provide evidence for the etiologic research and early detection of ASD.Methods: Methylated microarray data GSE131706(17 normal controls and 17 patients)and GSE80017(9 normal controls and 9 patients)from brain tissue were downloaded from the GEO database.Rn Beads 2.0 software was applied for integrated analysis of DNA methylation data and identification of differentially methylated sites in the case group and control group(|Δβ| > 0.1,P<0.05).GO enrichment analysis and KEGG pathway enrichment analysis were performed for differentially methylated genes corresponding to the differentially methylated sites using David 6.8 online software.GSE109905(31 normal controls and 38 patients from peripheral blood samples)was downloaded from the GEO database and used to further validate the methylation status of differentially methylated genes,and ROC analysis was performed using the p ROC package in R language.GSE53162 and GSE53924(brain tissue samples)from the GEO database were used to verify the expressions of some differentially methylated genes,and ROC analysis was performed to study whether differentially methylated genes have diagnostic value.Results:1.A total of 136 differentially methylated sites were identified in ASD,including 64 hypermethylated sites and 72 hypomethylated sites.Differentially methylated sites were mainly distributed in the open sea of the Cp G region and the intergenic region of the genome.2.According to the 450 K official annotation,74 differentially methylated genes corresponding to differentially methylated sites were obtained,including 38 hypermethylated genes and 36 hypomethylated genes.GO and KEGG enrichment results showed that the functions of these differentially methylated genes were mainly enriched in the biological processes and pathways of antigen processing and presentation.Among the major differentially methylated genes,HLA-DRB1,HLA-DRB5,and HLA-DQA1 were involved in antigen processing and presentation,cell adhesion molecules,Epstein-Barr virus infection,interferon-γ mediated signaling,and MHC II receptor activity.3.The validation results of GSE109905 data set showed that the methylation status of 15 differentially methylated genes was in line with the integrated analysis.These 15 genes were FMN2,ACTR3,BAZ2 B,ABI3BP,HTR4,DUSP22,RNF39,HLA-DRB5,HLA-DQA1,TSPAN18,NTM,DGKH,IL-16,CALCOCO2,COL5A3.ROC curve analysis showed that FMN2,BAZ2 B,and HLA-DRB5(AUC > 0.6)had potential diagnostic values for ASD.The validation results of GSE53162 data set and GSE53924 data set showed that the expressions of ABI3 BP,NTM,and HLA-DRB5 were upregulated,and the expression of HLA-DQA1 was downregulated in ASD patients.CALCOCO2,DUSP22,BAZ2 B and HLA-DRB5(AUC > 0.6)had potential diagnostic value for ASD.Summary:1.Abnormal DNA methylation exists in ASD patients,and some differentially methylated genes have potential diagnostic values for ASD,suggesting that differentially methylated genes are involved in the occurrence of ASD.2.Differentially methylated genes are mainly enriched in the process of antigen processing and presentation,and the main differentially methylated genes involved are HLA genes.These biological processes and genes are mainly involved in the immune function and process of the body,suggesting that the immune process may play an important role in the pathogenesis of ASD.Part Two Research on m RNA expressions of differentially methylated genes in ASDObjective: This part aimed to identify the differentially methylated genes closely related to ASD and analyze the expressions of these genes at the m RNA level.To explore the pathogenesis of ASD,we also established the PPI network of differentially methylated genes.Methods: Combined with the results of part one,online Pubmed literature retrieval was conducted to find genes closely related to the pathogenesis of ASD or mental disorders as the target genes.11 children with ASD in Hebei Children’s Hospital from January to June 2020 were included,7healthy children who underwent physical examination in the hospital during the same period were included as the normal control group.The two groups were matched in terms of gender and age.RT-PCR was used to detect the m RNA expressions of target genes in the whole blood of the ASD group and the normal control group.SPSS software was used to analyze and compare the expression differences of the target genes between the two groups.With m RNA expression level of the target genes as test variable and ASD as status variable,the ROC curves were obtained and the diagnostic values were evaluated.The PPI network of 15 differentially methylated genes was established by using the online String database.Then we imported the results into Cytoscape software to filter core differentially methylated genes by using the Cyto Hubba plug-in.Results:1.Ten differentially methylated genes were closely associated with ASD,including FMN2,ACTR3,BAZ2 B,HLA-DRB1,HLA-DRB5,HLA-DQA1,NTM,IL-16,CALCOCO2,and COL5A3.2.Compared with the control group,the expressions of FMN2,BAZ2 B,HLA-DRB1,NTM,IL-16,and CALCOCO2 in ASD group were significantly upregulated(P <0.05),while the expressions of HLA-DRB5,HLA-DQA1,ACTR3,and COL5A3 were upregulated,but there were no significant differences(P > 0.05).The AUC of FMN2,BAZ2 B,NTM,and IL-16 for predicting ASD were 0.896,0.831,0.948,and 0.831(P <0.05)respectively,which had potential diagnostic values.3.PPI analysis revealed three groups of highly interactive differentially methylated genes,HLA-DRB5 and HLA-DQA1 were proteins with the highest interaction,followed by the interaction between FMN2 and ACTR3 and the interaction between CALCOCO2 and BAZ2 B.Summary:1.DNA methylation leads to abnormal gene expression at the m RNA level,suggesting that DNA methylation may be involved in the pathogenesis of ASD by affecting the expressions of related genes.2.Differentially methylated genes can be directly or indirectly involved in the immune and inflammatory responses,suggesting that abnormally expressed methylated genes can promote the development of ASD by regulating immune and inflammatory processes.Part Three Research on immune function of ASDObjective: To analyze the immune function-related indicators in peripheral blood of children with ASD and provide new ideas for exploring the pathogenesis and intervention methods of ASD.Methods: A total of 165 children diagnosed with ASD in Hebei Children’s Hospital from January 2019 to December 2020 were included,and86 healthy children who underwent physical examination in the hospital during the same period were included as the normal control group.The two groups were matched in terms of gender and age.Blood samples of all enrolled individuals were collected for detection of immune function-related indicators,including lymphocyte subsets detection(CD3+,CD4+,CD8+,CD4+/CD8+,CD19+,CD56+,double-negative T cells,double-positive T cells),immunoglobulin and complement detection(Ig A,Ig G,Ig M,Ig E,complement C3,complement C4),cytokine detection(IL-2,IL-4,IL-6,IL-10,IFN-γ,TNF-α).To understand the immune condition of ASD,the differences of these indicators between the ASD group and the control group were analyzed,and the results of food-specific Ig G antibody detection of ASD patients were analyzed.The ASD group was divided into the severe group and the mild-moderate group according to CARS score,and the differences of immune function-related indicators among the severe group,the mild-moderate group and the control group were analyzed.With immune function-related indicators independently or in combination as test variable and ASD as status variable,the ROC curves were obtained and the diagnostic values were evaluated.Results:1.Compared with the normal control group,the levels of CD3+,CD4+,CD8+,CD56+,Ig A,Ig G,and complement C3 in the ASD group were significantly decreased(P<0.05).The levels of CD19+,double-negative T cells,Ig E,IL-2,IL-4,IL-6,IL-10,IFN-γ,and TNF-α were significantly increased(P<0.001),while there were no significant differences in CD4+/CD8+,double-positive T cells,Ig M,and complement C4(P > 0.05).2.Compared with the mild-moderate ASD group,the levels of IL-2,IL-4,IL-6,IL-10,IFN-γ,and TNF-α were significantly increased(P<0.05)in the severe group,but there were no significant differences in other indicators(P >0.05).3.The AUC of CD19+,double-negative T cells,Ig E,IL-2,IL-4,IL-6,IL-10,IFN-γ,and TNF-α for predicting ASD were 0.840,0.654,0.766,0.761,0.799,0.848,0.899,0.877,0.765 respectively(P<0.001),The AUC of lymphocyte subsets,immunoglobulin and complement,cytokines combined for predicting ASD were 0.910,0.917,0.917 respectively(P<0.001),which had potential diagnostic values.4.The detection rate of food-specific Ig G antibody in ASD patients was98.79%,mainly in eggs,milk,and wheat.Summary:1.Abnormalities of lymphocyte subsets,immunoglobulin,complement,and cytokines in ASD may be the basis of immune abnormalities in ASD patients,thus affecting the balance of immune regulation and inflammatory process.2.Some abnormal immunological indicators have certain predictive values for ASD,suggesting that immune abnormalities are related to the occurrence of ASD.Conclusions:1.Abnormal DNA methylation exists in ASD patients,the functions of differentially methylated genes are mainly enriched in the biological processes and pathways of antigen processing and presentation.Some differentially methylated genes have abnormal expressions,and have diagnostic values for ASD,suggesting differentially methylated genes will affect antigen processing and presentation through affecting gene expression and regulation,which will promote the occurrence and development of ASD.2.Abnormal DNA methylation leads to abnormal gene expression at the m RNA level.Core differentially methylated genes can directly promote or participate in immune and inflammatory responses.Suggesting that abnormally expressed methylated genes can promote the pathological process of ASD by regulating immune and inflammatory processes.3.Lymphocyte subsets,immunoglobulin,complement,and cytokines are abnormal in ASD patients.Cytokines are related to the severity of symptoms.Some abnormal immunological indicators have certain diagnostic values for ASD,suggesting that immune abnormalities and inflammatory response are related to the pathogenesis of ASD.