Establishment Of Visual Database For Meta-analysis In Autism Gene Expression Profiles And Preliminary Validation Of Its Candidate Genes

BackgroundAutism spectrum disorders(ASD)are hereditary,heterogeneous and biologically complex neurodevelopmental disorders.Each of the individual studies on gene expression in ASD lack a clear consensus.Therefore,the systematic review to synthesize the current findings in brain tissue and a search tool to share the meta-analysis results are eagerly needed.Meanwhile,The candidate genes that secleted from the visual database were validated experimentally to explore and discover the possible genetic risk factors of autism,and to provide evidence for the prevention and treatment of autism.Methods(1)Establishment of visual database:Here we conducted a meta-analysis of brain gene expression profiles in the current reported human ASD expression datasets(with 84 collective male cortex frozen samples,18 female cortex samples,32 cerebellum samples and 4 formalin fixed samples)and knock out mouse ASD models expression datasets(with 80 collective brain samples).Then,we applied the R language software and developed an interactive shared and update database(dbMDEGA)with meta-analysis results of differentially expressed genes(DEGs)in the brain of ASD studies.(2)Experimental verification of candidate genes: The candidate genes with P value less than 10-5 and associated with the nervous system were selected from meta analysis database to carry out the later experimental verification.In this study,nerve growth factor(NGF)was used to induce the abnormal dendrite growth of PC12 cells as the ASD model,and the candidate gene was verified in this cell model.PC12 cells were cultured in incubator with 37℃ and 5% CO2,and then treated with NGF(0 ng/ml and 50 ng/ml)after 24 h,48h,72 h,96h and 118 h.The Rhodamine Phalloidin was used to perform staining of the cytoskeleton F-actin,and changes of the cells morphology was observed under inverted fluorescence microscope.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression level of candidate genes in PC12 cells after different concentration and time of NGF.Results(1)Establishment of visual database for meta-analysis of ASD gene expression profile: Based on the reported gene expression database of autistic brain tissue,R language software was used to conduct meta-analysis on the gene expression profiles.Then the database dbMDEGA,which is the result of meta-analysis of differentially expressed genes in autistic brain tissue,was successfully established by R language software.This database,dbMDEGA(https://dbmdega.shinyapps.io/dbMDEGA/),is a publicly available web-portal for manual annotation and visualization of DEGs in the brain of ASD studies.In dbMDEGA,which presented the meta-analysis statistics of differentially expressed genes in different sex,brain tissue status and brain tissue regions,as well as Forest plot and beanplot.This database is to provide the searchable and update meta-analysis results based on the current reported brain gene expression datasets of ASD for helping to detect the underlying candidate gene of this disorder.(the results of this study have been submitted under review).(2)Establishment of ASD cell model with abnormal dendrite growth: PC12 cells induced by different time and concentration of NGF,F-actin staining showed that compared with the control group,50 ng/ml NGF treatment could cause the average number and length of dendrite of PC12 cells increased(P<0.01).Additionally,the average number and length of dendrite increased with the increase of induction time(P<0.05).The results suggest that ASD cell model with abnormal dendrite growth was successfully established.(3)The level of candidate genes in the ASD cell model with abnormal dendrite growth: The candidate genes that screened from meta-analysis visualization database and associated with the nervous system include regulator of G-protein signaling 2(RGS2)、transmembrane protein 132A(TMEM132A)、THO complex 5(THOC5)and DnaJ heat shock protein family(Hsp40)member B1(DNAJB1).RT-PCR results showed that the expression level of RGS2 gene increased in 50 ng/ml NGF treated group after 96 h and 118 h compared with control group(P<0.05).50 ng/ml NGF treatment for 96 h and 118 h can also increase the expression level of TMEM132 A gene(P<0.05).Additionally,the expression of TMEM132 A gene in PC12 cells was increased with the increase of NGF treatment time,but the difference was not statistically significant(P>0.05).However,there was no difference in the expression level of DNAJB1 and THOC5 gene in different treatment groups(P>0.05).Conclusion(1)This study through meta analysis of brain gene expression profile date successfully established a visual database of meta analysis results for user query for the first time.As a new query tool,this database can provide valuable help for the exploration of etiology and the discovery of differentially expressed genes in ASD.At the same time,it provides a method for establishing meta-analysis results visualization database for other diseases.(2)In this study,NGF was used to induce the growth of dendrite of PC12 cells,and the ASD cell model with abnormal dendrite growth was successfully established.(3)In this study,four candidate genes,RGS2,TMEM132 A,DNAJB1 and THOC5 were selected based on the visual database,and the gene level was detected in the ASD cell model with abnormal dendrite growth.It was found that the expression level of RGS2 and TMEM132 A gene in the cell model was changed,which provided the basis for the later research and verification of the relationship between RGS2 and TMEM132 A gene and the etiology of autism.