Genome-Wide Microarray Based Study Of Gene Copy Number Variation Associated With Autism Spectrum Disorder

Research background and purpose Autism spectrum disorder(ASD)is a complex neurodevelopmental disorder characterized by persistent impairment in reciprocal social communication and social interaction and restricted,repetitive patterns of behavior,interests,or activities.These symptoms are present from early childhood and limit or impair everyday functioning.The prevalence has increased significantly since it was first reported in 1943,now it is closer to 1%of the population.The etiology and pathogenesis of autism spectrum disorders are not clear,most scholars believe that are the result of genetiic and environmental factors interaction effect,but genetic factors play a more important role,heritability estimates for autism spectrum disorder have ranged from 37%to higher than 90%.Many studys showed that a variety of rare genetic variations with high penetrance lead to the occurrence of ASD,and CNVs is an important form of such genetic variations.Autism spectrum disorder is one of the earliest cases of copy number variations.Up to September 2018,the AutDB database has collected 2,274 about copy number variants associated with autism,and the trend is increasing.But the heterogeneity of these copy number variants is high,most of them have not been verified,especially in Chinese population.For this purpose,this study will further explore the characteristics of copy number variation in autistic children of Shandong Province and screen the possible pathogenic genes by using Genome-wide microarray chip technology.Subjects and methods 100 ASD children and their core families were collected from Qilu Children’s Hospital of Shandong University.The children with ASD were diagnosed based on criteria defined in the Diagnostic and Statistical Manual of Mental Disorder,4th Edition,Text Revision(DSM-Ⅳ-TR)by two designated specialists.These patients are eliminated severe nervous system diseases,physical disease,metabolic diseases and abnormal chromosome karyotype.We used Genome-Wide Human SNP Array 6.0 perform genome-wide genotyping for 100 ASD samples.The genome-wide CNVs analysis was performed with the software Genotyping Console.The results were compared with public database to identify suspicious pathogenic CNVs.Real-time fluorescence quantitative PCR was used to verify and analyze the suspicious pathogenic CNVs region to determine whether CNVs was a new mutation or a genetic mutation,and false positive results of microarray were excluded.Finally,the genes in these CNVs regions were further screened by bioinformatics analysis to evaluate the correlation between the gene locus and the onset of autism and its contribution to the risk of onset.Research results Ten suspected pathogenic CNVs were found in 100 ASD children,with a detection rate of 10%,including seven micromisses and three duplicates.There are 5 de novo CNVs and 5 inherited CNVs(2 from father and 3 from mother).After further bioinformatics analysis,a total of 11 suspected pathogenic genes were involved in these 10 suspected pathogenic CNVs,include IMMP2L,AUTS2,GRM7,GBE1,NEO1,BBS4,ADPGK,HCN4,LRRC4C,CTNNA2 and IGBP1.The microdeletions located at 7q31.1 was found at 3 ASD children and effected IMMP2L gene.In the first case,there were 182kb missing fragments from chr7:111105975 to chr7:111288089.In the second case,there were 245kb missing fragments from chr7:110963723 to chr7:111209092.In the third case,there was a missing fragment of size 201 kb on chromosome 7 from chr7:111077519 to chr7:111278265.These deletions destroyed exon regions located in exon 1-3 of IMMP2L gene on chromosome 7q31.1,which were verified by real-time quantitative PCR.Although IMMP2L gene has a high incidence,it is found that there is no significant difference between the ASD group and the control group after comparison with multiple database data,suggesting that there is no significant correlation between IMMP2L gene and ASDThe other two microdeletions were all new mutations,the deletionsof one case was from chr7:68254845 to chr7:69084409,with a size of 830Kb.The deletion is located in the upstream distal A UTS2 gene and affects the transcription and translation promoter of AUTS2 gene.In another case,from chr3:7221090 to chr3:7524552 was deleted,the size of the deletion is 303Kb.This deletion disrupts the five coding exons(exons 3 to 7)of the GRM7 gene,with a break point in the lateral introns 2 and 7.The correlation between the deletion of GRM7 gene and AUTS2 gene and ASD has been reported in foreign countries,our data supported the pathogenic role of GRM7 and AUTS2 in neurodevelopmental disorders especially ASDThe boy with complete deletion of GBE1 gene were severe ASD cases with typical social dysfunction and stereotypical behavior accompanied by delayed language development.Follow-up showed poor recovery effect.The deletion located at 3p12.2 from chr3:80780361 to chr3:83213281,with a size of2433Kb,is heterozygous deletion GBE1 genotype is glycogen storage disease Ⅳ disease-causing genes,with Recessive inheritance.At present,there is no report on its association with ASD.In this study,GBE1 gene deletion was detected for the first time in patients with ASD.Based on the functional analysis,it was preliminarily concluded that GBE1 gene deletion played a role in the pathogenesis of ASD by affecting the key transcription factor of the Wnt/β-catenin classical signaling pathway,more detailed pathogenic mechanism needs to be further studiedThis study also detected a deletion at 15q24.1q24.2 in a 2 years old ASD boy.The deletion from chrl5:72964144 to chr15:75544822.Thorough review of the cases and this new patient suggests that the second critical region of 15q24 and genes,particularly NEO1,play an etiologic role on the ASD phenotype of the syndromeAll three regions of repeated fragment were inherited from their parents,the first region is located in X chromosome Xq13.1,from chrX:69368049 to chrX:69768574,size 401Kb,with maternal inheritance.The other two CNVs are both paternal,one is located on 11p12,with a size of 208Kb from chrl 1:40746589 to chrl 1:40954326,and the other located on 2p12,with a size of 1197Kb from chr2:78657154-79854651.The repeat sequence at Xq13.1 contains exon 5-7 and part of intron 4 of the IGBP1 gene,the repeat sequence at 11 p12 contains intron 2 of LRRC4C gene and the repeat sequence at 2p12 contains exon 1 and part of intron 1 of CTNNA2 gene.At present,there are seldom reports of the association about these three genes with ASD,all three children with ASD had healthy parents who did not have a broad ASD phenotype,so whether these three genes are related to ASD remains to be further studied in ASD cases.Research conclusion1.The incidence of suspected pathogenic copy number variations in ASD children in a hospital in Shandong province was 10%.2.There was no significant correlation between IMMP2L gene deletion and ASD.3.Gene mutations of GRM7,AUTS2 and NEO1 have causative role in ASD.4.GBE1 gene may be a new candidate pathogenic gene for ASD,it play a role in the pathogenesis of ASD by affecting the Wnt/β-catenin classical signaling pathway.