Studies On Genetic Etiology Of Autism Spectrum Disorder And Neurological Mechanisms Underlying Cognitive Impairment Related To SH2B1 Gene

Objectives: The aim of this study was to assess the diagnostic yield and the clinical practicality of children with autism spectrum disorder(ASD)by chromosomal microarray analysis(CMA)and Trio-based whole exome sequencing(Trio-WES).And to further investigate neurobiological mechanisms of cognitive deficits related to ASD risk gene SH2B1.Methods: From 2014 to 2017,high resolution CMA was applied among Chinese cohort of ASD(n = 414)to identified associated genetic etiology.After quality checks,children diagnosed as ASD(n=401)together with an ancestry-matched control cohort(n = 197)were enrolled to examine the rare copy number variants(CNVs)burden.The severity categorization was based on CARS scores.Rare CNV burden analysis was performed to examine the rare CNVs burden between mildly and severely affected subgroups,and to compare different rare CNVs burden between children with ASD and controls.Trio-WES was further utilized among families of 65 children classified with negative findings of CNVs and complex co-occurring conditions.Clinical managements of ASD children affected by CMA and WES were evaluated.Association between ASD risk gene SH2B1 and cognitive function,and regions of brain(focusing on the hippocampus)involved were investigated based on data of UK Biobank.Data of cognitive function tests at initial assessment visit were used in the study,including fluid intelligence(FI)test(n=165,460),prospective memory test(n=171,549),pair matching test(n=497,882)and reaction time(n=496,680).In addition,quality controlled genetic data(n=337,199),voxel-based morphometry(VBM)based MR Imaging volumetric data(n=9,888),and FI in the imaging visit(N=11,176)were applied in the analysis.Based on these data,correlation of SNPs within SH2B1 and cognitive function was assessed,and only FI was correlated with SH2B1.Then the mediation effect of hippocampus on the association between SNPs within SH2B1 was evaluated by the mediation analyses.To further validate the findings in the population,AAV-hsyn-cre-EGFP was injected into CA1 region of bilateral hippocampus among SH2B1 flox+/+ mice and SH2B1 flox-/-mice.Behavior tests including Y-maze and novel object recognition(NOR)were performed in 4weeks after that.As a measure of working memory,Y-maze alternations and error rate was calculated.Also,and the preference during NOR task were also analyzed to assess the short memory.Results: The diagnostic yield of CMA was approximately 7.2% in the ASD cohort,and that of trio-WES was 9.2% among children with negative findings of CNVs and complex co-occurring conditions.Among children with earlier molecular diagnosis,about 75.0% children with ASD benefited from the subsequent changes in clinical managements.Compared with controls,rare loss events were more common in ASD cohort(p=0.021),whereas the rate of rare gain events correlated with the phenotypic severity(p=0.003).Among these highly constrained genes disrupted by rare losses,RIMS2 is a promising candidate risk gene for ASD.All of the six presumed causative genes(CHD8,AFF2,ADNP,POGZ,SHANK3 and IL1RAPL1)identified by WES were related to the low-functioning ASD.By using data from UK Biobank,we found 3 SNPs(rs4788102,rs7359397 and rs7498665)within SH2B1 locus was only associated with FI scores(FDR p<5*10-8).More importantly,bilateral hippocampus mediated the correlation between the 3SNPs and FI scores,with age at tests,sex,intracranial volume and top 15 PCs of the genotype data adjusted as covariates.In addition,clusters in the bilateral hippocampus mediated the association between 3 SNPs and FI(Alpha Sim corrected cluster level p<0.02).Meanwhile,the mediation by the bilateral hippocampus was still significant when BMI was adjusted in the model.To further validate the role of the hippocampus in the association between SH2B1 and FI,SH2B1 knock-down mice were applied.In Y-maze test,in comparison with wild-type mice,those with SH2B1 knock-down in CA1 of hippocampus showed reduced rated of alternation(p<0.05).And in NOR test,SH2B1 knock-down in CA1 of hippocampus showed significant decreased exploring capability compared with controls(p<0.05).Conclusions: In the Chinese ASD cohort,CMA is the first-tier diagnostic assay.And trio-WES can be applied among children with negative findings of CNVs and complex co-occurring conditions,making it useful in complementing CMA and improving the diagnostic yield.Rare deletion of CNVs might contribute more to the molecular etiology of ASD,while rare duplication of CNVs could enhance the severity of ASD.Additionally,the association between ASD risk gene SH2B1 and FI could be mediated by the hippocampus.Further research is warranted to investigate the underlying mechanisms of modulation.