Analysis of Genomic Copy Number Variations and Exome Sequencing in Japanese Autism Spectrum Disorder Subjects

Background: Copy number variation(CNV) studies have revealed the importance of rare de novo and inherited CNVs in autism spectrum disorder (ASD). Family-based studies have reported a high rate of de novo CNVs in idiopathic ASD cases from simplex families compared to that of multiple families. Recently, exome sequencing studies have revealed the importance of rare single nucleotide variations in ASD risk.

Objectives: To date, almost of the CNV and exome sequencing studies in ASD have focused predominantly on the Caucasian populations, with little reprensentation of the Asians and Africans. In this study, we have examined the global CNV in Japanese sampled and also sequenced the exomes of ASD and their parents samples.

Methods: ASD family samples were recruited in collaboration with the Asperger Society Japan. All the subjects were Japanese; diagnosis was made according to DSM-IV criteria and supported by Autism Diagnostic Interview-Revised scores. Among the 203 familes, 191 were simplex, while the remaining 12 were multiple. The samples were analyzed using Affymetrix Genome-Wide Human SNP Nsp/Sty 6.0 microarray. Three different calling algorithms, PennCNV, Birdseye and Canary, were used to identify the autosomal CNVs.Twenty families which met the highest quality control parameters in array studies, and with at least one rare/interestiog CNV were selected for exome sequencing. Genomic DNA was captured using SureSelect Human All Exon v5 kit, and sequenced on HiSeq2000.

Results: CNV analysis:above 90% of the CNVs in children are found to be inherited; almost equally from both the parents. Among the CNVs which are spanning genes, 71% are deletions in the case of de novo events; it’s only 53% in inherited cases. 281 highly confident de novo CNVs were identified; and hence supposed to have some functional significance. Among these 281 CNVs, 23 are found be rare de novo events. These 23 rare de novo CNVc were validated with qPCR using SyBr Green. A few potential candidate genes like ABR, PITPNA, YWHAE, TRAPPC9, CSMD3, BRD1, MAPK8IP2, PANX2 etc with a possible role in neurodevelopment were identified.Exome Sequencing: Based on GERP and Grantham scores, 15 de novo events that resulted in missense, nonsense, frameshift or splice site mutations were considered to be of major impact. These de novo events occurred in the following genes: DPP6, MATN2, AVPRIA, C6orf32, PELI3, ATAD3B, CNKSR3, ARHGAP8, PRPF8, SF3B2, TRIP12, ADAMTS18, DLX1, NEK8, NALCN. Majority of these mutations were predicted to have a damaging effect on the respective protein structure/function. Several of these genes have been implicated in neural functions, while a few of them have been previously reported to be associated with ASD.

Conclusions: We found several novel/ultra-rare de novo and inherited CNVs and SNVs, many of them are potentially deleterious, in Japanese ASD subjects.