Towards a molecular characterization of autism spectrum disorders: an exome sequencing and systems approach

Abstract

The hypothetical 'AXAS' gene network model that profiles functional patterns of heterogeneous DNA variants overrepresented in autism spectrum disorder (ASD), X-linked intellectual disability, attention deficit and hyperactivity disorder and schizophrenia was used in this current study to analyze whole exome sequencing data from an Australian ASD cohort. An optimized DNA variant filtering pipeline was used to identify loss-of-function DNA variations. Inherited variants from parents with a broader autism phenotype and de novo variants were found to be significantly associated with ASD. Gene ontology analysis revealed that putative rare causal variants cluster in key neurobiological processes and are overrepresented in functions involving neuronal development, signal transduction and synapse development including the neurexin trans-synaptic complex. We also show how a complex gene network model can be used to fine map combinations of inherited and de novo variations in families with ASD that converge in the L1CAM pathway. Our results provide an important step forward in the molecular characterization of ASD with potential for developing a tool to analyze the pathogenesis of individual affected families.

Figure 1

Genome distribution of DNA variants. Circle plots show the type and localization of filtered DNA variants detected by exome sequence analysis. The location of variants was annotated using the Reference Sequence hg19 database with ANNOVAR software. (a) SNPs were equally distributed in both intronic and exonic regions, whereas most indels were observed in intronic and 3'-UTR regions of gene transcripts. (b) DNA variants were also found to occur in noncoding RNA sequences. SNP, single-nucleotide polymorphism; UTR, untranslated region.

Figure 2

Overview of variant filtering pipeline. (a) A detailed diagram of the computational pipeline and databases used to filter DNA variants found in WES data. Shown are the average number of variants per individual. (b) Comparison of databases showing the association of filtered variants with ASD. The level of association was measured using a binomial distribution with a standardized Z-score as determined by the AXAS model. ADHD, attention deficit and hyperactivity disorder; ASD, autism spectrum disorder; MAF, minor allele frequency; PPI, protein–protein interaction; SZ, schizophrenia; WES, whole exome sequencing; XLID, X-linked intellectual disorder.

Figure 3

Variant classification using the AXAS model. Radar plots showing binomial distributions with standardized Z-scores used to examine the association of putative causal DNA variants with the AXAS-PPI model. Z-scores ⩾1.65 show significant association with a neurodevelopmental disorder. Inherited variants from BAP parents (g) and de novo variants (c) show the greatest association with ASD; see also Supplementary Table S1. ADHD, attention deficit and hyperactivity disorder; ASD, autism spectrum disorder; BAP, broader autism phenotype; SZ, schizophrenia; XLID, X-linked intellectual disorder.

Figure 4

Clustering analysis in ASD cases. PPI networks were constructed using putative causal variants and their first-degree protein interactors that result in three types of affected networks. Two possible schemes (a and b) show how maternally inherited variants (yellow), paternally inherited variants (green) and de novo variants (blue) converge and cluster in unique functional pathways (gray). Functional ontology of three proband cases: (c) 06.s1, neuronal migration; (d) 30.s1, long-term potentiation, (e) 38.s1, axon guidance and inherited variants that merge with de novo variants that converge in (f) 18.s1, ankyrin and adhesive related proteins involved the synapse development and neurodevelopment. Of the 48 ASD cases, there were four unique combinations (pedigrees) of variants that cluster in the L1CAM interaction pathway (REACTOME:22205; see Supplementary Table 5 for details of gene names). ASD, autism spectrum disorder; PPI, protein–protein interaction.