Identification of Convergent Molecular Pathways during the Development of Neurons Derived from Patients with Idiopathic Autism Using Rnaseq Analysis

Background:  Recent studies have shown that genes harboring ASD risk loci are highly enriched in sets of genes expressed during early neocortical development and genes encoding proteins that function in specific biological pathways involving regulation of transcription, chromatin remodeling, cell adhesion, signaling complexes, and synapse function. However, the impact that these genetic variants have on ASD pathophysiology remains largely unknown. This is due in large part to a lack of genetically-relevant human disease models. The advent of human induced pluripotent stem cell (iPSC) technology and advances in neural differentiation techniques, have made it possible to study the molecular mechanisms that underlie ASD pathology. These stem cell-based differentiation approaches mimic in vivo neurogenesis and, as such, provide the opportunity to study the neurodevelopmental nature of ASD.

Objectives:  Patient-specific induced pluripotent stem cells (iPSCs) present a unique opportunity to examine the hypothesis that heterogeneous ASD loci converge on specific molecular pathways during the early neural development.  

Methods:  Therefore, we generated patient-specific iPSC lines from 6 unrelated ASD individuals bearing rare variants identified through exome sequencing in extended multiplex families. The patient-specific iPSCs, in addition to iPSCs derived from unrelated control individuals, were differentiated into cortical neurons and transcriptome analysis using RNAseq was performed on the neurons at three time points over a 135 day time course of their in vitro development. Pathway and gene ontology (GO) analysis was performed on the set of identified differentially expressed (DE) genes.

Results:  Transcriptome analysis implicate disturbances in the regulation of transcription, WNT signaling, chromatin remodeling, cell adhesion and migration, and synapse development across all time points analyzed. Over the course of neuronal differentiation, we observe key changes in gene associated with neural development and synaptic functionality. The greatest number of differentially expressed genes occurred at the earliest time point. To highlight some of these findings, specific pathways enriched in early neurons (day 35 in vitro) were identified that include WNT/MET signaling (p = 4.9510-36) and collagen catabolism (p = 7.41-09). Diffferentially expressed genes in midpoint neurons (day 85 in vitro) map to pathways involving cell migration (p = 1.34-06) and GABAergic neuron signaling transmission (p = 2.86-09). In later time point neurons (day 135 in vitro), differentially expressed genes are enriched in pathways involving WNT-mediated axon guidance (p = 4.71-05), calcium signaling (p = 8.92-20), and chromatin remodeling (p = 1.49-24).

Conclusions:  The findings of this study show that common molecular etiologies underlie pathogenesis in subsets of individuals with idiopathic autism, including neuronal differentiation, axon growth, and synapse function.