Convergence of Causal Paths across Epigenetic Regulators in ASD

Background: Autism spectrum disorders (ASD) are a range of neurodevelopmental disorders affecting 1 in 59 children in the US. A large number of risk genes have been identified by whole exome sequencing studies of patients. Previous studies provided evidence for both common themes in idiopathic ASD as well as common pathways affected by ASD risk genes, giving rise to the concept of convergence across different genetic etiologies. This concept states that different causes, such as mutations at the DNA level converge onto similar changes along the causal path from mutation to phenotype, for example at the level of transcriptional regulation.

Objectives: The aim of this study is to provide evidence for convergence across multiple ASD risk genes associated with chromatin regulation, as this process has been implicated to play an important role in the pathogenesis of ASD. For this reason we study the molecular consequences for neurodevelopment of heterozygous mutations in these genes using cortical spheroids derived from genetically modified human embryonic stem cells (hESCs) as an in vitro human model system.

Methods: Heterozygous mutant hESC lines were established by insertion of a GFP cassette on one allele using CRISPR/Cas9 and the use of a FACS based selection strategy. Multiple independent clones were generated per gene and verified by PCR and Southern Blot including off-target effects. We then used both wild type and mutant hESC to generate cortical spheroids and study the transcriptomic and epigenomic consequences through RNA- and ATAC-seq at different stages of development, starting with the stem cell state.

Results: We successfully generated and verified clones for five genes (KDM5B, SETD5, CHD8, KMT5B and ASH1L). Sequencing showed correct insertion and an intact wild type allele, Southern blot and PCR confirmed the absence of random integrations and off-target effects. Cortical spheroids were generated and showed the expected cell-types across differentiation. RNA-sequencing and ATAC-seq of hESC and cortical spheroids are ongoing at the time of abstract submission.

Conclusions: The genetic heterogeneity present in ASD hinders the progress in understanding the underlying pathogenic mechanisms. Convergence across causal paths in the pathogenesis of the disorder is a useful paradigm to narrow down the list of possible causative mechanisms. The focus on common downstream effects of mutations in different risk genes allows the delineation of the processes important for ASD pathogenesis from confounding coincidental effects. This might ultimately lead to a way to develop common treatments for patients with mutations in different genes.