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Yasin et al. J Transl Genet Genom 2020;4:307-19 I https://doi.org/10.20517/jtgg.2020.30 Page 315
[75]
the same source . They observed 1,248 and 3,248 genes differentially expressed in NPCs and neurons,
respectively, with an enrichment for genes involved in neuronal development and β-catenin/Wnt signaling,
[76]
including known ASD and SCZ candidate genes . Interestingly, they highlight the involvement of genes
associated with brain volume among their differentially expressed genes, speculating a connection to the
macrocephaly phenotype observed for patients with ZFS. The subsequent publication from this group of
[75]
their findings for brain organoids showed a much lower number of differentially expressed genes, i.e., 559 .
Nevertheless, neuronal development was once more enriched as was Wnt/β-catenin signaling. Of note
from this study was the finding of a non-coding RNA, DLX6-AS1, as their topmost differentially expressed
[75]
gene with a staggering +39 fold change in expression level , a gene gaining increasing importance in ASD
[78]
pathophysiology .
We note that while each of the above studies followed rigorous established experimental protocols, there is a
remarkable variation in the resulting differentially expressed gene datasets, with overall non-robust overlap,
which could be the result of variation in both biological and bioinformatics experimental methods. However,
in this respect, a meta-analysis conducted by running all the raw data from these studies through the same
[64]
bioinformatics processing pipeline failed to eliminate or reduce the discordance in a meaningful manner .
In summary, the lack of any one or handful of consistent downstream target gene(s) for CHD8 from these
studies is an observation that only further emphasizes that CHD8 indeed does have widespread downstream
targeting, suggesting it is a possible master-regulator of transcription that displays both genomic and cellular
context-dependent variation. It also underlines the importance of pathway-based data analyses when
deciphering the results of gene expression studies for CHD8 deficiency models.

Studies in animal models
The earliest animal model for CHD8 was presented as a functional study in zebrafish that cemented the
[73]
pathogenicity of the gene for macrocephaly . However, the development of mouse models of Chd8+/- by
several groups now dominates the characterization of haploinsufficiency of this gene in a complete organism,
and significantly, from a mammalian system. While the sum characterizations from the mouse models offer a
rich trove of information, we still note that all of the studies we are aware of [65-70] , investigated the gene defect
only as a major causative gene for ASD with the additional noted phenotype of macrocephaly, and not as one
causing syndromic NDD. This bias must be kept in mind, as it greatly impacts the characterization results,
since only ASD or related phenotypes were investigated. Nonetheless, observations from the mouse model
studies offer key insights into the molecular physiology of Chd8 haploinsufficiency.

[65]
The first mouse model report in 2016 involved in utero-targeted cortical knockdown of Chd8 . Consistent
with findings from the cellular model studies, a key role in the regulation of Wnt signaling (as a positive
regulator) was observed. A distinct role in mammalian cortical development was proposed, acting via
cell cycle regulation and PRC complex targeting. And behavioral deficits, in keeping with ASD equivalent
[65]
phenotyping, were also noted . In the same year, another group reported the characterization of a Chd8
[67]
heterozygous knockout mouse model . They also demonstrated behavioral defects and further noted
[79]
macrocephaly. Two subsequent publications from the same group showed impairment in adipogenesis
[80]
and in oligodendrocyte myelination , causing a slender habitus and behavioral defects, respectively.
In 2017, a CRISPER-mediated knockout mouse study also showed macrocephaly and widespread gene
[66]
expression profile changes, enriched for neurogenesis and synaptic processing among others . However,
this study was not able to recapitulate the repetitive behaviors observed in other mouse models, but did
[66]
show cognitive impairment . Another CRISPER-mediated Chd8 heterozygous mouse study showed a
similar widespread gene expression perturbation in the brain, macrocephaly, and in this case also learning
[69]
[68]
deficits . Macrocephaly was a key observation in another mouse model published the following year , but
no behavioral defects were noted. Finally sex-specific behavioral traits in male mice but not female mice were
[70]
reported , probing the known sex bias for ASD presentation.