Initial Characterization of a New Loss-of-Function Mouse Model of the Autism Susceptibility Gene Chd8

Background:   Autism spectrum disorder (ASD) is a heterogeneous disease in which prominent efforts are being made to define subtypes with genetics and behavior.  Recent successes using genetic based subtype identification have identified the chromodomain helicase DNA-binding protein 8 (CHD8) as a likely candidate for a specific subtype of ASD (Bernier et al., 2014, O’Roak et al., 2012, Talkowski et al., 2012 and Neale et al., 2012).  CHD8, located on 14q11.2, binds to β-catenin and has major chromatin remodeling functions (Thompson et al., 2008).  However, in vivo studies of the functional and phenotypic consequences of heterozygous mutation are lacking.  To evaluate biological and behavioral consequences of Chd8 mutations, we used targeted genomic engineering, to generate a mouse line harboring a loss-of-function mutation in Chd8.

Objectives:   The present experiments in Chd8 mice were designed to: 1) validate our genetic engineering of mice heterozygous for the Chd8 mutation, 2) begin a behavioral characterization of phenotypes of high relevance to ASD, and 3) demonstrate the power of integrated genomic, neuroanatomical, and behavioral approaches towards revealing the effects of heterozygous Chd8 mutation using a novel preclinical model of ASD.

Methods:   Genetic engineering via the Cas9/CRISPR system was employed to generate one of the first Chd8 mutant mouse lines. Briefly, synthetic guide RNA was injected along with Cas9 mRNA to mouse oocytes, and F0s carrying mutations were genotyped and bred to expand lines that harbored a mutation.  We selected a line that harbors a short deletion in the fifth exon of Chd8 causing a frameshift resulting in a predicted loss-of-function allele. A battery of behavioral assays relevant to ASD, as well as numerous control assays to detect confounds in physical health or ability to evaluate complex behaviors were conducted as previously described (Crawley et al., 2007; Silverman et al., 2010, 2012).  This inventory focused on domains that are affected in ASD, such as sociability, social communication, repetitive behavior, cognition, sensorimotor function and anxiety-like behaviors.

Results:  Consistent with an earlier study, Chd8 mutations are associated with early embryonic lethality in homozygous carriers, with no homozygous mice observed in the first five litters examined. In preliminary studies comparing mRNA generated via deep RNA sequencing performed on forebrains of heterozygous and wildtype littermates at different embryonic and postnatal ages, we have identified Chd8 haploinsufficiency and associated changes of gene expression.  Female mice with the Chd8 haploinsufficient mutations exhibited motor deficits, anxiety-like behavior on the elevated plus-maze, and learning and memory deficits on both cued and contextual fear conditioning compared to wildtype control mice.   Mutant males exhibited a trend toward anxiety-like behavior on another anxiety-related task, light↔dark transitions.  Lower acoustic startle responses were also noted in the Chd8 mutant males compared to wildtype littermate control mice. 

Conclusions:   We discovered important clinically relevant behavioral phenotypes in our novel Chd8 mutant model of ASD. These preliminary studies also observed substantial sex differences in behavioral phenotypes, analogous to the clinic ASD literature.  Our findings highlight the need to further examine this unique Chd8 mouse model with extensive pathophysiology and behavioral phenotyping efforts.