Abstract
BackgroundDisruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions, and its targets are enriched with ASD-associated genes.MethodsTo further understand the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs.ResultsTranscriptome profiling revealed that CHD8 hemizygosity (CHD8+/−) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development, β-catenin/Wnt signaling, extracellular matrix, and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia, as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly, we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g., HGMA2) were dysregulated in CHD8+/− neural progenitors or neurons.ConclusionsWe have established a renewable source of CHD8+/− iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0048-6) contains supplementary material, which is available to authorized users.
Highlights
Disruptive mutation in the Chromodomain helicase DNA binding protein 8 (CHD8) gene is one of the top genetic risk factors in autism spectrum disorders (ASDs)
We designed two separate CRISPR Single guide RNA (sgRNA) sequences to target the N-terminal of CHD8 protein to generate truncated mutations (Fig. 1a). Induced pluripotent stem cell (iPSC) derived from a healthy male subject were transduced with CRISPR/Cas9 vectors containing each of the two target sequences
Differentially expressed gene (DEG) with ASD-risk gene sets from multiple sources, we found that upregulated genes in CHD8+/− Neural progenitor cell (NPC) and downregulated genes in CHD8+/− neurons were significantly enriched with ASD-risk genes (Fig. 5b, Additional file 7: Table S6)
Summary
Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). The genetic risk factors for ASD are heterogeneous, and up to 1 thousand genes are estimated to be involved [2]. Recent whole exome and genome-sequencing projects, focused on the identification of rare de novo mutation in the probands of ASD family “trios” or “quads”, have discovered hundreds of genes with functionally disruptive mutations [3,4,5,6,7,8,9,10,11,12], some of which map to ASD-associated de novo copy number variations (CNVs) [13]. Chromodomain helicase DNA binding protein 8 (CHD8) emerged as a top ASD-candidate gene from multiple exome-sequencing studies [6, 8], which altogether have analyzed thousands of ASD probands and in some cases their families too [15, 16]. Macrocephaly is often found in ASD caused by disruption of other candidate genes [17]
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