TRANSCRIPTOME ANALYSIS OF WNT7A-MEDIATED GENE EXPRESSION IN HUMAN NEURAL PROGENITOR CELLS DERIVED FROM CONTROL AND CHD8 HAPLOINSUFFICIENT INDUCED PLURIPOTENT STEM CELLS

Background CHD8 (Chromodomain Helicase DNA Binding Protein 8) codes for a member of the CHD family of chromatin-remodeling factors and is one of the most commonly mutated genes in Autism Spectrum Disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in Schizophrenia (SZ) and intellectual disabilities. We previously reported RNA-seq analyses carried out on Neural Progenitor Cells (NPCs), monolayer neurons and cerebral organoids derived from control induced Pluripotent Stem (iPS) cells that were made haploinsufficient for CHD8 (CHD8+/-) using CRISPR-Cas9 gene editing. Several hundred Differentially Expressed Genes (DEGs) were found in comparisons between the CHD8+/- and isogenic controls (CHD8+/+), including a significant number of ASD and SZ candidate genes, such as the SZ and Bipolar Disorder (BD) candidate gene TCF4. Pathway analysis of DEGs showed enrichment for genes involved in Wnt-signaling. A number of Wnt receptors (members of the FZD family) and Wnt ligands were differentially expressed, such as Wnt7A and its receptor FZD7, which were down-regulated in CHD8+/- NPCs. Methods We tested the hypothesis that Wnt7A-mediated gene expression is deficient in CHD8+/- NPCs by carrying out RNA-seq on two CHD8+/- lines and two controls, including the isogenic control sample. The NPCs were treated with vehicle (control) or Wnt7A (1 microgram/ml) for 24 hours. Biological duplicates were prepared for each sample. RNA was extracted and converted to cDNA for paired-end sequencing. The RNA-seq reads were aligned to the human genes annotated in the GENCODE database (v18). Gene expression levels were determined as transcripts per million (TPM). Of the 12,898 expressed genes, with an average TPM >1 in either controls or CHD8+/− samples, DESeq. 2 was used to identify differentially expressed genes (DEGs) at false discovery rate [FDR] Results We obtained 892 DEGs in the isogenic control NPCs, from a comparison of the Wnt7A treated samples and controls, but only 410 among the CHD8+/- samples, suggesting a potential reduction in Wnt7A signaling in the CHD8+/- samples. Among the DEGs affected by Wnt7A in the isogenic controls, but not in CHD8+/- samples was TCF4. Pathway analysis showed that DEGs from the isogenic controls were enriched for Wnt-beta catenin signaling pathway and acute phase response signaling. In CHD8+/- NPCs, there was less enrichment of both. RNA-seq findings were confirmed for selected genes by quantitative real time PCR. Discussion These preliminary findings suggest that CHD8 haploinsufficiency can attenuate Wnt7A signaling. Wnt/β-catenin signaling plays a key role in brain development and in post-mitotic brain functions. Thus, the attenuation of Wnt7A-inducible genes in CHD8 haploinsufficient NPCs could be mediating some of the effects CHD8 loss-of-function mutations have on brain structures and patient behaviors. Understanding the role of Wnt-signaling on neurodevelopmental and neuropsychiatric disorders is important because of the insight provided on pathogenesis and from the therapeutic perspective; canonical Wnt-signaling is a target of lithium salts, for example. These preliminary studies are currently being expanded to include three additional control and CHD8+/- NPC lines, and whether gene expression changes are affected by lithium.