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Schizophrenia Genes Prioritized in Zebrafish Mutant Study

NEW YORK (GenomeWeb) – A team led by investigators at Harvard University, the Stanley Center for Psychiatric Research, and the University of Basel has characterized the whole-brain activity, brain structure, and behavioral features found in zebrafish carrying mutations in the orthologs of genes suspected of influencing schizophrenia in humans.  

"[T]he phenotype atlas of schizophrenia-associated genes lays the foundation for deciphering the molecular, cellular, and physiological pathways underlying neuropsychiatric disease and for high-throughput screens for genetic or small-molecule suppressors of mutant phenotypes," first and co-corresponding author Summer Thyme, a molecular and cellular biology researcher at Harvard University, and her colleagues wrote.

Thyme and her colleagues focused in on a few dozen genes for future studies through detailed anatomical and behavioral analyses on 132 zebrafish mutants, each carrying a distinct alteration affecting the zebrafish version of a gene implicated in schizophrenia in human genome-wide association studies. The phenotypic consequences of these mutations ranged from unusual brain activity to developmental shifts in specific brain regions of the engineered zebrafish larvae, they reported today in Cell.

Many SNPs, loci, and corresponding genes have been implicated in schizophrenia and related neuropsychiatric conditions through prior GWAS, the team noted. For example, common variants at some 108 loci were linked to schizophrenia by members of the Psychiatric Genomics Consortium's Schizophrenia Working Group in 2014, leading to candidate genes in the major histocompatibility complex and beyond.

Although some of these genes are believed to contribute to synaptic pruning and other processes that might affect brain structure and activity, the researchers noted that additional functional studies are needed to uncover the neurodevelopmental effects of mutations in other candidate genes.

"To discover the mechanisms underlying polygenic illnesses such as schizophrenia and other neuropsychiatric disease, it would be useful to identify the in vivo functions of many associated genes," the authors explained, adding that "such analysis could also prioritize candidate genes for further analysis."

The investigators used CRISPR-Cas9 and a series of guide RNAs to engineer 132 zebrafish larvae mutants, each containing truncations that affected an individual gene. The genes targeted were informed by the 108 loci identified by the Schizophrenia Working Group, post-mortem human gene expression data insights from the CommonMind database, and other previous research.

"Criteria for choosing genes to mutate included nervous system expression and human data that further implicated them in neuropsychiatric illness, such as patient exome sequencing or post-mortem brain analyses of gene expression or chromatin conformation," the authors explained.

The team assessed more than 15,000 of the resulting mutant larvae with high-throughput genotyping, anatomical, brain activity mapping, and behavioral analyses — from larval brain imaging, morphometry, and whole-brain activity assays to experiments tracking developing zebrafish movement in response to light, heat, and other sensory stimuli.

The researchers identified brain morphology changes in 16 of the zebrafish mutants, while more than half of the mutants showed shifts in activity involving brain regions such as the hypothalamus, pallium, and tectum. They noted that zebrafish with mutations in a histone methyltransferase enzyme-coding gene previously linked to schizophrenia and autism spectrum disorder typically had larger-than-usual brain sizes, for example.

With the brain activity data, meanwhile, the team saw significant brain effects for both well-characterized synaptic pruning, neurotransmitter, and calcium channel genes for zebrafish with mutations affecting genes with murkier or unknown functions.

All told, the team's analyses pointed to more than 30 genes that appear to be particularly promising candidates for future schizophrenia research, including a transcription factor-coding gene called znf536 with an apparent role in forebrain neuron development in the single-cell RNA-seq experiments presented in the paper.

Based on their results in the schizophrenia context, the authors noted that a similar pipeline "can be used to screen genes that are candidates for other disorders, and future mechanistic studies of these genes will provide insights into multiple disorders."