Patient Brain Organoids Identify a Link between the 16p11.2 Copy Number Variant and the RBFOX1 Gene.

Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.2 CNVs lead to disease are unclear. Relevant models, such as human cortical organoids (hCOs), are needed to understand the human-specific mechanisms of neurodevelopmental disease. We generated hCOs from 17 patients and controls, profiling 167,958 cells with single-cell RNA-sequencing analysis, which revealed neuronal-specific differential expression of genes outside the 16p11.2 region that are related to cell-cell adhesion, neuronal projection growth, and neurodevelopmental disorders. Furthermore, 16p11.2 deletion syndrome organoids exhibited reduced mRNA and protein levels of RBFOX1, a gene that can also harbor CNVs linked to neurodevelopmental phenotypes. We found that the genes previously shown to be regulated by RBFOX1 are also perturbed in organoids from patients with the 16p11.2 deletion syndrome and thus identified a novel link between independent CNVs associated with neuronal development and autism. Overall, this work suggests convergent signaling, which indicates the possibility of a common therapeutic mechanism across multiple rare neuronal diseases.

Natural variation in gene expression and viral susceptibility revealed by neural progenitor cell villages.

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes.