Highly Parallel Analysis of High-Confidence ASD and Schizophrenia Risk Gene Mechanisms

About This Grant

Advances in defining the genetic contributions to neuropsychiatric disorders (NPDs), such as autism spectrum disorder (ASD) and schizophrenia (SCZ), have led to the identification of over 200 risk genes harboring rare, protein-disrupting heterozygous mutations. Understanding how mutations in these genes alter brain development or function is essential for elucidating disease mechanisms. Here, we address the substantial challenges posed by genetic heterogeneity and pleiotropy in these two NPDs by studying approximately 50 risk genes in a highly parallel manner. This approach allows us to efficiently identify areas of overlap and convergence among mutations within and across disorders. We leverage our scalable human iPSC engineering platform alongside high-throughput, high-content phenotyping to conduct multi-layered analyses of molecular and cellular alterations caused by heterozygous mutations in these risk genes. Mutant and control lines will be differentiated into 2D neuronal cultures, where high-content imaging will assess proliferation and morphological features. RNA sequencing and high-sensitivity proteomics will be performed to measure gene expression and identify perturbed pathways. These data will be augmented by automated calcium imaging to assess spontaneous and evoked neuronal activity and network synchrony. To model early cortical development in vitro, complementing the 2D system, we will also generate 3D human cortical organoids (hCOs), enabling investigation of cell type specification, maturation, and tissue architecture. We will apply advanced platforms including the Phenocycler for multiplexed spatial proteomics and mononucleotide Repeat Frameshift (MORF) technology coupled with CLARITY imaging to examine cell and dendritic morphology in detail in maturing 3D hCOs. Proteomic and transcriptomic data will be integrated using weighted gene co-expression network analysis (WGCNA) to identify shared and distinct biological pathways affected by the various mutations and to relate these changes to alterations in cellular morphology and physiology in parallel across genes. Our work will clarify whether there are convergent molecular pathways disrupted within and across disorders, and how such disruptions propagate from the molecular to the physiological level, illuminating underlying disease mechanisms. In addition, the resulting ≈200 highly characterized iPSC lines representing ASD and SCZ risk genes will serve as a valuable resource for the field, supporting in-depth follow-up studies, replication, and data integration across platforms.