Reverse engineering neurodevelopmental disorders

Intellectual disability (ID) is a common feature in many neurodevelopmental diseases (NDDs) and has an estimated prevalence of approximately 2-3% of the Western population. While clinical observa- tions and genetics underlining ID are highly multiplexed, its diagnostic yield has nevertheless im- mensely benefitted from improvement and increased availability of next generation sequencing strat- egies. While mutations in over 1000 genes are known to cause a monogenetic form of ID, it is esti- mated that over 40% of ID await genetic discovery, underscoring the critical need for functional work- up necessary for identification of genetic causality. Our goal is to identify common molecular pathways in known ID-related genes, as well as developing network-based approach to predict genes highly likely to play a role in similar pathology of their ge- netic neighbors. To gain mechanistic understanding of how ID-causative genes might functionally relate to each other, we created the ID-interactome using a systems-based computational method- ology, representing an integrated network of all physical interactions of gene products identified to be causative of ID in human patients. We will use CRISPR-Cas9 genome editing strategies and phar- macological manipulation to determine contribution of identified common pathways or newly pre- dicted genes to cellular pathology. We therefore, reverse the usual workflow, by first identifying and functionally validating possible disease causing genes, and then searching for already sequenced but still undiagnosed patients with high scoring variants within our validated genes. This approach pro- vides an opportunity to rapidly diagnose many patients and generate a database of potentially causa- tive genes for future cases. Additionally, these studies will provide insight into fundamental cellular neurobiology that may have implications to other unrelated diseases as well.

A rare disease is defined by the European Union as one that affects less than 5 in 10,000 of the general population which amounts to approximately ~30 million people across the European Union that suffers from a rare disorder. There are currently ~7,000 identified rare diseases to date, many of which are inherited, of monogenetic origin, and are diagnosed early in life, therefore considered chronic. Unfortunately, many rare disorders remain undiagnosed, and most remain without direct therapeutic interventions. Recent technological advances have significantly increased discovery of genetic causes for rare diseases, however, detailed characterization and therapeutic options for these vulnerable patients have been lagging behind. Comprehensive clinical characterization is often not enough to inform clinicians of the genetic origins or proper treatment avenues, therefore, genetic and molecular characterization of the disease pathology must be clearly defined. From a basic biology standpoint, studying diseases of monogenic origin also gives us valuable insight into genotype-to-phenotype relationship relevant to human physiology. A large portion or rare diseases affects the nervous system. We are particularly interested in causes of unexplained rare forms of intellectual disabilities (ID), to identify common molecular pathways and uncover potential therapeutic targets that may be relevant to more than one disorder. We therefore combined state-of-the-art "dry" and "wet" bench techniques, including systems biology and CRISPR-Cas9 edited stem cells to identify and validate a subgroup of rare IDs that may converge on common molecular pathways. We focused on a sub-network of ID-causing genes surrounding a highly interactive gene RAC1, referred to as the "RAC-ome" for proof-of concept. As part of our efforts we have identified a highly interconnected disease gene network surrounding RAC1 that presents with similar clinical pathologies. We have generated knock-outs of approximately 100 genes in this network, in three transformed cell lines to analyze morphological deviations and other predicted pathological hallmarks caused by individual gene perturbations. We have additionally performed a small compound screen of FDA and EMA approved drugs for neurological diseases to further confirm involved pathways and determine gene-drug interactions. To validate our findings, we have generated a dox-inducible, stably expressing Cas9 iPSC line, for rapid generation of neurons with select gene perturbations. Additionally, we have obtained 3 individual patient-derived iPSCs containing deleterious variants in one gene within this network for detailed molecular analysis. Upon completion, these data are expected to shed light on a critical unmed medical need by not only identifying and validating novel disease genes in cell lines and neurons, determining common molecular pathways, but also creating a platform for therapeutic intervention screen that may lead to novel therapies.