MULTI-OMIC DECONVOLUTION OF THE REGULATORY NETWORKS UNDERLYING NEURODEVELOPMENTAL AND AUTISM SPECTRUM DISORDERS: A MULTIDIMENTIONAL ANALYSIS FOR A NEW DISEASE MODELLING PARADIGM

Recent literature has highlighted that mutations causing neurodevelopmental syndromes are particularly enriched in genes related to chromatin regulation and synaptic functions. While the latter could be easily predicted, the former shed seeds to the flourishing of epigenomic studies focused on this type of disorders. Intriguingly, most of such disorders couple different shades of intellectual disabilities with peculiar cranio-facial features and systemic defects which are shared, opposite or unique across them. I have set up a dynamic framework of analysis, that encompasses the comparison of multiple disorders, cell types and cultures, to highlight cell-type specific and developmentally-relevant paths of transcriptional deregulation. Building on my lab’s expertise to harness potency and stability of induced pluripotent stem cells (iPSCs), I identified two main axes of development through which we could characterize on the one hand cerebral cortex related dysregulations and on the other hand cranio-facial features associated traits, peripheral nervous system- and cardiovascular system-related dysregulations. The former is based on the production of adult glutamatergic cortical neurons through ectopic expression of NGN2 in iPSCs and, in parallel, through production of brain organoids: 3D cultures obtained by neuronal differentiation and patterning via sequential exposure to small molecules. The latter is based on differentiation of iPSCs to neural crest stem cells (NCSCs) and mesenchymal stem cells (MSCs). I collected and standardised transcriptomic data coming from controls- and patient-derived iPSCs accounting for six disorders, NCSCs for five disorders and MSCs for two disorders; NGN2 neurons for two disorders and brain organoid for one disorder. During my research I helped define new standards for RNA-seq experiments tailored for differential-expression analysis and developed or implemented tools to make cross-disorder and cross-tissue comparisons in a connectable way. This work let me identify regulatory circuitries shared by all disorders or by subgroups characterized by shared phenotypes; symmetric deregulations in disorders caused by mutation of opposite histone modifiers; unexpected subgroups that will require further investigation. For most disorders, my work confirms previously published evidence that dysregulations identified at the pluripotent stage can be inherited and amplified in disease-relevant tissues in a tissue-specific fashion. Thus, I was capable of identifying disease-specific dysregulations at the pluripotent stage and in disease-relevant tissues; I drew conclusions on iPSCs cross-disorder transcriptional dysregulations through the definition of transcriptional modules; I implemented an analytical framework to boost the ability of identifying the effect of knocking down a certain gene on transcriptional and epigenetic landscapes; I identified sets of genes whose deregulation at the pluripotent stage reverberates and amplifies along development, funnelling and filtering several analyses to converge on a small set of actionable targets; I identified a small set of potential direct targets of PRC2 complex involved in brain development and on the onset of Weaver Syndrome; I identified BAZ1B-specific transcriptional dysregulations in NCSCs that confirm its importance for migration and craniofacial morphogenesis but more in general for chromatin remodelling and human evolution; I helped in the molecular characterization of YY1 mutations, which led to the identification of Gabriele-de Vries Syndrome; I contributed to the molecular characterization of Kabuki Syndrome in neural crest and adult cortical neurons.