Using iPSCs to evaluate pathogenicity and define disease mechanisms for gene variants associated with rare neurodevelopmental disorders

Neurodevelopmental disorders (NDDs) are characterized by complex behavioral conditions, like speech abnormalities, intellectual disability, learning and motor dysfunctions, and affect about 5% of children. They include autism spectrum disorder, attention deficit hyperactivity disorder, and epilepsy. NDDs have complex etiology involving environmental risk and a substantial genetic contribution. Assessing the pathophysiological mechanisms underlying brain disorders is challenging due to limitations in accessing human brain and the complexity of the central nervous system. Although studies in animal models have provided insights into the various genetic and environmental conditions that influence neurodevelopment and neurological function, rarely achieved to recapitulate precisely the most complex human neurological phenotypes. Therefore, advanced in vitro models are needed to understand the complex scenario behind pathogenetic mechanisms associated to NDDs. For this reason, the aim of this study was to use physiologically relevant heterogeneous iPSC-derived neural models to investigate both the association of DOCK1 variants with a new rare neurological syndrome and the physio-pathological mechanisms of Mowat-Wilson syndrome, which was already associated with mutations in the ZEB2 gene. DOCK1 encodes a member of the Dedicator of Cytokinesis protein family, which are guanine nucleotide exchange factors involved in activating RHO GTPases, and regulate a range of cellular events, including cell motility, adhesion and proliferation. ZEB2 encodes a transcription factor, mainly acting as a transcription repressor, which leads cell fate decisions, differentiation, and maturation in multiple cell lineages in embryos and after birth. We started exploring the potential of human iPSCs to generate mixed neural cultures by immunofluorescence detection of specific markers and by single-cell RNA sequencing. Thus, the outline of our study was focused on generation of iPSC lines derived from patients, then differentiated in neural progenitors, and for DOCK1, subsequent differentiation in mature neuronal networks. The fundamental prerequisite for the development of advanced neural models is the generation of high-quality iPSC lines. Therefore, high-quality of patients-derived iPSC lines was verified applying standardized characterization. Next, each differentiation step was assessed by evaluation of specific stage markers. Transcriptomic analysis of neural progenitors and mixed neuronal cultures carrying ZEB2 and DOCK1 variants, respectively, allowed us to reveal an alteration in neural induction and cell lineage specification caused by ZEB2 variants and an altered neuronal network composition caused by DOCK1 variants.