TBRS-associated mutations disrupt cortical interneuron differentiation and neuronal networks.

Pathogenic mutations cause Tatton-Brown-Rahman Syndrome (TBRS), a disorder characterized by intellectual disability and overgrowth of multiple somatic tissues including the brain. However, the functions of DNMT3A during human cortical development remain poorly understood. Here, we utilized newly developed human pluripotent stem cell models of TBRS-associated mutation to define DNMT3A requirements and consequences of mutation during human cortical neuron development. Profiling changes to epigenetic gene regulation across both GABAergic and glutamatergic neuron development, we identified GABAergic cortical interneurons as particularly sensitive to TBRS-associated mutation. During GABAergic neuron development, TBRS-associated DNMT3A mutations resulted in reduced DNA methylation and were associated with concomitant de-repression of gene expression, causing precocious neuronal differentiation. By contrast, the consequences of mutation on glutamatergic neuron development were less pronounced, due in part to compensatory repressive histone methylation, and resulted in increased expression of early neurodevelopmental genes during glutamatergic neuron differentiation. Assessing the consequences of these molecular phenotypes by patch-clamp electrophysiology, we found that DNMT3A deficient GABAergic neurons were hyperactive, while glutamatergic neuron function was largely unaffected by these DNMT3A loss of function mutations. Finally, we used both low density and high density multi electrode array techniques in conjunction with glutamatergic-GABAergic neuron co-cultures to assess how TBRS-associated GABAergic neuron hyperactivity affected the emergence and development of neuronal networks. We found that TBRS GABAergic neuron hyperactivity was sufficient to drive abnormal neuronal network development, increasing the neuronal activity consolidated into neuronal bursting and networks. Ultimately, this work elucidated new roles for DNMT3A-mediated repression in human cortical development, identifying critical requirements in regulating neuronal and synaptic gene expression during GABAergic differentiation, with these TBRS-associated molecular changes driving alterations of neuronal network function likely to contribute to TBRS etiology.