Identifying the Pathogenicity of a Novel NPRL3 Missense Mutation Using Personalized Cortical Organoid Model of Focal Cortical Dysplasia

Focal cortical dysplasia (FCD) II is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, with or without balloon cells. Here, we systematically explored the pathophysiological role of the GATOR1 subunit NPRL3 variants including a novel mutation from iPSCs derived from one FCD II patient. Three FCD II children aged 0.5-7 years who underwent cerebral lesion resection in our hospital from March 2019 to October 2019 were included in this study. We generated patient-derived iPSCs and performed whole-exome sequencing to accurately identify somatic cells with mutations. The effect of the newly identified NPRL3 mutation found in one of our FCD II patients was evaluated using the personalized cortical organoid model and the NPRL3 knockout HEK293T cells. Whole-exome sequencing of iPSCs derived from FCD II patients revealed a novel NPRL3 C.767G > C (p.R256P) heterozygous mutation. Cortical organoids generated from iPSCs of FCD II patients were larger than control iPSCs, with increased number of p-S6(+) cells and NeuN(+) neurons. In NPRL3 knockout HEK293T cells, overexpression of NPRL3 together with NPRL2 protein is necessary to reduce p-S6 level upon amino acid starvation. The reduced binding between NPRL3 (Arg256Pro) and NPRL2 protein leads to downregulation of the relative total protein amount of both proteins in the cell. Our study describes a novel cortical organoid model generated from iPSCs of the FCD patients to investigate the underlying mechanism of NPRL3-related epilepsy. The mutation of NPRL3 (Arg256Pro) impaired the function of NPRL3 protein via affecting the binding with NPRL2 protein, which resulted in unstable protein monomer.