A Brain Somatic RHEB Doublet Mutation Causes Focal Cortical Dysplasia Type Ⅱ

Focal cortical dysplasia type Ⅱ(FCDⅡ)is a malformation of the cerebral cortex characterized by local cortical structure disorganization,dysmorphic neurons and refractory epilepsy.In recent years,somatic mutations in several genes involved in the PI3K/AKT/mTOR pathway have been identified to cause FCDⅡ.In this study,we aimed to identify novel somatic mutations that also contribute to FCDⅡ onset.Firstly,we carried out deep whole exome sequencing(WES)in paired brain-blood DNAs from four FCDⅡ subjects.WES identified a brain somatic doublet mutation c.(A104T,C105A)in the RHEB gene in one FCDⅡ patient;and this mutation results in a p.Y35L change of the RHEB protein.We confirmed the existence of this doublet mutation by T-vector cloning and Sanger sequencing.Next,we evaluated the effect of this RHEB doublet mutation on activating the mTOR pathway by immunoblotting and immunohistochemistry of patient brain samples,and through in vitro transfection and in utero electroporation.Biochemical studies revealed that the RHEB mutation-carrier had dramatically increased phosphorylation in ribosomal protein S6 compared to controls,indicating activation of the mTOR signaling pathway.Overexpression of RHEB p.Y35L mutation in cultured cells resulted in elevated S6 phosphorylation compared to wild type RHEB.Importantly,in utero electroporation of RHEB p.Y35L mutation in mice not onlyinduced S6 phosphorylation,cytomegalic neurons and dysregulated neuron migration,but also led to abnormal electroencephalogram and seizures,all of which reminiscent those found in FCDⅡ patients.These results demonstrate that brain somatic mutations in RHEB are also responsible for FCDⅡ pathogenesis,further indicating that aberrant activation of mTOR signaling is a primary driver for FCDⅡ.