Abstract
Focal cortical dysplasia type II (FCDII) is a cerebral cortex malformation characterized by local cortical structure disorganization, neuronal dysmorphology, and refractory epilepsy. Brain somatic mutations in several genes involved in the PI3K/AKT/mTOR pathway are associated with FCDII, but they are only found in a proportion of patients with FCDII. The genetic causes underlying the development FCDII in other patients remain unclear. Here, we carried out whole exome sequencing and targeted sequencing in paired brain–blood DNA from patients with FCDII and identified a brain somatic doublet mutation c.(A104T, C105A) in the Ras homolog, mTORC1 binding (RHEB) gene, which led to the RHEB p.Y35L mutation in one patient with FCDII. This RHEB mutation carrier had a dramatic increase of ribosomal protein S6 phosphorylation, indicating mTOR activation in the region of the brain lesion. The RHEB p.Y35L mutant protein had increased GTPλS-binding activity compared with wild-type RHEB. Overexpression of the RHEB p.Y35L variant in cultured cells also resulted in elevated S6 phosphorylation compared to wild-type RHEB. Importantly, in utero electroporation of the RHEB p.Y35L variant in mice induced S6 phosphorylation, cytomegalic neurons, dysregulated neuron migration, abnormal electroencephalogram, and seizures, all of which are found in patients with FCDII. Rapamycin treatment rescued abnormal electroencephalograms and alleviated seizures in these mice. These results demonstrate that brain somatic mutations in RHEB are also responsible for the pathogenesis of FCDII, indicating that aberrant activation of mTOR signaling is a primary driver and potential drug target for FCDII.
Highlights
Focal cortical dysplasia (FCD) is a cortical developmental malformation associated with intractable epilepsy in children
We focused on brain somatic single nucleotide variants (SNVs) and indels that fulfilled the high-priority screening criteria, which identified two adjacent SNV mutations (C105A and A104T) in the Ras homolog enriched in brain (RHEB) gene in patient Focal cortical dysplasia type II (FCDII)-1 (Fig. 1a and Table 2) and one SNV in DPP7 and one indel in MED16 in patient with FCDII-2 (Table 2)
The RHEB gene encodes Ras homolog enriched in brain (RHEB), a GTP-binding protein involved in cell cycle regulation and an important activator of the mTOR pathway[6,28]
Summary
Focal cortical dysplasia (FCD) is a cortical developmental malformation associated with intractable epilepsy in children. The PI3K/AKT/mTOR signaling pathway plays important roles in cell proliferation, migration, and metabolism during brain development[5,6]. Recent studies have identified brain somatic mutations in several genes in this pathway in association with FCDII, including PIK3CA7, PTEN8, DEPDC59, TSC110,11, TSC210,11, and MTOR11–15. Somatic mutations in MTOR were only identified in 7.711, 46.212, 15.613, 13.814, and 37.5%15 of participants in different studies. Somatic mutations in TSC1 were identified in 10.0% of participants in one study[10]. Somatic mutations in other genes are even scarcer, with only one case identified in each study. We describe a new pathogenic mutation in the Ras homolog, mTORC1-binding (RHEB) gene associated with mTOR pathway activation, providing evidence of an additional somatic mTOR modulator in FCDII
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