Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy

Abstract

Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412-668×) validated by site-specific amplicon sequencing (100-347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.

Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia

“…These findings also suggest that hyperactivated mTOR kinase is a therapeutic and genetically validated target in individuals with FCD. 8 Although the genetic etiology and molecular pathogenesis of FCD remain to be fully defined, our study has not only revealed additional somatic mosaic mutations that lead to FCD but also described the generation of a mouse model with brain somatic mutations via in utero somatic genome-editing technology. This study will provide a foundation for future clinical and scientific research of FCD.…”

“…To minimize erroneous and false-positive mutations that could mimic low-level somatic mutations, we performed cross-platform replications by adopting both hybrid capture and PCRbased amplicon sequencing of five genes. 8,39,40 To detect true de novo somatic mutations, we selected overlapping mutations in both sequencing platforms and then performed simple filtering steps ( Figure S2). The subjects were considered to have a mutation when the percentage of mutated reads exceeded 103 (1%) the expected base miscall rate (0.1%).…”

“…Because the molecular genetic etiology of FCD has remained elusive, genetically validated drug targets in individuals with FCD are poorly understood. Recently, we and another group have shown that somatic activating mutations in MTOR (MIM: 601231) in the brain account for 15%-25% of individuals with FCDII 8,9 and that inhibition of hyperactivated mTOR kinase reverses epilepsy and the development of cytomegalic neurons. 8 However, the genetic causes underlying the development of FCDII in individuals who are negative for MTOR mutations remain elusive.…”

Impaired oligodendroglial turnover is associated with myelin pathology in focal cortical dysplasia and tuberous sclerosis complex

“…TSC is naturally a mutation of either TSC1 or TSC2 in 85%, whereas more than half of those cases show also mosaicism 43. Recent studies found somatic (mosaic) mutations affecting the mTOR signaling also in focal lesions of FCD II patients 4, 8, 21, 25, 32, 36. This is a very important aspect since as far as we know, in FCD, the mutations are localized and show profound variability in cellular expression.…”

Dysregulation of NEUROG2 plays a key role in focal cortical dysplasia

“…Although recent literature points to the involvement of the mTOR pathway in FCD type II,9, 10, 11, 12 the exact molecular mechanism leading to this type of cortical malformation, especially to the presence of aberrant cells, remains undetermined 13, 14. As the development of the cerebral cortex is a sophisticated process, requiring the fine‐tuning of gene expression, we decided to analyze the expression of microRNAs as a shortcut to gain a better understanding of the mechanisms involved in the pathogenesis of FCD type II.…”