Abstract
Brain Somatic Mutations in MTOR Cause Focal Cortical Dysplasia Type II Leading to Intractable Epilepsy
Lim JS, Kim WI, Kang HC, Kim SH, Park AH, Park EK, Cho YW, Kim S, Kim HM, Kim JA, Kim J, Rhee H, Kang SG, Kim HD, Kim D, Kim DS, Lee JH. Nat Med 2015;21:395–400.
Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy (1, 2). It has been hypothesized that FCD is caused by somatic mutations in affected regions (3, 4). 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.
Commentary
Rapid advances in whole genome sequencing (WGS) technology have led to a transformation in clinical medicine to diagnose and treat genetic disorders (1). For epilepsy, a population-based twin study suggests that an estimated 70% to 80% of epilepsies are derived from genetic factors (2, 3). While strong evidence suggests many of these factors are polygenic with complex inheritance (4), emerging research indicates that de novo genetic mutations contribute to epilepsy pathogenesis (5).
In the current study, the authors use deep whole exome sequencing (WES) of paired blood–brain DNA samples from four identified subjects with focal cortical dysplasia type II (FCDII) to search for low-frequency de novo somatic mutations. Three variants of mechanistic target of rapamycin (MTOR) were initially identified. They further validated these low-frequency mutations to exclude any sequencing artifacts with site-specific amplicon sequencing of all three variants on the same brain tissue. Two of the four FCDII patients had a mutant variant of MTOR (c.7280T > C) detected in the brain tissue that was not found in the paired blood samples. The authors expanded their detection of somatic MTOR mutations by combining both hybrid capture sequencing and deep site–specific amplicon sequencing of the original three candidate mutations to brain samples from a cohort of 73 FCDII subjects. These two sequencing approaches ensured that mutations detected by both platforms would mitigate sequence artifacts and erroneous calls. With this level of scrutiny, they were able to detect an additional eight MTOR mutation variants from ten more subjects. These sequencing results suggest that de novo somatic mutations of the MTOR gene in brain tissue are associated with FCDII.
The authors switched to an in vitro cell culture system to determine how the identified MTOR mutations alter endogenous mTOR signaling activity. Wild-type or mutant MTOR was transfected into human embryonic kidney 293T (HEK293T) cells, and endogenous S6 kinase phosphorylation activity was measured as an assay for mTOR activity. Surprisingly, mutant MTOR lead to hyperactivation of the mTOR pathway compared with wild-type MTOR. They further validated these results in brain sections from FCDII patients with immunostaining of phospho-S6 and found an increase in the size and number of neurons expressing phospho-S6 compared with non-FCD samples. Thus, FCDII patients with mutations in MTOR display hyperactive mTOR signaling in cortical neurons. Of interest, loss-of-function mutations of upstream regulators in the mTOR signaling pathway (e.g., PI3K, AKT, PTEN, TSC1/2, KRAS) are strongly associated with mTOR hyperactivity in various cancers (6). While it is surprising that the identified MTOR mutations drive a hyperactivity of the mTOR pathway, there is evidence suggesting a similar mechanism driving dysregulated cellular function and rapamycin sensitivity in cancer (7).
If mutations in MTOR in FCDII patients associate with hyperactivation of the mTOR pathway, is this mutation sufficient to drive spontaneous seizure development? To address this question, the authors electroporated mice with mutant or wild-type MTOR in utero at E14 and monitored seizure development with video-EEG as they aged. Seizure onset was observed at about 6 weeks of age, which the authors suggest (arguably) is analogous to 4 years of age in a human. These spontaneous seizures subsided following treatment with rapamycin, a well-known inhibitor of the mTOR pathway. Thus, generating a de novo mutation in the MTOR gene early in utero causes somatic mutations that are sufficient to induce spontaneous seizures in young mice.
Lim et al. have elegantly identified key de novo mutations in the MTOR gene that disrupt cortical development and cause spontaneous seizures in FCDII. Independently, another group also identified somatic mutations of the MTOR gene in patients with FCDII (8). Furthermore, a mutation in MTOR and activation of MTOR pathway was found in a patient with hemispheric cortical dysplasia possibly extending MTOR dysregulation to nonsyndromic cortical dysplasias (9). As many intractable epilepsies have unknown etiologies, understanding the molecular genetics underlying de novo somatic mutations provides novel mechanisms for treating epilepsy. This study presents evidence of a somatic mutation in a gene not associated with the “canonical” inheritable channelopathies (10), thereby opening many questions for further investigation:
Are there other genes with de novo mutations accountable for the remaining FCDII patients? At what developmental stage(s) does the identified MTOR mutations arise, and are specific brain regions affected? Can genome editing, such as CRISPR/Cas9 technology, be used to correct somatic mutations and treat the disease?
While there is still much work to be done in identifying genetic factors for epilepsy, this study provides a promising step forward.