Molecular genetic decoding of focal cortical dysplasia leading to intractable epilepsy

Abstract

Part I. Brain somatic mutations of MTOR cause focal cortical dysplasia type II leading to intractable focal epilepsy in human and mouse.

Focal cortical dysplasia type II (FCDII) is a developmental malformation of cerebral cortex and an important cause of medically refractory epilepsy. FCDII sporadically occurs, and this condition is characterized by dysmorphic neurons and disrupted cortical lamination in affected cortical regions. It has been hypothesized that FCD is caused by somatic mutations in affected regions. However, no such mutations have been identified. Here, I report de no-vo somatic mutations of MTOR in the affected brains of FCDII patients. Deep whole exome sequencing (read depth, 412-668×) validated with the use of site-specific amplicon se-quencing (read depth, 100-347,499×) in paired brain-blood DNA from 4 FCDII patients revealed a brain somatic mutation, MTOR c.7280T>C (p.Leu2427Pro) in 2 patients. I then performed deep sequencing of MTOR gene in additional 73 FCDII patients using two dif-ferent sequencing platforms such as hybrid capture (read depth, 100-17,700×) and PCR amplicon sequencing (read depth, 100-20,210×). In total, I identified 9 different somatic missense mutations of MTOR in 12 FCDII patients, which were reproducibly reported in both sequencing platforms and in multi-samples from the brain tissue block of each patient. Identified mutations accounted for 15.6% of all FCDII participants (12 of 77). The preva-lence of the mutant allele in affected brain tissues ranged from 1.26% to 12.6%. The identi-fied mutations induced the constitutive activation of mTOR kinase and cytomegalic neu-rons in affected brains carrying these mutations. In addition, the focal cortical expression of MTOR mutant in in utero electroporated mice was sufficient to interfere with proper neu-ronal migration and cause spontaneous seizures with epileptic discharge and cytomegalic neurons. Furthermore, rapamycin, an inhibitor of mTOR, suppressed cytomegalic neurons and epileptic seizures. Therefore, this study provides the first evidence that brain somatic activating mutations in MTOR cause FCD as well as the potential drug target for intractable epilepsy in FCD patients.

Keywords: focal cortical dysplasia, somatic mutation, mechanistic target of rapamycin (MTOR)

Part II. Brain somatic mutations in TSC1 and TSC2 lead to FCDII patients negative for mTOR mutations and in utero somatic genome editing with CRISPR/Cas9 system recapitulates TSC1 and TSC2 mutations in mouse models.

Together with my finding in Part I, it was recently reported that the brain somatic mutations in MTOR accounted for 15-25% of FCD type II (FCDII) characterized by cortical dyslami-nation and dysmorphic neurons. However, molecular genetic etiologies of remaining FCDII patients negative for MTOR mutation remain unclear. Here, I performed deep hybrid-capture and amplicon sequencing (100 - 20012X, read depth) of five important mTOR pathway genes including PIK3CA, PIK3R2, AKT3, TSC1, and TSC2 in paired brain-blood (or saliva) tissues from 47 FCDII patients negative for MTOR mutations. I found that 5 out of 47 patients (10.2%) have brain somatic mutations in TSC1 and TSC2 genes such as TSC1 c.64C>T (p.Arg22Trp), TSC1 c.610C>T (p.Arg204Cys), and TSC2 c.4639C>T (p.Val1547Ile) reproducibly reported in two different sequencing platforms. Especially, TSC1 c.64C>T (p.Arg22Trp) was recurrently found in 3 patients. Their mutational burdens ranged 1.0 to 2.8% in affected brains. All identified mutations induced the hyperactivation of mTOR pathway by disrupting the formation or function of TSC1/TSC2 complex. Fur-thermore, to recapitulate low-level somatic alterations in TSC1 or TSC2 in vivo, I estab-lished the one-step generation of brain somatic alterations in mouse using in utero electro-poration of CRISPR/Cas9 system (in utero CRISPR/Cas9 system). In utero CRISPR/Cas9 system mediated disruptions of TSC1 or TSC2 gene in focal cortical regions were sufficient to induce all pathological and symptomatic FCDII phenotypes, such as severe spontaneous seizures, cytomegalic neurons, and cortical dyslamination. This study shows that that brain somatic mutation in TSC1 and TSC2 cause FCDII and in utero CRISPR/Cas9 system is use-ful for generating faithful animal models of neurodevelopmental disorders with brain so-matic mutations.

Keywords: mTOR pathway, tuberous sclerosis complex, CRISPR/Cas9 system, mouse model