title: Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia.
abstract: Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.
authors.affiliation: Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea.
authors.forename: Jae Seok
authors.initials: JS
authors.lastname: Lim
authors.affiliation: Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea; Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Ramu
authors.initials: R
authors.lastname: Gopalappa
authors.affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Se Hoon
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea.
authors.forename: Suresh
authors.initials: S
authors.lastname: Ramakrishna
authors.affiliation: Department of Integrated OMICS for Biomedical Science, Yonsei University, Seoul 03722, South Korea.
authors.forename: Minji
authors.initials: M
authors.lastname: Lee
authors.affiliation: Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea.
authors.forename: Woo-Il
authors.initials: WI
authors.lastname: Kim
authors.affiliation: Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Junho
authors.initials: J
authors.lastname: Kim
authors.affiliation: Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea.
authors.forename: Sang Min
authors.initials: SM
authors.lastname: Park
authors.affiliation: Biomedical HPC Technology Research Center, Korea Institute of Science and Technology Information, Daejeon 34141, South Korea.
authors.forename: Junehawk
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon 34114, South Korea.
authors.forename: Jung-Hwa
authors.initials: JH
authors.lastname: Oh
authors.affiliation: Division of Pediatric Neurology, Department of Pediatrics, Pediatric Epilepsy Clinics, Severance Children's Hospital, Epilepsy Research Institute, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Heung Dong
authors.initials: HD
authors.lastname: Kim
authors.affiliation: Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea.
authors.forename: Chang-Hwan
authors.initials: CH
authors.lastname: Park
authors.affiliation: Division of Pediatric Neurology, Department of Pediatrics, Pediatric Epilepsy Clinics, Severance Children's Hospital, Epilepsy Research Institute, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Joon Soo
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Sangwoo
authors.initials: S
authors.lastname: Kim
authors.affiliation: Pediatric Neurosurgery, Severance Children's Hospital, Department of Neurosurgery, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Dong Seok
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Department of Integrated OMICS for Biomedical Science, Yonsei University, Seoul 03722, South Korea; College of Pharmacy, Yonsei University, Seoul 03722, South Korea.
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Han
authors.affiliation: Division of Pediatric Neurology, Department of Pediatrics, Pediatric Epilepsy Clinics, Severance Children's Hospital, Epilepsy Research Institute, Yonsei University College of Medicine, Seoul 03722, South Korea.
authors.forename: Hoon-Chul
authors.initials: HC
authors.lastname: Kang
authors.affiliation: Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, South Korea; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, South Korea; Center for Nanomedicine, Institute for Basic Science, Yonsei University, Seoul 03722, South Korea. Electronic address: hkim1@yuhs.ac.
authors.forename: Hyongbum Henry
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea. Electronic address: jhlee4246@kaist.ac.kr.
authors.forename: Jeong Ho
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000293:Adolescent; D000818:Animals; D001921:Brain / Q000378:metabolism; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D002648:Child; D058534:Class I Phosphatidylinositol 3-Kinases; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D004827:Epilepsy / Q000235:genetics*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D065703:Malformations of Cortical Development, Group I / Q000235:genetics*; D051379:Mice; D009154:Mutation; D009474:Neurons; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics; D012463:Saliva / Q000737:chemistry; D017422:Sequence Analysis, DNA; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000235:genetics; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; C000624651:Tsc1 protein, mouse; C000624654:Tsc2 protein, mouse; C560566:phosphoinositol-3 kinase regulatory subunit 2, human; C546842:MTOR protein, human; C484760:PIK3CA protein, human; C491754:AKT3 protein, human
keywords: CRISPR-Cas9 genome editing; TSC1; TSC2; brain mosaicism; brain somatic mutation; focal cortical dysplasia; intractable epilepsy