Mitsuko Nakashima1, Hirotomo Saitsu1, Nobuyuki Takei2, Jun Tohyama3, Mitsuhiro Kato4, Hiroki Kitaura5, Masaaki Shiina6, Hiroshi Shirozu7, Hiroshi Masuda7, Keisuke Watanabe8, Chihiro Ohba1, Yoshinori Tsurusaki1, Noriko Miyake1, Yingjun Zheng5, Tatsuhiro Sato9, Hirohide Takebayashi8, Kazuhiro Ogata6, Shigeki Kameyama7, Akiyoshi Kakita5, Naomichi Matsumoto1. 1. Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan. 2. Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan. 3. Department of Child Neurology, Nishi-Niigata Chuo National Hospital, Niigata, Japan. 4. Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan. 5. Department of Pathology, Brain Research Institute, University of Niigata, Niigata, Japan. 6. Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan. 7. Department of Functional Neurosurgery, Epilepsy Center, Nishi-Niigata Chuo National Hospital, Niigata, Japan. 8. Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan. 9. Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Abstract
OBJECTIVE: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb. METHODS: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments. RESULTS: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase. INTERPRETATION: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb.
OBJECTIVE: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb. METHODS: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments. RESULTS: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase. INTERPRETATION: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb.
Authors: Jang Keun Kim; Jun Cho; Se Hoon Kim; Hoon-Chul Kang; Dong-Seok Kim; V Narry Kim; Jeong Ho Lee Journal: J Clin Invest Date: 2019-10-01 Impact factor: 14.808
Authors: Michael S Hildebrand; Nicole G Griffin; John A Damiano; Elisa J Cops; Rosemary Burgess; Ezgi Ozturk; Nigel C Jones; Richard J Leventer; Jeremy L Freeman; A Simon Harvey; Lynette G Sadleir; Ingrid E Scheffer; Heather Major; Benjamin W Darbro; Andrew S Allen; David B Goldstein; John F Kerrigan; Samuel F Berkovic; Erin L Heinzen Journal: Am J Hum Genet Date: 2016-07-21 Impact factor: 11.025