乳児重症ミオクロニーてんかんにおける変異イオンチャネル蓄積病態の実証

• Project/Area Number: 19659272
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Pediatrics
• Research Institution: Fukuoka University
• Principal Investigator: 廣瀬 伸一 Fukuoka University, 医学部, 教授 (60248515)
• Project Period (FY): 2007 – 2009
• Project Status: Completed (Fiscal Year 2009)
• Budget Amount: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2009: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2008: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2007: ¥1,500,000 (Direct Cost: ¥1,500,000)
• Keywords: チャネル / 受容体 / てんかん / 中枢神経 / 遺伝子

Research Abstract

乳児重症ミオクロニーてんかん(SMEI)などの重症てんかんに変異イオンチャネル蓄積病態が関係していることを以下方法であきらかにした。まず、細胞生物学的に変異GABA_A受容体やNa+チャネルの細胞局在、小胞体(ER)輸送障害とERストレス・アポトーシスを検証した。
ラットGABRG2に蛍光蛋白標識し、ヒトSMEIの変異とGEFS+の変異を導入し、培養細胞上に発現させ、α1β 2γ2よりなる5量体のGABA_A受容体を再構成させた。この細胞を用いて変異GABA_A受容体標識蛍光蛋白を指標に、免疫染色とともに蛍光顕微鏡を用いる方法、共焦点レーザー顕微鏡による方法、さらに、GABAA受容体にある糖鎖を利用しパルスチェイスにてその細胞内局在を調査した。これらの実験により変異を導入したGABA_A受容体のオルガネラ局在が小胞体であることが明らかになった。この細胞を用いてERストレスによる小胞体やアポトーシス関連分子の同定を行った。今回はカスパーゼ、DNAフラグメンテーション、アネキシンV、抗PARP等の複数の指標の応用によりアポトーシスの証明を行った。これによりGABAA受容体を発現させた、培養細胞での変異アポトーシスが実証された。さらに分子病態をin vivoで明らかにするため、ヒトてんかんで発見された遺伝子変異と相同変異を持つ動物の作出を試みた。このためヒトと同じ遺伝子異常を持つモデル動物をすでに実績のあるPDGFプロモーターを用いた組換え法により遺伝子組換えラットで作成した。今回はヒト家族性特発性てんかん(常染色体優性夜間前頭葉てんかん)と同じ遺伝子異常をCHRNA4遺伝子に持つ遺伝子組換えラットを作出することに成功した。(特許取得済、J.Neuroscience 2008)

Research Products

• [Journal Article] Current Proceedings of Febrile Seizures and Related Epileptic Syndromes in SCN1A : from Bedside to Bench (2009)
  • Author(s): Chen S-J, Hirose S.
  • Journal Title: J Med Sci 29(4) Pages: 167-172
  • Peer Reviewed
• [Journal Article] A de novo KCNQ2 mutation detected in non-familial benign neonatal convulsions (2009)
  • Author(s): Hirose S., et al.
  • Journal Title: Brain Dev 31(1) Pages: 27-33
  • NAID: 10025578186
  • Peer Reviewed
• [Journal Article] Physicochemical property changes of amino acid residues that accompany missense mutations in SCNIA affect epilepsy phenotype severity (2009)
  • Author(s): Hirose S., et al.
  • Journal Title: J Med Genet 46(10) Pages: 671-9
  • Peer Reviewed
• [Journal Article] Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome (2009)
  • Author(s): Hirose S., et al
  • Journal Title: Brain Dev 31 Pages: 758-62
  • NAID: 10026413050
  • Peer Reviewed
• [Journal Article] Lack of potassium current in W309R mutant KCNQ3 channel causing benign familial neonatal convulsions(BFNC) (2009)
  • Author(s): Hirose S., et al
  • Journal Title: Epilepsy Res 84(1) Pages: 82-5
  • Peer Reviewed
• [Journal Article] Hemiconvulsion-hemiplegia syndrome in a patient with severe myoclonic enilensv in infancy (2009)
  • Author(s): Hirose S., et al.
  • Journal Title: Epilepsia 50(9) Pages: 2158-62
  • Peer Reviewed
• [Journal Article] Novel de novo splice-site mutation of SCN1A in a patient with partial epilepsy with febrile seizures plus (2009)
  • Author(s): Hirose S., et al.
  • Journal Title: Brain Dev 31(2) Pages: 179-82
  • NAID: 10025578995
  • Peer Reviewed
• [Journal Article] Deletions involving both KCNQ2 and CHRNA4 present with benign familial neonatal seizures (2009)
  • Author(s): Hirose S., et al
  • Journal Title: Neurology 73(15) Pages: 1214-7
  • Peer Reviewed
• [Journal Article] Developmental changes in KCNQ2 and KCNQ3 expression in humanbrain : possible contribution to the age-dependent etiology of benign familial neonatal convulsions (2008)
  • Author(s): Hirose S., et al
  • Journal Title: Brain Dev 30(5) Pages: 362-9
  • NAID: 10025577003
  • Peer Reviewed
• [Journal Article] Microchromosomal deletions involving SCN1A and adjacent genes in severe myoclonic epilepsy in infancy. (2008)
  • Author(s): Hirose S., et al
  • Journal Title: Epilepsia 49(9) Pages: 1528-9
  • Peer Reviewed
• [Journal Article] Rats Harboring S284L Chrna4 Mutation Show Attenuation of Synaptic and Extrasynaptic GABAergic Transmission and Exhibit the Nocturnal Frontal Lobe Epilepsy Phenotype. (2008)
  • Author(s): Hirose S., et al
  • Journal Title: J Neurosci 28(47) Pages: 12465-76
  • Peer Reviewed
• [Journal Article] Altered KCNQ3 potassium channel function caused by the W309R pore-helix mutation found in human epilepsy. (2008)
  • Author(s): Hirose S., et al
  • Journal Title: J Membr Biol 222(2) Pages: 55-63
  • Peer Reviewed
• [Journal Article] Mutation screening of AP3M2 in Japanese epilepsy patients (2007)
  • Author(s): Hirose S, et. al.
  • Journal Title: Brain Dev 29(8) Pages: 462-467
  • NAID: 10024136602
  • Peer Reviewed
• [Journal Article] Type I split cord malfomlation is a significant pathomechanism of spinabifida aerta in neonates (2007)
  • Author(s): Hirose S, et. al.
  • Journal Title: Med.Bull.Fukuoka Univ 34(4) Pages: 261-266
  • Peer Reviewed
• [Journal Article] Midazolam for the treatment of neonatal electroencephalography-confirmed seizures (2007)
  • Author(s): Hirose S, et. al.
  • Journal Title: Med.Bull.Fukuoka Univ 34(4) Pages: 267-271
  • Peer Reviewed
• [Presentation] てんかん遺伝子研究の進歩とその臨床応用 (2010)
  • Author(s): 廣瀬伸一
  • Organizer: 南大阪小児疾患研究会
  • Place of Presentation: 大阪 近畿大学大阪狭山キャンパス
  • Year and Date: 2010-02-06
• [Presentation] てんかんの遺伝子研究の進歩とその臨床応用 (2009)
  • Author(s): 廣瀬伸一
  • Organizer: 第21回山梨神経学研究会学術講演会
  • Place of Presentation: 山梨 古奈屋ホテル
  • Year and Date: 2009-06-02
• [Presentation] Genetics of febrile Serizures and Related Conditions (2009)
  • Author(s): 廣瀬伸一
  • Organizer: 10^<th> Asian & Oseanian Cogress of Child Nuerology
  • Place of Presentation: Daegu, Korea Interburgu-EXCO Hotel
• [Presentation] Benign childhood epilepsy with centrotemporal spikes following benign neonatal seizures resulting from a mutation of KCNQ2 (2008)
  • Author(s): Hirose S.
  • Organizer: 8th European Congress on Epileptology
  • Place of Presentation: Berlin
  • Year and Date: 2008-09-22
• [Presentation] Mutational analysis for repetitive febrile seizures and sporadic febrile seizures plus in Japanese (2008)
  • Author(s): Hirose S.
  • Organizer: 8th European Congress on Epileptology
  • Place of Presentation: Berlin
  • Year and Date: 2008-09-20
• [Presentation] Recent advances in the genetics of epilepsy Joint (2008)
  • Author(s): Hirose S.
  • Organizer: 7^<th> Human Genome Organization (HUGO)
  • Place of Presentation: セブ、フィリピン
  • Year and Date: 2008-04-05
• [Presentation] MICRO DELETION OF SCNIA IS ASSOCIATED NOT ONLY、 WITH SEVERE MYOCLONIC EPILEPSY IN INFANCY BUT ALSO、 WITH ITS BODERLINE PHENOTYPE (2007)
  • Author(s): Hirose S., et. al.
  • Organizer: the International congress of Pediatrics
  • Place of Presentation: Athens Greece
• [Presentation] Mutation screening of AP3M2 in Japanese epilepsy/Ming (2007)
  • Author(s): Hirose S., et. al.
  • Organizer: HUGO s 12th Human Genome Meeting
  • Place of Presentation: Montreal
• [Patent(Industrial Property Rights)] 変異導入遺伝子およびそれを導入したノックイン非ヒト動物 (2008)
  • Inventor(s): 廣瀬伸一, 弟子丸正伸, 荒木喜美
  • Industrial Property Rights Holder: 福岡大学熊本大学
  • Industrial Property Number: 2008-273446
  • Filing Date: 2008-10-23