Development of mutagenesis to the mitochondrial DNA using artificial restriction enzyme and application to definitive diagnosis.

• Project/Area Number: 25670261
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Applied pharmacology
• Research Institution: Saitama Medical University
• Principal Investigator: YATSUKA YUKIKO 埼玉医科大学, 医学部, 助手 (20458524)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: ミトコンドリアDNA / 遺伝子診断・治療 / 人工制限酵素 / 遺伝子工学

Outline of Final Research Achievements

We performed whole mitochondrial DNA (mtDNA) sequence analysis in patients with mitochondrial respiratory chain disorder, and identified several plausible causative mutations. Here, we tried to produce mutant cell lines harboring the variants of unknown significance (VUSs) from normal cells using artificial nucleases in the aim for establishing a new method for definitive diagnosis for VUSs.
We applied TALENs (Transcription activator-like effector nucleases) system, and engineered so that they localize to mitochondria and recognize the specific target sequence on mtDNA. Actually, the engineered mitochondria-targeted TALENs correctly recognized one-base -difference in the specific target sequence, and cut the proper site.
Although we’ve not achieved producing mutant cell lines from normal cells, our TALEN system cut the specific mtDNA site. This system can control the heteroplasmy rate inside the mitochondria, and will pave the way for establishing a new method for definitive diagnosis.

Research Products

• [Journal Article] A comprehensive genomic analysis reveals the genetic landscape of mitochondrial respiratory chain deficiency. (2016)
  • Author(s): Kohda M, Mizuno Y, Hirata T, Yatsuka Y, Yamashita, Okuda A, Borna NN, Banshoya K, Ohnuma K, Suzuki T, Nagao A, Maehata H, Matsuda F, Higasa K, Nagasaki M, Yasuda J, Yamamoto M, Fushimi T, Shimura M, Kaiho-Ichimoto K, Harashima H, Yamazaki T, Mori M, Murayama K, Ohtake A, Okazaki Y.
  • Journal Title: PLoS Genetics: Volume: 12(1) Issue: 1 Pages: e1005679-e1005679
  • DOI: 10.1371/journal.pgen.1005679
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Myocerebrohepatopathy spectrum disorder due to POLG mutations: A clinicopathological report. (2014)
  • Author(s): Montassir H, Maegaki Y, Murayama K, Yamazaki T, Kohda M, Ohtake A, Iwasa H, Yatsuka Y, Okazaki Y, Sugiura C, Nagata I, Toyoshima M, Saito Y, Itoh M, Nishino I, Ohno K.
  • Journal Title: Brain & development Volume: S0387-7604(14) Issue: 7 Pages: 261-267
  • DOI: 10.1016/j.braindev.2014.10.013
  • Peer Reviewed / Open Access
• [Presentation] A comprehensive genomic analysis reveals the genetic landscape of mitochondrial respiratory chain complex deficiencies. (2015)
  • Author(s): Masakzu Kohda, Yoshimi Tokuzawa, Yoshihito Kishita, Hiromi Nyuzuki, Yosuke Mizuno, Tomoko Hirata, Yukiko Yatsuka, et al.
  • Organizer: 第15回日本ミトコンドリア学会年会
  • Place of Presentation: 福井県国際交流会館（福井県福井市）
  • Year and Date: 2015-11-19
  • Int'l Joint Research
• [Presentation] 全エキソーム解析で病因を同定したミトコンドリア呼吸鎖異常症の出生前遺伝子診断 (2015)
  • Author(s): 山崎太郎、原嶋宏子、伏見拓矢、志村 優、八塚由紀子、木下善仁、神田将和、岡崎康司、村山 圭、大竹 明
  • Organizer: 第13回 アジア先天代謝異常症シンポジウム
  • Place of Presentation: 大阪国際会議場（大阪府大阪市）
  • Year and Date: 2015-11-12
  • Int'l Joint Research
• [Presentation] ミトコンドリア呼吸鎖複合体異常症のゲノム解析と新規原因遺伝子の発見 (2015)
  • Author(s): 神田 将和、徳澤 佳美、木下 善仁、森山 陽介、水野 洋介、平田 智子、八塚 由紀子、菅原 泉、仲地 豊、加藤 英政、田丸 俊輔、入月 浩美、Nurun Nahar Borna、原嶋 宏子、山崎 太郎、森雅人、村山 圭、大竹 明、岡崎 康司
  • Organizer: 日本人類遺伝学会 第60回大会
  • Place of Presentation: 京王プラザホテル（東京都新宿区）
  • Year and Date: 2015-10-14
• [Presentation] Large-scale comprehensive identification and analysis of causative genes in mitochondrial respiratory chain disorder (2014)
  • Author(s): Y Kishita, Y Tokuzawa, M Kohda, Y Moriyama, Y Yamashita-Sugahara, S Tamaru, H Tochigi, Y Mizuno, N Uehara, Y Nakachi, Y Yatsuka, H Nyuzuki, S Suzuki, NN Borna, T Hirata, N Matoba, H Kato, A Okuda, M Mori, M Ajima, H Harashima, T Yamazaki, K Murayama, A Ohtake, Y Okazaki
  • Organizer: 第37回日本分子生物学会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] A comprehensive genomic analysis of 144 patients with mitochondrial respiratory chain deficiencies. (2014)
  • Author(s): Masakazu Kohda, Yoshimi Tokuzawa, Yoshihito Kishita, Yzumi Yamashita-Sugahara, Yosuke Mizuno, Yutaka Nakachi, Tomoko Hirata, Yukiko Kanesaki-Yatsuka, Hiroko Harashima, Taro Yamazaki, Masato Mori, Kei Murayama, Hidemasa Kato, Akira Ohtake, Yasushi Okazaki
  • Organizer: 日本人類遺伝学会第59回大会
  • Place of Presentation: タワーホール船堀（東京都江戸川区）
  • Year and Date: 2014-11-19 – 2014-11-22
• [Presentation] A comprehensive genomic analysis for mitochondrial respiratory chain disorder. (2014)
  • Author(s): M. Kohda, Y. Tokuzawa, Y. Kishita, Y. Yamashita-Sugahara, Y. Mizuno, Y. Nakachi, Y. Moriyama, T. Hirata, Y. Kanesaki-Yatsuka, H. Harashima, M. Ajima, T. Yamazaki, M. Mori, K. Murayama, H. Kato, A. Ohtake, Y. Okazaki
  • Organizer: アメリカ人類遺伝学会（ASHG2014）
  • Place of Presentation: サンディエゴ（米カリフォルニア）
  • Year and Date: 2014-10-18 – 2014-10-22
• [Remarks] MO Bank
  • URL: http://mo-bank.com/index.html
• [Remarks] 文部科学省 革新的細胞解析研究プログラム（セルイノベーション）先導研究 課題A
  • URL: http://www.cell-innovation.org/html/program/theme_010_okazaki.html
• [Remarks] 埼玉医科大学ゲノム医学研究センター
  • URL: http://www.saitama-med.ac.jp/genome/
• [Remarks] 埼玉医科大学ゲノム医学研究センター(ゲノム科学部門）
  • URL: http://www.saitama-med.ac.jp/genome/Div05_FGSM/index.html
• [Remarks] 文部科学省 革新的細胞解析研究プログラム（セルイノベーション）先導研究 課題A
  • URL: http://www.cell-innovation.org/html/program/theme_010_okazaki.html
• [Remarks] 埼玉医科大学ゲノム医学研究センター
  • URL: http://www.saitama-med.ac.jp/genome/
• [Remarks] 埼玉医科大学ゲノム医学研究センター(ゲノム科学部門）
  • URL: http://www.saitama-med.ac.jp/genome/Div05_FGSM/index.html