Research on defects in ketone body metabolism: Establishment of defective cell lines and expression systems of mutant enzymes .

• Project/Area Number: 16K09962
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Pediatrics
• Research Institution: Gifu University
• Principal Investigator: Fukao Toshiyuki 岐阜大学, 大学院医学系研究科, 教授 (70260578)
• Research Collaborator: SASAI Hideo ; AGO Yasuhiko ; OTSUKA Hiroki ; MATSUMOTO Hideki ; MAKAMA Mina
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 遺伝子 / 遺伝学 / 先天代謝異常症 / ケトン体 / 発現実験

Outline of Final Research Achievements

I have been studying inborn errors of ketone body metabolism. In this study, we tried to establish expression systems of wild-type and mutant cDNAs for HMGCS2 deficiency and HMGCL deficiency of which patients have already been identified in Japan, MCT1 deficiency of which patients have not been identified yet in Japan, and 3HBD deficiency of which patients have never identified in the world. In case of HMGCS2 expression, we failed to get enough expression level for characterization, but we then successfully showed that 4 HMGCS2 mutants reduced enzyme activities significantly using bacterial expression system. In case of 3HBD expression, we established expression system using fetal fibroblast cell line from 3hBDH knock-out mice. We are now trying to establish knock-out cell lines of MCT1 or HMGCL from wild-type fetal fibroblasts.

Academic Significance and Societal Importance of the Research Achievements

HMG-CoA合成酵素欠損症の変異タンパクの発現系の確立をできたことで、日本の症例の確定診断ができ、日本に本症が存在することを論文として報告が可能となった。また日本で症例が同定されていないMCT１欠損症、世界でまだ欠損症の報告がない３HBD欠損症における変異タンパクの評価をできるようにしておくことは今後の迅速な確定診断において重要である。

Research Products

• [Int'l Joint Research] Sohag University(Egypt)
• [Int'l Joint Research] National Children's Hospital, Hanoi/Vinmec International Hospital(ベトナム)
• [Int'l Joint Research] University of the Sacred Heart(Italy)
• [Int'l Joint Research] Memorial University of Newfoundland(Canada)
• [Int'l Joint Research] Manipal University(India)
• [Int'l Joint Research] Uludag University(Turkey)
• [Int'l Joint Research] Medical Universty og Vienna(Austria)
• [Int'l Joint Research] raibow hospital for women and children(India)
• [Int'l Joint Research] Sohag University(Egypt)
• [Int'l Joint Research] University of Applied Science(Germany)
• [Journal Article] Recent advances in understanding beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency (2019)
  • Author(s): Fukao T, Sasai H, Aoyama Y, Otsuka H, Ago Y, Matsumoto H, Abdelkreem E.
  • Journal Title: Journal of Human Genetics Volume: 64 Issue: 2 Pages: 99-111
  • DOI: 10.1038/s10038-018-0524-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry (2018)
  • Author(s): Kiema Tiila-Riikka、Thapa Chandan J.、Laitaoja Mikko、Schmitz Werner、Maksimainen Mirko M.、Fukao Toshiyuki、Rouvinen Juha、J?nis Janne、Wierenga Rik K.
  • Journal Title: Biochemical Journal Volume: 476 Issue: 2 Pages: 307-332
  • DOI: 10.1042/bcj20180788
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Heterozygous carriers of succinyl-CoA:3-oxoacid CoA transferase deficiency can develop severe ketoacidosis (2017)
  • Author(s): Sasai H, Aoyama Y, Otsuka H, Abdelkreem E, Naiki Y, Kubota M, Sekine Y, Itoh M, Nakama M, Ohnishi H, Fujiki R, Ohara O, Fukao T
  • Journal Title: Journal of Inherited Metabolic Disease Volume: 40 Issue: 6 Pages: 845-852
  • DOI: 10.1007/s10545-017-0065-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A novel mutation (c.121-13T>A) in the polypyrimidine tract of the splice acceptor site of intron 2 causes exon 3 skipping in mitochondrial acetoacetyl-CoA thiolase gene (2017)
  • Author(s): Aoyama Y, Sasai H, Abdelkreem E, Otsuka H, Nakama M, Kumar S, Aroor S,Shukla A, Fukao T
  • Journal Title: Molecular Medicine Reports Volume: 印刷中 Issue: 6 Pages: 3879-3884
  • DOI: 10.3892/mmr.2017.6434
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Characterization and outcome of 41 patients with beta-ketothiolase deficiency: 10 years’ experience of a medical center in northern Vietnam (2017)
  • Author(s): Nguyen KN, Abdelkreem E, Colombo R, Hasegawa Y, Can NT, Bui TP, Le HT, Tran MT, Nguyen HT, Trinh HT, Aoyama Y, Sasai H, Yamaguchi S, Fukao T, Vu DC
  • Journal Title: Journal of Inherited Metabolic Disease Volume: 40 Issue: 3 Pages: 395-401
  • DOI: 10.1007/s10545-017-0026-6
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Single-nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in the ACAT1 gene. Molecular Genetics and Genomic Medicine (2017)
  • Author(s): Sasai H, Aoyama Y, Otsuka H, Abdelkreem E, Nakama M, Hori T, Ohnishi H, Turner L, Fukao T
  • Journal Title: Molecular Genetics and Genomic Medicine Volume: 5 Issue: 2 Pages: 177-184
  • DOI: 10.1002/mgg3.275
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Japanese Male Siblings with 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase Deficiency (HSD10 Disease) Without Neurological Regression (2017)
  • Author(s): Akagawa S, Fukao T, Akagawa Y, Sasai H, Kohdera U, Kino M, Shigematsu Y, Aoyama Y, Kaneko K
  • Journal Title: JIMD reports Volume: 32 Pages: 81-85
  • DOI: 10.1007/8904_2016_570
  • ISBN: 9783662543849, 9783662543856
  • Peer Reviewed / Open Access
• [Journal Article] Two Libyan siblings with beta-ketothiolase deficiency: A case report and review of literature (2017)
  • Author(s): Abdelkreem E, Alobaidy H, Aoyama Y, Mahmoud S, El Aal MA, Fukao T
  • Journal Title: The Egyptian Journal of Medical Human Genetics Volume: 18 Issue: 2 Pages: 100-203
  • DOI: 10.1016/j.ejmhg.2016.11.001
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Clinical and Mutational Characterizations of Ten Indian Patients with Beta-Ketothiolase Deficiency (2017)
  • Author(s): Abdelkreem E, Akella RR, Dave U, Sane S, Otsuka H, Sasai H, Aoyama Y, Nakama M, Ohnishi H, Mahmoud S, Abd El Aal M, Fukao T
  • Journal Title: JIMD reports Volume: 印刷中 Pages: 59-65
  • DOI: 10.1007/8904_2016_26
  • ISBN: 9783662558324, 9783662558331
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Turkish patient with Succinyl-CoA:3-oxoacid CoA transferase deficiency mimicking diabetic ketoacidosis. (2016)
  • Author(s): Erdol S, Tre M, Yakut T, Saglam H, Sasai H, Abdelkreem E, Ohtsuka H, Fukao T
  • Journal Title: Journal of Inborn Errors of Metabolism and Screening Volume: 4 Pages: 232640981665128-232640981665128
  • DOI: 10.1177/2326409816651281
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Exon 10 skipping in ACAT1 caused by a novel mutation (c.949G>A) located at an exonic splice enhancer site (2016)
  • Author(s): Otsuka H, Sasai H, Nakama M, Aoyama Y, Abdelkreem E, Ohnishi H, Konstantopoulou V, Sass JO, Fukao T
  • Journal Title: Molecular Medicine Reports Volume: 14 Pages: 4906-4910
  • Acknowledgement Compliant
• [Presentation] Defects in Ketone body metabolism and their newborn screening (2018)
  • Author(s): Fukao T
  • Organizer: The 18th Annual Meeting of Korean Society of Inherited Metabolic Diseases
  • Invited
• [Presentation] Recent advances of defect in ketone body metabolism (2018)
  • Author(s): Fukao T
  • Organizer: The 5th Asian Congress of Inherited Metabolic Diseases
  • Int'l Joint Research / Invited
• [Presentation] Beta-Ketothiolase deficiency: unusual clinical presentation of non-ketotic hypoglycemic episodes due to secondary carnitine deficiency (2018)
  • Author(s): Alijanpour M, Sasai H, Abdelkreem E, Ago Y, Soleimani S, Moslem L, Yamaguchi S, Rezapour M, Taghi M, Matsumoto H, Fukao T
  • Organizer: Annual symposium of the society for the study of inborn errors of metabolism
  • Int'l Joint Research
• [Presentation] Urinary organic acid profiles in mitochondrial HMG-CoA synthase deficiency (2018)
  • Author(s): Watanabe Y, Fukui K, Tashiro K, Sasai H, Fukao T, Hasegawa K, Y, Uchimura N, Yamashita Y
  • Organizer: Annual symposium of the society for the study of inborn errors of metabolism
• [Presentation] 絶食負荷による3ヒドロキシ酪酸脱水素酵素(Bdh1)KOマウスの病態解析 (2018)
  • Author(s): 大塚 博樹, 木村 豪, 吾郷 耕彦, 仲間 美奈, Abdelkreem Elsayed, 青山 友佳, 笹井 英雄, 大西 秀典, 大沢 匡毅, 川島 祐介, 小原 収, 山口 清次, 深尾 敏幸
  • Organizer: 日本小児科学会学術集会(第121回)
• [Presentation] Characterization of HMGCS2 identified in Japanese patients with its deficiency (2018)
  • Author(s): Ago Y, Otsuka H, Abdelkreem E, Sasai H, Nakama M, Aoyama Y, Nishimura Y, Watanabe Y, Fukui K, Akiyama K, Lee T, Nakajima Y, Ito T, Ohnishi H, Fukao T
  • Organizer: 日本先天代謝異常学会総会(第60回)
• [Presentation] 乳児期の3ヒドロキシ酪酸脱水素酵素(Bdh1）KOマウスにおける絶食負荷試験 (2018)
  • Author(s): 大塚 博樹, 木村 豪, 吾郷 耕彦, 仲間 美奈, 青山 友佳, Elsayed Abdelkreem, 松本 英樹, 笹井 英雄, 大西 秀典, 深尾 敏幸
  • Organizer: 日本先天代謝異常学会総会(第60回)
• [Presentation] Genetic Diseases of Ketone Body Metabolism (2017)
  • Author(s): Fukao T
  • Organizer: The 12th Asia-Pacific Conference on Human Genetics
  • Int'l Joint Research / Invited
• [Presentation] Insufficient Ketogenesis in 3-hydroxybutyrate Dehydrogenase (Bdh1) KO Mice in Fasting Test (2017)
  • Author(s): Otsuka H., Kimura T., Ago Y., Sasai H., Nakama M., Aoyama Y., Abrelkreem E., Ohnishi H., Osawa M., Yamaguchi S., Kawashima Y., Ohara O., Fukao T
  • Organizer: 3th the International Congress of Inborn Errors of Metabolism
  • Int'l Joint Research
• [Presentation] Heterozygous Carriers of Succinyl-CoA:3-oxoacid CoA Transferase Deficiency Can Develop Severe Ketoacidosis (2017)
  • Author(s): Sasai H., Aoyama Y., Otsuka H., Abdelkreem E., Naiki Y., Kubota M., Sekine Y., Itoh M., Nakama M., Ohnishi H., Fujiki R., Ohara O., Fukao T
  • Organizer: 13th the International Congress of Inborn Errors of Metabolism
  • Int'l Joint Research
• [Presentation] 絶食負荷試験において3ヒドロキシ酪酸脱水素酵素（Bdh1）KOマウスではケトン体産生が障害される (2017)
  • Author(s): 大塚 博樹, 木村 豪, 吾郷 耕彦, 仲間 美奈, Abdelkreem Elsayed, 青山 友佳, 笹井 英雄, 大西 秀典, 大沢 匡毅, 川島 祐介, 小原 収, 山口 清次, 深尾 敏幸
  • Organizer: 日本先天代謝異常学会(第59回)
• [Presentation] リコンビナントHSD17B10タンパクを用いたHSD10病の病態解析 (2017)
  • Author(s): 笹井 英雄, 大西 秀典, 赤川 翔平, 秋葉 和壽, 長谷川 行洋, 小林 正久, 大塚 博樹, 青山 友佳, 深尾 敏幸
  • Organizer: 日本先天代謝異常学会(第59回)
• [Presentation] Japan-wide gene panel study for target metabolic diseases in newborn mass screening using tandem mass spectrometry (2016)
  • Author(s): Sasai H, Otsuka H, Fujiki R, Ohara O, Nakajima Y, Ito T, Kobayashi M, Tajima G, Sakamoto O, Matsumoto S, Nakamura K, Hamazaki T, Kobayashi H, Hasegawa Y, Fukao
  • Organizer: Annual symposium of the society for the study of inborn erroros of metabolism
  • Place of Presentation: ローマ
  • Year and Date: 2016-09-06
  • Int'l Joint Research
• [Presentation] An IVS9-9T>A substitution identified in beta-ketothiolase deficient patients results in exon 10 skipping in most transcripts of ACAT1 gene (2016)
  • Author(s): Fukao T, Sasai H, Aoyama Y, Otsuka H, Abdelkreem E, Nakama M, Ohnishi H, Turner L, Sweetman L
  • Organizer: Annual symposium of the society for the study of inborn erroros of metabolism
  • Place of Presentation: ローマ
  • Year and Date: 2016-09-06
  • Int'l Joint Research
• [Presentation] マススクリーニング対象疾患の診療ネットワーク体制：分野別シンポジウム 先天代謝異常症の早期診断、治療に向けた診療ネットワーク (2016)
  • Author(s): 深尾敏幸
  • Organizer: 日本小児科学会学術集会 （第１１９回）
  • Place of Presentation: 札幌
  • Year and Date: 2016-05-13
  • Invited
• [Presentation] 治療法向上を目的としたマススクリーニング対象疾患の遺伝型評価：分野別シンポジウム 今後検討すべき新生児マススクリーニング対象疾患． (2016)
  • Author(s): 深尾敏幸
  • Organizer: 日本小児科学会学術集会 （第１１９回）
  • Place of Presentation: 札幌
  • Year and Date: 2016-05-13
  • Invited
• [Presentation] Disorders of Ketone Body Synthesis (2016)
  • Author(s): Fukao T
  • Organizer: The International Network for Fatty Acid Oxidation Research and Management (INFORM)
  • Place of Presentation: ボストン
  • Year and Date: 2016-05-09
  • Int'l Joint Research / Invited