Genetic analysis and investigation of molecular pathogenesis for mitochondrial diseases

• Project/Area Number: 17F17714
• Research Category: Grant-in-Aid for JSPS Fellows
• Allocation Type: Single-year Grants
• Section: 外国
• Research Field: Pathological medical chemistry
• Research Institution: Juntendo University (2018); Saitama Medical University (2017)
• Host Researcher: 岡崎 康司 順天堂大学, 医学(系)研究科(研究院), 教授 (80280733)
• Foreign Research Fellow: LIM SZE CHERN 順天堂大学, 医学(系)研究科(研究院), 外国人特別研究員
• Project Period (FY): 2017-07-26 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥2,300,000 (Direct Cost: ¥2,300,000); Fiscal Year 2018: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2017: ¥1,200,000 (Direct Cost: ¥1,200,000)
• Keywords: Mitochondria / Human genetics / Molecular biology

Outline of Annual Research Achievements

Whole exome sequencing of mitochondrial disease patients was performed in our laboratory. In fiscal year 2018, I investigated candidate mutations in 4 genes/gene cluster for their pathogenicity in 9 patients. Bioinformatic, molecular genetic and biochemical analyses including RNA sequencing, Sanger sequencing, DNA cloning, qPCR, SDS-PAGE and BN-PAGE western blotting, respiration rate analysis and OXPHOS enzyme assays were performed.
We detected large chromosomal deletions and rearrangements of the gene region in several patients with confirmed or suspected mitochondrial disease. My research highlighted the technical challenge in elucidating the exact gDNA mutations in the gene cluster region. Molecular diagnosis was confirmed in 2 of the 5 patients investigated. This is an on-going research in collaboration with Prof. David Thorburn from Murdoch Children’s Research Institute in Australia.
One of the novel disease genes was investigated in collaboration with Dr Diana Stojanovski from the University of Melbourne in Australia. As a result, the variant was excluded from further analysis in two patients due to the lack of supportive evidence for pathogenicity.
Two novel DNA variants in a nuclear gene encoding an OXPHOS complex III subunit were identified in a patient with Leigh Syndrome. Genomic DNA, RNA and protein analyses were performed to identify evidence for pathogenicity caused by those 2 compound heterozygous variants in the patient. This study led to an on-going collaboration with Prof. David Thorburn. A joint-publication is in preparation.

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Murdoch Children’s Research Institute/The University of Melbourne(オーストラリア)
• [Int'l Joint Research] University of Melbourne(Australia)
• [Journal Article] Mitochondrial ribosomal protein PTCD3 mutations cause oxidative phosphorylation defects with Leigh syndrome. (2019)
  • Author(s): Borna NN, Kishita Y, Kohda M, Lim SC, Shimura M, Wu Y, Mogushi K, Yatsuka Y, Harashima H, Hisatomi Y, Fushimi T, Ichimoto K, Murayama K, Ohtake A, Okazaki Y.
  • Journal Title: neurogenetics Volume: 20(1) Pages: 9-25
  • DOI: 10.1007/s10048-018-0561-9
  • Peer Reviewed
• [Presentation] Genetic causes of mitochondrial oxidative phosphorylation (OXPHOS) disorders. (2018)
  • Author(s): Sze Chern Lim
  • Organizer: Wellcome Genome Campus Advanced Course: NGS Bioinformatics, Hinxton in UK.
  • Int'l Joint Research
• [Presentation] Providing molecular diagnosis to patients with mitochondrial oxidative phosphorylation disease using functional studies following next generation sequencing. (2018)
  • Author(s): Sze Chern Lim, Yoshihito Kishita, Masakazu Kohda, Tomoko Hirata, Yukiko Yatsuka, Hiroko Harashima, Kei Murayama, Akira Ohtake and Yasushi Okazaki.
  • Organizer: Variant Effect Prediction Training Course, Johor in Malaysia.
  • Int'l Joint Research
• [Presentation] Identification of the genetic causes of mitochondrial oxidative phosphorylation (OXPHOS) disease (2017)
  • Author(s): Sze Chern Lim, Yoshihito Kishita, Masakazu Kohda, Yosuke Mizuno, Tomoko Hirata, Yukiko Yatsuka, Nurun Nahar Borna, Hiroko Harashima, Kei Murayama, Akira Ohtake and Yasushi Okazaki.
  • Organizer: Annual Meeting of the American Society of Human Genetics 2017
  • Int'l Joint Research
• [Presentation] Investigation of causative genes for mitochondrial oxidative phosphorylation (OXPHOS) disorders (2017)
  • Author(s): Sze Chern Lim, Yoshihito Kishita, Masakazu Kohda, Tomoko Hirata, Yukiko Yatsuka, Atsuko Imai-Okazaki, Hiroko Harashima, Masaru Shimura, Kei Murayama, Akira Ohtake and Yasushi Okazaki.
  • Organizer: The 59th Annual Meeting of the Japanese Society for Inherited Metabolic Diseases/The 15th Asian Symposium of Inherited Metabolic Diseases
  • Int'l Joint Research