Pyrosequencing using liver biopsy specimens for evaluating pathogenesis of cholestatic liver diseases.

• Project/Area Number: 16H05283
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Yamagata University
• Principal Investigator: Ueno Yoshiyuki 山形大学, 医学部, 教授 (70282126)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2018: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2017: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2016: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000)
• Keywords: 外来性遺伝子 / レーザーキャプチャーマイクロダイセクション法 / 次世代シーケンサー / 胆汁うっ滞性肝疾患 / 腸内細菌 / 次世代シーケンシング / 胆汁うっ滞 / 原発性胆汁性胆管炎 / 16S rRNA / 16SRNA / ピロシーケンス / LCM / 内科 / 肝臓病学

Outline of Final Research Achievements

We have extracted DNAs from paraffin embedded pathological specimens from patients with cholestatic liver diseases such as primary biliary cholangitis. We have used laser micro-dissection methods to extract DNAs, and constructed the libraries from deep sequencing. Similarly, we extracted DNAs from serum samples with identical patients, and compared their libraries along with their clinical backgrounds. After adequate adjustments, we have found novel external 16rRNAs from both liver and serum samples. We have identified these pathogens at phyrum levels. To further identify these exogenous genes, adequate primer designing seems to be feasible.

Academic Significance and Societal Importance of the Research Achievements

厚労省の指定難病となっている原発性胆汁性胆管炎などの胆汁うっ滞性肝疾患の病態形成期所を明らかにするために肝病理組織標本から微量の核酸検体を抽出し、次世代シーケンサーで外来性遺伝子を検出する系を作成した。さらに、同様の手法を用いて、血液中からの検体も抽出し、簡素織病本からのものと整合性がとれるかについても検討した。その結果これまでに報告されていない、「門」レベルでの外来性微生物由来の遺伝子を再現性を持って検出した。さらに疾患特異性を検証するため、適切な疾患コントロールを用いて検討し、今回の手法の特異性も確認した。こんごは「種」レベルで外来遺伝子を同定することが必要と考えられた。

Research Products

• [Journal Article] Symptoms and health-related quality of life in Japanese patients with primary biliary cholangitis (2018)
  • Author(s): Yagi, M. Tanaka, A. Abe, M. Namisaki, T. Yoshiji, H. Takahashi, A. Ohira, H. Komori, A. Yamagiwa, S. Kikuchi, K. Yasunaka, T. Takaki, A. Ueno, Y. Honda, A. Matsuzaki, Y. Takikawa, H.
  • Journal Title: Sci Rep Volume: 8 Issue: 1 Pages: 12542-12542
  • DOI: 10.1038/s41598-018-31063-8
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Activation of Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholestrol Gallstones. (2017)
  • Author(s): Asai Y, Yamada T, Tsukita S, Takahashi K, Maekawa M, Honma M, Ikeda M, Murakami K, Munakata Y, Shirai Y, Kodama S, Sugisawa T, Chiba Y, Kondo Y, Kaneko K, Uno K, Sawada S, Imai J, Nakamura Y, Yamaguchi H, Tanaka K, Sasano H, Mano N, Ueno Y, Shimosegawa T, Katagiri H
  • Journal Title: Gastroenterology Volume: 印刷中 Issue: 6 Pages: 30012-4
  • DOI: 10.1053/j.gastro.2017.01.001
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Autonomic Nervous System and the Liver (2017)
  • Author(s): Kei Mizuno
  • Journal Title: Hepatology Research Volume: 47 Issue: 2 Pages: 160-165
  • DOI: 10.1111/hepr.12760
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population. (2017)
  • Author(s): Kawashima M, Honda M, Nakamura M. et al.
  • Journal Title: Hum Mol Genet. Volume: - Pages: 650-659
  • DOI: 10.1093/hmg/ddw406
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] NGSを用いたPBCの病型診断 (2016)
  • Author(s): 勝見智大、上野義之
  • Journal Title: 肝胆膵 Volume: 73 Pages: 733-739