Development of a new meaningful clinical bacteriological test based on metagenomic and DNA methylation analysis

• Project/Area Number: 18K07408
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 52010:General internal medicine-related
• Research Institution: Chiba University
• Principal Investigator: Nishimura Motoi 千葉大学, 医学部附属病院, 講師 (80400969)
• Co-Investigator(Kenkyū-buntansha): 佐藤 守 千葉大学, 医学部附属病院, 特任准教授 (20401002) ; 田中 知明 千葉大学, 大学院医学研究院, 教授 (50447299) ; 別府 美奈子 千葉大学, 大学院医学研究院, 助教 (70623669) ; 松下 一之 千葉大学, 医学部附属病院, 准教授 (90344994) ; 土田 祥央 日本大学, 医学部, 助手 (90410422)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2019: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2018: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: 臨床検査 / microbiome / bacterial epigenetics / 細菌検査 / メタゲノム / メチル化解析 / 新型コロナウイルス感染症 / COVID-19 / SARS-CoV-2

Outline of Final Research Achievements

DNA methylation analysis can now be combined with conventional (Sanger method) 16S rDNA analysis and biome analysis of cross-species 16S rRNA genes using a next-generation sequencer.
This analysis in drug-resistant strains and bacterial flora revealed the presence of previously unreported DNA methylation and novel DNA-modifying enzymes. We have also succeeded in analyzing the microbiome of the Fecalis-positive clinical flora, and have obtained results that can be interpreted as a mixture of multiple strains with diverse DNA methylation patterns.

Academic Significance and Societal Importance of the Research Achievements

次世代シーケンシングの普及に伴って、検体中の核酸断片を網羅的に解析することによるバイオーム解析、いわゆるメタゲノム解析が可能となり、もっとも先進的な解析では１分子シーケンサーなどによるDNAメチル化解析と並行的にお紺われるようになっている。。しかしながらこれら核酸断片に由来する配列情報は、多く由来する菌種が不明で、細菌種同定を柱とする感染症臨床検査としては非効率的で相性が悪かった。
本研究成果は臨床検査と相性が良くその延長として可能な従来なかった"16S rDNA meta-epigenetics"とでも呼ぶべき分野の登場を示している。

Research Products

• [Journal Article] Tracking SARS-CoV-2 variants by entire S-gene analysis using long-range RT-PCR and Sanger sequencing (2022)
  • Author(s): Matsubara Mirai、Imaizumi Yuri、Fujikawa Tatsuki、Ishige Takayuki、Nishimura Motoi、Miyabe Akiko、Murata Shota、Kawasaki Kenji、Taniguchi Toshibumi、Igari Hidetoshi、Matsushita Kazuyuki
  • Journal Title: Clinica Chimica Acta Volume: 530 Pages: 94-98
  • DOI: 10.1016/j.cca.2022.03.014
  • Peer Reviewed
• [Journal Article] DRD2 Taq1A Polymorphism-Related Brain Volume Changes in Parkinson’s Disease: Voxel-Based Morphometry (2022)
  • Author(s): Ohira K, Yokota H, Hirano S, Nishimura M, Mukai H, Horikoshi T, Sawai S, Yamanaka Y, Yamamoto T, Kakeda S, Kuwabara S, Tanaka T, Uno T.
  • Journal Title: Parkinson's Disease Volume: - Pages: 1-7
  • DOI: 10.1155/2022/8649195
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive mutational analysis of background mucosa in patients with Lugol‐voiding lesions (2021)
  • Author(s): Akizue Naoki、Okimoto Kenichiro、Arai Makoto、Hirotsu Yosuke、Amemiya Kenji、Oura Hirotaka、Kaneko Tatsuya et al
  • Journal Title: Cancer Medicine Volume: 10 Issue: 11 Pages: 3545-3555
  • DOI: 10.1002/cam4.3905
  • Peer Reviewed / Open Access
• [Journal Article] Genetic profiles of Barrett’s esophagus and esophageal adenocarcinoma in Japanese patients (2021)
  • Author(s): Tokunaga Mamoru、Okimoto Kenichiro、Akizue Naoki、Ishikawa Kentaro、Hirotsu Yosuke、Amemiya Kenji、Ota Masayuki、Matsusaka Keisuke、et al.
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 17671-17671
  • DOI: 10.1038/s41598-021-97249-9
  • Peer Reviewed / Open Access
• [Journal Article] TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia. (2021)
  • Author(s): Ochiiwa H, Ailiken G, Yokoyama M, Yamagata K, Nagano H, Yoshimura C, Muraoka H, Ishida K, Haruma T, Nakayama A, Hashimoto N, Murata K, Nishimura M, Kawashima, Y, Ohara O, Okubo S, Tanaka T
  • Journal Title: Oncogene Volume: 40 Issue: 7 Pages: 1217-1230
  • DOI: 10.1038/s41388-020-01586-4
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive mutational analysis of background mucosa in patients with Lugol-voiding lesions (2021)
  • Author(s): Akizue, N; Okimoto, K; Arai, M; Hirotsu, Y; Amemiya, K; Oura, H; Kaneko, T; Tokunaga, M; Ishikawa, K; Ohta, Y Taida, T; Saito, K; Maruoka, D; Matsumura, T; Nakagawa, T; Nishimura, M ; Chiba, T; Matsushita, K; Mochizuki, H; Yokosuka, O; Omata, M; Kato, N
  • Journal Title: Cancer Medicine Volume: in press
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive Analysis of Barrett's Esophagus: Focused on Carcinogenic Potential for Barrett's Cancer in Japanese Patients (2020)
  • Author(s): Kentaro Ishikawa , Kenichiro Okimoto, et al.
  • Journal Title: Dig Dis Sci . Volume: epub ahead of printing Issue: 8 Pages: 2674-2681
  • DOI: 10.1007/s10620-020-06563-1
  • Peer Reviewed / Open Access
• [Journal Article] Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories (2020)
  • Author(s): Ishige Takayuki、Murata Shota、Taniguchi Toshibumi、Miyabe Akiko、Kitamura Kouichi、Kawasaki Kenji、Nishimura Motoi、Igari Hidetoshi、Matsushita Kazuyuki
  • Journal Title: Clinica Chimica Acta Volume: 507 Pages: 139-142
  • DOI: 10.1016/j.cca.2020.04.023
  • Peer Reviewed / Open Access
• [Journal Article] Monoamine Oxidase B rs1799836 G Allele Polymorphism Is a Risk Factor for Early Development of Levodopa-Induced Dyskinesia in Parkinson's Disease. (2020)
  • Author(s): Kakinuma S, Beppu M, Sawai S, Nakayama A, Hirano S, Yamanaka Y, Yamamoto T, Chigusa M, Aisihaer X, Aersilan A, Gao Y, Sato K, Itoga S, Ishige T, Nishimura M, Matsushita K, Satoh M, Nomura F, Kuwabara S, Tanaka T
  • Journal Title: eNeurologicalSci. Volume: 19 Pages: 100239-100239
  • DOI: 10.1016/j.ensci.2020.100239
  • Peer Reviewed / Open Access
• [Journal Article] Monoamine oxidase B rs1799836 G allele polymorphism is a risk factor for early development of levodopa-induced dyskinesia in Parkinson's disease. (2020)
  • Author(s): Kakinuma S, BeppuM, Sawai S, Akitoshi Nakayama A, Hirano S, Yamanaka Y, Yamamoto T, Chigusa M, Aisihaer X, Aersilan, A,Y Gao Y, Sato K, Itoga S, Ishige T, Nishimura M, Matsushita K, Satoh M, Nomura F, Kuwabara S, Tanaka T.
  • Journal Title: eNeurologicalSci. Volume: in press
  • Peer Reviewed / Open Access
• [Journal Article] Central blood pressure and pulse wave velocity in young and middle-aged Japanese adults with isolated systolic hypertension (2019)
  • Author(s): Nakagomi Atsushi、Imazeki Fumio、Nishimura Motoi、Sawabe Yuji、Matsushita Kazuyuki、Murata Akitoshi、Watase Mariko、Okada Sho、Kobayashi Yoshio
  • Journal Title: Hypertension Research Volume: 43 Issue: 3 Pages: 207-212
  • DOI: 10.1038/s41440-019-0364-x
  • Peer Reviewed / Open Access
• [Journal Article] Multiplex PCR and multicolor probes melting for the simultaneous detection of five UGT1A1 variants (2019)
  • Author(s): Ishige Takayuki、Itoga Sakae、Kawasaki Kenji、Sawai Setsu、Nishimura Motoi、Nomura Fumio、Matsushita Kazuyuki
  • Journal Title: Analytical Biochemistry Volume: 587 Pages: 113448-113448
  • DOI: 10.1016/j.ab.2019.113448
  • Peer Reviewed / Open Access
• [Journal Article] A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing (2019)
  • Author(s): Yao Yue、Nishimura Motoi、Murayama Kei、Kuranobu Naomi、Tojo Satomi、Beppu Minako、Ishige Takayuki、Itoga Sakae、Tsuchida Sachio、Mori Masato、Takayanagi Masaki、Yokoyama Masataka、Yamagata Kazuyuki、Kishita Yoshihito、Okazaki Yasushi、Nomura Fumio、Matsushita Kazuyuki、Tanaka Tomoaki
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 17411-17421
  • DOI: 10.1038/s41598-019-53449-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Development and validation of the simultaneous measurement of estrone and 17-β estradiol in serum by LC-MS/MS for clinical laboratory applications. (2019)
  • Author(s): Mamoru Satoh, Yui Miyabayashi, Masanori Seimiya, Takahiro Kazami, Takayuki Ishige, Toshihiko Yoshida, Motoi Nishimura, Kazuyuki Matsushita, Makio Shozu, Fumio Nomura.
  • Journal Title: Medical Mass Spectrometry Volume: 3 Pages: 19-30
  • NAID: 130007897964
  • Peer Reviewed
• [Journal Article] High-throughput genotyping of GC (vitamin D-binding protein) by melting analysis with locked nucleic acid-incorporating dual hybridization probe for improving mismatch discrimination. (2018)
  • Author(s): Ishige T, Satoh M, Itoga S, Nishimura M, Matsushita K, Nomura F.
  • Journal Title: Clin Chim Acta Volume: 487 Pages: 126-132
  • DOI: 10.1016/j.cca.2018.09.034
  • Peer Reviewed / Open Access
• [Journal Article] Surveillance evaluation of the standardization of assay values for serum total 25-hydroxyvitamin D concentration in Japan. (2018)
  • Author(s): Ihara H, Kiuchi S, Ishige T, Nishimura M, Matsushita K, Satoh M, Nomura F, Yamashita M, Kitajima I, Tsugawa N, Okano T, Hirota K, Miura M, Totani M, Hashizume N; Nutrition Division, Japan Society of Clinical Chemistry.
  • Journal Title: Ann Clin Biochem. Volume: Epub ahead of print Issue: 6 Pages: 0-0
  • DOI: 10.1177/0004563218765570
  • Peer Reviewed / Open Access
• [Journal Article] Assessment by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long-Term Sample Storage. (2018)
  • Author(s): Tsuchida S, Satoh M, Umemura H, Sogawa K, Takiwaki M, Ishige T, Miyabayashi Y, Iwasawa Y, Kobayashi S, Beppu M, Nishimura M, Kodera Y, Matsushita K, Nomura F
  • Journal Title: Proteomics. Clinical applications Volume: 15 Issue: 3 Pages: 47-53
  • DOI: 10.1002/prca.201700047
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Variant in C-terminal region of intestinal alkaline phosphatase associated with benign familial hyperphosphatasaemia. (2018)
  • Author(s): Ishige T, Itoga S, Utsuno E, Nishimura M, Yoshikawa M, Kato N, Matsushita K, Yokosuka O, Nomura F
  • Journal Title: Journal of medical genetics Volume: 10 Issue: 10 Pages: 47-53
  • DOI: 10.1136/jmedgenet-2017-104964
  • Peer Reviewed / Open Access
• [Presentation] 変異株同定のためのサンガー法によるSARS-CoV-2のS遺伝子の塩基配列解析 (2022)
  • Author(s): 松原未来、藤川樹、石毛崇之、西村基、村田正太、谷口俊文、猪狩英俊、川崎健治、松下一之
  • Organizer: 日本臨床微生物学会
• [Presentation] 小児横紋筋肉腫に対するがん遺伝子パネル検査(NCCオンコパネル)でLi-Fraumeni症候群と診断された２例 (2021)
  • Author(s): 力石浩志、日野もえ子、山下喜晴、奥主朋子、宇津野恵美、西村基、松下一之、服部真也、市川智彦、濱田洋通
  • Organizer: 日本人類遺伝学会
• [Presentation] 当院で検出された新型コロナウイルス(SARS-CoV-2)のRNA配列の解析 (2021)
  • Author(s): 西村 基、村田 正太、石毛 崇之、松原 未来、藤川 樹、宮部 安規子、北村 浩一、根川 真実、古家 若葉、梅谷 友輔、堀田 恵海、吉田 俊彦、川崎 健治、松下 一之
  • Organizer: 日本遺伝子診療学会
• [Presentation] SARS-CoV-2 S-gene sequencing and its mutational dynamics at the Chiba University Hospital (2021)
  • Author(s): 松原未来、藤川樹、石毛崇之、西村基、村田正太、谷口俊文、猪狩英俊、川崎健治、松下一之
  • Organizer: 日本遺伝子診療学会
• [Presentation] 当院で検出された新型コロナウイルス(SARS-CoV-2)のRNA配列の解析 (2021)
  • Author(s): 西村 基、石毛 崇之、吉田 俊彦、川崎 健治、松下 一之
  • Organizer: 日本臨床化学会
• [Presentation] SARS-CoV-2抗原定性検査(イムノクロマト法) (2021)
  • Author(s): 村田正太、宮部安規子、斎藤知子、瀬川俊介、堀田恵海、山下晃司、松原未来、鈴木眞、川崎健治、西村基、松下一之
  • Organizer: 日本感染症学会
• [Presentation] 新型コロナウイルスの全ゲノム解析におけるMinIONの性能評価 (2021)
  • Author(s): 藤川樹、石毛崇之、西村基、松原未来、村田正太、谷口俊文、猪狩英俊、川崎健治、松下一之
  • Organizer: 日本医療検査科学会
• [Presentation] デヂエを用いた多職種連携のためのがん遺伝子プロファイリング検査管理システムの構築 (2021)
  • Author(s): 北村 浩一, 藤澤陽子, 川崎 健治, 西村 基, 松下 一之
  • Organizer: 日本医療検査科学会
• [Presentation] 当院で検出された新型コロナウイルス(SARS-CoV-2)のRNA配列の解析 (2020)
  • Author(s): 西村基、村田正太、石毛崇之、宮部安規子、北村浩一、根川真実、藤川樹、古家若葉、梅谷友輔、堀田恵海、吉田俊彦、川崎健治、松下一之
  • Organizer: 第32回日本臨床微生物学会総会・学術集会
• [Presentation] 腫瘍部組織のみを調べるがん遺伝子パネル検査で二次的所見が疑われた３症例の当院における取り扱い (2020)
  • Author(s): 西村基、楯真一、錦見恭子、松岡歩、生水真紀夫、太和田暁之、新井誠人、滝口裕一、藤木亮次、金田篤志、宇津野恵美、市川 智彦、太田昌幸、松坂恵介、佐伯 宏美、藤澤陽子、石毛崇之、北村浩一、小林崇平、中津川智子、清水直美、松下一之
  • Organizer: 第44回日本遺伝カウンセリング学会学術集会
• [Presentation] 千葉大学医学部附属病院検査部における教育業務の担当と、そこからバンキングされた検体の研究への活用 (2019)
  • Author(s): 西村基、土田祥央、石毛崇之、浅野はるな、吉田俊彦、川崎健治 、糸賀栄 、澤部祐司 、野村文夫 、松下一
  • Organizer: 日本臨床検査医学会
• [Presentation] 遺伝学的にdefinitiveなQT延長症候群とてんかん様症状の合併症例の３例とそれらの考察 (2019)
  • Author(s): 西村 基, 江畑 亮太, 小林 弘信, 近藤 祐介, 仲野 美代,上田 希彦, 石川 亜貴, 水上 都, 澤井 摂,下条 直樹, 小林 欣夫, 野村 文夫, 松下 一之, 市川 智彦
  • Organizer: 日本遺伝カウンセリング学会
• [Presentation] 血液培養陽性検体よりの複数菌由来の核酸検出 (2018)
  • Author(s): 西村 基, 村田 正太, 姚 躍, 土田 祥央, 田中 知明, 松下 一之
  • Organizer: 第25回日本遺伝子診療学会大会