コラーゲン遺伝子異常による心臓刺激伝導障害の新たな分子病態の解明

2016 Fiscal Year Research-status Report

• Project/Area Number: 15K15311
• Research Institution: Nagasaki University
• Principal Investigator: 蒔田 直昌 長崎大学, 医歯薬学総合研究科(医学系), 教授 (00312356)
• Co-Investigator(Kenkyū-buntansha): 牧山 武 京都大学, 医学研究科, 助教 (30528302)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: コラーゲン / 心臓伝導障害 / CRISPR/cas9 / ノックインマウス

Outline of Annual Research Achievements

1. Purkinje線維標識マウス心筋におけるCOL6A2の局在・経年変化：
Purkinje線維に特異的に発現するContactin2-EGFP融合タンパクのトランスジェニックマウスの左室をVI型コラーゲンで免疫組織染色した。胎生時にはVI型コラーゲンは心室筋細胞の全層にわたって発現していたが、年齢依存性に心内膜側に集簇するようになり、生後１月ではcontactin2と同様、ほぼ刺激伝導系に強く発現していた。
2. 進行性心臓伝導障害(PCCD)の網羅的遺伝子解析：
4世代日本人PCCD家系の全エクソン解析でCOL6A2, TTN, NUPL2変異が候補遺伝子と考えられた、さらに日本人PCCD 53家系に関して、200遺伝子のターゲットエクソン解析を行ったところ、COL6A2 変異キャリアが2人確認された。その他に、LMNA、SCN5A, GJC1などの遺伝子変異を確認した。COL6A2はPCCDの新たな疾患遺伝子の可能性が高くなった。
3.Col6A2ノックアウトマウスおよびCRISPR/Cas Col6a2ノックインマウスの心電図計測:
Col6a2 KO マウスを購入し、心電図を計測したがCRISPR/Casシステムを用いて上記PCCD家系に同定されたCOL66A2-変異P402Aノックインマウスを作製した。年齢6カ月のF2マウス(WT n=9, ヘテロn=17, ホモn=13)で心電図計測したが、心拍数、 PR時間, QRS幅, QTc時間いずれも差がなかった。伝導障害が強いとされるSvjストレインと交配したマウスでもその傾向は変わらなかった。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

遺伝子解析（エクソーム）、組織染色、ノックインマウス作成およびin vivo解析を完了した。想定と異なりネガティブな結果となったが、可能な限りの手段を取って解析を行った。

Strategy for Future Research Activity

心臓伝導障害の網羅的遺伝子解析で、COL6A2と同時にギャップジャンクション遺伝子GJC1（コネキシン45)の変異を同定した。房室ブロックの3世代日本人家系とフランス人新規発症例に同一の変異R75Hを同定し、その結果をin vitro,in vino解析し、論文に投稿した(Seki, Makita et al JACC, in revision)。

Causes of Carryover

H28年９月, iPS心筋細胞の機能解析に用いる予定の多電極細胞電気解析装置(MED64)が故障した。古いOSを使用していたため修理に時間を要し、その間のiPSの培養も中断しており、実験がおよそ３か月遅れた。H29年度にiPSの実験を行い、成果を年度内にまとめる。

Expenditure Plan for Carryover Budget

伝導障害の機能を見るために、すでに作成したiPS心筋細胞の培養に用いる試薬に充てる予定である。

Remarks

コラーゲンの仮説は否定的な結果になったが、同じ心臓伝導障害の遺伝子解析で重要な結果がでて、現在J Am Coll Cardiolにrevision中である。

Research Products

• [Int'l Joint Research] INSERM(フランス)
  • Country Name: FRANCE
  • Counterpart Institution: INSERM
• [Journal Article] Sick sinus syndrome with HCN4 mutations shows early onset and frequent association with atrial fibrillation and left ventricular noncompaction (2017)
  • Author(s): Ishikawa T, Ohno S, Murakami T, Yoshida K, Mishima H, Fukuoka T, Kimoto H, Sakamoto R, Ohkusa T, Aiba T, Nogami A, Sumitomo N, Shimizu W, Yoshiura KI, Horigome H, Horie M, Makita N
  • Journal Title: Heart Rhythm Volume: 14 Pages: 717-724.
  • DOI: 10.1016/j.hrthm.2017.01.020
  • Peer Reviewed
• [Journal Article] Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes (2017)
  • Author(s): Kuroda Y, Yuasa S, Watanabe Y, Ito S, Egashira T, Seki T, Hattori T, Ohno S, Kodaira M, Suzuki T, Hashimoto H, Okata S, Tanaka A, Aizawa Y, Murata M, Aiba T, Makita N, Furukawa T, Shimizu W, Kodama I, Ogawa S, Kokubun N, Horigome H, Horie M, Kamiya K, Fukuda K
  • Journal Title: Biochemistry and Biophysics Reports Volume: 9 Pages: 245-256.
  • DOI: 10.1016/j.bbrep.2017.01.002
  • Peer Reviewed
• [Journal Article] A novel de novo calmodulin mutation in a 6-year-old boy who experienced an aborted cardiac arrest (2017)
  • Author(s): Takahashi K, Ishikawa T, Makita N, Takefuta K, Nabeshima T, Nakayashiro M
  • Journal Title: HeartRhythm Case Reports Volume: 3 Pages: 69-72.
  • DOI: 10.1016/j.hrcr.2016.09.004
  • Peer Reviewed / Open Access
• [Journal Article] The Phenotypic Spectrum of a Mutation Hotspot Responsible for the Short QT Syndrome (2017)
  • Author(s): Hu D, Li Y, Zhang J, Pfeiffer R, Gollob MH, Healey J, Harrell DT, Makita N, Abe H, Sun Y, Guo J, Zhang L, Yan G, Mah D, Walsh EP, Leopold HB, Giustetto C, Gaita F, Zienciuk-Krajka A, Mazzanti A, Priori SG, Antzelevitch C, Barajas-Martinez H.
  • Journal Title: JACC: Clinical Electrophysiology Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1016/j.jacep.2016.11.013
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Genotype-Phenotype Correlation of SCN5A Mutation for the Clinical and Electrocardiographic Characteristics of Probands with Brugada Syndrome: A Japanese Multicenter Registry (2017)
  • Author(s): Yamagata K, Horie M, Aiba T, Ogawa S, Aizawa Y, Ohe T, Yamagishi M, Makita N, Sakurada H, Tanaka T, Shimizu A, Hagiwara N, Kishi R, Nakano Y, Takagi M, Makiyama T, Ohno S, Fukuda K, Watanabe H, Morita H, Hayashi K, Fukushima-Kusano K, Kamakura S, Yasuda S, Ogawa H, Y.
  • Journal Title: Circulation Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1161/CIRCULATIONAHA.117.027983
  • Peer Reviewed
• [Journal Article] Allele-specific Ablation Rescues Electrophysiological Abnormalities in a Human iPS Cell Model of Long-QT Syndrome with a CALM2 Mutation (2017)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Sasaki K, Wuriyanghai Y, Hayano M, Nishiuchi S, Kohjitani H, Hirose S, Chen J, Yokoi F, Ishikawa T, Ohno S, Chonabayashi K, Motomura H, Yoshida Y, Horie M, Makita N, Kimura T.
  • Journal Title: Hum Mol Genet Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1093/hmg/ddx073
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Progressive atrial conduction system defects associated with bone malformation caused by a connexin45 mutation (2017)
  • Author(s): Seki A, Ishikawa T, Daumy X, Mishima H, Barc J, Sasaki R, Nishii K, Saito K, Urano M, Ohno S, Otsuki S, Kimoto H, Baruteau A, Thollet A, Fouchard S, Bonnaud S, Parent P, Shibata Y, Perrin JP, Le Marec H, Hagiwara N, Mercier S, Horie M, Probst V, Yoshiura KI, Redon R, Schott JJ, Makita N.
  • Journal Title: J Am Coll Cardiol Volume: 印刷中 Pages: 印刷中
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Embryonic type Na+ channel beta-subunit, SCN3B masks the disease phenotype of Brugada syndrome. (2016)
  • Author(s): Okata S, Yuasa S, Suzuki T, Ito S, Makita N, Yoshida T, Li M, Kurokawa J, Seki T, Egashira T, Aizawa Y, Kodaira M, Motoda C, Yozu G, Shimojima M, Hayashiji N, Hashimoto H, Kuroda Y, Tanaka A, Murata M, Aiba T, Shimizu W, Horie M, Kamiya K, Furukawa T, Fukuda K.
  • Journal Title: Sci Rep Volume: 6 Pages: 34198
  • DOI: 10.1038/srep34198
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I (2016)
  • Author(s): Daumy, X.Amarouch MY, Lindenbaum P, Bonnaud S, Charpentier E, Bianchi B, Nafzger S, Baron E, Fouchard S, Thollet A, Kyndt F, Barc J, Le Scouarnec S, Makita N, Le Marec H, Dina C, Gourraud JB, Probst V, Abriel H, Redon R, Schott JJ.
  • Journal Title: Int J Cardiol Volume: Int J Cardiol Pages: 349-358
  • DOI: 10.1016/j.ijcard.2016.01.052
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Inherited bradyarrhythmia: A diverse genetic background (2016)
  • Author(s): Ishikawa T, Tsuji Y, Makita N.
  • Journal Title: J Arrhythmia Volume: 32 Pages: 352-358
  • DOI: 10.1016/j.joa.2015.09.009
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Variants in the SCN5A Promoter Associated With Various Arrhythmia Phenotypes (2016)
  • Author(s): Yagihara N, Watanabe H, Barnett P, Makita N. et. al
  • Journal Title: J Am Heart Assoc Volume: 5 Pages: e003644
  • DOI: 10.1161/JAHA.116.003644
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Reply: Search for Evidence-Based Medicine for Brugada Syndrome: The Complex Network of the Brugada Syndrome (2016)
  • Author(s): Nademanee K, Raju H, De Noronha S, Papadakis M, Robinson L, Rothery S, Makita N, Kowase S, Boonmee N, Vitayakritsirikul V, Ratanarapee S, Sharma S, van der Wal A. C. Christiansen M, Tan H. L, Wilde A. A, Nogami A, Sheppard M. N, Veerakul G, Behr E. R.
  • Journal Title: J Am Coll Cardiol Volume: 67 Pages: 1658-1659
  • DOI: 10.1016/j.jacc.2016.01.032
  • Peer Reviewed / Int'l Joint Research
• [Presentation] Multinational genome-wide association study in long QT syndrome identifies a role for common genetic variation in disease susceptibility and points to a polygenic architecture in mutation-negative cases (2017)
  • Author(s): Lahrouchi N, Tadros R, Makita N. et al
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Place of Presentation: Chicago, USA
  • Year and Date: 2017-05-12 – 2017-05-12
  • Int'l Joint Research
• [Presentation] Sick sinus syndrome caused by HCN4 mutations shows early onset and frequent association with atrial fibrillation and left ventricular non-compaction (2017)
  • Author(s): Ishikawa T, Ohno S, Murakami T, Yoshida K, Fukuoka T, Mishima H, Kimoto H, Aiba T, Nogami A, Sumitomo N, Shimizu W, Horigome H, Yoshiura K. I, Horie M, Makita N.
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Place of Presentation: Chicago, USA
  • Year and Date: 2017-05-11 – 2017-05-11
  • Int'l Joint Research
• [Presentation] De novo and Familial Connexin45 Mutant R75H Causes Progressive Atrioventricular Block Associated with Craniofacial and Dentodigital Dysmorphisms (2017)
  • Author(s): Makita N, Seki A, Ishikawa T, Schott JJ. et al.
  • Organizer: Printemps de la Cardiologie Recherche Fondamentale et Clinique
  • Place of Presentation: La Cite des Congres, Nantes, France
  • Year and Date: 2017-04-06 – 2017-04-06
  • Invited
• [Presentation] Calmodulin Mutations Responsible for Long QT Syndrome and Functional Rescue using a CRISPR/Cas9 System (2017)
  • Author(s): Makita N.
  • Organizer: 第94回日本生理学会大会
  • Place of Presentation: アクトシティ浜松（静岡県浜松市）
  • Year and Date: 2017-03-28 – 2017-03-28
  • Invited
• [Presentation] Mutations in Desmin Gene Uncover Phenotypic Overlap between Progressive Cardiac Conduction Defect with Muscular Dystrophy and Cardiomyopathy (2017)
  • Author(s): 木本浩樹, 町田紘子, 森田宏, 住友直方, 中村一文, 伊藤浩, バーク・ジュリアン, ショット・ジョンジャック, 蒔田直昌.
  • Organizer: 第81回日本循環器学会学術集会
  • Place of Presentation: 石川県立音楽堂音楽堂（石川県金沢市）
  • Year and Date: 2017-03-17 – 2017-03-17
• [Presentation] Genetic Mutation of Brugada Syndrome (2017)
  • Author(s): Makita N.
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Presentation] Brugada Syndrome: Basic and Clinical Updates, Advancement of Basic Research (2017)
  • Author(s): Makita N.
  • Organizer: 13th Annual Congress European Cardiac Arrhythmia Society
  • Int'l Joint Research / Invited
• [Presentation] Genetics of Familial Atrial Fibrillation (2017)
  • Author(s): Ishikawa T, Makita N.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Genetic-based risk stratification for cardiac disorders in LMNA mutation carriers (2017)
  • Author(s): Nishiuchi S, Aiba T, Nakajima K, Hirose S, Kohjitani H, Yamamoto Y, Harita T, Hayano M, Sasaki K, Ishikawa T, Onoue K, Saito Y, Kusano K, Makita N, Shimizu W, Horie M, Kimura T.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Genotype-Phenotype Correlation of SCN5A Mutations for the Clinical and Electrocardiographic Characteristics of Probands with Brugada Syndrome: A Japanese Multicenter Registry (2017)
  • Author(s): Yamagata K, Horie M, Aiba T, Ogawa S, Aizawa Y, Ohe T, Yamagishi M, Makita N, Sakurada H, Tanaka T, Shimizu A, Hagiwara N, Kishi R, Nakano Y, Takagi M, Makiyama T, Ohno S, Fukuda K, Watanabe H, Morita H, Hayashi K, Fukushima-Kusano K, Kamakura S, Yasuda S, Ogawa H, Y. M, Kapplinger J, Ackerman M, Shimizu W.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation (2017)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Sasaki K, Wuriyanghai Y, Nishiuchi S, Kohjitani H, Hirose S, Ishikawa T, Motomura H, Ohno S, Yoshida Y, Horie M, Makita N, Kimura T.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Mutation Spot-sensitive Clinical Features and Risk of Life-threatening Arrhythmia in Long QT Syndrome Type 1 in Japan (2017)
  • Author(s): Aiba T, Makimoto H, Yagihara N, Watanabe H, Ohno S, Hayashi K, Sumitomo N, Yoshinaga M, Morita H, Miyamoto Y, Makita N, Horie M, Yasuda S, Kusano K, Shimizu W.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] International Calmodulinopathy Registry (ICaMR) (2016)
  • Author(s): Crotti L, Makita N.
  • Organizer: American Heart Association's Scientific Sessions
  • Place of Presentation: New Orleands, USA
  • Year and Date: 2016-11-15 – 2016-11-15
• [Presentation] ECG Screening of 1-month-old Infants May Prevent Out-of-hospital Cardiac Arrest in Infancy (2016)
  • Author(s): Yoshinaga M, Ohno S, Ushinohama H, Sato S, Miyamoto T, Tauchi N, Horigome H, Sumitomo N, Shiraishi H, Ichida F, Hata T, Nomura Y, Horie M, Makita N, Nagashima M.
  • Organizer: American Heat Association Scientific Meeting 2016
  • Place of Presentation: New Orleans Convention Center, USA
  • Year and Date: 2016-11-13 – 2016-11-13
• [Presentation] 歯牙骨格形成異常を合併する洞不全症候群に固定されたコネキシン45遺伝子変異と機能異常. (2016)
  • Author(s): 木本浩樹, 石川泰輔, 西井明子, 斎藤加代子, 三嶋博之, 大槻早紀, 辻幸臣, 吉浦孝一郎, 萩原誠久, 蒔田直昌.
  • Organizer: 第67回西日本生理学会
  • Place of Presentation: レインボー桜島（鹿児島県鹿児島市）
  • Year and Date: 2016-10-07 – 2016-10-07
• [Presentation] Catheter Ablation of Brugada Syndrome：Further Evidence of Conduction Delay in the Right Ventricular Subepicardium as Mechanism of Brugada ECG and Ventricular Fibrillation (2016)
  • Author(s): Nakagawa H, Sakamoto Y, Yamashiro K, Takagi M, Kusano K, Noda T, Yamazaki M, Honjo H, Makita N, Tsuchiya T, Hoogeudiik MG, Nademanee K.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-17 – 2016-07-17
• [Presentation] Novel de novo Calmodulin Mutation in a Pre-school boy Experiencing Aborted Cardiac Arrest (2016)
  • Author(s): Takahashi K, Makita N.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-17 – 2016-07-17
• [Presentation] Common genetic variants at SCN5A, SCN10A, and HEY2 are associated with cardiac conduction disturbance in patients with Brugada syndrome (2016)
  • Author(s): Murakoshi N, Xu D, Nogami A, Makita N, Sekiguchi Y, Kurosaki K, Kowase S, Naruse Y, Aonuma K.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-16 – 2016-07-16
• [Presentation] Modeling of long-QT syndrome associated with a calmodulin mutation using human induced pluripotent stem cells (2016)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Sasaki K, Hayano M, Nishiuchi S, Wuriyanghai Y, Kohjitani H, Hirose S, Cheng J, Ishikawa T, Ohno S, Yoshida Y, Horie M, Makita N, Kimura T.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-16 – 2016-07-16
• [Presentation] Selective therapeutic targeting of ion channel rare variants predisposing to lone atrial fibrillation (2016)
  • Author(s): Hayashi K, Fujino N, Tsuda T, Tanaka Y, Ino H, Makita N, Yamagishi M.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-15 – 2016-07-15
• [Presentation] Broader Genetic Spectrum of Familial Atrial Arrhythmias Involving Rare Variations in the Common Arrhythmia-Susceptible Genes (2016)
  • Author(s): Ishikawa T, Mishima H, Ohno S, Harrell DT, Tsuji Y, Yoshiura K, Horie M, Makita N.
  • Organizer: 第63回日本不整脈心電学会学術大会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2016-07-15 – 2016-07-15
• [Presentation] Overview of Genes Related to Cardiac Conduction (2016)
  • Author(s): Makita N.
  • Organizer: Korean Heart Rhythm Society 8th Annual Scientific Session
  • Place of Presentation: KINTEX,Korea
  • Year and Date: 2016-07-08 – 2016-07-08
  • Int'l Joint Research / Invited
• [Presentation] Genetic Background of Inherited Bradyarrhythmia (2016)
  • Author(s): Makita N.
  • Organizer: Korean Heart Rhythm Society 8th Annual Scientific Session
  • Place of Presentation: KINTEX,Korea
  • Year and Date: 2016-07-08 – 2016-07-08
  • Int'l Joint Research / Invited