Disease-related genome analyses by long-read sequencers

2017 Fiscal Year Annual Research Report

• Project/Area Number: 17H01539
• Research Institution: Yokohama City University
• Principal Investigator: 松本 直通 横浜市立大学, 医学研究科, 教授 (80325638)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: 次世代シーケンス解析 / ロングリードシーケンス / 全ゲノム解析 / CNV / SNV

Outline of Annual Research Achievements

1. Sequelで得られた全ゲノムシーケンスデータの検討:18XカバレージのSequelシーケンスを1例で行い,7X,13X,18Xカバレージのデータでそれぞれ>50 bpの欠失・重複CNVをPBhoneyを用いて検討した。欠失CNVはそれぞれのカバレージで、4000個,7000個,8000個程度,重複CNVはそれぞれ6000個,10000個,11000個程度検出された。現状のSMRT cell(1枚20万円程度)を5枚程度使用し,コストはおよそ1サンプル当たり100万円で10xカバレージのシーケンス産出が可能であった。初年度予算では少なくても5症例のSequel解析を10Xカバレージで行った。
2. CNV検出に必要なゲノムカバレージ量とCNV検出系の確立：10XレベルのSequel算出ゲノムデータからランダムにCNV検出を進めた。特にUCSC遺伝子をinvolveするCNVに焦点を当てた。異なる疾患の症例間比較で重複しないCNVは1例当たり10個を超えなかった。カバレージが上がると検出CNVの数が上昇するため,コスト面と検出数のバランスから現状10Xカバレージを基準に解析を進めているが,コストが下がればさらにカバレージを挙げることで制度の向上を期待できる。
3. 高精度疾患ゲノム解析系の確立:ショート/ロングリードハイブリッドシーケンス:CNVを好感度に検出できるミニマルカバレージを10X程度としCNV解析を進めたが,これにショートリードシーケンスデータに組合わせて高感度なCNV検出系の確立を次年度より目指す予定である(ハイブリッドシーケンス)。

Current Status of Research Progress

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

Reason

当初想定していたSequelのコストであるが1 SMRT cell(1枚20万円程度)当たりのシーケンス産出量は5~10 Gbに到達し10x カバレージの全ゲノムシーケンスが100万円程度で可能となった。これで コントロール18xと原因未解明症例5サンプルの10xカバレージシーケンスを終了している。このデータ解析から判明したのは、検出するCNVはカバレージが上昇するほど増加する。検出する大量の7000個の欠失CNVと10000個の重複CNVの解釈に多数のコントロールデータが必要であることは明らかとなった。コスト面での低減を期待するが、現状容易でないことも予想されるため、異なるプラットフォームOxford NanoporeのPromethIONシーケンサーの導入も検討することが必要である。PromethIONで1 フローセル当たり50~80 GBものシーケンス産出が期待できる。

Strategy for Future Research Activity

大量の検出CNVの解釈には多数のコントロールデータが必要であることは明らかとなった。コスト面での低減を期待するが、現状容易でないことも予想されるため、異なるプラットフォームOxford NanoporeのPromethIONシーケンサーの導入も検討することが必要である。PromethIONで1 フローセル（1枚20万円程度）当たり50~80 GBものシーケンス産出が期待できる。次年度はSequelのシーケンス産出量の向上に期待しながらPromethIONをトライアルで検証し、十分なスループットが臨めればこちらに主軸を移すことも検討する。

Research Products

• [Journal Article] Integrative Analyses of De Novo Mutations Provide Deeper Biological Insights into Autism Spectrum Disorder. (2018)
  • Author(s): *Takata A, *Miyake N, *Tsurusaki Y (*: equal contribution), et al., Matsumoto N# (#: corresponding).
  • Journal Title: Cell Rep Volume: 22(3) Pages: 734-747
  • DOI: 10.1016/j.celrep.2017.12.074.
  • Peer Reviewed
• [Journal Article] A novel mutation in SLC1A3 causes episodic ataxia. (2018)
  • Author(s): Iwama K, Iwata A, Shiina M, Mitsuhashi S, Miyatake S, Takata A, Miyake N, Ogata K, Ito S, Mizuguchi T, Matsumoto N*.
  • Journal Title: J Hum Genet. Volume: 63(2) Pages: 207-211
  • DOI: 10.1038/s10038-017-0365-z.
  • Peer Reviewed
• [Journal Article] A familial case of PDE10A-associated childhood-onset chorea with bilateral striatal lesions. (2018)
  • Author(s): Miyatake S, Koshimizu E, Shirai I, Kumada S, Nakata Y, Kamemaru A, Nakashima M, Mizuguchi T, Miyake N, Saitsu H, Matsumoto N*.
  • Journal Title: Mov Disord Volume: 33(1) Pages: 177-179
  • DOI: 10.1002/mds.27219.
  • Peer Reviewed
• [Journal Article] De novo variants in SETD1B are associated with intellectual disability, epilepsy and autism. (2018)
  • Author(s): Hiraide T, Nakashima M, Yamoto K, Fukuda T, Kato M, Ikeda H, Sugie Y, Aoto K, Kaname T, Nakabayashi K, Ogata T, Matsumoto N*, Saitsu H* (*: co-correspondence).
  • Journal Title: Hum Genet Volume: 137(1) Pages: :95-104
  • DOI: 10.1007/s00439-017-1863-y.
  • Peer Reviewed
• [Journal Article] Epileptic apnea in a patient with inherited glycosylphosphatidylinositol anchor deficiency and PIGT mutations. (2018)
  • Author(s): Kohashi K, Ishiyama A*, Yuasa S, Tanaka T, Miya K, Adachi Y, Sato N, Saitsu H, Ohba C, Matsumoto N, Murakami Y, Kinoshita T, Sugai K, Sasaki M.
  • Journal Title: Brain Dev Volume: 40(1) Pages: 53-57
  • DOI: 10.1016/j.braindev.2017.06.005.
  • Peer Reviewed
• [Journal Article] A patient with early myoclonic encephalopathy (EME) with a de novo KCNQ2 mutation. (2018)
  • Author(s): Kojima K, Shirai K, Kobayashi M, Miyauchi A, Saitsu H, Matsumoto N, Osaka H, Yamagata T.
  • Journal Title: Brain Dev Volume: 40(1) Pages: 69-73
  • DOI: 10.1016/j.braindev.2017.06.004.
  • Peer Reviewed
• [Journal Article] Heterozygous Mutations in OAS1 Cause Infantile-Onset Pulmonary Alveolar Proteinosis with Hypogammaglobulinemia. (2018)
  • Author(s): Cho K#,*, Yamada M# (# denotes equal contribution) (*: correspondence), Agematsu K, Kanegane H, Miyake N, Ueki M, Akimoto T, Kobayashi N, Ikemoto S, Tanino M, Fujita A, Hayasaka I, Miyamoto S, Tanaka-Kubota M, Nakata K, Shiina M, Ogata K, Minakami H, Matsumoto N, Ariga T.
  • Journal Title: Am J Hum Genet Volume: 1;102(3) Pages: 480-486
  • DOI: 10.1016/j.ajhg.2018.01.019.
  • Peer Reviewed
• [Journal Article] Three patients with Schaaf-Yang syndrome exhibiting arthrogryposis and endocrinological abnormalities. (2018)
  • Author(s): Enya T*, Okamoto N, Iba Y, Miyazawa T, Okada M, Ida S, Naruto T, Imoto I, Fujita A, Miyake N, Matsumoto N, Sugimoto K, Takemura T.
  • Journal Title: Am J Med Genet A Volume: 176(3) Pages: :707-711
  • DOI: doi: 10.1002/ajmg.a.38606.
  • Peer Reviewed
• [Journal Article] Novel recessive mutations in MSTO1 cause cerebellar atrophy with pigmentary retinopathy. (2018)
  • Author(s): Iwama K, Takaori T, Fukushima A, Tohyama J, Ishiyama A, Ohba C, Mitsuhashi S, Miyatake S, Takata A, Miyake N, Ito S, Saitsu H, Mizuguchi T, Matsumoto N*.
  • Journal Title: J Hum Genet Volume: 63(3) Pages: 263-270
  • DOI: 10.1038/s10038-017-0405-8.
  • Peer Reviewed
• [Journal Article] A novel PGAP3 mutation in a Croatian boy with brachytelephalangy and a thin corpus callosum. (2018)
  • Author(s): Sakaguchi T, Zigman T, Petkovic; Ramadza D, Omerza L, Puseljic; S, Eres Hrvacanin Z, Miyake N, Matsumoto N, Bariic I.
  • Journal Title: Hum Genome Var Volume: 8;5 Pages: 18005
  • DOI: 10.1038/hgv.2018.5.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment. (2017)
  • Author(s): Satake T, Yamashita K, Hayashi K, Miyatake S, Tamura-Nakano M, Doi H, Furuta Y, Shioi G, Miura E, Takeo YH, Yoshida K, Yahikozawa H, Matsumoto N, Yuzaki M, Suzuki A*.
  • Journal Title: EMBO J Volume: 36(9) Pages: 1227-1242
  • DOI: 10.15252/embj.201695630.
  • Peer Reviewed
• [Journal Article] Mutations in genes encoding polycomb repressive complex 2 subunits cause Weaver syndrome. (2017)
  • Author(s): Imagawa E, Higashimoto K, Sakai Y, Numakura C, Okamoto N, Matsunaga S, Ryo A, Sato Y, Sanefuji M, Ihara K, Takada Y, Nishimura G, Saitsu H, Mizuguchi T, Miyatake S, Nakashima M, Miyake N, Soejima H, Matsumoto N*.
  • Journal Title: Hum Mut Volume: 38(6) Pages: 637-648
  • DOI: 10.1002/humu.23200.
  • Peer Reviewed
• [Journal Article] Defects in autophagosome-lysosome fusion underlie Vici syndrome, a neurodevelopmental disorder with multisystem involvement. (2017)
  • Author(s): #Hori I, #Otomo T, (# denotes equal contribution) et al., Matsumoto N, et al..
  • Journal Title: Sci Rep Volume: 7 Pages: 3552
  • DOI: 10.1038/s41598-017-02840-8.
  • Peer Reviewed / Open Access
• [Journal Article] Nephron development and extrarenal features in a child with congenital nephrotic syndrome caused by null LAMB2 mutations. (2017)
  • Author(s): Kino J, *Tsukaguchi H, Kimata T, Nguyen HT, Nakano Y, Miyake N, Matsumoto N, Kaneko K.
  • Journal Title: BMC Nephrol Volume: 6;18(1) Pages: 220
  • DOI: 10.1186/s12882-017-0632-4.
  • Peer Reviewed
• [Journal Article] Dystonia due to bilateral caudate hemorrhage associated with a COL4A1 mutation. (2017)
  • Author(s): *Hatano T, Daida K, Hoshino Y, Li Y, Saitsu H, Matsumoto N, Hattori N.
  • Journal Title: Parkinsonism Relat Disord Volume: 40 Pages: 80-82
  • DOI: 10.1016/j.parkreldis.2017.04.009.
  • Peer Reviewed
• [Journal Article] ANKRD11 variants cause variable clinical features associated with KBG syndrome and Coffin-Siris-like syndrome. (2017)
  • Author(s): Miyatake S, Okamoto N, Stark Z, Nabetani M, Tsurusaki Y, Nakashima M, Miyake N, Mizuguchi T, Ohtake A, Saitsu H, Matsumoto N*.
  • Journal Title: J Hum Genet Volume: 62(8) Pages: 741-746
  • DOI: 10.1038/jhg.2017.24.
  • Peer Reviewed
• [Journal Article] Delayed myelination is a unique clinical marker for CYP2U1-related spastic paraplegia. (2017)
  • Author(s): Minase G, Miyatake S, Nabatame S, Arai H, Koshimizu E, Mizuguchi T, Nakashima M, Miyake N, Saitsu H, Miyamoto T, Sengoku K, Matsumoto N*.
  • Journal Title: J Hum Genet Volume: 62(11) Pages: 997-1000
  • DOI: 10.1038/jhg.2017.77.
  • Peer Reviewed
• [Journal Article] A patient with Muenke syndrome manifesting migrating neonatal seizures. (2017)
  • Author(s): Okubo Y*, Kitamura T, Anzai M, Endo W, Inui T, Takezawa Y, Suzuki-Muromoto S, Miyabayashi T, Togashi N, Oba H, Saitsu H, Matsumoto N, Haginoya K.
  • Journal Title: Brain Dev Volume: 39(10) Pages: 873-876
  • DOI: 10.1016/j.braindev.2017.05.007.
  • Peer Reviewed
• [Journal Article] Equivalent missense variant in the FOXP2 and FOXP1 transcription factors causes distinct neurodevelopmental disorders. (2017)
  • Author(s): Sollis E, Deriziotis P, Saitsu H, Miyake N, Matsumoto N, Hoffer MJV, Ruivenkamp CAL, Alders M, Okamoto N, Bijlsma EK, Plomp AS, Fisher SE.
  • Journal Title: Hum Mutat Volume: 38(11) Pages: 1542-1554
  • DOI: 10.1002/humu.23303.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Identification of biallelic EXTL3 mutations in a novel type of spondylo-epi-metaphyseal dysplasia. (2017)
  • Author(s): Guo L#, Elcioglu NH# (# denotes equal contribution), Mizumoto S, Wang Z, Noyan B, Albayrak HM, Yamada S, Matsumoto N, Miyake N, Nishimura G, Ikegawa S*.
  • Journal Title: J Hum Genet Volume: 62(8) Pages: 797-801
  • DOI: 10.1038/jhg.2017.38.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Disturbed chromosome segregation and multipolar spindle formation in a patient with CHAMP1 mutation. (2017)
  • Author(s): Okamoto N*, Tsuchiya Y, Kuki I, Yamamoto T, Saitsu H, Kitagawa D*, Matsumoto N* (*: co-correspondence).
  • Journal Title: Mol Genet Genomic Med Volume: 12;5(5) Pages: 585-591
  • DOI: 10.1002/mgg3.303.
  • Peer Reviewed
• [Journal Article] Succinate dehydrogenase B-deficient renal cell carcinoma: A case report with novel germline mutation. (2017)
  • Author(s): Iwashita H, Okudela K, Matsumura M, Yamanaka S, Sawazumi T, Enaka M, Udaka N, Miyake A, Hibiya T, Miyake N, Matsumoto N, Makiyama K, Yao M, Nagashima Y, Ohashi K.
  • Journal Title: Pathol Int Volume: 67(11) Pages: 585-589
  • DOI: 10.1111/pin.12587.
  • Peer Reviewed
• [Journal Article] Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy. (2017)
  • Author(s): Syrbe S#, Harms FL# (# denotes equal contribution), et al., Matsumoto N, et al..
  • Journal Title: Brain Volume: 1;140(9) Pages: 2322-2336
  • DOI: 10.1093/brain/awx195.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A novel DARS2 mutation in a Japanese patient with leukoencephalopathy with brainstem and spinal cord involvement but no lactate elevation. (2017)
  • Author(s): Shimojima K, Higashiguchi T, Kishimoto K, Miyatake S, Miyake N, Takanashi JI, Matsumoto N, Yamamoto T*.
  • Journal Title: Hum Genome Var Volume: 9;4 Pages: 17051
  • DOI: 10.1038/hgv.2017.51.
  • Peer Reviewed / Open Access
• [Journal Article] Novel and recurrent COL11A1 and COL2A1 mutations in the Marshall-Stickler syndrome spectrum. (2017)
  • Author(s): Guo L, Elcioglu NH, Wang Z, Demirkol YK, Isguven P, Matsumoto N, Nishimura G, Miyake N, Ikegawa S*.
  • Journal Title: Hum Genome Var Volume: 5;4 Pages: 17040
  • DOI: 10.1038/hgv.2017.40.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Three cases of KCNT1 mutations: malignant migrating partial seizures in infancy with massive systemic to pulmonary collateral arteries. (2017)
  • Author(s): *Kawasaki Y, Kuki I, Ehara E, Murakami Y, Okazaki S, Kawawaki H, Hara M, Watanabe Y, Kishimoto S, Suda K, Saitsu H, Matsumoto N.
  • Journal Title: J Pediatr Volume: 191 Pages: :270-274
  • DOI: 10.1016/j.jpeds.2017.08.057.
  • Peer Reviewed
• [Journal Article] X-linked hypomyelination with spondylometaphyseal dysplasia (H-SMD) associated with mutations in AIFM1. (2017)
  • Author(s): *Miyake N, *Wolf N# (# correspondence), *Cayami F (*: equal contribution), et al., Matsumoto N, et al..
  • Journal Title: Neurogenetics Volume: 18(4) Pages: 185-194
  • DOI: 10.1007/s10048-017-0520-x.
  • Peer Reviewed / Int'l Joint Research
• [Presentation] 「タンパク質翻訳後修飾拠点におけるゲノム解析研究」 (2018)
  • Author(s): 松本直通
  • Organizer: 第８回国際公開シンポジウム
• [Presentation] “Rare genomic variants in human diseases” (2018)
  • Author(s): Naomichi Matsumoto
  • Organizer: 14.International Symposium on Approaching from model organisms to rare and undiagnosed diseases
• [Presentation] 「次世代シーケンス研究で直面する様々な問題点に対する取り組み」 (2017)
  • Author(s): 松本直通
  • Organizer: NGS現場の会・スポンサードセッション
  • Invited
• [Presentation] “Biallelic mutations in the myopalladin gene, MYPN, are associated with childhood-onset, slowly progressive nemaline myopathy” (2017)
  • Author(s): Naomichi Matsumoto and Satoko Miyatake
  • Organizer: ESHG 2017
  • Invited
• [Presentation] 「周産期異常とゲノム解析」 (2017)
  • Author(s): 松本直通
  • Organizer: 九州大学医学部講義（受胎・成長・発達）
  • Invited
• [Presentation] 「ヒト疾患とRare Variants」 (2017)
  • Author(s): 松本直通
  • Organizer: 第20回山梨神経先端セミナー
• [Presentation] “Rare variants in human diseases” (2017)
  • Author(s): Naomichi Matsumoto
  • Organizer: IX International Congress Cornelia de Lange Syndrome,
• [Presentation] “Rare variants in human diseases” (2017)
  • Author(s): Naomichi Matsumoto
  • Organizer: Lecture for department of Medical Genetics, University of Sao Paulo
• [Presentation] 「最新のNGS 研究の動向と新しい展開」 (2017)
  • Author(s): 松本直通
  • Organizer: 第22回日本ライソゾーム病研究会特別講演
• [Presentation] 「体細胞モザイク変異とヒト疾患」 (2017)
  • Author(s): 松本直通
  • Organizer: 日本環境変異原学会（JEMS）第46回大会シンポジウム
• [Presentation] “Rare variants in Rare and Intractable diseases” (2017)
  • Author(s): Naomichi Matsumoto
  • Organizer: 9.International Symposium on Genomic Medicine-Genomics of Rare and Intractable Diseases
• [Presentation] 「ロングリードシーケンサーSequelを用いた疾患ゲノム解析の試み」 (2017)
  • Author(s): 松本直通
  • Organizer: 第62回日本人類遺伝学会学術集会ランチョンセミナー
• [Presentation] 「次世代シーケンサーがもたらした希少”遺伝性”疾患解析の現状と展望」 (2017)
  • Author(s): 松本直通
  • Organizer: 協和発酵キリン㈱富士リサーチパークセミナー
• [Presentation] “How to Detect Ultra-Low-Level Somatic Mutations.” (2017)
  • Author(s): Naomichi Matsumoto
  • Organizer: AES Annual Meeting 2017, Investigator Workshop 2: Somatic mutation: the ‘hidden genetics’ of brain malformations.