Research Projects -
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Research on neurodegenerative disorders
Grant number：23H02823 2023.04 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
石浦 浩之, 角元 利行
Grant amount：\18460000 （ Direct expense: \14200000 、 Indirect expense：\4260000 ）
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comprehensive search for expanded repeats in neurodegenerative diseaess
Grant number：22H02823 2022.04 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
辻 省次, 池内 健, 田中 真生, 石浦 浩之
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
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comprehensive search for expanded repeats in neurodegenerative diseaess
Grant number：23K24085 2022.04 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
辻 省次, 池内 健, 田中 真生, 石浦 浩之, 三井 純
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
本研究では、封入体形成が病理学的特徴である神経変性疾患に着目して、非翻訳領域の伸長リピート配列を探索し、神経変性疾患の発症機構を解明することを目的とする。この考え方は、非翻訳領域のリピート伸長配列は、Repeat Associated Non-AUG translation (RAN translation) を引き起こし、異常なアミノ酸配列を有するタンパクへの翻訳が行われ、その結果、封入体を形成するという仮説に基づいている。リピート伸長配列の検出は、short read sequencerでは困難であることから、long read sequencer による全ゲノムシーケンス解析を実施し、3-6塩基の繰り返し配列に焦点を当てて、伸長リピート配列を検出する独自に開発したプログラムを用いて、伸長リピート配列をゲノム全域から検出し、神経変性疾患の発症に関連する伸長リピート配列の発見を進めた。解析対象として、発症年齢の早いextreme phenotype を示すアルツハイマー病、家族歴を有する家族性アルツハイマー病症例を中心に7例を選択して、long read sequencer (Pacific Bioscience s 社のSequel II) を用いて、ロングリードシーケンス解析を実施した。long read sequencerは、error readを生じやすく精度が十分でないことから、circular consensus sequencing (CCS) というアルゴリズムにより、得られた全ゲノム配列データを用いた。その結果，これまでに報告のない、伸長リピート配列がさらに縦列に重複している例を見出した。本研究で開発したプログラムを用いて、伸長リピート配列の解析を進め、その疾患発症における意義の検討を進める。
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Revealing pathogenesis of noncoding repeat expansion diseases using long-read sequencing
Grant number：20H03588 2020.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
石浦 浩之
Grant amount：\17680000 （ Direct expense: \13600000 、 Indirect expense：\4080000 ）
ミオクローヌスてんかん患者でリピート伸長変異を有さない症例については全ゲノム配列解析を行い、通常のbasecallingに加えて、ExpansionHunterDenovoも使用してリピート配列のプロファイリングも行っている。これらの解析により、全ゲノム配列解析の有用性を含め、検討中である。
ロングリード全ゲノム配列解析データについても現在データ解析中であるが、非常に高精度に塩基配列解析が出来ていることが判明している。特に、circular consensus sequencingにより実際高い精度を出せることがわかっている。
伸長リピートを効率良く増幅することが可能になったため、ロングリードシーケンサーによる全長解読を行うために現在検体を準備している。また、PCR以外の方法を用いたリピート配列のクローニングも可能となり、この技術を用いて、機能解析に使用出来るコンストラクト作成を進めている。 -
Role of immune checkpoint molecules and macrophages in inflammatory myopathies
Grant number：19K07956 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
SHIMIZU Jun
Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）
The clinical and pathological features of myositis as a side effect of immune checkpoint inhibitors (irAE myositis) were analyzed in 12 cases to clarify the role of immune checkpoint molecules and macrophages. Similar pathology to irAE myositis was shown to be present in Graft-versus-host disease (GvHD) related myositis, revealing that immune checkpoint abnormalities are also involved in chronic GvHD myositis.In RNA-sequencing analysis of muscle samples from patients with inclusion body myositis (IBM) or polymyositis, it was revealed that, CDH1, which encodes the epidermal cell junction protein cadherin 1, was ectopically expressed in the muscles of IBM.
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Exploring rare variants associated with Alzheimer disease
Grant number：19H03425 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
Tsuji Shoji
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
To improve the power of association studies for relatively small sample size, we employed permutation testing to determine the p-value to be used for association studies. Employing exome sequence data obtained from 446 cases of sporadic Alzheimer disease (AD) and 446 aged controls with preserved cognitive function, we identified a candidate gene associated with AD. Since the candidate gene contains GC-rich regions, we optimized the methods for exon capture and library construction. We newly conducted exome sequence analysis of 262 cases of AD and 260 aged controls with preserved cognitive function. We confirmed that sequence data with high quality were obtained with the improved procedures. Although significant association of the candidate gene was not replicated, we found several candidate genes possibly associated with AD. The current study confirmed the utility of permutation testing for exome association studies.
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Toward pathogenesis of noncoding repeat expansion disease focusing on RNA toxicity
Grant number：18K19506 2018.06 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
Ishiura Hiroyuki
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
Benign adult familial myoclonic epilepsy type 1 (BAFME1) is an autosomal dominant disease which is characterized by infrequent seizures and myoclonic tremor. The cause of the disease have been identified as TTTTA and TTTCA repeat expansion mutation in intron 4 of SAMD12.
We focused on RNA-mediated pathomechanism in BAFME1 in this study. We identified RNA foci in autopsied brain in patients with BAFME1. RNA foci were not found in lymphoblastoid cell lines from patients with BAFME1. RNA-seq using autopsied brains revealed differentially expressed genes (40 downregulated and 44 upregulated). There seems no alternatively transcribed genes. In addition we found abortive transcription around the expanded repeats in SAMD12. We also cloned TTTTA, TTTCA, and TTTTA/TTTCA repeats in vectors and successfully expressed in HEK293 cells. Collectively, RNA-mediated toxicity is considered to play an important role in pathogenesis of BAFME and more investigation focusing on RNA metabolism should be paid. -
Grant number：17H05085 2017.04 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A) Grant-in-Aid for Young Scientists (A)
Ishiura Hiroyuki
Grant amount：\25090000 （ Direct expense: \19300000 、 Indirect expense：\5790000 ）
In benign adult familial myoclonic epilepsy (BAFME), expansions of intronic TTTCA and TTTTA repeats were identified in SAMD12, TNRC6A, and RAPGEF2. A new concept of repeat motif-phenotype correlation was proposed. The finding strongly indicates the gain-of-function of the expanded repeats is pathomechanism of BAFME.
The study also indicate the importance of genetic analysis focusing on repeat expansions. By applying this strategy to neuronal intranuclear inclusion disease, oculopharyngeal myopathy with leukoencephalopathy, and oculopharyngodistal myopathy, CGG repeat expansions were identified in NOTCH2NLC, LOC642361/NUTM2B-AS1, LRP12, respectively. -
Gene expression profiling of inflammatory myopathy in correlation with autoantibodies
Grant number：15K09347 2015.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Shimizu Jun
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
We performed mRNA microarray analysis of biopsied muscles from patients with IIMs and analyzed the relation with myositis specific antibodies (MSAs). In analysis of muscles with perifascicular atrophy, specific sets of mRNAs allow for molecular classification of patients with anti-Jo1 (n=4), -TIF1γ (n=5), and -Mi-2 (n=5) antibodies. In analysis of muscles with early pathological findings with anti-Jo1(n=4), -TIF1γ(n=4), and -MDA5 (n=4) antibodies, a total of 1705 mRNAs were significantly expressed (fold change>3, p<0.05). Pathway analysis for the 192 mRNAs commonly significantly changed in three groups were Toll-like and NOD-like receptor pathways. In analysis of mRNAs with significant changes, specific sets of mRNAs allow for molecular classification of patients with MSAs, respectively. Our findings showed that there are characteristic mRNAs expression profiles in relation with the types of MSAs even in muscles with mild pathological changes.
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Grant number：15K20941 2015.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
Ishiura Hiroyuki
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
Several kinds of mutations including rearrangements and repeat expansions as well as single nucleotide variants and short insertion/deletions cause neurodegenerative and neuromuscular disorders. Usual exome sequencing, however, overlook some of the mutations. To overcome this, I performed whole genome sequencing using a short read sequencer and a long read sequencer. Some of the mutations can only be delineated by the long read sequencer, indicating the importance of long read sequencing. This strategy will help us to understand the molecular mechanism of neurodegenerative disorders.
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Expression profiling of inclusion body myositis by massive parallel sequencing
Grant number：26461265 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Goto Jun, ISHIURA Hiroyuki, IKENAGA Chiseko
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
The aim of this study was to elucidate pathophysiology of inclusion body myositis by comprehensive expression profiling. Seventy-nine samples (11 controls, 11 PM, 47 IBM and 10 non-PM, non-IBM MHC-1 complex positive cases) were subjected to RNA-seq analysis. Seventy-one cases, of which RIN were more than 5, were statistically analyzed. Cluster analysis suggested that there was correlation between morphological classification and expression profile cluster. In IBM expression of cell adhesion molecules, inflammatory system, autophagy-lysosome system, and splicesome system were up-regulated. Expression of oxidative phosphorylation, citric cycle and electron transport genes was down-regilated.
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Identification of causative gene for autosomal recessive hereditary spastic paraplegia
Grant number：24890044 2012.08 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
ISHIURA Hiroyuki
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
Exome analysis for 16 patients with autosomal recessive hereditary spastic paraplegia was performed. Among them, I found a patient with SPG15, a rare form of autosomal recessive spastic paraplegia. Three homozygous mutations was found in a gene in 3 patients with hereditary spastic paraplegia with retinitis pigmentosa. Further studies are needed to characterize the mutations.
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遺伝性痙性対麻痺と紀伊筋萎縮性側索硬化症・パーキンソン認知症複合の遺伝子解析研究
Grant number：10J05639 2010 - 2011
日本学術振興会 科学研究費助成事業 特別研究員奨励費 特別研究員奨励費
石浦 浩之
Grant amount：\1400000 （ Direct expense: \1400000 ）
遺伝性痙性対麻痺:に関しては、SPG12に関する国際共同研究に参画し、遺伝子同定に寄与することができた(Montenegro G,Ishiura H,et al,J Clin Invest 2012)。また、リソース収集を進め、引き続き遺伝性痙性対麻痺の分子病態の解明を進める予定である。
紀伊筋萎縮性側索硬化症・パーキンソン認知症複合(ALS/PDC)に関しては、紀伊半島の最南端のALS3症例においてC90RF72内の6塩基反復配列の伸長を認め、9番染色体に連鎖する筋萎縮性側索硬化症であることが判明した。本地域における9番染色体に連鎖する筋萎縮性側索硬化症の頻度は、本邦の他の地域(Majounie E,Ishiura H et al. 2012)に比較して有意に頻度が高いことを見出し、紀伊半島におけるALSの高罹病率を部分的に説明できると考えられた(Ishiura H,et al.Archives of Neurology in press)。その他、紀伊半島最南端地域においてはOPTNのヘテロ接合性新規アミノ酸置換を持つALS症例も見出したが、他のALS症例においては観察されず、病原性については検討の余地が残った(Naruse H,Ishiura H,et al.Amyotroph Lateral Scler in press)。パーキンソン認知症複合(PDC)に関しては、本研究では遺伝子同定には至らず、今後の研究が必要であると考えられた。