Publications by authors named "Eigo Shimizu"

22 Publications

  • Page 1 of 1

Comprehensive molecular analysis of genomic profiles and PD-L1 expression in lung adenocarcinoma with a high-grade fetal adenocarcinoma component.

Transl Lung Cancer Res 2021 Mar;10(3):1292-1304

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan.

Background: Fetal adenocarcinoma of the lung is a rare variant of lung adenocarcinoma and is subcategorized into low-grade and high-grade (H-FLAC) fetal adenocarcinoma. We previously reported poor prognosis in pulmonary adenocarcinomas with an H-FLAC component; however, the genetic abnormalities involved in H-FLAC remain unclear. Therefore, this study aimed to elucidate molecular abnormalities as potential therapeutic targets for H-FLACs.

Methods: We performed immunohistochemical analysis and comprehensive genetic analyses using whole-exome sequencing in 16 lung cancer samples with an H-FLAC component. DNA was extracted from formalin-fixed paraffin-embedded tissues after macrodissection of the H-FLAC component.

Results: Cancer-related mutations were identified in (7/16 cases), (6/16 cases), (4/16 cases), (3/16 cases), (3/16 cases), (2/16 cases), and (1/16 cases). A high tumor mutation burden of ≥10 mutations per megabase was observed in 3/16 cases. A high microsatellite instability was not detected in any case. Based on the cosine similarity with the Catalogue of Somatic Mutations in Cancer mutational signatures, H-FLACs were hierarchically clustered into three types: common adenocarcinoma-like (five cases), surfactant-deficient (ten cases), and signatures 2 and 13-related (one case). All common adenocarcinoma-like cases presented thyroid transcription factor-1 (TTF-1) expression, whereas surfactant-deficient cases often presented loss of TTF-1 and surfactant protein expression and included cases with mutations in the surfactant system genes and . H-FLACs displayed low programmed death ligand-1 (PD-L1) expression (1-49% of tumor cells) in 5/16 cases, and no case displayed high PD-L1 expression (≥50% of tumor cells).

Conclusions: This study indicates that lung cancers with an H-FLAC component rarely harbor currently targetable driver gene mutations for lung cancer but display a high frequency of mutations. The microsatellite instability, tumor mutation burden, and PD-L1 expression status suggest a poor response to immune checkpoint therapy.
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http://dx.doi.org/10.21037/tlcr-20-1158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044470PMC
March 2021

Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders.

Science 2021 01;371(6526):265-270

Division of Cancer Cell Biology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 () as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.
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http://dx.doi.org/10.1126/science.abb5916DOI Listing
January 2021

Neoantimon: a multifunctional R package for identification of tumor-specific neoantigens.

Bioinformatics 2020 09;36(18):4813-4816

Division of Health Medical Data Science, Health Intelligence Center, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Summary: It is known that some mutant peptides, such as those resulting from missense mutations and frameshift insertions, can bind to the major histocompatibility complex and be presented to antitumor T cells on the surface of a tumor cell. These peptides are termed neoantigen, and it is important to understand this process for cancer immunotherapy. Here, we introduce an R package termed Neoantimon that can predict a list of potential neoantigens from a variety of mutations, which include not only somatic point mutations but insertions, deletions and structural variants. Beyond the existing applications, Neoantimon is capable of attaching and reflecting several additional information, e.g. wild-type binding capability, allele specific RNA expression levels, single nucleotide polymorphism information and combinations of mutations to filter out infeasible peptides as neoantigen.

Availability And Implementation: The R package is available at http://github/hase62/Neoantimon.
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http://dx.doi.org/10.1093/bioinformatics/btaa616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750962PMC
September 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 Nov 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Association of single nucleotide polymorphisms in the NRF2 promoter with vascular stiffness with aging.

PLoS One 2020 11;15(8):e0236834. Epub 2020 Aug 11.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Purpose: Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (-617C>A; hereafter called SNP-617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP-617 affects vascular stiffness with aging in apparently healthy people.

Methods: Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP-617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP-617 on other cardiovascular and blood test measurements.

Results: We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP-617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP-617 accelerates the incremental ratio of baPWV with age.

Conclusions: This study is the first to show that SNP-617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236834PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418968PMC
October 2020

Variant analysis of prostate cancer in Japanese patients and a new attempt to predict related biological pathways.

Oncol Rep 2020 Mar 27;43(3):943-952. Epub 2020 Jan 27.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Kanagawa 241‑8515, Japan.

There are regional and/or ethnic differences in tumorigenic pathways among several types of cancer, including prostate cancer (PCa). However, information on genome‑wide gene alterations and the transcriptome is currently only available for PCa patients from Western countries. In order to profile the genetic alterations in Japanese patients with PCa, new panels were created to examine nucleotide sequence variations in 71 selected PCa‑related genes (KCC71) and to detect all fusion RNA transcripts known in PCa (PCaFusion). An analysis of 21 Japanese PCa cases identified 33 different somatic variants in 24 genes in the KCC71 panel, including 2 in SPOP (F102V and F133L), 2 in BRCA2 (I1859fs and R2318ter, resulting in premature termination of the polypeptide), and 1 each in BRAF (K601E), CDH1 (E880K) and RB1 (R621S), as pathogenic alterations. Unexpectedly, the TMPRSS2‑ERG fusion transcript was detected in only 1 case, although the SLC45A3‑ELK4 and USP9Y‑TTTY15 fusion transcripts, known as transcription‑mediated chimeric RNAs, were detected in all examined cases. A new pathway analysis with The Cancer Network Galaxy (TCNG), a cancer gene regulatory network database, was also applied in an attempt to predict molecular pathways implicated in PCa in the Japanese population. Based on the 24 genes having somatic variants identified by the panel analysis as initial seed genes, a putative core network was finally established, including 5 identified genes, namely TNK2, SOX9, CDH1, FOXA1 and TP53, with high commonality from TCNG datasets. These genes are expected to be involved in tumor development, as revealed by the results of an enrichment analysis with Gene Ontology terms. This analysis must be further extended to include more cases in order to verify this method and also to elucidate the characteristics of PCa in Japanese patients.
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http://dx.doi.org/10.3892/or.2020.7481DOI Listing
March 2020

An Unusually Short Latent Period of Therapy-Related Myeloid Neoplasm Harboring a Rare MLL-EP300 Rearrangement: Case Report and Literature Review.

Case Rep Hematol 2019 2;2019:4532434. Epub 2019 Oct 2.

Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Therapy-related myeloid neoplasm (t-MN) is a late and lethal complication induced by chemotherapy and/or radiation therapy. Hematological malignancy is one of the most common primary diseases in patients with t-MN. However, the occurrence of t-MN in adult T-cell leukemia/lymphoma (ATL) patients is rarely reported, possibly due to the dismal prognosis of ATL per se. Here, we report a 62-year-old female who developed t-MN only three months after the completion of conventional chemotherapy and anti-CCR4 antibody for ATL acute type. The patient presented with persistent fever and monocytosis without any evidence of infectious diseases. Bone marrow examinations revealed chronic myelomonocytic leukemia-like disease with a chromosomal translocation of t(11;22)(q23;q13) as a solo cytogenetic abnormality, resulting in the diagnosis of t-MN. Next-generation sequencing analysis identified a rare chimeric transcript, MLL-EP300, without any additional somatic mutations. Although the patient underwent allogenic hematopoietic stem cell transplantation, she died of viral encephalomyelitis at 7 months after diagnosis of t-MN. Since recent therapeutic advances have extended the survival of patients with ATL, further evaluation of the long-term risks of developing t-MN in these patients is warranted.
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http://dx.doi.org/10.1155/2019/4532434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791222PMC
October 2019

Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient.

J Hum Genet 2019 Aug 14;64(8):729-740. Epub 2019 May 14.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.
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http://dx.doi.org/10.1038/s10038-019-0611-7DOI Listing
August 2019

The first case of elderly -positive B-cell acute lymphoblastic leukemia.

Leuk Lymphoma 2019 11 6;60(11):2821-2824. Epub 2019 May 6.

Department of Hematology/Oncology, Research Hospital, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

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http://dx.doi.org/10.1080/10428194.2019.1602267DOI Listing
November 2019

Prognostic impact of circulating tumor DNA status post-allogeneic hematopoietic stem cell transplantation in AML and MDS.

Blood 2019 06 1;133(25):2682-2695. Epub 2019 Apr 1.

Division of Molecular Therapy, Advanced Clinical Research Center.

This study was performed to assess the utility of tumor-derived fragmentary DNA, or circulating tumor DNA (ctDNA), for identifying high-risk patients for relapse of acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively collected tumor and available matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML/MDS. After identifying driver mutations in 51 patients using next-generation sequencing, we designed at least 1 personalized digital polymerase chain reaction assay per case. Diagnostic ctDNA and matched tumor DNA exhibited excellent correlations with variant allele frequencies. Sixteen patients relapsed after a median of 7 months post-alloSCT. Both mutation persistence (MP) in bone marrow (BM) at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in the matched serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year cumulative incidence of relapse (CIR) rates (MP1 vs non-MP1, 72.9% vs 13.8% [ = .0012]; CP1 vs non-CP1, 65.6% vs 9.0% [ = .0002]; MP3 vs non-MP3, 80% vs 11.6% [ = .0002]; CP3 vs non-CP3, 71.4% vs 8.4% [ < .0001]). We subsequently evaluated whether subset analysis of patients with 3 genes associated with clonal hematopoiesis, , , and (DTA), could also be helpful in relapse prediction. As a result, CP based on DTA gene mutations also had the prognostic effect on CIR. These results, for the first time, support the utility of ctDNA as a noninvasive prognostic biomarker in patients with AML/MDS undergoing alloSCT.
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http://dx.doi.org/10.1182/blood-2018-10-880690DOI Listing
June 2019

Cell-lineage level-targeted sequencing to identify acute myeloid leukemia with myelodysplasia-related changes.

Blood Adv 2018 10;2(19):2513-2521

Department of Hematology/Oncology, Research Hospital, Institute of Medical Science.

Acute myeloid leukemia (AML) is a clonal myeloid neoplasm that typically arises de novo; however, some cases evolve from a preleukemic state, such as myelodysplastic syndrome (MDS). Such secondary AMLs and those with typical MDS-related clinical features are known as AMLs with myelodysplasia-related changes (AML-MRC). Because patients with AML-MRC have poor prognosis, more accurate diagnostic approaches are required. In this study, we performed targeted sequencing of 54 genes in 3 cell populations (granulocyte, blast, and T-cell fractions) using samples from 13 patients with MDS, 16 patients with clinically diagnosed AML-MRC, 4 patients with suspected AML-MRC but clinically diagnosed as AML not otherwise specified (AML-NOS), and 11 patients with de novo AML. We found that overlapping mutations, defined as those shared at least by the blast and granulocyte fractions, were significantly enriched in patients with MDS and AML-MRC, including those with suspected AML-MRC, indicating a substantial history of clonal hematopoiesis. In contrast, blast-specific nonoverlapping mutations were significantly enriched in patients with de novo AML. Furthermore, the presence of overlapping mutations, excluding , , and , effectively segregated patients with MDS and AML-MRC or suspected AML-MRC from patients with de novo AML. Additionally, the presence of ≥3 mutations in the blast fraction was useful for distinguishing patients with AML-MRC from those with MDS. In conclusion, our approach is useful for classifying clinically diagnosable AML-MRC and identifying clinically diagnosed AML-NOS as latent AML-MRC. Additional prospective studies are needed to confirm the utility of this approach.
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http://dx.doi.org/10.1182/bloodadvances.2017010744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177645PMC
October 2018

Circulating tumor DNA dynamically predicts response and/or relapse in patients with hematological malignancies.

Int J Hematol 2018 Oct 29;108(4):402-410. Epub 2018 Jun 29.

Division of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

A growing body of evidence suggests that tumor-derived fragmentary DNA, known as circulating tumor DNA (ctDNA), has the potential to serve as a non-invasive biomarker for disease monitoring. However, in the setting of hematological malignancy, few published studies support the utility of ctDNA. We retrospectively investigated ctDNA levels of 17 patients with various hematological malignancies who had achieved remission after first-line therapy. We identified somatic driver mutations by next-generation sequencing, and designed droplet digital PCR assays for each mutation to measure ctDNA. Variant allele frequencies of ctDNA changed in association with clinical response in all patients. Eight patients clinically relapsed after a median of 297 days post-first-line therapy (termed, "relapsed group"); the remaining nine patients remained disease-free for a median of 332 days (termed, "remission group"). Among patients in the relapsed group, ctDNA levels increased more than twofold at paired serial time points. In marked contrast, ctDNA levels of all patients in the remission group remained undetectable or stable during clinical remission. Notably, ctDNA-based molecular relapse demonstrated a median 30-day lead time over clinical relapse. In summary, ctDNA monitoring may help identify hematologic cancer patients at risk for relapse in advance of established clinical parameters.
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http://dx.doi.org/10.1007/s12185-018-2487-2DOI Listing
October 2018

Azacitidine effectively reduces -mutant leukemic cell burden in secondary acute myeloid leukemia after cord blood transplantation.

Leuk Lymphoma 2018 11 12;59(11):2755-2756. Epub 2018 Apr 12.

a Division of Molecular Therapy, Advanced Clinical Research Center, the Institute of Medical Science , University of Tokyo , Tokyo , Japan.

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http://dx.doi.org/10.1080/10428194.2018.1443335DOI Listing
November 2018

[Autologous peripheral blood stem cell transplantation for double-refractory myeloma with K-RAS and N-RAS mutations].

Rinsho Ketsueki 2017;58(12):2380-2385

Department of Hematology/Oncology, Research Hospital, the Institute of Medical Science, the University of Tokyo.

The prognosis of multiple myeloma (MM) has been improved due to the introduction of novel agents like proteasome inhibitors and immunomodulatory drugs (IMiDs). However, some cases are refractory to the use of novel agents, and the prognosis of such cases is poor. A 53-year-old male was diagnosed with MM and categorized as follows: Bence-Jones protein lambda type MM, Durie-Salmon IIIA, international staging system (ISS) stage II, and revised ISS stage II. Mutations in K-RAS and IGH/FGFR3 translocation were detected at diagnosis. His tumor was refractory to seven therapeutic regimens including bortezomib, IMiDs (lenalidomide, thalidomide, pomalidomide), conventional chemotherapy, and radiation therapy. N-RAS mutations, CKS1B gains, and C-MYC split signals were detected after treatment. We performed high-dose melphalan/autologous stem cell transplantation (HD-MEL/ASCT) as a salvage therapy and achieved very good partial response. The correlation between K-RAS mutations and poor prognosis or between N-RAS mutations and reduced sensitivity to bortezomib is reported. However, RAS mutations are reported as a favorable factor for HD-MEL/ASCT. In general, mutations of both the K-RAS and N-RAS are known to be mutually exclusive. This rare MM case has mutations in both K-RAS and N-RAS, and the possible relevance of these mutations to both the refractoriness to novel therapies and sensitivity to HD-MEL/ASCT is suggested.
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http://dx.doi.org/10.11406/rinketsu.58.2380DOI Listing
June 2018

Different clonal dynamics of chronic myeloid leukaemia between bone marrow and the central nervous system.

Br J Haematol 2018 12 19;183(5):842-845. Epub 2017 Dec 19.

Department of Haematology/Oncology, Research Hospital, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

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http://dx.doi.org/10.1111/bjh.15065DOI Listing
December 2018

Requirement of glycosylation machinery in TLR responses revealed by CRISPR/Cas9 screening.

Int Immunol 2017 08;29(8):347-355

Division of Innate Immunity, Department of Microbiology and Immunology.

The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.
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http://dx.doi.org/10.1093/intimm/dxx044DOI Listing
August 2017

Phenotype-based gene analysis allowed successful diagnosis of X-linked neutropenia associated with a novel WASp mutation.

Ann Hematol 2018 Feb 27;97(2):367-369. Epub 2017 Sep 27.

Division of Molecular Therapy, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

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http://dx.doi.org/10.1007/s00277-017-3134-3DOI Listing
February 2018

Reduced expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis.

Sci Rep 2016 05 24;6:26011. Epub 2016 May 24.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39-45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing.
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http://dx.doi.org/10.1038/srep26011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877598PMC
May 2016

Detection of APC mosaicism by next-generation sequencing in an FAP patient.

J Hum Genet 2015 May 26;60(5):227-31. Epub 2015 Feb 26.

Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Familial adenomatous polyposis (FAP) of the colon is characterized by multiple polyps in the intestine and extra-colonic manifestations. Most FAP cases are caused by a germline mutation in the tumor-suppressor gene APC, but some cases of adenomatous polyposis result from germline mutations in MUTYH, POLD1 or POLE. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic FAP patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. Whole-genome sequencing and subsequent deep sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in ~12% of his peripheral leukocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data implied that genetic analysis by next-generation sequencing is an effective strategy to identify genetic mosaicism in hereditary diseases.
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http://dx.doi.org/10.1038/jhg.2015.14DOI Listing
May 2015

Attenuated familial adenomatous polyposis with desmoids caused by an APC mutation.

Hum Genome Var 2015 26;2:15011. Epub 2015 Mar 26.

Division of Clinical Genome Research, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan.

We present here a case of attenuated familial adenomatous polyposis (AFAP) with a family history of desmoids and thyroid tumors. This patient had no colonic polyps but did have multiple desmoids. Genetic analysis identified a 4-bp deletion in codon 2644 (c.7932_7935delTTAT: p.Tyr2645LysfsX14) of the adenomatous polyposis coli (APC) gene. In cases with limited numbers of colonic polyps and desmoids, AFAP may be caused by a mutation in the 3' region of APC.
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http://dx.doi.org/10.1038/hgv.2015.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785566PMC
April 2016

Fluctuation of global gene expression by endogenous miRNA response to the introduction of an exogenous miRNA.

Int J Mol Sci 2013 May 27;14(6):11171-89. Epub 2013 May 27.

Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan.

Most of the intracellular endogenous microRNAs (endo-miRNAs) are considered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells, endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, and endo-miRNAs might also compete for the position in the newly synthesized RISC with each other. This would lead to the fluctuation of global gene expression not only by repression of exo-miRNA target gene expression, but also by the increase of the endo-miRNA target gene expression. In the present study, we quantified the changes in the expression levels of target genes of exo-miRNA and endo-miRNA in the cells transfected with fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs increased ratios of expression levels of target genes of a given endo-miRNA to different extents, suggesting that the replacement efficiencies might differ according to the exo-miRNA types. However, the increased ratios in the expression levels of each endo-miRNA target genes by the transfection of any particular exo-miRNA were mostly equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced with excessive exo-miRNAs at similar levels, probably because they exist in single-stranded forms in the RISC.
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http://dx.doi.org/10.3390/ijms140611171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709726PMC
May 2013

Stability of miRNA 5'terminal and seed regions is correlated with experimentally observed miRNA-mediated silencing efficacy.

Sci Rep 2012 18;2:996. Epub 2012 Dec 18.

Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo , 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan.

MicroRNAs (miRNAs) are key regulators of sequence-specific gene silencing. However, crucial factors that determine the efficacy of miRNA-mediated target gene silencing are poorly understood. Here we mathematized base-pairing stability and showed that miRNAs with an unstable 5' terminal duplex and stable seed-target duplex exhibit strong silencing activity. The results are consistent with the previous findings that an RNA strand with unstable 5' terminal in miRNA duplex easily loads onto the RNA-induced silencing complex (RISC), and miRNA recognizes target mRNAs with seed-complementary sequences to direct posttranscriptional repression. Our results suggested that both the unwinding and target recognition processes of miRNAs could be proficiently controlled by the thermodynamics of base-pairing in protein-free condition. Interestingly, such thermodynamic parameters might be evolutionarily well adapted to the body temperatures of various species.
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http://dx.doi.org/10.1038/srep00996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524778PMC
May 2013