Publications by authors named "Kyoko Watanabe"

70 Publications

Alternative Evaluation of an ELISPOT Assay Using Cytokine Activity as a Novel Parameter.

Anticancer Res 2021 Aug;41(8):3825-3831

Immunotherapy Division, Shizuoka Cancer Center Research Institute

Background/aim: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before.

Materials And Methods: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated.

Results: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes.

Conclusion: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter.
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http://dx.doi.org/10.21873/anticanres.15175DOI Listing
August 2021

Pan-ancestry exome-wide association analyses of COVID-19 outcomes in 586,157 individuals.

Am J Hum Genet 2021 07 3;108(7):1350-1355. Epub 2021 Jun 3.

Centre for Genomics Research, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge CB2 0AA, UK.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a respiratory illness that can result in hospitalization or death. We used exome sequence data to investigate associations between rare genetic variants and seven COVID-19 outcomes in 586,157 individuals, including 20,952 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome wide or when specifically focusing on (1) 13 interferon pathway genes in which rare deleterious variants have been reported in individuals with severe COVID-19, (2) 281 genes located in susceptibility loci identified by the COVID-19 Host Genetics Initiative, or (3) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, and results are publicly available through the Regeneron Genetics Center COVID-19 Results Browser.
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http://dx.doi.org/10.1016/j.ajhg.2021.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173480PMC
July 2021

Large-scale association analyses identify host factors influencing human gut microbiome composition.

Nat Genet 2021 02 18;53(2):156-165. Epub 2021 Jan 18.

Department of Twin Research & Genetic Epidemiology, King's College London, London, UK.

To study the effect of host genetics on gut microbiome composition, the MiBioGen consortium curated and analyzed genome-wide genotypes and 16S fecal microbiome data from 18,340 individuals (24 cohorts). Microbial composition showed high variability across cohorts: only 9 of 410 genera were detected in more than 95% of samples. A genome-wide association study of host genetic variation regarding microbial taxa identified 31 loci affecting the microbiome at a genome-wide significant (P < 5 × 10) threshold. One locus, the lactase (LCT) gene locus, reached study-wide significance (genome-wide association study signal: P = 1.28 × 10), and it showed an age-dependent association with Bifidobacterium abundance. Other associations were suggestive (1.95 × 10 < P < 5 × 10) but enriched for taxa showing high heritability and for genes expressed in the intestine and brain. A phenome-wide association study and Mendelian randomization identified enrichment of microbiome trait loci in the metabolic, nutrition and environment domains and suggested the microbiome might have causal effects in ulcerative colitis and rheumatoid arthritis.
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http://dx.doi.org/10.1038/s41588-020-00763-1DOI Listing
February 2021

Genome-wide meta-analysis of brain volume identifies genomic loci and genes shared with intelligence.

Nat Commun 2020 11 5;11(1):5606. Epub 2020 Nov 5.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

The phenotypic correlation between human intelligence and brain volume (BV) is considerable (r ≈ 0.40), and has been shown to be due to shared genetic factors. To further examine specific genetic factors driving this correlation, we present genomic analyses of the genetic overlap between intelligence and BV using genome-wide association study (GWAS) results. First, we conduct a large BV GWAS meta-analysis (N = 47,316 individuals), followed by functional annotation and gene-mapping. We identify 18 genomic loci (14 not previously associated), implicating 343 genes (270 not previously associated) and 18 biological pathways for BV. Second, we use an existing GWAS for intelligence (N = 269,867 individuals), and estimate the genetic correlation (r) between BV and intelligence to be 0.24. We show that the r is partly attributable to physical overlap of GWAS hits in 5 genomic loci. We identify 92 shared genes between BV and intelligence, which are mainly involved in signaling pathways regulating cell growth. Out of these 92, we prioritize 32 that are most likely to have functional impact. These results provide information on the genetics of BV and provide biological insight into BV's shared genetic etiology with intelligence.
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http://dx.doi.org/10.1038/s41467-020-19378-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644755PMC
November 2020

Alpha-type-1 Polarized Dendritic Cell-based Vaccination in Newly Diagnosed High-grade Glioma: A Phase II Clinical Trial.

Anticancer Res 2020 Nov;40(11):6473-6484

Office of the President, Shizuoka Cancer Center Hospital, Shizuoka, Japan.

Background/aim: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy.

Patients And Methods: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients.

Results: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients.

Conclusion: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.
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http://dx.doi.org/10.21873/anticanres.14669DOI Listing
November 2020

A case of pneumatosis cystoides intestinalis complicated by intussusception.

Pediatr Int 2020 Aug 16;62(8):987-988. Epub 2020 Jul 16.

Department of Pediatric Surgery, NHO Kokura Medical Center, Kitakyushu, Japan.

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http://dx.doi.org/10.1111/ped.14219DOI Listing
August 2020

Biochemical profiles of rat primary cultured hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid.

J Toxicol Sci 2020 ;45(6):339-347

Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd.

The metabolomic profiles of rat primary hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid were determined using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Significant and similar changes in the levels of 283 biochemical metabolites were associated with the three treatments compared with solvent control samples. Overall, the three treatments generated similar global biochemical profiles, with some minor differences associated with rotenone treatment. All three treatments resulted in a shift in energy metabolism as demonstrated by decreased glycogen stores and glycolysis. A reduced antioxidant response was detected in cells following all treatments. In addition, bile acid biosynthesis decreased as a potential consequence of increased oxidative stress by all three treatments. Conversely, rotenone treatment induced a number of changes after 1 hr, which were not detected in FCCP- or (+)-usnic acid-treated samples; these changes were not sustained over time and included increased NAD+ salvage and lysine degradation. In conclusion, these biochemical profiles could provide new insights into the mechanism(s) of mitochondrial toxicity.
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http://dx.doi.org/10.2131/jts.45.339DOI Listing
September 2020

Author Correction: Genetic mapping of cell type specificity for complex traits.

Nat Commun 2020 Apr 1;11(1):1718. Epub 2020 Apr 1.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, Amsterdam, The Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-15365-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113291PMC
April 2020

Author Correction: A global overview of pleiotropy and genetic architecture in complex traits.

Nat Genet 2020 03;52(3):353

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, Amsterdam, the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41588-019-0571-zDOI Listing
March 2020

Fluoroquinolones suppress gluconeogenesis by inhibiting fructose 1,6-bisphosphatase in primary monkey hepatocytes.

Toxicol In Vitro 2020 Jun 29;65:104786. Epub 2020 Jan 29.

Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address:

Dysglycemia is one of the most serious adverse events associated with the clinical use of certain fluoroquinolones. The purpose of this study was to investigate the effects of the representative fluoroquinolones moxifloxacin and gatifloxacin on hepatic gluconeogenesis using primary monkey hepatocytes. Glucose production was induced after the cells were incubated for 4 h with 10 mM sodium lactate and 1 mM sodium pyruvate as gluconeogenic substrates. Under these conditions, moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis at concentrations of 100 μM or higher. Transcriptome analysis of rate-limiting enzymes involved in hepatic gluconeogenesis revealed that moxifloxacin and gatifloxacin at a concentration of 1000 μM did not affect the expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase, glucose 6-phosphatase, and fructose 1,6-bisphosphatase. Furthermore, metabolome analysis, in vitro glucose production assay using additional gluconeogenic substrates, and fructose 1,6-bisphosphatase assay using the cell extracts showed that fluoroquinolones enzymatically suppressed hepatic gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. These inhibitory effects may involve in the clinically relevant dysglycemia associated with fluoroquinolones in human.
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http://dx.doi.org/10.1016/j.tiv.2020.104786DOI Listing
June 2020

Genetic mapping and evolutionary analysis of human-expanded cognitive networks.

Nat Commun 2019 10 24;10(1):4839. Epub 2019 Oct 24.

Connectome Lab, Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HV, Amsterdam, The Netherlands.

Cognitive brain networks such as the default-mode network (DMN), frontoparietal network, and salience network, are key functional networks of the human brain. Here we show that the rapid evolutionary cortical expansion of cognitive networks in the human brain, and most pronounced the DMN, runs parallel with high expression of human-accelerated genes (HAR genes). Using comparative transcriptomics analysis, we present that HAR genes are differentially more expressed in higher-order cognitive networks in humans compared to chimpanzees and macaques and that genes with high expression in the DMN are involved in synapse and dendrite formation. Moreover, HAR and DMN genes show significant associations with individual variations in DMN functional activity, intelligence, sociability, and mental conditions such as schizophrenia and autism. Our results suggest that the expansion of higher-order functional networks subserving increasing cognitive properties has been an important locus of genetic changes in recent human brain evolution.
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http://dx.doi.org/10.1038/s41467-019-12764-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813316PMC
October 2019

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter.

Ann Neurol 2019 11 10;86(5):780-792. Epub 2019 Sep 10.

Department of Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Neuroscience, Amsterdam, the Netherlands.

Objective: Astrocytes have gained attention as important players in neurological disease. In line with their heterogeneous character, defects in specific astrocyte subtypes have been identified. Leukodystrophy vanishing white matter (VWM) shows selective vulnerability in white matter astrocytes, but the underlying mechanisms remain unclear. Induced pluripotent stem cell technology is being extensively explored in studies of pathophysiology and regenerative medicine. However, models for distinct astrocyte subtypes for VWM are lacking, thereby hampering identification of disease-specific pathways.

Methods: Here, we characterize human and mouse pluripotent stem cell-derived gray and white matter astrocyte subtypes to generate an in vitro VWM model. We examined morphology and functionality, and used coculture methods, high-content microscopy, and RNA sequencing to study VWM cultures.

Results: We found intrinsic vulnerability in specific astrocyte subpopulations in VWM. When comparing VWM and control cultures, white matter-like astrocytes inhibited oligodendrocyte maturation, and showed affected pathways in both human and mouse cultures, involving the immune system and extracellular matrix. Interestingly, human white matter-like astrocytes presented additional, human-specific disease mechanisms, such as neuronal and mitochondrial functioning.

Interpretation: Astrocyte subtype cultures revealed disease-specific pathways in VWM. Cross-validation of human- and mouse-derived protocols identified human-specific disease aspects. This study provides new insights into VWM disease mechanisms, which helps the development of in vivo regenerative applications, and we further present strategies to study astrocyte subtype vulnerability in neurological disease. ANN NEUROL 2019;86:780-792.
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http://dx.doi.org/10.1002/ana.25585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856690PMC
November 2019

A global overview of pleiotropy and genetic architecture in complex traits.

Nat Genet 2019 09 19;51(9):1339-1348. Epub 2019 Aug 19.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, Amsterdam, the Netherlands.

After a decade of genome-wide association studies (GWASs), fundamental questions in human genetics, such as the extent of pleiotropy across the genome and variation in genetic architecture across traits, are still unanswered. The current availability of hundreds of GWASs provides a unique opportunity to address these questions. We systematically analyzed 4,155 publicly available GWASs. For a subset of well-powered GWASs on 558 traits, we provide an extensive overview of pleiotropy and genetic architecture. We show that trait-associated loci cover more than half of the genome, and 90% of these overlap with loci from multiple traits. We find that potential causal variants are enriched in coding and flanking regions, as well as in regulatory elements, and show variation in polygenicity and discoverability of traits. Our results provide insights into how genetic variation contributes to trait variation. All GWAS results can be queried and visualized at the GWAS ATLAS resource ( https://atlas.ctglab.nl ).
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http://dx.doi.org/10.1038/s41588-019-0481-0DOI Listing
September 2019

Genetic mapping of cell type specificity for complex traits.

Nat Commun 2019 07 19;10(1):3222. Epub 2019 Jul 19.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam, Amsterdam, The Netherlands.

Single-cell RNA sequencing (scRNA-seq) data allows to create cell type specific transcriptome profiles. Such profiles can be aligned with genome-wide association studies (GWASs) to implicate cell type specificity of the traits. Current methods typically rely only on a small subset of available scRNA-seq datasets, and integrating multiple datasets is hampered by complex batch effects. Here we collated 43 publicly available scRNA-seq datasets. We propose a 3-step workflow with conditional analyses within and between datasets, circumventing batch effects, to uncover associations of traits with cell types. Applying this method to 26 traits, we identify independent associations of multiple cell types. These results lead to starting points for follow-up functional studies aimed at gaining a mechanistic understanding of these traits. The proposed framework as well as the curated scRNA-seq datasets are made available via an online platform, FUMA, to facilitate rapid evaluation of cell type specificity by other researchers.
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http://dx.doi.org/10.1038/s41467-019-11181-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642112PMC
July 2019

Correction: Genome-wide analysis reveals extensive genetic overlap between schizophrenia, bipolar disorder, and intelligence.

Mol Psychiatry 2020 Apr;25(4):914

NORMENT, KG Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo and Division of Mental Health and Addiction, Oslo University Hospital, 0407, Oslo, Norway.

A correction to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41380-019-0456-7DOI Listing
April 2020

Feasibility of a cryopreservation of cultured human corneal endothelial cells.

PLoS One 2019 21;14(6):e0218431. Epub 2019 Jun 21.

Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.

Transparency of the cornea is essential for vision and is maintained by the corneal endothelium. Consequently, corneal endothelial decompensation arising from irreversible damage to the corneal endothelium causes severe vision impairment. Until recently, transplantation of donor corneas was the only therapeutic choice for treatment of endothelial decompensation. In 2013, we initiated clinical research into cell-based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs), in combination with Rho kinase inhibitor, into the anterior chamber. The aim of the present study was to establish a protocol for cryopreservation of HCECs to allow large-scale commercial manufacturing of these cells. This study focused on the effects of various cryopreservation reagents on HCEC viability. Screening of several commercially available cryopreservation reagents identified Bambanker hRM as an effective agent that maintained a cell viability of 89.4% after 14 days of cryopreservation, equivalent to the cell viability of 89.2% for non-cryopreserved control cells. The use of Bambanker hRM and HCECs at a similar grade to that used clinically for cell based therapy (passage 3-5 and a cell density higher than 2000 cells/mm2) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm2 and 2111 cells/mm2, respectively). HCECs preserved using Bambanker hRM grew in a similar fashion to non-preserved control HCECs and formed a monolayer sheet-like structure. Cryopreservation of HCECs has multiple advantages including the ability to accumulate stocks of master cells, to transport HCEC stocks, and to manufacture HCECs on demand for use in cell-based treatment of endothelial decompensation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218431PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588235PMC
February 2020

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Neuron 2019 07 3;103(2):217-234.e4. Epub 2019 Jun 3.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, 08025 Barcelona, Spain; Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain.

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).
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http://dx.doi.org/10.1016/j.neuron.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764089PMC
July 2019

Chrysin reduces the activity and protein level of mature forms of sterol regulatory element-binding proteins.

Biosci Biotechnol Biochem 2019 Sep 25;83(9):1740-1746. Epub 2019 Apr 25.

a Food Biochemistry laboratory, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Tokyo , Japan.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of genes involved in fatty acid and cholesterol biosynthetic pathways. The present study showed that the flavonoid chrysin impairs the fatty acid synthase promoter. Chrysin reduces the expression of SREBP target genes, such as fatty acid synthase, in human hepatoma Huh-7 cells and impairs synthesis of fatty acids and cholesterol. Moreover, it reduces the endogenous mature, transcriptionally active forms of SREBPs, which are generated by the proteolytic processing of precursor forms. In addition, chrysin reduces the enforced expressing mature forms of SREBPs and their transcriptional activity. The ubiquitin-proteasome system is not involved in the chrysin-mediated reduction of SREBPs mature forms. These results suggest that chrysin suppresses SREBP activity, at least partially, via the degradation of SREBPs mature forms. ACC1: acetyl-CoA carboxylase 1; DMEM: Dulbecco's modified Eagle's medium; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 25-HC: 25-hydroxycholesterol; HMGCS: HMG-CoA synthase; LDH: lactate dehydrogenase; LPDS: lipoprotein-deficient serum; PI3K: phosphatidylinositol 3-kinase; SCD1: stearoyl-CoA desaturase; SREBPs: sterol regulatory element-binding proteins.
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http://dx.doi.org/10.1080/09168451.2019.1608806DOI Listing
September 2019

Development of the VIGS System in the Dioecious Plant .

Int J Mol Sci 2019 Feb 27;20(5). Epub 2019 Feb 27.

Laboratory of Plant Pathology, Faculty of Agriculture, Tokyo University of Agriculture and Technology (TUAT), 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.

(1) Background: is a dioecious plant, whose sex is determined by XY-type sex chromosomes. is a smut fungus that infects plants and causes masculinization in female flowers, as if were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in plants, we selected (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in plants. Successful VIGS (virus-induced gene silencing) in plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in plants, which can unveil gene functions and networks of plants, such as the mechanisms of sex determination and fungal-induced masculinization.
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http://dx.doi.org/10.3390/ijms20051031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429067PMC
February 2019

Genome-wide analysis of insomnia in 1,331,010 individuals identifies new risk loci and functional pathways.

Nat Genet 2019 03 25;51(3):394-403. Epub 2019 Feb 25.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, the Netherlands.

Insomnia is the second most prevalent mental disorder, with no sufficient treatment available. Despite substantial heritability, insight into the associated genes and neurobiological pathways remains limited. Here, we use a large genetic association sample (n = 1,331,010) to detect novel loci and gain insight into the pathways, tissue and cell types involved in insomnia complaints. We identify 202 loci implicating 956 genes through positional, expression quantitative trait loci, and chromatin mapping. The meta-analysis explained 2.6% of the variance. We show gene set enrichments for the axonal part of neurons, cortical and subcortical tissues, and specific cell types, including striatal, hypothalamic, and claustrum neurons. We found considerable genetic correlations with psychiatric traits and sleep duration, and modest correlations with other sleep-related traits. Mendelian randomization identified the causal effects of insomnia on depression, diabetes, and cardiovascular disease, and the protective effects of educational attainment and intracranial volume. Our findings highlight key brain areas and cell types implicated in insomnia, and provide new treatment targets.
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http://dx.doi.org/10.1038/s41588-018-0333-3DOI Listing
March 2019

Effect of inter-individual variability in human liver cytochrome P450 isozymes on cyclophosphamide-induced micronucleus formation.

Mutat Res Genet Toxicol Environ Mutagen 2019 Feb 5;838:37-45. Epub 2018 Dec 5.

Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo, 134-8630, Japan.

We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 μg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system. The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.
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http://dx.doi.org/10.1016/j.mrgentox.2018.11.016DOI Listing
February 2019

Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer's disease risk.

Nat Genet 2019 03 7;51(3):404-413. Epub 2019 Jan 7.

MRC Biostatistics Unit, Cambridge Institute of Public Health, University of Cambridge, Cambridge, UK.

Alzheimer's disease (AD) is highly heritable and recent studies have identified over 20 disease-associated genomic loci. Yet these only explain a small proportion of the genetic variance, indicating that undiscovered loci remain. Here, we performed a large genome-wide association study of clinically diagnosed AD and AD-by-proxy (71,880 cases, 383,378 controls). AD-by-proxy, based on parental diagnoses, showed strong genetic correlation with AD (r = 0.81). Meta-analysis identified 29 risk loci, implicating 215 potential causative genes. Associated genes are strongly expressed in immune-related tissues and cell types (spleen, liver, and microglia). Gene-set analyses indicate biological mechanisms involved in lipid-related processes and degradation of amyloid precursor proteins. We show strong genetic correlations with multiple health-related outcomes, and Mendelian randomization results suggest a protective effect of cognitive ability on AD risk. These results are a step forward in identifying the genetic factors that contribute to AD risk and add novel insights into the neurobiology of AD.
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http://dx.doi.org/10.1038/s41588-018-0311-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836675PMC
March 2019

Genome-wide analysis reveals extensive genetic overlap between schizophrenia, bipolar disorder, and intelligence.

Mol Psychiatry 2020 04 4;25(4):844-853. Epub 2019 Jan 4.

NORMENT, KG Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo and Division of Mental Health and Addiction, Oslo University Hospital, 0407, Oslo, Norway.

Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders associated with cognitive impairment, which is considered a major determinant of functional outcome. Despite this, the etiology of the cognitive impairment is poorly understood, and no satisfactory cognitive treatments exist. Increasing evidence indicates that genetic risk for SCZ may contribute to cognitive impairment, whereas the genetic relationship between BD and cognitive function remains unclear. Here, we combined large genome-wide association study data on SCZ (n = 82,315), BD (n = 51,710), and general intelligence (n = 269,867) to investigate overlap in common genetic variants using conditional false discovery rate (condFDR) analysis. We observed substantial genetic enrichment in both SCZ and BD conditional on associations with intelligence indicating polygenic overlap. Using condFDR analysis, we leveraged this enrichment to increase statistical power and identified 75 distinct genomic loci associated with both SCZ and intelligence, and 12 loci associated with both BD and intelligence at conjunctional FDR < 0.01. Among these loci, 20 are novel for SCZ, and four are novel for BD. Most SCZ risk alleles (61 of 75, 81%) were associated with poorer cognitive performance, whereas most BD risk alleles (9 of 12, 75%) were associated with better cognitive performance. A gene set analysis of the loci shared between SCZ and intelligence implicated biological processes related to neurodevelopment, synaptic integrity, and neurotransmission; the same analysis for BD was underpowered. Altogether, the study demonstrates that both SCZ and BD share genetic influences with intelligence, albeit in a different manner, providing new insights into their genetic architectures.
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http://dx.doi.org/10.1038/s41380-018-0332-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609490PMC
April 2020

Generation of Isogenic Controls for In Vitro Disease Modelling of X-Chromosomal Disorders.

Stem Cell Rev Rep 2019 04;15(2):276-285

Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Boelelaan 1085, 1081HV, Amsterdam, The Netherlands.

Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome.
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http://dx.doi.org/10.1007/s12015-018-9851-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441401PMC
April 2019

The cup fungus Pestalopezia brunneopruinosa is Pestalotiopsis gibbosa and belongs to Sordariomycetes.

PLoS One 2018 27;13(6):e0197025. Epub 2018 Jun 27.

Pacific Forestry Centre, Natural Resources Canada, Victoria, British Columbia, Canada.

Pestalopezia brunneopruinosa, the type species of Pestalopezia in Leotiomycetes, produces typical cup-shaped ascomata. Because its asexual morph has conidia comprised of five cells including apical and basal appendages and three pigmented median cells, it was first described as Pestalotia gibbosa, which belongs to Sordariomycetes. This contradiction has not been resolved due to the difficulty in isolating this fungus in culture. In this study, we isolated separate strains from the sexual morph and the asexual morph for molecular analysis. Phylogenetic trees of Sporocadaceae based on internal transcribed spacer, partial β-tubulin, and partial translation elongation factor 1-alpha sequence datasets revealed that both strains fall into the same taxon, in a clade in Pestalotiopsis sensu stricto alongside P. gaultheriae and P. spathulata. We provide the first evidence that fungi producing cup-shaped ascomata in Pestalotiopsis belong to Sordariomycetes, and we have proposed the transfer of Pestalopezia brunneopruinosa to Pestalotiopsis gibbosa.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197025PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021046PMC
November 2018

Genome-wide association meta-analysis in 269,867 individuals identifies new genetic and functional links to intelligence.

Nat Genet 2018 07 25;50(7):912-919. Epub 2018 Jun 25.

Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

Intelligence is highly heritable and a major determinant of human health and well-being. Recent genome-wide meta-analyses have identified 24 genomic loci linked to variation in intelligence, but much about its genetic underpinnings remains to be discovered. Here, we present a large-scale genetic association study of intelligence (n = 269,867), identifying 205 associated genomic loci (190 new) and 1,016 genes (939 new) via positional mapping, expression quantitative trait locus (eQTL) mapping, chromatin interaction mapping, and gene-based association analysis. We find enrichment of genetic effects in conserved and coding regions and associations with 146 nonsynonymous exonic variants. Associated genes are strongly expressed in the brain, specifically in striatal medium spiny neurons and hippocampal pyramidal neurons. Gene set analyses implicate pathways related to nervous system development and synaptic structure. We confirm previous strong genetic correlations with multiple health-related outcomes, and Mendelian randomization analysis results suggest protective effects of intelligence for Alzheimer's disease and ADHD and bidirectional causation with pleiotropic effects for schizophrenia. These results are a major step forward in understanding the neurobiology of cognitive function as well as genetically related neurological and psychiatric disorders.
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http://dx.doi.org/10.1038/s41588-018-0152-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411041PMC
July 2018

Meta-analysis of genome-wide association studies for neuroticism in 449,484 individuals identifies novel genetic loci and pathways.

Nat Genet 2018 07 25;50(7):920-927. Epub 2018 Jun 25.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.

Neuroticism is an important risk factor for psychiatric traits, including depression, anxiety, and schizophrenia. At the time of analysis, previous genome-wide association studies (GWAS) reported 16 genomic loci associated to neuroticism. Here we conducted a large GWAS meta-analysis (n = 449,484) of neuroticism and identified 136 independent genome-wide significant loci (124 new at the time of analysis), which implicate 599 genes. Functional follow-up analyses showed enrichment in several brain regions and involvement of specific cell types, including dopaminergic neuroblasts (P = 3.49 × 10), medium spiny neurons (P = 4.23 × 10), and serotonergic neurons (P = 1.37 × 10). Gene set analyses implicated three specific pathways: neurogenesis (P = 4.43 × 10), behavioral response to cocaine processes (P = 1.84 × 10), and axon part (P = 5.26 × 10). We show that neuroticism's genetic signal partly originates in two genetically distinguishable subclusters ('depressed affect' and 'worry'), suggesting distinct causal mechanisms for subtypes of individuals. Mendelian randomization analysis showed unidirectional and bidirectional effects between neuroticism and multiple psychiatric traits. These results enhance neurobiological understanding of neuroticism and provide specific leads for functional follow-up experiments.
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http://dx.doi.org/10.1038/s41588-018-0151-7DOI Listing
July 2018

MIR137 schizophrenia-associated locus controls synaptic function by regulating synaptogenesis, synapse maturation and synaptic transmission.

Hum Mol Genet 2018 06;27(11):1879-1891

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Amsterdam Neuroscience, VU University Amsterdam and VU Medical Center, 1081 HV Amsterdam, The Netherlands.

The MIR137 locus is a replicated genetic risk factor for schizophrenia. The risk-associated allele is reported to increase miR-137 expression and miR-137 overexpression alters synaptic transmission in mouse hippocampus. We investigated the cellular mechanisms underlying these observed effects in mouse hippocampal neurons in culture. First, we correlated the risk allele to expression of the genes in the MIR137 locus in human postmortem brain. Some evidence for increased MIR137HG expression was observed, especially in hippocampus of the disease-associated genotype. Second, in mouse hippocampal neurons, we confirmed previously observed changes in synaptic transmission upon miR-137 overexpression. Evoked synaptic transmission and spontaneous release were 50% reduced. We identified defects in release probability as the underlying cause. In contrast to previous observations, no evidence was obtained for selective synaptic vesicle docking defects. Instead, ultrastructural morphometry revealed multiple effects of miR-137 overexpression on docking, active zone length and total vesicle number. Moreover, proteomic analyses of neuronal protein showed that expression of Syt1 and Cplx1, previously reported as downregulated upon miR-137 overexpression, was unaltered. Immunocytochemistry of synapses overexpressing miR-137 showed normal Synaptotagmin1 and Complexin1 protein levels. Instead, our proteomic analyses revealed altered expression of genes involved in synaptogenesis. Concomitantly, synaptogenesis assays revealed 31% reduction in synapse formation. Taken together, these data show that miR-137 regulates synaptic function by regulating synaptogenesis, synaptic ultrastructure and synapse function. These effects are plausible contributors to the increased schizophrenia risk associated with miR-137 overexpression.
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http://dx.doi.org/10.1093/hmg/ddy089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961183PMC
June 2018

Item-level analyses reveal genetic heterogeneity in neuroticism.

Nat Commun 2018 03 2;9(1):905. Epub 2018 Mar 2.

Department of Clinical Genetics, Section Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU Medical Centre, Amsterdam, 1081 HV, The Netherlands.

Genome-wide association studies (GWAS) of psychological traits are generally conducted on (dichotomized) sums of items or symptoms (e.g., case-control status), and not on the individual items or symptoms themselves. We conduct large-scale GWAS on 12 neuroticism items and observe notable and replicable variation in genetic signal between items. Within samples, genetic correlations among the items range between 0.38 and 0.91 (mean r = .63), indicating genetic heterogeneity in the full item set. Meta-analyzing the two samples, we identify 255 genome-wide significant independent genomic regions, of which 138 are item-specific. Genetic analyses and genetic correlations with 33 external traits support genetic differences between the items. Hierarchical clustering analysis identifies two genetically homogeneous item clusters denoted depressed affect and worry. We conclude that the items used to measure neuroticism are genetically heterogeneous, and that biological understanding can be gained by studying them in genetically more homogeneous clusters.
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http://dx.doi.org/10.1038/s41467-018-03242-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834468PMC
March 2018