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1. A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Author

Ieva Keraite, Philipp Becker, Davide Canevazzi, Cristina Frias-López, Marc Dabad, Raúl Tonda-Hernandez, Ida Paramonov, Matthew John Ingham, Isabelle Brun-Heath, Jordi Leno, Anna Abulí, Elena Garcia-Arumí, Simon Charles Heath, Marta Gut, and Ivo Glynne Gut

Subjects
Science
Abstract

Accurate analysis of mitochondrial DNA is important for mitochondrial disease clinical research and diagnostics. Here, authors present a method using Cas9 cleavage, nanopore sequencing and a custom pipeline to identify pathogenic variants, deletions and accurately quantify heteroplasmy to below 1%.

Published
2022
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2. Clinical, genetic, epidemiologic, evolutionary, and functional delineation of TSPEAR-related autosomal recessive ectodermal dysplasia 14

Author

Adam Jackson, Sheng-Jia Lin, Elizabeth A. Jones, Kate E. Chandler, David Orr, Celia Moss, Zahra Haider, Gavin Ryan, Simon Holden, Mike Harrison, Nigel Burrows, Wendy D. Jones, Mary Loveless, Cassidy Petree, Helen Stewart, Karen Low, Deirdre Donnelly, Simon Lovell, Konstantina Drosou, Gaurav K. Varshney, Siddharth Banka, J.C. Ambrose, P. Arumugam, R. Bevers, M. Bleda, F. Boardman-Pretty, C.R. Boustred, H. Brittain, M.A. Brown, M.J. Caulfield, G.C. Chan, A. Giess, J.N. Griffin, A. Hamblin, S. Henderson, T.J.P. Hubbard, R. Jackson, L.J. Jones, D. Kasperaviciute, M. Kayikci, A. Kousathanas, L. Lahnstein, A. Lakey, S.E.A. Leigh, I.U.S. Leong, F.J. Lopez, F. Maleady-Crowe, M. McEntagart, F. Minneci, J. Mitchell, L. Moutsianas, M. Mueller, N. Murugaesu, A.C. Need, P. O‘Donovan, C.A. Odhams, C. Patch, D. Perez-Gil, M.B. Pereira, J. Pullinger, T. Rahim, A. Rendon, T. Rogers, K. Savage, K. Sawant, R.H. Scott, A. Siddiq, A. Sieghart, S.C. Smith, A. Sosinsky, A. Stuckey, M. Tanguy, A.L. Taylor Tavares, E.R.A. Thomas, S.R. Thompson, A. Tucci, M.J. Welland, E. Williams, K. Witkowska, S.M. Wood, M. Zarowiecki, Olaf Riess, Tobias B. Haack, Holm Graessner, Birte Zurek, Kornelia Ellwanger, Stephan Ossowski, German Demidov, Marc Sturm, Julia M. Schulze-Hentrich, Rebecca Schüle, Christoph Kessler, Melanie Wayand, Matthis Synofzik, Carlo Wilke, Andreas Traschütz, Ludger Schöls, Holger Hengel, Peter Heutink, Han Brunner, Hans Scheffer, Nicoline Hoogerbrugge, Alexander Hoischen, Peter A.C. ’t Hoen, Lisenka E.L.M. Vissers, Christian Gilissen, Wouter Steyaert, Karolis Sablauskas, Richarda M. de Voer, Erik-Jan Kamsteeg, Bart van de Warrenburg, Nienke van Os, Iris te Paske, Erik Janssen, Elke de Boer, Marloes Steehouwer, Burcu Yaldiz, Tjitske Kleefstra, Anthony J. Brookes, Colin Veal, Spencer Gibson, Marc Wadsley, Mehdi Mehtarizadeh, Umar Riaz, Greg Warren, Farid Yavari Dizjikan, Thomas Shorter, Ana Töpf, Volker Straub, Chiara Marini Bettolo, Sabine Specht, Jill Clayton-Smith, Elizabeth Alexander, Laurence Faivre, Christel Thauvin, Antonio Vitobello, Anne-Sophie Denommé-Pichon, Yannis Duffourd, Emilie Tisserant, Ange-Line Bruel, Christine Peyron, Aurore Pélissier, Sergi Beltran, Ivo Glynne Gut, Steven Laurie, Davide Piscia, Leslie Matalonga, Anastasios Papakonstantinou, Gemma Bullich, Alberto Corvo, Carles Garcia, Marcos Fernandez-Callejo, Carles Hernández, Daniel Picó, Ida Paramonov, Hanns Lochmüller, Gulcin Gumus, Virginie Bros-Facer, Ana Rath, Marc Hanauer, Annie Olry, David Lagorce, Svitlana Havrylenko, Katia Izem, Fanny Rigour, Giovanni Stevanin, Alexandra Durr, Claire-Sophie Davoine, Léna Guillot-Noel, Anna Heinzmann, Giulia Coarelli, Gisèle Bonne, Teresinha Evangelista, Valérie Allamand, Isabelle Nelson, Rabah Ben Yaou, Corinne Metay, Bruno Eymard, Enzo Cohen, Antonio Atalaia, Tanya Stojkovic, Milan Macek, Jr., Marek Turnovec, Dana Thomasová, Radka Pourová Kremliková, Vera Franková, Markéta Havlovicová, Vlastimil Kremlik, Helen Parkinson, Thomas Keane, Dylan Spalding, Alexander Senf, Peter Robinson, Daniel Danis, Glenn Robert, Alessia Costa, Christine Patch, Mike Hanna, Henry Houlden, Mary Reilly, Jana Vandrovcova, Francesco Muntoni, Irina Zaharieva, Anna Sarkozy, Vincent Timmerman, Jonathan Baets, Liedewei Van de Vondel, Danique Beijer, Peter de Jonghe, Vincenzo Nigro, Sandro Banfi, Annalaura Torella, Francesco Musacchia, Giulio Piluso, Alessandra Ferlini, Rita Selvatici, Rachele Rossi, Marcella Neri, Stefan Aretz, Isabel Spier, Anna Katharina Sommer, Sophia Peters, Carla Oliveira, Jose Garcia Pelaez, Ana Rita Matos, Celina São José, Marta Ferreira, Irene Gullo, Susana Fernandes, Luzia Garrido, Pedro Ferreira, Fátima Carneiro, Morris A. Swertz, Lennart Johansson, Joeri K. van der Velde, Gerben van der Vries, Pieter B. Neerincx, Dieuwke Roelofs-Prins, Sebastian Köhler, Alison Metcalfe, Alain Verloes, Séverine Drunat, Caroline Rooryck, Aurelien Trimouille, Raffaele Castello, Manuela Morleo, Michele Pinelli, Alessandra Varavallo, Manuel Posada De la Paz, Eva Bermejo Sánchez, Estrella López Martín, Beatriz Martínez Delgado, F. Javier Alonso García de la Rosa, Andrea Ciolfi, Bruno Dallapiccola, Simone Pizzi, Francesca Clementina Radio, Marco Tartaglia, Alessandra Renieri, Elisa Benetti, Peter Balicza, Maria Judit Molnar, Ales Maver, Borut Peterlin, Alexander Münchau, Katja Lohmann, Rebecca Herzog, Martje Pauly, Alfons Macaya, Anna Marcé-Grau, Andres Nascimiento Osorio, Daniel Natera de Benito, Rachel Thompson, Kiran Polavarapu, David Beeson, Judith Cossins, Pedro M. Rodriguez Cruz, Peter Hackman, Mridul Johari, Marco Savarese, Bjarne Udd, Rita Horvath, Gabriel Capella, Laura Valle, Elke Holinski-Feder, Andreas Laner, Verena Steinke-Lange, Evelin Schröck, and Andreas Rump

Subjects
TSPEAR, Ectodermal dysplasia, Enamel knot, WNT10A, Hypodontia, Conical teeth, Genetics, QH426-470
Abstract

Summary: TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A-related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the β-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca, an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara−/−;tspearb−/− double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants.

Published
2023
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4. A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers

Author

Anthony Ferrari, Anne Vincent-Salomon, Xavier Pivot, Anne-Sophie Sertier, Emilie Thomas, Laurie Tonon, Sandrine Boyault, Eskeatnaf Mulugeta, Isabelle Treilleux, Gaëtan MacGrogan, Laurent Arnould, Janice Kielbassa, Vincent Le Texier, Hélène Blanché, Jean-François Deleuze, Jocelyne Jacquemier, Marie-Christine Mathieu, Frédérique Penault-Llorca, Frédéric Bibeau, Odette Mariani, Cécile Mannina, Jean-Yves Pierga, Olivier Trédan, Thomas Bachelot, Hervé Bonnefoi, Gilles Romieu, Pierre Fumoleau, Suzette Delaloge, Maria Rios, Jean-Marc Ferrero, Carole Tarpin, Catherine Bouteille, Fabien Calvo, Ivo Glynne Gut, Marta Gut, Sancha Martin, Serena Nik-Zainal, Michael R. Stratton, Iris Pauporté, Pierre Saintigny, Daniel Birnbaum, Alain Viari, and Gilles Thomas

Subjects
Science
Abstract

Breast cancer is separated into multiple subtypes based on the expression of HER2 and hormone receptors. Here, the authors report the whole genome sequence of 64 HER2 positive tumours and show that these can be further separated into four groups with different gene expression profiles and genomic features.

Published
2016
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5. Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome.

Author

Marion Engel, David Endesfelder, Brigitte Schloter-Hai, Susanne Kublik, Michael S Granitsiotis, Piera Boschetto, Mariarita Stendardo, Imre Barta, Balazs Dome, Jean-François Deleuze, Anne Boland, Joachim Müller-Quernheim, Antje Prasse, Tobias Welte, Jens Hohlfeld, Deepak Subramanian, David Parr, Ivo Glynne Gut, Timm Greulich, Andreas Rembert Koczulla, Adam Nowinski, Dorota Gorecka, Dave Singh, Sumit Gupta, Christopher E Brightling, Harald Hoffmann, Marion Frankenberger, Thomas P Hofer, Dorothe Burggraf, Marion Heiss-Neumann, Loems Ziegler-Heitbrock, Michael Schloter, and Wolfgang Zu Castell

Subjects
Medicine, Science
Abstract

Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited.Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons.We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype.Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.

Published
2017
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7. Erratum to: Making sense of big data in health research: towards an EU action plan

Author

Charles Auffray, Rudi Balling, Inês Barroso, László Bencze, Mikael Benson, Jay Bergeron, Enrique Bernal-Delgado, Niklas Blomberg, Christoph Bock, Ana Conesa, Susanna Del Signore, Christophe Delogne, Peter Devilee, Alberto Di Meglio, Marinus Eijkemans, Paul Flicek, Norbert Graf, Vera Grimm, Henk-Jan Guchelaar, Yi-Ke Guo, Ivo Glynne Gut, Allan Hanbury, Shahid Hanif, Ralf-Dieter Hilgers, Ángel Honrado, D. Rod Hose, Jeanine Houwing-Duistermaat, Tim Hubbard, Sophie Helen Janacek, Haralampos Karanikas, Tim Kievits, Manfred Kohler, Andreas Kremer, Jerry Lanfear, Thomas Lengauer, Edith Maes, Theo Meert, Werner Müller, Dörthe Nickel, Peter Oledzki, Bertrand Pedersen, Milan Petkovic, Konstantinos Pliakos, Magnus Rattray, Josep Redón i Màs, Reinhard Schneider, Thierry Sengstag, Xavier Serra-Picamal, Wouter Spek, Lea A. I. Vaas, Okker van Batenburg, Marc Vandelaer, Peter Varnai, Pablo Villoslada, Juan Antonio Vizcaíno, John Peter Mary Wubbe, and Gianluigi Zanetti

Subjects
Medicine, Genetics, QH426-470
Published
2016
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9. ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing.

Author

Spyros Darmanis, Rachel Yuan Nong, Johan Vänelid, Agneta Siegbahn, Olle Ericsson, Simon Fredriksson, Christofer Bäcklin, Marta Gut, Simon Heath, Ivo Glynne Gut, Lars Wallentin, Mats G Gustafsson, Masood Kamali-Moghaddam, and Ulf Landegren

Subjects
Medicine, Science
Abstract

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.

Published
2011
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11. Analyses of non-coding somatic drivers in 2,658 cancer whole genomes.

Author

Esther Rheinbay, Morten Muhlig Nielsen, Federico Abascal, Jeremiah Wala, Ofer Shapira, Grace Tiao, Henrik Hornshøj, Julian M. Hess, Randi Istrup Juul, Ziao Lin, Lars Feuerbach, Radhakrishnan Sabarinathan, Tobias Madsen, Jaegil Kim, Loris Mularoni, Shimin Shuai, Andrés Lanzós, Carl Herrmann, Yosef E. Maruvka, Ciyue Shen, Samirkumar B. Amin, Pratiti Bandopadhayay, Johanna Bertl, Keith A. Boroevich, John Busanovich, Joana Carlevaro-Fita, Dimple Chakravarty, Calvin Wing Yiu Chan, David Craft, Priyanka Dhingra, Klev Diamanti, Nuno A. Fonseca, Abel Gonzalez-Perez, Qianyun Guo, Mark P. Hamilton, Nicholas J. Haradhvala, Chen Hong, Keren Isaev, Todd A. Johnson, Malene Juul, André Kahles, Abdullah Kahraman, Youngwook Kim, Jan Komorowski, Kiran Kumar, Sushant Kumar, Donghoon Lee 0006, Kjong-Van Lehmann, Yilong Li, Eric Minwei Liu, Lucas Lochovsky, Keunchil Park, Oriol Pich, Nicola D. Roberts, Gordon Saksena, Steven E. Schumacher, Nikos Sidiropoulos, Lina Sieverling, Nasa Sinnott-Armstrong, Chip Stewart, David Tamborero, Jose M. C. Tubio, Husen M. Umer, Liis Uusküla-Reimand, Claes Wadelius, Lina Wadi, Xiaotong Yao, Cheng-Zhong Zhang, Jing Zhang 0062, James E. Haber, Asger Hobolth, Marcin Imielinski, Manolis Kellis, Michael S. Lawrence, Christian von Mering, Hidewaki Nakagawa, Benjamin J. Raphael, Mark A. Rubin, Chris Sander, Lincoln D. Stein, Joshua M. Stuart, Tatsuhiko Tsunoda, David A. Wheeler, Rory Johnson, Jüri Reimand, Mark Gerstein, Ekta Khurana, Peter J. Campbell, Núria López-Bigas, Gary D. Bader, Jonathan Barenboim, Rameen Beroukhim, Søren Brunak, Ken Chen, Jung Kyoon Choi, Jordi Deu-Pons, J. Lynn Fink, Joan Frigola, Carlo Gambacorti Passerini, Dale W. Garsed, Gad Getz, Ivo Glynne Gut, David Haan, Arif Ozgun Harmanci, Mohamed Helmy, Ermin Hodzic, José M. G. Izarzugaza, Jong K. Kim, Jan O. Korbel, Erik Larsson, Shantao Li, Xiaotong Li, Shaoke Lou, Kathleen Marchal, Iñigo Martincorena, Alexander Martínez-Fundichely, Patrick D. McGillivray, William Meyerson, Ferran Muiños, Marta Paczkowska, Kiejung Park, Jakob Skou Pedersen, Tirso Pons, Sergio Pulido-Tamayo, Iker Reyes-Salazar, Matthew A. Reyna, Carlota Rubio-Perez, Süleyman Cenk Sahinalp, Leonidas Salichos, Mark Shackleton, Raunak Shrestha, Alfonso Valencia, Miguel Vazquez, Lieven P. C. Verbeke, Jiayin Wang, Jonathan Warrell, Sebastian M. Waszak, Joachim Weischenfeldt, Guanming Wu, Jun Yu, Xuanping Zhang, Yan Zhang 0032, Zhongming Zhao, Lihua Zou, Kadir C. Akdemir, Eva G. Alvarez, Adrian Baez-Ortega, Paul C. Boutros, David D. L. Bowtell, Benedikt Brors, Kathleen H. Burns, Kin Chan, Isidro Cortés-Ciriano, Ana Dueso-Barroso, Andrew J. Dunford, Paul A. Edwards, Xavier Estivill, Dariush Etemadmoghadam, Milana Frenkel-Morgenstern, Dmitry A. Gordenin, Barbara Hutter, David T. W. Jones, Young Seok Ju, Marat D. Kazanov, Leszek J. Klimczak, Youngil Koh, Eunjung Alice Lee, Jake June-Koo Lee, Andy G. Lynch, Geoff MacIntyre, Florian Markowetz, Matthew Meyerson, Satoru Miyano, Fabio C. P. Navarro, Stephan Ossowski, Peter J. Park, John V. Pearson, Montserrat Puiggròs, Karsten Rippe, Steven A. Roberts, Bernardo Rodriguez-Martin, Ralph Scully, David Torrents, Izar Villasante, Nicola Waddell, Jeremiah A. Wala, Lixing Yang, Sung-Soo Yoon, and Jorge Zamora

Published
2020
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14. An Atlas of Cells in the Human Tonsil

Author

Ramon Massoni-Badosa, Paula Soler-Vila, Sergio Aguilar-Fernández, Juan C. Nieto, Marc Elosua-Bayes, Domenica Marchese, Marta Kulis, Amaia Vilas-Zornoza, Marco Matteo Bühler, Sonal Rashmi, Clara Alsinet, Ginevra Caratù, Catia Moutinho, Sara Ruiz, Patricia Lorden, Giulia Lunazzi, Dolors Colomer, Gerard Frigola, Will Blevins, Sara Palomino, David Gomez-Cabrero, Xabier Agirre, Marc A. Weniger, Federico Marini, Francisco Javier Cervera-Paz, Peter M. Baptista, Isabel Vilaseca, Felipe Prosper, Ralf Küppers, Ivo Glynne Gut, Elias Campo, José Ignacio Martin-Subero, and Holger Heyn

Abstract

Palatine tonsils are secondary lymphoid organs representing the first line of immunological defense against inhaled or ingested pathogens. Here, we present a comprehensive census of cell types forming the human tonsil by applying single-cell transcriptome, epigenome, proteome and adaptive immune repertoire sequencing as well as spatial transcriptomics, resulting in an atlas of >357,000 cells. We provide a glossary of 121 annotated cell types and states, and disentangle gene regulatory mechanisms that drive cells through specialized lineage trajectories. Exemplarily, we stratify multiple tonsil-resident myeloid slancyte subtypes, establish a distant BCL6 superenhancer as locally active in both follicle-associated T and B cells, and describe SIX5 as a potentially novel transcriptional regulator of plasma cell maturation. Further, our atlas is a reference map to understand alterations observed in disease. Here, we discover immune-phenotype plasticity in tumoral cells and microenvironment shifts of mantle cell lymphomas (MCL). To facilitate such reference-based analysis, we develop HCATonsilData and SLOcatoR, a computational framework that provides programmatic and modular access to our dataset; and allows the straightforward annotation of future single-cell profiles from secondary lymphoid organs.

Published
2022
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15. Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Author

Ieva Keraite, Philipp Becker, Davide Canevazzi, Maria C. Frias-López, Marc Dabad, Raúl Tonda-Hernandez, Ida Paramonov, Matthew John Ingham, Isabelle Brun-Heath, Jordi Leno, Anna Abulí, Elena Garcia-Arumí, Simon Heath, Marta Gut, and Ivo Glynne Gut

Abstract

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Long-read whole genome sequencing is prohibitively expensive for mtDNA heteroplasmy detection and often does not recapitulate the full mtDNA length.Here we describe a method to target, multiplex and sequence full-length, native single-molecule the human mitochondrial genome utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks as barcodes with long-read sequencing, we implemented a protocol in an optimal setting for both high or low integrity genomic DNA to target the circular mitochondrial genome with extremely high coverage. Our analytical pipeline efficiently detects single nucleotide heteroplasmy, physically determines phase and can accurately disentangle complex deletion patterns. This workflow is a unique tool for studying mtDNA variation in health and disease, and will accelerate mitochondrial research.

Published
2022
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16. Quantitative DNA Methylation Analysis at Single-Nucleotide Resolution by Pyrosequencing®

Author

Florence, Busato, Emelyne, Dejeux, Hafida, El Abdalaoui, Ivo Glynne, Gut, and Jörg, Tost

Subjects
DNA Repair Enzymes, Nucleotides, Tumor Suppressor Proteins, DNA, Single-Stranded, Humans, Sulfites, CpG Islands, Sequence Analysis, DNA, DNA Methylation, Promoter Regions, Genetic, DNA Modification Methylases, Polymerase Chain Reaction
Abstract

Many protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative and/or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by PCR with one of the two primers being biotinylated. The PCR generated template is rendered single-stranded and a pyrosequencing primer is annealed to analyze quantitatively cytosine methylation. In comparative studies, pyrosequencing has been shown to be among the most accurate and reproducible technologies for locus-specific DNA methylation analyses and has become a widely used tool for the validation of DNA methylation changes identified in genome-wide studies as well as for locus-specific analyses with clinical impact such as methylation analysis of the MGMT promoter. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity.

Published
2017

18. New sequencing technologies

Author

Ivo Glynne Gut

Subjects
Epigenomics, Cancer Research, 2 base encoding, Computational biology, Biology, DNA sequencing, Translational Research, Biomedical, 03 medical and health sciences, symbols.namesake, 0302 clinical medicine, Neoplasms, Biomarkers, Tumor, Humans, Illumina dye sequencing, 030304 developmental biology, Whole genome sequencing, Genetics, Sanger sequencing, 0303 health sciences, Massive parallel sequencing, Genetic Variation, High-Throughput Nucleotide Sequencing, General Medicine, DNA sequencer, Oncology, symbols, 030217 neurology & neurosurgery, ABI Solid Sequencing
Abstract

Nucleic acid sequencing is one of the most important tools of biological research with very broad application. Four generations of DNA sequencing technologies can be distinguished by their nature and the kind of output they provide. Sanger sequencing dominated for 30 years and was the workhorse of the Human Genome Project. In 2005 the first 2nd generation sequencer was presented with an output orders of magnitude higher than Sanger sequencing and dramatically reduced cost per base. Currently, we are at the dawn of third generation with nanopore systems that are being developed for DNA sequencing. Meanwhile, the field is broadening applications that complement first, second and third generation sequencing systems to get high-resolution genetic information and fourth generation sequencing is on the horizon. Second generation nucleic acid sequencers are systematically applied in many large-scale international projects such as the International Cancer Genome Consortium and the International Human Epigenome Project.

Published
2013
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19. Applications of second generation sequencing technologies in complex disorders

Author

Mònica, Bayés, Simon, Heath, and Ivo Glynne, Gut

Subjects
Genetic Research, Genome, Human, Computational Biology, Humans, Genomics, Sequence Analysis, DNA, Epigenesis, Genetic
Abstract

Second generation sequencing (2ndGS) technologies generate unprecedented amounts of sequence data very rapidly and at relatively limited costs, allowing the sequence of a human genome to be completed in a few weeks. The principle is on the basis of generating millions of relatively short reads from amplified single DNA fragments using iterative cycles of nucleotide extensions. However, the data generated on this scale present new challenges in interpretation, data analysis and data management. 2ndGS technologies are becoming widespread and are profoundly impacting biomedical research. Common applications include whole-genome sequencing, target resequencing, characterization of structural and copy number variation, profiling epigenetic modifications, transcriptome sequencing and identification of infectious agents. New methodologies and instruments that will enable to sequence the complete human genome in less than a day at a cost of less than $1,000 are currently in development.

Published
2012

20. Mutation discovery in mice by whole exome sequencing

Author

Frank J. Probst, Federica Di Palma, Wenhui Huang, Joel E. Richardson, Rebecca R. Corrigan, Yueming Ding, Laura G. Reinholdt, Heather Fairfield, Stephen A. Murray, Michelle Curtain, Mary Barter, Daniel J. Gerhardt, Jay Shendure, Jeffrey A. Jeddeloh, Carol J. Bult, Kerstin Lindblad-Toh, Leah Rae Donahue, Todd Richmond, David E. Bergstrom, Mona S. Spector, Snaevar Sigurdsson, Chao He, Griffith J Gilbert, Evan Mauceli, Jorg Hager, Mark D'Ascenzo, Benjamin T. Kile, Lucy B. Rowe, Ivo Glynne Gut, Scott W. Lowe, Timothy C. Cox, Michael L. Cunningham, Thomas J. Albert, and Monica J. Justice

Subjects
Genotype, Mutant, DNA Mutational Analysis, Method, Genomics, Mice, Inbred Strains, Biology, medicine.disease_cause, 03 medical and health sciences, Mice, 0302 clinical medicine, Inbred strain, Gene Frequency, INDEL Mutation, medicine, Animals, Exome, Collagen Type II, Exome sequencing, 030304 developmental biology, Genetics, 0303 health sciences, Mutation, Chromosome Mapping, Exons, MAP Kinase Kinase Kinases, Chromosomes, Mammalian, Human genetics, 3. Good health, Phenotype, Indicators and Reagents, 030217 neurology & neurosurgery
Abstract

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

Published
2011

21. Identification and quantification of differentially methylated loci by the pyrosequencing technology

Author

Emelyne, Dejeux, Hafida, El abdalaoui, Ivo Glynne, Gut, and Jörg, Tost

Subjects
Carcinoma, Hepatocellular, Genes, p16, Liver Neoplasms, Humans, CpG Islands, DNA, DNA, Neoplasm, Genomics, Sequence Analysis, DNA, DNA Methylation, Nucleic Acid Amplification Techniques, Polymerase Chain Reaction, DNA Primers
Abstract

Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated. The PCR-generated template is rendered single stranded and a pyrosequencing primer is annealed to analyze quantitatively CpGs within 120 bases. Advantages of the pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.

Published
2008

22. Analysis of gene-specific DNA methylation patterns by pyrosequencing technology

Author

Jörg, Tost and Ivo Glynne, Gut

Subjects
HLA-G Antigens, Base Sequence, Genome, Human, Histocompatibility Antigens Class I, Molecular Sequence Data, DNA, Exons, Sequence Analysis, DNA, Templates, Genetic, DNA Methylation, Polymerase Chain Reaction, Diphosphates, HLA Antigens, Humans, Sulfites
Abstract

As the sequence of the human genome is now nearly finished, genome research turns to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the pathogenesis of various diseases. The real-time luminometric detection of pyrophosphate release upon nucleotide incorporation in the Pyrosequencing technology is ideally suited for the simultaneous analysis and quantification of the methylation degree of several CpG positions in close proximity. We developed and improved this analysis to obtain reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 80 nt. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity, which may be used as DNA methylation markers.

Published
2006

26. DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras

Author

Cassandra D. Calloway, Thomas W. Myers, Tetsuya Nishimoto, Jérémy Semhoun, David Harrow Gelfand, Ivo Glynne Gut, Keith A. Bauer, and Florence Mauger

Subjects
Polymorphism, Genetic, biology, DNA polymerase, Base pair, Oligonucleotide, Deoxyribonucleotides, DNA, DNA-Directed DNA Polymerase, Sequence Analysis, DNA, Ribonucleotides, Molecular biology, DNA, Mitochondrial, DNA sequencing, Sequencing by ligation, Biochemistry, Sequencing by hybridization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Genetics, biology.protein, Methods Online, RNA, Sodium Hydroxide, Primase, In vitro recombination
Abstract

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3' of all ribonucleotides to form a terminal 3' phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

Published
2007

27. Flash photolytic generation of two keto tautomers of 1-naphthol in aqueous solution: kinetics and equilibria of enolizationThis article is published as part of a themed issue in appreciation of the many important contributions made to the field of molecular photophysics by Jan Verhoeven.Electronic supplementary information (ESI) available: Tables of rate constants. See DOI: 10.1039/c0pp00034e

Author

Ivo Glynne Gut, Lukas C. Scheibler, and Jakob Wirz

Subjects
*FLASH photolysis, *TAUTOMERISM, *CHEMICAL kinetics, *CYCLOHEXANE, *SOLUTION (Chemistry), *THERMODYNAMIC cycles, *CHEMICAL equilibrium
Abstract

Benzo[b]cyclohexa-2,4-dien-1-one (4) and benzo[b]cyclohexa-2,5-dien-1-one (5), the two most stable keto tautomers of 1-naphthol (1), were generated in aqueous solution by Norrish Type II fission of 4- and 2-phenacyl-1-tetralone, respectively, and the pH–rate profiles of their enolization were measured by flash photolysis. Several isotopic exchange rates of 1were measured in aqueous acid to determine the corresponding rate constants of ketonization. The resulting equilibrium constants for enolization are pKE(4) = −7.1 and pKE(5) = −6.2. The acidity constants of the carbon acids 4and 5, pKa(4) = 2.1 and pKa(5) = 3.0, were then obtained from a thermodynamic cycle using pKa(1) = 9.25. [ABSTRACT FROM AUTHOR]

Published
2010
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