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1. Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

Author

Hillary Elrick, Kevin A. Peterson, Brandon J. Willis, Denise G. Lanza, Elif F. Acar, Edward J. Ryder, Lydia Teboul, Petr Kasparek, Marie-Christine Birling, David J. Adams, Allan Bradley, Robert E. Braun, Steve D. Brown, Adam Caulder, Gemma F. Codner, Francesco J. DeMayo, Mary E. Dickinson, Brendan Doe, Graham Duddy, Marina Gertsenstein, Leslie O. Goodwin, Yann Hérault, Lauri G. Lintott, K. C. Kent Lloyd, Isabel Lorenzo, Matthew Mackenzie, Ann-Marie Mallon, Colin McKerlie, Helen Parkinson, Ramiro Ramirez-Solis, John R. Seavitt, Radislav Sedlacek, William C. Skarnes, Damien Smedley, Sara Wells, Jacqueline K. White, Joshua A. Wood, International Mouse Phenotyping Consortium, Stephen A. Murray, Jason D. Heaney, and Lauryl M. J. Nutter

Subjects
Cas9, Genome editing, Mouse, Knockout, Medicine, Science
Abstract

Abstract The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Published
2024
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2. Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Author

Kevin A. Peterson, Sam Khalouei, Nour Hanafi, Joshua A. Wood, Denise G. Lanza, Lauri G. Lintott, Brandon J. Willis, John R. Seavitt, Robert E. Braun, Mary E. Dickinson, Jacqueline K. White, K. C. Kent Lloyd, Jason D. Heaney, Stephen A. Murray, Arun Ramani, and Lauryl M. J. Nutter

Subjects
Biology (General), QH301-705.5
Abstract

Abstract Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for “correcting” variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Published
2023
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3. Delayed skeletal development and IGF-1 deficiency in a mouse model of lysinuric protein intolerance

Author

Bridget M. Stroup, Xiaohui Li, Sara Ho, Haonan Zhouyao, Yuqing Chen, Safa Ani, Brian Dawson, Zixue Jin, Ronit Marom, Ming-Ming Jiang, Isabel Lorenzo, Daniel Rosen, Denise Lanza, Nathalie Aceves, Sara Koh, John R. Seavitt, Jason D. Heaney, Brendan Lee, and Lindsay C. Burrage

Subjects
osteoporosis, osteoblast, igf-1, lysinuric protein intolerance, slc7a7, arginine, Medicine, Pathology, RB1-214
Published
2023
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4. Human and mouse essentiality screens as a resource for disease gene discovery

Author

Pilar Cacheiro, Violeta Muñoz-Fuentes, Stephen A. Murray, Mary E. Dickinson, Maja Bucan, Lauryl M. J. Nutter, Kevin A. Peterson, Hamed Haselimashhadi, Ann M. Flenniken, Hugh Morgan, Henrik Westerberg, Tomasz Konopka, Chih-Wei Hsu, Audrey Christiansen, Denise G. Lanza, Arthur L. Beaudet, Jason D. Heaney, Helmut Fuchs, Valerie Gailus-Durner, Tania Sorg, Jan Prochazka, Vendula Novosadova, Christopher J. Lelliott, Hannah Wardle-Jones, Sara Wells, Lydia Teboul, Heather Cater, Michelle Stewart, Tertius Hough, Wolfgang Wurst, Radislav Sedlacek, David J. Adams, John R. Seavitt, Glauco Tocchini-Valentini, Fabio Mammano, Robert E. Braun, Colin McKerlie, Yann Herault, Martin Hrabě de Angelis, Ann-Marie Mallon, K. C. Kent Lloyd, Steve D. M. Brown, Helen Parkinson, Terrence F. Meehan, Damian Smedley, The Genomics England Research Consortium, and The International Mouse Phenotyping Consortium

Subjects
Science
Abstract

Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery.

Published
2020
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5. Untargeted Metabolomics of Slc13a5 Deficiency Reveal Critical Liver–Brain Axis for Lipid Homeostasis

Author

Sofia Milosavljevic, Kevin E. Glinton, Xiqi Li, Cláudia Medeiros, Patrick Gillespie, John R. Seavitt, Brett H. Graham, and Sarah H. Elsea

Subjects
SLC13A5, lipid synthesis, untargeted metabolomics, SLC13A5 deficiency, citrate transport, bile acid metabolism, Microbiology, QR1-502
Abstract

Though biallelic variants in SLC13A5 are known to cause severe encephalopathy, the mechanism of this disease is poorly understood. SLC13A5 protein deficiency reduces citrate transport into the cell. Downstream abnormalities in fatty acid synthesis and energy generation have been described, though biochemical signs of these perturbations are inconsistent across SLC13A5 deficiency patients. To investigate SLC13A5-related disorders, we performed untargeted metabolic analyses on the liver, brain, and serum from a Slc13a5-deficient mouse model. Metabolomic data were analyzed using the connect-the-dots (CTD) methodology and were compared to plasma and CSF metabolomics from SLC13A5-deficient patients. Mice homozygous for the Slc13a5tm1b/tm1b null allele had perturbations in fatty acids, bile acids, and energy metabolites in all tissues examined. Further analyses demonstrated that for several of these molecules, the ratio of their relative tissue concentrations differed widely in the knockout mouse, suggesting that deficiency of Slc13a5 impacts the biosynthesis and flux of metabolites between tissues. Similar findings were observed in patient biofluids, indicating altered transport and/or flux of molecules involved in energy, fatty acid, nucleotide, and bile acid metabolism. Deficiency of SLC13A5 likely causes a broader state of metabolic dysregulation than previously recognized, particularly regarding lipid synthesis, storage, and metabolism, supporting SLC13A5 deficiency as a lipid disorder.

Published
2022
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6. Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Author

Denise G. Lanza, Angelina Gaspero, Isabel Lorenzo, Lan Liao, Ping Zheng, Ying Wang, Yu Deng, Chonghui Cheng, Chuansheng Zhang, John R. Seavitt, Francesco J. DeMayo, Jianming Xu, Mary E. Dickinson, Arthur L. Beaudet, and Jason D. Heaney

Subjects
CRISPR/Cas9, Conditional null allele, High-throughput production, Genome editing, Single-stranded DNA oligonucleotides, Homology directed repair, Biology (General), QH301-705.5
Abstract

Abstract Background The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene–phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). Results Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. Conclusions Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.

Published
2018
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7. Prevalence of sexual dimorphism in mammalian phenotypic traits

Author

Natasha A. Karp, Jeremy Mason, Arthur L. Beaudet, Yoav Benjamini, Lynette Bower, Robert E. Braun, Steve D.M. Brown, Elissa J. Chesler, Mary E. Dickinson, Ann M. Flenniken, Helmut Fuchs, Martin Hrabe de Angelis, Xiang Gao, Shiying Guo, Simon Greenaway, Ruth Heller, Yann Herault, Monica J. Justice, Natalja Kurbatova, Christopher J. Lelliott, K.C. Kent Lloyd, Ann-Marie Mallon, Judith E. Mank, Hiroshi Masuya, Colin McKerlie, Terrence F. Meehan, Richard F. Mott, Stephen A. Murray, Helen Parkinson, Ramiro Ramirez-Solis, Luis Santos, John R. Seavitt, Damian Smedley, Tania Sorg, Anneliese O. Speak, Karen P. Steel, Karen L. Svenson, International Mouse Phenotyping Consortium, Shigeharu Wakana, David West, Sara Wells, Henrik Westerberg, Shay Yaacoby, and Jacqueline K. White

Subjects
Science
Abstract

Systemic dissection of sexually dimorphic phenotypes in mice is lacking. Here, Karp and the International Mouse Phenotype Consortium show that approximately 10% of qualitative traits and 56% of quantitative traits in mice as measured in laboratory setting are sexually dimorphic.

Published
2017
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8. Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts

Author

Bret A. Moore, Brian C. Leonard, Lionel Sebbag, Sydney G. Edwards, Ann Cooper, Denise M. Imai, Ewan Straiton, Luis Santos, Christopher Reilly, Stephen M. Griffey, Lynette Bower, David Clary, Jeremy Mason, Michel J. Roux, Hamid Meziane, Yann Herault, International Mouse Phenotyping Consortium, Colin McKerlie, Ann M. Flenniken, Lauryl M. J. Nutter, Zorana Berberovic, Celeste Owen, Susan Newbigging, Hibret Adissu, Mohammed Eskandarian, Chih-Wei Hsu, Sowmya Kalaga, Uchechukwu Udensi, Chinwe Asomugha, Ritu Bohat, Juan J. Gallegos, John R. Seavitt, Jason D. Heaney, Arthur L. Beaudet, Mary E. Dickinson, Monica J. Justice, Vivek Philip, Vivek Kumar, Karen L. Svenson, Robert E. Braun, Sara Wells, Heather Cater, Michelle Stewart, Sharon Clementson-Mobbs, Russell Joynson, Xiang Gao, Tomohiro Suzuki, Shigeharu Wakana, Damian Smedley, J. K. Seong, Glauco Tocchini-Valentini, Mark Moore, Colin Fletcher, Natasha Karp, Ramiro Ramirez-Solis, Jacqueline K. White, Martin Hrabe de Angelis, Wolfgang Wurst, Sara M. Thomasy, Paul Flicek, Helen Parkinson, Steve D. M. Brown, Terrence F. Meehan, Patsy M. Nishina, Stephen A. Murray, Mark P. Krebs, Ann-Marie Mallon, K. C. Kent Lloyd, Christopher J. Murphy, and Ala Moshiri

Subjects
Biology (General), QH301-705.5
Abstract

In the original published version of the article, Valerie Vancollie was mistakenly omitted from the list of members of the International Mouse Phenotyping Consortium. In addition, recognition of funding from Wellcome Trust grant WT098051 was mistakenly omitted from the Acknowledgements.The errors have been corrected in both the PDF and HTML versions of the paper.

Published
2019
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9. Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Author

Mary E. Dickinson, Yu Deng, Chonghui Cheng, Angelina Gaspero, Jianming Xu, John R. Seavitt, Denise G. Lanza, Lan Liao, Isabel Lorenzo, Ying Wang, Francesco J. DeMayo, Ping Zheng, Jason D. Heaney, Arthur L. Beaudet, and Chuansheng Zhang

Subjects
0301 basic medicine, Single-stranded DNA oligonucleotides, Physiology, DNA, Single-Stranded, Cre recombinase, Plant Science, Biology, Mouse models, Genome, General Biochemistry, Genetics and Molecular Biology, Homology directed repair, Mice, 03 medical and health sciences, Loss of Function Mutation, Structural Biology, Conditional null allele, Conditional gene knockout, Animals, CRISPR, Allele, Gene, CRISPR/Cas9, lcsh:QH301-705.5, Alleles, Ecology, Evolution, Behavior and Systematics, Gene Editing, Mice, Knockout, Genetics, Exons, Cell Biology, Null allele, 030104 developmental biology, lcsh:Biology (General), High-throughput production, CRISPR-Cas Systems, General Agricultural and Biological Sciences, Research Article, RNA, Guide, Kinetoplastida, Developmental Biology, Biotechnology, Genome editing
Abstract

Background The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene–phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). Results Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. Conclusions Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome. Electronic supplementary material The online version of this article (10.1186/s12915-018-0529-0) contains supplementary material, which is available to authorized users.

Published
2018
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10. The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities

Author

Bonnie L. Lyons, Juan Antonio Aguilar-Pimentel, Colin McKerlie, Sara Wells, Michelle Stewart, Eleanor Herbert, John R. Seavitt, Rosalinda A Doty, Jean-Paul Wiegand, Liane Hobson, Ann M Flenniken, Lauryl M. J. Nutter, Brenda Kick, Dawei Qu, Jacqueline K. White, Martin Hrabe de Angelis, Marie Hutchison, Mary E Dickinson, and Cheryl L. Scudamore

Subjects
Male, 0301 basic medicine, Internationality, Adult male, Physiology, Inbred Strains, Social Sciences, Male mice, Hindlimb, Diagnostic Radiology, Tendons, Mice, 0302 clinical medicine, Skeletal Joints, Medicine and Health Sciences, Psychology, Medicine, Animal Husbandry, Musculoskeletal System, Animal Management, Histological examination, Mammals, 0303 health sciences, Multidisciplinary, Animal Behavior, Radiology and Imaging, Eukaryota, Agriculture, Anatomy, Bone Imaging, 3. Good health, Experimental Organism Systems, Connective Tissue, Lameness, Vertebrates, Gait Analysis, Research Article, animal structures, Imaging Techniques, Science, C57bl 6n, Research and Analysis Methods, Animal Welfare, Rodents, 03 medical and health sciences, Diagnostic Medicine, Animals, 030304 developmental biology, Behavior, Biological Locomotion, business.industry, Organisms, Biology and Life Sciences, Tarsal Bones, X-Ray Radiography, Mice, Inbred C57BL, body regions, Biological Tissue, 030104 developmental biology, Amniotes, Animal Studies, Calcaneus, Tomography, X-Ray Computed, business, Zoology, 030217 neurology & neurosurgery, Sudden onset
Abstract

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneous-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.

Published
2020

11. Prevalence of sexual dimorphism in mammalian phenotypic traits

Author

Damian Smedley, Colin McKerlie, Xiang Gao, Henrik Westerberg, Simon Greenaway, Monica J. Justice, Hiroshi Masuya, Elissa J. Chesler, Robert E. Braun, Mary E. Dickinson, Shay Yaacoby, Stephen A. Murray, Karen L. Svenson, Jeremy Mason, Martin Hrabé de Angelis, Luis Santos, Tania Sorg, Christopher J. Lelliott, Sara Wells, Ann M. Flenniken, Ruth Heller, Ann-Marie Mallon, Lynette Bower, Karen P. Steel, Helen Parkinson, Judith E. Mank, Arthur L. Beaudet, Kevin C K Lloyd, Richard Mott, Yann Herault, Yoav Benjamini, Jacqueline K. White, Steve D.M. Brown, Shiying Guo, John R. Seavitt, Helmut Fuchs, Natalja Kurbatova, Anneliese O. Speak, Natasha A. Karp, Ramiro Ramirez-Solis, Terrence F. Meehan, David B. West, Shigeharu Wakana, The Wellcome Trust Sanger Institute [Cambridge], AstraZeneca [Cambridge, UK], European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Baylor College of Medicine (BCM), Baylor University, Tel Aviv University (TAU), University of California [Davis] (UC Davis), University of California (UC), The Jackson Laboratory [Bar Harbor] (JAX), MRC Harwell Institute [UK], Helmholtz Zentrum München = German Research Center for Environmental Health, Technische Universität München = Technical University of Munich (TUM), German Center for Diabetes Research - Deutsches Zentrum für Diabetesforschung [Neuherberg] (DZD), Nanjing University (NJU), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), French National Infrastructure for Mouse Phenogenomics (PHENOMIN), Institut Clinique de la Souris (ICS), The Hospital for sick children [Toronto] (SickKids), University College of London [London] (UCL), RIKEN BioResource Research Center [Tsukuba, Japan] (RIKEN BRC), Queen Mary University of London (QMUL), King‘s College London, Children's Hospital Oakland Research Institute (CHORI), International Mouse Phenotyping Consortium: Yuichi Obata, Tomohiro Suzuki, Masaru Tamura, Hideki Kaneda, Tamio Furuse, Kimio Kobayashi, Ikuo Miura, Ikuko Yamada, Nobuhiko Tanaka, Atsushi Yoshiki, Shinya Ayabe, David A Clary, Heather A Tolentino, Michael A Schuchbauer, Todd Tolentino, Joseph Anthony Aprile, Sheryl M Pedroia, Lois Kelsey, Igor Vukobradovic, Zorana Berberovic, Celeste Owen, Dawei Qu, Ruolin Guo, Susan Newbigging, Lily Morikawa, Napoleon Law, Xueyuan Shang, Patricia Feugas, Yanchun Wang, Mohammad Eskandarian, Yingchun Zhu, Lauryl M J Nutter, Patricia Penton, Valerie Laurin, Shannon Clarke, Qing Lan, Khondoker Sohel, David Miller, Greg Clark, Jane Hunter, Jorge Cabezas, Mohammed Bubshait, Tracy Carroll, Sandra Tondat, Suzanne MacMaster, Monica Pereira, Marina Gertsenstein, Ozge Danisment, Elsa Jacob, Amie Creighton, Gillian Sleep, James Clark, Lydia Teboul, Martin Fray, Adam Caulder, Jorik Loeffler, Gemma Codner, James Cleak, Sara Johnson, Zsombor Szoke-Kovacs, Adam Radage, Marina Maritati, Joffrey Mianne, Wendy Gardiner, Susan Allen, Heather Cater, Michelle Stewart, Piia Keskivali-Bond, Caroline Sinclair, Ellen Brown, Brendan Doe, Hannah Wardle-Jones, Evelyn Grau, Nicola Griggs, Mike Woods, Helen Kundi, Mark N D Griffiths, Christian Kipp, David G Melvin, Navis P S Raj, Simon A Holroyd, David J Gannon, Rafael Alcantara, Antonella Galli, Yvette E Hooks, Catherine L Tudor, Angela L Green, Fiona L Kussy, Elizabeth J Tuck, Emma J Siragher, Simon A Maguire, David T Lafont, Valerie E Vancollie, Selina A Pearson, Amy S Gates, Mark Sanderson, Carl Shannon, Lauren F E Anthony, Maksymilian T Sumowski, Robbie S B McLaren, Agnieszka Swiatkowska, Christopher M Isherwood, Emma L Cambridge, Heather M Wilson, Susana S Caetano, Cecilia Icoresi Mazzeo, Monika H Dabrowska, Charlotte Lillistone, Jeanne Estabel, Anna Karin B Maguire, Laura-Anne Roberson, Guillaume Pavlovic, Marie-Christine Birling, Wattenhofer-Donze Marie, Sylvie Jacquot, Abdel Ayadi, Dalila Ali-Hadji, Philippe Charles, Philippe André, Elise Le Marchand, Amal El Amri, Laurent Vasseur, Antonio Aguilar-Pimentel, Lore Becker, Irina Treise, Kristin Moreth, Tobias Stoeger, Oana V Amarie, Frauke Neff, Wolfgang Wurst, Raffi Bekeredjian, Markus Ollert, Thomas Klopstock, Julia Calzada-Wack, Susan Marschall, Robert Brommage, Ralph Steinkamp, Christoph Lengger, Manuela A Östereicher, Holger Maier, Claudia Stoeger, Stefanie Leuchtenberger, AliÖ Yildrim, Lillian Garrett, Sabine M Hölter, Annemarie Zimprich, Claudia Seisenberger, Antje Bürger, Jochen Graw, Oliver Eickelberg, Andreas Zimmer, Eckhard Wolf, Dirk H Busch, Martin Klingenspor, Carsten Schmidt-Weber, Valérie Gailus-Durner, Johannes Beckers, Birgit Rathkolb, Jan Rozman, univOAK, Archive ouverte, Mason, Jeremy [0000-0002-2796-5123], Chesler, Elissa J [0000-0002-5642-5062], Angelis, Martin Hrabe de [0000-0002-7898-2353], Herault, Yann [0000-0001-7049-6900], Lelliott, Christopher J [0000-0001-8087-4530], McKerlie, Colin [0000-0002-2232-0967], Wakana, Shigeharu [0000-0001-8532-0924], Yaacoby, Shay [0000-0002-2583-4170], and Apollo - University of Cambridge Repository

Subjects
0301 basic medicine, Genotype, Science, Mutant, General Physics and Astronomy, [SDV.GEN] Life Sciences [q-bio]/Genetics, Quantitative trait locus, Biology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Mice, Quantitative Trait, Quantitative Trait, Heritable, Genetics, Animals, Modifier, Gene, Heritable, Mammals, Sex Characteristics, [SDV.GEN]Life Sciences [q-bio]/Genetics, Multidisciplinary, Genes, Modifier, Body Weight, General Chemistry, Phenotypic trait, Phenotype, Sexual dimorphism, 030104 developmental biology, Genes, Evolutionary biology, Female, International Mouse Phenotyping Consortium, Sex characteristics
Abstract

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans., Systemic dissection of sexually dimorphic phenotypes in mice is lacking. Here, Karp and the International Mouse Phenotype Consortium show that approximately 10% of qualitative traits and 56% of quantitative traits in mice as measured in laboratory setting are sexually dimorphic.

Published
2017
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12. Biallelic variants in OTUD6B cause an intellectual disability syndrome associated with seizures and dysmorphic features

Author

Christine M. Eng, Thomas Besnard, Ingo Helbig, Holger Lerche, Bülent Kara, H. Hjalgrim, Peter-Michael Kloetzel, Mahmoud Y. Issa, Frédéric Ebstein, Isabelle Hazart, M. Cecilia Ljungberg, Eric LeGuern, Yaping Yang, Jill A. Rosenfeld, Rikke S. Møller, Alicia Braxton, Lindsay C. Burrage, Sarah Weckhuysen, Felix Rosenow, Peter De Jonghe, Johannes R. Lemke, Hiltrud Muhle, Elizabeth Roeder, Mary E. Dickinson, Nina Barišić, Bassam Abu-Libdeh, Federico Zara, Sara Wells, Carla Marini, Jennifer L. Silhavy, Marcus J. Miller, Maha S. Zaki, Bertrand Isidor, Teresa Santiago-Sim, LaDonna Immken, Andrea Lehmann, Joseph G. Gleeson, Karl Martin Klein, Mari Tokita, A Suls, Honey Nagakura, Rudi Balling, Rosa Guerrero-López, Katalin Sterbova, Benjamin Cogné, John R. Seavitt, Pasquale Striano, Maher Shahrour, Stéphanie Baulac, Renzo Guerrini, Sara Johnson, Lydia Teboul, Stéphane Bézieau, Kiely N. James, Katia Hardies, Orly Elpeleg, Dana Craiu, Jason D. Heaney, Sébastien Küry, Yvonne G. Weber, Zaid Afawi, Weimin Bi, Rebecca O. Littlejohn, Patrick May, Jose Serratosa, Magdalena Walkiewicz, Johanna A. Jähn, and EuroEPINOMICS RES Consortium Autos

Subjects
0301 basic medicine, Male, Microcephaly, Proteasome Endopeptidase Complex, Adolescent, Protein degradation, Deubiquitinating enzyme, 03 medical and health sciences, Mice, Ubiquitin, Seizures, Report, Intellectual Disability, Intellectual disability, Endopeptidases, Genetics, medicine, Journal Article, Animals, Humans, Abnormalities, Multiple, Global developmental delay, Child, Genetics (clinical), biology, Syndrome, medicine.disease, Hypotonia, Musculoskeletal Abnormalities, Pedigree, Disease Models, Animal, 030104 developmental biology, Proteasome, Child, Preschool, biology.protein, Female, Human medicine, medicine.symptom, Gene Deletion
Abstract

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain(OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.

Published
2017
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13. COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay

Author

Shan Chen, Yi-Chien Lee, Bernardo Blanco-Sánchez, Nitesh R. Mehta, Xiaohui Li, Brendan Lee, Catherine DeVile, Jill A. Rosenfeld, Daryl A. Scott, Ming-Ming Jiang, V. Reid Sutton, I-Wen Song, Kyu Sang Joeng, Rossella Venditti, Yuqing Chen, Richard A. Gibbs, John Hicks, Alistair Calder, Ghayda Mirzaa, David R. Murdock, Jeremy Wegner, Gladys Zapata, Tashunka Taylor-Miller, David R. Eyre, Aurélie Clément, Brian Dawson, Joseph M. Sliepka, Jason D. Heaney, Marwan Shinawi, Shalini N. Jhangiani, Donna M. Muzny, Dominyka Batkovskyte, Lisa Emrick, John R. Seavitt, Ronit Marom, Brenna A. Tremp, Maria Antonietta De Matteis, Zixue Jin, Lindsay C. Burrage, Denise G. Lanza, Monte Westerfield, Jeremy Allgrove, Rolf W. Stottmann, Jennifer B. Phillips, Rowenna Roberts, Abbey A. Scott, Neil A. Hanchard, Mahim Jain, Megan Washington, Alyssa A. Tran, Mary E. Dickinson, Ingo Grafe, MaryAnn Weis, Marom, R., Burrage, L. C., Venditti, R., Clement, A., Blanco-Sanchez, B., Jain, M., Scott, D. A., Rosenfeld, J. A., Sutton, V. R., Shinawi, M., Mirzaa, G., Devile, C., Roberts, R., Calder, A. D., Allgrove, J., Grafe, I., Lanza, D. G., Li, X., Joeng, K. S., Lee, Y. -C., Song, I. -W., Sliepka, J. M., Batkovskyte, D., Washington, M., Dawson, B. C., Jin, Z., Jiang, M. -M., Chen, S., Chen, Y., Tran, A. A., Emrick, L. T., Murdock, D. R., Hanchard, N. A., Zapata, G. E., Mehta, N. R., Weis, M. A., Scott, A. A., Tremp, B. A., Phillips, J. B., Wegner, J., Taylor-Miller, T., Gibbs, R. A., Muzny, D. M., Jhangiani, S. N., Hicks, J., Stottmann, R. W., Dickinson, M. E., Seavitt, J. R., Heaney, J. D., Eyre, D. R., Westerfield, M., De Matteis, M. A., and Lee, B.

Subjects
Male, Embryo, Nonmammalian, Developmental Disabilities, Golgi Apparatus, Ascorbic Acid, Haploinsufficiency, Endoplasmic Reticulum, Coatomer Protein, Severity of Illness Index, Bone and Bones, Collagen Type I, Article, Coat Protein Complex I, Mice, symbols.namesake, Intellectual Disability, Genetics, Animals, Humans, COPI complex, RNA, Small Interfering, Child, Zebrafish, Genetics (clinical), Chemistry, Endoplasmic reticulum, collagen trafficking, Brain, Gene Expression Regulation, Developmental, COPB2, juvenile osteoporosis, COPI, Fibroblasts, Golgi apparatus, Ascorbic acid, Cell biology, Coatomer, Child, Preschool, Unfolded protein response, symbols, Osteoporosis, coatopathy, Female, Type I collagen
Abstract

Summary Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects’ fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/− mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/− mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/− mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

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14. Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes

Author

Lindsay C. Burrage, John J. Reynolds, Nissan Vida Baratang, Jennifer B. Phillips, Jeremy Wegner, Ashley McFarquhar, Martin R. Higgs, Audrey E. Christiansen, Denise G. Lanza, John R. Seavitt, Mahim Jain, Xiaohui Li, David A. Parry, Vandana Raman, David Chitayat, Ivan K. Chinn, Alison A. Bertuch, Lefkothea Karaviti, Alan E. Schlesinger, Dawn Earl, Michael Bamshad, Ravi Savarirayan, Harsha Doddapaneni, Donna Muzny, Shalini N. Jhangiani, Christine M. Eng, Richard A. Gibbs, Weimin Bi, Lisa Emrick, Jill A. Rosenfeld, John Postlethwait, Monte Westerfield, Mary E. Dickinson, Arthur L. Beaudet, Emmanuelle Ranza, Celine Huber, Valérie Cormier-Daire, Wei Shen, Rong Mao, Jason D. Heaney, Jordan S. Orange, Débora Bertola, Guilherme L. Yamamoto, Wagner A.R. Baratela, Merlin G. Butler, Asim Ali, Mehdi Adeli, Daniel H. Cohn, Deborah Krakow, Andrew P. Jackson, Melissa Lees, Amaka C. Offiah, Colleen M. Carlston, John C. Carey, Grant S. Stewart, Carlos A. Bacino, Philippe M. Campeau, Brendan Lee, David R. Adams, Aaron Aday, Mercedes E. Alejandro, Patrick Allard, Euan A. Ashley, Mahshid S. Azamian, Eva Baker, Ashok Balasubramanyam, Hayk Barseghyan, Gabriel F. Batzli, Alan H. Beggs, Babak Behnam, Hugo J. Bellen, Jonathan A. Bernstein, Gerard T. Berry, Anna Bican, David P. Bick, Camille L. Birch, Devon Bonner, Braden E. Boone, Bret L. Bostwick, Lauren C. Briere, Elly Brokamp, Donna M. Brown, Matthew Brush, Elizabeth A. Burke, Manish J. Butte, Shan Chen, Gary D. Clark, Terra R. Coakley, Joy D. Cogan, Heather A. Colley, Cynthia M. Cooper, Heidi Cope, William J. Craigen, Precilla D’Souza, Mariska Davids, Jean M. Davidson, Jyoti G. Dayal, Esteban C. Dell’Angelica, Shweta U. Dhar, Katrina M. Dipple, Laurel A. Donnell-Fink, Naghmeh Dorrani, Daniel C. Dorset, Emilie D. Douine, David D. Draper, Annika M. Dries, Laura Duncan, David J. Eckstein, Lisa T. Emrick, Gregory M. Enns, Ascia Eskin, Cecilia Esteves, Tyra Estwick, Liliana Fernandez, Carlos Ferreira, Elizabeth L. Fieg, Paul G. Fisher, Brent L. Fogel, Noah D. Friedman, William A. Gahl, Emily Glanton, Rena A. Godfrey, Alica M. Goldman, David B. Goldstein, Sarah E. Gould, Jean-Philippe F. Gourdine, Catherine A. Groden, Andrea L. Gropman, Melissa Haendel, Rizwan Hamid, Neil A. Hanchard, Frances High, Ingrid A. Holm, Jason Hom, Ellen M. Howerton, Yong Huang, Fariha Jamal, Yong-hui Jiang, Jean M. Johnston, Angela L. Jones, David M. Koeller, Isaac S. Kohane, Jennefer N. Kohler, Donna M. Krasnewich, Susan Korrick, Mary Koziura, Joel B. Krier, Jennifer E. Kyle, Seema R. Lalani, C. Christopher Lau, Jozef Lazar, Kimberly LeBlanc, Brendan H. Lee, Hane Lee, Shawn E. Levy, Richard A. Lewis, Sharyn A. Lincoln, Sandra K. Loo, Joseph Loscalzo, Richard L. Maas, Ellen F. Macnamara, Calum A. MacRae, Valerie V. Maduro, Marta M. Majcherska, May Christine V. Malicdan, Laura A. Mamounas, Teri A. Manolio, Thomas C. Markello, Ronit Marom, Martin G. Martin, Julian A. Martínez-Agosto, Shruti Marwaha, Thomas May, Allyn McConkie-Rosell, Colleen E. McCormack, Alexa T. McCray, Jason D. Merker, Thomas O. Metz, Matthew Might, Paolo M. Moretti, Marie Morimoto, John J. Mulvihill, David R. Murdock, Jennifer L. Murphy, Donna M. Muzny, Michele E. Nehrebecky, Stan F. Nelson, J. Scott Newberry, John H. Newman, Sarah K. Nicholas, Donna Novacic, James P. Orengo, J. Carl Pallais, Christina G.S. Palmer, Jeanette C. Papp, Neil H. Parker, Loren D.M. Pena, John A. Phillips, Jennifer E. Posey, John H. Postlethwait, Lorraine Potocki, Barbara N. Pusey, Genecee Renteria, Chloe M. Reuter, Lynette Rives, Amy K. Robertson, Lance H. Rodan, Jacinda B. Sampson, Susan L. Samson, Kelly Schoch, Daryl A. Scott, Lisa Shakachite, Prashant Sharma, Vandana Shashi, Rebecca Signer, Edwin K. Silverman, Janet S. Sinsheimer, Kevin S. Smith, Rebecca C. Spillmann, Joan M. Stoler, Nicholas Stong, Jennifer A. Sullivan, David A. Sweetser, Queenie K.-G. Tan, Cynthia J. Tifft, Camilo Toro, Alyssa A. Tran, Tiina K. Urv, Eric Vilain, Tiphanie P. Vogel, Daryl M. Waggott, Colleen E. Wahl, Nicole M. Walley, Chris A. Walsh, Melissa Walker, Jijun Wan, Michael F. Wangler, Patricia A. Ward, Katrina M. Waters, Bobbie-Jo M. Webb-Robertson, Matthew T. Wheeler, Anastasia L. Wise, Lynne A. Wolfe, Elizabeth A. Worthey, Shinya Yamamoto, John Yang, Yaping Yang, Amanda J. Yoon, Guoyun Yu, Diane B. Zastrow, Chunli Zhao, and Allison Zheng

Subjects
Adult, Pathology, medicine.medical_specialty, Adolescent, DNA repair, Osteochondrodysplasias, Short stature, Article, 03 medical and health sciences, Mice, Young Adult, 0302 clinical medicine, Chromosomal Instability, Exome Sequencing, Genetics, medicine, Homologous chromosome, Animals, Humans, Allele, Child, Zebrafish, Genetics (clinical), Exome sequencing, Alleles, Cells, Cultured, Genetic Association Studies, 030304 developmental biology, Mice, Knockout, 0303 health sciences, Spondyloepimetaphyseal dysplasia, biology, NF-kappa B, Genetic Variation, Fibroblasts, medicine.disease, biology.organism_classification, 3. Good health, Musculoskeletal Abnormalities, Dysplasia, Child, Preschool, Female, medicine.symptom, 030217 neurology & neurosurgery, DNA Damage
Abstract

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

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15. Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

Author

Anna L Swan, Christine Schütt, Jan Rozman, Maria Del Mar Muñiz Moreno, Stefan Brandmaier, Michelle Simon, Stefanie Leuchtenberger, Mark Griffiths, Robert Brommage, Piia Keskivali-Bond, Harald Grallert, Thomas Werner, Raffaele Teperino, Lore Becker, Gregor Miller, Ala Moshiri, John R Seavitt, Derek D Cissell, Terrence F Meehan, Elif F Acar, Christopher J Lelliott, Ann M Flenniken, Marie-France Champy, Tania Sorg, Abdel Ayadi, Robert E Braun, Heather Cater, Mary E Dickinson, Paul Flicek, Juan Gallegos, Elena J Ghirardello, Jason D Heaney, Sylvie Jacquot, Connor Lally, John G Logan, Lydia Teboul, Jeremy Mason, Nadine Spielmann, Colin McKerlie, Stephen A Murray, Lauryl M J Nutter, Kristian F Odfalk, Helen Parkinson, Jan Prochazka, Corey L Reynolds, Mohammed Selloum, Frantisek Spoutil, Karen L Svenson, Taylor S Vales, Sara E Wells, Jacqueline K White, Radislav Sedlacek, Wolfgang Wurst, K C Kent Lloyd, Peter I Croucher, Helmut Fuchs, Graham R Williams, J H Duncan Bassett, Valerie Gailus-Durner, Yann Herault, Ann-Marie Mallon, Steve D M Brown, Philipp Mayer-Kuckuk, Martin Hrabe de Angelis, and IMPC Consortium

Subjects
Genetics, QH426-470
Abstract

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Published
2020
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