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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. 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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10. Identifying genetic determinants of inflammatory pain in mice using a large-scale gene-targeted screen

Author: Sara Wells, Marc Parisien, Abigail J. D’Souza, Rasneer Sonia Bains, Hamed Haselimashhadi, Mary E. Dickinson, Matthew Mckay, Amelia M. Willett, Christopher S. Ward, Robert Braun, Jeremy Mason, Stephen A. Murray, Emma Peterson, Michayla A. Moore, Damian Smedley, Erin E. Young, Luis Santos, Elissa L. Chesler, Laura C. Anderson, Rachel Urban, Helen Parkinson, Lynette Bower, John R. Seavitt, Dawei Qu, Leslie O. Goodwin, Vivek Kumar, Jason D. Heaney, Subhiksha Srinivasan, Kevin C K Lloyd, Mark E. Harrison, Steve D.M. Brown, Colin McKerlie, Ann M. Flenniken, Jacqueline K. White, Lauryl M. J. Nutter, Ziyue Huang, Jason A. Bubier, Daniel J. Delbarre, Robert P. Bonin, Kyle M. Baumbauer, Alexandr Bezginov, Dave Clary, Igor Vukobradovic, Ann-Marie Mallon, Silvia Mandillo, Luda Diatchenko, Surabi Veeraragavan, Michelle Stewart, Rodney C. Samaco, and Janine M Wotton
Subjects: Nociception, Scale (ratio), Autism, Freund's Adjuvant, Pain, Comorbidity, Computational biology, Biology, Sensitization, Mice, Hargreaves, IMPC, Animals, Single-gene knockout mouse, Gene, Pain Measurement, Mice, Knockout, Inflammatory pain, Formalin, Anesthesiology and Pain Medicine, Neurology, Screen, Complete Freund's adjuvant, Nocifensive behavior, Neurology (clinical), von Frey
Abstract: Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.
Published: 2021
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11. The production of 4,182 mouse lines identifies experimental and biological variables impacting Cas9-mediated mutant mouse line production

Author: Elif F. Acar, Luis Santos, Graham Duddy, Adam Caulder, Yann Herault, Joshua A. Wood, Jing Zhao, John R. Seavitt, Masaru Tamura, Martin Hrabĕ de Angelis, Gemma F. Codner, Helen Parkinson, Marie-Christine Birling, Lauryl M. J. Nutter, Jason D. Heaney, Radislav Sedlacek, Brandon J. Willis, Susan Marschall, Kevin A. Peterson, Marina Gertsenstein, Alba Gomez-Segura, Allan Bradley, Je Kyung Seong, Lydia Teboul, Kevin C K Lloyd, Fabio Mammano, Jacqueline K. White, Mary E. Dickinson, Francesco Chiani, Matthew Mackenzie, Robert Braun, Isabel Lorenzo, Colin McKerlie, Wolfgang Wurst, Edward Ryder, Cunxiang Ju, Denise G. Lanza, Ruairidh King, Alessia Gambadoro, Ho Lee, Zhiwei Liu, Ramiro Ramirez-Solis, Ann-Marie Mallon, William C. Skarnes, Xiang Gao, Stephen A. Murray, Francesco J. DeMayo, Lauri G. Lintott, Terrence F. Meehan, Fei Zhou, Hannah Wardle-Jones, Shinya Ayabe, Mark T. Ruhe, Atsushi Yoshiki, Brendan Doe, Peter Matthews, Sara Wells, Hillary Elrick, Claudia Seisenberger, David J. Adams, Jie Zhang, Damien Smedley, Petr Kasparek, Daekee Lee, and Leslie O. Goodwin
Subjects: Genetics, Mutant, Null (mathematics), Knockout mouse, Allele, Biology, Null allele, Genome, Gene, Germline
Abstract: The International Mouse Phenotyping Consortium (IMPC) is generating and phenotyping null mutations for every protein-coding gene in the mouse1,2. The IMPC now uses Cas9, a programmable RNA-guided nuclease that has revolutionized mouse genome editing3 and 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 >3,300 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo null allele 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. Our analysis identified that whether a gene is essential for viability was the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for generating null alleles in mice using Cas9; these recommendations may be applicable to other allele types and species.
Published: 2021
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12. Whole genome analysis for 163 guide RNAs in Cas9 edited mice reveals minimal off-target activity

Author: Denise G. Lanza, John R. Seavitt, Lauryl M J Nutter, Mary E. Dickinson, Brandon J. Willis, K. A. Peterson, Robert Braun, Stephen A. Murray, S. Khalouei, Kent Lloyd, Arun K. Ramani, Jason D. Heaney, L. G. Lintott, J. A. Woodd, N. Hanafi, and Jacqui White
Subjects: Whole genome sequencing, Genetics, Inbred strain, Genetic heterogeneity, Strain (biology), Knockout mouse, Mutagenesis (molecular biology technique), Biology, Genome, Null allele
Abstract: Introductory ParagraphThe Knockout Mouse Phenotyping Program (KOMP2) uses CRISRPR/Cas9 for high-throughput mouse line production to generate null alleles in the inbred C57BL/6N strain for broad-based in vivo phenotyping. In order to assess the risk of spurious S. pyogenes Cas9-induced off-target mutagenesis, we applied whole genome sequencing to compare the genomes of 50 Cas9-derived founder mice representing 163 different gRNAs to 28 untreated inbred control mice. Our analysis pipeline detected 28 off-target sequence variants associated with 21 guides. These potential off-targets were identified in 18/50 (36%) founders with 9/28 (32%) independently validated corresponding to 8 founder animals. In total, only 4.9% (8/163) of all guides exhibited off-target activity resulting in a rate of 0.16 Cas9 off-target mutations per founder analyzed. In comparison, we observed ~1225 unique variants in each mouse regardless of whether or not it was exposed to Cas9. These findings indicate that Cas9-mediated off-target mutagenesis is rare in founder knockout mice generated using guide RNAs designed to minimize off-target risk. Overall, bona fide off-target variants comprise a small fraction of the genetic heterogeneity found in carefully maintained colonies of inbred strains.
Published: 2021
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13. Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density

Author: Paul Flicek, Raffaele Teperino, Lydia Teboul, Thomas Werner, Marie-France Champy, Christopher J. Lelliott, Graham R. Williams, Jacqueline K. White, Gregor Miller, Mary E. Dickinson, Ann M Flenniken, Radislav Sedlacek, Martin Hrabé de Angelis, John R. Seavitt, Peter I. Croucher, Maria del Mar Muniz Moreno, Sara Wells, Jan Rozman, Terrence F. Meehan, Kristian F Odfalk, Juan Gallegos, J. H. Duncan Bassett, Mohammed Selloum, John G. Logan, Sylvie Jacquot, Elena J. Ghirardello, Robert Braun, Frantisek Spoutil, Kevin C K Lloyd, Lore Becker, Stephen A. Murray, Jan Prochazka, Elif F. Acar, Taylor S Vales, Michelle Simon, Helmut Fuchs, Nadine Spielmann, Mark Griffiths, Piia Keskivali-Bond, Valerie Gailus-Durner, Tania Sorg, Christine Schütt, Jeremy Mason, Helen Parkinson, Karen L. Svenson, Abdel Ayadi, Anna L Swan, Jason D. Heaney, Colin McKerlie, Wolfgang Wurst, Ann-Marie Mallon, Heather Cater, Stefanie Leuchtenberger, Harald Grallert, Steve D.M. Brown, Stefan Brandmaier, Yann Herault, Philipp Mayer-Kuckuk, Corey L. Reynolds, Ala Moshiri, Robert Brommage, Derek D. Cissell, Lauryl M J Nutter, Connor Lally, MRC Harwell Institute [UK], German Research Center for Environmental Health - Helmholtz Center München (GmbH), German Center for Diabetes Research - Deutsches Zentrum für Diabetesforschung [Neuherberg] (DZD), Institute of Molecular Genetics of the Czech Academy of Sciences (IMG / CAS), Czech Academy of Sciences [Prague] (CAS), 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), Helmholtz-Zentrum München (HZM), The Wellcome Trust Sanger Institute [Cambridge], University of Michigan [Ann Arbor], University of Michigan System, University of California [Davis] (UC Davis), University of California, Baylor College of Medicine (BCM), Baylor University, European Molecular Biology Laboratory [Hinxton], University of Toronto, University of Manitoba [Winnipeg], Lunenfeld-Tanenbaum Research Institute [Toronto, Canada], French National Infrastructure for Mouse Phenogenomics (PHENOMIN), The Jackson Laboratory [Bar Harbor] (JAX), European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Imperial College London, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Deutsches Zentrum für Neurodegenerative Erkrankungen [Ulm] (DZNE), German Research Center for Neurodegenerative Diseases - Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich Cluster for systems neurology [Munich] (SyNergy), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Ludwig-Maximilians-Universität München (LMU), Garvan Institute of Medical Research [Sydney, Australia], St. Vincent’s Clinical School [Sydney, Australia], UNSW Faculty of Medicine [Sydney], University of New South Wales [Sydney] (UNSW)-University of New South Wales [Sydney] (UNSW), Université de Nouvelle-Galles du Sud - UNSW [Sydney, Australia], Mundlos, Stefan, Wellcome Trust, Commission of the European Communities, European Commission, Herault, Yann, Helmholtz Zentrum München = German Research Center for Environmental Health, University of California (UC), Garvan Institute of medical research, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Technische Universität München [München] (TUM), Technische Universität München [München] (TUM)-Ludwig-Maximilians-Universität München (LMU), Swan, Anna L [0000-0003-1810-3756], Rozman, Jan [0000-0002-8035-8904], Del Mar Muñiz Moreno, Maria [0000-0002-2662-890X], Leuchtenberger, Stefanie [0000-0003-2475-0810], Brommage, Robert [0000-0002-9947-3822], Grallert, Harald [0000-0002-6876-9655], Werner, Thomas [0000-0003-0402-4539], Teperino, Raffaele [0000-0001-8815-1409], Becker, Lore [0000-0002-6890-4984], Miller, Gregor [0000-0002-4281-4905], Seavitt, John R [0000-0003-3209-3187], Cissell, Derek D [0000-0002-6450-422X], Acar, Elif F [0000-0003-2908-7691], Lelliott, Christopher J [0000-0001-8087-4530], Braun, Robert E [0000-0003-3856-9465], Cater, Heather [0000-0002-8696-6070], Flicek, Paul [0000-0002-3897-7955], Ghirardello, Elena J [0000-0002-1100-9217], Heaney, Jason D [0000-0001-8475-8828], Lally, Connor [0000-0002-3801-1966], Logan, John G [0000-0003-2801-700X], Mason, Jeremy [0000-0002-2796-5123], Nutter, Lauryl MJ [0000-0001-9619-146X], Odfalk, Kristian F [0000-0003-4152-4583], Prochazka, Jan [0000-0003-4675-8995], Selloum, Mohammed [0000-0003-4057-3519], Spoutil, Frantisek [0000-0002-7310-3487], Svenson, Karen L [0000-0002-7928-1911], Vales, Taylor S [0000-0001-9751-5681], Wells, Sara E [0000-0002-0572-0600], White, Jacqueline K [0000-0001-6268-2826], Sedlacek, Radislav [0000-0002-3352-392X], Wurst, Wolfgang [0000-0003-4422-7410], Lloyd, KC Kent [0000-0002-5318-4144], Williams, Graham R [0000-0002-8555-8219], Herault, Yann [0000-0001-7049-6900], Brown, Steve DM [0000-0002-0617-4824], Hrabe de Angelis, Martin [0000-0002-7898-2353], and Apollo - University of Cambridge Repository
Subjects: Male, Osteoporosis, genetics [Gene Expression Regulation], Transgenic, Transcriptome, Mice, 0302 clinical medicine, Animal Cells, Aetiology, Musculoskeletal System, Connective Tissue Cells, Genetics & Heredity, 0303 health sciences, Genomics, 3. Good health, Cellular Types, musculoskeletal diseases, Genotype, In silico, 1.1 Normal biological development and functioning, 03 medical and health sciences, genetics [Osteoporosis], Rheumatology, pathology [Osteoblasts], Genetic, Genome-Wide Association Studies, Genetics, GENOME-WIDE ASSOCIATION, Molecular Biology, Skeleton, Ecology, Evolution, Behavior and Systematics, METAANALYSIS, metabolism [Osteoblasts], 0604 Genetics, [SDV.GEN]Life Sciences [q-bio]/Genetics, Science & Technology, Osteoblasts, IDENTIFICATION, Biology and Life Sciences, Computational Biology, medicine.disease, COLLAGEN, Biological Tissue, OSTEOGENESIS IMPERFECTA, DISCOVERY, IMPC Consortium, Mutation, Animal Studies, Developmental Biology, Candidate gene, Cancer Research, Bone density, Gene Expression, Osteoclasts, [SDV.GEN] Life Sciences [q-bio]/Genetics, QH426-470, Bone remodeling, Bone Density, Medicine and Health Sciences, 2.1 Biological and endogenous factors, Protein Interaction Maps, Connective Tissue Diseases, Promoter Regions, Genetic, Genetics (clinical), Bone mineral, Sex Characteristics, genetics [Bone Density], metabolism [Osteoclasts], Genetic Pleiotropy, Animal Models, pathology [Osteoclasts], Phenotype, DIFFERENTIATION, Experimental Organism Systems, ANIMAL-MODELS, Connective Tissue, SEX, Female, Anatomy, Technology Platforms, Life Sciences & Biomedicine, Research Article, metabolism [Osteoporosis], Mouse Models, 030209 endocrinology & metabolism, Mice, Transgenic, Biology, Research and Analysis Methods, Promoter Regions, Model Organisms, Underpinning research, medicine, Animals, ddc:610, Bone, 030304 developmental biology, Human Genetics, Cell Biology, Genome Analysis, Gene Ontology, Gene Expression Regulation, Musculoskeletal, Genome-Wide Association Study
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., Author summary Patients affected by osteoporosis frequently present with decreased BMD and increased fracture risk. Genes are known to control the onset and progression of bone diseases such as osteoporosis. Therefore, we aimed to identify osteoporosis-related genes using BMD measures obtained from a large pool of mutant mice genetically modified for deletion of individual genes (knockout mice). In a collaborative endeavor involving several research sites world-wide, we generated and phenotyped 3,823 knockout mice and identified 200 genes which regulated BMD. Of the 200 BMD genes, 141 genes were previously not known to affect BMD. The discovery and study of novel BMD genes will help to better understand the causes and therapeutic options for patients with low BMD. In the long run, this will improve the clinical management of osteoporosis.
Published: 2021
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14. DELAYED SKELETAL DEVELOPMENT IN A MOUSE MODEL OF GLOBAL DEFICIENCY

Author: Bridget M. Stroup, Ronit Marom, Jason D. Heaney, Xiaohui Li, Safa Ani, Yuqing Chen, Jennie Rose Green, Audrey Christiansen, Mary E. Dickinson, John R. Seavitt, Brendan Lee, and Lindsay C. Burrage
Subjects: Endocrinology, Endocrinology, Diabetes and Metabolism, Genetics, Molecular Biology, Biochemistry
Published: 2022
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15. COPB2haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Author: Gladys Zapata, Mahim Jain, Maria Antonella De Matteis, Jeremy Allgrove, David R. Murdock, Jeremy Wegner, Daryl A. Scott, Nitesh R. Mehta, Yuqing Chen, Alyssa A. Tran, Brian Dawson, Shan Chen, David R. Eyre, Xiaohui Li, Brendan Lee, Elda Munivez, Aurélie Clément, Rolf W. Stottmann, Richard A. Gibbs, Jill A. Rosenfeld, Jennifer B. Phillips, Yi-Chien Lee, Alistair Calder, Zixue Jin, V. Reid Sutton, Rossella Venditti, Ronit Marom, Kyu Sang Joeng, Ming-Ming Jiang, Lindsay C. Burrage, Rowenna Roberts, Marwan Shinawi, Denise G. Lanza, Joseph M. Sliepka, Shalini N. Jhangiani, Brenna A. Tremp, John R. Seavitt, Mary E. Dickinson, Ingo Grafe, Donna M. Muzny, Lisa Emrick, Tashunka Taylor-Miller, MaryAnn Weis, Abbey A. Scott, Neil A. Hanchard, Bernardo Blanco-Sánchez, Catherine DeVile, Cole D Kuzawa, Dominyka Batkovskyte, I-Wen Song, Ghayda Mirzaa, Jason D. Heaney, and Catherine G. Ambrose
Subjects: symbols.namesake, Coatomer, Endoplasmic reticulum, symbols, COPI, Biology, Golgi apparatus, Haploinsufficiency, Ascorbic acid, COPB2, Type I collagen, Cell biology
Abstract: 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 inCOPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect ofCOPB2deficiency on skeletal development in mice and zebrafish.Copb2+/−mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying acopb2heterozygous frameshift variant showed delayed mineralization.copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality.COPB2siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest thatCOPB2haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due toCOPB2deficiency.
Published: 2020
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16. A global Slc7a7 knockout mouse model demonstrates characteristic phenotypes of human lysinuric protein intolerance

Author: John R. Seavitt, J. P. Barrish, Luan D. Truong, Lindsay C. Burrage, Farrah Kheradmand, Cheng Yen Chang, Denise G. Lanza, Yuqing Chen, Brian Dawson, Qin Sun, Jason D. Heaney, Xiaohui Li, Brendan Lee, Audrey E. Christiansen, Chih-Wei Hsu, Jennie R. Green, Ronit Marom, Safa Ani, Mary E. Dickinson, Ingo Grafe, Bridget M. Stroup, and Ming Ming Jiang
Subjects: medicine.medical_specialty, Arginine, Renal cortex, Biology, Kidney, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Internal medicine, Genetics, medicine, Animals, Humans, Amino Acids, Molecular Biology, Amino Acid Metabolism, Inborn Errors, Genetics (clinical), 030304 developmental biology, Mice, Knockout, 0303 health sciences, Amino Acid Transport System y+L, General Medicine, Exons, X-Ray Microtomography, Ornithine, medicine.disease, Phenotype, Lysinuric protein intolerance, Disease Models, Animal, medicine.anatomical_structure, Endocrinology, chemistry, Aminoaciduria, Knockout mouse, General Article, Pulmonary alveolar proteinosis, 030217 neurology & neurosurgery
Abstract: Lysinuric protein intolerance (LPI) is an inborn error of cationic amino acid (arginine, lysine, ornithine) transport caused by biallelic pathogenic variants in SLC7A7, which encodes the light subunit of the y+LAT1 transporter. Treatments for the complications of LPI, including growth failure, renal disease, pulmonary alveolar proteinosis, autoimmune disorders and osteoporosis, are limited. Given the early lethality of the only published global Slc7a7 knockout mouse model, a viable animal model to investigate global SLC7A7 deficiency is needed. Hence, we generated two mouse models with global Slc7a7 deficiency (Slc7a7em1Lbu/em1Lbu; Slc7a7Lbu/Lbu and Slc7a7em1(IMPC)Bay/em1(IMPC)Bay; Slc7a7Bay/Bay) using CRISPR/Cas9 technology by introducing a deletion of exons 3 and 4. Perinatal lethality was observed in Slc7a7Lbu/Lbu and Slc7a7Bay/Bay mice on the C57BL/6 and C57BL/6NJ inbred genetic backgrounds, respectively. We noted improved survival of Slc7a7Lbu/Lbu mice on the 129 Sv/Ev × C57BL/6 F2 background, but postnatal growth failure occurred. Consistent with human LPI, these Slc7a7Lbu/Lbu mice exhibited reduced plasma and increased urinary concentrations of the cationic amino acids. Histopathological assessment revealed loss of brush border and lipid vacuolation in the renal cortex of Slc7a7Lbu/Lbu mice, which combined with aminoaciduria suggests proximal tubular dysfunction. Micro-computed tomography of L4 vertebrae and skeletal radiographs showed delayed skeletal development and suggested decreased mineralization in Slc7a7Lbu/Lbu mice, respectively. In addition to delayed skeletal development and delayed development in the kidneys, the lungs and liver were observed based on histopathological assessment. Overall, our Slc7a7Lbu/Lbu mouse model on the F2 mixed background recapitulates multiple human LPI phenotypes and may be useful for future studies of LPI pathology.
Published: 2020

17. Soft windowing application to improve analysis of high-throughput phenotyping data

Author: Alexandr Bezginov, Paul Flicek, Masaru Tamura, Masuya Hiroshi, Mohammed Selloum, Yeon Kyung Kang, Herault Yann, Rodney C. Samaco, Ann-Marie Mallon, Helen Parkinson, Soo Young Cho, Ghina Bou About, Nadine Spielmann, Arturo Garza, Glauco P. Tocchini-Valentini, Gregor Miller, Obata Yuichi, John R. Seavitt, Surabi Veeraragavan, Jennie R. Green, Ewan Straiton, Marie-France Champy, Ann M Flenniken, Je Kyung Seong, Kyung Dong Soo, Isabel Lorenzo, Kevin C K Lloyd, Terrence F. Meehan, Vivek Kumar, Lillian Garrett, Damian Smedley, Elif F. Acar, Hamid Meziane, Ritu Bohat, Lore Becker, Sophie Leblanc, Denise G. Lanza, Angelina Gaspero, Audrey E. Christianson, Juan Gallegos, Wurst Wolfgang, Chih-Wei Hsu, Hamed Haselimashhadi, Colin McKerlie, Ruairidh King, Stephen D.M. Brown, Dave Clary, Nobuhiko Tanaka, Tania Sorg, Helmut Fuchs, Jason D. Heaney, Jacqui White, Jeremy Mason, Martin Hrabé de Angelis, Federico López-Gómez, Laurent Vasseur, Valerie Gailus-Durner, Holger Maier, Corey L. Reynolds, Violeta Muñoz-Fuentes, Mary E Dickinson, K. O. Babalola, Patrick T. Reilly, Jong Kyoung Kim, Schwartz, Russell, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
Subjects: Statistics and Probability, Computer science, Bioinformatics, Pipeline (computing), [SDV]Life Sciences [q-bio], Disease, computer.software_genre, 01 natural sciences, Biochemistry, Article, Mathematical Sciences, Set (abstract data type), Reduction (complexity), 010104 statistics & probability, 03 medical and health sciences, Mice, 0302 clinical medicine, Resampling, Information and Computing Sciences, False positive paradox, Genetics, Animals, Humans, 0101 mathematics, Molecular Biology, Throughput (business), ComputingMilieux_MISCELLANEOUS, Genetic Association Studies, 030304 developmental biology, Linkage (software), 0303 health sciences, Population Health, Biological Sciences, Computer Science Applications, Computational Mathematics, Phenotype, Computational Theory and Mathematics, Data mining, computer, 030217 neurology & neurosurgery, Software
Abstract: Motivation High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. Results Here we introduce ‘soft windowing’, a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype–phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. Availability and implementation The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. Supplementary information Supplementary data are available at Bioinformatics online.
Published: 2020
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18. 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

19. eP090: Precision medicine modelling for undiagnosed and rare disease

Author: Lindsay Burrage, Jill Rosenfeld, Shinya Yamamoto, Michael Wangler, Hugo Bellen, Denise Lanza, Matthew Roth, Ramin Zahedi Darshoori, John R. Seavitt, Mary Dickinson, Brendan Lee, Aleksandar Milosavljevic, and Jason Heaney
Subjects: Genetics (clinical)
Published: 2022
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20. Rapid and Integrative Discovery of Retina Regulatory Molecules

Author: Melanie A. Samuel, Danye Jiang, Courtney A. Burger, Julia Wang, Jonathan Alevy, Fenge Li, Seon Young Kim, Peter D. Westenskow, Brian I. Liu, Ritu Bohat, Angelina Gaspero, Shinya Yamamoto, Uchechukwu Udensi, Chih-Wei Hsu, John R. Seavitt, Nicholas E. Albrecht, Sowmya Kalaga, Arthur L. Beaudet, Mary E. Dickinson, Monica J. Justice, and Chinwe Asomugha
Subjects: Neurons, 0301 basic medicine, Nervous system, Retina, Retinal, Biology, Article, General Biochemistry, Genetics and Molecular Biology, Regulatory molecules, Synapse, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, medicine.anatomical_structure, chemistry, Synapses, medicine, Humans, Neuron, Gene, Neuroscience, Regulator gene
Abstract: Retinal function relies on precisely organized neurons and synapses and a properly patterned vasculature to support them. Alterations in these features can result in vision loss. However, our understanding of retinal organization pathways remains incomplete because of a lack of methods to rapidly identify neuron and vasculature regulators in mammals. Here we developed a pipeline for the identification of neural and synaptic integrity genes by high-throughput retinal screening (INSiGHT) that analyzes candidate expression, vascular patterning, cellular organization, and synaptic arrangement. Using this system, we examined 102 mutant mouse lines and identified 16 unique retinal regulatory genes. Fifteen of these candidates are identified as novel retina regulators, and many (9 of 16) are associated with human neural diseases. These results expand the genetic landscape involved in retinal circuit organization and provide a road map for continued discovery of mammalian retinal regulators and disease-causing alleles.
Published: 2018
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21. 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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22. CRIPTexonic deletion and a novel missense mutation in a female with short stature, dysmorphic features, microcephaly, and pigmentary abnormalities

Author: Christine M. Eng, Theodore Chiang, Weimin Bi, John R. Seavitt, Audrey Chan, Stephen A. Murray, Magalie S. Leduc, Wenmiao Zhu, Haley Streff, Seema R. Lalani, Zhiyv Niu, Yaping Yang, and Irene Miloslavskaya
Subjects: 0301 basic medicine, Proband, Microcephaly, DNA Mutational Analysis, Mutation, Missense, Dwarfism, Biology, Article, 03 medical and health sciences, Exon, 0302 clinical medicine, Genetics, medicine, Humans, Missense mutation, Abnormalities, Multiple, Global developmental delay, Syndactyly, Alleles, Genetic Association Studies, Genetics (clinical), Exome sequencing, Adaptor Proteins, Signal Transducing, Sequence Deletion, Facies, Exons, medicine.disease, Pedigree, Phenotype, 030104 developmental biology, Amino Acid Substitution, Child, Preschool, Female, Primordial dwarfism, 030217 neurology & neurosurgery
Abstract: Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc.
Published: 2016
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23. Correction to: The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation

Author: Hugh P. Morgan, Hamed Haseli Mashhadi, Ann M. Flenniken, Kevin C K Lloyd, Violeta Muñoz-Fuentes, Jason D. Heaney, Stephen A. Murray, Mary E. Dickinson, Paul Flicek, Steve D.M. Brown, Hannah Wardle-Jones, John R. Seavitt, Michelle Simon, Damian Smedley, Juan Antonio Aguilar-Pimentel, Helen Parkinson, Colin McKerlie, Je Kyung Seong, Martin Hrabě de Angelis, Ann-Marie Mallon, Lauryl M. J. Nutter, Terrence F. Meehan, Antonella Galli, Jong Kyoung Kim, Patrick T. Reilly, Arthur L. Beaudet, and Pilar Cacheiro
Subjects: Ecology (disciplines), Genetics, Computational biology, Mammalian genome, Biology, Ecology, Evolution, Behavior and Systematics
Published: 2019
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24. The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation

Author: Michelle Simon, Pilar Cacheiro, John R. Seavitt, Je Kyung Seong, Lauryl M. J. Nutter, Terrence F. Meehan, Martin Hrabě de Angelis, Patrick T. Reilly, Helen Parkinson, Juan Antonio Aguilar-Pimentel, Antonella Galli, Colin McKerlie, Steve D.M. Brown, Jong Kyoung Kim, Stephen A. Murray, Ann M. Flenniken, Hugh P. Morgan, Hamed Haseli Mashhadi, Damian Smedley, Kevin C K Lloyd, Hannah Wardle-Jones, Ann-Marie Mallon, Paul Flicek, and Violeta Muñoz-Fuentes
Subjects: Wolf, 0301 basic medicine, Candidate gene, Mouse, IMPC consortium, Life on Land, 1.1 Normal biological development and functioning, Cheetah, Endangered Species, Essential Genes, Impc, Knockout, Loss-of-function, Non-model Species, Panda, Phenotype, Polar Bear, Computational biology, Biology, Essential genes, Endangered species, 03 medical and health sciences, 0302 clinical medicine, Underpinning research, IMPC, Genetics, 2.1 Biological and endogenous factors, Aetiology, Non-model species, Gene, Genotyping, Ecology, Evolution, Behavior and Systematics, Loss function, 2. Zero hunger, Evolutionary Biology, Human Genome, Biological Sciences, ddc, Polar bear, 030104 developmental biology, Knockout mouse, Identification (biology), Generic health relevance, Environmental Sciences, 030217 neurology & neurosurgery, Function (biology), Biotechnology, Research Article
Abstract: The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied. Electronic supplementary material The online version of this article (10.1007/s10592-018-1072-9) contains supplementary material, which is available to authorized users.
Published: 2017

25. Corrigendum: High-throughput discovery of novel developmental phenotypes

Author: Mary E, Dickinson, Ann M, Flenniken, Xiao, Ji, Lydia, Teboul, Michael D, Wong, Jacqueline K, White, Terrence F, Meehan, Wolfgang J, Weninger, Henrik, Westerberg, Hibret, Adissu, Candice N, Baker, Lynette, Bower, James M, Brown, L Brianna, Caddle, Francesco, Chiani, Dave, Clary, James, Cleak, Mark J, Daly, James M, Denegre, Brendan, Doe, Mary E, Dolan, Sarah M, Edie Helmut Fuchs, Valerie, Gailus-Durner, Antonella, Galli, Alessia, Gambadoro, Juan, Gallegos, Shiying, Guo, Neil R, Horner, Chih-Wei, Hsu, Sara J, Johnson, Sowmya, Kalaga, Lance C, Keith, Louise, Lanoue, Thomas N, Lawson, Monkol, Lek, Manuel, Mark, Susan, Marschall, Jeremy, Mason, Melissa L, McElwee, Susan Newbigging Lauryl M J, Nutter, Kevin A, Peterson, Ramiro, Ramirez-Solis, Douglas J, Rowland, Edward, Ryder, Kaitlin E, Samocha, John R, Seavitt, Mohammed, Selloum, Zsombor, Szoke-Kovacs, Masaru, Tamura, Amanda G, Trainor, Ilinca, Tudose, Shigeharu, Wakana, Jonathan, Warren, Olivia, Wendling, David B, West, Leeyean, Wong, Atsushi, Yoshiki, Wolfgang, Wurst, Daniel G, MacArthur, Glauco P, Tocchini-Valentini, Xiang, Gao, Paul, Flicek, Allan, Bradley, William C, Skarnes, Monica J, Justice, Helen E, Parkinson, Mark, Moore, Sara, Wells, Robert E, Braun, Karen L, Svenson, Martin Hrabe, de Angelis, Yann, Herault, Tim, Mohun, Ann-Marie, Mallon, R Mark, Henkelman, Steve D M, Brown, David J, Adams, K C Kent, Lloyd, Colin, McKerlie, Arthur L, Beaudet, and Maja Bućan Stephen A, Murray
Subjects: IMPC, KOMP, EUCOMM, knockout, embryonic lethal, Article, mouse
Abstract: Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts.
Published: 2017

26. Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

Author: Jianming Xu, Lan Liao, Denise G. Lanza, Matthew N. Rasband, Yu Deng, Chuansheng Zhang, Isabel Lorenzo, Chonghui Cheng, Ying Wang, Angelina Gaspero, Francisco J. DeMayo, Mary E. Dickinson, Arthur L. Beaudet, John R. Seavitt, Ping Zheng, and Jason D. Heaney
Subjects: Genetics, 0303 health sciences, Cre recombinase, Biology, Genome, Null allele, 03 medical and health sciences, 0302 clinical medicine, Genome editing, Conditional gene knockout, CRISPR, Allele, Gene, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: 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. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-mediated homology driven repair (HDR) with short and long single-stranded oligodeoxynucleotides (ssODNs and lssODNs). Using pairs of guides and ssODNs donating loxP sites, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 of 30 genes targeted. LoxP sites integrated in cis in at least one F0 for 18 of 23 targeted genes. However, loxP sites were mutagenized in 4 of 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was not influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and a single lssODN to introduce a loxP-flanked exon, conditional allele founders were generated for all 4 genes targeted. 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, lssODNs are amenable to high-throughput production of conditional alleles when they can be employed.
Published: 2017
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27. 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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28. 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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29. Response to 'Unexpected mutations after CRISPR–Cas9 editing in vivo'

Author: Stephen A. Murray, Jason D. Heaney, John R. Seavitt, William C. Skarnes, Lydia Teboul, Steve D.M. Brown, Mark W. Moore, Kevin C K Lloyd, and Lauryl M. J. Nutter
Subjects: 0301 basic medicine, Genetics, Extramural, Cell Biology, Biology, Biochemistry, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Genome editing, In vivo, Mutation (genetic algorithm), CRISPR, Molecular Biology, 030217 neurology & neurosurgery, Biotechnology
Published: 2018
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30. High-throughput discovery of novel developmental phenotypes

Author: Henrik Westerberg, Yann Herault, Colin McKerlie, Candice N. Baker, Dave Clary, William C. Skarnes, Hibret A. Adissu, Thomas N. Lawson, Monica J. Justice, R. Mark Henkelman, Monkol Lek, Helen Parkinson, Timothy J. Mohun, Sarah M. Edie, Paul Flicek, Francesco Chiani, Steve D.M. Brown, Martin Hrabé de Angelis, Mary E. Dolan, Chih-Wei Hsu, L. Brianna Caddle, Sara Wells, John R. Seavitt, Wolfgang Weninger, James M. Brown, Neil R. Horner, Zsombor Szoke-Kovacs, Louise Lanoue, Jacqueline K. White, Lauryl M. J. Nutter, Shiying Guo, Allan Bradley, Jonathan Warren, Ann M. Flenniken, Manuel Mark, Kevin A. Peterson, Kaitlin E. Samocha, Douglas J. Rowland, Daniel G. MacArthur, Ann-Marie Mallon, Maja Bucan, Amanda G. Trainor, Susan Newbigging, Ramiro Ramirez-Solis, Glauco P. Tocchini-Valentini, Shigeharu Wakana, Ilinca Tudose, Olivia Wendling, Edward Ryder, Lydia Teboul, Melissa L. McElwee, Kevin C K Lloyd, Terrence F. Meehan, David B. West, Stephen A. Murray, Valerie Gailus-Durner, Lance C. Keith, Mark J. Daly, Lynette Bower, Juan Gallegos, Masaru Tamura, Helmut Fuchs, Susan Marschall, Mark W. Moore, Karen L. Svenson, Sara Johnson, David J. Adams, Xiang Gao, Robert E. Braun, Mohammed Selloum, Xiao Ji, Michael D. Wong, Atsushi Yoshiki, Alessia Gambadoro, James M. Denegre, Leeyean Wong, Jeremy Mason, Antonella Galli, Sowmya Kalaga, Arthur L. Beaudet, James Cleak, Brendan Doe, and Mary E. Dickinson
Subjects: 0301 basic medicine, Mouse, Mammalian embryology, Sequence Homology, Genome-wide association study, Penetrance, Development, Biology, medicine.disease_cause, Polymorphism, Single Nucleotide, Article, 03 medical and health sciences, Mice, Imaging, Three-Dimensional, medicine, Lethal allele, Animals, Humans, Disease, Gene, Gene knockout, Conserved Sequence, Genetics, Mice, Knockout, Mutation, Multidisciplinary, Genes, Essential, Embryo, Mammalian, Phenotype, High-Throughput Screening Assays, Mice, Inbred C57BL, 030104 developmental biology, Mutagenesis, Genes, Lethal, Genome-Wide Association Study
Abstract: Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
Published: 2015
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31. Erratum: Corrigendum: High-throughput discovery of novel developmental phenotypes

Author: Steve D.M. Brown, L. Brianna Caddle, Valerie Gailus-Durner, Xiao Ji, Sowmya Kalaga, Mark J. Daly, Kevin C K Lloyd, Susan Newbigging Lauryl M. J. Nutter, Jeremy Mason, Martin Hrabé de Angelis, Ann-Marie Mallon, Helen Parkinson, Paul Flicek, Timothy J. Mohun, Lynette Bower, Shiying Guo, Edward Ryder, Ramiro Ramirez-Solis, Ilinca Tudose, William C. Skarnes, Hibret A. Adissu, Francesco Chiani, Lance C. Keith, Maja Bućan Stephen A. Murray, Susan Marschall, John R. Seavitt, Henrik Westerberg, Chih-Wei Hsu, Terrence F. Meehan, David B. West, Ann M. Flenniken, Mohammed Selloum, Karen L. Svenson, Daniel G. MacArthur, Allan Bradley, Zsombor Szoke-Kovacs, Alessia Gambadoro, Glauco P. Tocchini-Valentini, James M. Denegre, Leeyean Wong, Neil R. Horner, Amanda G. Trainor, Mary E. Dickinson, James M. Brown, Shigeharu Wakana, Kevin A. Peterson, Lydia Teboul, Michael D. Wong, Atsushi Yoshiki, Robert Braun, Mark W. Moore, Masaru Tamura, Jacqueline K. White, Mary E. Dolan, Manuel Mark, David J. Adams, Kaitlin E. Samocha, Thomas N. Lawson, Monica J. Justice, Candice N. Baker, Wolfgang Weninger, Melissa L. McElwee, Sara Wells, Olivia Wendling, Douglas J. Rowland, R. Mark Henkelman, Juan Gallegos, Sara Johnson, Colin McKerlie, Wolfgang Wurst, Dave Clary, Xiang Gao, Louise Lanoue, Monkol Lek, Sarah M. Edie Helmut Fuchs, Jonathan Warren, Yann Herault, Arthur L. Beaudet, James Cleak, Brendan Doe, and Antonella Galli
Subjects: 0301 basic medicine, German, 03 medical and health sciences, 030104 developmental biology, Multidisciplinary, Developmental genetics, language, Library science, Sociology, language.human_language
Abstract: Nature 537, 508–514 (2016); doi:10.1038/nature19356 In this Article, the author Wolfgang Wurst was erroneously omitted from the author list. They are associated with the affiliations: HelmholtzZentrum Munich, Institute of Developmental Genetics, 85764 Munich-Neuherberg, Germany; Technical Universityof Munich, Chair of Developmental Genetics, 85764 Munich-Neuherberg, Germany; German Center for Neurodegenerative Diseases (DZNE) Site Munich, 81377 Munich, Germany; and Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany.
Published: 2017
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32. Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development

Author: Roger M. Perlmutter, Kenneth M. Murphy, Lynn S. White, Matthew L. Thomas, Dennis Y. Loh, and John R. Seavitt
Subjects: CD4-Positive T-Lymphocytes, Time Factors, Transgene, Recombinant Fusion Proteins, Immunoblotting, Receptors, Antigen, T-Cell, chemical and pharmacologic phenomena, Apoptosis, Mice, Transgenic, Protein tyrosine phosphatase, Thymus Gland, Biology, CD8-Positive T-Lymphocytes, Dephosphorylation, Mice, Antigen, Animals, Molecular Biology, Cell Growth and Development, Crosses, Genetic, Regulation of gene expression, CD69, T-cell receptor, Phosphotransferases, hemic and immune systems, Cell Biology, Flow Cytometry, Molecular biology, Thymocyte, Gene Expression Regulation, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Leukocyte Common Antigens, Electrophoresis, Polyacrylamide Gel, Spleen
Abstract: Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.
Published: 1999

33. 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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34. 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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34 results on '"John R. Seavitt"'

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

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