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1. 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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2. 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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3. 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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4. 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

5. 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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6. 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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7. 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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8. 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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9. 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

10. 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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11. Prevalence of sexual dimorphism in mammalian phenotypic traits

Author

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

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

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

Published
2017
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12. 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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13. 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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14. 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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15. 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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16. 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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17. 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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18. 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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