Publications by authors named "Lydia Teboul"

52 Publications

Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis.

J Biol Chem 2021 Feb 18:100439. Epub 2021 Feb 18.

Division of Gene Regulation and Expression, School of Life Sciences,. Electronic address:

O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal O-GlcNAcase catalytic domain and a C-terminal pseudo histone acetyl transferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of O-GlcNAcase catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue specific changes in O-GlcNAc homeostasis regulation to compensate for loss of O-GlcNAcase activity. Using X-ray micro computed tomography on late gestation embryos, we identified defects in the kidney, brain, liver and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically due to loss of O-GlcNAcase catalytic activity and independent of the function of the HAT domain.
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http://dx.doi.org/10.1016/j.jbc.2021.100439DOI Listing
February 2021

LAMA: Automated image analysis for developmental phenotyping of mouse embryos.

Development 2021 Feb 11. Epub 2021 Feb 11.

Medical Research Council Harwell Institute, Harwell, Oxfordshire OX11 0RD, UK

Advanced 3D imaging modalities, such as micro computed tomography (micro-CT), have been incorporated into the high-throughput embryo pipeline of the International Mouse Phenotyping Consortium (IMPC). This project generates large volumes of raw data that cannot be immediately exploited without significant resources of manpower and expertise. Thus, rapid automated annotation is critical to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline, which harnesses the large amount of wild type control data available in the IMPC embryo pipeline in order to address issues of low mutant sample number as well as incomplete penetrance and variable expressivity. We also investigate the effect of developmental substage on automated phenotyping results. Designed primarily for developmental biologists, our software performs image pre-processing, registration, statistical analysis and segmentation of embryo images. We also present a novel anatomical E14.5 embryo atlas average and using it with LAMA show that we can uncover known and novel dysmorphology from two IMPC knockout lines.
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http://dx.doi.org/10.1242/dev.192955DOI Listing
February 2021

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

PLoS Genet 2020 Dec 28;16(12):e1009190. Epub 2020 Dec 28.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany.

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.
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http://dx.doi.org/10.1371/journal.pgen.1009190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822523PMC
December 2020

Anticipating and Identifying Collateral Damage in Genome Editing.

Trends Genet 2020 12 8;36(12):905-914. Epub 2020 Oct 8.

The Mary Lyon Centre, Medical Research Council Harwell Institute, Harwell Campus, Didcot, OX11 0RD, UK. Electronic address:

Genome editing has powerful applications in research, healthcare, and agriculture. However, the range of possible molecular events resulting from genome editing has been underestimated and the technology remains unpredictable on, and away from, the target locus. This has considerable impact in providing a safe approach for therapeutic genome editing, agriculture, and other applications. This opinion article discusses how to anticipate and detect those editing events by a combination of assays to capture all possible genomic changes. It also discusses strategies for preventing unwanted effects, critical to appraise the benefit or risk associated with the use of the technology. Anticipating and verifying the result of genome editing are essential for the success for all applications.
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http://dx.doi.org/10.1016/j.tig.2020.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658041PMC
December 2020

Diverse species-specific phenotypic consequences of loss of function sorting nexin 14 mutations.

Sci Rep 2020 08 13;10(1):13763. Epub 2020 Aug 13.

Genetics and Genomic Medicine, UCL GOS Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
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http://dx.doi.org/10.1038/s41598-020-70797-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427099PMC
August 2020

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination.

Methods 2020 Jun 26. Epub 2020 Jun 26.

The Mary Lyon Centre, MRC Harwell Institute, Harwell Campus, Didcot, Oxon OX11 0RD, UK. Electronic address:

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.
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http://dx.doi.org/10.1016/j.ymeth.2020.06.011DOI Listing
June 2020

Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety.

Mol Ther 2020 06 20;28(6):1422-1431. Epub 2020 Mar 20.

Université de Strasbourg, CNRS, INSERM, IGBMC, PHENOMIN-Institut Clinique de la Souris, Celphedia, Strasbourg 67404, France. Electronic address:

Genome editing tools have already revolutionized biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off and on target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity does not preclude the use of these tools but requires that molecular evidence to underpin the relevant risk:benefit evaluation is available. Safe and successful clinical application will also depend on the mode of delivery and the cellular context.
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http://dx.doi.org/10.1016/j.ymthe.2020.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264426PMC
June 2020

Human and mouse essentiality screens as a resource for disease gene discovery.

Nat Commun 2020 01 31;11(1):655. Epub 2020 Jan 31.

Clinical Pharmacology, William Harvey Research Institute, School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.
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http://dx.doi.org/10.1038/s41467-020-14284-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994715PMC
January 2020

Male mice lacking ADAMTS-16 are fertile but exhibit testes of reduced weight.

Sci Rep 2019 11 20;9(1):17195. Epub 2019 Nov 20.

Mammalian Genetics Unit, Medical Research Council, Harwell Institute, Oxfordshire, OX11 0RD, UK.

Adamts16 encodes a disintegrin-like and metalloproteinase with thrombospondin motifs, 16, a member of a family of multi-domain, zinc-binding proteinases. ADAMTS-16 is implicated in a number of pathological conditions, including hypertension, cancer and osteoarthritis. A large number of observations, including a recent report of human ADAMTS16 variants in cases of 46,XY disorders/differences of sex development (DSD), also implicate this gene in human testis determination. We used CRISPR/Cas9 genome editing to generate a loss-of-function allele in the mouse in order to examine whether ADAMTS-16 functions in mouse testis determination or testicular function. Male mice lacking Adamts16 on the C57BL/6N background undergo normal testis determination in the fetal period. However, adult homozygotes have an average testis weight that is around 10% lower than age-matched controls. Cohorts of mutant males tested at 3-months and 6-months of age were fertile. We conclude that ADAMTS-16 is not required for testis determination or male fertility in mice. We discuss these phenotypic data and their significance for our understanding of ADAMTS-16 function.
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http://dx.doi.org/10.1038/s41598-019-53900-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868159PMC
November 2019

The development of a high throughput drug-responsive model of white adipose tissue comprising adipogenic 3T3-L1 cells in a 3D matrix.

Biofabrication 2019 12 11;12(1):015018. Epub 2019 Dec 11.

OxSyBio Ltd, Building R27, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0QX, United Kingdom.

Adipose models have been applied to mechanistic studies of metabolic diseases (such as diabetes) and the subsequent discovery of new therapeutics. However, typical models are either insufficiently complex (2D cell cultures) or expensive and labor intensive (mice/in vivo). To bridge the gap between these models and in order to better inform pre-clinical studies we have developed a drug-responsive 3D model of white adipose tissue (WAT). Here, spheroids (680 ± 60 μm) comprising adipogenic 3T3-L1 cells encapsulated in 3D matrix were fabricated manually on a 96 well scale. Spheroids were highly characterised for lipid morphology, selected metabolite and adipokine secretion, and gene expression; displaying significant upregulation of certain adipogenic-specific genes compared with a 2D model. Furthermore, induction of lipolysis and promotion of lipogenesis in spheroids could be triggered by exposure to 8-br-cAMP and oleic-acid respectively. Metabolic and high content imaging data of spheroids exposed to an adipose-targeting drug, rosiglitazone, resulted in dose-responsive behavior. Thus, our 3D WAT model has potential as a powerful scalable tool for compound screening and for investigating adipose biology.
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http://dx.doi.org/10.1088/1758-5090/ab56feDOI Listing
December 2019

Targeted Mutations in the Mouse via Embryonic Stem Cells.

Methods Mol Biol 2020 ;2066:59-82

The Centre for Phenogenomics (TCP), Toronto, ON, Canada.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.
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http://dx.doi.org/10.1007/978-1-4939-9837-1_5DOI Listing
December 2020

When all is not lost: considering genetic compensation in laboratory animals.

Lab Anim (NY) 2019 10;48(10):282-284

The Mary Lyon Centre, Medical Research Council Harwell Institute, Oxon, United Kingdom.

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http://dx.doi.org/10.1038/s41684-019-0397-4DOI Listing
October 2019

Microhomologies are prevalent at Cas9-induced larger deletions.

Nucleic Acids Res 2019 08;47(14):7402-7417

MRC Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK.

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.
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http://dx.doi.org/10.1093/nar/gkz459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698657PMC
August 2019

CRISPR-Cas9-Mediated Mutagenesis: Mind the Gap?

CRISPR J 2018 08;1:263-264

2 Mammalian Genetics Unit, Medical Research Council Harwell Institute, Oxfordshire, United Kingdom.

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http://dx.doi.org/10.1089/crispr.2018.29027.greDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636871PMC
August 2018

Characterisation and use of a functional Gadd45g bacterial artificial chromosome.

Sci Rep 2018 11 23;8(1):17318. Epub 2018 Nov 23.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire, OX11 0RD, UK.

Bacterial artificial chromosomes (BACs) offer a means of manipulating gene expression and tagging gene products in the mammalian genome without the need to alter endogenous gene structure and risk deleterious phenotypic consequences. However, for a BAC clone to be useful for such purposes it must be shown to contain all the regulatory elements required for normal gene expression and allow phenotypic rescue in the absence of an endogenous gene. Here, we report identification of a functional BAC containing Gadd45g, a gene implicated in DNA repair, DNA demethylation and testis determination in mice and exhibiting a broad pattern of embryonic expression. Mouse fetuses lacking the endogenous Gadd45g gene undergo normal testis development in the presence of the Gadd45g BAC transgene. Moreover, a survey of embryonic Gadd45g expression from the BAC reveals that all reported sites of expression are maintained. This functional BAC can now be used for subsequent manipulation of the Gadd45g gene with the confidence that regulatory elements required for embryonic expression, including testis determination, are present. We describe the generation and characterisation of a Gadd45g-mCherry fluorescent reporter exhibiting strong expression in developing gonads and neural tissue, recapitulating endogenous gene expression, as evidence of this.
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http://dx.doi.org/10.1038/s41598-018-35458-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251886PMC
November 2018

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

BMC Biol 2018 06 21;16(1):70. Epub 2018 Jun 21.

The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon, OX11 0RD, UK.

Background: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.

Results: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors.

Conclusion: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.
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http://dx.doi.org/10.1186/s12915-018-0530-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011369PMC
June 2018

High-resolution μCT of a mouse embryo using a compact laser-driven X-ray betatron source.

Proc Natl Acad Sci U S A 2018 06 5;115(25):6335-6340. Epub 2018 Jun 5.

The John Adams Institute for Accelerator Science, Blackett Laboratory, Imperial College London, London SW7 2AZ, United Kingdom.

In the field of X-ray microcomputed tomography (μCT) there is a growing need to reduce acquisition times at high spatial resolution (approximate micrometers) to facilitate in vivo and high-throughput operations. The state of the art represented by synchrotron light sources is not practical for certain applications, and therefore the development of high-brightness laboratory-scale sources is crucial. We present here imaging of a fixed embryonic mouse sample using a compact laser-plasma-based X-ray light source and compare the results to images obtained using a commercial X-ray μCT scanner. The radiation is generated by the betatron motion of electrons inside a dilute and transient plasma, which circumvents the flux limitations imposed by the solid or liquid anodes used in conventional electron-impact X-ray tubes. This X-ray source is pulsed (duration <30 fs), bright (>10 photons per pulse), small (diameter <1 μm), and has a critical energy >15 keV. Stable X-ray performance enabled tomographic imaging of equivalent quality to that of the μCT scanner, an important confirmation of the suitability of the laser-driven source for applications. The X-ray flux achievable with this approach scales with the laser repetition rate without compromising the source size, which will allow the recording of high-resolution μCT scans in minutes.
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http://dx.doi.org/10.1073/pnas.1802314115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016801PMC
June 2018

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

EMBO J 2018 06 15;37(11). Epub 2018 May 15.

MRC Mammalian Genetics Unit, Harwell, UK

TDP-43 (encoded by the gene ) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous to dissect TDP-43 function at physiological levels both and Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. , this gain-of-function mutation in endogenous causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.
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http://dx.doi.org/10.15252/embj.201798684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983119PMC
June 2018

Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".

Nat Methods 2018 04 30;15(4):235-236. Epub 2018 Mar 30.

IMPC Cas9 Working Group and the IMPC Steering Committee and executive director of the IMPC (www.mousephenotype.org).

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http://dx.doi.org/10.1038/nmeth.4559DOI Listing
April 2018

N-ethyl-N-nitrosourea-Induced Adaptor Protein 2 Sigma Subunit 1 () Mutations Establish Loss-of-Function Mice.

JBMR Plus 2017 May;1(1):3-15

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, Oxford, UK.

The adaptor protein-2 sigma subunit (AP2σ), encoded by , forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for mutations and identified 5 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide to . Three-dimensional modeling and cellular expression of the missense variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-β3 loop, and β3 strand. Heterozygous mutant mice ( ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. mice, when compared with mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)) concentrations. However, homozygous mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.
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http://dx.doi.org/10.1002/jbm4.10001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824975PMC
May 2017

FTO demethylase activity is essential for normal bone growth and bone mineralization in mice.

Biochim Biophys Acta Mol Basis Dis 2018 Mar 2;1864(3):843-850. Epub 2017 Dec 2.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bbadis.2017.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798602PMC
March 2018

Corrigendum: High-throughput discovery of novel developmental phenotypes.

Nature 2017 11 8;551(7680):398. Epub 2017 Nov 8.

This corrects the article DOI: 10.1038/nature19356.
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http://dx.doi.org/10.1038/nature24643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849394PMC
November 2017

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

Nat Commun 2017 10 12;8(1):886. Epub 2017 Oct 12.

Medical Research Council Harwell Institute (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, Oxfordshire, OX11 0RD, UK.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.
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http://dx.doi.org/10.1038/s41467-017-00595-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638796PMC
October 2017

Phenotyping first-generation genome editing mutants: a new standard?

Mamm Genome 2017 08 29;28(7-8):377-382. Epub 2017 Jul 29.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Science Campus, Didcot, Oxfordshire, UK.

The unprecedented efficiency of the CRISPR/Cas9 system in genome engineering has opened the prospect of employing mutant founders for phenotyping cohorts, thus accelerating research projects by circumventing the requirement to generate cohorts using conventional two- or three-generation crosses. However, these first-generation mutants are often genetic mosaics, with a complex and difficult to define genetic make-up. Here, we discuss the potential benefits, challenges and scientific validity of such models.
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http://dx.doi.org/10.1007/s00335-017-9711-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569115PMC
August 2017

Introduction to Mammalian Genome Special Issue: Genome Editing.

Mamm Genome 2017 08;28(7-8):235-236

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, UK.

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http://dx.doi.org/10.1007/s00335-017-9708-5DOI Listing
August 2017

Analysing the outcome of CRISPR-aided genome editing in embryos: Screening, genotyping and quality control.

Methods 2017 05 28;121-122:68-76. Epub 2017 Mar 28.

The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK. Electronic address:

The application of CRISPR/Cas9 technology has revolutionised genetics by greatly enhancing the efficacy of genome editing in the early embryo. Furthermore, the system has enabled the generation of allele types previously incompatible with in vivo mutagenesis. Despite its versatility and ease of implementation, CRISPR/Cas9 editing outcome is unpredictable and can generate mosaic founders. Therefore, careful genotyping and characterisation of new mutants is proving essential. The literature presents a wide range of protocols for molecular characterisation, each representing different levels of investment. We present strategies and protocols for designing, producing and screening CRISPR/Cas9 edited founders and genotyping their offspring according to desired allele type (indel, point mutation and deletion).
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http://dx.doi.org/10.1016/j.ymeth.2017.03.016DOI Listing
May 2017

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.

Am J Hum Genet 2017 Apr 23;100(4):676-688. Epub 2017 Mar 23.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Baylor Miraca Genetics Laboratories, Baylor College of Medicine, Houston, TX 77021, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajhg.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384096PMC
April 2017

A bioimage informatics platform for high-throughput embryo phenotyping.

Brief Bioinform 2018 01;19(1):41-51

High-throughput phenotyping is a cornerstone of numerous functional genomics projects. In recent years, imaging screens have become increasingly important in understanding gene-phenotype relationships in studies of cells, tissues and whole organisms. Three-dimensional (3D) imaging has risen to prominence in the field of developmental biology for its ability to capture whole embryo morphology and gene expression, as exemplified by the International Mouse Phenotyping Consortium (IMPC). Large volumes of image data are being acquired by multiple institutions around the world that encompass a range of modalities, proprietary software and metadata. To facilitate robust downstream analysis, images and metadata must be standardized to account for these differences. As an open scientific enterprise, making the data readily accessible is essential so that members of biomedical and clinical research communities can study the images for themselves without the need for highly specialized software or technical expertise. In this article, we present a platform of software tools that facilitate the upload, analysis and dissemination of 3D images for the IMPC. Over 750 reconstructions from 80 embryonic lethal and subviable lines have been captured to date, all of which are openly accessible at mousephenotype.org. Although designed for the IMPC, all software is available under an open-source licence for others to use and develop further. Ongoing developments aim to increase throughput and improve the analysis and dissemination of image data. Furthermore, we aim to ensure that images are searchable so that users can locate relevant images associated with genes, phenotypes or human diseases of interest.
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http://dx.doi.org/10.1093/bib/bbw101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862285PMC
January 2018

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

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.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

BMC Cell Biol 2016 08 5;17(1):30. Epub 2016 Aug 5.

The Mary Lyon Centre, Medical Research Council Harwell Institute, Harwell Science and Innovation Campus, Didcot, OX11 0RD, Oxon, UK.

Background: Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes.

Results: As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.

Conclusion: We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).
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http://dx.doi.org/10.1186/s12860-016-0108-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974727PMC
August 2016