Publications by authors named "Ann-Marie Mallon"

64 Publications

LAMA: automated image analysis for the developmental phenotyping of mouse embryos.

Development 2021 Mar 24;148(18). Epub 2021 Mar 24.

Medical Research Council Harwell Institute, Harwell 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 personnel and expertise. Thus, rapid automated annotation is crucial to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline that 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015254PMC
March 2021

OpenStats: A robust and scalable software package for reproducible analysis of high-throughput phenotypic data.

PLoS One 2020 30;15(12):e0242933. Epub 2020 Dec 30.

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.

Reproducibility in the statistical analyses of data from high-throughput phenotyping screens requires a robust and reliable analysis foundation that allows modelling of different possible statistical scenarios. Regular challenges are scalability and extensibility of the analysis software. In this manuscript, we describe OpenStats, a freely available software package that addresses these challenges. We show the performance of the software in a high-throughput phenomic pipeline in the International Mouse Phenotyping Consortium (IMPC) and compare the agreement of the results with the most similar implementation in the literature. OpenStats has significant improvements in speed and scalability compared to existing software packages including a 13-fold improvement in computational time to the current production analysis pipeline in the IMPC. Reduced complexity also promotes FAIR data analysis by providing transparency and benefiting other groups in reproducing and re-usability of the statistical methods and results. OpenStats is freely available under a Creative Commons license at www.bioconductor.org/packages/OpenStats.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242933PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773254PMC
January 2021

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

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

Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

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

A holistic view of mouse enhancer architectures reveals analogous pleiotropic effects and correlation with human disease.

BMC Genomics 2020 Nov 2;21(1):754. Epub 2020 Nov 2.

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

Background: Efforts to elucidate the function of enhancers in vivo are underway but their vast numbers alongside differing enhancer architectures make it difficult to determine their impact on gene activity. By systematically annotating multiple mouse tissues with super- and typical-enhancers, we have explored their relationship with gene function and phenotype.

Results: Though super-enhancers drive high total- and tissue-specific expression of their associated genes, we find that typical-enhancers also contribute heavily to the tissue-specific expression landscape on account of their large numbers in the genome. Unexpectedly, we demonstrate that both enhancer types are preferentially associated with relevant 'tissue-type' phenotypes and exhibit no difference in phenotype effect size or pleiotropy. Modelling regulatory data alongside molecular data, we built a predictive model to infer gene-phenotype associations and use this model to predict potentially novel disease-associated genes.

Conclusion: Overall our findings reveal that differing enhancer architectures have a similar impact on mammalian phenotypes whilst harbouring differing cellular and expression effects. Together, our results systematically characterise enhancers with predicted phenotypic traits endorsing the role for both types of enhancers in human disease and disorders.
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http://dx.doi.org/10.1186/s12864-020-07109-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607678PMC
November 2020

Genetic background influences tumour development in heterozygous Men1 knockout mice.

Endocr Connect 2020 May;9(5):426-437

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

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5-26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.
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http://dx.doi.org/10.1530/EC-20-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274560PMC
May 2020

TarGo: network based target gene selection system for human disease related mouse models.

Lab Anim Res 2019 13;35:23. Epub 2019 Nov 13.

3Laboratory of Developmental Biology and Genomics, Research Institute for Veterinary Science, and BK21 Plus Program for Creative Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826 Republic of Korea.

Genetically engineered mouse models are used in high-throughput phenotyping screens to understand genotype-phenotype associations and their relevance to human diseases. However, not all mutant mouse lines with detectable phenotypes are associated with human diseases. Here, we propose the "Target gene selection system for Genetically engineered mouse models" (TarGo). Using a combination of human disease descriptions, network topology, and genotype-phenotype correlations, novel genes that are potentially related to human diseases are suggested. We constructed a gene interaction network using protein-protein interactions, molecular pathways, and co-expression data. Several repositories for human disease signatures were used to obtain information on human disease-related genes. We calculated disease- or phenotype-specific gene ranks using network topology and disease signatures. In conclusion, TarGo provides many novel features for gene function prediction.
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http://dx.doi.org/10.1186/s42826-019-0023-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081697PMC
November 2019

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

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

Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.

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

High-throughput discovery of genetic determinants of circadian misalignment.

PLoS Genet 2020 01 13;16(1):e1008577. Epub 2020 Jan 13.

SKL of Pharmaceutical Biotechnology and Model Animal Research Center, Collaborative Innovation Center for Genetics and Development, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, China.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.
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http://dx.doi.org/10.1371/journal.pgen.1008577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980734PMC
January 2020

Protection Against XY Gonadal Sex Reversal by a Variant Region on Mouse Chromosome 13.

Genetics 2020 02 13;214(2):467-477. Epub 2019 Dec 13.

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

XY C57BL/6J (B6) mice harboring a -type Y chromosome (Y ), known as B6.Y mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.Y males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.Y colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.Y sex reversal. Here. we show that a B6.Y colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call , protects against B6.Y sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss 's influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.Y sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.
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http://dx.doi.org/10.1534/genetics.119.302786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017026PMC
February 2020

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

Bioinformatics 2020 03;36(5):1492-1500

Korea Mouse Phenotyping Center (KMPC), Korea.

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.
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http://dx.doi.org/10.1093/bioinformatics/btz744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115897PMC
March 2020

Genomic Mutation Identification in Mice Using Illumina Sequencing and Linux-Based Computational Methods.

Curr Protoc Mouse Biol 2019 Sep;9(3):e64

MRC Harwell Institute, Mammalian Genetics Unit, Harwell Campus, Oxfordshire, United Kingdom.

Genetically modified mice are an essential tool for modeling disease-causing mechanisms and discovering gene function. SNP genotyping was traditionally used to associate candidate regions with traits in the mouse, but failed to reveal novel variants without further targeted sequencing. Using a robust set of computational protocols, we present a platform to enable scientists to detect variants arising from whole-genome and exome sequencing experiments. This article guides researchers on aligning reads to the mouse genome, quality-assurance strategies, mutation discovery, comparing mutations to previously discovered mouse SNPs, and the annotation of novel variants, in order to predict mutation consequences on the protein level. Challenges unique to the mouse are discussed, and two protocols use self-contained containers to maintain version control and allow users to adapt our approach to new techniques by upgrading container versions. Our protocols are suited for servers or office workstations and are usable by non-bioinformatics specialists. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.64DOI Listing
September 2019

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

J Bone Miner Res 2019 07 4;34(7):1324-1335. Epub 2019 Mar 4.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4 ) mice, when compared with wild-type (Brd4 ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4 and Brd4 mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4 mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658219PMC
July 2019

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Commun Biol 2018 21;1:236. Epub 2018 Dec 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
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http://dx.doi.org/10.1038/s42003-018-0226-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303268PMC
December 2018

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice.

J Bone Miner Res 2019 03 14;34(3):497-507. Epub 2018 Dec 14.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2 ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2 ). The Polg2 and Polg2 mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 kidneys was reduced compared to Polg2 mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 mice, compared to Polg2 littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446808PMC
March 2019

An -Ethyl--Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion.

JBMR Plus 2018 May 8;2(3):154-163. Epub 2018 Mar 8.

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

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen -ethyl--nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as . Micro-computed tomography (μCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. μCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124210PMC
May 2018

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

Conserv Genet 2018 19;19(4):995-1005. Epub 2018 May 19.

1European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD UK.

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.
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http://dx.doi.org/10.1007/s10592-018-1072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061128PMC
May 2018

High-throughput mouse phenomics for characterizing mammalian gene function.

Nat Rev Genet 2018 06;19(6):357-370

MRC Harwell Institute, Harwell, UK.

We are entering a new era of mouse phenomics, driven by large-scale and economical generation of mouse mutants coupled with increasingly sophisticated and comprehensive phenotyping. These studies are generating large, multidimensional gene-phenotype data sets, which are shedding new light on the mammalian genome landscape and revealing many hitherto unknown features of mammalian gene function. Moreover, these phenome resources provide a wealth of disease models and can be integrated with human genomics data as a powerful approach for the interpretation of human genetic variation and its relationship to disease. In the future, the development of novel phenotyping platforms allied to improved computational approaches, including machine learning, for the analysis of phenotype data will continue to enhance our ability to develop a comprehensive and powerful model of mammalian gene-phenotype space.
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http://dx.doi.org/10.1038/s41576-018-0005-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582361PMC
June 2018

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Nat Commun 2018 01 18;9(1):288. Epub 2018 Jan 18.

Monterotondo Mouse Clinic, Italian National Research Council (CNR), Institute of Cell Biology and Neurobiology, Adriano Buzzati-Traverso Campus, Via E. Ramarini 32, Monterotondo Scalo, RM, 00015, Italy.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.
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http://dx.doi.org/10.1038/s41467-017-01995-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773596PMC
January 2018

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.

RIKEN BioResource Center, Tsukuba, Ibaraki, 305-0074, Japan.

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

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

PLoS Genet 2017 Aug 14;13(8):e1006969. Epub 2017 Aug 14.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell, Oxfordshire, United Kingdom.

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.
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http://dx.doi.org/10.1371/journal.pgen.1006969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570507PMC
August 2017

Prevalence of sexual dimorphism in mammalian phenotypic traits.

Nat Commun 2017 06 26;8:15475. Epub 2017 Jun 26.

Mouse Genetics Project, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

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.
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http://dx.doi.org/10.1038/ncomms15475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203PMC
June 2017

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Nat Genet 2017 Aug 26;49(8):1231-1238. Epub 2017 Jun 26.

CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), Illkirch-Graffenstaden, France.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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http://dx.doi.org/10.1038/ng.3901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546242PMC
August 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.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, 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

Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair.

Genome Med 2016 Feb 15;8(1):16. Epub 2016 Feb 15.

Mammalian Genetics Unit, MRC Harwell, Harwell, Oxford, OX11 0RD, UK.

Background: Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote. These approaches provide an opportunity to modify the genomes of inbred mice, and allow the removal of strain-specific mutations that confound phenotypic assessment. One such mutation is the Cdh23 (ahl) allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss.

Results: We have used targeted CRISPR/Cas9-mediated homology directed repair (HDR) to correct the Cdh23 (ahl) allele directly in C57BL/6NTac zygotes. Employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide donor template we show that allele repair was successfully achieved. To investigate potential Cas9-mediated 'off-target' mutations in our corrected mouse, we undertook whole-genome sequencing and assessed the 'off-target' sites predicted for the guide RNAs (≤4 nucleotide mis-matches). No induced sequence changes were identified at any of these sites. Correction of the progressive hearing loss phenotype was demonstrated using auditory-evoked brainstem response testing of mice at 24 and 36 weeks of age, and rescue of the progressive loss of sensory hair cell stereocilia bundles was confirmed using scanning electron microscopy of dissected cochleae from 36-week-old mice.

Conclusions: CRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23 (ahl) allele in C57BL/6NTac mice, and rescue the associated auditory phenotype. The corrected mice described in this report will allow age-related auditory phenotyping studies to be undertaken using C57BL/6NTac-derived models, such as those generated by the International Mouse Phenotyping Consortium (IMPC) programme.
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http://dx.doi.org/10.1186/s13073-016-0273-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753642PMC
February 2016

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing.

Mamm Genome 2015 Oct 8;26(9-10):486-500. Epub 2015 Oct 8.

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

Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.
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http://dx.doi.org/10.1007/s00335-015-9603-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602060PMC
October 2015