Publications by authors named "Andrew Bassett"

54 Publications

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

CRISPR J 2021 Feb;4(1):132-146

Department of Physiology, Faculty of Veterinary, University of Murcia, Campus Mare Nostrum, Murcia, Spain.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.
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http://dx.doi.org/10.1089/crispr.2020.0078DOI Listing
February 2021

Unraveling the developmental roadmap toward human brown adipose tissue.

Stem Cell Reports 2021 Feb 6. Epub 2021 Feb 6.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK; Metabolic Research Laboratories, Addenbrooke's Treatment Centre, Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK; Cambridge University Nanjing Centre of Technology and Innovation, Jiangbei Area, Nanjing, P.R. China. Electronic address:

Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development.
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http://dx.doi.org/10.1016/j.stemcr.2021.01.013DOI Listing
February 2021

Genome-wide meta-analysis, fine-mapping and integrative prioritization implicate new Alzheimer's disease risk genes.

Nat Genet 2021 Feb 15. Epub 2021 Feb 15.

Open Targets, Wellcome Genome Campus, Cambridge, UK.

Genome-wide association studies have discovered numerous genomic loci associated with Alzheimer's disease (AD); yet the causal genes and variants are incompletely identified. We performed an updated genome-wide AD meta-analysis, which identified 37 risk loci, including new associations near CCDC6, TSPAN14, NCK2 and SPRED2. Using three SNP-level fine-mapping methods, we identified 21 SNPs with >50% probability each of being causally involved in AD risk and others strongly suggested by functional annotation. We followed this with colocalization analyses across 109 gene expression quantitative trait loci datasets and prioritization of genes by using protein interaction networks and tissue-specific expression. Combining this information into a quantitative score, we found that evidence converged on likely causal genes, including the above four genes, and those at previously discovered AD loci, including BIN1, APH1B, PTK2B, PILRA and CASS4.
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http://dx.doi.org/10.1038/s41588-020-00776-wDOI Listing
February 2021

INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing.

Sci Adv 2021 02 12;7(7). Epub 2021 Feb 12.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

We present INSIGHT [isothermal NASBA (nucleic acid sequence-based amplification) sequencing-based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing-based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.
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http://dx.doi.org/10.1126/sciadv.abe5054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880595PMC
February 2021

The professional development and career journey into musculoskeletal first contact physiotherapy: a telephone interview study.

Physiother Theory Pract 2021 Jan 11:1-16. Epub 2021 Jan 11.

School of Sport, University of Essex, Rehabilitation and Exercise Sciences (SRES) , Colchester, UK.

Musculoskeletal (MSK) first contact physiotherapy (FCP) is being rolled out in the National Health Service, but limited research exists on career pathways into MSK FCP, or on pre-and-post-registration educational preparation for the knowledge and skills that are required for musculoskeletal first contact physiotherapy.  From the perspectives of existing MSK FCPs, the study sought to understand the pre-and-post-registration professional developmental journey into musculoskeletal first contact physiotherapy.  Semi-structured interviews over the telephone were conducted with a self-selected and snowball sample of 15 MSK FCPs from across Britain. Framework analysis was used to analyze the interview transcripts.  Four overarching themes were identified: (1) Decision to choose a career path as a MSK FCP; (2) Relevancy of pre-registration physiotherapy (PT) education for MSK FCP; (3) Relevancy of post-registration continuing professional development for MSK FCP, and; (4) Improving pre-registration PT education for the foundational knowledge and skills required to work in musculoskeletal first contact physiotherapy. Each overarching theme generated several subthemes.  The research contributes to understanding the career pathway into the MSK FCP role and showed what relevant knowledge and skills were acquired for this role at pre-and-post registration levels. Findings will inform guidance for pre-registration PT curriculum development.
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http://dx.doi.org/10.1080/09593985.2021.1872127DOI Listing
January 2021

The Ncoa7 locus regulates V-ATPase formation and function, neurodevelopment and behaviour.

Cell Mol Life Sci 2020 Dec 19. Epub 2020 Dec 19.

MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK.

Members of the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) protein family are associated with multiple neurodevelopmental disorders, although their exact roles in disease remain unclear. For example, nuclear receptor coactivator 7 (NCOA7) has been associated with autism, although almost nothing is known regarding the mode-of-action of this TLDc protein in the nervous system. Here we investigated the molecular function of NCOA7 in neurons and generated a novel mouse model to determine the consequences of deleting this locus in vivo. We show that NCOA7 interacts with the cytoplasmic domain of the vacuolar (V)-ATPase in the brain and demonstrate that this protein is required for normal assembly and activity of this critical proton pump. Neurons lacking Ncoa7 exhibit altered development alongside defective lysosomal formation and function; accordingly, Ncoa7 deletion animals exhibited abnormal neuronal patterning defects and a reduced expression of lysosomal markers. Furthermore, behavioural assessment revealed anxiety and social defects in mice lacking Ncoa7. In summary, we demonstrate that NCOA7 is an important V-ATPase regulatory protein in the brain, modulating lysosomal function, neuronal connectivity and behaviour; thus our study reveals a molecular mechanism controlling endolysosomal homeostasis that is essential for neurodevelopment.
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http://dx.doi.org/10.1007/s00018-020-03721-6DOI Listing
December 2020

Screening for functional transcriptional and splicing regulatory variants with GenIE.

Nucleic Acids Res 2020 12;48(22):e131

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK.

Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.
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http://dx.doi.org/10.1093/nar/gkaa960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736817PMC
December 2020

Single-Cell Transcriptomics of Parkinson's Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses.

Cell Rep 2020 10;33(2):108263

UK Dementia Research Institute, Department of Clinical Neurosciences, Cambridge Biomedical Campus, University of Cambridge, Cambridge, CB2 0AH, UK. Electronic address:

The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest single-cell transcriptomic study of human iPSC-derived dopaminergic neurons to elucidate gene expression dynamics in response to cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles and differential sensitivity to stress, highlighting cellular heterogeneity in dopamine in vitro models. We validate this disease model by showing robust expression of PD GWAS genes and overlap with postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, we find perturbations in glycolysis, cholesterol metabolism, synaptic signaling, and ubiquitin-proteasomal degradation. Overall, our study reveals cell type-specific perturbations in human dopamine neurons, which will further our understanding of PD and have implications for cell replacement therapies.
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http://dx.doi.org/10.1016/j.celrep.2020.108263DOI Listing
October 2020

Challenges and Learning Opportunities of Pre-Registration Physiotherapy Placements in First Contact Settings: The Perspectives of Musculoskeletal First Contact Physiotherapists.

Musculoskeletal Care 2020 06 27;18(2):140-149. Epub 2020 Jan 27.

University of Essex, School of Sport, Rehabilitation and Exercise Sciences (SRES), UK.

Objectives: As musculoskeletal first contact physiotherapy is rolled out into primary healthcare in Britain, this could offer up new practice-based educational opportunities for pre-registration physiotherapy students. Thus, the present study sought to explore the perceived challenges and learning opportunities of pre-registration physiotherapy placements in musculoskeletal first contact physiotherapy settings from first contact physiotherapists' perspectives.

Methods: Using a qualitative strategy, 15 musculoskeletal first contact physiotherapists from different geographical locations in Britain, participated in telephone mediated semi-structured interviews. Participants were self-selected through a Chartered Society of Physiotherapy fortnightly bulletin and online forum for first contact physiotherapists, or recruited via snowball sampling. Interview transcripts were analysed according to framework analysis - and the findings were member-checked by proxy.

Results: Three core themes emerged: operational challenges, challenges for pre-registration physiotherapy students and learning opportunities for pre-registration physiotherapy students. Operational challenges included: ensuring sufficient support from first contact physiotherapy practice educators; financial cost implications of placements, and; lack of capacity within the existing first contact physiotherapy workforce to provide placements. Challenges for physiotherapy students involved: time pressures and stressors of a first contact physiotherapy placement; identifying red flags, and; complexity of patient presentations. Identified learning opportunities for physiotherapy students were: experience of a specialised physiotherapy role in a primary healthcare setting; bringing awareness of first contact physiotherapy as a potential career pathway, and; experience multidisciplinary team working in primary care.

Conclusions: By seeking the perspectives of first contact physiotherapists, this study provides the first step for the development of placements in an emerging practice area.
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http://dx.doi.org/10.1002/msc.1446DOI Listing
June 2020

The MITF-SOX10 regulated long non-coding RNA DIRC3 is a melanoma tumour suppressor.

PLoS Genet 2019 12 27;15(12):e1008501. Epub 2019 Dec 27.

Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom.

The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis. Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated. Here we identify 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions. Our work suggests that one of these, Disrupted In Renal Carcinoma 3 (DIRC3), may be a clinically important MITF-SOX10 regulated tumour suppressor. DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival. DIRC3 is a nuclear lncRNA that activates expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus. In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in cancer associated processes and is needed for DIRC3 control of anchorage-independent growth. Our work indicates that lncRNA components of MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets that can act not only as downstream mediators of MITF-SOX10 function but as feedback regulators of MITF-SOX10 activity.
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http://dx.doi.org/10.1371/journal.pgen.1008501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934268PMC
December 2019

A causal role for TRESK loss of function in migraine mechanisms.

Brain 2019 12;142(12):3852-3867

Translational Molecular Neuroscience Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.
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http://dx.doi.org/10.1093/brain/awz342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906598PMC
December 2019

Author Correction: Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Nat Commun 2019 Jun 4;10(1):2530. Epub 2019 Jun 4.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-10536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547739PMC
June 2019

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Nat Commun 2019 04 18;10(1):1817. Epub 2019 Apr 18.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.
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http://dx.doi.org/10.1038/s41467-019-09494-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472390PMC
April 2019

Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes.

Methods Mol Biol 2019 ;1961:153-183

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.

Genome editing using the CRISPR/Cas9 system has rapidly established itself as an essential tool in the genetic manipulation of many organisms, including human cell lines. Its application to human induced pluripotent stem cells (hiPSCs) allows for the generation of isogenic cell pairs that differ in a single genetic lesion, and therefore the identification and characterization of causal genetic variants. We describe a simple, effective approach to perform delicate manipulations of the genome of hiPSCs through delivery of Cas9 RNPs along with ssDNA oligonucleotide repair templates that can generate mutations in up to 98% of single cell clones and introduce single nucleotide changes at an efficiency of up to 40%. We describe our use of a T7 endonuclease assay to identify active guide RNAs, and a high-throughput sequencing genotyping strategy that allows the identification of correctly edited clones. We also present our experiences of generating single nucleotide changes at 15 sites, which show considerable variability between both guides and target sites in the efficiency at which such changes can be introduced.
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http://dx.doi.org/10.1007/978-1-4939-9170-9_11DOI Listing
July 2019

Rheumatoid Arthritis Portrayal by UK National Newspapers 2011-2016: A Service User - Led Thematic Analysis of Language Used.

BMC Rheumatol 2018 20;2. Epub 2018 Feb 20.

King's College London, Weston Education Centre, 10 Cutcombe Road, London, SE5 9RJ UK.

Background: An important source of knowledge, beliefs, and attitudes about illness is the mass media. Research has established the often negative and emotive language utilised by journalists to report on physical and mental long-term illnesses. The limited amount of research on rheumatological conditions has largely focused on the extent of, and/or accuracy of media coverage. This is the first published study to examine systematically the language used by the United Kingdom (UK) popular press to specifically describe rheumatoid arthritis (RA).

Methods: A patient and public involvement (PPI) approach, involving academics and service users with RA, was used to conduct the research. LexisNexis online repository of print media was searched for articles within a defined five year time frame, which included RA in the headline and/or lead paragraph of 15 UK national non-specialist newspapers. Resultant articles were uploaded to NVivo, and a realist perspective aided a thematic analysis of the data set.

Results: A search of LexisNexis produced 413 newspaper articles, of which 147 met the inclusion criteria. Three themes emerged: (1) language used to describe RA; (2) language used to refer to those who live with RA and; (3) language used to report on potential new treatments for RA. Negative and emotive terms such as 'attack', 'painful', 'crippling', and 'agony' were the most frequently used to describe the experience of RA. People diagnosed with RA were often portrayed as 'sufferers' or 'victims', though neutral language was also deployed. 'Hope' and 'breakthrough' were the most reported terms for potential new treatments for RA. Across the three themes, tabloid and middle market newspaper articles applied more sensationalised language with attention grabbing headlines and news stories. By contrast, such emotive terminology was less apparent in broadsheets.

Conclusions: The media is a source of information about RA for the general population, but the quality of newspaper journalism about the condition requires improvement. The findings may act as a stimulus for a national public awareness initiative and/or social marketing campaign. How the language currently constructed to describe RA in the press is received by people with RA would be an important area for future research.
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http://dx.doi.org/10.1186/s41927-018-0013-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390613PMC
February 2018

Generation of gene-corrected human induced pluripotent stem cell lines derived from retinitis pigmentosa patient with Ser331Cysfs*5 mutation in MERTK.

Stem Cell Res 2019 01 16;34:101341. Epub 2018 Nov 16.

Stem Cells Therapies in Neurodegenerative Diseases Lab, Centro de Investigacion Principe Felipe (CIPF), Valencia, Spain; National Stem Cell Bank-Valencia Node, Proteomics, Genotyping and Cell Line Platform, PRB3, ISCIII, Research Centre Principe Felipe, c/Eduardo Primo Yúfera 3, 46012, Valencia, Spain. Electronic address:

The human induced pluripotent stem cell (hiPSC) line RP1-FiPS4F1 generated from the patient with autosomal recessive retinitis pigmentosa (arRP) caused by homozygous Ser331Cysfs*5 mutation in Mer tyrosine kinase receptor (MERTK) was genetically corrected using CRISPR/Cas9 system. Two isogenic hiPSCs lines, with heterozygous and homozygous correction of c.992_993delCA mutation in the MERTK gene were generated. These cell lines demonstrate normal karyotype, maintain a pluripotent state, and can differentiate toward three germ layers in vitro. These genetically corrected hiPSCs represent accurate controls to study the contribution of the specific genetic change to the disease, and potentially therapeutic material for cell-replacement therapy.
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http://dx.doi.org/10.1016/j.scr.2018.11.003DOI Listing
January 2019

GPR35 promotes glycolysis, proliferation, and oncogenic signaling by engaging with the sodium potassium pump.

Sci Signal 2019 01 1;12(562). Epub 2019 Jan 1.

Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.
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http://dx.doi.org/10.1126/scisignal.aau9048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364804PMC
January 2019

Predicting the mutations generated by repair of Cas9-induced double-strand breaks.

Nat Biotechnol 2018 Nov 27. Epub 2018 Nov 27.

Wellcome Sanger Institute, Hinxton, UK.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >10 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.
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http://dx.doi.org/10.1038/nbt.4317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949135PMC
November 2018

Quantifying the contribution of recessive coding variation to developmental disorders.

Science 2018 12 8;362(6419):1161-1164. Epub 2018 Nov 8.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK.

We estimated the genome-wide contribution of recessive coding variation in 6040 families from the Deciphering Developmental Disorders study. The proportion of cases attributable to recessive coding variants was 3.6% in patients of European ancestry, compared with 50% explained by de novo coding mutations. It was higher (31%) in patients with Pakistani ancestry, owing to elevated autozygosity. Half of this recessive burden is attributable to known genes. We identified two genes not previously associated with recessive developmental disorders, and , and functionally validated them with mouse and cellular models. Our results suggest that recessive coding variants account for a small fraction of currently undiagnosed nonconsanguineous individuals, and that the role of noncoding variants, incomplete penetrance, and polygenic mechanisms need further exploration.
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http://dx.doi.org/10.1126/science.aar6731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726470PMC
December 2018

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons.

Hum Mol Genet 2019 02;28(4):584-597

Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford, UK.

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.
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http://dx.doi.org/10.1093/hmg/ddy370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360273PMC
February 2019

Transitional journeys into, and through medical education for First-in-Family (FiF) students: a qualitative interview study.

BMC Med Educ 2018 May 9;18(1):102. Epub 2018 May 9.

Kings College, London, Weston Education Centre, Third Floor-Room 3.52, 10 Cutcombe Road, London, SE5 9RJ, UK.

Background: There has been much interest in the transitions along the medical education continuum. However, little is known about how students from non-traditional backgrounds experience both the move to, and through Medical School, and their ambitions post-graduation. This research sought to understand the transitional journey into, and through undergraduate medical education, and future career aspirations for first-in-family (FiF) medical students.

Methods: Based on a interpretivist epistemological perspective, 20 FiF students from one English Medical School participated in semi-structured interviews. Participants were identified according to purposive inclusion criteria and were contacted by email via the student association at the Medical School and academic year leaders. The team approach to the thematic analysis enhanced the findings credibility. This research was part of an international collaboration.

Results: In the first transition, 'The Road to Medical School', a passion for science with an interest in people was a motivator to study medicine. Participants' parents' shared the elation of acceptance into Medical School, however, the support from school/college teachers was a mixed experience. In 'The Medical School Journey' transition, knowledge about the medical curriculum was variable. 'Fitting' in at Medical School was a problem for some, but studying for an elite degree elevated social status for many study participants. A source of support derived from senior medical student peers, but a medical degree could sacrifice students' own health. In the final transition, 'Future Plans', a medical career was perceived to have intrinsic value. Clarity about future aspirations was related to clinical experience. For some, career trajectories were related to a work-life balance and future NHS working conditions for Junior Doctors.

Conclusions: The transitions highlighted in this article have important implications for those educators interested in a life cycle approach to widening participation in medical education. Future research should explore the post-graduation transitions for doctors from first-in-family University backgrounds.
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http://dx.doi.org/10.1186/s12909-018-1217-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944111PMC
May 2018

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

Nat Commun 2017 12 13;8(1):2109. Epub 2017 Dec 13.

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

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.
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http://dx.doi.org/10.1038/s41467-017-00686-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727397PMC
December 2017

Alpha-synuclein induces the unfolded protein response in Parkinson's disease SNCA triplication iPSC-derived neurons.

Hum Mol Genet 2017 11;26(22):4441-4450

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.

The recent generation of induced pluripotent stem cells (iPSCs) from a patient with Parkinson's disease (PD) resulting from triplication of the α-synuclein (SNCA) gene locus allows unprecedented opportunities to explore its contribution to the molecular pathogenesis of PD. We used the double-nicking CRISPR/Cas9 system to conduct site-specific mutagenesis of SNCA in these cells, generating an isogenic iPSC line with normalized SNCA gene dosage. Comparative gene expression analysis of neuronal derivatives from these iPSCs revealed an ER stress phenotype, marked by induction of the IRE1α/XBP1 axis of the unfolded protein response (UPR) and culminating in terminal UPR activation. Neuropathological analysis of post-mortem brain tissue demonstrated that pIRE1α is expressed in PD brains within neurons containing elevated levels of α-synuclein or Lewy bodies. Having used this pair of isogenic iPSCs to define this phenotype, these cells can be further applied in UPR-targeted drug discovery towards the development of disease-modifying therapeutics.
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http://dx.doi.org/10.1093/hmg/ddx331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886237PMC
November 2017

PLCζ is the physiological trigger of the Ca oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Development 2017 08 10;144(16):2914-2924. Epub 2017 Jul 10.

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca oscillations in eggs and debilitating mutations in the gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca oscillations in eggs. Here, we show that sperm derived from male mice fail to trigger Ca oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.
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http://dx.doi.org/10.1242/dev.150227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592814PMC
August 2017

Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

Mol Biol Evol 2017 09;34(9):2285-2306

Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom.

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations. We find that reducing the amount of epigenetic variation available to populations can reduce adaptation in environments where it otherwise happens. From genomic and epigenomic sequences from a subset of the populations, we see changes in methylation patterns between the evolved populations over-represented in some functional categories of genes, which is consistent with some of these differences being adaptive. Based on whole genome sequencing of evolved clones, the majority of DNA methylation changes do not appear to be linked to cis-acting genetic mutations. Our results show that transgenerational epigenetic effects play a role in adaptive evolution, and suggest that the relationship between changes in methylation patterns and differences in evolutionary outcomes, at least for quantitative traits such as cell division rates, is complex.
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http://dx.doi.org/10.1093/molbev/msx166DOI Listing
September 2017

Editing the genome of hiPSC with CRISPR/Cas9: disease models.

Authors:
Andrew R Bassett

Mamm Genome 2017 Aug 16;28(7-8):348-364. Epub 2017 Mar 16.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

The advent of human-induced pluripotent stem cell (hiPSC) technology has provided a unique opportunity to establish cellular models of disease from individual patients, and to study the effects of the underlying genetic aberrations upon multiple different cell types, many of which would not normally be accessible. Combining this with recent advances in genome editing techniques such as the clustered regularly interspaced short palindromic repeat (CRISPR) system has provided an ability to repair putative causative alleles in patient lines, or introduce disease alleles into a healthy "WT" cell line. This has enabled analysis of isogenic cell pairs that differ in a single genetic change, which allows a thorough assessment of the molecular and cellular phenotypes that result from this abnormality. Importantly, this establishes the true causative lesion, which is often impossible to ascertain from human genetic studies alone. These isogenic cell lines can be used not only to understand the cellular consequences of disease mutations, but also to perform high throughput genetic and pharmacological screens to both understand the underlying pathological mechanisms and to develop novel therapeutic agents to prevent or treat such diseases. In the future, optimising and developing such genetic manipulation technologies may facilitate the provision of cellular or molecular gene therapies, to intervene and ultimately cure many debilitating genetic disorders.
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http://dx.doi.org/10.1007/s00335-017-9684-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569153PMC
August 2017

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2.

Nat Commun 2016 11 28;7:13661. Epub 2016 Nov 28.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.
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http://dx.doi.org/10.1038/ncomms13661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133711PMC
November 2016

Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons.

Sci Rep 2016 Jun 24;6:28420. Epub 2016 Jun 24.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3QX, UK.

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases. The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases. Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons. This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis.
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http://dx.doi.org/10.1038/srep28420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920027PMC
June 2016

Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs.

Genome Res 2016 Apr 11;26(4):519-29. Epub 2016 Mar 11.

Department of Plant Sciences, University of Cambridge CB2 3EA, Cambridge CB2 3EA, United Kingdom.

We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species,Chlamydomonas reinhardtii Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.
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http://dx.doi.org/10.1101/gr.199703.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817775PMC
April 2016