Enhancer evolution across 20 mammalian species.

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.

Pervasive lesion segregation shapes cancer genome evolution.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Mutational landscape of a chemically-induced mouse model of liver cancer.

BACKGROUND & AIMS: Carcinogen-induced mouse models of liver cancer are used extensively to study the pathogenesis of the disease and are critical for validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Herein, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC). METHODS: We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). Mutational signatures were compared between liver tumours from DEN-treated and untreated mice, and human HCCs. RESULTS: DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/ß-catenin signalling in cancer progression. CONCLUSIONS: Our study provides detailed insight into the mutational landscape of tumours arising in a commonly used carcinogen model of HCC, facilitating the future use of this model to better understand the human disease. LAY SUMMARY: Mouse models are widely used to study the biology of cancer and to test potential therapies. Herein, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer.

Genetically distinct subsets within ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis. METHODS: A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria. RESULTS: We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8)). CONCLUSIONS: This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

Genome-wide methylation analyses of primary human leukocyte subsets identifies functionally important cell-type-specific hypomethylated regions.

DNA methylation is an important mechanism by which gene transcription and hence cellular function are regulated. Here, we provide detailed functional genome-wide methylome maps of 5 primary peripheral blood leukocyte subsets including T cells, B cells, monocytes/macrophages, and neutrophils obtained from healthy individuals. A comparison of these methylomes revealed highly specific cell-lineage and cell-subset methylation profiles. DNA hypomethylation is known to be permissive for gene expression and we identified cell-subset-specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels suggesting these HMRs may regulate corresponding cell functions. Single-nucleotide polymorphisms associated with immune-mediated disease in genome-wide association studies preferentially localized to these cell-specific regulatory HMRs, offering insight into pathogenesis by highlighting cell subsets in which specific epigenetic changes may drive disease. Our data provide a valuable reference tool for researchers aiming to investigate the role of DNA methylation in regulating primary leukocyte function in health and immune-mediated disease.

MiR-210 is induced by Oct-2, regulates B cells, and inhibits autoantibody production.

MicroRNAs (MiRs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we show that MiR-210 is induced by Oct-2, a key transcriptional mediator of B cell activation. Germline deletion of MiR-210 results in the development of autoantibodies from 5 mo of age. Overexpression of MiR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice overexpressing MiR-210 exhibited impaired class-switched Ab responses, a finding confirmed in wild-type B cells transfected with a MiR-210 mimic. In vitro studies demonstrated defects in cellular proliferation and cell cycle entry, which were consistent with the transcriptomic analysis demonstrating downregulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of MiR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.

Sleep disorder risk, perceived control over sleep, and mental health symptoms in paramedicine students.

Paramedics commonly experience both poor sleep and mental health symptoms. Clarifying whether sleep or mental health symptoms are a challenge prior to commencement of employment is important, as early prevention and intervention initiatives during training could support these workers. Paramedicine students (n=53) were included, with sleep disorder screening (obstructive sleep apnea, insomnia and restless legs syndrome), and mental health outcomes (depressive symptoms: Patient Health Questionnaire-9, and anxiety symptoms: General Anxiety Disorder-7). Data were analysed using robust regression models, adjusted for age, sex, and shift work status. Meeting criteria for a sleep disorder (n=21) was associated with higher scores for anxiety (8.2 [95% CI: 5.9-10.5] v 4.6, [3.4-5.8]) and depressive symptoms (11.1 [8.6-13.6] v 4.4 [3.1-5.7)] compared to those who did not meet the criteria for a sleep disorder (n=32). Depressive symptoms were lower in those with perceived control over sleep (5.2 [3.2-7.2] v 9.8 [7.7-11.8]). There was no interaction between sleep disorder risk and perceived control over sleep on mental health symptoms. Investigation and management of factors contributing to low perceived control over sleep, together with early screening and management of sleep disorders, are likely to be important priorities to support paramedic student wellbeing prior to commencing shift work.

A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease more prevalent in people of African and Asian origin than Caucasian origin. FcgammaRIIb is an inhibitory Fc receptor with a critical role in immune regulation. Mouse data suggest that FcgammaRIIb deficiency increases susceptibility to autoimmune disease but protects against infection. We show that a SNP in human FCGR2B that abrogates receptor function is strongly associated with susceptibility to SLE in both Caucasians and Southeast Asians. The minor allele of this SNP is more common in Southeast Asians and Africans, populations from areas where malaria is endemic, than in Caucasians. We show that homozygosity for the minor allele is associated with substantial protection against severe malaria in an East African population (odds ratio = 0.56; P = 7.1 x 10(-5)). This protective effect against malaria may contribute to the higher frequency of this SNP and hence, SLE in Africans and Southeast Asians.

What and how can we learn from complex global problems for antimicrobial resistance policy? A comparative study combining historical and foresight approaches.

OBJECTIVES: To (i) develop a methodology for using historical and comparative perspectives to inform policy and (ii) provide evidence for antimicrobial-resistance (AMR) policymaking by drawing on lessons from climate change and tobacco control. METHODS: Using a qualitative design, we systematically examined two other complex, large-scale policy issues-climate change and tobacco control-to identify what relevance to AMR can be learned from how these issues have evolved over time. During 2018-2020, we employed a five-stage approach to conducting an exploratory study involving a review of secondary historical analysis, identification of drivers of change, prioritisation of the identified drivers, scenario generation and elicitation of possible policy responses. We sought to disrupt more 'traditional' policy and research spaces to create an alternative where, stimulated by historical analysis, academics (including historians) and policymakers could come together to challenge norms and practices and think creatively about AMR policy design. RESULTS: An iterative process of analysis and engagement resulted in lessons for AMR policy concerning persistent evidence gaps and uncertainty, the need for cross-sector involvement and a collective effort through global governance, the demand for new interventions through more investment in research and innovation, and recognising the dynamic relationship between social change and policy to change people's attitudes and behaviours are crucial towards tackling AMR. CONCLUSION: We draw on new methodological lessons around the pragmatism of future- and policy-oriented approaches incorporating robust historical and comparative analysis. The study demonstrates proof of concept and offers a reproducible method to advance further methodology, including transferrable policies that could tackle health problems, such as AMR.

Copy number, linkage disequilibrium and disease association in the FCGR locus.

The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08-2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25-2.18), P = 4 x 10(-4)]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.

ArrayExpress update--from an archive of functional genomics experiments to the atlas of gene expression.

ArrayExpress http://www.ebi.ac.uk/arrayexpress consists of three components: the ArrayExpress Repository--a public archive of functional genomics experiments and supporting data, the ArrayExpress Warehouse--a database of gene expression profiles and other bio-measurements and the ArrayExpress Atlas--a new summary database and meta-analytical tool of ranked gene expression across multiple experiments and different biological conditions. The Repository contains data from over 6000 experiments comprising approximately 200,000 assays, and the database doubles in size every 15 months. The majority of the data are array based, but other data types are included, most recently-ultra high-throughput sequencing transcriptomics and epigenetic data. The Warehouse and Atlas allow users to query for differentially expressed genes by gene names and properties, experimental conditions and sample properties, or a combination of both. In this update, we describe the ArrayExpress developments over the last two years.

Importing ArrayExpress datasets into R/Bioconductor.

SUMMARY: ArrayExpress is one of the largest public repositories of microarray datasets. R/Bioconductor provides a comprehensive suite of microarray analysis and integrative bioinformatics software. However, easy ways for importing datasets from ArrayExpress into R/Bioconductor have been lacking. Here, we present such a tool that is suitable for both interactive and automated use. AVAILABILITY: The ArrayExpress package is available from the Bioconductor project at http://www.bioconductor.org. A users guide and examples are provided with the package.

MAGETabulator, a suite of tools to support the microarray data format MAGE-TAB.

SUMMARY: The MAGE-TAB format for microarray data representation and exchange has been proposed by the microarray community to replace the more complex MAGE-ML format. We present a suite of tools to support MAGE-TAB generation and validation, conversion between existing formats for data exchange, visualization of the experiment designs encoded by MAGE-TAB documents and the mining of such documents for semantic content.

Novel expression signatures identified by transcriptional analysis of separated leucocyte subsets in systemic lupus erythematosus and vasculitis.

OBJECTIVE: To optimise a strategy for identifying gene expression signatures differentiating systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody-associated vasculitis that provide insight into disease pathogenesis and identify biomarkers. METHODS: 44 vasculitis patients, 13 SLE patients and 25 age and sex-matched controls were enrolled. CD4 and CD8 T cells, B cells, monocytes and neutrophils were isolated from each patient and, together with unseparated peripheral blood mononuclear cells (PBMC), were hybridised to spotted oligonucleotide microarrays. RESULTS: Using expression data obtained from purified cells a substantial number of differentially expressed genes were identified that were not detectable in the analysis of PBMC. Analysis of purified T cells identified a SLE-associated, CD4 T-cell signature consistent with type 1 interferon signalling driving the generation and survival of tissue homing T cells and thereby contributing to disease pathogenesis. Moreover, hierarchical clustering using expression data from purified monocytes provided significantly improved discrimination between the patient groups than that obtained using PBMC data, presumably because the differentially expressed genes reflect genuine differences in processes underlying disease pathogenesis. CONCLUSION: Analysis of leucocyte subsets enabled the identification of gene signatures of both pathogenic relevance and with better disease discrimination than those identified in PBMC. This approach thus provides substantial advantages in the search for diagnostic and prognostic biomarkers in autoimmune disease.

The first RSBI (ISA-TAB) workshop: "can a simple format work for complex studies?".

This article summarizes the motivation for, and the proceedings of, the first ISA-TAB workshop held December 6-8, 2007, at the EBI, Cambridge, UK. This exploratory workshop, organized by members of the Microarray Gene Expression Data (MGED) Society's Reporting Structure for Biological Investigations (RSBI) working group, brought together a group of developers of a range of collaborative systems to discuss the use of a common format to address the pressing need of reporting and communicating data and metadata from biological, biomedical, and environmental studies employing combinations of genomics, transcriptomics, proteomics, and metabolomics technologies along with more conventional methodologies. The expertise of the participants comprised database development, data management, and hands-on experience in the development of data communication standards. The workshop's outcomes are set to help formalize the proposed Investigation, Study, Assay (ISA)-TAB tab-delimited format for representing and communicating experimental metadata. This article is part of the special issue of OMICS on the activities of the Genomics Standards Consortium (GSC).

Paramedic Application of a Triage Sieve: A Paper-Based Exercise.

Introduction Triage is the systematic prioritization of casualties when there is an imbalance between the needs of these casualties and resource availability. The triage sieve is a recognized process for prioritizing casualties for treatment during mass-casualty incidents (MCIs). While the application of a triage sieve generally is well-accepted, the measurement of its accuracy has been somewhat limited. Obtaining reliable measures for triage sieve accuracy rates is viewed as a necessity for future development in this area. OBJECTIVE: The goal of this study was to investigate how theoretical knowledge acquisition and the practical application of an aide-memoir impacted triage sieve accuracy rates. METHOD: Two hundred and ninety-two paramedics were allocated randomly to one of four separate sub-groups, a non-intervention control group, and three intervention groups, which involved them receiving either an educational review session and/or an aide-memoir. Participants were asked to triage sieve 20 casualties using a previously trialed questionnaire. RESULTS: The study showed the non-intervention control group had a correct accuracy rate of 47%, a similar proportion of casualties found to be under-triaged (37%), but a significantly lower number of casualties were over-triaged (16%). The provision of either an educational review or aide-memoir significantly increased the correct triage sieve accuracy rate to 77% and 90%, respectively. Participants who received both the educational review and aide-memoir had an overall accuracy rate of 89%. Over-triaged rates were found not to differ significantly across any of the study groups. CONCLUSION: This study supports the use of an aide-memoir for maximizing MCI triage accuracy rates. A "just-in-time" educational refresher provided comparable benefits, however its practical application to the MCI setting has significant operational limitations. In addition, this study provides some guidance on triage sieve accuracy rate measures that can be applied to define acceptable performance of a triage sieve during a MCI. Cuttance G , Dansie K , Rayner T . Paramedic application of a triage sieve: a paper-based exercise. Prehosp Disaster Med. 2017;32(1):3-13.

Interplay of cis and trans mechanisms driving transcription factor binding and gene expression evolution.

Noncoding regulatory variants play a central role in the genetics of human diseases and in evolution. Here we measure allele-specific transcription factor binding occupancy of three liver-specific transcription factors between crosses of two inbred mouse strains to elucidate the regulatory mechanisms underlying transcription factor binding variations in mammals. Our results highlight the pre-eminence of cis-acting variants on transcription factor occupancy divergence. Transcription factor binding differences linked to cis-acting variants generally exhibit additive inheritance, while those linked to trans-acting variants are most often dominantly inherited. Cis-acting variants lead to local coordination of transcription factor occupancies that decay with distance; distal coordination is also observed and may be modulated by long-range chromatin contacts. Our results reveal the regulatory mechanisms that interplay to drive transcription factor occupancy, chromatin state, and gene expression in complex mammalian cell states.

A simple spreadsheet-based, MIAME-supportive format for microarray data: MAGE-TAB.

BACKGROUND: Sharing of microarray data within the research community has been greatly facilitated by the development of the disclosure and communication standards MIAME and MAGE-ML by the MGED Society. However, the complexity of the MAGE-ML format has made its use impractical for laboratories lacking dedicated bioinformatics support. RESULTS: We propose a simple tab-delimited, spreadsheet-based format, MAGE-TAB, which will become a part of the MAGE microarray data standard and can be used for annotating and communicating microarray data in a MIAME compliant fashion. CONCLUSION: MAGE-TAB will enable laboratories without bioinformatics experience or support to manage, exchange and submit well-annotated microarray data in a standard format using a spreadsheet. The MAGE-TAB format is self-contained, and does not require an understanding of MAGE-ML or XML.

AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation.

ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days.