Sanger Institute - Publications 2017

Number of papers published in 2017: 401

  • Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome.

    Abdi A, Yu L, Goulding D, Rono MK, Bejon P, Choudhary J and Rayner J

    Pwani University Bioscience Research Centre, Pwani University, Kilifi, Kenya.

    Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence.

    Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925).

    Results: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs.

    Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

    Wellcome open research 2017;2;50

  • Rapid identification of genes controlling virulence and immunity in malaria parasites.

    Abkallo HM, Martinelli A, Inoue M, Ramaprasad A, Xangsayarath P, Gitaka J, Tang J, Yahata K, Zoungrana A, Mitaka H, Acharjee A, Datta PP, Hunt P, Carter R, Kaneko O, Mustonen V, Illingworth CJR, Pain A and Culleton R

    Malaria Unit, Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

    Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

    PLoS pathogens 2017;13;7;e1006447

  • Phylogenetic characterisation of circulating, clinical influenza isolates from Bali, Indonesia: preliminary report from the BaliMEI project.

    Adisasmito W, Budayanti SN, Aisyah DN, Gallo Cassarino T, Rudge JW, Watson SJ, Kozlakidis Z, Smith GJD and Coker R

    Universitas Indonesia, Depok, Indonesia.

    Background: Human influenza represents a major public health concern, especially in south-east Asia where the risk of emergence and spread of novel influenza viruses is particularly high. The BaliMEI study aims to conduct a five year active surveillance and characterisation of influenza viruses in Bali using an extensive network of participating healthcare facilities.

    Methods: Samples were collected during routine diagnostic treatment in healthcare facilities. In addition to standard clinical and molecular methods for influenza typing, next generation sequencing and subsequent de novo genome assembly were performed to investigate the phylogeny of the collected patient samples.

    Results: The samples collected are characteristic of the seasonally circulating influenza viruses with indications of phylogenetic links to other samples characterised in neighbouring countries during the same time period.

    Conclusions: There were some strong phylogenetic links with sequences from samples collected in geographically proximal regions, with some of the samples from the same time-period resulting to small clusters at the tree-end points. However this work, which is the first of its kind completely performed within Indonesia, supports the view that the circulating seasonal influenza in Bali reflects the strains circulating in geographically neighbouring areas as would be expected to occur within a busy regional transit centre.

    BMC infectious diseases 2017;17;1;583

  • Enhanced Nasopharyngeal Infection and Shedding Associated with an Epidemic Lineage of emm3 group A Streptococcus.

    Afshar B, Turner CE, Lamagni T, Smith K, Al-Shahib A, Underwood A, Holden MTG, Efstratiou A and Sriskandan S

    a Department of Medicine , Imperial College London , London , United Kingdom.

    Background A group A Streptococcus (GAS) lineage of genotype emm3, sequence type 15 (ST15) was associated with a six month upsurge in invasive GAS disease in the UK. The epidemic lineage (Lineage C) had lost two typical emm3 prophages, Φ315.1 and Φ315.2 associated with the superantigen ssa, but gained a different prophage (ΦUK-M3.1) associated with a different superantigen, speC and a DNAse spd1. Methods and Results The presence of speC and spd1 in Lineage C ST15 strains enhanced both in vitro mitogenic and DNAse activities over non-Lineage C ST15 strains. Invasive disease models in Galleria mellonella and SPEC-sensitive transgenic mice, revealed no difference in overall invasiveness of Lineage C ST15 strains compared to non-Lineage C ST15 strains, consistent with clinical and epidemiological analysis. Lineage C strains did however markedly prolong murine nasal infection with enhanced nasal and airborne shedding compared to non-Lineage C strains. Deletion of speC or spd1 in two Lineage C strains identified a possible role for spd1 in airborne shedding from the murine nasopharynx. Conclusions Nasopharyngeal infection and shedding of Lineage C strains was enhanced compared to non-Lineage C strains and this was, in part, mediated by the gain of the DNase spd1 through prophage acquisition.

    Virulence 2017;0

  • The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

    Akay A, Di Domenico T, Suen KM, Nabih A, Parada GE, Larance M, Medhi R, Berkyurek AC, Zhang X, Wedeles CJ, Rudolph KLM, Engelhardt J, Hemberg M, Ma P, Lamond AI, Claycomb JM and Miska EA

    Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

    Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.

    Developmental cell 2017;42;3;241-255.e6

  • Genetic association analysis identifies variants associated with disease progression in primary sclerosing cholangitis.

    Alberts R, de Vries EMG, Goode EC, Jiang X, Sampaziotis F, Rombouts K, Böttcher K, Folseraas T, Weismüller TJ, Mason AL, Wang W, Alexander G, Alvaro D, Bergquist A, Björkström NK, Beuers U, Björnsson E, Boberg KM, Bowlus CL, Bragazzi MC, Carbone M, Chazouillères O, Cheung A, Dalekos G, Eaton J, Eksteen B, Ellinghaus D, Färkkilä M, Festen EAM, Floreani A, Franceschet I, Gotthardt DN, Hirschfield GM, Hoek BV, Holm K, Hohenester S, Hov JR, Imhann F, Invernizzi P, Juran BD, Lenzen H, Lieb W, Liu JZ, Marschall HU, Marzioni M, Melum E, Milkiewicz P, Müller T, Pares A, Rupp C, Rust C, Sandford RN, Schramm C, Schreiber S, Schrumpf E, Silverberg MS, Srivastava B, Sterneck M, Teufel A, Vallier L, Verheij J, Vila AV, Vries B, Zachou K, International PSC Study Group, The UK PSC Consortium, Chapman RW, Manns MP, Pinzani M, Rushbrook SM, Lazaridis KN, Franke A, Anderson CA, Karlsen TH, Ponsioen CY and Weersma RK

    Department of Gastroenterology and Hepatology, University of Groningen and University Medical Centre Groningen, Groningen, The Netherlands.

    Objective: Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications.

    Design: We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes.

    Results: We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10(-9)). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells.

    Conclusion: We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.

    Gut 2017

  • Genetic markers associated with dihydroartemisinin-piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype-phenotype association study.

    Amato R, Lim P, Miotto O, Amaratunga C, Dek D, Pearson RD, Almagro-Garcia J, Neal AT, Sreng S, Suon S, Drury E, Jyothi D, Stalker J, Kwiatkowski DP and Fairhurst RM

    Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK. Electronic address:

    Background: As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond.

    Methods: We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC50s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin-piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin-piperaquine treatment.

    Findings: Piperaquine IC50s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC50s: 20·0 nmol/L [IQR 13·7-29·0] vs 39·2 nmol/L [32·8-48·1] for Ratanakiri, 19·3 nmol/L [15·1-26·2] vs 66·2 nmol/L [49·9-83·0] for Preah Vihear, and 19·6 nmol/L [11·9-33·9] vs 81·1 nmol/L [61·3-113·1] for Pursat; all p≤10(-3); Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC50s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin-piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the plasmepsin 2 and plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC50s. After adjusting for covariates, both exo-E415G and plasmepsin 2-3 markers significantly associate (p=3·0 × 10(-8) and p=1·7 × 10(-7), respectively) with decreased treatment efficacy (survival rates 0·38 [95% CI 0·25-0·51] and 0·41 [0·28-0·53], respectively).

    Interpretation: The exo-E415G SNP and plasmepsin 2-3 amplification are markers of piperaquine resistance and dihydroartemisinin-piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2-3 amplification probably mediates piperaquine resistance.

    Funding: Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.

    Funded by: Medical Research Council: MR/M006212/1; Wellcome Trust

    The Lancet. Infectious diseases 2017;17;2;164-173

  • Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration.

    Ambrosi TH, Scialdone A, Graja A, Gohlke S, Jank AM, Bocian C, Woelk L, Fan H, Logan DW, Schürmann A, Saraiva LR and Schulz TJ

    German Institute of Human Nutrition Potsdam-Rehbrücke, 14558 Nuthetal, Germany.

    Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

    Cell stem cell 2017

  • One-step generation of conditional and reversible gene knockouts.

    Andersson-Rolf A, Mustata RC, Merenda A, Kim J, Perera S, Grego T, Andrews K, Tremble K, Silva JC, Fink J, Skarnes WC and Koo BK

    Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.

    Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.

    Nature methods 2017;14;3;287-289

  • Identifying cell populations with scRNASeq.

    Andrews TS and Hemberg M

    Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK.

    Single-cell RNASeq (scRNASeq) has emerged as a powerful method for quantifying the transcriptome of individual cells. However, the data from scRNASeq experiments is often both noisy and high dimensional, making the computational analysis non-trivial. Here we provide an overview of different experimental protocols and the most popular methods for facilitating the computational analysis. We focus on approaches for identifying biologically important genes, projecting data into lower dimensions and clustering data into putative cell-populations. Finally we discuss approaches to validation and biological interpretation of the identified cell-types or cell-states.

    Molecular aspects of medicine 2017

  • DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning.

    Angermueller C, Lee HJ, Reik W and Stegle O

    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.

    Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells. We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variability.

    Genome biology 2017;18;1;67

  • Heterogeneity of the Epstein-Barr virus major internal repeat reveals evolutionary mechanisms of EBV and a functional defect in the prototype EBV strain B95-8.

    Ba Abdullah M, Palermo R, Palser A, Grayson NE, Kellam P, Correia S, Szymula A and White R

    Section of Virology, Imperial College Faculty of Medicine, St Mary's Hospital, London, UK.

    Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified both through co-evolution with its host, and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging, because of the large number and length of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat of EBV (IR1 or BamW repeats) from over 70 strains.Diversity of the latency protein EBNA-LP resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 ORF is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp), and one zone upstream of and two within BWRF1.IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as spontaneous mutation, with inter-strain recombination more common in tumour-derived viruses. This genetic exchange often incorporates regions of <1kb, and allelic gene conversion changes the frequency of small regions within the repeat, but not close to the flanks. These observations suggest that IR1 - and by extension EBV - diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four non-consensus variants within a single IR1 repeat unit, including a STOP codon in EBNA-LP. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 BAC.IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population, but only causes illness in a small minority. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity, to see if different strains have different disease impacts, have excluded regions of repeating sequence, as they are more technically challenging. Here we analyse the sequence of the largest repeat in EBV (IR1). We first characterised the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and suggest that tumour-associated viruses may be more likely to contain DNA mixed from two strains. Patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

    Journal of virology 2017

  • Whole genome sequencing of Shigella sonnei through PulseNet Latin America and Caribbean: advancing global surveillance of foodborne illnesses.

    Baker KS, Campos J, Pichel M, Della Gaspera A, Duarte-Martínez F, Campos-Chacón E, Bolaños-Acuña HM, Guzman-Verri C, Mather AE, Velasco SD, Zamudio Rojas ML, Forbester J, Connor TR, Keddy KH, Smith AM, Lopez de Delgado EA, Angiolillo G, Cuaical N, Fernandez J, Aguayo C, Aguilar MM, Valenzuela C, Morales Medrano AJ, Esteve AS, Gustafson NW, Diaz Guevara PL, Montaño LA, Perez E and Thomson NR

    University of Liverpool, Department of Functional and Comparative Genomics, Liverpool, United Kingdom, L69 7ZB; Wellcome Trust Sanger Institute, Pathogen Variation Programme, Hinxton, United Kingdom, CB10 1SA. Electronic address:

    Objective: Shigella sonnei is a globally-important diarrhoeal pathogen tracked through the surveillance network PulseNet Latin America and Caribbean (PNLA&C), which participates in PulseNet International. PNLA&C laboratories use common molecular techniques to track pathogens causing foodborne illness. We aimed to demonstrate the possibility and advantages of transitioning to whole genome sequencing (WGS) for surveillance within existing networks across a continent where S. sonnei is endemic.

    Methods: We applied WGS to representative archive isolates of S. sonnei (n=323) from laboratories in nine PNLA&C countries to generate a regional phylogenomic reference for S. sonnei and put this in the global context. We used this reference to contextualise 16 S. sonnei from three Argentinian outbreaks, using locally-generated sequence data. Assembled genome sequences were used to predict antimicrobial resistance (AMR) phenotypes and identify AMR determinants.

    Results: S. sonnei isolates clustered in five Latin American sublineages in the global phylogeny, with many (46%, 149 of 323) belonging to previously undescribed sublineages. Predicted multiple drug resistance was common (77%, 249 of 323) and clinically-relevant differences in AMR were found among sublineages. The regional overview showed that Argentinian outbreak isolates belonged to distinct sublineages and had different epidemiological origins.

    Conclusions: Latin America contains novel genetic diversity of S. sonnei that is relevant on a global scale and commonly exhibits multiple drug resistance. Retrospective passive surveillance with WGS has utility for informing treatment , identifying regionally-epidemic sublineages and providing a framework for interpretation of prospective, locally-sequenced outbreaks.

    Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 2017

  • Phylogeography of human Y-chromosome haplogroup Q3-L275 from an academic/citizen science collaboration.

    Balanovsky O, Gurianov V, Zaporozhchenko V, Balaganskaya O, Urasin V, Zhabagin M, Grugni V, Canada R, Al-Zahery N, Raveane A, Wen SQ, Yan S, Wang X, Zalloua P, Marafi A, Koshel S, Semino O, Tyler-Smith C and Balanovska E

    Vavilov Institute of General Genetics, Moscow, Russia.

    Background: The Y-chromosome haplogroup Q has three major branches: Q1, Q2, and Q3. Q1 is found in both Asia and the Americas where it accounts for about 90% of indigenous Native American Y-chromosomes; Q2 is found in North and Central Asia; but little is known about the third branch, Q3, also named Q1b-L275. Here, we combined the efforts of population geneticists and genetic genealogists to use the potential of full Y-chromosome sequencing for reconstructing haplogroup Q3 phylogeography and suggest possible linkages to events in population history.

    Results: We analyzed 47 fully sequenced Y-chromosomes and reconstructed the haplogroup Q3 phylogenetic tree in detail. Haplogroup Q3-L275, derived from the oldest known split within Eurasian/American haplogroup Q, most likely occurred in West or Central Asia in the Upper Paleolithic period. During the Mesolithic and Neolithic epochs, Q3 remained a minor component of the West Asian Y-chromosome pool and gave rise to five branches (Q3a to Q3e), which spread across West, Central and parts of South Asia. Around 3-4 millennia ago (Bronze Age), the Q3a branch underwent a rapid expansion, splitting into seven branches, some of which entered Europe. One of these branches, Q3a1, was acquired by a population ancestral to Ashkenazi Jews and grew within this population during the 1st millennium AD, reaching up to 5% in present day Ashkenazi.

    Conclusions: This study dataset was generated by a massive Y-chromosome genotyping effort in the genetic genealogy community, and phylogeographic patterns were revealed by a collaboration of population geneticists and genetic genealogists. This positive experience of collaboration between academic and citizen science provides a model for further joint projects. Merging data and skills of academic and citizen science promises to combine, respectively, quality and quantity, generalization and specialization, and achieve a well-balanced and careful interpretation of the paternal-side history of human populations.

    BMC evolutionary biology 2017;17;Suppl 1;18

  • Compound heterozygous variants in NBAS as a cause of atypical osteogenesis imperfecta.

    Balasubramanian M, Hurst J, Brown S, Bishop NJ, Arundel P, DeVile C, Pollitt RC, Crooks L, Longman D, Caceres JF, Shackley F, Connolly S, Payne JH, Offiah AC, Hughes D, DDD Study, Parker MJ, Hide W and Skerry TM

    Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundation Trust, UK; Highly Specialised Service for Severe, Complex and Atypical OI, UK. Electronic address:

    Background: Osteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit.

    Report: Whole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency.

    Discussion: Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from 'Classical' OI.

    Conclusions: Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients.

    Funded by: Medical Research Council: MC_PC_15018, MC_PC_U127584479

    Bone 2017;94;65-74

  • Chitayat syndrome: hyperphalangism, characteristic facies, hallux valgus and bronchomalacia results from a recurrent c.266A>G p.(Tyr89Cys) variant in the ERF gene.

    Balasubramanian M, Lord H, Levesque S, Guturu H, Thuriot F, Sillon G, Wenger AM, Sureka DL, Lester T, Johnson DS, Bowen J, Calhoun AR, Viskochil DH, DDD Study, Bejerano G, Bernstein JA and Chitayat D

    Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, UK.

    Background: In 1993, Chitayat et al., reported a newborn with hyperphalangism, facial anomalies, and bronchomalacia. We identified three additional families with similar findings. Features include bilateral accessory phalanx resulting in shortened index fingers; hallux valgus; distinctive face; respiratory compromise.

    Objectives: To identify the genetic aetiology of Chitayat syndrome and identify a unifying cause for this specific form of hyperphalangism.

    Methods: Through ongoing collaboration, we had collected patients with strikingly-similar phenotype. Trio-based exome sequencing was first performed in Patient 2 through Deciphering Developmental Disorders study. Proband-only exome sequencing had previously been independently performed in Patient 4. Following identification of a candidate gene variant in Patient 2, the same variant was subsequently confirmed from exome data in Patient 4. Sanger sequencing was used to validate this variant in Patients 1, 3; confirm paternal inheritance in Patient 5.

    Results: A recurrent, novel variant NM_006494.2:c.266A>G p.(Tyr89Cys) in ERF was identified in five affected individuals: de novo (patient 1, 2 and 3) and inherited from an affected father (patient 4 and 5). p.Tyr89Cys is an aromatic polar neutral to polar neutral amino acid substitution, at a highly conserved position and lies within the functionally important ETS-domain of the protein. The recurrent ERF c.266A>C p.(Tyr89Cys) variant causes Chitayat syndrome.

    Discussion: ERF variants have previously been associated with complex craniosynostosis. In contrast, none of the patients with the c.266A>G p.(Tyr89Cys) variant have craniosynostosis.

    Conclusions: We report the molecular aetiology of Chitayat syndrome and discuss potential mechanisms for this distinctive phenotype associated with the p.Tyr89Cys substitution in ERF.

    Journal of medical genetics 2017;54;3;157-165

  • Delineating the phenotypic spectrum of Bainbridge-Ropers syndrome: 12 new patients with de novo, heterozygous, loss-of-function mutations in ASXL3 and review of published literature.

    Balasubramanian M, Willoughby J, Fry AE, Weber A, Firth HV, Deshpande C, Berg JN, Chandler K, Metcalfe KA, Lam W, Pilz DT and Tomkins S

    Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, UK.

    Background: Bainbridge-Ropers syndrome (BRPS) is a recently described developmental disorder caused by de novo truncating mutations in the additional sex combs like 3 (ASXL3) gene. To date, there have been fewer than 10 reported patients.

    Objectives: Here, we delineate the BRPS phenotype further by describing a series of 12 previously unreported patients identified by the Deciphering Developmental Disorders study.

    Methods: Trio-based exome sequencing was performed on all 12 patients included in this study, which found a de novo truncating mutation in ASXL3. Detailed phenotypic information and patient images were collected and summarised as part of this study.

    Results: By obtaining genotype:phenotype data, we have been able to demonstrate a second mutation cluster region within ASXL3. This report expands the phenotype of older patients with BRPS; common emerging features include severe intellectual disability (11/12), poor/ absent speech (12/12), autistic traits (9/12), distinct face (arched eyebrows, prominent forehead, high-arched palate, hypertelorism and downslanting palpebral fissures), (9/12), hypotonia (11/12) and significant feeding difficulties (9/12) when young.

    Discussion: Similarities in the patients reported previously in comparison with this cohort included their distinctive craniofacial features, feeding problems, absent/limited speech and intellectual disability. Shared behavioural phenotypes include autistic traits, hand-flapping, rocking, aggressive behaviour and sleep disturbance.

    Conclusions: This series expands the phenotypic spectrum of this severe disorder and highlights its surprisingly high frequency. With the advent of advanced genomic screening, we are likely to identify more variants in this gene presenting with a variable phenotype, which this study will explore.

    Journal of medical genetics 2017;54;8;537-543

  • Evaluation of applicability of DNA microarray-based characterization of bovine Shiga toxin-producing Escherichia coli isolates using whole genome sequence analysis.

    Barth SA, Menge C, Eichhorn I, Semmler T, Pickard D and Geue L

    Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany (Barth, Menge, Geue).

    We assessed the ability of a commercial DNA microarray to characterize bovine Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results using in silico hybridization of the microarray probes within whole genome sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178 isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico probe hybridization. Results were more congruent when detecting the genoserotype (209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes (VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the microarray, only 37.2% of the isolates could be genoserotyped. However, the microarray proved suitable to rapidly screen bovine STEC strains for the occurrence of high numbers of VAGs and AMRGs and is suitable for molecular surveillance workflows.

    Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 2017;1040638717700689

  • Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes.

    Bassano I, Ong SH, Lawless N, Whitehead T, Fife M and Kellam P

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

    Background: Interferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4 genome, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases.

    Methods: We re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and we mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, namely a White Leghorn, and a turkey.

    Results: We confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. The pull down revealed a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. RNA-seq shows chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues.

    Conclusions: Locus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.

    BMC genomics 2017;18;1;419

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

    Bassett AR

    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.

    Mammalian genome : official journal of the International Mammalian Genome Society 2017

  • A Family Based Study of Carbon Monoxide and Nitric Oxide Signalling Genes and Preeclampsia.

    Bauer AE, Avery CL, Shi M, Weinberg CR, Olshan AF, Harmon QE, Luo J, Yang J, Manuck TA, Wu MC, Williams N, McGinnis R, Morgan L, Klungsøyr K, Trogstad L, Magnus P and Engel SM

    Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA.

    Background: Preeclampsia is thought to originate during placentation, with incomplete remodelling and perfusion of the spiral arteries leading to reduced placental vascular capacity. Nitric oxide (NO) and carbon monoxide (CO) are powerful vasodilators that play a role in the placental vascular system. Although family clustering of preeclampsia has been observed, the existing genetic literature is limited by a failure to consider both mother and child.

    Methods: We conducted a nested case-control study within the Norwegian Mother and Child Birth Cohort of 1545 case-pairs and 995 control-pairs from 2540 validated dyads (2011 complete pairs, 529 missing mother or child genotype). We selected 1518 single-nucleotide polymorphisms (SNPs) with minor allele frequency >5% in NO and CO signalling pathways. We used log-linear Poisson regression models and likelihood ratio tests to assess maternal and child effects.

    Results: One SNP met criteria for a false discovery rate Q-value <0.05. The child variant, rs12547243 in adenylate cyclase 8 (ADCY8), was associated with an increased risk (relative risk [RR] 1.42, 95% confidence interval [CI] 1.20, 1.69 for AG vs. GG, RR 2.14, 95% CI 1.47, 3.11 for AA vs. GG, Q = 0.03). The maternal variant, rs30593 in PDE1C was associated with a decreased risk for the subtype of preeclampsia accompanied by early delivery (RR 0.45, 95% CI 0.27, 0.75 for TC vs. CC; Q = 0.02). None of the associations were replicated after correction for multiple testing.

    Conclusions: This study uses a novel approach to disentangle maternal and child genotypic effects of NO and CO signalling genes on preeclampsia.

    Paediatric and perinatal epidemiology 2017

  • Evolution of complexity in the zebrafish synapse proteome.

    Bayés À, Collins MO, Reig-Viader R, Gou G, Goulding D, Izquierdo A, Choudhary JS, Emes RD and Grant SG

    Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Maria Claret 167, 08025 Barcelona, Spain.

    The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.

    Nature communications 2017;8;14613

  • The evolving craniofacial phenotype of a patient with Sensenbrenner syndrome caused by IFT140 compound heterozygous mutations.

    Bayat A, Kerr B, Douzgou S and DDD Study

    aDepartment of Pediatrics, University Hospital of Hvidovre, Hvidovre, Denmark bManchester Centre for Genomic Medicine, St Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre cSchool of Biological Sciences, Division of Evolution and Genomic Sciences, University of Manchester, Manchester dWellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    Clinical dysmorphology 2017;26;4;247-251

  • Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma.

    Behjati S, Tarpey PS, Haase K, Ye H, Young MD, Alexandrov LB, Farndon SJ, Collord G, Wedge DC, Martincorena I, Cooke SL, Davies H, Mifsud W, Lidgren M, Martin S, Latimer C, Maddison M, Butler AP, Teague JW, Pillay N, Shlien A, McDermott U, Futreal PA, Baumhoer D, Zaikova O, Bjerkehagen B, Myklebost O, Amary MF, Tirabosco R, Van Loo P, Stratton MR, Flanagan AM and Campbell PJ

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

    Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation, we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. It may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.

    Funded by: Wellcome Trust

    Nature communications 2017;8;15936

  • Prospects of Fine-Mapping Trait-Associated Genomic Regions by Using Summary Statistics from Genome-wide Association Studies.

    Benner C, Havulinna AS, Järvelin MR, Salomaa V, Ripatti S and Pirinen M

    Institute for Molecular Medicine Finland, University of Helsinki, 00014 Helsinki, Finland; Department of Public Health, University of Helsinki, 00014 Helsinki, Finland. Electronic address:

    During the past few years, various novel statistical methods have been developed for fine-mapping with the use of summary statistics from genome-wide association studies (GWASs). Although these approaches require information about the linkage disequilibrium (LD) between variants, there has not been a comprehensive evaluation of how estimation of the LD structure from reference genotype panels performs in comparison with that from the original individual-level GWAS data. Using population genotype data from Finland and the UK Biobank, we show here that a reference panel of 1,000 individuals from the target population is adequate for a GWAS cohort of up to 10,000 individuals, whereas smaller panels, such as those from the 1000 Genomes Project, should be avoided. We also show, both theoretically and empirically, that the size of the reference panel needs to scale with the GWAS sample size; this has important consequences for the application of these methods in ongoing GWAS meta-analyses and large biobank studies. We conclude by providing software tools and by recommending practices for sharing LD information to more efficiently exploit summary statistics in genetics research.

    American journal of human genetics 2017

  • Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

    Berger CN, Crepin VF, Roumeliotis TI, Wright JC, Carson D, Pevsner-Fischer M, Furniss RCD, Dougan G, Bachash M, Yu L, Clements A, Collins JW, Elinav E, Larrouy-Maumus GJ, Choudhary JS and Frankel G

    MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK.

    The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

    Cell metabolism 2017

  • A Neolithic expansion, but strong genetic structure, in the independent history of New Guinea.

    Bergström A, Oppenheimer SJ, Mentzer AJ, Auckland K, Robson K, Attenborough R, Alpers MP, Koki G, Pomat W, Siba P, Xue Y, Sandhu MS and Tyler-Smith C

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

    New Guinea shows human occupation since ~50 thousand years ago (ka), independent adoption of plant cultivation ~10 ka, and great cultural and linguistic diversity today. We performed genome-wide single-nucleotide polymorphism genotyping on 381 individuals from 85 language groups in Papua New Guinea and find a sharp divide originating 10 to 20 ka between lowland and highland groups and a lack of non-New Guinean admixture in the latter. All highlanders share ancestry within the last 10 thousand years, with major population growth in the same period, suggesting population structure was reshaped following the Neolithic lifestyle transition. However, genetic differentiation between groups in Papua New Guinea is much stronger than in comparable regions in Eurasia, demonstrating that such a transition does not necessarily limit the genetic and linguistic diversity of human societies.

    Science (New York, N.Y.) 2017;357;6356;1160-1163

  • Cross-Species Y Chromosome Function Between Malaria Vectors of the Anopheles gambiae Species Complex.

    Bernardini F, Galizi R, Wunderlich M, Taxiarchi C, Kranjc N, Kyrou K, Hammond A, Nolan T, Lawniczak MNK, Papathanos PA, Crisanti A and Windbichler N

    Imperial College London.

    Y chromosome function, structure and evolution is poorly understood in many species including the Anopheles genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the Anopheles gambiae complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing confirmed that the A. gambiae Y retained its original sequence content in the A. arabiensis genomic background. In contrast to comparable experiments in Drosophila, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of A. arabiensis genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the A. gambiae Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85Myr ago function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whether intentionally or contingent, between the major malaria vector species.

    Genetics 2017

  • Cracking Ali Baba's code.

    Billker O

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    A protein called P36 holds the key to how different species of malaria parasite invade liver cells.

    eLife 2017;6

  • Variants of AbGRI3 carrying the armA gene in extensively antibiotic-resistant Acinetobacter baumannii from Singapore.

    Blackwell GA, Holt KE, Bentley SD, Hsu LY and Hall RM

    School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia.

    Objectives: To investigate the context of the ribosomal RNA methyltransferase gene armA in carbapenem-resistant global clone 2 (GC2) Acinetobacter baumannii isolates from Singapore.

    Methods: Antibiotic resistance was determined using disc diffusion; PCR was used to identify resistance genes. Whole genome sequences were determined and contigs were assembled and ordered using PCR. Resistance regions in unsequenced isolates were mapped.

    Results: Fifteen GC2 A. baumannii isolated at Singapore General Hospital over the period 2004-11 and found to carry the armA gene were resistant to carbapenems, third-generation cephalosporins, fluoroquinolones and most aminoglycosides. In these isolates, the armA gene was located in a third chromosomal resistance island, previously designated AbGRI3. In four isolates, armA was in a 19 kb IS26-bounded transposon, designated Tn6180 In three of them, a 2.7 kb transposon carrying the aphA1b gene, designated Tn6179, was found adjacent to and sharing an IS26 with Tn6180. However, in these four isolates a 3.1 kb segment of the adjacent chromosomal DNA has been inverted by an IS26-mediated event. The remaining 11 isolates all contained a derivative of Tn6180 that had lost part of the central segment and only one retained Tn6179 The chromosomal inversion was present in four of these and in seven the deletion extended beyond the inversion into adjacent chromosomal DNA. AbGRI3 forms were found in available GC2 sequences carrying armA.

    Conclusions: In GC2 A. baumannii, the armA gene is located in various forms of a third genomic resistance island named AbGRI3. An aphA1b transposon is variably present in AbGRI3.

    The Journal of antimicrobial chemotherapy 2017

  • Galleria mellonella is low cost and suitable surrogate host for studying virulence of human pathogenic Vibrio cholerae.

    Bokhari H, Ali A, Noreen Z, Thomson N and Wren BW

    Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. Electronic address:

    Vibrio cholerae causes a severe diarrheal disease affecting millions of people worldwide, particularly in low income countries. V. cholerae successfully persist in aquatic environment and its pathogenic strains results in sever enteric disease in humans. This dual life style contributes towards its better survival and persistence inside host gut and in the environment. Alternative animal replacement models are of great value in studying host-pathogen interaction and for quick screening of various pathogenic strains. One such model is Galleria mellonella, a wax worm which has a complex innate immune system and here we investigate its suitability as a model for clinical human isolates of O1 El TOR, Ogawa serotype belonging to two genetically distinct subclades found in Pakistan (PSC-1 and PSC-2). We demonstrate that the PSC-2 strain D59 frequently isolated from inland areas, was more virulent than PSC-1 strain K7 mainly isolated from coastal areas (p=0.0001). In addition, we compared the relative biofilm capability of the representative strains as indicators of their survival and persistence in the environment and K7 showed enhanced biofilm forming capabilities (p=0.004). Finally we present the annotated genomes of the strains D59 and K7, and compared them with the reference strain N16961.

    Gene 2017

  • The impact of rare and low-frequency genetic variants in common disease.

    Bomba L, Walter K and Soranzo N

    Human Genetics, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, CB10 1HH, UK.

    Despite thousands of genetic loci identified to date, a large proportion of genetic variation predisposing to complex disease and traits remains unaccounted for. Advances in sequencing technology enable focused explorations on the contribution of low-frequency and rare variants to human traits. Here we review experimental approaches and current knowledge on the contribution of these genetic variants in complex disease and discuss challenges and opportunities for personalised medicine.

    Funded by: Wellcome Trust

    Genome biology 2017;18;1;77

  • Revealing hidden complexities of genomic rearrangements generated with Cas9.

    Boroviak K, Fu B, Yang F, Doe B and Bradley A

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

    Modelling human diseases caused by large genomic rearrangements has become more accessible since the utilization of CRISPR/Cas9 in mammalian systems. In a previous study, we showed that genomic rearrangements of up to one million base pairs can be generated by direct injection of CRISPR/Cas9 reagents into mouse zygotes. Although these rearrangements are ascertained by junction PCR, we describe here a variety of anticipated structural changes often involving reintegration of the region demarcated by the gRNAs in the vicinity of the edited locus. We illustrate here some of this diversity detected by high-resolution fibre-FISH and conclude that extensive molecular analysis is required to fully understand the structure of engineered chromosomes generated by Cas9.

    Scientific reports 2017;7;1;12867

  • Whole Genome Sequencing for Surveillance of Antimicrobial Resistance in Actinobacillus pleuropneumoniae.

    Bossé JT, Li Y, Rogers J, Fernandez Crespo R, Li Y, Chaudhuri RR, Holden MT, Maskell DJ, Tucker AW, Wren BW, Rycroft AN and Langford PR

    Section of Paediatrics, Department of Medicine, Imperial College London London, UK.

    The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.

    Frontiers in microbiology 2017;8;311

  • Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

    Botthof JG, Bielczyk-Maczyńska E, Ferreira L and Cvejic A

    Department of Haematology, University of Cambridge, Addenbrookes Hospital, Cambridge CB2 0XY, United Kingdom.

    RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

    Proceedings of the National Academy of Sciences of the United States of America 2017

  • Semantic prioritization of novel causative genomic variants.

    Boudellioua I, Mahamad Razali RB, Kulmanov M, Hashish Y, Bajic VB, Goncalves-Serra E, Schoenmakers N, Gkoutos GV, Schofield PN and Hoehndorf R

    King Abdullah University of Science and Technology, Computer, Electrical & Mathematical Sciences and Engineering Division, Computational Bioscience Research Center, Thuwal, Saudi Arabia.

    Discriminating the causative disease variant(s) for individuals with inherited or de novo mutations presents one of the main challenges faced by the clinical genetics community today. Computational approaches for variant prioritization include machine learning methods utilizing a large number of features, including molecular information, interaction networks, or phenotypes. Here, we demonstrate the PhenomeNET Variant Predictor (PVP) system that exploits semantic technologies and automated reasoning over genotype-phenotype relations to filter and prioritize variants in whole exome and whole genome sequencing datasets. We demonstrate the performance of PVP in identifying causative variants on a large number of synthetic whole exome and whole genome sequences, covering a wide range of diseases and syndromes. In a retrospective study, we further illustrate the application of PVP for the interpretation of whole exome sequencing data in patients suffering from congenital hypothyroidism. We find that PVP accurately identifies causative variants in whole exome and whole genome sequencing datasets and provides a powerful resource for the discovery of causal variants.

    PLoS computational biology 2017;13;4;e1005500

  • Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations.

    Brammeld JS, Petljak M, Martincorena I, Williams SP, Alonso LG, Dalmases A, Bellosillo B, Robles-Espinoza CD, Price S, Barthorpe S, Tarpey P, Alifrangis C, Bignell G, Vidal J, Young J, Stebbings L, Beal K, Stratton MR, Saez-Rodriguez J, Garnett M, Montagut C, Iorio F and McDermott U

    Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom.

    Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients.

    Genome research 2017;27;4;613-625

  • Artificial and natural RNA interactions between bacteria and C. elegans.

    Braukmann F, Jordan D and Miska E

    a Gurdon Institute, University of Cambridge , Tennis Court Road, Cambridge , CB2 1QN , United Kingdom.

    19 years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental setting [Timmons and Fire, 1998 ] the biological role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA.

    RNA biology 2017;0

  • Longitudinal genomic surveillance of multidrug-resistant Escherichia coli carriage in a long-term care facility in the United Kingdom.

    Brodrick HJ, Raven KE, Kallonen T, Jamrozy D, Blane B, Brown NM, Martin V, Török ME, Parkhill J and Peacock SJ

    Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK.

    Background: Residents of long-term care facilities (LTCF) may have high carriage rates of multidrug-resistant pathogens, but are not currently included in surveillance programmes for antimicrobial resistance or healthcare-associated infections. Here, we describe the value derived from a longitudinal epidemiological and genomic surveillance study of drug-resistant Escherichia coli in a LTCF in the United Kingdom (UK).

    Methods: Forty-five of 90 (50%) residents were recruited and followed for six months in 2014. Participants were screened weekly for carriage of extended-spectrum beta-lactamase (ESBL) producing E. coli. Participants positive for ESBL E. coli were also screened for ESBL-negative E. coli. Phenotypic antibiotic susceptibility of E. coli was determined using the Vitek2 instrument and isolates were sequenced on an Illumina HiSeq2000 instrument. Information was collected on episodes of clinical infection and antibiotic consumption.

    Results: Seventeen of 45 participants (38%) carried ESBL E. coli. Twenty-three of the 45 participants (51%) had 63 documented episodes of clinical infection treated with antibiotics. Treatment with antibiotics was associated with higher risk of carrying ESBL E. coli. ESBL E. coli was mainly sequence type (ST)131 (16/17, 94%). Non-ESBL E. coli from these 17 cases was more genetically diverse, but ST131 was found in eight (47%) cases. Whole-genome analysis of 297 ST131 E. coli from the 17 cases demonstrated highly related strains from six participants, indicating acquisition from a common source or person-to-person transmission. Five participants carried highly related strains of both ESBL-positive and ESBL-negative ST131. Genome-based comparison of ST131 isolates from the LTCF study participants with ST131 associated with bloodstream infection at a nearby acute hospital and in hospitals across England revealed sharing of highly related lineages between the LTCF and a local hospital.

    Conclusions: This study demonstrates the power of genomic surveillance to detect multidrug-resistant pathogens and confirm their connectivity within a healthcare network.

    Genome medicine 2017;9;1;70

  • Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.

    Brown JS, O'Carrigan B, Jackson SP and Yap TA

    Royal Marsden NHS Foundation Trust, London, United Kingdom.

    Germline aberrations in critical DNA-repair and DNA damage-response (DDR) genes cause cancer predisposition, whereas various tumors harbor somatic mutations causing defective DDR/DNA repair. The concept of synthetic lethality can be exploited in such malignancies, as exemplified by approval of poly(ADP-ribose) polymerase inhibitors for treating BRCA1/2-mutated ovarian cancers. Herein, we detail how cellular DDR processes engage various proteins that sense DNA damage, initiate signaling pathways to promote cell-cycle checkpoint activation, trigger apoptosis, and coordinate DNA repair. We focus on novel therapeutic strategies targeting promising DDR targets and discuss challenges of patient selection and the development of rational drug combinations.

    Significance: Various inhibitors of DDR components are in preclinical and clinical development. A thorough understanding of DDR pathway complexities must now be combined with strategies and lessons learned from the successful registration of PARP inhibitors in order to fully exploit the potential of DDR inhibitors and to ensure their long-term clinical success. Cancer Discov; 7(1); 20-37. ©2016 AACR.

    Cancer discovery 2017;7;1;20-37

  • Transmission of the gut microbiota: spreading of health.

    Browne HP, Neville BA, Forster SC and Lawley TD

    Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK.

    Transmission of commensal intestinal bacteria between humans could promote health by establishing, maintaining and replenishing microbial diversity in the microbiota of an individual. Unlike pathogens, the routes of transmission for commensal bacteria remain unappreciated and poorly understood, despite the likely commonalities between both. Consequently, broad infection control measures that are designed to prevent pathogen transmission and infection, such as oversanitation and the overuse of antibiotics, may inadvertently affect human health by altering normal commensal transmission. In this Review, we discuss the mechanisms and factors that influence host-to-host transmission of the intestinal microbiota and examine how a better understanding of these processes will identify new approaches to nurture and restore transmission routes that are used by beneficial bacteria.

    Nature reviews. Microbiology 2017;15;9;531-543

  • Chromosome contacts in activated T cells identify autoimmune disease candidate genes.

    Burren OS, Rubio García A, Javierre BM, Rainbow DB, Cairns J, Cooper NJ, Lambourne JJ, Schofield E, Castro Dopico X, Ferreira RC, Coulson R, Burden F, Rowlston SP, Downes K, Wingett SW, Frontini M, Ouwehand WH, Fraser P, Spivakov M, Todd JA, Wicker LS, Cutler AJ and Wallace C

    Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0SP, UK.

    Background: Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4(+) T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes.

    Results: Within 4 h, activation of CD4(+) T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach.

    Conclusions: Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.

    Genome biology 2017;18;1;165

  • Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes.

    Bushell E, Gomes AR, Sanderson T, Anar B, Girling G, Herd C, Metcalf T, Modrzynska K, Schwach F, Martin RE, Mather MW, McFadden GI, Parts L, Rutledge GG, Vaidya AB, Wengelnik K, Rayner JC and Billker O

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

    The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.

    Funded by: NIAID NIH HHS: R01 AI028398

    Cell 2017;170;2;260-272.e8

  • Guideline for the investigation and management of eosinophilia.

    Butt NM, Lambert J, Ali S, Beer PA, Cross NC, Duncombe A, Ewing J, Harrison CN, Knapper S, McLornan D, Mead AJ, Radia D, Bain BJ and British Committee for Standards in Haematology

    Royal Liverpool and Broadgreen University Teaching Hospitals NHS Trust, Liverpool, UK.

    British journal of haematology 2017

  • 11,670 whole-genome sequences representative of the Han Chinese population from the CONVERGE project.

    Cai N, Bigdeli TB, Kretzschmar WW, Li Y, Liang J, Hu J, Peterson RE, Bacanu S, Webb BT, Riley B, Li Q, Marchini J, Mott R, Kendler KS and Flint J

    Wellcome Trust Centre for Human Genetics, OX3 7BN Oxford, UK.

    The China, Oxford and Virginia Commonwealth University Experimental Research on Genetic Epidemiology (CONVERGE) project on Major Depressive Disorder (MDD) sequenced 11,670 female Han Chinese at low-coverage (1.7X), providing the first large-scale whole genome sequencing resource representative of the largest ethnic group in the world. Samples are collected from 58 hospitals from 23 provinces around China. We are able to call 22 million high quality single nucleotide polymorphisms (SNP) from the nuclear genome, representing the largest SNP call set from an East Asian population to date. We use these variants for imputation of genotypes across all samples, and this has allowed us to perform a successful genome wide association study (GWAS) on MDD. The utility of these data can be extended to studies of genetic ancestry in the Han Chinese and evolutionary genetics when integrated with data from other populations. Molecular phenotypes, such as copy number variations and structural variations can be detected, quantified and analysed in similar ways.

    Scientific data 2017;4;170011

  • The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

    Cambridge EL, McIntyre Z, Clare S, Arends MJ, Goulding D, Isherwood C, Caetano SS, Reviriego CB, Swiatkowska A, Kane L, Harcourt K, Sanger Mouse Genetics Project, Adams DJ, White JK and Speak AO

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

    Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance.

    Experimental hematology 2017;45;64-68.e5

  • Cliques and Schisms of Cancer Genes.

    Campbell PJ

    Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Department of Haematology, University of Cambridge, Cambridge CB2 2XY, UK. Electronic address:

    With a few exceptions, cancers typically carry more than one driver mutation, sometimes five, ten, or more, and these driver mutations do not necessarily assort randomly. In this issue of Cancer Cell, Mina et al. systematically characterize patterns of co-mutation and mutual exclusivity in 6,456 cancers across 23 tumor types.

    Cancer cell 2017;32;2;129-130

  • CamOptimus: a tool for exploiting complex adaptive evolution to optimize experiments and processes in biotechnology.

    Cankorur-Cetinkaya A, Dias JML, Kludas J, Slater NKH, Rousu J, Oliver SG and Dikicioglu D

    1​Cambridge Systems Biology Centre and Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.

    Multiple interacting factors affect the performance of engineered biological systems in synthetic biology projects. The complexity of these biological systems means that experimental design should often be treated as a multiparametric optimization problem. However, the available methodologies are either impractical, due to a combinatorial explosion in the number of experiments to be performed, or are inaccessible to most experimentalists due to the lack of publicly available, user-friendly software. Although evolutionary algorithms may be employed as alternative approaches to optimize experimental design, the lack of simple-to-use software again restricts their use to specialist practitioners. In addition, the lack of subsidiary approaches to further investigate critical factors and their interactions prevents the full analysis and exploitation of the biotechnological system. We have addressed these problems and, here, provide a simple-to-use and freely available graphical user interface to empower a broad range of experimental biologists to employ complex evolutionary algorithms to optimize their experimental designs. Our approach exploits a Genetic Algorithm to discover the subspace containing the optimal combination of parameters, and Symbolic Regression to construct a model to evaluate the sensitivity of the experiment to each parameter under investigation. We demonstrate the utility of this method using an example in which the culture conditions for the microbial production of a bioactive human protein are optimized. CamOptimus is available through: (

    Microbiology (Reading, England) 2017

  • Adipose Tissue Function and Expandability as Determinants of Lipotoxicity and the Metabolic Syndrome.

    Carobbio S, Pellegrinelli V and Vidal-Puig A

    Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Addenbrooke's Hospital, Box 289, Cambridge, CB2 OQQ, UK.

    The adipose tissue organ is organised as distinct anatomical depots located all along the body axis and it is constituted of three different types of adipocytes : white, beige and brown which are integrated with vascular, immune, neural and extracellular stroma cells. These distinct adipocytes serve different specialised functions. The main function of white adipocytes is to ensure healthy storage of excess nutrients/energy and its rapid mobilisation to supply the demand of energy imposed by physiological cues in other organs, whereas brown and beige adipocytes are designed for heat production through uncoupling lipid oxidation from energy production. The concert action of the three type of adipocytes/tissues has been reported to ensure an optimal metabolic status in rodents. However, when one or multiple of these adipose depots become dysfunctional as a consequence of sustained lipid/nutrient overload, then insulin resistance and associated metabolic complications ensue. These metabolic alterations negatively affects the adipose tissue functionality and compromises global metabolic homeostasis. Optimising white adipose tissue expandability and its functional metabolic flexibility and/or promoting brown/beige mediated thermogenic activity counteracts obesity and its associated lipotoxic metabolic effects. The development of these therapeutic approaches requires a deep understanding of adipose tissue in all broad aspects. In this chapter we will discuss the characteristics of the different adipose tissue depots with respect to origins and precursors recruitment, plasticity, cellular composition and expandability capacity as well as molecular and metabolic signatures in both physiological and pathophysiological conditions.

    Advances in experimental medicine and biology 2017;960;161-196

  • Genome-wide association study of nevirapine hypersensitivity in a sub-Saharan African HIV-infected population.

    Carr DF, Bourgeois S, Chaponda M, Takeshita LY, Morris AP, Castro EM, Alfirevic A, Jones AR, Rigden DJ, Haldenby S, Khoo S, Lalloo DG, Heyderman RS, Dandara C, Kampira E, van Oosterhout JJ, Ssali F, Munderi P, Novelli G, Borgiani P, Nelson MR, Holden A, Deloukas P and Pirmohamed M

    Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK

    Background: The antiretroviral nevirapine is associated with hypersensitivity reactions in 6%-10% of patients, including hepatotoxicity, maculopapular exanthema, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).

    Objectives: To undertake a genome-wide association study (GWAS) to identify genetic predisposing factors for the different clinical phenotypes associated with nevirapine hypersensitivity.

    Methods: A GWAS was undertaken in a discovery cohort of 151 nevirapine-hypersensitive and 182 tolerant, HIV-infected Malawian adults. Replication of signals was determined in a cohort of 116 cases and 68 controls obtained from Malawi, Uganda and Mozambique. Interaction with ERAP genes was determined in patients positive for HLA-C*04:01 In silico docking studies were also performed for HLA-C*04:01 RESULTS: Fifteen SNPs demonstrated nominal significance (P < 1 × 10(-5)) with one or more of the hypersensitivity phenotypes. The most promising signal was seen in SJS/TEN, where rs5010528 (HLA-C locus) approached genome-wide significance (P < 8.5 × 10(-8)) and was below HLA-wide significance (P < 2.5 × 10(-4)) in the meta-analysis of discovery and replication cohorts [OR 4.84 (95% CI 2.71-8.61)]. rs5010528 is a strong proxy for HLA-C*04:01 carriage: in silico docking showed that two residues (33 and 123) in the B pocket were the most likely nevirapine interactors. There was no interaction between HLA-C*04:01 and ERAP1, but there is a potential protective effect with ERAP2 [P = 0.019, OR 0.43 (95% CI 0.21-0.87)].

    Conclusions: HLA-C*04:01 predisposes to nevirapine-induced SJS/TEN in sub-Saharan Africans, but not to other hypersensitivity phenotypes. This is likely to be mediated via binding to the B pocket of the HLA-C peptide. Whether this risk is modulated by ERAP2 variants requires further study.

    The Journal of antimicrobial chemotherapy 2017

  • TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa.

    Castaneda JM, Hua R, Miyata H, Oji A, Guo Y, Cheng Y, Zhou T, Guo X, Cui Y, Shen B, Wang Z, Hu Z, Zhou Z, Sha J, Prunskaite-Hyyrylainen R, Yu Z, Ramirez-Solis R, Ikawa M, Matzuk MM and Liu M

    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030.

    Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.

    Proceedings of the National Academy of Sciences of the United States of America 2017

  • Adaptation... that's what you need?

    Chaguza C and Bentley SD

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

    Nature reviews. Microbiology 2017;15;8;452

  • Population genetic structure, antibiotic resistance, capsule switching and evolution of invasive pneumococci before conjugate vaccination in Malawi.

    Chaguza C, Cornick JE, Andam CP, Gladstone RA, Alaerts M, Musicha P, Peno C, Bar-Zeev N, Kamng'ona AW, Kiran AM, Msefula CL, McGee L, Breiman RF, Kadioglu A, French N, Heyderman RS, Hanage WP, Bentley SD and Everett DB

    Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, UK; Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi.

    Introduction: Pneumococcal infections cause a high death toll in Sub Saharan Africa (SSA) but the recently rolled out pneumococcal conjugate vaccines (PCV) will reduce the disease burden. To better understand the population impact of these vaccines, comprehensive analysis of large collections of pneumococcal isolates sampled prior to vaccination is required. Here we present a population genomic study of the invasive pneumococcal isolates sampled before the implementation of PCV13 in Malawi.

    Materials and methods: We retrospectively sampled and whole genome sequenced 585 invasive isolates from 2004 to 2010. We determine the pneumococcal population genetic structure and assessed serotype prevalence, antibiotic resistance rates, and the occurrence of serotype switching.

    Results: Population structure analysis revealed 22 genetically distinct sequence clusters (SCs), which consisted of closely related isolates. Serotype 1 (ST217), a vaccine-associated serotype in clade SC2, showed highest prevalence (19.3%), and was associated with the highest MDR rate (81.9%) followed by serotype 12F, a non-vaccine serotype in clade SC10 with an MDR rate of 57.9%. Prevalence of serotypes was stable prior to vaccination although there was an increase in the PMEN19 clone, serotype 5 ST289, in clade SC1 in 2010 suggesting a potential undetected local outbreak. Coalescent analysis revealed recent emergence of the SCs and there was evidence of natural capsule switching in the absence of vaccine induced selection pressure. Furthermore, majority of the highly prevalent capsule-switched isolates were associated with acquisition of vaccine-targeted capsules.

    Conclusions: This study provides descriptions of capsule-switched serotypes and serotypes with potential to cause serotype replacement post-vaccination such as 12F. Continued surveillance is critical to monitor these serotypes and antibiotic resistance in order to design better infection prevention and control measures such as inclusion of emerging replacement serotypes in future conjugate vaccines.

    Vaccine 2017

  • The evolutionary and phylogeographic history of woolly mammoths: a comprehensive mitogenomic analysis.

    Chang D, Knapp M, Enk J, Lippold S, Kircher M, Lister A, MacPhee RD, Widga C, Czechowski P, Sommer R, Hodges E, Stümpel N, Barnes I, Dalén L, Derevianko A, Germonpré M, Hillebrand-Voiculescu A, Constantin S, Kuznetsova T, Mol D, Rathgeber T, Rosendahl W, Tikhonov AN, Willerslev E, Hannon G, Lalueza-Fox C, Joger U, Poinar H, Hofreiter M and Shapiro B

    Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

    Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

    Scientific reports 2017;7;44585

  • THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.

    Chang HH, Worby CJ, Yeka A, Nankabirwa J, Kamya MR, Staedke SG, Dorsey G, Murphy M, Neafsey DE, Jeffreys AE, Hubbart C, Rockett KA, Amato R, Kwiatkowski DP, Buckee C and Greenhouse B

    Center for Communicable Disease Dynamics, Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States.

    As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

    PLoS computational biology 2017;13;1;e1005348

  • The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

    Charnaud SC, Dixon MWA, Nie CQ, Chappell L, Sanders PR, Nebl T, Hanssen E, Berriman M, Chan JA, Blanch AJ, Beeson JG, Rayner JC, Przyborski JM, Tilley L, Crabb BS and Gilson PR

    Burnet Institute, Melbourne, Victoria, Australia.

    Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

    PloS one 2017;12;7;e0181656

  • "Like sugar in milk": reconstructing the genetic history of the Parsi population.

    Chaubey G, Ayub Q, Rai N, Prakash S, Mushrif-Tripathy V, Mezzavilla M, Pathak AK, Tamang R, Firasat S, Reidla M, Karmin M, Rani DS, Reddy AG, Parik J, Metspalu E, Rootsi S, Dalal K, Khaliq S, Mehdi SQ, Singh L, Metspalu M, Kivisild T, Tyler-Smith C, Villems R and Thangaraj K

    Evolutionary Biology Group, Estonian Biocentre, Riia23b, Tartu, 51010, Estonia.

    Background: The Parsis are one of the smallest religious communities in the world. To understand the population structure and demographic history of this group in detail, we analyzed Indian and Pakistani Parsi populations using high-resolution genetic variation data on autosomal and uniparental loci (Y-chromosomal and mitochondrial DNA). Additionally, we also assayed mitochondrial DNA polymorphisms among ancient Parsi DNA samples excavated from Sanjan, in present day Gujarat, the place of their original settlement in India.

    Results: Among present-day populations, the Parsis are genetically closest to Iranian and the Caucasus populations rather than their South Asian neighbors. They also share the highest number of haplotypes with present-day Iranians and we estimate that the admixture of the Parsis with Indian populations occurred ~1,200 years ago. Enriched homozygosity in the Parsi reflects their recent isolation and inbreeding. We also observed 48% South-Asian-specific mitochondrial lineages among the ancient samples, which might have resulted from the assimilation of local females during the initial settlement. Finally, we show that Parsis are genetically closer to Neolithic Iranians than to modern Iranians, who have witnessed a more recent wave of admixture from the Near East.

    Conclusions: Our results are consistent with the historically-recorded migration of the Parsi populations to South Asia in the 7th century and in agreement with their assimilation into the Indian sub-continent's population and cultural milieu "like sugar in milk". Moreover, in a wider context our results support a major demographic transition in West Asia due to the Islamic conquest.

    Funded by: Wellcome Trust

    Genome biology 2017;18;1;110

  • Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification.

    Chen L, Jenjaroenpun P, Pillai AM, Ivshina AV, Ow GS, Efthimios M, Zhiqun T, Tan TZ, Lee SC, Rogers K, Ward JM, Mori S, Adams DJ, Jenkins NA, Copeland NG, Ban KH, Kuznetsov VA and Thiery JP

    Institute of Molecular and Cell Biology, Singapore 138673.

    Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.

    Proceedings of the National Academy of Sciences of the United States of America 2017;114;11;E2215-E2224

  • Whole-genome view of the consequences of a population bottleneck using 2926 genome sequences from Finland and United Kingdom.

    Chheda H, Palta P, Pirinen M, McCarthy S, Walter K, Koskinen S, Salomaa V, Daly M, Durbin R, Palotie A, Aittokallio T and Ripatti S

    Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.

    Isolated populations with enrichment of variants due to recent population bottlenecks provide a powerful resource for identifying disease-associated genetic variants and genes. As a model of an isolate population, we sequenced the genomes of 1463 Finnish individuals as part of the Sequencing Initiative Suomi (SISu) Project. We compared the genomic profiles of the 1463 Finns to a sample of 1463 British individuals that were sequenced in parallel as part of the UK10K Project. Whereas there were no major differences in the allele frequency of common variants, a significant depletion of variants in the rare frequency spectrum was observed in Finns when comparing the two populations. On the other hand, we observed >2.1 million variants that were twice as frequent among Finns compared with Britons and 800 000 variants that were more than 10 times more frequent in Finns. Furthermore, in Finns we observed a relative proportional enrichment of variants in the minor allele frequency range between 2 and 5% (P<2.2 × 10(-16)). When stratified by their functional annotations, loss-of-function variants showed the highest proportional enrichment in Finns (P=0.0291). In the non-coding part of the genome, variants in conserved regions (P=0.002) and promoters (P=0.01) were also significantly enriched in the Finnish samples. These functional categories represent the highest a priori power for downstream association studies of rare variants using population isolates.

    Funded by: Wellcome Trust

    European journal of human genetics : EJHG 2017;25;4;477-484

  • Pathways to understanding the genomic aetiology of osteoarthritis.

    Cibrián Uhalte E, Wilkinson JM, Southam L and Zeggini E

    Human Genetics and Cellular Genetics, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK.

    Osteoarthritis is a common, complex disease with no curative therapy. In this review, we summarize current knowledge on disease aetiopathogenesis and outline genetics and genomics approaches that are helping catalyse a much-needed improved understanding of the biological underpinning of disease development and progression.

    Human molecular genetics 2017;26;R2;R193-R201

  • Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

    Claessens A, Affara M, Assefa SA, Kwiatkowski DP and Conway DJ

    London School of Hygiene and Tropical Medicine, London, UK.

    Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

    Scientific reports 2017;7;41303

  • Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

    Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W and Kelsey G

    Epigenetics Programme, Babraham Institute, Cambridge, UK.

    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

    Nature protocols 2017;12;3;534-547

  • Global, site-specific analysis of neuronal protein S-acylation.

    Collins MO, Woodley KT and Choudhary JS

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

    Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.

    Funded by: Wellcome Trust

    Scientific reports 2017;7;1;4683

  • Clonal haematopoiesis is not prevalent in survivors of childhood cancer.

    Collord G, Park N, Podestà M, Dagnino M, Cilloni D, Jones D, Varela I, Frassoni F and Vassiliou GS

    Wellcome Trust Sanger Institute, Cambridge, UK.

    British journal of haematology 2017

  • The driver mutational landscape of ovarian squamous cell carcinomas arising in mature cystic teratoma.

    Cooke SL, Ennis D, Evers L, Dowson S, Chan MY, Paul J, Hirschowitz L, Glasspool RM, Singh N, Bell S, Day EK, Kochman A, Wilkinson N, Beer P, Martin S, Millan DW, Biankin AV and McNeish IA

    Institute of Cancer Sciences, University of Glasgow.

    Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynaecological malignancy of poor prognosis.

    Experimental design: We performed copy number, mutational state and zygosity analysis of 151 genes in SCC arising in MCT (n=25) using next-generation sequencing. The presence of high/intermediate risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome Results. MCT had a low mutation burden with a mean of only 1 mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%) and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8/20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic.

    Conclusions: Ovarian SCC arising in MCT has a high mutational burden with TP53 mutation the most common abnormality. The presence TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2017

  • From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

    Cornelissen M, Gall A, Vink M, Zorgdrager F, Binter Š, Edwards S, Jurriaans S, Bakker M, Ong SH, Gras L, van Sighem A, Bezemer D, de Wolf F, Reiss P, Kellam P, Berkhout B, Fraser C, van der Kuyl AC and BEEHIVE Consortium

    Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

    The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

    Virus research 2017;239;10-16

  • Natural variation of Epstein-Barr virus genes, proteins and pri-miRNA (revised).

    Correia S, Palser A, Elgueta Karstegl C, Middeldorp JM, Ramayanti O, Cohen JI, Hildesheim A, Fellner MD, Wiels J, White RE, Kellam P and Farrell PJ

    Section of Virology, Imperial College Faculty of Medicine, Norfolk Place, London W2 1PG, UK.

    Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains including many primary isolates have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains named QCIGP results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through SNPs in the pri-miRNA outside of the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future more directed analysis of association of specific EBV variation with EBV biology and EBV associated diseases.IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Relationships between EBV genome sequence variation and health, disease, geography and ethnicity of the host may thus be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focussing on variation in LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2, new relationships to the known type 1/type 2 strains are demonstrated and novel classification of EBNA1 and the BART miRNAs is proposed.

    Journal of virology 2017

  • The Expanding World of Human Leishmaniasis.

    Cotton JA

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambs, CB10 1SA, UK. Electronic address:

    New Leishmania isolates form a novel group of human parasites related to Leishmania enrietti, with cases in Ghana, Thailand, and Martinique; other relatives infect Australian and South American wildlife. These parasites apparently cause both cutaneous and visceral disease, and may have evolved a novel transmission mechanism exploiting blood-feeding midges.

    Trends in parasitology 2017

  • The genome of Leishmania adleri from a mammalian host highlights chromosome fission in Sauroleishmania.

    Coughlan S, Mulhair P, Sanders M, Schonian G, Cotton JA and Downing T

    School of Mathematics, Applied Mathematics and Statistics, National University of Ireland, Galway, Ireland.

    Control of pathogens arising from humans, livestock and wild animals can be enhanced by genome-based investigation. Phylogenetically classifying and optimal construction of these genomes using short sequence reads are key to this process. We examined the mammal-infecting unicellular parasite Leishmania adleri belonging to the lizard-infecting Sauroleishmania subgenus. L. adleri has been associated with cutaneous disease in humans, but can be asymptomatic in wild animals. We sequenced, assembled and investigated the L. adleri genome isolated from an asymptomatic Ethiopian rodent (MARV/ET/75/HO174) and verified it as L. adleri by comparison with other Sauroleishmania species. Chromosome-level scaffolding was achieved by combining reference-guided with de novo assembly followed by extensive improvement steps to produce a final draft genome with contiguity comparable with other references. L. tarentolae and L. major genome annotation was transferred and these gene models were manually verified and improved. This first high-quality draft Leishmania adleri reference genome is also the first Sauroleishmania genome from a non-reptilian host. Comparison of the L. adleri HO174 genome with those of L. tarentolae Parrot-TarII and lizard-infecting L. adleri RLAT/KE/1957/SKINK-7 showed extensive gene amplifications, pervasive aneuploidy, and fission of chromosomes 30 and 36. There was little genetic differentiation between L. adleri extracted from mammals and reptiles, highlighting challenges for leishmaniasis surveillance.

    Scientific reports 2017;7;43747

  • Using whole genome sequencing to investigate transmission in a multi-host system: bovine tuberculosis in New Zealand.

    Crispell J, Zadoks RN, Harris SR, Paterson B, Collins DM, de-Lisle GW, Livingstone P, Neill MA, Biek R, Lycett SJ, Kao RR and Price-Carter M

    Institute of Biodiversity, Animal Health, and Comparative Medicine, University of Glasgow, Glasgow, Scotland, G61 1QH, UK.

    Background: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is an important livestock disease raising public health and economic concerns around the world. In New Zealand, a number of wildlife species are implicated in the spread and persistence of bTB in cattle populations, most notably the brushtail possum (Trichosurus vulpecula). Whole Genome Sequenced (WGS) M. bovis isolates sourced from infected cattle and wildlife across New Zealand were analysed. Bayesian phylogenetic analyses were conducted to estimate the substitution rate of the sampled population and investigate the role of wildlife. In addition, the utility of WGS was examined with a view to these methods being incorporated into routine bTB surveillance.

    Results: A high rate of exchange was evident between the sampled wildlife and cattle populations but directional estimates of inter-species transmission were sensitive to the sampling strategy employed. A relatively high substitution rate was estimated, this, in combination with a strong spatial signature and a good agreement to previous typing methods, acts to endorse WGS as a typing tool.

    Conclusions: In agreement with the current knowledge of bTB in New Zealand, transmission of M. bovis between cattle and wildlife was evident. Without direction, these estimates are less informative but taken in conjunction with the low prevalence of bTB in New Zealand's cattle population it is likely that, currently, wildlife populations are acting as the main bTB reservoir. Wildlife should therefore continue to be targeted if bTB is to be eradicated from New Zealand. WGS will be a considerable aid to bTB eradication by greatly improving the discriminatory power of molecular typing data. The substitution rates estimated here will be an important part of epidemiological investigations using WGS data.

    BMC genomics 2017;18;1;180

  • Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening.

    Croucher NJ, Campo JJ, Le TQ, Liang X, Bentley SD, Hanage WP and Lipsitch M

    Department of Infectious Disease Epidemiology, Imperial College London, London W2 1PG, United Kingdom;

    Characterizing the immune response to pneumococcal proteins is critical in understanding this bacterium's epidemiology and vaccinology. Probing a custom-designed proteome microarray with sera from 35 healthy US adults revealed a continuous distribution of IgG affinities for 2,190 potential antigens from the species-wide pangenome. Reproducibly elevated IgG binding was elicited by 208 "antibody binding targets" (ABTs), which included 109 variants of the diverse pneumococcal surface proteins A and C (PspA and PspC) and zinc metalloprotease A and B (ZmpA and ZmpB) proteins. Functional analysis found ABTs were enriched in motifs for secretion and cell surface association, with extensive representation of cell wall synthesis machinery, adhesins, transporter solute-binding proteins, and degradative enzymes. ABTs were associated with stronger evidence for evolving under positive selection, although this varied between functional categories, as did rates of diversification through recombination. Particularly rapid variation was observed at some immunogenic accessory loci, including a phage protein and a phase-variable glycosyltransferase ubiquitous among the diverse set of genomic islands encoding the serine-rich PsrP glycoprotein. Nevertheless, many antigens were conserved in the core genome, and strains' antigenic profiles were generally stable. No strong evidence was found for any epistasis between antigens driving population dynamics, or redundancy between functionally similar accessory ABTs, or age stratification of antigen profiles. These results highlight the paradox of why substantial variation is observed in only a subset of epitopes. This result may indicate only some interactions between immunoglobulins and ABTs clear pneumococcal colonization or that acquired immunity to pneumococci is an accumulation of individually weak responses to ABTs evolving under different levels of functional constraint.

    Proceedings of the National Academy of Sciences of the United States of America 2017

  • BCFtools/csq: haplotype-aware variant consequences.

    Danecek P and McCarthy SA

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

    Motivation: Prediction of functional variant consequences is an important part of sequencing pipelines, allowing the categorization and prioritization of genetic variants for follow up analysis. However, current predictors analyze variants as isolated events, which can lead to incorrect predictions when adjacent variants alter the same codon, or when a frame-shifting indel is followed by a frame-restoring indel. Exploiting known haplotype information when making consequence predictions can resolve these issues.

    Results: BCFtools/csq is a fast program for haplotype-aware consequence calling which can take into account known phase. Consequence predictions are changed for 501 of 5019 compound variants found in the 81.7M variants in the 1000 Genomes Project data, with an average of 139 compound variants per haplotype. Predictions match existing tools when run in localized mode, but the program is an order of magnitude faster and requires an order of magnitude less memory.

    Availability and implementation: The program is freely available for commercial and non-commercial use in the BCFtools package which is available for download from .


    Supplementary information: Supplementary data are available at Bioinformatics online.

    Bioinformatics (Oxford, England) 2017;33;13;2037-2039

  • The STRATAA study protocol: a programme to assess the burden of enteric fever in Bangladesh, Malawi and Nepal using prospective population census, passive surveillance, serological studies and healthcare utilisation surveys.

    Darton TC, Meiring JE, Tonks S, Khan MA, Khanam F, Shakya M, Thindwa D, Baker S, Basnyat B, Clemens JD, Dougan G, Dolecek C, Dunstan SJ, Gordon MA, Heyderman RS, Holt KE, Pitzer VE, Qadri F, Zaman K, Pollard AJ and STRATAA Study Consortium

    The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.

    Introduction: Invasive infections caused by Salmonella enterica serovar Typhi and Paratyphi A are estimated to account for 12-27 million febrile illness episodes worldwide annually. Determining the true burden of typhoidal Salmonellae infections is hindered by lack of population-based studies and adequate laboratory diagnostics.The Strategic Typhoid alliance across Africa and Asia study takes a systematic approach to measuring the age-stratified burden of clinical and subclinical disease caused by typhoidal Salmonellae infections at three high-incidence urban sites in Africa and Asia. We aim to explore the natural history of Salmonella transmission in endemic settings, addressing key uncertainties relating to the epidemiology of enteric fever identified through mathematical models, and enabling optimisation of vaccine strategies.

    Methods/design: Using census-defined denominator populations of ≥100 000 individuals at sites in Malawi, Bangladesh and Nepal, the primary outcome is to characterise the burden of enteric fever in these populations over a 24-month period. During passive surveillance, clinical and household data, and laboratory samples will be collected from febrile individuals. In parallel, healthcare utilisation and water, sanitation and hygiene surveys will be performed to characterise healthcare-seeking behaviour and assess potential routes of transmission. The rates of both undiagnosed and subclinical exposure to typhoidal Salmonellae (seroincidence), identification of chronic carriage and population seroprevalence of typhoid infection will be assessed through age-stratified serosurveys performed at each site. Secondary attack rates will be estimated among household contacts of acute enteric fever cases and possible chronic carriers.

    Ethics and dissemination: This protocol has been ethically approved by the Oxford Tropical Research Ethics Committee, the icddr,b Institutional Review Board, the Malawian National Health Sciences Research Committee and College of Medicine Research Ethics Committee and Nepal Health Research Council. The study is being conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Informed consent was obtained before study enrolment. Results will be submitted to international peer-reviewed journals and presented at international conferences.

    Trial registration number: ISRCTN 12131979.

    Ethics references: Oxford (Oxford Tropical Research Ethics Committee 39-15).Bangladesh (icddr,b Institutional Review Board PR-15119).Malawi (National Health Sciences Research Committee 15/5/1599).Nepal (Nepal Health Research Council 306/2015).

    BMJ open 2017;7;6;e016283

  • Seeding and Establishment of Legionella pneumophila in Hospitals: Implications for Genomic Investigations of Nosocomial Legionnaires' Disease.

    David S, Afshar B, Mentasti M, Ginevra C, Podglajen I, Harris SR, Chalker VJ, Jarraud S, Harrison TG and Parkhill J

    Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.

    Background: Legionnaires' disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1.

    Methods: WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires' disease.

    Results: Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1.

    Conclusions: WGS can be used to support or refute suspected links between hospitals and Legionnaires' disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations.

    Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2017;64;9;1251-1259

  • Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

    David S, Sánchez-Busó L, Harris SR, Marttinen P, Rusniok C, Buchrieser C, Harrison TG and Parkhill J

    Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, United Kingdom.

    Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination.

    PLoS genetics 2017;13;6;e1006855

  • Whole-Genome Sequencing Reveals Breast Cancers with Mismatch Repair Deficiency.

    Davies H, Morganella S, Purdie CA, Jang SJ, Borgen E, Russnes H, Glodzik D, Zou X, Viari A, Richardson AL, Børresen-Dale AL, Thompson A, Eyfjord JE, Kong G, Stratton MR and Nik-Zainal S

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    Mismatch repair (MMR)-deficient cancers have been discovered to be highly responsive to immune therapies such as PD-1 checkpoint blockade, making their definition in patients, where they may be relatively rare, paramount for treatment decisions. In this study, we utilized patterns of mutagenesis known as mutational signatures, which are imprints of the mutagenic processes associated with MMR deficiency, to identify MMR-deficient breast tumors from a whole-genome sequencing dataset comprising a cohort of 640 patients. We identified 11 of 640 tumors as MMR deficient, but only 2 of 11 exhibited germline mutations in MMR genes or Lynch Syndrome. Two additional tumors had a substantially reduced proportion of mutations attributed to MMR deficiency, where the predominant mutational signatures were related to APOBEC enzymatic activity. Overall, 6 of 11 of the MMR-deficient cases in this cohort were confirmed genetically or epigenetically as having abrogation of MMR genes. However, IHC analysis of MMR-related proteins revealed all but one of 10 samples available for testing as MMR deficient. Thus, the mutational signatures more faithfully reported MMR deficiency than sequencing of MMR genes, because they represent a direct pathophysiologic readout of repair pathway abnormalities. As whole-genome sequencing continues to become more affordable, it could be used to expose individually abnormal tumors in tissue types where MMR deficiency has been rarely detected, but also rarely sought. Cancer Res; 77(18); 1-8. ©2017 AACR.

    Cancer research 2017

  • A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability

    Davies,B., Brown,L. A., Cais,O et al.

    Human Molecular Genetics 2017;26;20;3869-3882

  • Cytogenetic Resources and Information.

    De Braekeleer E, Huret JL, Mossafa H and Dessen P

    Haematological Cancer Genetics & Stem Cell Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

    The main databases devoted stricto sensu to cancer cytogenetics are the "Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer" ( ), the "Atlas of Genetics and Cytogenetics in Oncology and Haematology" ( ), and COSMIC ( ).However, being a complex multistep process, cancer cytogenetics are broadened to "cytogenomics," with complementary resources on: general databases (nucleic acid and protein sequences databases; cartography browsers: GenBank, RefSeq, UCSC, Ensembl, UniProtKB, and Entrez Gene), cancer genomic portals associated with recent international integrated programs, such as TCGA or ICGC, other fusion genes databases, array CGH databases, copy number variation databases, and mutation databases. Other resources such as the International System for Human Cytogenomic Nomenclature (ISCN), the International Classification of Diseases for Oncology (ICD-O), and the Human Gene Nomenclature Database (HGNC) allow a common language.Data within the scientific/medical community should be freely available. However, most of the institutional stakeholders are now gradually disengaging, and well-known databases are forced to beg or to disappear (which may happen!).

    Methods in molecular biology (Clifton, N.J.) 2017;1541;311-331

  • A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes.

    de la Rosa J, Weber J, Friedrich MJ, Li Y, Rad L, Ponstingl H, Liang Q, de Quirós SB, Noorani I, Metzakopian E, Strong A, Li MA, Astudillo A, Fernández-García MT, Fernández-García MS, Hoffman GJ, Fuente R, Vassiliou GS, Rad R, López-Otín C, Bradley A and Cadiñanos J

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

    The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.

    Funded by: Wellcome Trust

    Nature genetics 2017;49;5;730-741

  • Disentangling PTEN-cooperating tumor suppressor gene networks in cancer.

    de la Rosa J, Weber J, Rad R, Bradley A and Cadiñanos J

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

    We have recently performed a whole-body, genome-wide screen in mice using a single-copy inactivating transposon for the identification of Pten (phosphatase and tensin homolog)-cooperating tumor suppressor genes (TSGs). We identified known and putative TSGs in multiple cancer types and validated the functional and clinical relevance of several promising candidates for human prostate cancer.

    Molecular & cellular oncology 2017;4;4;e1325550

  • Identifying transposon insertions and their effects from RNA-sequencing data.

    de Ruiter JR, Kas SM, Schut E, Adams DJ, Koudijs MJ, Wessels LFA and Jonkers J

    Division of Molecular Pathology and Cancer Genomics Netherlands, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands.

    Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes.

    Nucleic acids research 2017

  • Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

    De Sousa PA, Steeg R, Wachter E, Bruce K, King J, Hoeve M, Khadun S, McConnachie G, Holder J, Kurtz A, Seltmann S, Dewender J, Reimann S, Stacey G, O'Shea O, Chapman C, Healy L, Zimmermann H, Bolton B, Rawat T, Atkin I, Veiga A, Kuebler B, Serano BM, Saric T, Hescheler J, Brüstle O, Peitz M, Thiele C, Geijsen N, Holst B, Clausen C, Lako M, Armstrong L, Gupta SK, Kvist AJ, Hicks R, Jonebring A, Brolén G, Ebneth A, Cabrera-Socorro A, Foerch P, Geraerts M, Stummann TC, Harmon S, George C, Streeter I, Clarke L, Parkinson H, Harrison PW, Faulconbridge A, Cherubin L, Burdett T, Trigueros C, Patel MJ, Lucas C, Hardy B, Predan R, Dokler J, Brajnik M, Keminer O, Pless O, Gribbon P, Claussen C, Ringwald A, Kreisel B, Courtney A and Allsopp TE

    Centre for Clinical Brain Sciences, Chancellors Building, 49 Little France Crescent, University of Edinburgh, Edinburgh EH16 4SB, UK; Roslin Cells Ltd(1), Head office, Nine Edinburgh Bioquarter, 9 Little France Rd, Edinburgh EH16 4UX, UK; EBiSC banking facility, Babraham Research Campus, B260 Meditrina, Cambridge CB22 3AT, UK. Electronic address:

    A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

    Stem cell research 2017;20;105-114

  • Phase-variable methylation and epigenetic regulation by type I restriction-modification systems.

    De Ste Croix M, Vacca I, Kwun MJ, Ralph JD, Bentley SD, Haigh R, Croucher NJ and Oggioni MR

    Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.

    Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that , through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria.

    FEMS microbiology reviews 2017;41;Supp_1;S3-S15

  • Prevalence and architecture of de novo mutations in developmental disorders.

    Deciphering Developmental Disorders Study

    The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year.

    Funded by: Medical Research Council: MC_PC_U127561093, MR/M014568/1; Wellcome Trust Sanger Institute: WT098051

    Nature 2017;542;7642;433-438

  • Environmental DNA metabarcoding: transforming how we survey animal and plant communities.

    Deiner K, Bik HM, Mächler E, Seymour M, Lacoursière-Roussel A, Altermatt F, Creer S, Bista I, Lodge DM, de Vere N, Pfrender ME and Bernatchez L

    Cornell University, Atkinson Center for a Sustainable Future, and Department of Ecology and Evolutionary Biology, Ithaca, NY, 14850, USA.

    The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ('HTS') platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed 'environmental DNA' or 'eDNA'). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called 'eDNA metabarcoding' and offers a powerful molecular tool capable of non-invasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine, and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education. This article is protected by copyright. All rights reserved.

    Molecular ecology 2017

  • Principles of Reconstructing the Subclonal Architecture of Cancers.

    Dentro SC, Wedge DC and Van Loo P

    Wellcome Trust Sanger Institute, Cambridge CB10 1HH, United Kingdom.

    Most cancers evolve from a single founder cell through a series of clonal expansions that are driven by somatic mutations. These clonal expansions can lead to several coexisting subclones sharing subsets of mutations. Analysis of massively parallel sequencing data can infer a tumor's subclonal composition through the identification of populations of cells with shared mutations. We describe the principles that underlie subclonal reconstruction through single nucleotide variants (SNVs) or copy number alterations (CNAs) from bulk or single-cell sequencing. These principles include estimating the fraction of tumor cells for SNVs and CNAs, performing clustering of SNVs from single- and multisample cases, and single-cell sequencing. The application of subclonal reconstruction methods is providing key insights into tumor evolution, identifying subclonal driver mutations, patterns of parallel evolution and differences in mutational signatures between cellular populations, and characterizing the mechanisms of therapy resistance, spread, and metastasis.

    Cold Spring Harbor perspectives in medicine 2017

  • Using reference-free compressed data structures to analyze sequencing reads from thousands of human genomes.

    Dolle DD, Liu Z, Cotten M, Simpson JT, Iqbal Z, Durbin R, McCarthy SA and Keane TM

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

    We are rapidly approaching the point where we have sequenced millions of human genomes. There is a pressing need for new data structures to store raw sequencing data and efficient algorithms for population scale analysis. Current reference-based data formats do not fully exploit the redundancy in population sequencing nor take advantage of shared genetic variation. In recent years, the Burrows-Wheeler transform (BWT) and FM-index have been widely employed as a full-text searchable index for read alignment and de novo assembly. We introduce the concept of a population BWT and use it to store and index the sequencing reads of 2705 samples from the 1000 Genomes Project. A key feature is that, as more genomes are added, identical read sequences are increasingly observed, and compression becomes more efficient. We assess the support in the 1000 Genomes read data for every base position of two human reference assembly versions, identifying that 3.2 Mbp with population support was lost in the transition from GRCh37 with 13.7 Mbp added to GRCh38. We show that the vast majority of variant alleles can be uniquely described by overlapping 31-mers and show how rapid and accurate SNP and indel genotyping can be carried out across the genomes in the population BWT. We use the population BWT to carry out nonreference queries to search for the presence of all known viral genomes and discover human T-lymphotropic virus 1 integrations in six samples in a recognized epidemiological distribution.

    Genome research 2017;27;2;300-309

  • Wounding induces dedifferentiation of epidermal Gata6(+) cells and acquisition of stem cell properties.

    Donati G, Rognoni E, Hiratsuka T, Liakath-Ali K, Hoste E, Kar G, Kayikci M, Russell R, Kretzschmar K, Mulder KW, Teichmann SA and Watt FM

    King's College London Centre for Stem Cells and Regenerative Medicine, 28th Floor, Tower Wing, Guy's Campus, Great Maze Pond, London SE1 9RT, UK.

    The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6(+) cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

    Nature cell biology 2017

  • No Functional Role for microRNA-342 in a Mouse Model of Pancreatic Acinar Carcinoma.

    Dooley J, Lagou V, Pasciuto E, Linterman MA, Prosser HM, Himmelreich U and Liston A

    Translational Immunology Laboratory, VIB, Leuven, Belgium.

    The intronic microRNA (miR)-342 has been proposed as a potent tumor-suppressor gene. miR-342 is found to be downregulated or epigenetically silenced in multiple different tumor sites, and this loss of expression permits the upregulation of several key oncogenic pathways. In several different cell lines, lower miR-342 expression results in enhanced proliferation and metastasis potential, both in vitro and in xenogenic transplant conditions. Here, we sought to determine the function of miR-342 in an in vivo spontaneous cancer model, using the Ela1-TAg transgenic model of pancreatic acinar carcinoma. Through longitudinal magnetic resonance imaging monitoring of Ela1-TAg transgenic mice, either wild-type or knockout for miR-342, we found no role for miR-342 in the development, growth rate, or pathogenicity of pancreatic acinar carcinoma. These results indicate the importance of assessing miR function in the complex physiology of in vivo model systems and indicate that further functional testing of miR-342 is required before concluding it is a bona fide tumor-suppressor-miR.

    Frontiers in oncology 2017;7;101

  • Control of virulence gene transcription by indirect readout in Vibrio cholerae and Salmonella enterica serovar Typhimurium.

    Dorman CJ and Dorman MJ

    Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland.

    Indirect readout mechanisms of transcription control rely on the recognition of DNA shape by transcription factors (TFs). TFs may also employ a direct readout mechanism that involves the reading of the base sequence in the DNA major groove at the binding site. TFs with winged helix-turn-helix (wHTH) motifs use an alpha helix to read the base sequence in the major groove while inserting a beta sheet 'wing' into the adjacent minor groove. Such wHTH proteins are important regulators of virulence gene transcription in many pathogens; they also control housekeeping genes. This article considers the cases of the non-invasive Gram-negative pathogen Vibrio cholerae and the invasive pathogen Salmonella enterica serovar Typhimurium. Both possess clusters of A+T-rich horizontally acquired virulence genes that are silenced by the nucleoid-associated protein H-NS and regulated positively or negatively by wHTH TFs: for example, ToxR and LeuO in V. cholerae; HilA, LeuO, SlyA and OmpR in S. Typhimurium. Because of their relatively relaxed base sequence requirements for target recognition, indirect readout mechanisms have the potential to engage regulatory proteins with many more targets than might be the case using direct readout, making indirect readout an important, yet often ignored, contributor to the expression of pathogenic phenotypes. This article is protected by copyright. All rights reserved.

    Environmental microbiology 2017

  • Genome watch: Klebsiella pneumoniae: when a colonizer turns bad.

    Dorman MJ and Short FL

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

    Nature reviews. Microbiology 2017;15;7;384

  • Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity.

    Doyle SR, Bourguinat C, Nana-Djeunga HC, Kengne-Ouafo JA, Pion SDS, Bopda J, Kamgno J, Wanji S, Che H, Kuesel AC, Walker M, Basáñez MG, Boakye DA, Osei-Atweneboana MY, Boussinesq M, Prichard RK and Grant WN

    Department of Animal, Plant and Soil Sciences, La Trobe University, Bundoora, Australia.

    Background: Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.

    Methodology/principal findings: Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.

    Conclusions/significance: This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations.

    Funded by: Wellcome Trust

    PLoS neglected tropical diseases 2017;11;7;e0005816

  • Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer.

    Drost J, van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Sasselli V, de Ligt J, Behjati S, Grolleman JE, van Wezel T, Nik-Zainal S, Kuiper RP, Cuppen E and Clevers H

    Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, Netherlands.

    Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology, and hold diagnostic and prognostic value. Here, we develop a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR/Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed sub-cloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations.

    Science (New York, N.Y.) 2017

  • The Experimental Design Assistant.

    du Sert NP, Bamsey I, Bate ST, Berdoy M, Clark RA, Cuthill IC, Fry D, Karp NA, Macleod M, Moon L, Stanford SC and Lings B

    National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), London, UK.

    Nature methods 2017

  • Strategies for managing rival bacterial communities: lessons from burying beetles.

    Duarte A, Welch M, Swannack C, Wagner J and Kilner RM

    Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, UK.

    1.The role of bacteria in animal development, ecology and evolution is increasingly well-understood, yet little is known of how animal behaviour affects bacterial communities. Animals that benefit from defending a key resource from microbial competitors are likely to evolve behaviours to control or manipulate the animal's associated external microbiota. 2.We describe four possible mechanisms by which animals could gain a competitive edge by disrupting a rival bacterial community: 'weeding', 'seeding', 'replanting' and 'preserving'. By combining detailed behavioural observations with molecular and bioinformatic analyses, we then test which of these mechanisms best explains how burying beetles, Nicrophorus vespilloides, manipulate the bacterial communities on their carcass breeding resource. 3.Burying beetles are a suitable species to study how animals manage external microbiota because reproduction revolves around a small vertebrate carcass. Parents shave a carcass and apply antimicrobial exudates on its surface, shaping it into an edible nest for their offspring. We compared bacterial communities in mice carcasses that were either fresh, prepared by beetles or unprepared but buried underground for the same length of time. We also analysed bacterial communities in the burying beetle's gut, during and after breeding, to understand whether beetles could be 'seeding' the carcass with particular microbes. 4.We show that burying beetles do not 'preserve' the carcass by reducing bacterial load, as is commonly supposed. Instead, our results suggest they 'seed' the carcass with bacterial groups which are part of the Nicrophorus core microbiome. They may also 'replant' other bacteria from the carcass gut onto the surface of their carrion nest. Both these processes may lead to the observed increase in bacterial load on the carcass surface in the presence of beetles. Beetles may also 'weed' the bacterial community by eliminating some groups of bacteria on the carcass, perhaps through the production of antimicrobials themselves. 5.Whether these alterations to the bacterial community are adaptive from the beetle's perspective, or are simply a by-product of the way in which the beetles prepare the carcass for reproduction, remains to be determined in future work. In general, our work suggests that animals might use more sophisticated techniques for attacking and disrupting rival microbial communities than is currently appreciated. This article is protected by copyright. All rights reserved.

    The Journal of animal ecology 2017

  • Population genetic structure and adaptation of malaria parasites on the edge of endemic distribution.

    Duffy CW, Ba H, Assefa S, Ahouidi AD, Deh YB, Tandia A, Kirsebom FC, Kwiatkowski DP and Conway DJ

    Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, Keppel St, UK.

    To determine whether the major human malaria parasite Plasmodium falciparum exhibits fragmented population structure or local adaptation at the northern limit of its African distribution where the dry Sahel zone meets the Sahara, samples were collected from diverse locations within Mauritania over a range of ~ 1000 kilometres. Microsatellite genotypes were obtained for 203 clinical infection samples from eight locations, and Illumina paired-end sequences were obtained to yield high coverage genome-wide single nucleotide polymorphism (SNP) data for 65 clinical infection samples from four locations. Most infections contained single parasite genotypes, reflecting low rates of transmission and superinfection locally, in contrast to the situation seen in population samples from countries further south. A minority of infections shared related or identical genotypes locally, indicating some repeated transmission of parasite clones without recombination. This caused some multi-locus linkage disequilibrium and local divergence, but aside from the effect of repeated genotypes there was minimal differentiation between locations. Several chromosomal regions had elevated integrated haplotype scores (|iHS|) indicating recent selection, including those containing drug resistance genes. A genome-wide FST scan comparison with previous sequence data from an area in West Africa with higher infection endemicity indicates that regional gene flow prevents genetic isolation, but revealed allele frequency differentiation at three drug resistance loci and an erythrocyte invasion ligand gene. Contrast of extended haplotype signatures revealed none to be unique to Mauritania. Discrete foci of infection on the edge of the Sahara are genetically highly connected to the wider continental parasite population, and local elimination would be difficult to achieve without very substantial reduction in malaria throughout the region. This article is protected by copyright. All rights reserved.

    Molecular ecology 2017

  • Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

    Dumetz F, Imamura H, Sanders M, Seblova V, Myskova J, Pescher P, Vanaerschot M, Meehan CJ, Cuypers B, De Muylder G, Späth GF, Bussotti G, Vermeesch JR, Berriman M, Cotton JA, Volf P, Dujardin JC and Domagalska MA

    Molecular Parasitology, Institute of Tropical Medicine, Antwerp, Belgium.

    Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

    mBio 2017;8;3

  • "Matching" consent to purpose: The example of the Matchmaker Exchange.

    Dyke SOM, Knoppers BM, Hamosh A, Firth HV, Hurles M, Brudno M, Boycott KM, Philippakis AA and Rehm HL

    Centre of Genomics and Policy, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.

    The Matchmaker Exchange (MME) connects rare disease clinicians and researchers to facilitate the sharing of data from undiagnosed patients for the purpose of novel gene discovery. Such sharing raises the odds that two or more similar patients with candidate genes in common may be found, thereby allowing their condition to be more readily studied and understood. Consent considerations for data sharing in MME included both the ethical and legal differences between clinical and research settings and the level of privacy risk involved in sharing varying amounts of rare disease patient data to enable patient matches. In this commentary, we discuss these consent considerations and the resulting MME Consent Policy as they may be relevant to other international data sharing initiatives.

    Human mutation 2017

  • Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types.

    Ecker S, Chen L, Pancaldi V, Bagger FO, Fernández JM, Carrillo de Santa Pau E, Juan D, Mann AL, Watt S, Casale FP, Sidiropoulos N, Rapin N, Merkel A, BLUEPRINT Consortium, Stunnenberg HG, Stegle O, Frontini M, Downes K, Pastinen T, Kuijpers TW, Rico D, Valencia A, Beck S, Soranzo N and Paul DS

    Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.

    Background: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability.

    Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14(+)CD16(-) monocytes, CD66b(+)CD16(+) neutrophils, and CD4(+)CD45RA(+) naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers.

    Conclusions: Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: .

    Genome biology 2017;18;1;18

  • Drug resistance mechanisms in colorectal cancer dissected with cell type-specific dynamic logic models.

    Eduati F, Doldàn-Martelli V, Klinger B, Cokelaer T, Sieber A, Kogera F, Dorel M, Garnett MJ, Blüthgen N and Saez-Rodriguez J

    European Bioinformatics Institute, European Molecular Biology Laboratory.

    Genomic features are used as biomarkers of sensitivity to kinase inhibitors used widely to treat human cancer, but effective patient stratification based on these principles remains limited in impact. Insofar as kinase inhibitors interfere with signaling dynamics, and, in turn, signaling dynamics affects inhibitor responses, we investigated associations in this study between cell-specific dynamic signaling pathways and drug sensitivity. Specifically, we measured 14 phosphoproteins under 43 different perturbed conditions (combinations of 5 stimuli and 7 inhibitors) in 14 colorectal cancer cell lines, building cell line-specific dynamic logic models of underlying signaling networks. Model parameters representing pathway dynamics were used as features to predict sensitivity to a panel of 27 drugs. Specific parameters of signaling dynamics correlated strongly with drug sensitivity for 14 of the drugs, 9 of which had no genomic biomarker. Following one of these associations, we validated a drug combination predicted to overcome resistance to MEK inhibitors by co-blockade of GSK3, which was not found based on associations with genomic data. These results suggest that, in order to better understand cancer resistance and move toward personalized medicine, it is essential to consider signaling network dynamics that can not be inferred from static genotypes.

    Cancer research 2017

  • A reversible haploid mouse embryonic stem cell biobank resource for functional genomics.

    Elling U, Wimmer RA, Leibbrandt A, Burkard T, Michlits G, Leopoldi A, Micheler T, Abdeen D, Zhuk S, Aspalter IM, Handl C, Liebergesell J, Hubmann M, Husa AM, Kinzer M, Schuller N, Wetzel E, van de Loo N, Martinez JAZ, Estoppey D, Riedl R, Yang F, Fu B, Dechat T, Ivics Z, Agu CA, Bell O, Blaas D, Gerhardt H, Hoepfner D, Stark A and Penninger JM

    Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria.

    The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

    Nature 2017

  • A non-endoscopic device to sample the oesophageal microbiota: a case-control study.

    Elliott DR, Walker AW, O'Donovan M, Parkhill J and Fitzgerald RC

    Medical Research Centre Cancer Unit, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK.

    Background: The strongest risk factor for oesophageal adenocarcinoma is reflux disease, and the rising incidence of this coincides with the eradication of Helicobacter pylori, both of which might alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett's carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota.

    Methods: In this case-control study, 16S rRNA gene amplicon sequencing was done on 210 oesophageal samples from 86 patients representing the Barrett's oesophagus progression sequence (normal squamous controls [n=20], non-dysplastic [n=24] and dysplastic Barrett's oesophagus [n=23], and oesophageal adenocarcinoma [n=19]), relevant negative controls, and replicates on the Illumina MiSeq platform. Samples were taken from patients enrolled in the BEST2 study at five UK hospitals and the OCCAMS study at six UK hospitals. We compared fresh frozen tissue, fresh frozen endoscopic brushings, and the Cytosponge device for microbial DNA yield (qPCR), diversity, and community composition.

    Findings: There was decreased microbial diversity in oesophageal adenocarcinoma tissue compared with tissue from healthy control patients as measured by the observed operational taxonomic unit (OTU) richness (p=0·0012), Chao estimated total richness (p=0·0004), and Shannon diversity index (p=0·0075). Lactobacillus fermentum was enriched in oesophageal adenocarcinoma (p=0·028), and lactic acid bacteria dominated the microenvironment in seven (47%) of 15 cases of oesophageal adenocarcinoma. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-times higher quantities of microbial DNA than did endoscopic brushes or biopsies using quantitative PCR (p<0·0001). The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga, and Dialister. The Cytosponge detected decreased microbial diversity in patients with high-grade dysplasia in comparison to control patients, as measured by the observed OTU richness (p=0·0147), Chao estimated total richness (p=0·023), and Shannon diversity index (p=0·0085).

    Interpretation: Alterations in microbial communities occur in the lower oesophagus in Barrett's carcinogenesis, which can be detected at the pre-invasive stage of high-grade dysplasia with the novel Cytosponge device. Our findings are potentially applicable to early disease detection, and future test development should focus on longitudinal sampling of the microbiota to monitor for changes in microbial diversity in a larger cohort of patients.

    Funding: Cancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).

    The lancet. Gastroenterology & hepatology 2017;2;1;32-42

  • Phenotypic Consequences of a Genetic Predisposition to Enhanced Nitric Oxide Signaling.

    Emdin CA, Khera AV, Klarin D, Natarajan P, Zekavat SM, Nomura A, Haas ME, Aragam K, Ardissino D, Wilson JG, Schunkert H, McPherson R, Watkins H, Elosua R, Bown MJ, Samani NJ, Baber U, Erdmann J, Gormley P, Palotie A, Stitziel N, Gupta N, Danesh JN, Saleheen D, Gabriel SB and Kathiresan S

    Center for Genomic Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; Cardiology Division Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA.

    Background -Nitric oxide signaling plays a key role in regulation of vascular tone and platelet activation. Here, we seek to understand the impact of a genetic predisposition to enhanced nitric oxide signaling on risk for cardiovascular diseases, thus informing the potential utility of pharmacologic stimulation of the nitric oxide pathway as a therapeutic strategy. Methods -We analyzed the association of common and rare genetic variants in two genes that mediate nitric oxide signaling [Nitric Oxide Synthase 3 (NOS3) and Guanylate Cyclase 1, Soluble, Alpha 3 (GUCY1A3)] with a range of human phenotypes. We selected two common variants (rs3918226 in NOS3 and rs7692387 in GUCY1A3) known to associate with increased NOS3 and GUCY1A3 expression and reduced mean arterial pressure, combined them into a genetic score, and standardized this exposure to a 5 mm Hg reduction in mean arterial pressure. Using individual-level data from 335,464 participants in the UK Biobank and summary association results from seven large-scale genome wide association studies, we examined the effect of this nitric oxide signaling score on cardiometabolic and other diseases. We also examined whether rare loss-of-function mutations in NOS3 and GUCY1A3 were associated with coronary heart disease using gene sequencing data from the Myocardial Infarction Genetics Consortium (n=27,815). Results -A genetic predisposition to enhanced nitric oxide signaling was associated with reduced risks of coronary heart disease [OR 0.37 95% CI 0.31, 0.45; p=5.5*10(-26)], peripheral arterial disease (OR 0.42 CI 0.26, 0.68; p=0.0005) and stroke (OR 0.53 CI 0.37, 0.76; p=0.0006). In a mediation analysis, the effect of the genetic score on decreased coronary heart disease risk extended beyond its effect on blood pressure. Conversely, rare variants that inactivate the NOS3 or GUCY1A3 genes were associated with a 23 mm Hg higher systolic blood pressure (CI 12, 34 mm Hg; p=5.6*10(-5)) and a three-fold higher risk of coronary heart disease (OR 3.03 CI 1.29, 7.12, p=0.01). Conclusions -A genetic predisposition to enhanced nitric oxide signaling is associated with reduced risks of coronary heart disease, peripheral arterial disease and stroke. Pharmacologic stimulation of nitric oxide signaling may prove useful in the prevention or treatment of cardiovascular disease.

    Circulation 2017

  • Application of rare variant transmission disequilibrium tests to epileptic encephalopathy trio sequence data.

    Epi4K Consortium, EuroEPINOMICS-RES Consortium and Epilepsy Phenome Genome Project

    The classic epileptic encephalopathies, including infantile spasms (IS) and Lennox-Gastaut syndrome (LGS), are severe seizure disorders that usually arise sporadically. De novo variants in genes mainly encoding ion channel and synaptic proteins have been found to account for over 15% of patients with IS or LGS. The contribution of autosomal recessive genetic variation, however, is less well understood. We implemented a rare variant transmission disequilibrium test (TDT) to search for autosomal recessive epileptic encephalopathy genes in a cohort of 320 outbred patient-parent trios that were generally prescreened for rare metabolic disorders. In the current sample, our rare variant transmission disequilibrium test did not identify individual genes with significantly distorted transmission over expectation after correcting for the multiple tests. While the rare variant transmission disequilibrium test did not find evidence of a role for individual autosomal recessive genes, our current sample is insufficiently powered to assess the overall role of autosomal recessive genotypes in an outbred epileptic encephalopathy population.European Journal of Human Genetics advance online publication, 17 May 2017; doi:10.1038/ejhg.2017.61.

    European journal of human genetics : EJHG 2017

  • Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt).

    Erickson T, Morgan CP, Olt J, Hardy K, Busch-Nentwich EM, Maeda R, Clemens-Grisham R, Krey JF, Nechiporuk AV, Barr-Gillespie PG, Marcotti W and Nicolson T

    Oregon Hearing Research Center and the Vollum Institute, Oregon Health and Science University, Portland, United States.

    Transmembrane O-methyltransferase (TOMT / LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.

    Funded by: NICHD NIH HHS: R01 HD072844

    eLife 2017;6

  • A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells.

    Ersing I, Nobre L, Wang LW, Soday L, Ma Y, Paulo JA, Narita Y, Ashbaugh CW, Jiang C, Grayson NE, Kieff E, Gygi SP, Weekes MP and Gewurz BE

    Division of Infectious Disease, Department of Medicine, Brigham & Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA; Institut für Klinische und Molekulare Virologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

    Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

    Cell reports 2017;19;7;1479-1493

  • Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.

    Esson D, Gupta S, Bailey D, Wigley P, Wedley A, Mather AE, Méric G, Mastroeni P, Sheppard SK, Thomson NR, Parkhill J, Maskell DJ, Christie G and Grant AJ

    Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK.

    Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped 'wild type' C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape.

    Microbial pathogenesis 2017;104;202-211

  • Structural analysis of pathogenic mutations in the DYRK1A gene in patients with developmental disorders.

    Evers JM, Laskowski RA, Bertolli M, Clayton-Smith J, Deshpande C, Eason J, Elmslie F, Flinter F, Gardiner C, Hurst JA, Kingston H, Kini U, Lampe AK, Lim D, Male A, Naik S, Parker MJ, Price S, Robert L, Sarkar A, Straub V, Woods G, Thornton JM, DDD Study and Wright CF

    European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, UK.

    Haploinsufficiency in DYRK1A is associated with a recognizable developmental syndrome, though the mechanism of action of pathogenic missense mutations is currently unclear. Here we present 19 de novo mutations in this gene, including five missense mutations, identified by the Deciphering Developmental Disorder study. Protein structural analysis reveals that the missense mutations are either close to the ATP or peptide binding-sites within the kinase domain, or are important for protein stability, suggesting they lead to a loss of the protein's function mechanism. Furthermore, there is some correlation between the magnitude of the change and the severity of the resultant phenotype. A comparison of the distribution of the pathogenic mutations along the length of DYRK1A with that of natural variants, as found in the ExAC database, confirms that mutations in the N-terminal end of the kinase domain are more disruptive of protein function. In particular, pathogenic mutations occur in significantly closer proximity to the ATP and the substrate peptide than the natural variants. Overall, we suggest that de novo dominant mutations in DYRK1A account for nearly 0.5% of severe developmental disorders due to substantially reduced kinase function.

    Funded by: Wellcome Trust

    Human molecular genetics 2017;26;3;519-526

  • Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

    Evrony GD, Cordero DR, Shen J, Partlow JN, Yu TW, Rodin RE, Hill RS, Coulter ME, Lam AN, Jayaraman D, Gerrelli D, Diaz DG, Santos C, Morrison V, Galli A, Tschulena U, Wiemann S, Martel MJ, Spooner B, Ryu SC, Elhosary PC, Richardson JM, Tierney D, Robinson CA, Chibbar R, Diudea D, Folkerth R, Wiebe S, Barkovich AJ, Mochida GH, Irvine J, Lemire EG, Blakley P and Walsh CA

    Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts 02115, USA.

    While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

    Funded by: NICHD NIH HHS: K12 HD001255; NIDCD NIH HHS: R03 DC013866; NIGMS NIH HHS: T32 GM007753; NIMH NIH HHS: U24 MH081810; NINDS NIH HHS: R01 NS035129

    Genome research 2017;27;8;1323-1335

  • Multiple short windows of Calcium-Dependent Protein Kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis.

    Fang H, Klages N, Baechler B, Hillner E, Yu L, Pardo M, Choudhary J and Brochet M

    Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

    Malaria transmission relies on the production of gametes following ingestion by a mosquito. Here, we show that (Ca2+)-dependent protein kinase 4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by (Ca2+) to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. Altogether, this study reveals how a kinase integrates and transduces multiple signals to control key cell-cycle transitions during Plasmodium gametogenesis.

    eLife 2017;6

  • Neutrophil-mediated IL-6 receptor trans-signaling and the risk of chronic obstructive pulmonary disease and asthma.

    Farahi N, Paige E, Balla J, Prudence E, Ferreira RC, Southwood M, Appleby SL, Bakke P, Gulsvik A, Litonjua AA, Sparrow D, Silverman EK, Cho MH, Danesh J, Paul DS, Freitag DF and Chilvers ER

    Division of Respiratory Medicine, Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge CB2 0QQ, UK.

    The Asp358Ala variant in the interleukin-6 receptor (IL-6R) gene has been implicated in asthma, autoimmune and cardiovascular disorders, but its role in other respiratory conditions such as chronic obstructive pulmonary disease (COPD) has not been investigated. The aims of this study were to evaluate whether there is an association between Asp358Ala and COPD or asthma risk, and to explore the role of the Asp358Ala variant in sIL-6R shedding from neutrophils and its pro-inflammatory effects in the lung. We undertook logistic regression using data from the UK Biobank and the ECLIPSE COPD cohort. Results were meta-analyzed with summary data from a further three COPD cohorts (7,519 total cases and 35,653 total controls), showing no association between Asp358Ala and COPD (OR = 1.02 [95% CI: 0.96, 1.07]). Data from the UK Biobank showed a positive association between the Asp358Ala variant and atopic asthma (OR = 1.07 [1.01, 1.13]). In a series of in vitro studies using blood samples from 37 participants, we found that shedding of sIL-6R from neutrophils was greater in carriers of the Asp358Ala minor allele than in non-carriers. Human pulmonary artery endothelial cells cultured with serum from homozygous carriers showed an increase in MCP-1 release in carriers of the minor allele, with the difference eliminated upon addition of tocilizumab. In conclusion, there is evidence that neutrophils may be an important source of sIL-6R in the lungs, and the Asp358Ala variant may have pro-inflammatory effects in lung cells. However, we were unable to identify evidence for an association between Asp358Ala and COPD.

    Funded by: NHLBI NIH HHS: R01 HL089856, R01 HL089897; NIEHS NIH HHS: R25 ES011080

    Human molecular genetics 2017;26;8;1584-1596

  • How to use… lymph node biopsy in paediatrics.

    Farndon S, Behjati S, Jonas N and Messahel B

    Cancer Genome Project, Wellcome Trust Sanger Institute, Cambridge, UK.

    Lymphadenopathy is a common finding in children. It often causes anxiety among parents and healthcare professionals because it can be a sign of cancer. There is limited high-quality evidence to guide clinicians as to which children should be referred for lymph node biopsy. The gold standard method for evaluating lymphadenopathy of unknown cause is an excision biopsy. In this Interpretation, we discuss the use of lymph node biopsy in children.

    Archives of disease in childhood. Education and practice edition 2017

  • Association of Genetic Variants Related to CETP Inhibitors and Statins With Lipoprotein Levels and Cardiovascular Risk.

    Ference BA, Kastelein JJP, Ginsberg HN, Chapman MJ, Nicholls SJ, Ray KK, Packard CJ, Laufs U, Brook RD, Oliver-Williams C, Butterworth AS, Danesh J, Smith GD, Catapano AL and Sabatine MS

    Division of Cardiovascular Medicine, Wayne State University School of Medicine, Detroit, Michigan.

    Importance: Some cholesteryl ester transfer protein (CETP) inhibitors lower low-density lipoprotein cholesterol (LDL-C) levels without reducing cardiovascular events, suggesting that the clinical benefit of lowering LDL-C may depend on how LDL-C is lowered.

    Objective: To estimate the association between changes in levels of LDL-C (and other lipoproteins) and the risk of cardiovascular events related to variants in the CETP gene, both alone and in combination with variants in the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) gene.

    Design, setting, and participants: Mendelian randomization analyses evaluating the association between CETP and HMGCR scores, changes in lipid and lipoprotein levels, and the risk of cardiovascular events involving 102 837 participants from 14 cohort or case-control studies conducted in North America or the United Kingdom between 1948 and 2012. The associations with cardiovascular events were externally validated in 189 539 participants from 48 studies conducted between 2011 and 2015.

    Exposures: Differences in mean high-density lipoprotein cholesterol (HDL-C), LDL-C, and apolipoprotein B (apoB) levels in participants with CETP scores at or above vs below the median.

    Main outcomes and measures: Odds ratio (OR) for major cardiovascular events.

    Results: The primary analysis included 102 837 participants (mean age, 59.9 years; 58% women) who experienced 13 821 major cardiovascular events. The validation analyses included 189 539 participants (mean age, 58.5 years; 39% women) with 62 240 cases of coronary heart disease (CHD). Considered alone, the CETP score was associated with higher levels of HDL-C, lower LDL-C, concordantly lower apoB, and a corresponding lower risk of major vascular events (OR, 0.946 [95% CI, 0.921-0.972]) that was similar in magnitude to the association between the HMGCR score and risk of major cardiovascular events per unit change in levels of LDL-C (and apoB). When combined with the HMGCR score, the CETP score was associated with the same reduction in LDL-C levels but an attenuated reduction in apoB levels and a corresponding attenuated nonsignificant risk of major cardiovascular events (OR, 0.985 [95% CI, 0.955-1.015]). In external validation analyses, a genetic score consisting of variants with naturally occurring discordance between levels of LDL-C and apoB was associated with a similar risk of CHD per unit change in apoB level (OR, 0.782 [95% CI, 0.720-0.845] vs 0.793 [95% CI, 0.774-0.812]; P = .79 for difference), but a significantly attenuated risk of CHD per unit change in LDL-C level (OR, 0.916 [95% CI, 0.890-0.943] vs 0.831 [95% CI, 0.816-0.847]; P < .001) compared with a genetic score associated with concordant changes in levels of LDL-C and apoB.

    Conclusions and relevance: Combined exposure to variants in the genes that encode the targets of CETP inhibitors and statins was associated with discordant reductions in LDL-C and apoB levels and a corresponding risk of cardiovascular events that was proportional to the attenuated reduction in apoB but significantly less than expected per unit change in LDL-C. The clinical benefit of lowering LDL-C levels may therefore depend on the corresponding reduction in apoB-containing lipoprotein particles.

    JAMA 2017

  • An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

    Fisher CL, Marks H, Cho LT, Andrews R, Wormald S, Carroll T, Iyer V, Tate P, Rosen B, Stunnenberg HG, Fisher AG and Skarnes WC

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

    Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.

    Nucleic acids research 2017

  • Genome editing reveals a role for OCT4 in human embryogenesis.

    Fogarty NME, McCarthy A, Snijders KE, Powell BE, Kubikova N, Blakeley P, Lea R, Elder K, Wamaitha SE, Kim D, Maciulyte V, Kleinjung J, Kim JS, Wells D, Vallier L, Bertero A, Turner JMA and Niakan KK

    Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

    Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

    Nature 2017

  • Deletion of the MAD2L1 spindle assembly checkpoint gene is tolerated in mouse models of acute T-cell lymphoma and hepatocellular carcinoma.

    Foijer F, Albacker LA, Bakker B, Spierings DC, Yue Y, Xie SZ, Davis SH, Lutum-Jehle A, Takemoto D, Hare B, Furey B, Bronson RT, Lansdorp PM, Bradley A and Sorger PK

    European Research Institute for the Biology of Ageing, University Medical Center Groningen, Groningen, Netherlands.

    Chromosome instability (CIN) is deleterious to normal cells because of the burden of aneuploidy. However, most human solid tumors have an abnormal karyotype implying that gain and loss of chromosomes by cancer cells confers a selective advantage. CIN can be induced in the mouse by inactivating the spindle assembly checkpoint. This is lethal in the germline but we show here that adult T cells and hepatocytes can survive conditional inactivation of the Mad2l1 SAC gene and resulting CIN. This causes rapid onset of acute lymphoblastic leukemia (T-ALL) and progressive development of hepatocellular carcinoma (HCC), both lethal diseases. The resulting DNA copy number variation and patterns of chromosome loss and gain are tumor-type specific, suggesting differential selective pressures on the two tumor cell types.

    eLife 2017;6

  • Conservation and diversification of small RNA pathways within flatworms.

    Fontenla S, Rinaldi G, Smircich P and Tort JF

    Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), Gral. Flores 2125, CP11800, Montevideo, MVD, Uruguay.

    Background: Small non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum.

    Results: Overall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages.

    Conclusion: These findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.

    BMC evolutionary biology 2017;17;1;215

  • COSMIC: somatic cancer genetics at high-resolution.

    Forbes SA, Beare D, Boutselakis H, Bamford S, Bindal N, Tate J, Cole CG, Ward S, Dawson E, Ponting L, Stefancsik R, Harsha B, Kok CY, Jia M, Jubb H, Sondka Z, Thompson S, De T and Campbell PJ

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

    COSMIC, the Catalogue of Somatic Mutations in Cancer ( is a high-resolution resource for exploring targets and trends in the genetics of human cancer. Currently the broadest database of mutations in cancer, the information in COSMIC is curated by expert scientists, primarily by scrutinizing large numbers of scientific publications. Over 4 million coding mutations are described in v78 (September 2016), combining genome-wide sequencing results from 28 366 tumours with complete manual curation of 23 489 individual publications focused on 186 key genes and 286 key fusion pairs across all cancers. Molecular profiling of large tumour numbers has also allowed the annotation of more than 13 million non-coding mutations, 18 029 gene fusions, 187 429 genome rearrangements, 1 271 436 abnormal copy number segments, 9 175 462 abnormal expression variants and 7 879 142 differentially methylated CpG dinucleotides. COSMIC now details the genetics of drug resistance, novel somatic gene mutations which allow a tumour to evade therapeutic cancer drugs. Focusing initially on highly characterized drugs and genes, COSMIC v78 contains wide resistance mutation profiles across 20 drugs, detailing the recurrence of 301 unique resistance alleles across 1934 drug-resistant tumours. All information from the COSMIC database is available freely on the COSMIC website.

    Nucleic acids research 2017;45;D1;D777-D783

  • Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells.

    Forment JV, Herzog M, Coates J, Konopka T, Gapp BV, Nijman SM, Adams DJ, Keane TM and Jackson SP

    The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Biochemistry, University of Cambridge, Cambridge, UK.

    In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.

    Nature chemical biology 2017;13;1;12-14

  • Illuminating microbial diversity.

    Forster SC

    Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK and the Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia.

    Nature reviews. Microbiology 2017;15;10;578

  • Identification of highly-protective combinations of Plasmodium vivax recombinant proteins for vaccine development.

    França CT, White MT, He WQ, Hostetler JB, Brewster J, Frato G, Malhotra I, Gruszczyk J, Huon C, Lin E, Kiniboro B, Yadava A, Siba P, Galinski MR, Healer J, Chitnis C, Cowman AF, Takashima E, Tsuboi T, Tham WH, Fairhurst RM, Rayner JC, King CL and Mueller I

    Division of Population Health and Immunity, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

    The study of antigenic targets of naturally-acquired immunity is essential to identify and prioritize antigens for further functional characterization. We measured total IgG antibodies to 38 P. vivax antigens, investigating their relationship with prospective risk of malaria in a cohort of 1-3 years old Papua New Guinean children. Using simulated annealing algorithms, the potential protective efficacy of antibodies to multiple antigen-combinations, and the antibody thresholds associated with protection were investigated for the first time. High antibody levels to multiple known and newly identified proteins were strongly associated with protection (IRR 0.44-0.74, P<0.001-0.041). Among five-antigen combinations with the strongest protective effect (>90%), EBP, DBPII, RBP1a, CyRPA, and PVX_081550 were most frequently identified; several of them requiring very low antibody levels to show a protective association. These data identify individual antigens that should be prioritized for further functional testing and establish a clear path to testing a multicomponent P. vivax vaccine.

    eLife 2017;6

  • Document retrieval on repetitive string collections.

    Gagie T, Hartikainen A, Karhu K, Kärkkäinen J, Navarro G, Puglisi SJ and Sirén J

    CeBiB - Center of Biotechnology and Bioengineering, School of Computer Science and Telecommunications, Diego Portales University, Santiago, Chile.

    Most of the fastest-growing string collections today are repetitive, that is, most of the constituent documents are similar to many others. As these collections keep growing, a key approach to handling them is to exploit their repetitiveness, which can reduce their space usage by orders of magnitude. We study the problem of indexing repetitive string collections in order to perform efficient document retrieval operations on them. Document retrieval problems are routinely solved by search engines on large natural language collections, but the techniques are less developed on generic string collections. The case of repetitive string collections is even less understood, and there are very few existing solutions. We develop two novel ideas, interleaved LCPs and precomputed document lists, that yield highly compressed indexes solving the problem of document listing (find all the documents where a string appears), top-k document retrieval (find the k documents where a string appears most often), and document counting (count the number of documents where a string appears). We also show that a classical data structure supporting the latter query becomes highly compressible on repetitive data. Finally, we show how the tools we developed can be combined to solve ranked conjunctive and disjunctive multi-term queries under the simple [Formula: see text] model of relevance. We thoroughly evaluate the resulting techniques in various real-life repetitiveness scenarios, and recommend the best choices for each case.

    Funded by: Wellcome Trust

    Information retrieval 2017;20;3;253-291

  • P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5.

    Galaway F, Drought LG, Fala M, Cross N, Kemp AC, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.

    Invasion of erythrocytes by Plasmodium falciparum merozoites is necessary for malaria pathogenesis and is therefore a primary target for vaccine development. RH5 is a leading subunit vaccine candidate because anti-RH5 antibodies inhibit parasite growth and the interaction with its erythrocyte receptor basigin is essential for invasion. RH5 is secreted, complexes with other parasite proteins including CyRPA and RIPR, and contains a conserved N-terminal region (RH5Nt) of unknown function that is cleaved from the native protein. Here, we identify P113 as a merozoite surface protein that directly interacts with RH5Nt. Using recombinant proteins and a sensitive protein interaction assay, we establish the binding interdependencies of all the other known RH5 complex components and conclude that the RH5Nt-P113 interaction provides a releasable mechanism for anchoring RH5 to the merozoite surface. We exploit these findings to design a chemically synthesized peptide corresponding to RH5Nt, which could contribute to a cost-effective malaria vaccine.

    Nature communications 2017;8;14333

  • Epigenetic germline inheritance in mammals: looking to the past to understand the future.

    Gapp K and Bohacek J

    Gurdon Institute, University of Cambridge, Cambridge, UK.

    Life experiences can induce epigenetic changes in mammalian germ cells, which can influence the developmental trajectory of the offspring and impact health and disease across generations. While this concept of epigenetic germline inheritance has long been met with skepticism, evidence in support of this route of information transfer is now overwhelming, and some key mechanisms underlying germline transmission of acquired information are emerging. This review focuses specifically on sperm RNAs as causal vectors of inheritance. We examine how they might become altered in the germline, and how different classes of sperm RNAs might interact with other epimodifications in germ cells or in the zygote. We integrate the latest findings with earlier pioneering work in this field, point out major questions and challenges, and suggest how new experiments could address them.

    Genes, brain, and behavior 2017

  • Minimal genetic change in Vibrio cholerae in Mozambique over time: Multilocus variable number tandem repeat analysis and whole genome sequencing.

    Garrine M, Mandomando I, Vubil D, Nhampossa T, Acacio S, Li S, Paulson JN, Almeida M, Domman D, Thomson NR, Alonso P and Stine OC

    Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.

    Although cholera is a major public health concern in Mozambique, its transmission patterns remain unknown. We surveyed the genetic relatedness of 75 Vibrio cholerae isolates from patients at Manhiça District Hospital between 2002-2012 and 3 isolates from river using multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). MLVA revealed 22 genotypes in two clonal complexes and four unrelated genotypes. WGS revealed i) the presence of recombination, ii) 67 isolates descended monophyletically from a single source connected to Wave 3 of the Seventh Pandemic, and iii) four clinical isolates lacking the cholera toxin gene. This Wave 3 strain persisted for at least eight years in either an environmental reservoir or circulating within the human population. Our data raises important questions related to where these isolates persist and how identical isolates can be collected years apart despite our understanding of high change rate of MLVA loci and the V. cholerae molecular clock.

    PLoS neglected tropical diseases 2017;11;6;e0005671

  • No genetic association between attention-deficit/hyperactivity disorder (ADHD) and Parkinson's disease in nine ADHD candidate SNPs.

    Geissler JM, International Parkinson Disease Genomics Consortium members, Romanos M, Gerlach M, Berg D and Schulte C

    Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Center of Mental Health, University Hospital of Würzburg, Margarete-Höppel-Platz 1, 97080, Würzburg, Germany.

    Attention-deficit/hyperactivity disorder (ADHD) and Parkinson's disease (PD) involve pathological changes in brain structures such as the basal ganglia, which are essential for the control of motor and cognitive behavior and impulsivity. The cause of ADHD and PD remains unknown, but there is increasing evidence that both seem to result from a complicated interplay of genetic and environmental factors affecting numerous cellular processes and brain regions. To explore the possibility of common genetic pathways within the respective pathophysiologies, nine ADHD candidate single nucleotide polymorphisms (SNPs) in seven genes were tested for association with PD in 5333 cases and 12,019 healthy controls: one variant, respectively, in the genes coding for synaptosomal-associated protein 25 k (SNAP25), the dopamine (DA) transporter (SLC6A3; DAT1), DA receptor D4 (DRD4), serotonin receptor 1B (HTR1B), tryptophan hydroxylase 2 (TPH2), the norepinephrine transporter SLC6A2 and three SNPs in cadherin 13 (CDH13). Information was extracted from a recent meta-analysis of five genome-wide association studies, in which 7,689,524 SNPs in European samples were successfully imputed. No significant association was observed after correction for multiple testing. Therefore, it is reasonable to conclude that candidate variants implicated in the pathogenesis of ADHD do not play a substantial role in PD.

    Attention deficit and hyperactivity disorders 2017

  • Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Escherichia coli Strains Colonizing Cattle.

    Geue L, Menge C, Eichhorn I, Semmler T, Wieler LH, Pickard D, Berens C and Barth SA

    Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis Jena, Germany.

    Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5' and 3' flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain's pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.

    Frontiers in microbiology 2017;8;424

  • A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5).

    Geyer SH, Reissig L, Rose J, Wilson R, Prin F, Szumska D, Ramirez-Solis R, Tudor C, White J, Mohun TJ and Weninger WJ

    Centre for Anatomy and Cell Biology & MIC, Medical University of Vienna, Vienna, Austria.

    We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub-stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal null mutant lines from the DMDD research programme ( and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage-specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.

    Journal of anatomy 2017

  • Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21-S23.

    Geyer SH, Reissig LF, Hüsemann M, Höfle C, Wilson R, Prin F, Szumska D, Galli A, Adams DJ, White J, Mohun TJ and Weninger WJ

    Division of Anatomy & MIC, Medical University of Vienna, Vienna, Austria.

    Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage-matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high-resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21-S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.

    Journal of anatomy 2017

  • De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms.

    Giordano F, Aigrain L, Quail MA, Coupland P, Bonfield JK, Davies RM, Tischler G, Jackson DK, Keane TM, Li J, Yue JX, Liti G, Durbin R and Ning Z

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

    Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.

    Scientific reports 2017;7;1;3935

  • Immunogenomic approaches to understand the function of immune disease variants.

    Glinos DA, Soskic B and Trynka G

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

    Mapping hundreds of genetic variants through genome wide association studies provided an opportunity to gain insights into the pathobiology of immune mediated diseases. However, since most of the disease variants fall outside the gene coding sequences, thereby implicating gene expression regulation as the causal mechanism, the functional interpretation of the exact role of the associated variants remains to be determined. Despite these challenges, integration of disease associated variants with large scale genomic maps of cell type specific gene regulation both at chromatin and transcript levels deliver examples of functionally prioritised causal variants and genes. Particularly, the enrichment of disease variants with histone marks can point towards cell types most relevant to disease development. Furthermore, chromatin contact maps that link enhancers to promoter regions in a direct way allow identification of genes that can be regulated by the disease variants. Candidate genes implicated with such approaches can be further examined through the gene expression correlation with genotypes. Additionally, in the context of immune mediated diseases it is important to combine genomics with immunology approaches. Genotype correlations with the immune system as a whole, as well as with cellular responses to different stimuli, provides a valuable platform for the understanding of functional impact of disease associated variants. Intersection of immunogenomic resources with disease associated variants paints a detailed picture of disease causal mechanisms. Here, we provide an overview of recent studies that combine these approaches to identify disease vulnerable pathways. This article is protected by copyright. All rights reserved.

    Immunology 2017

  • A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers.

    Glodzik D, Morganella S, Davies H, Simpson PT, Li Y, Zou X, Diez-Perez J, Staaf J, Alexandrov LB, Smid M, Brinkman AB, Rye IH, Russnes H, Raine K, Purdie CA, Lakhani SR, Thompson AM, Birney E, Stunnenberg HG, van de Vijver MJ, Martens JW, Børresen-Dale AL, Richardson AL, Kong G, Viari A, Easton D, Evan G, Campbell PJ, Stratton MR and Nik-Zainal S

    Wellcome Trust Sanger Institute, Cambridge, UK.

    Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. The transcriptomic consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.

    Nature genetics 2017;49;3;341-348

  • Genetic diversity of next generation antimalarial targets: A baseline for drug resistance surveillance programmes.

    Gomes AR, Ravenhall M, Benavente ED, Talman A, Sutherland C, Roper C, Clark TG and Campino S

    Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.

    Drug resistance is a recurrent problem in the fight against malaria. Genetic and epidemiological surveillance of antimalarial resistant parasite alleles is crucial to guide drug therapies and clinical management. New antimalarial compounds are currently at various stages of clinical trials and regulatory evaluation. Using ∼2000 Plasmodium falciparum genome sequences, we investigated the genetic diversity of eleven gene-targets of promising antimalarial compounds and assessed their potential efficiency across malaria endemic regions. We determined if the loci are under selection prior to the introduction of new drugs and established a baseline of genetic variance, including potential resistant alleles, for future surveillance programmes.

    International journal for parasitology. Drugs and drug resistance 2017;7;2;174-180

  • Gastrointestinal carriage is a major reservoir of K. pneumoniae infection in intensive care patients.

    Gorrie CL, Mirceta M, Wick RR, Edwards DJ, Thomson NR, Strugnell RA, Pratt N, Garlick J, Watson K, Pilcher D, McGloughlin S, Spelman DW, Jenney AW and Holt KE

    Centre for Systems Genomics, The University of Melbourne, Victoria, Australia.

    Background: Klebsiella pneumoniae (Kp) is an opportunistic pathogen and leading cause of hospital-associated infections. Intensive care unit (ICU) patients are particularly at risk. Kp is part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of gut colonization and its contribution to infections are not well characterized.

    Methods: We conducted a one-year prospective cohort study in which 498 ICU patients were screened for rectal and throat carriage of Kp shortly after admission. Kp isolated from screening swabs and clinical diagnostic samples were characterized using whole genome sequencing and combined with epidemiological data to identify likely transmission events.

    Results: Kp carriage frequencies were estimated at 6% (95% CI, 3%-8%) amongst ICU patients admitted direct from the community, and 19% (95% CI, 14% - 51%) amongst those with recent healthcare contact. Gut colonisation on admission was significantly associated with subsequent infection (infection risk 16% vs 3%, OR=6.9, p<0.001), and genome data indicated matching carriage and infection isolates in 80% of isolate pairs. Five likely transmission chains were identified, responsible for 12% of Kp infections in ICU. 49% of Kp infections were caused by the patients' own unique strain, and 48% of screened patients with infections were positive for prior colonisation.

    Conclusions: These data confirm Kp colonisation is a significant risk factor for infection in ICU, and indicate ~50% of Kp infections result from patients' own microbiota. Screening for colonisation on admission could limit risk of infection in the colonised patient and others.

    Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2017

  • De novo SETD5 loss-of-function variant as a cause for intellectual disability in a 10-year old boy with an aberrant blind ending bronchus.

    Green C, Willoughby J, DDD Study and Balasubramanian M

    Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, UK.

    Although rare, 3p microdeletion cases have been well described in the clinical literature. The clinical phenotype includes; intellectual disability (ID), growth retardation, facial dysmorphism, and cardiac malformations. Advances in chromosome microarray (CMA) testing narrowed the 3p25 critical region to a 124 kb region, and recent Whole Exome Sequencing (WES) studies have suggested that the SETD5 gene contributes significantly to the 3p25 phenotype. Loss-of-Function (LoF) variants in SETD5 are now considered a likely cause of ID. We report here a patient with a frameshift LoF variant in exon 12 of SETD5. This patient has features overlapping with other patients described with LoF SETD5 variants to include; similar facial morphology, feeding difficulties, ID, behavioral abnormalities and leg length discrepancy. In addition, he presents with an aberrant blind ending bronchus. This report adds to publications describing intragenic mutations in SETD5 and supports the assertion that de novo LoF mutations in SETD5 present with an overlapping but distinct phenotype in comparison with 3p25 microdeletion syndromes.

    American journal of medical genetics. Part A 2017

  • Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations.

    Guo BB, Nigel Allcock RJ, Mirzai B, Jacobus Malherbe JA, Choudry FA, Frontini M, Chuah H, Liang J, Kavanagh SE, Howman R, Ouwehand WH, Fuller KA and Erber WN

    School of Biomedical Sciences, University of Western Australia, Crawley, Western Australia, Australia.

    Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2(V617F), CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Eleven of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

    The American journal of pathology 2017

  • First Draft Genome Sequence of the Dourine Causative Agent: Trypanosoma Equiperdum Strain OVI.

    Hébert L, Moumen B, Madeline A, Steinbiss S, Lakhdar L, Van Reet N, Büscher P, Laugier C, Cauchard J and Petry S

    ANSES, Dozulé Laboratory for Equine Diseases, Bacteriology and Parasitology Unit, 14430 Goustranville, France.

    Trypanosoma equiperdum is the causative agent of dourine, a sexually-transmitted infection of horses. This parasite belongs to the subgenus Trypanozoon that also includes the agent of sleeping sickness (Trypanosoma brucei) and surra (Trypanosoma evansi). We herein report the genome sequence of a T. equiperdum strain OVI, isolated from a horse in South-Africa in 1976. This is the first genome sequence of the T. equiperdum species, and its availability will provide important insights for future studies on genetic classification of the subgenus Trypanozoon.

    Journal of genomics 2017;5;1-3

  • Dietary restriction protects from age-associated DNA methylation and induces epigenetic reprogramming of lipid metabolism.

    Hahn O, Grönke S, Stubbs TM, Ficz G, Hendrich O, Krueger F, Andrews S, Zhang Q, Wakelam MJ, Beyer A, Reik W and Partridge L

    Max Planck Institute for Biology of Ageing, 50931, Cologne, Germany.

    Background: Dietary restriction (DR), a reduction in food intake without malnutrition, increases most aspects of health during aging and extends lifespan in diverse species, including rodents. However, the mechanisms by which DR interacts with the aging process to improve health in old age are poorly understood. DNA methylation could play an important role in mediating the effects of DR because it is sensitive to the effects of nutrition and can affect gene expression memory over time.

    Results: Here, we profile genome-wide changes in DNA methylation, gene expression and lipidomics in response to DR and aging in female mouse liver. DR is generally strongly protective against age-related changes in DNA methylation. During aging with DR, DNA methylation becomes targeted to gene bodies and is associated with reduced gene expression, particularly of genes involved in lipid metabolism. The lipid profile of the livers of DR mice is correspondingly shifted towards lowered triglyceride content and shorter chain length of triglyceride-associated fatty acids, and these effects become more pronounced with age.

    Conclusions: Our results indicate that DR remodels genome-wide patterns of DNA methylation so that age-related changes are profoundly delayed, while changes at loci involved in lipid metabolism affect gene expression and the resulting lipid profile.

    Genome biology 2017;18;1;56

  • The Y chromosomes of the great apes.

    Hallast P and Jobling MA

    Institute of Molecular and Cell Biology, University of Tartu, Tartu, 51010, Estonia.

    The great apes (orangutans, gorillas, chimpanzees, bonobos and humans) descended from a common ancestor around 13 million years ago, and since then their sex chromosomes have followed very different evolutionary paths. While great-ape X chromosomes are highly conserved, their Y chromosomes, reflecting the general lability and degeneration of this male-specific part of the genome since its early mammalian origin, have evolved rapidly both between and within species. Understanding great-ape Y chromosome structure, gene content and diversity would provide a valuable evolutionary context for the human Y, and would also illuminate sex-biased behaviours, and the effects of the evolutionary pressures exerted by different mating strategies on this male-specific part of the genome. High-quality Y-chromosome sequences are available for human and chimpanzee (and low-quality for gorilla). The chromosomes differ in size, sequence organisation and content, and while retaining a relatively stable set of ancestral single-copy genes, show considerable variation in content and copy number of ampliconic multi-copy genes. Studies of Y-chromosome diversity in other great apes are relatively undeveloped compared to those in humans, but have nevertheless provided insights into speciation, dispersal, and mating patterns. Future studies, including data from larger sample sizes of wild-born and geographically well-defined individuals, and full Y-chromosome sequences from bonobos, gorillas and orangutans, promise to further our understanding of population histories, male-biased behaviours, mutation processes, and the functions of Y-chromosomal genes.

    Human genetics 2017

  • Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum.

    Hansen HB, Damgaard PB, Margaryan A, Stenderup J, Lynnerup N, Willerslev E and Allentoft ME

    Centre for GeoGenetics, Natural History Museum, University of Copenhagen, Copenhagen, Denmark.

    Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements.

    PloS one 2017;12;1;e0170940

  • A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.

    Haque A, Engel J, Teichmann SA and Lönnberg T

    QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland, 4006, Australia.

    RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample and is useful for studying cellular responses. RNA-seq has fueled much discovery and innovation in medicine over recent years. For practical reasons, the technique is usually conducted on samples comprising thousands to millions of cells. However, this has hindered direct assessment of the fundamental unit of biology-the cell. Since the first single-cell RNA-sequencing (scRNA-seq) study was published in 2009, many more have been conducted, mostly by specialist laboratories with unique skills in wet-lab single-cell genomics, bioinformatics, and computation. However, with the increasing commercial availability of scRNA-seq platforms, and the rapid ongoing maturation of bioinformatics approaches, a point has been reached where any biomedical researcher or clinician can use scRNA-seq to make exciting discoveries. In this review, we present a practical guide to help researchers design their first scRNA-seq studies, including introductory information on experimental hardware, protocol choice, quality control, data analysis and biological interpretation.

    Genome medicine 2017;9;1;75

  • The micro-evolution and epidemiology of Staphylococcus aureus colonization during atopic eczema disease flare.

    Harkins CP, Pettigrew KA, Oravcová K, Gardner J, Hearn RMR, Rice D, Mather AE, Parkhill J, Brown SJ, Proby CM and Holden MTG

    School of Medicine, University of St Andrews, St Andrews, KY11 9TF, UK; Department of Dermatology, Ninewells Hospital, Dundee, DD1 9SY, UK; School of Medicine, University of Dundee, Dundee, DD1 9SY, UK. Electronic address:

    Staphylococcus aureus is an opportunistic pathogen and variable component of the human microbiota. In atopic eczema (AE) a characteristic of the disease is colonization by S. aureus, with exacerbations associated with an increased bacterial burden of the organism. Despite this, the origins and genetic diversity of S. aureus colonizing individual patients during AE disease flares is poorly understood. To examine the micro-evolution of S. aureus colonization we have deep-sequenced S. aureus populations from nine children with moderate to severe AE, and 18 non-atopic children asymptomatically carrying S. aureus nasally. Colonization by clonal S. aureus populations was observed in both AE cases and controls, with all but one of the individuals containing colonies belonging to a single sequence type. Phylogenetic analysis revealed that disease flares were associated with the clonal expansion of the S. aureus population, occurring over a period of weeks to months. There was a significant difference in the genetic backgrounds of S. aureus colonizing AE patients versus controls (Fisher's Exact test, p=0.03). Examination of intra-host genetic heterogeneity of the colonizing S. aureus populations identified evidence of within-host selection in the AE patients, with AE variants being potentially selectively advantageous for intracellular persistence and treatment resistance.

    The Journal of investigative dermatology 2017

  • Methicillin-resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice.

    Harkins CP, Pichon B, Doumith M, Parkhill J, Westh H, Tomasz A, de Lencastre H, Bentley SD, Kearns AM and Holden MTG

    School of Medicine, University of St Andrews, St Andrews, KY16 9TF, UK.

    Background: The spread of drug-resistant bacterial pathogens poses a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA) was first observed in 1960, less than one year after the introduction of this second generation beta-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when the mecA gene encoding methicillin resistance carried on an SCCmec element, was horizontally transferred to an intrinsically sensitive strain of S. aureus.

    Results: Whole genome sequencing a collection of the first MRSA isolates allows us to reconstruct the evolutionary history of the archetypal MRSA. We apply Bayesian phylogenetic reconstruction to infer the time point at which this early MRSA lineage arose and when SCCmec was acquired. MRSA emerged in the mid-1940s, following the acquisition of an ancestral type I SCCmec element, some 14 years before the first therapeutic use of methicillin.

    Conclusions: Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation beta-lactams such as penicillin in the years prior to the introduction of methicillin, which selected for S. aureus strains carrying the mecA determinant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics.

    Funded by: Wellcome Trust

    Genome biology 2017;18;1;130

  • Genomic surveillance reveals low prevalence of livestock-associated methicillin-resistant Staphylococcus aureus in the East of England.

    Harrison EM, Coll F, Toleman MS, Blane B, Brown NM, Török ME, Parkhill J and Peacock SJ

    Department of Medicine, University of Cambridge, Box 157 Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, United Kingdom.

    Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging problem in many parts of the world. LA-MRSA has been isolated previously from animals and humans in the United Kingdom (UK), but the prevalence is unknown. The aim of this study was to determine the prevalence and to describe the molecular epidemiology of LA-MRSA isolated in the East of England (broadly Cambridge and the surrounding area). We accessed whole genome sequence data for 2,283 MRSA isolates from 1,465 people identified during a 12-month prospective study between 2012 and 2013 conducted in the East of England, United Kingdom. This laboratory serves four hospitals and 75 general practices. We screened the collection for multilocus sequence types (STs) and for host specific resistance and virulence factors previously associated with LA-MRSA. We identified 13 putative LA-MRSA isolates from 12 individuals, giving an estimated prevalence of 0.82% (95% CI 0.47% to 1.43%). Twelve isolates were mecC-MRSA (ten CC130, one ST425 and one ST1943) and single isolate was ST398. Our data demonstrate a low burden of LA-MRSA in the East of England, but the detection of mecC-MRSA and ST398 indicates the need for vigilance. Genomic surveillance provides a mechanism to detect and track the emergence and spread of MRSA clones of human importance.

    Scientific reports 2017;7;1;7406

  • Antimicrobial resistance determinants and susceptibility profiles of pneumococcal isolates recovered in Trinidad and Tobago.

    Hawkins PA, Akpaka PE, Nurse-Lucas M, Gladstone R, Bentley SD, Breiman RF, McGee L and Swanston WH

    Emory University, Atlanta, Georgia, USA; Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. Electronic address:

    Introduction: In Latin America and the Caribbean, pneumococcal infections were estimated to account for 12,000-18,000 deaths, 327,000 cases of pneumonia, 4,000 cases of meningitis and 1,229 cases of sepsis each year in children under five years old. Resistance of pneumococci to antimicrobial agents has evolved into a worldwide health problem in the last few decades.

    Objective: The aim of this study was to determine the antimicrobial susceptibility profiles of 98 pneumococcal isolates collected in Trinidad and Tobago and associated genetic determinants.

    Methods: Whole genome sequences were obtained from 98 pneumococcal isolates recovered at several regional hospitals, including 83 invasive and 15 non-invasive strains, recovered before (n=25) and after (n=73) the introduction of two pneumococcal conjugate vaccines. A bioinformatics pipeline was used to identify core genomic and accessory elements that conferred antimicrobial resistance phenotypes, including β-lactam non-susceptibility.

    Results: and discussion: Forty-one (41.8%) isolates were predicted as resistant to at least one antimicrobial class, including 13 (13.3%) isolates resistant to at least three classes. The most common serotypes associated with antimicrobial resistance were 23F (n=10), 19F (n=8), 6B (n=6), and 14 (n=5). The most common serotypes associated with penicillin non-susceptibility were 19F (n=7) and 14 (n=5). Thirty-nine (39.8%) isolates were positive for PI-1 or PI-2 type pili: 30 (76.9%) were PI-1+, 4 (10.3%) were PI-2+, and 5 (12.8%) were positive for both PI-1 and PI-2. Of the 13 isolates with multidrug resistance, 10 belonged to globally distributed clones PMEN3 and PMEN14 and were isolated in the post-PCV period, suggesting a clonal expansion.

    Journal of global antimicrobial resistance 2017

  • Circulating and Tissue-Resident CD4(+) T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation.

    Hegazy AN, West NR, Stubbington MJT, Wendt E, Suijker KIM, Datsi A, This S, Danne C, Campion S, Duncan SH, Owens BMJ, Uhlig HH, McMichael A and Oxford IBD Cohort Investigators

    Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, Experimental Medicine Division, John Radcliffe Hospital, University of Oxford, United Kingdom; Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, United Kingdom.

    Background &amp; aims: Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4(+) T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.

    Methods: We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4(+) T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4(+) T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.

    Results: Circulating and gut-resident CD4(+) T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4(+) T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4(+) T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.

    Conclusions: In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4(+) T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

    Gastroenterology 2017

  • Summing up the parts of the hypothalamus.

    Hemberg M

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Nature neuroscience 2017;20;3;378-379

  • Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity.

    Hendricks AE, Bochukova EG, Marenne G, Keogh JM, Atanassova N, Bounds R, Wheeler E, Mistry V, Henning E, Körner A, Muddyman D, McCarthy S, Hinney A, Hebebrand J, Scott RA, Langenberg C, Wareham NJ, Surendran P, Howson JM, Butterworth AS, Danesh J, Nordestgaard BG, Nielsen SF, Afzal S, Papadia S, Ashford S, Garg S, Millhauser GL, Palomino RI, Kwasniewska A, Tachmazidou I, O'Rahilly S, Zeggini E, Barroso I, Farooqi IS, Understanding Society Scientific Group, EPIC-CVD Consortium and UK10K Consortium

    Wellcome Trust Sanger Institute, Cambridge, UK.

    Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF~0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 × 10(-3)), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.

    Funded by: European Research Council: 268834; NIDDK NIH HHS: R01 DK064265; Wellcome Trust

    Scientific reports 2017;7;1;4394

  • Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form.

    Henke K, Daane JM, Hawkins MB, Dooley CM, Busch-Nentwich EM, Stemple DL and Harris MP

    Department of Orthopedic Research, Boston Children's Hospital, Massachusetts 02115

    Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

    Genetics 2017;207;2;609-623

  • HLA haplotypes in primary sclerosing cholangitis patients of admixed and non-European ancestry.

    Henriksen EKK, Viken MK, Wittig M, Holm K, Folseraas T, Mucha S, Melum E, Hov JR, Lazaridis KN, Juran BD, Chazouillères O, Färkkilä M, Gotthardt DN, Invernizzi P, Carbone M, Hirschfield GM, Rushbrook SM, Goode E, UK-PSC Consortium, Ponsioen CY, Weersma RK, Eksteen B, Yimam KK, Gordon SC, Goldberg D, Yu L, Bowlus CL, Franke A, Lie BA and Karlsen TH

    Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital Rikshospitalet, Oslo, Norway.

    Primary sclerosing cholangitis (PSC) is strongly associated with several human leukocyte antigen (HLA) haplotypes. Due to extensive linkage disequilibrium and multiple polymorphic candidate genes in the HLA complex, identifying the alleles responsible for these associations has proven difficult. We aimed to evaluate whether studying populations of admixed or non-European descent could help in defining the causative HLA alleles. When assessing haplotypes carrying HLA-DRB1*13:01 (hypothesized to specifically increase the susceptibility to chronic cholangitis), we observed that every haplotype in the Scandinavian PSC population carried HLA-DQB1*06:03. In contrast, only 65% of HLA-DRB1*13:01 haplotypes in an admixed/non-European PSC population carried this allele, suggesting that further assessments of the PSC-associated haplotype HLA-DRB1*13:01-DQA1*01:03-DQB1*06:03 in admixed or multi-ethnic populations could aid in identifying the causative allele.

    HLA 2017

  • Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya.

    Henson SP, Boinett CJ, Ellington MJ, Kagia N, Mwarumba S, Nyongesa S, Mturi N, Kariuki S, Scott JAG, Thomson NR and Morpeth SC

    KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya; Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, Oxford University, Oxford, United Kingdom.

    Multidrug resistant (MDR) Klebsiella pneumoniae is a common cause of nosocomial infections worldwide. Recent years have seen an explosion of resistance to extended-spectrum β-lactamases (ESBLs) and emergence of carbapenem resistance. Here, we examine 198 invasive K. pneumoniae isolates collected from over a decade in Kilifi County Hospital (KCH) in Kenya. We observe a significant increase in MDR K. pneumoniae isolates, particularly to third generation cephalosporins conferred by ESBLs. Using whole-genome sequences, we describe the population structure and the distribution of antimicrobial resistance genes within it. More than half of the isolates examined in this study were ESBL-positive, encoding CTX-M-15, SHV-2, SHV-12 and SHV-27, and 79% were MDR conferring resistance to at least three antimicrobial classes. Although no isolates in our dataset were found to be resistant to carbapenems we did find a plasmid with the genetic architecture of a known New Delhi metallo-β-lactamase-1 (NDM)-carrying plasmid in 25 isolates. In the absence of carbapenem use in KCH and because of the instability of the NDM-1 gene in the plasmid, the NDM-1 gene has been lost in these isolates. Our data suggests that isolates that encode NDM-1 could be present in the population; should carbapenems be introduced as treatment in public hospitals in Kenya, resistance is likely to ensue rapidly.

    International journal of medical microbiology : IJMM 2017;307;7;422-429

  • PGBD5 promotes site-specific oncogenic mutations in human tumors.

    Henssen AG, Koche R, Zhuang J, Jiang E, Reed C, Eisenberg A, Still E, MacArthur IC, Rodríguez-Fos E, Gonzalez S, Puiggròs M, Blackford AN, Mason CE, de Stanchina E, Gönen M, Emde AK, Shah M, Arora K, Reeves C, Socci ND, Perlman E, Antonescu CR, Roberts CWM, Steen H, Mullen E, Jackson SP, Torrents D, Weng Z, Armstrong SA and Kentsis A

    Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

    Nature genetics 2017

  • Ecto-5'-nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia.

    Holmes AP, Ray CJ, Pearson SA, Coney AM and Kumar P

    Institute of Cardiovascular Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

    Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The current study tested whether ecto-5'-nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of ecto-5'-nucleotidase by α,β-methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8-(p-sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (ΔV˙E control 74 ± 6%, ΔV˙E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as evidenced by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify ecto-5'-nucleotidase as a novel regulator of CB sensory activity. Future investigations are warranted to evaluate whether inhibition of ecto-5'-nucleotidase can effectively reduce CB activity in CB-mediated cardiovascular pathology. This article is protected by copyright. All rights reserved.

    The Journal of physiology 2017

  • Fine-mapping inflammatory bowel disease loci to single-variant resolution.

    Huang H, Fang M, Jostins L, Umićević Mirkov M, Boucher G, Anderson CA, Andersen V, Cleynen I, Cortes A, Crins F, D'Amato M, Deffontaine V, Dmitrieva J, Docampo E, Elansary M, Farh KK, Franke A, Gori AS, Goyette P, Halfvarson J, Haritunians T, Knight J, Lawrance IC, Lees CW, Louis E, Mariman R, Meuwissen T, Mni M, Momozawa Y, Parkes M, Spain SL, Théâtre E, Trynka G, Satsangi J, van Sommeren S, Vermeire S, Xavier RJ, International Inflammatory Bowel Disease Genetics Consortium, Weersma RK, Duerr RH, Mathew CG, Rioux JD, McGovern DPB, Cho JH, Georges M, Daly MJ and Barrett JC

    Analytic and Translational Genetics Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.

    Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

    Funded by: NCI NIH HHS: R01 CA141743; NIAID NIH HHS: U01 AI067068; NIDCR NIH HHS: U54 DE023789; NIDDK NIH HHS: P30 DK043351, R01 DK064869, R01 DK092235, R01 DK106593, U01 DK062413, U01 DK062420, U01 DK062422, U01 DK062429, U01 DK062432; Wellcome Trust

    Nature 2017;547;7662;173-178

  • Novel viral vectors in infectious diseases.

    Humphreys IR and Sebastian S

    Institute of Infection and Immunity/Systems Immunity, University Research Institute, Cardiff University, Cardiff, UK.

    Since the development of Vaccinia virus as a vaccine vector in 1984, the utility of numerous viruses in vaccination strategies has been explored. In recent years, key improvements to existing vectors such as those based on adenovirus have led to significant improvements in immunogenicity and efficacy. Furthermore, exciting new vectors that exploit viruses such as cytomegalovirus (CMV) and vesicular stomatitis virus (VSV) have emerged. Herein, we summarize these recent developments in viral vector technologies, focusing on novel vectors based on CMV, VSV, measles and modified adenovirus. We discuss the potential utility of these exciting approaches in eliciting protection against infectious diseases. This article is protected by copyright. All rights reserved.

    Immunology 2017

  • Pooling strategy and chromosome painting characterize a living zebroid for the first time.

    Iannuzzi A, Pereira J, Iannuzzi C, Fu B and Ferguson-Smith M

    Laboratory of Animal Cytogenetics and Genomics, National Research Council of Italy, Institute of Animal Production Systems in Mediterranean Environments (ISPAAM), Naples, Italy.

    We have investigated the complex karyotype of a living zebra-donkey hybrid for the first time using chromosome-specific painting probes produced from flow-sorted chromosomes from a zebra (Equus burchelli) and horse (Equus caballus). As the chromosomes proved difficult to distinguish from one another, a successful new strategy was devised to resolve the difficulty and characterize each chromosome. This was based on selecting five panels of whole chromosome painting probes that could differentiate zebra and donkey chromosomes by labelling the probes with either FITC or Cy3 fluorochromes. Each panel was hybridized sequentially to the same G-Q-banded metaphases and the results combined so that every zebra and donkey chromosome in each suitable metaphase could be identified. A diploid number of 2n = 53, XY was found, containing haploid sets of 22 chromosomes from the zebra and 31 chromosomes from the donkey, without evidence of chromosome rearrangement. This new strategy, developed for the first time, may have several applications in the resolution of other complex hybrid karyotypes and chromosomal aberrations.

    PloS one 2017;12;7;e0180158

  • Gene Expression in Leishmania Is Regulated Predominantly by Gene Dosage.

    Iantorno SA, Durrant C, Khan A, Sanders MJ, Beverley SM, Warren WC, Berriman M, Sacks DL, Cotton JA and Grigg ME

    Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal "somy" (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection.IMPORTANCELeishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy.

    mBio 2017;8;5

  • Variation in olfactory neuron repertoires is genetically controlled and environmentally modulated.

    Ibarra-Soria X, Nakahara TS, Lilue J, Jiang Y, Trimmer C, Souza MA, Netto PH, Ikegami K, Murphy NR, Kusma M, Kirton A, Saraiva LR, Keane TM, Matsunami H, Mainland J, Papes F and Logan DW

    Wellcome Trust Sanger Institute, Cambridge, United Kingdom.

    The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify cis-acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.

    eLife 2017;6

  • The role of sex and body weight on the metabolic effects of high-fat diet in C57BL/6N mice.

    Ingvorsen C, Karp NA and Lelliott CJ

    Mouse Pipelines, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.

    Background: Metabolic disorders are commonly investigated using knockout and transgenic mouse models on the C57BL/6N genetic background due to its genetic susceptibility to the deleterious metabolic effects of high-fat diet (HFD). There is growing awareness of the need to consider sex in disease progression, but limited attention has been paid to sexual dimorphism in mouse models and its impact in metabolic phenotypes. We assessed the effect of HFD and the impact of sex on metabolic variables in this strain.

    Methods: We generated a reference data set encompassing glucose tolerance, body composition and plasma chemistry data from 586 C57BL/6N mice fed a standard chow and 733 fed a HFD collected as part of a high-throughput phenotyping pipeline. Linear mixed model regression analysis was used in a dual analysis to assess the effect of HFD as an absolute change in phenotype, but also as a relative change accounting for the potential confounding effect of body weight.

    Results: HFD had a significant impact on all variables tested with an average absolute effect size of 29%. For the majority of variables (78%), the treatment effect was modified by sex and this was dominated by male-specific or a male stronger effect. On average, there was a 13.2% difference in the effect size between the male and female mice for sexually dimorphic variables. HFD led to a significant body weight phenotype (24% increase), which acts as a confounding effect on the other analysed variables. For 79% of the variables, body weight was found to be a significant source of variation, but even after accounting for this confounding effect, similar HFD-induced phenotypic changes were found to when not accounting for weight.

    Conclusion: HFD and sex are powerful modifiers of metabolic parameters in C57BL/6N mice. We also demonstrate the value of considering body size as a covariate to obtain a richer understanding of metabolic phenotypes.

    Nutrition & diabetes 2017;7;4;e261

  • Insecticide-induced leg loss does not eliminate biting and reproduction in Anopheles gambiae mosquitoes.

    Isaacs AT, Lynd A and Donnelly MJ

    Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK.

    Recent successes in malaria control have been largely attributable to the deployment of insecticide-based vector control tools such as bed nets and indoor residual spraying. Pyrethroid-treated bed nets are acutely neurotoxic to mosquitoes, inducing symptoms such as loss of coordination, paralysis, and violent spasms. One result of pyrethroid exposure often seen in laboratory tests is mosquito leg loss, a condition that has thus far been assumed to equate to mortality, as females are not expected to blood feed. However, whilst limb loss is unlikely to be adaptive, females with missing limbs may play a role in the propagation of both their species and pathogens. To test the hypothesis that leg loss inhibits mosquitoes from biting and reproducing, mosquitoes with one, two, or six legs were evaluated for their success in feeding upon a human. These experiments demonstrated that insecticide-induced leg loss had no significant effect upon blood feeding or egg laying success. We conclude that studies of pyrethroid efficacy should not discount mosquitoes that survive insecticide exposure with fewer than six legs, as they may still be capable of biting humans, reproducing, and contributing to malaria transmission.

    Scientific reports 2017;7;46674

  • DNA methylation homeostasis in human and mouse development.

    Iurlaro M, von Meyenn F and Reik W

    Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK.

    The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals. An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species.

    Current opinion in genetics & development 2017;43;101-109

  • Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease.

    Jaiswal S, Natarajan P, Silver AJ, Gibson CJ, Bick AG, Shvartz E, McConkey M, Gupta N, Gabriel S, Ardissino D, Baber U, Mehran R, Fuster V, Danesh J, Frossard P, Saleheen D, Melander O, Sukhova GK, Neuberg D, Libby P, Kathiresan S and Ebert BL

    From the Department of Medicine, Division of Hematology, Brigham and Women's Hospital (S.J., A.J.S., M.M.) and Harvard Medical School (B.L.E.), the Department of Medicine, Division of Cardiovascular Medicine, Brigham and Women's Hospital (E.S.) and Harvard Medical School (G.K.S., P.L.), the Department of Pathology (S.J.) and the Center for Genomic Medicine (P.N., S.K.), Massachusetts General Hospital, the Department of Medicine, Division of Cardiology, and Cardiovascular Research Center (P.N., S.K.), and the Department of Medicine (A.G.B.), Massachusetts General Hospital and Harvard Medical School, and the Departments of Medical Oncology (C.J.G.) and Biostatistics and Computational Biology (D.N.), Dana-Farber Cancer Institute, Boston, and the Program in Medical and Population Genetics, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge (P.N., A.G.B., N.G., S.G., S.K.) - all in Massachusetts; the Department of Cardiology, University Hospital, Parma, Italy (D.A.); the Department of Medicine, Division of Cardiology, Mt. Sinai School of Medicine, New York (U.B., R.M., V.F.); Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid (V.F.); Medical Research Council-British Heart Foundation Cardiovascular Epidemiology Unit and National Institute for Health Research Blood and Transplant Research Unit in Donor Health and Genomics, Department of Public Health and Primary Care, and the British Heart Foundation, Cambridge Centre of Excellence, Department of Medicine, University of Cambridge, Cambridge (J.D.), and the Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton (J.D.) - both in the United Kingdom; the Center for Non-Communicable Diseases, Karachi, Pakistan (P.F., D.S.); the Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia (D.S.); and the Department of Clinical Sciences Malmö, Lund University, Lund, Sweden (O.M.).

    Background: Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of an expanded somatic blood-cell clone in persons without other hematologic abnormalities, is common among older persons and is associated with an increased risk of hematologic cancer. We previously found preliminary evidence for an association between CHIP and atherosclerotic cardiovascular disease, but the nature of this association was unclear.

    Methods: We used whole-exome sequencing to detect the presence of CHIP in peripheral-blood cells and associated such presence with coronary heart disease using samples from four case-control studies that together enrolled 4726 participants with coronary heart disease and 3529 controls. To assess causality, we perturbed the function of Tet2, the second most commonly mutated gene linked to clonal hematopoiesis, in the hematopoietic cells of atherosclerosis-prone mice.

    Results: In nested case-control analyses from two prospective cohorts, carriers of CHIP had a risk of coronary heart disease that was 1.9 times as great as in noncarriers (95% confidence interval [CI], 1.4 to 2.7). In two retrospective case-control cohorts for the evaluation of early-onset myocardial infarction, participants with CHIP had a risk of myocardial infarction that was 4.0 times as great as in noncarriers (95% CI, 2.4 to 6.7). Mutations in DNMT3A, TET2, ASXL1, and JAK2 were each individually associated with coronary heart disease. CHIP carriers with these mutations also had increased coronary-artery calcification, a marker of coronary atherosclerosis burden. Hypercholesterolemia-prone mice that were engrafted with bone marrow obtained from homozygous or heterozygous Tet2 knockout mice had larger atherosclerotic lesions in the aortic root and aorta than did mice that had received control bone marrow. Analyses of macrophages from Tet2 knockout mice showed elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis.

    Conclusions: The presence of CHIP in peripheral-blood cells was associated with nearly a doubling in the risk of coronary heart disease in humans and with accelerated atherosclerosis in mice. (Funded by the National Institutes of Health and others.).

    Funded by: FIC NIH HHS: RC1 TW008485; NHGRI NIH HHS: U54 HG003067; NHLBI NIH HHS: R01 HL080472, R01 HL082945, RC2 HL101834, T32 HL116324

    The New England journal of medicine 2017;377;2;111-121

  • Evolution of mobile genetic element composition in an epidemic methicillin-resistant Staphylococcus aureus: temporal changes correlated with frequent loss and gain events.

    Jamrozy D, Coll F, Mather AE, Harris SR, Harrison EM, MacGowan A, Karas A, Elston T, Estée Török M, Parkhill J and Peacock SJ

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

    Background: Horizontal transfer of mobile genetic elements (MGEs) that carry virulence and antimicrobial resistance genes mediates the evolution of methicillin-resistant Staphylococcus aureus, and the emergence of new MRSA clones. Most MRSA lineages show an association with specific MGEs and the evolution of MGE composition following clonal expansion has not been widely studied.

    Results: We investigated the genomes of 1193 S. aureus bloodstream isolates, 1169 of which were MRSA, collected in the UK and the Republic of Ireland between 2001 and 2010. The majority of isolates belonged to clonal complex (CC)22 (n = 923), which contained diverse MGEs including elements that were found in other MRSA lineages. Several MGEs showed variable distribution across the CC22 phylogeny, including two antimicrobial resistance plasmids (pWBG751-like and SAP078A-like, carrying erythromycin and heavy metal resistance genes, respectively), a pathogenicity island carrying the enterotoxin C gene and two phage types Sa1int and Sa6int. Multiple gains and losses of these five MGEs were identified in the CC22 phylogeny using ancestral state reconstruction. Analysis of the temporal distribution of the five MGEs between 2001 and 2010 revealed an unexpected reduction in prevalence of the two plasmids and the pathogenicity island, and an increase in the two phage types. This occurred across the lineage and was not correlated with changes in the relative prevalence of CC22, or of any sub-lineages within in.

    Conclusions: Ancestral state reconstruction coupled with temporal trend analysis demonstrated that epidemic MRSA CC22 has an evolving MGE composition, and indicates that this important MRSA lineage has continued to adapt to changing selective pressure since its emergence.

    BMC genomics 2017;18;1;684

  • The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

    Jana B, Cain AK, Doerrler WT, Boinett CJ, Fookes MC, Parkhill J and Guardabassi L

    Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.

    Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.

    Scientific reports 2017;7;42483

  • Human Y-chromosome variation in the genome-sequencing era.

    Jobling MA and Tyler-Smith C

    Department of Genetics &Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.

    The properties of the human Y chromosome - namely, male specificity, haploidy and escape from crossing over - make it an unusual component of the genome, and have led to its genetic variation becoming a key part of studies of human evolution, population history, genealogy, forensics and male medical genetics. Next-generation sequencing (NGS) technologies have driven recent progress in these areas. In particular, NGS has yielded direct estimates of mutation rates, and an unbiased and calibrated molecular phylogeny that has unprecedented detail. Moreover, the availability of direct-to-consumer NGS services is fuelling a rise of 'citizen scientists', whose interest in resequencing their own Y chromosomes is generating a wealth of new data.

    Nature reviews. Genetics 2017

  • Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

    Ju YS, Martincorena I, Gerstung M, Petljak M, Alexandrov LB, Rahbari R, Wedge DC, Davies HR, Ramakrishna M, Fullam A, Martin S, Alder C, Patel N, Gamble S, O'Meara S, Giri DD, Sauer T, Pinder SE, Purdie CA, Borg Å, Stunnenberg H, van de Vijver M, Tan BK, Caldas C, Tutt A, Ueno NT, van 't Veer LJ, Martens JW, Sotiriou C, Knappskog S, Span PN, Lakhani SR, Eyfjörd JE, Børresen-Dale AL, Richardson A, Thompson AM, Viari A, Hurles ME, Nik-Zainal S, Campbell PJ and Stratton MR

    Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK.

    Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

    Funded by: Wellcome Trust: 077012/Z/05/Z

    Nature 2017;543;7647;714-718

  • Genetic Characterization of Vibrio cholerae O1 isolates from outbreaks between 2011 and 2015 in Tanzania.

    Kachwamba Y, Mohammed AA, Lukupulo H, Urio L, Majigo M, Mosha F, Matonya M, Kishimba R, Mghamba J, Lusekelo J, Nyanga S, Almeida M, Li S, Domman D, Massele SY and Stine OC

    Muhimbili University of Health and Allied Sciences, Dar es Salaam, United Republic of Tanzania.

    Background: Cholera outbreaks have occurred in Tanzania since 1974. To date, the genetic epidemiology of these outbreaks has not been assessed.

    Methods: 96 Vibrio cholerae O1 isolates from five regions were characterized, and their genetic relatedness assessed using multi-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS).

    Results: Of the 48 MLVA genotypes observed, 3 were genetically unrelated to any others, while the remaining 45 genotypes separated into three MLVA clonal complexes (CCs) - each comprised of genotypes differing by a single allelic change. In Kigoma, two separate outbreaks, 4 months apart (January and May, 2015), were each caused by genetically distinct strains by MLVA and WGS. Remarkably, one MLVA CC contained isolates from both the May outbreak and ones from the 2011/2012 outbreak in Dar-es-Salaam. However, WGS revealed the isolates from the two outbreaks to be distinct clades. The outbreak that started in August 2015 in Dar-es-Salaam and spread to Morogoro, Singida and Mara was comprised of a single MLVA CC and WGS clade. Isolates from within an outbreak were closely related differing at fewer than 5 nucleotides. All isolates were part of the 3(rd) wave of the 7(th) pandemic and were found in four clades related to isolates from Kenya and Asia.

    Conclusions: We conclude that genetically related V. cholerae cluster in outbreaks, and distinct strains circulate simultaneously.

    BMC infectious diseases 2017;17;1;157

  • Systematic longitudinal survey of invasive Escherichia coli in England demonstrates a stable population structure only transiently disturbed by the emergence of ST131.

    Kallonen T, Brodrick HJ, Harris SR, Corander J, Brown NM, Martin V, Peacock SJ and Parkhill J

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

    Escherichia coli associated with urinary tract infections and bacteremia has been intensively investigated, including recent work focusing on the virulent, globally disseminated, multidrug-resistant lineage ST131. To contextualize ST131 within the broader E. coli population associated with disease, we used genomics to analyze a systematic 11-yr hospital-based survey of E. coli associated with bacteremia using isolates collected from across England by the British Society for Antimicrobial Chemotherapy and from the Cambridge University Hospitals NHS Foundation Trust. Population dynamics analysis of the most successful lineages identified the emergence of ST131 and ST69 and their establishment as two of the five most common lineages along with ST73, ST95, and ST12. The most frequently identified lineage was ST73. Compared to ST131, ST73 was susceptible to most antibiotics, indicating that multidrug resistance was not the dominant reason for prevalence of E. coli lineages in this population. Temporal phylogenetic analysis of the emergence of ST69 and ST131 identified differences in the dynamics of emergence and showed that expansion of ST131 in this population was not driven by sequential emergence of increasingly resistant subclades. We showed that over time, the E. coli population was only transiently disturbed by the introduction of new lineages before a new equilibrium was rapidly achieved. Together, these findings suggest that the frequency of E. coli lineages in invasive disease is driven by negative frequency-dependent selection occurring outside of the hospital, most probably in the commensal niche, and that drug resistance is not a primary determinant of success in this niche.

    Genome research 2017

  • Flipping between Polycomb repressed and active transcriptional states introduces noise in gene expression.

    Kar G, Kim JK, Kolodziejczyk AA, Natarajan KN, Torlai Triglia E, Mifsud B, Elderkin S, Marioni JC, Pombo A and Teichmann SA

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

    Polycomb repressive complexes (PRCs) are important histone modifiers, which silence gene expression; yet, there exists a subset of PRC-bound genes actively transcribed by RNA polymerase II (RNAPII). It is likely that the role of Polycomb repressive complex is to dampen expression of these PRC-active genes. However, it is unclear how this flipping between chromatin states alters the kinetics of transcription. Here, we integrate histone modifications and RNAPII states derived from bulk ChIP-seq data with single-cell RNA-sequencing data. We find that Polycomb repressive complex-active genes have greater cell-to-cell variation in expression than active genes, and these results are validated by knockout experiments. We also show that PRC-active genes are clustered on chromosomes in both two and three dimensions, and interactions with active enhancers promote a stabilization of gene expression noise. These findings provide new insights into how chromatin regulation modulates stochastic gene expression and transcriptional bursting, with implications for regulation of pluripotency and development.Polycomb repressive complexes modify histones but it is unclear how changes in chromatin states alter kinetics of transcription. Here, the authors use single-cell RNAseq and ChIPseq to find that actively transcribed genes with Polycomb marks have greater cell-to-cell variation in expression.

    Nature communications 2017;8;1;36

  • Prevalence of sexual dimorphism in mammalian phenotypic traits.

    Karp NA, Mason J, Beaudet AL, Benjamini Y, Bower L, Braun RE, Brown SDM, Chesler EJ, Dickinson ME, Flenniken AM, Fuchs H, Angelis MH, Gao X, Guo S, Greenaway S, Heller R, Herault Y, Justice MJ, Kurbatova N, Lelliott CJ, Lloyd KCK, Mallon AM, Mank JE, Masuya H, McKerlie C, Meehan TF, Mott RF, Murray SA, Parkinson H, Ramirez-Solis R, Santos L, Seavitt JR, Smedley D, Sorg T, Speak AO, Steel KP, Svenson KL, International Mouse Phenotyping Consortium, Wakana S, West D, Wells S, Westerberg H, Yaacoby S and White JK

    Mouse Informatics Group, 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.

    Funded by: NHGRI NIH HHS: UM1 HG006348; NIH HHS: U42 OD011185, UM1 OD023222

    Nature communications 2017;8;15475

  • Insertional mutagenesis identifies drivers of a novel oncogenic pathway in invasive lobular breast carcinoma.

    Kas SM, de Ruiter JR, Schipper K, Annunziato S, Schut E, Klarenbeek S, Drenth AP, van der Burg E, Klijn C, Ten Hoeve JJ, Adams DJ, Koudijs MJ, Wesseling J, Nethe M, Wessels LFA and Jonkers J

    Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

    Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

    Nature genetics 2017

  • Single-cell epigenomics: Recording the past and predicting the future.

    Kelsey G, Stegle O and Reik W

    Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK.

    Single-cell multi-omics has recently emerged as a powerful technology by which different layers of genomic output-and hence cell identity and function-can be recorded simultaneously. Integrating various components of the epigenome into multi-omics measurements allows for studying cellular heterogeneity at different time scales and for discovering new layers of molecular connectivity between the genome and its functional output. Measurements that are increasingly available range from those that identify transcription factor occupancy and initiation of transcription to long-lasting and heritable epigenetic marks such as DNA methylation. Together with techniques in which cell lineage is recorded, this multilayered information will provide insights into a cell's past history and its future potential. This will allow new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease.

    Science (New York, N.Y.) 2017;358;6359;69-75

  • Identification of 153 new loci associated with heel bone mineral density and functional involvement of GPC6 in osteoporosis.

    Kemp JP, Morris JA, Medina-Gomez C, Forgetta V, Warrington NM, Youlten SE, Zheng J, Gregson CL, Grundberg E, Trajanoska K, Logan JG, Pollard AS, Sparkes PC, Ghirardello EJ, Allen R, Leitch VD, Butterfield NC, Komla-Ebri D, Adoum AT, Curry KF, White JK, Kussy F, Greenlaw KM, Xu C, Harvey NC, Cooper C, Adams DJ, Greenwood CMT, Maurano MT, Kaptoge S, Rivadeneira F, Tobias JH, Croucher PI, Ackert-Bicknell CL, Bassett JHD, Williams GR, Richards JB and Evans DM

    University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia.

    Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study (GWAS) in 142,487 individuals from the UK Biobank to identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.

    Nature genetics 2017

  • Fine-Scale Genetic Structure in Finland.

    Kerminen S, Havulinna AS, Hellenthal G, Martin AR, Sarin AP, Perola M, Palotie A, Salomaa V, Daly MJ, Ripatti S and Pirinen M

    Institute for Molecular Medicine Finland, University of Helsinki, 00014, Finland.

    Coupling dense genotype data with new computational methods offers unprecedented opportunities for individual-level ancestry estimation once geographically precisely defined reference data sets become available. We study such a reference data set for Finland containing 2376 such individuals from the FINRISK Study survey of 1997 both of whose parents were born close to each other. This sampling strategy focuses on the population structure present in Finland before the 1950s. By using the recent haplotype-based methods ChromoPainter (CP) and FineSTRUCTURE (FS) we reveal a highly geographically clustered genetic structure in Finland and report its connections to the settlement history as well as to the current dialectal regions of the Finnish language. The main genetic division within Finland shows striking concordance with the 1323 borderline of the treaty of Nöteborg. In general, we detect genetic substructure throughout the country, which reflects stronger regional genetic differences in Finland compared to, for example, the UK, which in a similar analysis was dominated by a single unstructured population. We expect that similar population genetic reference data sets will become available for many more populations in the near future with important applications, for example, in forensic genetics and in genetic association studies. With this in mind, we report those extensions of the CP + FS approach that we found most useful in our analyses of the Finnish data.

    G3 (Bethesda, Md.) 2017;7;10;3459-3468

  • Clinical features associated with CTNNB1 de novo loss of function mutations in ten individuals.

    Kharbanda M, Pilz DT, Tomkins S, Chandler K, Saggar A, Fryer A, McKay V, Louro P, Smith JC, Burn J, Kini U, De Burca A, FitzPatrick DR, Kinning E and DDD Study

    West of Scotland Clinical Genetics Service, Level 2A Laboratory Medicine Building, Queen Elizabeth University Hospital, Glasgow, UK. Electronic address:

    Loss of function mutations in CTNNB1 have been reported in individuals with intellectual disability [MIM #615075] associated with peripheral spasticity, microcephaly and central hypotonia, suggesting a recognisable phenotype associated with haploinsufficiency for this gene. Trio based whole exome sequencing via the Deciphering Developmental Disorders (DDD) study has identified eleven further individuals with de novo loss of function mutations in CTNNB1. Here we report detailed phenotypic information on ten of these. We confirm the features that have been previously described and further delineate the skin and hair findings, including fair skin and fair and sparse hair with unusual patterning.

    Funded by: Medical Research Council: MC_PC_U127561093

    European journal of medical genetics 2017;60;2;130-135

  • Common genetic variation drives molecular heterogeneity in human iPSCs.

    Kilpinen H, Goncalves A, Leha A, Afzal V, Alasoo K, Ashford S, Bala S, Bensaddek D, Casale FP, Culley OJ, Danecek P, Faulconbridge A, Harrison PW, Kathuria A, McCarthy D, McCarthy SA, Meleckyte R, Memari Y, Moens N, Soares F, Mann A, Streeter I, Agu CA, Alderton A, Nelson R, Harper S, Patel M, White A, Patel SR, Clarke L, Halai R, Kirton CM, Kolb-Kokocinski A, Beales P, Birney E, Danovi D, Lamond AI, Ouwehand WH, Vallier L, Watt FM, Durbin R, Stegle O and Gaffney DJ

    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.

    Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

    Funded by: Wellcome Trust: WT090851

    Nature 2017;546;7658;370-375

  • Detection of structural mosaicism from targeted and whole-genome sequencing data.

    King DA, Sifrim A, Fitzgerald TW, Rahbari R, Hobson E, Homfray T, Mansour S, Mehta SG, Shehla M, Tomkins SE, Vasudevan PC, Hurles ME and Deciphering Developmental Disorders Study

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

    Structural mosaic abnormalities are large post-zygotic mutations present in a subset of cells and have been implicated in developmental disorders and cancer. Such mutations have been conventionally assessed in clinical diagnostics using cytogenetic or microarray testing. Modern disease studies rely heavily on exome sequencing, yet an adequate method for the detection of structural mosaicism using targeted sequencing data is lacking. Here, we present a method, called MrMosaic, to detect structural mosaic abnormalities using deviations in allele fraction and read coverage from next-generation sequencing data. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) simulations were used to calculate detection performance across a range of mosaic event sizes, types, clonalities, and sequencing depths. The tool was applied to 4911 patients with undiagnosed developmental disorders, and 11 events among nine patients were detected. For eight of these 11 events, mosaicism was observed in saliva but not blood, suggesting that assaying blood alone would miss a large fraction, possibly >50%, of mosaic diagnostic chromosomal rearrangements.

    Genome research 2017

  • Proliferation Drives Aging-Related Functional Decline in a Subpopulation of the Hematopoietic Stem Cell Compartment.

    Kirschner K, Chandra T, Kiselev V, Flores-Santa Cruz D, Macaulay IC, Park HJ, Li J, Kent DG, Kumar R, Pask DC, Hamilton TL, Hemberg M, Reik W and Green AR

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, Cambridgeshire CB2 0XY, UK; Department of Haematology, University of Cambridge, Cambridge, Cambridgeshire CB2 0XY, UK; Stem Cell Institute, University of Cambridge, Cambridge, Cambridgeshire CB2 0XY, UK; Institute for Cancer Sciences, University of Glasgow, Glasgow, Lanarkshire G61 1BD, UK. Electronic address:

    Aging of the hematopoietic stem cell (HSC) compartment is characterized by lineage bias and reduced stem cell function, the molecular basis of which is largely unknown. Using single-cell transcriptomics, we identified a distinct subpopulation of old HSCs carrying a p53 signature indicative of stem cell decline alongside pro-proliferative JAK/STAT signaling. To investigate the relationship between JAK/STAT and p53 signaling, we challenged HSCs with a constitutively active form of JAK2 (V617F) and observed an expansion of the p53-positive subpopulation in old mice. Our results reveal cellular heterogeneity in the onset of HSC aging and implicate a role for JAK2V617F-driven proliferation in the p53-mediated functional decline of old HSCs.

    Cell reports 2017;19;8;1503-1511

  • SC3: consensus clustering of single-cell RNA-seq data.

    Kiselev VY, Kirschner K, Schaub MT, Andrews T, Yiu A, Chandra T, Natarajan KN, Reik W, Barahona M, Green AR and Hemberg M

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach ( We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

    Funded by: Wellcome Trust: 098051

    Nature methods 2017;14;5;483-486

  • The Influence of HIV on The Evolution of Mycobacterium tuberculosis.

    Koch A, Brites D, Stucki D, Evans JC, Seldon R, Heekes A, Mulder N, Nicol M, Oni T, Warner DF, Mizrahi V, Parkhill J, Gagneux S, Martin DP and Wilkinson RJ

    Wellcome Centre for Infectious Disease Research in Africa, Institute of Infectious Disease and Molecular Medicine, and Department of Medicine, University of Cape Town, South Africa.

    HIV significantly affects the immunological environment during tuberculosis co-infection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 co-infected and uninfected individuals and analysed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favouring and disfavouring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favouring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analysed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 co-infection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis dataset means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.

    Molecular biology and evolution 2017

  • Open Targets: a platform for therapeutic target identification and validation.

    Koscielny G, An P, Carvalho-Silva D, Cham JA, Fumis L, Gasparyan R, Hasan S, Karamanis N, Maguire M, Papa E, Pierleoni A, Pignatelli M, Platt T, Rowland F, Wankar P, Bento AP, Burdett T, Fabregat A, Forbes S, Gaulton A, Gonzalez CY, Hermjakob H, Hersey A, Jupe S, Kafkas Ş, Keays M, Leroy C, Lopez FJ, Magarinos MP, Malone J, McEntyre J, Munoz-Pomer Fuentes A, O'Donovan C, Papatheodorou I, Parkinson H, Palka B, Paschall J, Petryszak R, Pratanwanich N, Sarntivijal S, Saunders G, Sidiropoulos K, Smith T, Sondka Z, Stegle O, Tang YA, Turner E, Vaughan B, Vrousgou O, Watkins X, Martin MJ, Sanseau P, Vamathevan J, Birney E, Barrett J and Dunham I

    Open Targets, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK

    We have designed and developed a data integration and visualization platform that provides evidence about the association of known and potential drug targets with diseases. The platform is designed to support identification and prioritization of biological targets for follow-up. Each drug target is linked to a disease using integrated genome-wide data from a broad range of data sources. The platform provides either a target-centric workflow to identify diseases that may be associated with a specific target, or a disease-centric workflow to identify targets that may be associated with a specific disease. Users can easily transition between these target- and disease-centric workflows. The Open Targets Validation Platform is accessible at

    Nucleic acids research 2017;45;D1;D985-D994

  • Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

    Kronholm I, Bassett A, Baulcombe D and Collins S

    Department of Biological and Environmental Science, Centre of Excellence in Biological Interactions, University of Jyväskylä, P.O. Box 35, FI-40014 Jyväskylä, Finland.

    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 trangenerational 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.

    Molecular biology and evolution 2017

  • Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers.

    Kuchenbaecker KB, Hopper JL, Barnes DR, Phillips KA, Mooij TM, Roos-Blom MJ, Jervis S, van Leeuwen FE, Milne RL, Andrieu N, Goldgar DE, Terry MB, Rookus MA, Easton DF, Antoniou AC, BRCA1 and BRCA2 Cohort Consortium, McGuffog L, Evans DG, Barrowdale D, Frost D, Adlard J, Ong KR, Izatt L, Tischkowitz M, Eeles R, Davidson R, Hodgson S, Ellis S, Nogues C, Lasset C, Stoppa-Lyonnet D, Fricker JP, Faivre L, Berthet P, Hooning MJ, van der Kolk LE, Kets CM, Adank MA, John EM, Chung WK, Andrulis IL, Southey M, Daly MB, Buys SS, Osorio A, Engel C, Kast K, Schmutzler RK, Caldes T, Jakubowska A, Simard J, Friedlander ML, McLachlan SA, Machackova E, Foretova L, Tan YY, Singer CF, Olah E, Gerdes AM, Arver B and Olsson H

    Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, England2Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, England.

    Importance: The clinical management of BRCA1 and BRCA2 mutation carriers requires accurate, prospective cancer risk estimates.

    Objectives: To estimate age-specific risks of breast, ovarian, and contralateral breast cancer for mutation carriers and to evaluate risk modification by family cancer history and mutation location.

    Design, setting, and participants: Prospective cohort study of 6036 BRCA1 and 3820 BRCA2 female carriers (5046 unaffected and 4810 with breast or ovarian cancer or both at baseline) recruited in 1997-2011 through the International BRCA1/2 Carrier Cohort Study, the Breast Cancer Family Registry and the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, with ascertainment through family clinics (94%) and population-based studies (6%). The majority were from large national studies in the United Kingdom (EMBRACE), the Netherlands (HEBON), and France (GENEPSO). Follow-up ended December 2013; median follow-up was 5 years.

    Exposures: BRCA1/2 mutations, family cancer history, and mutation location.

    Main outcomes and measures: Annual incidences, standardized incidence ratios, and cumulative risks of breast, ovarian, and contralateral breast cancer.

    Results: Among 3886 women (median age, 38 years; interquartile range [IQR], 30-46 years) eligible for the breast cancer analysis, 5066 women (median age, 38 years; IQR, 31-47 years) eligible for the ovarian cancer analysis, and 2213 women (median age, 47 years; IQR, 40-55 years) eligible for the contralateral breast cancer analysis, 426 were diagnosed with breast cancer, 109 with ovarian cancer, and 245 with contralateral breast cancer during follow-up. The cumulative breast cancer risk to age 80 years was 72% (95% CI, 65%-79%) for BRCA1 and 69% (95% CI, 61%-77%) for BRCA2 carriers. Breast cancer incidences increased rapidly in early adulthood until ages 30 to 40 years for BRCA1 and until ages 40 to 50 years for BRCA2 carriers, then remained at a similar, constant incidence (20-30 per 1000 person-years) until age 80 years. The cumulative ovarian cancer risk to age 80 years was 44% (95% CI, 36%-53%) for BRCA1 and 17% (95% CI, 11%-25%) for BRCA2 carriers. For contralateral breast cancer, the cumulative risk 20 years after breast cancer diagnosis was 40% (95% CI, 35%-45%) for BRCA1 and 26% (95% CI, 20%-33%) for BRCA2 carriers (hazard ratio [HR] for comparing BRCA2 vs BRCA1, 0.62; 95% CI, 0.47-0.82; P=.001 for difference). Breast cancer risk increased with increasing number of first- and second-degree relatives diagnosed as having breast cancer for both BRCA1 (HR for ≥2 vs 0 affected relatives, 1.99; 95% CI, 1.41-2.82; P<.001 for trend) and BRCA2 carriers (HR, 1.91; 95% CI, 1.08-3.37; P=.02 for trend). Breast cancer risk was higher if mutations were located outside vs within the regions bounded by positions c.2282-c.4071 in BRCA1 (HR, 1.46; 95% CI, 1.11-1.93; P=.007) and c.2831-c.6401 in BRCA2 (HR, 1.93; 95% CI, 1.36-2.74; P<.001).

    Conclusions and relevance: These findings provide estimates of cancer risk based on BRCA1 and BRCA2 mutation carrier status using prospective data collection and demonstrate the potential importance of family history and mutation location in risk assessment.

    Funded by: NCI NIH HHS: R01 CA159868, UM1 CA164920

    JAMA 2017;317;23;2402-2416

  • The dental phenotype of hairless dogs with FOXI3 haploinsufficiency.

    Kupczik K, Cagan A, Brauer S and Fischer MS

    Max Planck Weizmann Center for Integrative Archaeology and Anthropology, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103, Leipzig, Germany.

    Hairless dog breeds show a form of ectodermal dysplasia characterised by a lack of hair and abnormal tooth morphology. This has been attributed to a semi-dominant 7-base-pair duplication in the first exon of the forkhead box I3 gene (FOXI3) shared by all three breeds. Here, we identified this FOXI3 variant in a historical museum sample of pedigreed hairless dog skulls by using ancient DNA extraction and present the associated dental phenotype. Unlike in the coated wild type dogs, the hairless dogs were characterised in both the mandibular and maxillary dentition by a loss of the permanent canines, premolars and to some extent incisors. In addition, the deciduous fourth premolars and permanent first and second molars consistently lacked the distal and lingual cusps; this resulted in only a single enlarged cusp in the basin-like heel (talonid in lower molars, talon in upper molars). This molar phenotype is also found among several living and fossil carnivorans and the extinct order Creodonta in which it is associated with hypercarnivory. We therefore suggest that FOXI3 may generally be involved in dental (cusp) development within and across mammalian lineages including the hominids which are known to exhibit marked variability in the presence of lingual cusps.

    Funded by: Wellcome Trust

    Scientific reports 2017;7;1;5459

  • Predicting evolution.

    Lässig M, Mustonen V and Walczak AM

    Institute of Theoretical Physics, University of Cologne, 50937 Cologne, Germany.

    The face of evolutionary biology is changing: from reconstructing and analysing the past to predicting future evolutionary processes. Recent developments include prediction of reproducible patterns in parallel evolution experiments, forecasting the future of individual populations using data from their past, and controlled manipulation of evolutionary dynamics. Here we undertake a synthesis of central concepts for evolutionary predictions, based on examples of microbial and viral systems, cancer cell populations, and immune receptor repertoires. These systems have strikingly similar evolutionary dynamics driven by the competition of clades within a population. These dynamics are the basis for models that predict the evolution of clade frequencies, as well as broad genetic and phenotypic changes. Moreover, there are strong links between prediction and control, which are important for interventions such as vaccine or therapy design. All of these are key elements of what may become a predictive theory of evolution.

    Nature ecology & evolution 2017;1;3;77

  • Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria.

    Lönnberg T, Svensson V, James KR, Fernandez-Ruiz D, Sebina I, Montandon R, Soon MS, Fogg LG, Nair AS, Liligeto U, Stubbington MJ, Ly LH, Bagger FO, Zwiessele M, Lawrence ND, Souza-Fonseca-Guimaraes F, Bunn PT, Engwerda CR, Heath WR, Billker O, Stegle O, Haque A and Teichmann SA

    European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, UK; Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.

    Differentiation of naïve CD4(+) T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates.

    Funded by: European Research Council: 260507; Wellcome Trust: 098051

    Science immunology 2017;2;9

  • Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T Cells.

    Lai Y, Weng J, Wei X, Qin L, Lai P, Zhao R, Jiang Z, Li B, Lin S, Wang S, Wu Q, Tang Z, Liu P, Pei D, Yao Y, Du X and Li P

    Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

    Chimeric antigen receptor (CAR) T-cell immunotherapies have shown unprecedented success in treating leukemia but limited clinical efficacy in solid tumors. Here, we generated 1928zT2 and m28zT2, targeting CD19 and mesothelin, respectively, by introducing the Toll/interleukin-1 receptor domain of Toll-like receptor 2 (TLR2) to 1928z and m28z. T cells expressing 1928zT2 or m28zT2 showed improved expansion, persistency and effector function against CD19(+) leukemia or mesothelin(+) solid tumors respectively in vitro and in vivo. In a patient with relapsed B-cell acute lymphoblastic leukemia, a single dose of 5 × 10(4)/kg 1928zT2 T cells resulted in robust expansion and leukemia eradication and led to complete remission. Hence, our results demonstrate that TLR2 signaling can contribute to the efficacy of CAR T cells. Further clinical trials are warranted to establish the safety and efficacy of this approach.Leukemia advance online publication, 25 August 2017; doi:10.1038/leu.2017.249.

    Leukemia 2017

  • Heterogeneity of hypothalamic pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing.

    Lam BYH, Cimino I, Polex-Wolf J, Nicole Kohnke S, Rimmington D, Iyemere V, Heeley N, Cossetti C, Schulte R, Saraiva LR, Logan DW, Blouet C, O'Rahilly S, Coll AP and Yeo GSH

    MRC Metabolic Diseases Unit, University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.

    Objective: Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture.

    Methods: Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons.

    Results: Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp expression and were considered "canonical" POMC neurons (P(+)). The other 45/163 expressed low levels of Pomc and high levels of Agrp (A(+)P+). Unbiased clustering analysis of P(+) neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P(+) neurons expressed the leptin receptor (Lepr) compared with 58% (26/45) of A(+)P+ neurons. In contrast, the insulin receptor (Insr) was expressed at similar frequency on P(+) and A(+)P+ neurons (64% and 55%, respectively).

    Conclusion: These data reveal arcuate POMC neurons to be a highly heterogeneous population. Accession Numbers: GSE92707.

    Molecular metabolism 2017;6;5;383-392

  • The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.

    Langie SA, Cameron KM, Ficz G, Oxley D, Tomaszewski B, Gorniak JP, Maas LM, Godschalk RW, van Schooten FJ, Reik W, von Zglinicki T and Mathers JC

    Centre for Ageing and Vitality, Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK.

    Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.

    Genes 2017;8;2

  • Obesity-associated gene TMEM18 has a role in the central control of appetite and body weight regulation.

    Larder R, Sim MFM, Gulati P, Antrobus R, Tung YCL, Rimmington D, Ayuso E, Polex-Wolf J, Lam BYH, Dias C, Logan DW, Virtue S, Bosch F, Yeo GSH, Saudek V, O'Rahilly S and Coll AP

    University of Cambridge Metabolic Research Laboratories, Level 4, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom.

    An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is TMEM18, although the molecular mechanisms mediating these effects remain entirely unknown. Tmem18 expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of Tmem18 in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of Tmem18 in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that TMEM18 itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.

    Proceedings of the National Academy of Sciences of the United States of America 2017

  • Separate and combined associations of obesity and metabolic health with coronary heart disease: a pan-European case-cohort analysis.

    Lassale C, Tzoulaki I, Moons KGM, Sweeting M, Boer J, Johnson L, Huerta JM, Agnoli C, Freisling H, Weiderpass E, Wennberg P, van der A DL, Arriola L, Benetou V, Boeing H, Bonnet F, Colorado-Yohar SM, Engström G, Eriksen AK, Ferrari P, Grioni S, Johansson M, Kaaks R, Katsoulis M, Katzke V, Key TJ, Matullo G, Melander O, Molina-Portillo E, Moreno-Iribas C, Norberg M, Overvad K, Panico S, Quirós JR, Saieva C, Skeie G, Steffen A, Stepien M, Tjønneland A, Trichopoulou A, Tumino R, van der Schouw YT, Verschuren WMM, Langenberg C, Di Angelantonio E, Riboli E, Wareham NJ, Danesh J and Butterworth AS

    Department of Epidemiology and Biostatistics, Imperial College London, London W2 1PG, UK.

    Aims: The hypothesis of 'metabolically healthy obesity' implies that, in the absence of metabolic dysfunction, individuals with excess adiposity are not at greater cardiovascular risk. We tested this hypothesis in a large pan-European prospective study.

    Methods and results: We conducted a case-cohort analysis in the 520 000-person European Prospective Investigation into Cancer and Nutrition study ('EPIC-CVD'). During a median follow-up of 12.2 years, we recorded 7637 incident coronary heart disease (CHD) cases. Using cut-offs recommended by guidelines, we defined obesity and overweight using body mass index (BMI), and metabolic dysfunction ('unhealthy') as ≥ 3 of elevated blood pressure, hypertriglyceridaemia, low HDL-cholesterol, hyperglycaemia, and elevated waist circumference. We calculated hazard ratios (HRs) and 95% confidence intervals (95% CI) within each country using Prentice-weighted Cox proportional hazard regressions, accounting for age, sex, centre, education, smoking, diet, and physical activity. Compared with metabolically healthy normal weight people (reference), HRs were 2.15 (95% CI: 1.79; 2.57) for unhealthy normal weight, 2.33 (1.97; 2.76) for unhealthy overweight, and 2.54 (2.21; 2.92) for unhealthy obese people. Compared with the reference group, HRs were 1.26 (1.14; 1.40) and 1.28 (1.03; 1.58) for metabolically healthy overweight and obese people, respectively. These results were robust to various sensitivity analyses.

    Conclusion: Irrespective of BMI, metabolically unhealthy individuals had higher CHD risk than their healthy counterparts. Conversely, irrespective of metabolic health, overweight and obese people had higher CHD risk than lean people. These findings challenge the concept of 'metabolically healthy obesity', encouraging population-wide strategies to tackle obesity.

    European heart journal 2017

  • Sources of error in measurement of minimal residual disease in childhood acute lymphoblastic leukemia.

    Latham S, Hughes E, Budgen B, Mechinaud F, Crock C, Ekert H, Campbell P and Morley A

    Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, SA, Australia.

    Introduction: The level of minimal residual disease (MRD) in marrow predicts outcome and guides treatment in childhood acute lymphoblastic leukemia (ALL) but accurate prediction depends on accurate measurement.

    Methods: Forty-one children with ALL were studied at the end of induction. Two samples were obtained from each iliac spine and each sample was assayed twice. Assay, sample and side-to-side variation were quantified by analysis of variance and presumptively incorrect decisions related to high-risk disease were determined using the result from each MRD assay, the mean MRD in the patient as the measure of the true value, and each of 3 different MRD cut-off levels which have been used for making decisions on treatment.

    Results: Variation between assays, samples and sides each differed significantly from zero and the overall standard deviation for a single MRD estimation was 0.60 logs. Multifocal residual disease seemed to be at least partly responsible for the variation between samples. Decision errors occurred at a frequency of 13-14% when the mean patient MRD was between 10-2 and 10-5. Decision errors were observed only for an MRD result within 1 log of the cut-off value used for assessing high risk. Depending on the cut-off used, 31-40% of MRD results were within 1 log of the cut-off value and 21-16% of such results would have resulted in a decision error.

    Conclusion: When the result obtained for the level of MRD is within 1 log of the cut-off value used for making decisions, variation in the assay and/or sampling may result in a misleading assessment of the true level of marrow MRD. This may lead to an incorrect decision on treatment.

    PloS one 2017;12;10;e0185556

  • Genome-wide association study identifies distinct genetic contributions to prognosis and susceptibility in Crohn's disease.

    Lee JC, Biasci D, Roberts R, Gearry RB, Mansfield JC, Ahmad T, Prescott NJ, Satsangi J, Wilson DC, Jostins L, Anderson CA, UK IBD Genetics Consortium, Traherne JA, Lyons PA, Parkes M and Smith KG

    Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK.

    For most immune-mediated diseases, the main determinant of patient well-being is not the diagnosis itself but instead the course that the disease takes over time (prognosis). Prognosis may vary substantially between patients for reasons that are poorly understood. Familial studies support a genetic contribution to prognosis, but little evidence has been found for a proposed association between prognosis and the burden of susceptibility variants. To better characterize how genetic variation influences disease prognosis, we performed a within-cases genome-wide association study in two cohorts of patients with Crohn's disease. We identified four genome-wide significant loci, none of which showed any association with disease susceptibility. Conversely, the aggregated effect of all 170 disease susceptibility loci was not associated with disease prognosis. Together, these data suggest that the genetic contribution to prognosis in Crohn's disease is largely independent of the contribution to disease susceptibility and point to a biology of prognosis that could provide new therapeutic opportunities.

    Nature genetics 2017

  • Complex Chromosomal Rearrangements by Single Catastrophic Pathogenesis in NUT Midline Carcinoma.

    Lee JK, Louzada S, An Y, Kim SY, Kim S, Youk J, Park S, Koo SH, Keam B, Jeon YK, Ku JL, Yang F, Kim TM and Ju YS

    Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea.

    Annals of oncology : official journal of the European Society for Medical Oncology 2017

  • Within-Host Sampling of a Natural Population Shows Signs of Selection on Pde1 during Bacterial Meningitis.

    Lees JA, Brouwer M, van der Ende A, Parkhill J, van de Beek D and Bentley SD

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    Infection and immunity 2017;85;3

  • Genome-wide identification of lineage and locus specific variation associated with pneumococcal carriage duration.

    Lees JA, Croucher NJ, Goldblatt D, Nosten F, Parkhill J, Turner C, Turner P and Bentley SD

    Infection Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    Streptococcus pneumoniae is a leading cause of invasive disease in infants, especially in low-income settings. Asymptomatic carriage in the nasopharynx is a prerequisite for disease, but variability in its duration is currently only understood at the serotype level. Here we developed a model to calculate the duration of carriage episodes from longitudinal swab data, and combined these results with whole genome sequence data. We estimated that pneumococcal genomic variation accounted for 63% of the phenotype variation, whereas the host traits considered here (age and previous carriage) accounted for less than 5%. We further partitioned this heritability into both lineage and locus effects, and quantified the amount attributable to the largest sources of variation in carriage duration: serotype (17%), drug-resistance (9%) and other significant locus effects (7%). A pan-genome-wide association study identified prophage sequences as being associated with decreased carriage duration independent of serotype, potentially by disruption of the competence mechanism. These findings support theoretical models of pneumococcal competition and antibiotic resistance.

    eLife 2017;6

  • Large scale genomic analysis shows no evidence for pathogen adaptation between the blood and cerebrospinal fluid niches during bacterial meningitis.

    Lees JA, Kremer PH, Manso AS, Croucher NJ, Ferwerda B, Serón MV, Oggioni MR, Parkhill J, Brouwer MC, van der Ende A, van de Beek D and Bentley SD

    Pathogen Genomics, Wellcome Trust Sanger Institute , Hinxton , UK.

    Recent studies have provided evidence for rapid pathogen genome diversification, some of which could potentially affect the course of disease. We have previously described such variation seen between isolates infecting the blood and cerebrospinal fluid (CSF) of a single patient during a case of bacterial meningitis. Here, we performed whole-genome sequencing of paired isolates from the blood and CSF of 869 meningitis patients to determine whether such variation frequently occurs between these two niches in cases of bacterial meningitis. Using a combination of reference-free variant calling approaches, we show that no genetic adaptation occurs in either invaded niche during bacterial meningitis for two major pathogen species, Streptococcus pneumoniae and Neisseria meningitidis. This study therefore shows that the bacteria capable of causing meningitis are already able to do this upon entering the blood, and no further sequence change is necessary to cross the blood-brain barrier. Our findings place the focus back on bacterial evolution between nasopharyngeal carriage and invasion, or diversity of the host, as likely mechanisms for determining invasiveness.

    Microbial genomics 2017;3;1;e000103

  • Stronger together.

    Lees JA, Tonkin-Hill G and Bentley SD

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

    Nature reviews. Microbiology 2017;15;9;516

  • Resistance to malaria through structural variation of red blood cell invasion receptors.

    Leffler EM, Band G, Busby GBJ, Kivinen K, Le QS, Clarke GM, Bojang KA, Conway DJ, Jallow M, Sisay-Joof F, Bougouma EC, Mangano VD, Modiano D, Sirima SB, Achidi E, Apinjoh TO, Marsh K, Ndila CM, Peshu N, Williams TN, Drakeley C, Manjurano A, Reyburn H, Riley E, Kachala D, Molyneux M, Nyirongo V, Taylor T, Thornton N, Tilley L, Grimsley S, Drury E, Stalker J, Cornelius V, Hubbart C, Jeffreys AE, Rowlands K, Rockett KA, Spencer CCA, Kwiatkowski DP and Malaria Genomic Epidemiology Network

    Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

    The malaria parasite Plasmodium falciparum invades human red blood cells via interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently risen in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

    Science (New York, N.Y.) 2017

  • Role of HIV-specific CD8+ T cells in pediatric HIV cure strategies after widespread early viral escape.

    Leitman,E. M., Thobakgale,C. F., Adland,E. et al.

    Journal of Experimental Medicine 2017

  • Skeletal Site-specific Changes in Bone Mass in a Genetic Mouse Model for Human 15q11-13 Duplication Seen in Autism.

    Lewis KE, Sharan K, Takumi T and Yadav VK

    Department of Mouse and Zebrafish Genetics, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, United Kingdom.

    Children suffering from autism have been reported to have low bone mineral density and increased risk for fracture, yet the cellular origin of the bone phenotype remains unknown. Here we have utilized a mouse model of autism that duplicates 6.3 Mb region of chromosome 7 (Dp/+) corresponding to a region of chromosome 15q11-13, duplication of which is recurrent in humans to characterize the bone phenotype. Paternally inherited Dp/+ (patDp/+) mice showed expected increases in the gene expression in bone, normal postnatal growth and body weight acquisition compared to the littermate controls. Four weeks-old patDp/+ mice develop a low bone mass phenotype in the appendicular but not the axial skeleton compared to the littermate controls. This low bone mass in the mutant mice was secondary to a decrease in the number of osteoblasts and bone formation rate while the osteoclasts remained relatively unaffected. Further in vitro cell culture experiments and gene expression analysis revealed a major defect in the proliferation, differentiation and mineralization abilities of patDp/+ osteoblasts while osteoclast differentiation remained unchanged compared to controls. This study therefore characterizes the structural and cellular bone phenotype in a mouse model of autism that can be further utilized to investigate therapeutic avenues to treat bone fractures in children with autism.

    Scientific reports 2017;7;1;9902

  • A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation.

    Li MA, Amaral PP, Cheung P, Bergmann JH, Kinoshita M, Kalkan T, Ralser M, Robson S, Meyenn FV, Paramor M, Yang F, Chen C, Nichols J, Spector DL, Kouzarides T, He L and Smith A

    Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.

    Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

    Funded by: NCI NIH HHS: P01 CA013106, R01 CA139067, R21 CA175560; Wellcome Trust

    eLife 2017;6

  • Ancient individuals from the North American Northwest Coast reveal 10,000 years of regional genetic continuity.

    Lindo J, Achilli A, Perego UA, Archer D, Valdiosera C, Petzelt B, Mitchell J, Worl R, Dixon EJ, Fifield TE, Rasmussen M, Willerslev E, Cybulski JS, Kemp BM, DeGiorgio M and Malhi RS

    Department of Human Genetics, University of Chicago, Chicago, IL 60637.

    Recent genomic studies of both ancient and modern indigenous people of the Americas have shed light on the demographic processes involved during the first peopling. The Pacific Northwest Coast proves an intriguing focus for these studies because of its association with coastal migration models and genetic ancestral patterns that are difficult to reconcile with modern DNA alone. Here, we report the low-coverage genome sequence of an ancient individual known as "Shuká Káa" ("Man Ahead of Us") recovered from the On Your Knees Cave (OYKC) in southeastern Alaska (archaeological site 49-PET-408). The human remains date to ∼10,300 calendar (cal) y B.P. We also analyze low-coverage genomes of three more recent individuals from the nearby coast of British Columbia dating from ∼6,075 to 1,750 cal y B.P. From the resulting time series of genetic data, we show that the Pacific Northwest Coast exhibits genetic continuity for at least the past 10,300 cal y B.P. We also infer that population structure existed in the late Pleistocene of North America with Shuká Káa on a different ancestral line compared with other North American individuals from the late Pleistocene or early Holocene (i.e., Anzick-1 and Kennewick Man). Despite regional shifts in mtDNA haplogroups, we conclude from individuals sampled through time that people of the northern Northwest Coast belong to an early genetic lineage that may stem from a late Pleistocene coastal migration into the Americas.

    Proceedings of the National Academy of Sciences of the United States of America 2017

  • An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

    Liu P, Yuan B, Carvalho CM, Wuster A, Walter K, Zhang L, Gambin T, Chong Z, Campbell IM, Coban Akdemir Z, Gelowani V, Writzl K, Bacino CA, Lindsay SJ, Withers M, Gonzaga-Jauregui C, Wiszniewska J, Scull J, Stankiewicz P, Jhangiani SN, Muzny DM, Zhang F, Chen K, Gibbs RA, Rautenstrauss B, Cheung SW, Smith J, Breman A, Shaw CA, Patel A, Hurles ME and Lupski JR

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

    De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.

    Cell 2017;168;5;830-842.e7

  • Human evolution: a tale from ancient genomes.

    Llamas B, Willerslev E and Orlando L

    Australian Centre for ADNA, School of Biological Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia.

    The field of human ancient DNA (aDNA) has moved from mitochondrial sequencing that suffered from contamination and provided limited biological insights, to become a fully genomic discipline that is changing our conception of human history. Recent successes include the sequencing of extinct hominins, and true population genomic studies of Bronze Age populations. Among the emerging areas of aDNA research, the analysis of past epigenomes is set to provide more new insights into human adaptation and disease susceptibility through time. Starting as a mere curiosity, ancient human genetics has become a major player in the understanding of our evolutionary history.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2017;372;1713

  • Asymptomatic Plasmodium vivax infections induce robust IgG responses to multiple blood-stage proteins in a low-transmission region of western Thailand

    Longley,R.J., Franca,C.T., White,M.T., Kumpitak,C., Sa-Angchai,P., Gruszczyk,J., Hostetler,J.B., Yadava,A., King,C.L., Fairhurst,R.M., Rayner,J.C., Tham,W.H., Nguitragool,W., Sattabongkot,J. and Mueller,I.

    Malaria journal 2017;16;1;178

  • Reclassification of the specialized metabolite producer Pseudomonas mesoacidophila ATCC 31433 as a member of the Burkholderia cepacia complex.

    Loveridge EJ, Jones C, Bull MJ, Moody SC, Kahl MW, Khan Z, Neilson L, Tomeva M, Adams SE, Wood AC, Rodriguez-Martin D, Pinel I, Parkhill J, Mahenthiralingam E and Crosby J

    Department of Chemistry, Swansea University, Singleton Park, Swansea SA2 8PP, UK

    Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, which produces the monobactam isosulfazecin and the β-lactam potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433 its complete genome was determined using single molecule real time DNA sequence analysis. The 7.8 Mb genome comprised four replicons: three chromosomal, each encoding ribosomal RNA, and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidopila ATCC 31433 was mis-classified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to B. ubonensis The sequenced genome shows considerable additional biosynthetic potential, with known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline and pyrrolnitrin biosynthesis, and several uncharacterized biosynthetic gene clusters for PKS, NRPS and other metabolites, identified. Furthermore P. mesoacidophila ATCC 31433 harbours many genes associated with environmental resilience and antibiotic resistance, and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433 pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. A large number of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidopila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species B. ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidopila ATCC 31433 is warranted.

    Journal of bacteriology 2017

  • Applications of CRISPR Genome Editing Technology in Drug Target Identification and Validation.

    Lu Q, Livi GP, Modha S, Yusa K, Macarrón R and Dow DJ

    a Target Sciences, GlaxoSmithKline R&D , 1250 South Collegeville Road, Collegeville , PA 19426 , USA.

    Structured Abstract Introduction: The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems. Areas Covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies. Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.

    Expert opinion on drug discovery 2017

  • A gene expression atlas of adult Schistosoma mansoni and their gonads.

    Lu Z, Sessler F, Holroyd N, Hahnel S, Quack T, Berriman M and Grevelding CG

    BFS, Institute of Parasitology, Justus Liebig University, 35392 Giessen, Germany.

    RNA-Seq has proven excellence in providing information about the regulation and transcript levels of genes. We used this method for profiling genes in the flatworm Schistosoma mansoni. This parasite causes schistosomiasis, an infectious disease of global importance for human and animals. The pathology of schistosomiasis is associated with the eggs, which are synthesized as a final consequence of male and female adults pairing. The male induces processes in the female that lead to the full development of its gonads as a prerequisite for egg production. Unpaired females remain sexually immature. Based on an organ-isolation method we obtained gonad tissue for RNA extraction from paired and unpaired schistosomes, with whole adults included as controls. From a total of 23 samples, we used high-throughput cDNA sequencing (RNA-Seq) on the Illumina platform to profile gene expression between genders and tissues, with and without pairing influence. The data obtained provide a wealth of information on the reproduction biology of schistosomes and a rich resource for exploitation through basic and applied research activities.

    Scientific data 2017;4;170118

  • Overcoming confounding plate effects in differential expression analyses of single-cell RNA-seq data.

    Lun ATL and Marioni JC

    Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, RobinsonWay, Cambridge CB2 0RE, UK.

    An increasing number of studies are using single-cell RNA-sequencing (scRNA-seq) to characterize the gene expression profiles of individual cells. One common analysis applied to scRNA-seq data involves detecting differentially expressed (DE) genes between cells in different biological groups. However, many experiments are designed such that the cells to be compared are processed in separate plates or chips, meaning that the groupings are confounded with systematic plate effects. This confounding aspect is frequently ignored in DE analyses of scRNA-seq data. In this article, we demonstrate that failing to consider plate effects in the statistical model results in loss of type I error control. A solution is proposed whereby counts are summed from all cells in each plate and the count sums for all plates are used in the DE analysis. This restores type I error control in the presence of plate effects without compromising detection power in simulated data. Summation is also robust to varying numbers and library sizes of cells on each plate. Similar results are observed in DE analyses of real data where the use of count sums instead of single-cell counts improves specificity and the ranking of relevant genes. This suggests that summation can assist in maintaining statistical rigour in DE analyses of scRNA-seq data with plate effects.

    Biostatistics (Oxford, England) 2017;18;3;451-464

  • Testing for differential abundance in mass cytometry data.

    Lun ATL, Richard AC and Marioni JC

    Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.

    When comparing biological conditions using mass cytometry data, a key challenge is to identify cellular populations that change in abundance. Here, we present a computational strategy for detecting 'differentially abundant' populations by assigning cells to hyperspheres, testing for significant differences between conditions and controlling the spatial false discovery rate. Our method ( outperforms other approaches in simulations and finds novel patterns of differential abundance in real data.

    Nature methods 2017

  • Exploring the genetic architecture of inflammatory bowel disease by whole-genome sequencing identifies association at ADCY7.

    Luo Y, de Lange KM, Jostins L, Moutsianas L, Randall J, Kennedy NA, Lamb CA, McCarthy S, Ahmad T, Edwards C, Serra EG, Hart A, Hawkey C, Mansfield JC, Mowat C, Newman WG, Nichols S, Pollard M, Satsangi J, Simmons A, Tremelling M, Uhlig H, Wilson DC, Lee JC, Prescott NJ, Lees CW, Mathew CG, Parkes M, Barrett JC and Anderson CA

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

    To further resolve the genetic architecture of the inflammatory bowel diseases ulcerative colitis and Crohn's disease, we sequenced the whole genomes of 4,280 patients at low coverage and compared them to 3,652 previously sequenced population controls across 73.5 million variants. We then imputed from these sequences into new and existing genome-wide association study cohorts and tested for association at ∼12 million variants in a total of 16,432 cases and 18,843 controls. We discovered a 0.6% frequency missense variant in ADCY7 that doubles the risk of ulcerative colitis. Despite good statistical power, we did not identify any other new low-frequency risk variants and found that such variants explained little heritability. We detected a burden of very rare, damaging missense variants in known Crohn's disease risk genes, suggesting that more comprehensive sequencing studies will continue to improve understanding of the biology of complex diseases.

    Funded by: Chief Scientist Office: CZB/4/540, ETM/137, ETM/75; Department of Health: NIHR-RP-R3-12-026; Medical Research Council: G0600329, G0800675, G0800759, MC_UU_12010/7, MR/J00314X/1; Wellcome Trust

    Nature genetics 2017;49;2;186-192

  • fCCAC: functional canonical correlation analysis to evaluate covariance between nucleic acid sequencing datasets.

    Madrigal P

    Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, UK.

    Summary: Computational evaluation of variability across DNA or RNA sequencing datasets is a crucial step in genomic science, as it allows both to evaluate reproducibility of biological or technical replicates, and to compare different datasets to identify their potential correlations. Here we present fCCAC, an application of functional canonical correlation analysis to assess covariance of nucleic acid sequencing datasets such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We show how this method differs from other measures of correlation, and exemplify how it can reveal shared covariance between histone modifications and DNA binding proteins, such as the relationship between the H3K4me3 chromatin mark and its epigenetic writers and readers.

    Availability and implementation: An R/Bioconductor package is available at .


    Supplementary information: Supplementary data are available at Bioinformatics online.

    Funded by: Medical Research Council: MC_PC_12009; Wellcome Trust

    Bioinformatics (Oxford, England) 2017;33;5;746-748

  • The AURORA pilot study for molecular screening of patients with advanced breast cancer-a study of the breast international group.

    Maetens M, Brown D, Irrthum A, Aftimos P, Viale G, Loibl S, Laes JF, Campbell PJ, Thompson A, Cortes J, Seiler S, Vinnicombe S, Oliveira M, Rothé F, Bareche Y, Fumagalli D, Zardavas D, Desmedt C, Piccart M, Loi S and Sotiriou C

    J.-C. Heuson Breast Cancer Translational Research Laboratory, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium.

    Several studies have demonstrated the feasibility of molecular screening of tumour samples for matching patients with cancer to targeted therapies. However, most of them have been carried out at institutional or national level. Herein, we report on the pilot phase of AURORA (NCT02102165), a European multinational collaborative molecular screening initiative for advanced breast cancer patients. Forty-one patients were prospectively enroled at four participating centres across Europe. Metastatic tumours were biopsied and profiled using an Ion Torrent sequencing platform at a central facility. Sequencing results were obtained for 63% of the patients in real-time with variable turnaround time stemming from delays between patient consent and biopsy. At least one clinically actionable mutation was identified in 73% of patients. We used the Illumina sequencing technology for orthogonal validation and achieved an average of 66% concordance of substitution calls per patient. Additionally, copy number aberrations inferred from the Ion Torrent sequencing were compared to single nucleotide polymorphism arrays and found to be 59% concordant on average. Although this study demonstrates that powerful next generation genomic techniques are logistically ready for international molecular screening programs in routine clinical settings, technical challenges remain to be addressed in order to ensure the accuracy and clinical utility of the genomic data.

    NPJ breast cancer 2017;3;23

  • Nutrient sensing modulates malaria parasite virulence.

    Mancio-Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, Sales-Dias J, Gomes AR, MacPherson CR, Crozet P, Adamo M, Baena-Gonzalez E, Tewari R, Llinás M, Billker O and Mota MM

    Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal.

    The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

    Funded by: NIAID NIH HHS: F32 AI104252; NIH HHS: DP2 OD001315; Wellcome Trust

    Nature 2017;547;7662;213-216

  • Visualization and Quantification of Browning Using a Ucp1-2A-Luciferase Knock-in Mouse Model.

    Mao L, Nie B, Nie T, Hui X, Gao X, Lin X, Liu X, Xu Y, Tang X, Yuan R, Li K, Li P, Ding K, Wang Y, Xu A, Fei J, Han W, Liu P, Madsen L, Kristiansen K, Zhou Z, Ding S and Wu D

    CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Medical University, and Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

    Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.

    Diabetes 2017;66;2;407-417

  • How Single-Cell Genomics Is Changing Evolutionary and Developmental Biology.

    Marioni JC and Arendt D

    Wellcome Genome Campus, EMBL-European Bioinformatics Institute, Cambridge CB10 1SD, United Kingdom; email:

    The recent flood of single-cell data not only boosts our knowledge of cells and cell types, but also provides new insight into development and evolution from a cellular perspective. For example, assaying the genomes of multiple cells during development reveals developmental lineage trees-the kinship lineage-whereas cellular transcriptomes inform us about the regulatory state of cells and their gradual restriction in potency-the Waddington lineage. Beyond that, the comparison of single-cell data across species allows evolutionary changes to be tracked at all stages of development from the zygote, via different kinds of stem cells, to the differentiating cells. We discuss recent insights into the evolution of stem cells and initial attempts to reconstruct the evolutionary cell type tree of the mammalian forebrain, for example, by the comparative analysis of neuron types in the mesencephalic floor. These studies illustrate the immense potential of single-cell genomics to open up a new era in developmental and evolutionary research. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 33 is October 6, 2017. Please see for revised estimates.

    Annual review of cell and developmental biology 2017

  • Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci.

    Martin J, Ponstingl H, Lefranc MP, Archer J, Sargan D and Bradley A

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Dogs are an excellent model for human disease. For example, the treatment of canine lymphoma has been predictive of the human response to that treatment. However, an incomplete picture of canine (Canis lupus familiaris) immunoglobulin (IG) and T cell receptor (TR)-or antigen receptor (AR)-gene loci has restricted their utility. This work advances the annotation of the canine AR loci and looks into breed-specific features of the loci. Bioinformatic analysis of unbiased RNA sequence data was used to complete the annotation of the canine AR genes. This annotation was used to query 107 whole genome sequences from 19 breeds and identified over 5500 alleles across the 550 genes of the seven AR loci: the IG heavy, kappa, and lambda loci; and the TR alpha, beta, gamma, and delta loci. Of note was the discovery that half of the IGK variable (V) genes were located downstream of, and inverted with respect to, the rest of the locus. Analysis of the germline sequences of all the AR V genes identified greater conservation between dog and human than mouse with either. This work brings our understanding of the genetic diversity and expression of AR in dogs to the same completeness as that of mice and men, making it the third species to have all AR loci comprehensively and accurately annotated. The large number of germline sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci.

    Immunogenetics 2017

  • Aging increases cell-to-cell transcriptional variability upon immune stimulation.

    Martinez-Jimenez CP, Eling N, Chen HC, Vallejos CA, Kolodziejczyk AA, Connor F, Stojic L, Rayner TF, Stubbington MJT, Teichmann SA, de la Roche M, Marioni JC and Odom DT

    University of Cambridge, Cancer Research UK Cambridge Institute, Robinson Way, Cambridge, CB2 0RE, UK.

    Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4(+) T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/I015914/3; Cancer Research UK: A15603, A22257; European Research Council: 615584, 646794; Medical Research Council: MC_UP_0801/1; Wellcome Trust: 098051, 107609, 202878ODOM

    Science (New York, N.Y.) 2017;355;6332;1433-1436

  • Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

    Martins WFS, Subramaniam K, Steen K, Mawejje H, Liloglou T, Donnelly MJ and Wilding CS

    Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK.

    Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

    Funded by: NIAID NIH HHS: R01 AI116811, U19 AI089674

    Scientific reports 2017;7;1;5821

  • Human Y chromosome copy number variation in the next generation sequencing era and beyond.

    Massaia A and Xue Y

    National Heart and Lung Institute, Imperial College London, London, SW7 2AZ, UK.

    The human Y chromosome provides a fertile ground for structural rearrangements owing to its haploidy and high content of repeated sequences. The methodologies used for copy number variation (CNV) studies have developed over the years. Low-throughput techniques based on direct observation of rearrangements were developed early on, and are still used, often to complement array-based or sequencing approaches which have limited power in regions with high repeat content and specifically in the presence of long, identical repeats, such as those found in human sex chromosomes. Some specific rearrangements have been investigated for decades; because of their effects on fertility, or their outstanding evolutionary features, the interest in these has not diminished. However, following the flourishing of large-scale genomics, several studies have investigated CNVs across the whole chromosome. These studies sometimes employ data generated within large genomic projects such as the DDD study or the 1000 Genomes Project, and often survey large samples of healthy individuals without any prior selection. Novel technologies based on sequencing long molecules and combinations of technologies, promise to stimulate the study of Y-CNVs in the immediate future.

    Human genetics 2017

  • Temporal Tracking of Microglia Activation in Neurodegeneration at Single-Cell Resolution.

    Mathys H, Adaikkan C, Gao F, Young JZ, Manet E, Hemberg M, De Jager PL, Ransohoff RM, Regev A and Tsai LH

    Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    Microglia, the tissue-resident macrophages in the brain, are damage sensors that react to nearly any perturbation, including neurodegenerative diseases such as Alzheimer's disease (AD). Here, using single-cell RNA sequencing, we determined the transcriptome of more than 1,600 individual microglia cells isolated from the hippocampus of a mouse model of severe neurodegeneration with AD-like phenotypes and of control mice at multiple time points during progression of neurodegeneration. In this neurodegeneration model, we discovered two molecularly distinct reactive microglia phenotypes that are typified by modules of co-regulated type I and type II interferon response genes, respectively. Furthermore, our work identified previously unobserved heterogeneity in the response of microglia to neurodegeneration, discovered disease stage-specific microglia cell states, revealed the trajectory of cellular reprogramming of microglia in response to neurodegeneration, and uncovered the underlying transcriptional programs.

    Cell reports 2017;21;2;366-380

  • Pro-inflammatory fatty acid profile and colorectal cancer risk: A Mendelian randomisation analysis.

    May-Wilson S, Sud A, Law PJ, Palin K, Tuupanen S, Gylfe A, Hänninen UA, Cajuso T, Tanskanen T, Kondelin J, Kaasinen E, Sarin AP, Eriksson JG, Rissanen H, Knekt P, Pukkala E, Jousilahti P, Salomaa V, Ripatti S, Palotie A, Renkonen-Sinisalo L, Lepistö A, Böhm J, Mecklin JP, Al-Tassan NA, Palles C, Farrington SM, Timofeeva MN, Meyer BF, Wakil SM, Campbell H, Smith CG, Idziaszczyk S, Maughan TS, Fisher D, Kerr R, Kerr D, Passarelli MN, Figueiredo JC, Buchanan DD, Win AK, Hopper JL, Jenkins MA, Lindor NM, Newcomb PA, Gallinger S, Conti D, Schumacher F, Casey G, Aaltonen LA, Cheadle JP, Tomlinson IP, Dunlop MG and Houlston RS

    Division of Genetics and Epidemiology, The Institute of Cancer Research, London, SW7 3RP, UK.

    Background: While dietary fat has been established as a risk factor for colorectal cancer (CRC), associations between fatty acids (FAs) and CRC have been inconsistent. Using Mendelian randomisation (MR), we sought to evaluate associations between polyunsaturated (PUFA), monounsaturated (MUFA) and saturated FAs (SFAs) and CRC risk.

    Methods: We analysed genotype data on 9254 CRC cases and 18,386 controls of European ancestry. Externally weighted polygenic risk scores were generated and used to evaluate associations with CRC per one standard deviation increase in genetically defined plasma FA levels.

    Results: Risk reduction was observed for oleic and palmitoleic MUFAs (OROA = 0.77, 95% CI: 0.65-0.92, P = 3.9 × 10(-3); ORPOA = 0.36, 95% CI: 0.15-0.84, P = 0.018). PUFAs linoleic and arachidonic acid had negative and positive associations with CRC respectively (ORLA = 0.95, 95% CI: 0.93-0.98, P = 3.7 × 10(-4); ORAA = 1.05, 95% CI: 1.02-1.07, P = 1.7 × 10(-4)). The SFA stearic acid was associated with increased CRC risk (ORSA = 1.17, 95% CI: 1.01-1.35, P = 0.041).

    Conclusion: Results from our analysis are broadly consistent with a pro-inflammatory FA profile having a detrimental effect in terms of CRC risk.

    European journal of cancer (Oxford, England : 1990) 2017;84;228-238

  • Draft Genome Sequence of an IMP-7-Producing Pseudomonas aeruginosa Bloodstream Infection Isolate from Australia.

    McCarthy KL, Jennison A, Wailan AM and Paterson DL

    University of Queensland, Centre for Clinical Research, Brisbane, Queensland, Australia

    IMP-7 is one of the two IMP-type carbapenemases that in Pseudomonas aeruginosa are not limited to a geographic area, but it has not been previously reported in the Australian setting. We report here the draft genome sequence of an Australian P. aeruginosa bloodstream infection isolate that contains IMP-7.

    Genome announcements 2017;5;27

  • Draft Genome Sequences of Two Pseudomonas aeruginosa Bloodstream Infection Isolates Associated with Rapid Patient Death.

    McCarthy KL, Jennison AV, Wailan AM and Paterson DL

    The University of Queensland, UQ Centre for Clinical Research, Brisbane, Queensland, Australia

    The morbidity and mortality associated with Pseudomonas aeruginosa bloodstream infections are significant. New strategies are required to treat such infections. We report here the draft genome sequences of two antibiotic-sensitive P. aeruginosa bloodstream infection isolates that were associated with rapid death in nonneutropenic patients.

    Genome announcements 2017;5;33

  • Sibling recurrence of total anomalous pulmonary venous drainage.

    McDermott JH, Study DD and Clayton-Smith J

    Manchester Centre for Genomic Medicine, St. Mary's Hospital, Central Manchester Foundation Trust, Oxford Road, Manchester, M13 9WL, United Kingdom. Electronic address:

    Many childhood syndromic disorders are associated with congenital heart defects, but few present specifically with total anomalous pulmonary venous drainage (TAPVD). Here, we report two siblings presenting with TAPVD, tracheo-oesophageal fistula and dysmorphic features in the neonatal period. Careful examination of the mother revealed subtle facial asymmetry and a pre-auricular tag, suggesting a potential variable expression of a dominant disorder. Whole exome sequencing identified a pathogenic heterozygous mutation in EFTUD2, a gene, normally associated with mandibulofacial dystosis Guion-Almedia type (MFDGA), in both siblings and the mother. This is the first report of TAPVD occurring as part of the MFDGA phenotype. It serves to highlight the importance of modern sequencing panels in identifying causative mutations for heterogeneous syndromes such as MFDGA and familial congenital heart defects whilst emphasising the relevance of variable expression when counselling parents.

    European journal of medical genetics 2017

  • Variants in the fetal genome near FLT1 are associated with risk of preeclampsia.

    McGinnis R, Steinthorsdottir V, Williams NO, Thorleifsson G, Shooter S, Hjartardottir S, Bumpstead S, Stefansdottir L, Hildyard L, Sigurdsson JK, Kemp JP, Silva GB, Thomsen LCV, Jääskeläinen T, Kajantie E, Chappell S, Kalsheker N, Moffett A, Hiby S, Lee WK, Padmanabhan S, Simpson NAB, Dolby VA, Staines-Urias E, Engel SM, Haugan A, Trogstad L, Svyatova G, Zakhidova N, Najmutdinova D, FINNPEC Consortium, GOPEC Consortium, Dominiczak AF, Gjessing HK, Casas JP, Dudbridge F, Walker JJ, Pipkin FB, Thorsteinsdottir U, Geirsson RT, Lawlor DA, Iversen AC, Magnus P, Laivuori H, Stefansson K and Morgan L

    Wellcome Trust Sanger Institute, Cambridge, UK.

    Preeclampsia, which affects approximately 5% of pregnancies, is a leading cause of maternal and perinatal death. The causes of preeclampsia remain unclear, but there is evidence for inherited susceptibility. Genome-wide association studies (GWAS) have not identified maternal sequence variants of genome-wide significance that replicate in independent data sets. We report the first GWAS of offspring from preeclamptic pregnancies and discovery of the first genome-wide significant susceptibility locus (rs4769613; P = 5.4 × 10(-11)) in 4,380 cases and 310,238 controls. This locus is near the FLT1 gene encoding Fms-like tyrosine kinase 1, providing biological support, as a placental isoform of this protein (sFlt-1) is implicated in the pathology of preeclampsia. The association was strongest in offspring from pregnancies in which preeclampsia developed during late gestation and offspring birth weights exceeded the tenth centile. An additional nearby variant, rs12050029, associated with preeclampsia independently of rs4769613. The newly discovered locus may enhance understanding of the pathophysiology of preeclampsia and its subtypes.

    Nature genetics 2017

  • Pro-death NMDA receptor signaling is promoted by the GluN2B C-terminus independently of DAPK1.

    McQueen J, Ryan TJ, McKay S, Marwick KF, Baxter PS, Carpanini SM, Wishart TM, Gillingwater TH, Manson JC, Wyllie DJ, Grant SG, McColl B, Komiyama N and Hardingham GE

    UK Dementia Research Institute, Edinburgh Medical School, University of Edinburgh, Edinburgh, United Kingdom.

    Aberrant NMDA receptor (NMDAR) activity contributes to several neurological disorders, but direct antagonism is poorly tolerated therapeutically. The GluN2B cytoplasmic C-terminal domain (CTD) represents an alternative therapeutic target since it potentiates excitotoxic signaling. The key GluN2B CTD-centred event in excitotoxicity is proposed to involve its phosphorylation at Ser-1303 by DAPK1, that is blocked by a neuroprotective cell-permeable peptide mimetic of the region. Contrary to this model, we find that excitotoxicity can proceed without increased Ser-1303 phosphorylation, and is unaffected by DAPK1 deficiency in vitro or following ischemia in vivo. Pharmacological analysis of the aforementioned neuroprotective peptide revealed that it acts in a sequence-independent manner as an open-channel NMDAR antagonist at or near the Mg(2+) site, due to its high net positive charge. Thus, GluN2B-driven excitotoxic signaling can proceed independently of DAPK1 or altered Ser-1303 phosphorylation.

    eLife 2017;6

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

    Meehan TF, Conte N, West DB, Jacobsen JO, Mason J, Warren J, Chen CK, Tudose I, Relac M, Matthews P, Karp N, Santos L, Fiegel T, Ring N, Westerberg H, Greenaway S, Sneddon D, Morgan H, Codner GF, Stewart ME, Brown J, Horner N, International Mouse Phenotyping Consortium, Haendel M, Washington N, Mungall CJ, Reynolds CL, Gallegos J, Gailus-Durner V, Sorg T, Pavlovic G, Bower LR, Moore M, Morse I, Gao X, Tocchini-Valentini GP, Obata Y, Cho SY, Seong JK, Seavitt J, Beaudet AL, Dickinson ME, Herault Y, Wurst W, de Angelis MH, Lloyd KCK, Flenniken AM, Nutter LMJ, Newbigging S, McKerlie C, Justice MJ, Murray SA, Svenson KL, Braun RE, White JK, Bradley A, Flicek P, Wells S, Skarnes WC, Adams DJ, Parkinson H, Mallon AM, Brown SDM and Smedley D

    European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK.

    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.

    Nature genetics 2017

  • The Typhoid Vaccine Acceleration Consortium (TyVAC): Vaccine effectiveness study designs: Accelerating the introduction of typhoid conjugate vaccines and reducing the global burden of enteric fever. Report from a meeting held on 26-27 October 2016, Oxford, UK.

    Meiring JE, Gibani M and TyVAC Consortium Meeting Group

    Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom. Electronic address:

    Typhoid fever is estimated to cause between 11.9-26.9 million infections globally each year with 129,000-216,510 deaths. Access to improved water sources have reduced disease incidence in parts of the world but the use of efficacious vaccines is seen as an important public health tool for countries with a high disease burden. A new generation of Vi typhoid conjugate vaccines (TCVs), licensed for use in young children and expected to provide longer lasting protection than previous vaccines, are now available. The WHO Strategic Advisory Group of Experts on Immunization (SAGE) has convened a working group to review the evidence on TCVs and produce an updated WHO position paper for all typhoid vaccines in 2018 that will inform Gavi, the Vaccine Alliance's future vaccine investment strategies for TCVs. The Typhoid Vaccine Acceleration Consortium (TyVAC) has been formed through a $36.9 million funding program from the Bill & Melinda Gates Foundation to accelerate the introduction of TCVs into Gavi-eligible countries. In October 2016, a meeting was held to initiate planning of TCV effectiveness studies that will provide the data required by policy makers and stakeholders to support decisions on TCV use in countries with a high typhoid burden. Discussion topics included (1) the latest evidence and data gaps in typhoid epidemiology; (2) WHO and Gavi methods and data requirements; (3) data on TCV efficacy; (4) cost effectiveness analysis for TCVs from mathematical models; (5) TCV delivery and effectiveness study design. Specifically, participants were asked to comment on study design in 3 sites for which population-based typhoid surveillance is underway. The conclusion of the meeting was that country-level decision making would best be informed by the respective selected sites in Africa and Asia vaccinating children aged from 9-months to 15-years-old, employing either an individual or cluster randomized design with design influenced by population characteristics, transmission dynamics, and statistical considerations.

    Vaccine 2017

  • Dietary (Poly)phenols, Brown Adipose Tissue Activation, and Energy Expenditure: A Narrative Review.

    Mele L, Bidault G, Mena P, Crozier A, Brighenti F, Vidal-Puig A and Del Rio D

    Laboratory of Phytochemicals in Physiology, Department of Food and Drugs, University of Parma, Parma, Italy.

    The incidence of overweight and obesity has reached epidemic proportions, making the control of body weight and its complications a primary health problem. Diet has long played a first-line role in preventing and managing obesity. However, beyond the obvious strategy of restricting caloric intake, growing evidence supports the specific antiobesity effects of some food-derived components, particularly (poly)phenolic compounds. The relatively new rediscovery of active brown adipose tissue in adult humans has generated interest in this tissue as a novel and viable target for stimulating energy expenditure and controlling body weight by promoting energy dissipation. This review critically discusses the evidence supporting the concept that the antiobesity effects ascribed to (poly)phenols might be dependent on their capacity to promote energy dissipation by activating brown adipose tissue. Although discrepancies exist in the literature, most in vivo studies with rodents strongly support the role of some (poly)phenol classes, particularly flavan-3-ols and resveratrol, in promoting energy expenditure. Some human data currently are available and most are consistent with studies in rodents. Further investigation of effects in humans is warranted.

    Advances in nutrition (Bethesda, Md.) 2017;8;5;694-704

  • Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47.

    Mentasti M, Cassier P, David S, Ginevra C, Gomez-Valero L, Underwood A, Afshar B, Etienne J, Parkhill J, Chalker V, Buchrieser C, Harrison TG, Jarraud S and ESCMID Study Group for Legionella Infections (ESGLI)

    Public Health England, London, UK.

    Objectives: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain.

    Methods: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47.

    Results: The gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO_1073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain.

    Conclusion: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

    Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 2017;23;4;264.e1-264.e9

  • Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

    Mentzer AV, Tobias J, Wiklund G, Nordqvist S, Aslett M, Dougan G, Sjöling Å and Svennerholm AM

    Department of Microbiology and Immunology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

    The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Although 22 antigenically different colonization factors (CFs) have been identified and characterized in ETEC at least 30% of clinical ETEC isolates lack known CFs. Ninety-four whole genome sequenced "CF negative" isolates were searched for novel CFs using a reverse genetics approach followed by phenotypic analyses. We identified a novel CF, CS30, encoded by a set of seven genes, csmA-G, related to the human CF operon CS18 and the porcine CF operon 987P (F6). CS30 was shown to be thermo-regulated, expressed at 37 °C, but not at 20 °C, by SDS-page and mass spectrometry analyses as well as electron microscopy imaging. Bacteria expressing CS30 were also shown to bind to differentiated human intestinal Caco-2 cells. The genes encoding CS30 were located on a plasmid (E873p3) together with the genes encoding LT and STp. PCR screening of ETEC isolates revealed that 8.6% (n = 13) of "CF negative" (n = 152) and 19.4% (n = 13) of "CF negative" LT + STp (n = 67) expressing isolates analyzed harbored CS30. Hence, we conclude that CS30 is common among "CF negative" LT + STp isolates and is associated with ETEC that cause diarrhea.

    Scientific reports 2017;7;1;12514

  • Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial.

    Mesa RA, Vannucchi AM, Mead A, Egyed M, Szoke A, Suvorov A, Jakucs J, Perkins A, Prasad R, Mayer J, Demeter J, Ganly P, Singer JW, Zhou H, Dean JP, Te Boekhorst PA, Nangalia J, Kiladjian JJ and Harrison CN

    Division of Hematology and Medical Oncology, Mayo Clinic, Scottsdale, AZ, USA. Electronic address:

    Background: Available therapies for myelofibrosis can exacerbate cytopenias and are not indicated for patients with severe thrombocytopenia. Pacritinib, which inhibits both JAK2 and FLT3, induced spleen responses with limited myelosuppression in phase 1/2 trials. We aimed to assess the efficacy and safety of pacritinib versus best available therapy in patients with myelofibrosis irrespective of baseline cytopenias.

    Methods: This international, multicentre, randomised, phase 3 trial (PERSIST-1) was done at 67 sites in 12 countries. Patients with higher-risk myelofibrosis (with no exclusions for baseline anaemia or thrombocytopenia) were randomly assigned (2:1) to receive oral pacritinib 400 mg once daily or best available therapy (BAT) excluding JAK2 inhibitors until disease progression or unacceptable toxicity. Randomisation was stratified by risk category, platelet count, and region. Treatment assignments were known to investigators, site personnel, patients, clinical monitors, and pharmacovigilance personnel. The primary endpoint was spleen volume reduction (SVR) of 35% or more from baseline to week 24 in the intention-to-treat population as assessed by blinded, centrally reviewed MRI or CT. We did safety analyses in all randomised patients who received either treatment. Here we present the final data. This trial is registered with, number NCT01773187.

    Findings: Between Jan 8, 2013, and Aug 1, 2014, 327 patients were randomly assigned to pacritinib (n=220) or BAT (n=107). Median follow-up was 23·2 months (IQR 14·8-28·7). At week 24, the primary endpoint of SVR of 35% or more was achieved by 42 (19%) patients in the pacritinib group versus five (5%) patients in the BAT group (p=0·0003). 90 patients in the BAT group crossed over to receive pacritinib at a median of 6·3 months (IQR 5·8-6·7). The most common grade 3-4 adverse events through week 24 were anaemia (n=37 [17%]), thrombocytopenia (n=26 [12%]), and diarrhoea (n=11 [5%]) in the pacritinib group, and anaemia (n=16 [15%]), thrombocytopenia (n=12 [11%]), dyspnoea (n=3 [3%]), and hypotension (n=3 [3%]) in the BAT group. The most common serious adverse events that occurred through week 24 were anaemia (10 [5%]), cardiac failure (5 [2%]), pyrexia (4 [2%]), and pneumonia (4 [2%]) with pacritinib, and anaemia (5 [5%]), sepsis (2 [2%]), and dyspnoea (2 [2%]) with BAT. Deaths due to adverse events were observed in 27 (12%) patients in the pacritinib group and 14 (13%) patients in the BAT group throughout the duration of the study.

    Interpretation: Pacritinib therapy was well tolerated and induced significant and sustained SVR and symptom reduction, even in patients with severe baseline cytopenias. Pacritinib could be a treatment option for patients with myelofibrosis, including those with baseline cytopenias for whom options are particularly limited.

    Funding: CTI BioPharma Corp.

    The Lancet. Haematology 2017

  • Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

    Messner CB, Driscoll PC, Piedrafita G, De Volder MFL and Ralser M

    The Molecular Biology of Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.

    The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

    Proceedings of the National Academy of Sciences of the United States of America 2017;114;28;7403-7407

  • Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

    Metzakopian E, Strong A, Iyer V, Hodgkins A, Tzelepis K, Antunes L, Friedrich MJ, Kang Q, Davidson T, Lamberth J, Hoffmann C, Davis GD, Vassiliou GS, Skarnes WC and Bradley A

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

    CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.

    Funded by: Wellcome Trust

    Scientific reports 2017;7;1;2244

  • Your DNA, Your Say.

    Middleton A

    a Society and Ethics Research, Connecting Science , Wellcome Genome Campus, Cambridge.

    Genomic and medical data sharing is pivotal if the promise of genomic medicine is to be fully realised. Social scientists working in the genomics arena ask the public 'how is the technology working for you?' Empirical studies on attitudes, values and beliefs are incredibly valuable; they offer a voice from those who are, or will be, directly affected. This is paramount if personalised medicine is to be truly personal. An International attitude study, Your DNA, Your Say, uses film to provide background information and an online survey to gather public views on donating one's own personal DNA and medical data for use by others. In this paper the rationale to the project is introduced together with an overview of the survey and film design. The project has been translated into multiple languages and the results will be used in policy for the Global Alliance for Genomics and Health.

    The New bioethics : a multidisciplinary journal of biotechnology and the body 2017;23;1;74-80

  • Gender Differences in Global but Not Targeted Demethylation in iPSC Reprogramming.

    Milagre I, Stubbs TM, King MR, Spindel J, Santos F, Krueger F, Bachman M, Segonds-Pichon A, Balasubramanian S, Andrews SR, Dean W and Reik W

    Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK. Electronic address:

    Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs) is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID)-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.

    Cell reports 2017;18;5;1079-1089

  • Deciphering the distance to antibiotic resistance for the pneumococcus using genome sequencing data.

    Mobegi FM, Cremers AJ, de Jonge MI, Bentley SD, van Hijum SA and Zomer A

    Laboratory of Pediatric Infectious Diseases, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen 6525 GA, The Netherlands.

    Advances in genome sequencing technologies and genome-wide association studies (GWAS) have provided unprecedented insights into the molecular basis of microbial phenotypes and enabled the identification of the underlying genetic variants in real populations. However, utilization of genome sequencing in clinical phenotyping of bacteria is challenging due to the lack of reliable and accurate approaches. Here, we report a method for predicting microbial resistance patterns using genome sequencing data. We analyzed whole genome sequences of 1,680 Streptococcus pneumoniae isolates from four independent populations using GWAS and identified probable hotspots of genetic variation which correlate with phenotypes of resistance to essential classes of antibiotics. With the premise that accumulation of putative resistance-conferring SNPs, potentially in combination with specific resistance genes, precedes full resistance, we retrogressively surveyed the hotspot loci and quantified the number of SNPs and/or genes, which if accumulated would confer full resistance to an otherwise susceptible strain. We name this approach the 'distance to resistance'. It can be used to identify the creep towards complete antibiotics resistance in bacteria using genome sequencing. This approach serves as a basis for the development of future sequencing-based methods for predicting resistance profiles of bacterial strains in hospital microbiology and public health settings.

    Scientific reports 2017;7;42808

  • Y-chromosomal sequences of diverse Indian populations and the ancestry of the Andamanese.

    Mondal M, Bergström A, Xue Y, Calafell F, Laayouni H, Casals F, Majumder PP, Tyler-Smith C and Bertranpetit J

    Institute of Evolutionary Biology (CSIC-UPF), Universitat Pompeu Fabra, Doctor Aiguader 88 (PRBB), 08003, Barcelona, Catalonia, Spain.

    We present 42 new Y-chromosomal sequences from diverse Indian tribal and non-tribal populations, including the Jarawa and Onge from the Andaman Islands, which are analysed within a calibrated Y-chromosomal phylogeny incorporating South Asian (in total 305 individuals) and worldwide (in total 1286 individuals) data from the 1000 Genomes Project. In contrast to the more ancient ancestry in the South than in the North that has been claimed, we detected very similar coalescence times within Northern and Southern non-tribal Indian populations. A closest neighbour analysis in the phylogeny showed that Indian populations have an affinity towards Southern European populations and that the time of divergence from these populations substantially predated the Indo-European migration into India, probably reflecting ancient shared ancestry rather than the Indo-European migration, which had little effect on Indian male lineages. Among the tribal populations, the Birhor (Austro-Asiatic-speaking) and Irula (Dravidian-speaking) are the nearest neighbours of South Asian non-tribal populations, with a common origin in the last few millennia. In contrast, the Riang (Tibeto-Burman-speaking) and Andamanese have their nearest neighbour lineages in East Asia. The Jarawa and Onge shared haplogroup D lineages with each other within the last ~7000 years, but had diverged from Japanese haplogroup D Y-chromosomes ~53000 years ago, most likely by a split from a shared ancestral population. This analysis suggests that Indian populations have complex ancestry which cannot be explained by a single expansion model.

    Human genetics 2017

  • Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes.

    Moradigaravand D, Jamrozy D, Mostowy R, Anderson A, Nickerson EK, Thaipadungpanit J, Wuthiekanun V, Limmathurotsakul D, Tandhavanant S, Wikraiphat C, Wongsuvan G, Teerawattanasook N, Jutrakul Y, Srisurat N, Chaimanee P, Eoin West T, Blane B, Parkhill J, Chantratita N and Peacock SJ

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, United Kingdom

    Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250.IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

    Funded by: Wellcome Trust

    mBio 2017;8;4

  • Evolution and Epidemiology of Multidrug-Resistant Klebsiella pneumoniae in the United Kingdom and Ireland.

    Moradigaravand D, Martin V, Peacock SJ and Parkhill J

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

    Klebsiella pneumoniae is a human commensal and opportunistic pathogen that has become a leading causative agent of hospital-based infections over the past few decades. The emergence and global expansion of hypervirulent and multidrug-resistant (MDR) clones of K. pneumoniae have been increasingly reported in community-acquired and nosocomial infections. Despite this, the population genomics and epidemiology of MDR K. pneumoniae at the national level are still poorly understood. To obtain insights into these, we analyzed a systematic large-scale collection of invasive MDR K. pneumoniae isolates from hospitals across the United Kingdom and Ireland. Using whole-genome phylogenetic analysis, we placed these in the context of previously sequenced K. pneumoniae populations from geographically diverse countries and identified their virulence and drug resistance determinants. Our results demonstrate that United Kingdom and Ireland MDR isolates are a highly diverse population drawn from across the global phylogenetic tree of K. pneumoniae and represent multiple recent international introductions that are mainly from Europe but in some cases from more distant countries. In addition, we identified novel genetic determinants underlying resistance to beta-lactams, gentamicin, ciprofloxacin, and tetracyclines, indicating that both increased virulence and resistance have emerged independently multiple times throughout the population. Our data show that MDR K. pneumoniae isolates in the United Kingdom and Ireland have multiple distinct origins and appear to be part of a globally circulating K. pneumoniae population.IMPORTANCEKlebsiella pneumoniae is a major human pathogen that has been implicated in infections in healthcare settings over the past few decades. Antimicrobial treatment of K. pneumoniae infections has become increasingly difficult as a consequence of the emergence and spread of strains that are resistant to multiple antimicrobials. To better understand the spread of resistant K. pneumoniae, we studied the genomes of a large-scale population of extensively antimicrobial-resistant K. pneumoniae in the United Kingdom and Ireland by utilizing the fine resolution that whole-genome sequencing of pathogen genomes provides. Our results indicate that the K. pneumoniae population is highly diverse and that, in some cases, resistant strains appear to have spread across the country over a few years. In addition, we found evidence that some strains have acquired antimicrobial resistance genes independently, presumably in response to antimicrobial treatment.

    mBio 2017;8;1

  • Pneumococcal capsule synthesis locus cps as evolutionary hotspot with potential to generate novel serotypes by recombination.

    Mostowy RJ, Croucher NJ, De Maio N, Chewapreecha C, Salter SJ, Turner P, Aanensen DM, Bentley SD, Didelot X and Fraser C

    Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, W2 1PG London, UK.

    Diversity of the polysaccharide capsule in Streptococcus pneumoniae -- main surface antigen and the target of the currently used pneumococcal vaccines -- constitutes a major obstacle in eliminating pneumococcal disease. Such diversity is genetically encoded by almost 100 variants of the capsule biosynthesis locus, cps. However, the evolutionary dynamics of the capsule remains not fully understood. Here, using genetic data from 4,519 bacterial isolates, we found cps to be an evolutionary hotspot with elevated substitution and recombination rates. These rates were a consequence of relaxed purifying selection and positive, diversifying selection acting at this locus, supporting the hypothesis that the capsule has an increased potential to generate novel diversity compared to the rest of the genome. Diversifying selection was particularly evident in the region of wzd/wze genes, which are known to regulate capsule expression and hence the bacterium's ability to cause disease. Using a novel, capsule-centred approach, we analysed the evolutionary history of twelve major serogroups. Such analysis revealed their complex diversification scenarios, which were principally driven by recombination with other serogroups and other streptococci. Patterns of recombinational exchanges between serogroups could not be explained by serotype frequency alone, thus pointing to non-random associations between co-colonising serotypes. Finally, we discovered a previously unobserved mosaic serotype 39X, which was confirmed to carry a viable and structurally novel capsule. Adding to previous discoveries of other mosaic capsules in densely sampled collections, these results emphasise the strong adaptive potential of the bacterium by its ability to generate novel antigenic diversity by recombination.

    Molecular biology and evolution 2017

  • Proteomic profiling of the brain of mice with experimental cerebral malaria.

    Moussa E, Huang H, Ahras M, Lall A, Thezenas ML, Fischer R, Kessler BM, Pain A, Billker O and Casals-Pascual C

    Wellcome Trust Centre for Human Genetics, Oxford, UK; King Abdulla University of Science and Technology, Saudi Arabia.

    Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma.

    Significance: Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM.

    Journal of proteomics 2017

  • Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

    Mukherjee A, Bopp S, Magistrado P, Wong W, Daniels R, Demas A, Schaffner S, Amaratunga C, Lim P, Dhorda M, Miotto O, Woodrow C, Ashley EA, Dondorp AM, White NJ, Wirth D, Fairhurst R and Volkman SK

    Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, 665 Huntington Avenue, I-704, Boston, MA, 02115, USA.

    Background: Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance.

    Methods: Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates.

    Results: Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance.

    Conclusions: This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

    Malaria journal 2017;16;1;195

  • Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts.

    Mupo A, Seiler M, Sathiaseelan V, Pance A, Yang Y, Agrawal AA, Iorio F, Bautista R, Pacharne S, Tzelepis K, Manes N, Wright P, Papaemmanuil E, Kent DG, Campbell PC, Buonamici S, Bolli N and Vassiliou GS

    Haematological Cancer Genetics, Wellcome Sanger Institute, Hinxton, Cambridge, UK.

    Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1(K700E/+) animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1(K700E/+) hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1(K700E/+) mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

    Funded by: Medical Research Council: MC_PC_12009; Wellcome Trust: 095663, 098051

    Leukemia 2017;31;3;720-727

  • Genomic landscape of extended-spectrum β-lactamase resistance in Escherichia coli from an urban African setting.

    Musicha P, Feasey NA, Cain AK, Kallonen T, Chaguza C, Peno C, Khonga M, Thompson S, Gray KJ, Mather AE, Heyderman RS, Everett DB, Thomson NR and Msefula CL

    Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Queen Elizabeth Central Hospital, Blantyre, Malawi.

    Objectives: Efforts to treat Escherichia coli infections are increasingly being compromised by the rapid, global spread of antimicrobial resistance (AMR). Whilst AMR in E. coli has been extensively investigated in resource-rich settings, in sub-Saharan Africa molecular patterns of AMR are not well described. In this study, we have begun to explore the population structure and molecular determinants of AMR amongst E. coli isolates from Malawi.

    Methods: Ninety-four E. coli isolates from patients admitted to Queen's Hospital, Malawi, were whole-genome sequenced. The isolates were selected on the basis of diversity of phenotypic resistance profiles and clinical source of isolation (blood, CSF and rectal swab). Sequence data were analysed using comparative genomics and phylogenetics.

    Results: Our results revealed the presence of five clades, which were strongly associated with E. coli phylogroups A, B1, B2, D and F. We identified 43 multilocus STs, of which ST131 (14.9%) and ST12 (9.6%) were the most common. We identified 25 AMR genes. The most common ESBL gene was bla CTX-M-15 and it was present in all five phylogroups and 11 STs, and most commonly detected in ST391 (4/4 isolates), ST648 (3/3 isolates) and ST131 [3/14 (21.4%) isolates].

    Conclusions: This study has revealed a high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi. The data highlight the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance.

    The Journal of antimicrobial chemotherapy 2017

  • Aboriginal Australian mitochondrial genome variation - an increased understanding of population antiquity and diversity.

    Nagle N, van Oven M, Wilcox S, van Holst Pellekaan S, Tyler-Smith C, Xue Y, Ballantyne KN, Wilcox L, Papac L, Cooke K, van Oorschot RA, McAllister P, Williams L, Kayser M, Mitchell RJ and Genographic Consortium

    Department of Biochemistry and Genetics, La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, Victoria, Australia.

    Aboriginal Australians represent one of the oldest continuous cultures outside Africa, with evidence indicating that their ancestors arrived in the ancient landmass of Sahul (present-day New Guinea and Australia) ~55 thousand years ago. Genetic studies, though limited, have demonstrated both the uniqueness and antiquity of Aboriginal Australian genomes. We have further resolved known Aboriginal Australian mitochondrial haplogroups and discovered novel indigenous lineages by sequencing the mitogenomes of 127 contemporary Aboriginal Australians. In particular, the more common haplogroups observed in our dataset included M42a, M42c, S, P5 and P12, followed by rarer haplogroups M15, M16, N13, O, P3, P6 and P8. We propose some major phylogenetic rearrangements, such as in haplogroup P where we delinked P4a and P4b and redefined them as P4 (New Guinean) and P11 (Australian), respectively. Haplogroup P2b was identified as a novel clade potentially restricted to Torres Strait Islanders. Nearly all Aboriginal Australian mitochondrial haplogroups detected appear to be ancient, with no evidence of later introgression during the Holocene. Our findings greatly increase knowledge about the geographic distribution and phylogenetic structure of mitochondrial lineages that have survived in contemporary descendants of Australia's first settlers.

    Scientific reports 2017;7;43041

  • Role of alanine racemase mutations in Mycobacterium tuberculosis D-cycloserine resistance.

    Nakatani Y, Opel-Reading HK, Merker M, Machado D, Andres S, Kumar SS, Moradigaravand D, Coll F, Perdigão J, Portugal I, Schön T, Nair D, Devi KRU, Kohl TA, Beckert P, Clark TG, Maphalala G, Khumalo D, Diel R, Klaos K, Aung HL, Cook GM, Parkhill J, Peacock SJ, Swaminathan S, Viveiros M, Niemann S, Krause KL and Köser CU

    University of Otago, Department of Microbiology and Immunology, Otago School of Medical Sciences, Dunedin, New Zealand.

    Screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to D-cycloserine.

    Antimicrobial agents and chemotherapy 2017

  • Estimating the human mutation rate from autozygous segments reveals population differences in human mutational processes.

    Narasimhan VM, Rahbari R, Scally A, Wuster A, Mason D, Xue Y, Wright J, Trembath RC, Maher ER, Heel DAV, Auton A, Hurles ME, Tyler-Smith C and Durbin R

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

    Heterozygous mutations within homozygous sequences descended from a recent common ancestor offer a way to ascertain de novo mutations across multiple generations. Using exome sequences from 3222 British-Pakistani individuals with high parental relatedness, we estimate a mutation rate of 1.45 ± 0.05 × 10(-8) per base pair per generation in autosomal coding sequence, with a corresponding non-crossover gene conversion rate of 8.75 ± 0.05 × 10(-6) per base pair per generation. This is at the lower end of exome mutation rates previously estimated in parent-offspring trios, suggesting that post-zygotic mutations contribute little to the human germ-line mutation rate. We find frequent recurrence of mutations at polymorphic CpG sites, and an increase in C to T mutations in a 5' CCG 3' to 5' CTG 3' context in the Pakistani population compared to Europeans, suggesting that mutational processes have evolved rapidly between human populations.Estimates of human mutation rates differ substantially based on the approach. Here, the authors present a multi-generational estimate from the autozygous segment in a non-European population that gives insight into the contribution of post-zygotic mutations and population-specific mutational processes.

    Nature communications 2017;8;1;303

  • Single cell transcriptomics of pluripotent stem cells: reprogramming and differentiation.

    Natarajan KN, Teichmann SA and Kolodziejczyk AA

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK; European Bioinformatics Institute, Wellcome Genome Campus, Cambridge, UK. Electronic address:

    Single-cell transcriptomics serves as a powerful tool to identify cell states within populations of cells, and to dissect underlying heterogeneity at high resolution. Single-cell transcriptomics on pluripotent stem cells has provided new insights into cellular variation, subpopulation structures and the interplay of cell cycle with pluripotency. The single-cell perspective has helped to better understand gene regulation and regulatory networks during exit from pluripotency, cell-fate determination as well as molecular mechanisms driving cellular reprogramming of somatic cells to induced pluripotent stage. Here we review the recent progress and significant findings from application of single-cell technologies on pluripotent stem cells along with a brief outlook on new combinatorial single-cell approaches that further unravel pluripotent stem cell states.

    Current opinion in genetics & development 2017;46;66-76

  • In Vivo Regulation of the Zebrafish Endoderm Progenitor Niche by T-Box Transcription Factors.

    Nelson AC, Cutty SJ, Gasiunas SN, Deplae I, Stemple DL and Wardle FC

    Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, UK; Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK; School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK. Electronic address:

    T-box transcription factors T/Brachyury homolog A (Ta) and Tbx16 are essential for correct mesoderm development in zebrafish. The downstream transcriptional networks guiding their functional activities are poorly understood. Additionally, important contributions elsewhere are likely masked due to redundancy. Here, we exploit functional genomic strategies to identify Ta and Tbx16 targets in early embryogenesis. Surprisingly, we discovered they not only activate mesodermal gene expression but also redundantly regulate key endodermal determinants, leading to substantial loss of endoderm in double mutants. To further explore the gene regulatory networks (GRNs) governing endoderm formation, we identified targets of Ta/Tbx16-regulated homeodomain transcription factor Mixl1, which is absolutely required in zebrafish for endoderm formation. Interestingly, we find many endodermal determinants coordinately regulated through common genomic occupancy by Mixl1, Eomesa, Smad2, Nanog, Mxtx2, and Pou5f3. Collectively, these findings augment the endoderm GRN and reveal a panel of target genes underlying the Ta, Tbx16, and Mixl1 mutant phenotypes.

    Cell reports 2017;19;13;2782-2795

  • Association analyses based on false discovery rate implicate new loci for coronary artery disease.

    Nelson CP, Goel A, Butterworth AS, Kanoni S, Webb TR, Marouli E, Zeng L, Ntalla I, Lai FY, Hopewell JC, Giannakopoulou O, Jiang T, Hamby SE, Di Angelantonio E, Assimes TL, Bottinger EP, Chambers JC, Clarke R, Palmer CNA, Cubbon RM, Ellinor P, Ermel R, Evangelou E, Franks PW, Grace C, Gu D, Hingorani AD, Howson JMM, Ingelsson E, Kastrati A, Kessler T, Kyriakou T, Lehtimäki T, Lu X, Lu Y, März W, McPherson R, Metspalu A, Pujades-Rodriguez M, Ruusalepp A, Schadt EE, Schmidt AF, Sweeting MJ, Zalloua PA, AlGhalayini K, Keavney BD, Kooner JS, Loos RJF, Patel RS, Rutter MK, Tomaszewski M, Tzoulaki I, Zeggini E, Erdmann J, Dedoussis G, Björkegren JLM, EPIC-CVD Consortium, CARDIoGRAMplusC4D, UK Biobank CardioMetabolic Consortium CHD working group, Schunkert H, Farrall M, Danesh J, Samani NJ, Watkins H and Deloukas P

    Department of Cardiovascular Sciences, University of Leicester, Leicester, UK.

    Genome-wide association studies (GWAS) in coronary artery disease (CAD) had identified 66 loci at 'genome-wide significance' (P < 5 × 10(-8)) at the time of this analysis, but a much larger number of putative loci at a false discovery rate (FDR) of 5% (refs. 1,2,3,4). Here we leverage an interim release of UK Biobank (UKBB) data to evaluate the validity of the FDR approach. We tested a CAD phenotype inclusive of angina (SOFT; ncases = 10,801) as well as a stricter definition without angina (HARD; ncases = 6,482) and selected cases with the former phenotype to conduct a meta-analysis using the two most recent CAD GWAS. This approach identified 13 new loci at genome-wide significance, 12 of which were on our previous list of loci meeting the 5% FDR threshold, thus providing strong support that the remaining loci identified by FDR represent genuine signals. The 304 independent variants associated at 5% FDR in this study explain 21.2% of CAD heritability and identify 243 loci that implicate pathways in blood vessel morphogenesis as well as lipid metabolism, nitric oxide signaling and inflammation.

    Nature genetics 2017

  • A Genome-wide Association Study of Dupuytren Disease Reveals 17 Additional Variants Implicated in Fibrosis.

    Ng M, Thakkar D, Southam L, Werker P, Ophoff R, Becker K, Nothnagel M, Franke A, Nürnberg P, Espirito-Santo AI, Izadi D, Hennies HC, Nanchahal J, Zeggini E and Furniss D

    Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Science, University of Oxford, Botnar Research Centre, Windmill Road, Oxford OX3 7HE, UK.

    Individuals with Dupuytren disease (DD) are commonly seen by physicians and surgeons across multiple specialties. It is an increasingly common and disabling fibroproliferative disorder of the palmar fascia, which leads to flexion contractures of the digits, and is associated with other tissue-specific fibroses. DD affects between 5% and 25% of people of European descent and is the most common inherited disease of connective tissue. We undertook the largest GWAS to date in individuals with a surgically validated diagnosis of DD from the UK, with replication in British, Dutch, and German individuals. We validated association at all nine previously described signals and discovered 17 additional variants with p ≤ 5 × 10(-8). As a proof of principle, we demonstrated correlation of the high-risk genotype at the statistically most strongly associated variant with decreased secretion of the soluble WNT-antagonist SFRP4, in surgical specimen-derived DD myofibroblasts. These results highlight important pathways involved in the pathogenesis of fibrosis, including WNT signaling, extracellular matrix modulation, and inflammation. In addition, many associated loci contain genes that were hitherto unrecognized as playing a role in fibrosis, opening up new avenues of research that may lead to novel treatments for DD and fibrosis more generally. DD represents an ideal human model disease for fibrosis research.

    American journal of human genetics 2017;101;3;417-427

  • Tracing the peopling of the world through genomics.

    Nielsen R, Akey JM, Jakobsson M, Pritchard JK, Tishkoff S and Willerslev E

    Department of Integrative Biology, University of California, Berkeley, Berkeley, California 94720, USA.

    Advances in the sequencing and the analysis of the genomes of both modern and ancient peoples have facilitated a number of breakthroughs in our understanding of human evolutionary history. These include the discovery of interbreeding between anatomically modern humans and extinct hominins; the development of an increasingly detailed description of the complex dispersal of modern humans out of Africa and their population expansion worldwide; and the characterization of many of the genetic adaptions of humans to local environmental conditions. Our interpretation of the evolutionary history and adaptation of humans is being transformed by analyses of these new genomic data.

    Nature 2017;541;7637;302-310

  • Mutational Signatures in Breast Cancer: The Problem at the DNA Level.

    Nik-Zainal S and Morganella S

    Wellcome Trust Sanger Institute, Hinxton Genome Campus, Cambridge, United Kingdom.

    A breast cancer genome is a record of the historic mutagenic activity that has occurred throughout the development of the tumor. Indeed, every mutation may be informative. Although driver mutations were the main focus of cancer research for a long time, passenger mutational signatures, the imprints of DNA damage and DNA repair processes that have been operative during tumorigenesis, are also biologically illuminating. This review is a chronicle of how the concept of mutational signatures arose and brings the reader up-to-date on this field, particularly in breast cancer. Mutational signatures have now been advanced to include mutational processes that involve rearrangements, and novel cancer biological insights have been gained through studying these in great detail. Furthermore, there are efforts to take this field into the clinical sphere. If validated, mutational signatures could thus form an additional weapon in the arsenal of cancer precision diagnostics and therapeutic stratification in the modern war against cancer. Clin Cancer Res; 23(11); 2617-29. ©2017 AACRSee all articles in this CCR Focus section, "Breast Cancer Research: From Base Pairs to Populations."

    Funded by: Wellcome Trust

    Clinical cancer research : an official journal of the American Association for Cancer Research 2017;23;11;2617-2629

  • Protein-Truncating Variants at the Cholesteryl Ester Transfer Protein Gene and Risk for Coronary Heart Disease.

    Nomura A, Won HH, Khera AV, Takeuchi F, Ito K, McCarthy S, Emdin CA, Klarin D, Natarajan P, Zekavat SM, Gupta N, Peloso GM, Borecki IB, Teslovich TM, Asselta R, Duga S, Merlini PA, Correa A, Kessler T, Wilson JG, Bown MJ, Hall AS, Braund PS, Carey DJ, Murray MF, Kirchner HL, Leader JB, Lavage DR, Manus JN, Hartze DN, Samani NJ, Schunkert H, Marrugat J, Elosua R, McPherson R, Farrall M, Watkins H, Juang JJ, Hsiung CA, Lin SY, Wang JS, Tada H, Kawashiri MA, Inazu A, Yamagishi M, Katsuya T, Nakashima E, Nakatochi M, Yamamoto K, Yokota M, Momozawa Y, Rotter JI, Lander ES, Rader DJ, Danesh J, Ardissino D, Gabriel S, Willer CJ, Abecasis GR, Saleheen D, Kubo M, Kato N, Ida Chen YD, Dewey FE and Kathiresan S

    Rationale: Therapies that inhibit CETP (cholesteryl ester transfer protein) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human DNA sequence variants that truncate the CETP gene may provide insight into the efficacy of CETP inhibition.

    Objective: To test whether protein-truncating variants (PTVs) at the CETP gene were associated with plasma lipid levels and CHD.

    Methods and results: We sequenced the exons of the CETP gene in 58 469 participants from 12 case-control studies (18 817 CHD cases, 39 652 CHD-free controls). We defined PTV as those that lead to a premature stop, disrupt canonical splice sites, or lead to insertions/deletions that shift frame. We also genotyped 1 Japanese-specific PTV in 27561 participants from 3 case-control studies (14 286 CHD cases, 13 275 CHD-free controls). We tested association of CETP PTV carrier status with both plasma lipids and CHD. Among 58 469 participants with CETP gene-sequencing data available, average age was 51.5 years and 43% were women; 1 in 975 participants carried a PTV at the CETP gene. Compared with noncarriers, carriers of PTV at CETP had higher high-density lipoprotein cholesterol (effect size, 22.6 mg/dL; 95% confidence interval, 18-27; P<1.0×10(-4)), lower low-density lipoprotein cholesterol (-12.2 mg/dL; 95% confidence interval, -23 to -0.98; P=0.033), and lower triglycerides (-6.3%; 95% confidence interval, -12 to -0.22; P=0.043). CETP PTV carrier status was associated with reduced risk for CHD (summary odds ratio, 0.70; 95% confidence interval, 0.54-0.90; P=5.1×10(-3)).

    Conclusions: Compared with noncarriers, carriers of PTV at CETP displayed higher high-density lipoprotein cholesterol, lower low-density lipoprotein cholesterol, lower triglycerides, and lower risk for CHD.

    Circulation research 2017;121;1;81-88

  • A graph extension of the positional Burrows-Wheeler transform and its applications.

    Novak AM, Garrison E and Paten B

    Genomics Institute, University of California Santa Cruz, CBSE, 501C Engineering 2, MS: CBSE, 1156 High St., Santa Cruz, CA 95064 USA.

    We present a generalization of the positional Burrows-Wheeler transform, or PBWT, to genome graphs, which we call the gPBWT. A genome graph is a collapsed representation of a set of genomes described as a graph. In a genome graph, a haplotype corresponds to a restricted form of walk. The gPBWT is a compressible representation of a set of these graph-encoded haplotypes that allows for efficient subhaplotype match queries. We give efficient algorithms for gPBWT construction and query operations. As a demonstration, we use the gPBWT to quickly count the number of haplotypes consistent with random walks in a genome graph, and with the paths taken by mapped reads; results suggest that haplotype consistency information can be practically incorporated into graph-based read mappers. We estimate that with the gPBWT of the order of 100,000 diploid genomes, including all forms structural variation, could be stored and made searchable for haplotype queries using a single large compute node.

    Algorithms for molecular biology : AMB 2017;12;18

  • A population-based analysis of germline BAP1 mutations in melanoma.

    O'Shea SJ, Robles-Espinoza CD, McLellan L, Harrigan J, Jacq X, Hewinson J, Iyer V, Merchant W, Elliott F, Harland M, Timothy Bishop D, Newton-Bishop J and Adams DJ

    Section of Epidemiology and Biostatistics, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, LS9 7TF.

    Germline mutation of the BRCA1 associated protein-1 (BAP1) gene has been linked to uveal melanoma, mesothelioma, meningioma, renal cell carcinoma and basal cell carcinoma. Germline variants have also been found in familial cutaneous melanoma pedigrees, but their contribution to sporadic melanoma has not been fully assessed. We sequenced BAP1 in 1,977 melanoma cases and 754 controls and used deubiquitinase assays, a pedigree analysis, and a histopathological review to assess the consequences of the mutations found. Sequencing revealed 30 BAP1 variants in total, of which 27 were rare (ExAc allele frequency <0.002). Of the 27 rare variants, 22 were present in cases (18 missense, one splice acceptor, one frameshift and two near splice regions) and 5 in controls (all missense). A missense change (S98R) in a case that completely abolished BAP1 deubiquitinase activity was identified. Analysis of cancers in the pedigree of the proband carrying the S98R variant and in two other pedigrees carrying clear loss-of-function alleles showed the presence of BAP1-associated cancers such as renal cell carcinoma, mesothelioma and meningioma, but not uveal melanoma. Two of these three probands carrying BAP1 loss-of-function variants also had melanomas with histopathological features suggestive of a germline BAP1 mutation. The remaining cases with germline mutations, which were predominantly missense mutations, were associated with less typical pedigrees and tumours lacking a characteristic BAP1-associated histopathological appearances, but may still represent less penetrant variants. Germline BAP1 alleles defined as loss-of-function or predicted to be deleterious/damaging are rare in melanoma.

    Human molecular genetics 2017

  • Micro-epidemiological structuring of Plasmodium falciparum parasite populations in regions with varying transmission intensities in Africa.

    Omedo I, Mogeni P, Bousema T, Rockett K, Amambua-Ngwa A, Oyier I, C Stevenson J, Y Baidjoe A, de Villiers EP, Fegan G, Ross A, Hubbart C, Jeffreys A, N Williams T, Kwiatkowski D and Bejon P

    KEMRI-Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, Kilifi, Kenya.

    Background: The first models of malaria transmission assumed a completely mixed and homogeneous population of parasites.  Recent models include spatial heterogeneity and variably mixed populations. However, there are few empiric estimates of parasite mixing with which to parametize such models. Methods: Here we genotype 276 single nucleotide polymorphisms (SNPs) in 5199 P. falciparum isolates from two Kenyan sites and one Gambian site to determine the spatio-temporal extent of parasite mixing, and use Principal Component Analysis (PCA) and linear regression to examine the relationship between genetic relatedness and relatedness in space and time for parasite pairs. Results: We show that there are no discrete geographically restricted parasite sub-populations, but instead we see a diffuse spatio-temporal structure to parasite genotypes.  Genetic relatedness of sample pairs is predicted by relatedness in space and time. Conclusions: Our findings suggest that targeted malaria control will benefit the surrounding community, but unfortunately also that emerging drug resistance will spread rapidly through the population.

    Wellcome open research 2017;2;10

  • Geographic-genetic analysis of Plasmodium falciparum parasite populations from surveys of primary school children in Western Kenya.

    Omedo I, Mogeni P, Rockett K, Kamau A, Hubbart C, Jeffreys A, Ochola-Oyier LI, de Villiers EP, Gitonga CW, Noor AM, Snow RW, Kwiatkowski D and Bejon P

    KEMRI-Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, Kilifi, Kenya.

    Background. Malaria control, and finally malaria elimination, requires the identification and targeting of residual foci or hotspots of transmission. However, the level of parasite mixing within and between geographical locations is likely to impact the effectiveness and durability of control interventions and thus should be taken into consideration when developing control programs. Methods. In order to determine the geographic-genetic patterns of Plasmodium falciparum parasite populations at a sub-national level in Kenya, we used the Sequenom platform to genotype 111 genome-wide distributed single nucleotide polymorphic (SNP) positions in 2486 isolates collected from children in 95 primary schools in western Kenya. We analysed these parasite genotypes for genetic structure using principal component analysis and assessed local and global clustering using statistical measures of spatial autocorrelation. We further examined the region for spatial barriers to parasite movement as well as directionality in the patterns of parasite movement. Results. We found no evidence of population structure and little evidence of spatial autocorrelation of parasite genotypes (correlation coefficients <0.03 among parasite pairs in distance classes of 1km, 2km and 5km; p value<0.01). An analysis of the geographical distribution of allele frequencies showed weak evidence of variation in distribution of alleles, with clusters representing a higher than expected number of samples with the major allele being identified for 5 SNPs. Furthermore, we found no evidence of the existence of spatial barriers to parasite movement within the region, but observed directional movement of parasites among schools in two separate sections of the region studied. Conclusions. Our findings illustrate a pattern of high parasite mixing within the study region. If this mixing is due to rapid gene flow, then "one-off" targeted interventions may not be currently effective at the sub-national scale in Western Kenya, due to the high parasite movement that is likely to lead to re-introduction of infection from surrounding regions. However repeated targeted interventions may reduce transmission in the surrounding regions.

    Wellcome open research 2017;2;29

  • The role of sapA and yfgA in susceptibility to antibody-mediated complement-dependent killing and virulence of Salmonella enterica Typhimurium.

    Ondari EM, Heath JN, Klemm EJ, Langridge G, Barquist L, Goulding DA, Clare S, Dougan G, Kingsley RA and MacLennan CA

    Swiss Tropical Public Health Institute, Basel, Switzerland.

    The ST313 pathovar of S Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S Typhimurium. Vaccination is an attractive disease-prevention strategy and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S Typhimurium to immune serum identified a repertoire of S Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation and metabolism. We generated defined mutant derivatives, using S Typhimurium SL1344 as host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S Typhimurium yfgA(-) mutant lost the characteristic Salmonella rod-shape appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes and demonstrated decreased ability to infect mice. In contrast, the S Typhimurium sapA(-) mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile, and an increased ability to infect enterocytes compared with wild-type, but had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence, and suggest the presence of different mechanisms of antibody resistance.

    Infection and immunity 2017

  • Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.

    Ong SH, Li Y, Koike-Yusa H and Yusa K

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

    Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.

    Scientific reports 2017;7;1;7384

  • Human Germline Genome Editing.

    Ormond KE, Mortlock DP, Scholes DT, Bombard Y, Brody LC, Faucett WA, Garrison NA, Hercher L, Isasi R, Middleton A, Musunuru K, Shriner D, Virani A and Young CE

    Department of Genetics and Stanford Center for Biomedical Ethics, School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

    With CRISPR/Cas9 and other genome-editing technologies, successful somatic and germline genome editing are becoming feasible. To respond, an American Society of Human Genetics (ASHG) workgroup developed this position statement, which was approved by the ASHG Board in March 2017. The workgroup included representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counselors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The statement includes the following positions. (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research. (3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.

    Funded by: NHGRI NIH HHS: ZIA HG200362

    American journal of human genetics 2017;101;2;167-176

  • Variability of human pluripotent stem cell lines.

    Ortmann D and Vallier L

    Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute @ Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, UK.

    Human pluripotent stem cells derived from embryos (human Embryonic Stem Cells or hESCs) or generated by direct reprogramming of somatic cells (human Induced Pluripotent Stem Cells or hiPSCs) can proliferate almost indefinitely in vitro while maintaining the capacity to differentiate into a broad diversity of cell types. These two properties (self-renewal and pluripotency) confers human pluripotent stem cells a unique interest for clinical applications since they could allow the production of infinite quantities of cells for disease modelling, drug screening and cell based therapy. However, recent studies have clearly established that human pluripotent stem cell lines can display variable capacity to differentiate into specific lineages. Consequently, the development of universal protocols of differentiation which could work efficiently with any human pluripotent cell line is complicated substantially. As a consequence, each protocol needs to be adapted to every cell line thereby limiting large scale applications and precluding personalised therapies. Here, we summarise our knowledge concerning the origin of this variability and describe potential solutions currently available to bypass this major challenge.

    Current opinion in genetics & development 2017;46;179-185

  • Transcriptome and proteome analysis of Salmonella enterica serovar Typhimurium systemic infection of wild type and immune-deficient mice.

    Oshota O, Conway M, Fookes M, Schreiber F, Chaudhuri RR, Yu L, Morgan FJE, Clare S, Choudhary J, Thomson NR, Lio P, Maskell DJ, Mastroeni P and Grant AJ

    Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.

    Salmonella enterica are a threat to public health. Current vaccines are not fully effective. The ability to grow in infected tissues within phagocytes is required for S. enterica virulence in systemic disease. As the infection progresses the bacteria are exposed to a complex host immune response. Consequently, in order to continue growing in the tissues, S. enterica requires the coordinated regulation of fitness genes. Bacterial gene regulation has so far been investigated largely using exposure to artificial environmental conditions or to in vitro cultured cells, and little information is available on how S. enterica adapts in vivo to sustain cell division and survival. We have studied the transcriptome, proteome and metabolic flux of Salmonella, and the transcriptome of the host during infection of wild type C57BL/6 and immune-deficient gp91-/-phox mice. Our analyses advance the understanding of how S. enterica and the host behaves during infection to a more sophisticated level than has previously been reported.

    PloS one 2017;12;8;e0181365

  • Melanoma: a global perspective.

    Ossio R, Roldán-Marín R, Martínez-Said H, Adams DJ and Robles-Espinoza CD

    Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Campus Juriquilla, Blvd Juriquilla 3001, Santiago de Querétaro 76230, México.

    Nature reviews. Cancer 2017

  • Emergence and clonal spread of colistin resistance due to multiple mutational mechanisms in carbapenemase-producing Klebsiella pneumoniae in London.

    Otter JA, Doumith M, Davies F, Mookerjee S, Dyakova E, Gilchrist M, Brannigan ET, Bamford K, Galletly T, Donaldson H, Aanensen DM, Ellington MJ, Hill R, Turton JF, Hopkins KL, Woodford N and Holmes A

    National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infection and Antimicrobial Resistance at Imperial College London & Public Health England, Hammersmith Hospital, Du Cane Road, W12 0HS, London, UK.

    Carbapenemase-producing Enterobacteriaceae (CPE) are emerging worldwide, limiting therapeutic options. Mutational and plasmid-mediated mechanisms of colistin resistance have both been reported. The emergence and clonal spread of colistin resistance was analysed in 40 epidemiologically-related NDM-1 carbapenemase producing Klebsiella pneumoniae isolates identified during an outbreak in a group of London hospitals. Isolates from July 2014 to October 2015 were tested for colistin susceptibility using agar dilution, and characterised by whole genome sequencing (WGS). Colistin resistance was detected in 25/38 (65.8%) cases for which colistin susceptibility was tested. WGS found that three potential mechanisms of colistin resistance had emerged separately, two due to different mutations in mgrB, and one due to a mutation in phoQ, with onward transmission of two distinct colistin-resistant variants, resulting in two sub-clones associated with transmission at separate hospitals. A high rate of colistin resistance (66%) emerged over a 10 month period. WGS demonstrated that mutational colistin resistance emerged three times during the outbreak, with transmission of two colistin-resistant variants.

    Scientific reports 2017;7;1;12711

  • Characterization of Posa and Posa-like virus genomes in fecal samples from humans, pigs, rats, and bats collected from a single location in Vietnam.

    Oude Munnink BB, Phan MVT, VIZIONS Consortium, Simmonds P, Koopmans MPG, Kellam P, van der Hoek L and Cotten M

    Department of Virus Genomics, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK.

    Porcine stool-associated RNA virus (posavirus), and Human stool-associated RNA virus (husavirus) are viruses in the order Picornavirales recently described in porcine and human fecal samples. The tentative group (Posa and Posa-like viruses: PPLVs) also includes fish stool-associated RNA virus (fisavirus) as well as members detected in insects (Drosophila subobscura and Anopheles sinensis) and parasites (Ascaris suum). As part of an agnostic deep sequencing survey of animal and human viruses in Vietnam, we detected three husaviruses in human fecal samples, two of which share 97-98% amino acid identity to Dutch husavirus strains and one highly divergent husavirus with only 25% amino acid identity to known husaviruses. In addition, the current study found forty-seven complete posavirus genomes from pigs, ten novel rat stool-associated RNA virus genomes (tentatively named rasavirus), and sixteen novel bat stool-associated RNA virus genomes (tentatively named basavirus). The five expected Picornavirales protein domains (helicase, 3C-protease, RNA-dependent RNA polymerase, and two Picornavirus capsid domain) were found to be encoded by all PPLV genomes. In addition, a nucleotide composition analysis revealed that the PPLVs shared compositional properties with arthropod viruses and predicted non-mammalian hosts for all PPLV lineages. The study adds seventy-six genomes to the twenty-nine PPLV genomes currently available and greatly extends our sequence knowledge of this group of viruses within the Picornavirales order.

    Virus evolution 2017;3;2;vex022

  • Accurate Classification of Protein Subcellular Localization from High Throughput Microscopy Images Using Deep Learning.

    Pärnamaa T and Parts L

    University of Tartu.

    High throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high throughput microscopy.

    G3 (Bethesda, Md.) 2017

  • An Ethnolinguistic and Genetic Perspective on the Origins of the Dravidian-Speaking Brahui in Pakistan.

    Pagani L, Colonna V, Tyler-Smith C and Ayub Q

    Department of Archaeology and Anthropology, University of Cambridge, United Kingdom, Department of Biological, Geological and Environmental Sciences, University of Bologna, Italy.

    Pakistan is a part of South Asia that modern humans encountered soon after they left Africa ~50 - 70,000 years ago. Approximately 9,000 years ago they began establishing cities that eventually expanded to represent the Harappan culture, rivalling the early city states of Mesopotamia. The modern state constitutes the north western land mass of the Indian sub-continent and is now the abode of almost 200 million humans representing many ethnicities and linguistic groups. Studies utilising autosomal, Y chromosomal and mitochondrial DNA markers in selected Pakistani populations revealed a mixture of Western Eurasian-, South- and East Asian-specific lineages, some of which were unequivocally associated with past migrations. Overall in Pakistan, genetic relationships are generally predicted more accurately by geographic proximity than linguistic origin. The Dravidian-speaking Brahui population are a prime example of this. They currently reside in south-western Pakistan, surrounded by Indo-Europeans speakers with whom they share a common genetic origin. In contrast, the Hazara share the highest affinity with East Asians, despite their Indo-European linguistic affiliation. In this report we reexamine the genetic origins of the Brahuis, and compare them with diverse populations from India, including several Dravidian-speaking groups, and present a genetic perspective on ethnolinguistic groups in present-day Pakistan. Given the high affinity of Brahui to the other Indo-European Pakistani populations and the absence of population admixture with any of the examined Indian Dravidian groups, we conclude that Brahui are an example of cultural (linguistic) retention following a major population replacement.

    Man in India 2017;97;1;267-278

  • Evolve and survive.

    Pance A

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

    Nature reviews. Microbiology 2017

  • Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

    Paquet T, Le Manach C, Cabrera DG, Younis Y, Henrich PP, Abraham TS, Lee MCS, Basak R, Ghidelli-Disse S, Lafuente-Monasterio MJ, Bantscheff M, Ruecker A, Blagborough AM, Zakutansky SE, Zeeman AM, White KL, Shackleford DM, Mannila J, Morizzi J, Scheurer C, Angulo-Barturen I, Martínez MS, Ferrer S, Sanz LM, Gamo FJ, Reader J, Botha M, Dechering KJ, Sauerwein RW, Tungtaeng A, Vanachayangkul P, Lim CS, Burrows J, Witty MJ, Marsh KC, Bodenreider C, Rochford R, Solapure SM, Jiménez-Díaz MB, Wittlin S, Charman SA, Donini C, Campo B, Birkholtz LM, Hanson KK, Drewes G, Kocken CHM, Delves MJ, Leroy D, Fidock DA, Waterson D, Street LJ and Chibale K

    Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

    As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

    Funded by: NIAID NIH HHS: R01 AI103058, R01 AI109023

    Science translational medicine 2017;9;387

  • Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling.

    Pardo M, Bode D, Yu L and Choudhary JS

    Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute;

    Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

    Journal of visualized experiments : JoVE 2017;122

  • Myst2/Kat7 histone acetyltransferase interaction proteomics reveals tumour-suppressor Niam as a novel binding partner in embryonic stem cells.

    Pardo M, Yu L, Shen S, Tate P, Bode D, Letney BL, Quelle DE, Skarnes W and Choudhary JS

    Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom.

    MYST histone acetyltransferases have crucial functions in transcription, replication and DNA repair and are hence implicated in development and cancer. Here we characterise Myst2/Kat7/Hbo1 protein interactions in mouse embryonic stem cells by affinity purification coupled to mass spectrometry. This study confirms that in embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2-associated protein, the tumour suppressor protein Niam (Nuclear Interactor of ARF and Mdm2). Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and Niam in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism early development and survival as well as cancer. Data are available via ProteomeXchange with identifier PXD005987.

    Scientific reports 2017;7;1;8157

  • Design and Application of Multiplex PCR Seq for the Detection of Somatic Mutations Associated with Myeloid Malignancies.

    Park N and Vassiliou G

    DNA Pipelines Research and Development, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.

    Targeted sequencing, in which only a selected set of genomic loci are sequenced, enables a much higher coverage of each target than what is obtained using whole genome or exome sequencing. Multiplex PCR offers a simple and affordable technique for specific capture of target regions and can be easily adapted to generate next-generation sequencing (NGS)-ready amplicons. Here we describe a multiplex PCR (MxPCR) approach for capturing 13 leukemia-associated mutation hotspots followed by MiSeq sequencing that enables robust detection of mutations with a variant allele fraction (VAF) as low as 0.8% (0.008) in blood DNA.

    Methods in molecular biology (Clifton, N.J.) 2017;1633;87-99

  • Physical enrichment of transposon mutants from saturation mutant libraries using the TraDISort approach.

    Paulsen IT, Cain AK and Hassan KA

    Department of Chemistry and Biomolecular Science, Macquarie University, North Ryde, NSW, Australia.

    Transposon-insertion sequencing methods are finding their way into the molecular toolbox of many fields of microbiology. These methods can identify the genomic locations and density of transposon insertions in saturated transposon mutant libraries and can be used to make inferences on gene function. For example, where no insertions or very few insertions are identified within a gene in a mutant library grown under permissive conditions, the gene may be essential. Furthermore, where mutations are enriched or lost in a gene after passaging the library through a selective process, the gene is likely to be involved in phenotypes linked to the process. Typically, a fitness based selection such as a stress condition is used in these experiments and the processed sequencing data are used to identify genes required for fitness under the selection. Our research team recently expanded the utility of the transposon directed insertion sequencing (TraDIS) method by applying a physical separation of a transposon mutant library mediated by fluorescence activated cell sorting, rather than a fitness-based selection. This approach, which we have named "TraDISort" is significant because it allows the study of phenotypes that are not linked to cell survival. The TraDISort approach has a broad range of future applications, in drug development, metabolic engineering and in studies of basic bacterial cell physiology.

    Mobile genetic elements 2017;7;3;1-7

  • Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes.

    Pawlowski M, Ortmann D, Bertero A, Tavares JM, Pedersen RA, Vallier L and Kotter MRN

    Anne McLaren Laboratory, Wellcome Trust-MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, UK; Department of Clinical Neuroscience, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address:

    The isolation or in vitro derivation of many human cell types remains challenging and inefficient. Direct conversion of human pluripotent stem cells (hPSCs) by forced expression of transcription factors provides a potential alternative. However, deficient inducible gene expression in hPSCs has compromised efficiencies of forward programming approaches. We have systematically optimized inducible gene expression in hPSCs using a dual genomic safe harbor gene-targeting strategy. This approach provides a powerful platform for the generation of human cell types by forward programming. We report robust and deterministic reprogramming of hPSCs into neurons and functional skeletal myocytes. Finally, we present a forward programming strategy for rapid and highly efficient generation of human oligodendrocytes.

    Stem cell reports 2017;8;4;803-812

  • Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies.

    Payne RO, Silk SE, Elias SC, Milne KH, Rawlinson TA, Llewellyn D, Shakri AR, Jin J, Labbé GM, Edwards NJ, Poulton ID, Roberts R, Farid R, Jørgensen T, Alanine DG, de Cassan SC, Higgins MK, Otto TD, McCarthy JS, de Jongh WA, Nicosia A, Moyle S, Hill AV, Berrie E, Chitnis CE, Lawrie AM and Draper SJ

    The Jenner Institute, University of Oxford, Oxford, United Kingdom.

    Background: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vaccination.

    Methods: Safety and immunogenicity of replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA) viral vectored vaccines targeting PvDBP_RII (Salvador I strain) were assessed in an open-label dose-escalation phase Ia study in 24 healthy UK adults. Vaccines were delivered by the intramuscular route in a ChAd63-MVA heterologous prime-boost regimen using an 8-week interval.

    Results: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. PvDBP_RII-specific ex-vivo IFN-γ T cell, antibody-secreting cell, memory B cell, and serum IgG responses were observed after the MVA boost immunization. Vaccine-induced antibodies inhibited the binding of vaccine homologous and heterologous variants of recombinant PvDBP_RII to the DARC receptor, with median 50% binding-inhibition titers greater than 1:100.

    Conclusion: We have demonstrated for the first time to our knowledge that strain-transcending antibodies can be induced against the PvDBP_RII antigen by vaccination in humans. These vaccine candidates warrant further clinical evaluation of efficacy against the blood-stage P. vivax parasite.

    Trial registration: NCT01816113.

    Funding: Support was provided by the UK Medical Research Council, UK National Institute of Health Research Oxford Biomedical Research Centre, and the Wellcome Trust.

    JCI insight 2017;2;12

  • SC83288 is a clinical development candidate for the treatment of severe malaria.

    Pegoraro S, Duffey M, Otto TD, Wang Y, Rösemann R, Baumgartner R, Fehler SK, Lucantoni L, Avery VM, Moreno-Sabater A, Mazier D, Vial HJ, Strobl S, Sanchez CP and Lanzer M

    4SC AG, Am Klopferspitz 19a, 82152 Martinsried, Germany.

    Severe malaria is a life-threatening complication of an infection with the protozoan parasite Plasmodium falciparum, which requires immediate treatment. Safety and efficacy concerns with currently used drugs accentuate the need for new chemotherapeutic options against severe malaria. Here we describe a medicinal chemistry program starting from amicarbalide that led to two compounds with optimized pharmacological and antiparasitic properties. SC81458 and the clinical development candidate, SC83288, are fast-acting compounds that can cure a P. falciparum infection in a humanized NOD/SCID mouse model system. Detailed preclinical pharmacokinetic and toxicological studies reveal no observable drawbacks. Ultra-deep sequencing of resistant parasites identifies the sarco/endoplasmic reticulum Ca(2+) transporting PfATP6 as a putative determinant of resistance to SC81458 and SC83288. Features, such as fast parasite killing, good safety margin, a potentially novel mode of action and a distinct chemotype support the clinical development of SC83288, as an intravenous application for the treatment of severe malaria.

    Nature communications 2017;8;14193

  • The Experimental Design Assistant.

    Percie du Sert N, Bamsey I, Bate ST, Berdoy M, Clark RA, Cuthill I, Fry D, Karp NA, Macleod M, Moon L, Stanford SC and Lings B

    National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), London, United Kingdom.

    Addressing the common problems that researchers encounter when designing and analysing animal experiments will improve the reliability of in vivo research. In this article, the Experimental Design Assistant (EDA) is introduced. The EDA is a web-based tool that guides the in vivo researcher through the experimental design and analysis process, providing automated feedback on the proposed design and generating a graphical summary that aids communication with colleagues, funders, regulatory authorities, and the wider scientific community. It will have an important role in addressing causes of irreproducibility.

    PLoS biology 2017;15;9;e2003779

  • Platelet function is modified by common sequence variation in megakaryocyte super enhancers.

    Petersen R, Lambourne JJ, Javierre BM, Grassi L, Kreuzhuber R, Ruklisa D, Rosa IM, Tomé AR, Elding H, van Geffen JP, Jiang T, Farrow S, Cairns J, Al-Subaie AM, Ashford S, Attwood A, Batista J, Bouman H, Burden F, Choudry FA, Clarke L, Flicek P, Garner SF, Haimel M, Kempster C, Ladopoulos V, Lenaerts AS, Materek PM, McKinney H, Meacham S, Mead D, Nagy M, Penkett CJ, Rendon A, Seyres D, Sun B, Tuna S, van der Weide ME, Wingett SW, Martens JH, Stegle O, Richardson S, Vallier L, Roberts DJ, Freson K, Wernisch L, Stunnenberg HG, Danesh J, Fraser P, Soranzo N, Butterworth AS, Heemskerk JW, Turro E, Spivakov M, Ouwehand WH, Astle WJ, Downes K, Kostadima M and Frontini M

    Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.

    Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

    Funded by: European Research Council: 268834; Wellcome Trust

    Nature communications 2017;8;16058

  • A Road Map for Precision Cancer Medicine Using Personalized Models.

    Picco G and Garnett MJ

    Wellcome Trust Sanger Institute, Cambridge, United Kingdom.

    A study by Pauli and colleagues in this issue of Cancer Discovery describes the creation of a precision cancer platform for patients with advanced disease, integrating DNA sequencing of patient tumors with the generation of patient-derived organoids and xenografts. They propose the use of this platform for drug testing to nominate therapeutic options for individual patients and for therapeutic biomarker discovery. Cancer Discov; 7(5); 456-8. ©2017 AACRSee related article by Pauli et al., p. 462.

    Cancer discovery 2017;7;5;456-458

  • Genome-wide profiling of humoral immunity and pathogen genes under selection identifies immune evasion tactics of Chlamydia trachomatis during ocular infection.

    Pickering H, Teng A, Faal N, Joof H, Makalo P, Cassama E, Nabicassa M, Last AR, Burr SE, Rowland-Jones SL, Thomson NR, Roberts CH, Mabey DCW, Bailey RL, Hayward RD, de la Maza LM and Holland MJ

    Clinical Research Department, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom.

    The frequency and duration of Chlamydia trachomatis (Ct) ocular infections decrease with age, suggesting development of partial immunity. However, there is a lack of clear correlates of immunity to Ct infection in humans. We screened sera from a cohort of Gambian children followed for six-months against a Ct-proteome microarray. At genome sequence level, we detected signatures of selection from a population of ocular Ct isolates from Guinea-Bissau. Together these approaches allowed us to highlight the focus of humoral responses and hypothesise new modes of pathogen immune evasion. Children who were susceptible to frequent and/or prolonged Ct infection had a less focussed antibody response, including preferential recognition of forty-two antigens. There was evidence of positive and purifying selection across the genome, but little balancing selection. In contrast, most antigens that were associated with susceptibility were under neutral selection. These data suggest an evasion strategy in which Ct presents a large panel of irrelevant antigens to the immune system to block or misdirect protective responses. Development of a focused immune response, possibly induced through vaccination, may be an effective strategy to promote protection to Ct infection.

    Scientific reports 2017;7;1;9634

  • Permeability-driven selection in a semi-empirical protocell model: the roots of prebiotic systems evolution.

    Piedrafita G, Monnard PA, Mavelli F and Ruiz-Mirazo K

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    The origin-of-life problem has been traditionally conceived as the chemical challenge to find the type of molecule and free-solution reaction dynamics that could have started Darwinian evolution. Different autocatalytic and 'self-replicative' molecular species have been extensively investigated, together with plausible synthetic pathways that might have led, abiotically, to such a minimalist scenario. However, in addition to molecular kinetics or molecular evolutionary dynamics, other physical and chemical constraints (like compartmentalization, differential diffusion, selective transport, osmotic forces, energetic couplings) could have been crucial for the cohesion, functional integration, and intrinsic stability/robustness of intermediate systems between chemistry and biology. These less acknowledged mechanisms of interaction and molecular control might have made the initial pathways to prebiotic systems evolution more intricate, but were surely essential for sustaining far-from-equilibrium chemical dynamics, given their functional relevance in all modern cells. Here we explore a protocellular scenario in which some of those additional constraints/mechanisms are addressed, demonstrating their 'system-level' implications. In particular, an experimental study on the permeability of prebiotic vesicle membranes composed of binary lipid mixtures allows us to construct a semi-empirical model where protocells are able to reproduce and undergo an evolutionary process based on their coupling with an internal chemistry that supports lipid synthesis.

    Funded by: Wellcome Trust

    Scientific reports 2017;7;1;3141

  • Mapping Post-Glacial expansions: The Peopling of Southwest Asia.

    Platt DE, Haber M, Dagher-Kharrat MB, Douaihy B, Khazen G, Ashrafian Bonab M, Salloum A, Mouzaya F, Luiselli D, Tyler-Smith C, Renfrew C, Matisoo-Smith E and Zalloua PA

    Computational Biology Center, IBM TJ Watson Research Centre, Yorktown Hgts, NY, USA.

    Archaeological, palaeontological and geological evidence shows that post-glacial warming released human populations from their various climate-bound refugia. Yet specific connections between these refugia and the timing and routes of post-glacial migrations that ultimately established modern patterns of genetic variation remain elusive. Here, we use Y-chromosome markers combined with autosomal data to reconstruct population expansions from regional refugia in Southwest Asia. Populations from three regions in particular possess distinctive autosomal genetic signatures indicative of likely refugia: one, in the north, centered around the eastern coast of the Black Sea, the second, with a more Levantine focus, and the third in the southern Arabian Peninsula. Modern populations from these three regions carry the widest diversity and may indeed represent the most likely descendants of the populations responsible for the Neolithic cultures of Southwest Asia. We reveal the distinct and datable expansion routes of populations from these three refugia throughout Southwest Asia and into Europe and North Africa and discuss the possible correlations of these migrations to various cultural and climatic events evident in the archaeological record of the past 15,000 years.

    Scientific reports 2017;7;40338

  • Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

    Poggi M, Canault M, Favier M, Turro E, Saultier P, Ghalloussi D, Baccini V, Vidal L, Mezzapesa A, Chelghoum N, Mohand-Oumoussa B, Falaise C, Favier R, Ouwehand WH, Fiore M, Peiretti F, Morange PE, Saut N, Bernot D, Greinacher A, BioResource N, Nurden AT, Nurden P, Freson K, Trégouët DA, Raslova H and Alessi MC

    Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.

    Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34(+) cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34(+) cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

    Haematologica 2017;102;2;282-294

  • Karyotype Evolution and Phylogenetic Relationships of Cricetulus sokolovi Orlov et Malygin 1988 (Cricetidae, Rodentia) Inferred from Chromosomal Painting and Molecular Data.

    Poplavskaya NS, Romanenko SA, Serdyukova NA, Trifonov VA, Yang F, Nie W, Wang J, Bannikova AA, Surov AV and Lebedev VS

    A.N. Severtsov Institute of Ecology and Evolution, RAS, Moscow, Russia.

    Sokolov's dwarf hamster (Cricetulus sokolovi) is the least studied representative of the striped hamsters (Cricetulus barabensis species group), the taxonomy of which remains controversial. The species was described based on chromosome morphology, but neither the details of the karyotype nor the phylogenetic relationships with other Cricetulus are known. In the present study, the karyotype of C. sokolovi was examined using cross-species chromosome painting. Molecular and cytogenetic data were employed to determine the phylogenetic position of Sokolov's hamster and to analyze the potential pathways of chromosome evolution in Cricetulus. Both the chromosome and molecular data support the species status of Sokolov's hamster. Phylogenetic analysis of the CYTB data placed C. sokolovi as sister to all other striped hamsters (sequence divergence of 8.1%). FISH data revealed that the karyotype of C. sokolovi is highly rearranged, with the most parsimonious scenario of its origin implying at least 4 robertsonian events and a centromere shift. Comparative cytogenetic data on Cricetinae suggest that their evolutionary history includes both periods of chromosomal conservatism and episodes of rapid chromosomal change.

    Cytogenetic and genome research 2017

  • Genome-wide analysis of health-related biomarkers in the UK Household Longitudinal Study reveals novel associations.

    Prins BP, Kuchenbaecker KB, Bao Y, Smart M, Zabaneh D, Fatemifar G, Luan J, Wareham NJ, Scott RA, Perry JRB, Langenberg C, Benzeval M, Kumari M and Zeggini E

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Serum biomarker levels are associated with the risk of complex diseases. Here, we aimed to gain insights into the genetic architecture of biomarker traits which can reflect health status. We performed genome-wide association analyses for twenty serum biomarkers involved in organ function and reproductive health. 9,961 individuals from the UK Household Longitudinal Study were genotyped using the Illumina HumanCoreExome array and variants imputed to the 1000 Genomes Project and UK10K haplotypes. We establish a polygenic heritability for all biomarkers, confirm associations of fifty-four established loci, and identify five novel, replicating associations at genome-wide significance. A low-frequency variant, rs28929474, (beta = 0.04, P = 2 × 10(-10)) was associated with levels of alanine transaminase, an indicator of liver damage. The variant is located in the gene encoding serine protease inhibitor, low levels of which are associated with alpha-1 antitrypsin deficiency which leads to liver disease. We identified novel associations (rs78900934, beta = 0.05, P = 6 × 10(-12); rs2911280, beta = 0.09, P = 6 × 10(-10)) for dihydroepiandrosterone sulphate, a precursor to major sex-hormones, and for glycated haemoglobin (rs12819124, beta = -0.03, P = 4 × 10(-9); rs761772, beta = 0.05, P = 5 × 10(-9)). rs12819124 is nominally associated with risk of type 2 diabetes. Our study offers insights into the genetic architecture of well-known and less well-studied biomarkers.

    Scientific reports 2017;7;1;11008

  • MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

    Protasio AV, van Dongen S, Collins J, Quintais L, Ribeiro DM, Sessler F, Hunt M, Rinaldi G, Collins JJ, Enright AJ and Berriman M

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, United Kingdom.

    Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware "miRNA-blind" bioinformatics tool Sylamer to analyse the 3'-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature "virgin" females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms' body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes.

    PLoS neglected tropical diseases 2017;11;5;e0005559

  • Chromatin determinants impart camptothecin sensitivity.

    Puddu F, Salguero I, Herzog M, Geisler NJ, Costanzo V and Jackson SP

    The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

    Camptothecin-induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes SIR1-4 suppresses much of the hypersensitivity of tof1∆ strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1-dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild-type and tof1∆ cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.

    EMBO reports 2017

  • Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells.

    Qin L, Lai Y, Zhao R, Wei X, Weng J, Lai P, Li B, Lin S, Wang S, Wu Q, Liang Q, Li Y, Zhang X, Wu Y, Liu P, Yao Y, Pei D, Du X and Li P

    Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.

    Background: Multiple iterations of chimeric antigen receptors (CARs) have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined.

    Methods: We generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo.

    Results: During in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv). In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities.

    Conclusions: Hence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.

    Journal of hematology & oncology 2017;10;1;68

  • Evaluating genetic drift in time-series evolutionary analysis.

    R Nené N, Mustonen V and J R Illingworth C

    Department of Genetics, University of Cambridge, Cambridge, UK.

    The Wright-Fisher model is the most popular population model for describing the behaviour of evolutionary systems with a finite population size. Approximations have commonly been used but the model itself has rarely been tested against time-resolved genomic data. Here, we evaluate the extent to which it can be inferred as the correct model under a likelihood framework. Given genome-wide data from an evolutionary experiment, we validate the Wright-Fisher drift model as the better option for describing evolutionary trajectories in a finite population. This was found by evaluating its performance against a Gaussian model of allele frequency propagation. However, we note a range of circumstances under which standard Wright-Fisher drift cannot be correctly identified.

    Journal of theoretical biology 2017

  • A recurrent de novo mutation in ACTG1 causes isolated ocular coloboma.

    Rainger J, Williamson KA, Soares DC, Truch J, Kurian D, Gillessen-Kaesbach G, Seawright A, Prendergast J, Halachev M, Wheeler A, McTeir L, Gill AC, van Heyningen V, Davey MG, UK10K and FitzPatrick DR

    The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, United Kingdom.

    Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole exome sequencing was used to analyse twelve trios (child affected with OC and both unaffected parents), This identified de novo mutations in ten different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1 and LCP1. Proband-only whole exome sequencing identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin-interactions and no further plausibly causative variants were identified in these genes on re-sequencing 380 unrelated individuals with OC. This article is protected by copyright. All rights reserved.

    Human mutation 2017

  • Addressing Beacon re-identification attacks: quantification and mitigation of privacy risks.

    Raisaro JL, Tramèr F, Ji Z, Bu D, Zhao Y, Carey K, Lloyd D, Sofia H, Baker D, Flicek P, Shringarpure S, Bustamante C, Wang S, Jiang X, Ohno-Machado L, Tang H, Wang X and Hubaux JP

    School of Computer and Communication Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

    The Global Alliance for Genomics and Health (GA4GH) created the Beacon Project as a means of testing the willingness of data holders to share genetic data in the simplest technical context-a query for the presence of a specified nucleotide at a given position within a chromosome. Each participating site (or "beacon") is responsible for assuring that genomic data are exposed through the Beacon service only with the permission of the individual to whom the data pertains and in accordance with the GA4GH policy and standards.While recognizing the inference risks associated with large-scale data aggregation, and the fact that some beacons contain sensitive phenotypic associations that increase privacy risk, the GA4GH adjudged the risk of re-identification based on the binary yes/no allele-presence query responses as acceptable. However, recent work demonstrated that, given a beacon with specific characteristics (including relatively small sample size and an adversary who possesses an individual's whole genome sequence), the individual's membership in a beacon can be inferred through repeated queries for variants present in the individual's genome.In this paper, we propose three practical strategies for reducing re-identification risks in beacons. The first two strategies manipulate the beacon such that the presence of rare alleles is obscured; the third strategy budgets the number of accesses per user for each individual genome. Using a beacon containing data from the 1000 Genomes Project, we demonstrate that the proposed strategies can effectively reduce re-identification risk in beacon-like datasets.

    Journal of the American Medical Informatics Association : JAMIA 2017

  • A Clostridium difficile lineage endemic to Costa Rican hospitals is multidrug-resistant by acquisition of chromosomal mutations and novel mobile genetic elements.

    Ramírez-Vargas G, Quesada-Gómez C, Acuña-Amador L, López-Ureña D, Murillo T, Gamboa-Coronado MD, Chaves-Olarte E, Thomson N, Rodríguez-Cavallini E and Rodríguez C

    Laboratorio de Investigación en Bacteriología Anaerobia, Centro de Investigación en Enfermedades Tropicales, and Facultad de Microbiología. Universidad de Costa Rica. San José 11051-2060, Costa Rica.

    The antimicrobial resistance rates and levels recorded for Clostridium difficile are on the rise. This study reports the nature, levels, diversity, and genomic context of the antimicrobial resistance of human C. difficile isolates from the NAPCR1/RT012/ST54 genotype, which caused an outbreak in 2009 and is endemic in Costa Rican hospitals. To this end, we determined the susceptibility of 38 NAPCR1 isolates to ten antibiotics from seven classes using E-tests or macrodilution tests and examined 31 NAPCR1 whole-genome sequences to identify single nucleotide polymorphisms and genes that could explain the observed resistance phenotypes. The NAPCR1 isolates were multidrug-resistant (MDR) and commonly exhibited very high resistance levels. From sequencing their genomes we showed that they possessed resistance-associated SNPs in gyrA and rpoB and carried between 8-9 acquired antimicrobial resistance (AMR) genes. Most of these genes were located on known or novel mobile genetic elements shared by isolates from different hospitals and time points. Metronidazole and vancomycin remain as first-line treatment options for these isolates. Overall, the NAPCR1 lineage showed an enhanced ability to acquire AMR genes through lateral gene transfer. This finding urges further vigilance and improved control measures to limit the dissemination of this lineage and the emergence of more C. difficile MDR-strains.

    Antimicrobial agents and chemotherapy 2017

  • Revisiting olfactory receptors as putative drivers of cancer.

    Ranzani M, Iyer V, Ibarra-Soria X, Del Castillo Velasco-Herrera M, Garnett M, Logan D and Adams DJ

    Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Olfactory receptors (ORs) recognize odorant molecules and activate a signal transduction pathway that ultimately leads to the perception of smell. This process also modulates the apoptotic cycle of olfactory sensory neurons in an olfactory receptor-specific manner. Recent reports indicate that some olfactory receptors are expressed in tissues other than the olfactory epithelium suggesting that they may have pleiotropic roles. Methods: We investigated the expression of 301 olfactory receptor genes in a comprehensive panel of 968 cancer cell lines. Results: Forty-nine per cent of cell lines show expression of at least one olfactory receptor gene. Some receptors display a broad pattern of expression across tumour types, while others were expressed in cell lines from a particular tissue. Additionally, most of the cancer cell lines expressing olfactory receptors express the effectors necessary for OR-mediated signal transduction. Remarkably, among cancer cell lines, OR2C3 is exclusively expressed in melanoma lines. We also confirmed the expression of OR2C3 in human melanomas, but not in normal melanocytes. Conclusions: The pattern of OR2C3 expression is suggestive of a functional role in the development and/or progression of melanoma. Some olfactory receptors may contribute to tumorigenesis.

    Wellcome open research 2017;2;9

  • Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1.

    Rathjen T, Yan X, Kononenko NL, Ku MC, Song K, Ferrarese L, Tarallo V, Puchkov D, Kochlamazashvili G, Brachs S, Varela L, Szigeti-Buck K, Yi CX, Schriever SC, Tattikota SG, Carlo AS, Moroni M, Siemens J, Heuser A, van der Weyden L, Birkenfeld AL, Niendorf T, Poulet JFA, Horvath TL, Tschöp MH, Heinig M, Trajkovski M, Haucke V and Poy MN

    Max Delbrück Center for Molecular Medicine, Berlin, Germany.

    Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

    Nature neuroscience 2017

  • Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression.

    Rocha NM, Bulger DA, Frontini A, Titheradge H, Gribsholt SB, Knox R, Page M, Harris J, Payne F, Adams C, Sleigh A, Crawford J, Gjesing AP, Bork-Jensen J, Pedersen O, Barroso I, Hansen T, Cox H, Reilly M, Rossor A, Brown RJ, Taylor SI, McHale D, Armstrong M, Oral EA, Saudek V, O'Rahilly SI, Maher ER, Richelsen B, Savage DB and Semple RK

    Wellcome Trust-MRC Institute of Metabolic Science, The University of Cambridge Metabolic Research Laboratories, Cambridge, United Kingdom.

    MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies.

    eLife 2017;6

  • Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

    Rodriguez-Broadbent H, Law PJ, Sud A, Palin K, Tuupanen S, Gylfe A, Hänninen UA, Cajuso T, Tanskanen T, Kondelin J, Kaasinen E, Sarin AP, Ripatti S, Eriksson JG, Rissanen H, Knekt P, Pukkala E, Jousilahti P, Salomaa V, Palotie A, Renkonen-Sinisalo L, Lepistö A, Böhm J, Mecklin JP, Al-Tassan NA, Palles C, Martin L, Barclay E, Farrington SM, Timofeeva MN, Meyer BF, Wakil SM, Campbell H, Smith CG, Idziaszczyk S, Maughan TS, Kaplan R, Kerr R, Kerr D, Passarelli MN, Figueiredo JC, Buchanan DD, Win AK, Hopper JL, Jenkins MA, Lindor NM, Newcomb PA, Gallinger S, Conti D, Schumacher F, Casey G, Aaltonen LA, Cheadle JP, Tomlinson IP, Dunlop MG and Houlston RS

    Division of Genetics and Epidemiology, The Institute of Cancer Research, London, SW7 3RP, UK.

    While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, P=1.68x10(-4) ). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, P=0.49), 0.94 (95% CI: 0.84-1.05, P= 0.27), and 0.98 (95% CI: 0.85-1.12, P=0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR=0.69, 95% CI: 0.49-0.99, P=0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia. This article is protected by copyright. All rights reserved.

    International journal of cancer 2017

  • Sphingolipids and glycerophospholipids - The "ying and yang" of lipotoxicity in metabolic diseases.

    Rodriguez-Cuenca S, Pellegrinelli V, Campbell M, Oresic M and Vidal-Puig A

    Metabolic Research Laboratories, Wellcome Trust MRC Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge. Cambridge, UK. Electronic address:

    Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of "ying/yang" interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.

    Progress in lipid research 2017

  • Expression of miR-618 in plasmacytoid dendritic cells from systemic sclerosis patients is associated with their altered frequency and activation.

    Rossato M, Affandi AJ, Thordardottir S, Wichers CGK, Cossu M, Broen JCA, Moret FM, Bossini-Castillo L, Chouri E, van Bon L, Wolters F, Marut W, van der Kroef M, Silva-Cardoso S, Bekker CPJ, Dolstra H, van Laar JM, Martin J, van Roon JAG, Reedquist KA, Beretta L and Radstake TRDJ

    Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

    Plasmacytoid dendritic cells (pDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to pDC dysregulation and persistent type I IFN signature are largely unexplored, especially in systemic sclerosis (SSc), a disease in which pDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα as compared to healthy controls. Objective To investigate potential microRNA-mediated epigenetic mechanisms underlying pDC dysregulation and type I IFN production in SSc. Methods microRNA expression profiling and validation was performed in highly purified pDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miR-618 on pDC biology were identified by overexpression in healthy pDCs. Results miR-618 expression was upregulated in pDCs of SSc patients, including those in the early stage of disease onset and not presenting with skin fibrosis. IRF8, a crucial transcription factor for pDC development and activation, was identified as a target of miR-618. miR-618 overexpression reduced the development of pDCs from CD34+ cells in-vitro and enhanced their ability to secrete IFNα, mimicking the pDC phenotype observed in SSc patients. Conclusion miR-618 upregulation suppresses pDC development and increases their ability to secrete IFNα, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of pDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions. This article is protected by copyright. All rights reserved.

    Arthritis & rheumatology (Hoboken, N.J.) 2017

  • Computational approaches for interpreting scRNA-seq data.

    Rostom R, Svensson V, Teichmann SA and Kar G

    Wellcome Trust Sanger Institute, Cambridge.

    The recent developments in high throughput single-cell RNA sequencing technology (scRNA-seq) have enabled the generation of vast amounts of transcriptomic data at cellular resolution. With these advances come new modes of data analysis, building on high-dimensional data mining techniques. Here, we consider biological questions for which scRNA-seq data is used, both at a cell and gene level, and describe tools available for these types of analyses. This is an exciting and rapidly evolving field, where clustering, pseudotime inference, branching inference and gene-level analyses are particularly informative areas of computational analysis. This article is protected by copyright. All rights reserved.

    FEBS letters 2017

  • You Are What You Eat: A Genomic Analysis of the Gut Microbiome of Captive and Wild Octopus vulgaris Paralarvae and Their Zooplankton Prey.

    Roura Á, Doyle SR, Nande M and Strugnell JM

    Department of Ecology, Environment and Evolution, La Trobe UniversityMelbourne, VIC, Australia.

    The common octopus (Octopus vulgaris) is an attractive species for aquaculture, however, several challenges inhibit sustainable commercial production. Little is known about the early paralarval stages in the wild, including diet and intestinal microbiota, which likely play a significant role in development and vitality of this important life stage. High throughput sequencing was used to characterize the gastrointestinal microbiome of wild O. vulgaris paralarvae collected from two different upwelling regions off the coast of North West Spain (n = 41) and Morocco (n = 35). These were compared to that of paralarvae reared with Artemia for up to 25 days in captivity (n = 29). In addition, the gastrointestinal microbiome of zooplankton prey (crabs, copepod and krill) was also analyzed to determine if the microbial communities present in wild paralarvae are derived from their diet. Paralarvae reared in captivity with Artemia showed a depletion of bacterial diversity, particularly after day 5, when almost half the bacterial species present on day 0 were lost and two bacterial families (Mycoplasmataceae and Vibrionaceae) dominated the microbial community. In contrast, bacterial diversity increased in wild paralarvae as they developed in the oceanic realm of both upwelling systems, likely due to the exposure of new bacterial communities via ingestion of a wide diversity of prey. Remarkably, the bacterial diversity of recently hatched paralarvae in captivity was similar to that of wild paralarvae and zooplankton, thus suggesting a marked effect of the diet in both the microbial community species diversity and evenness. This study provides a comprehensive overview of the bacterial communities inhabiting the gastrointestinal tract of O. vulgaris paralarvae, and reveals new research lines to challenge the current bottlenecks preventing sustainable octopus aquaculture.

    Frontiers in physiology 2017;8;362

  • Ensembl core software resources: storage and programmatic access for DNA sequence and genome annotation.

    Ruffier M, Kähäri A, Komorowska M, Keenan S, Laird M, Longden I, Proctor G, Searle S, Staines D, Taylor K, Vullo A, Yates A, Zerbino D and Flicek P

    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.

    The Ensembl software resources are a stable infrastructure to store, access and manipulate genome assemblies and their functional annotations. The Ensembl 'Core' database and Application Programming Interface (API) was our first major piece of software infrastructure and remains at the centre of all of our genome resources. Since its initial design more than fifteen years ago, the number of publicly available genomic, transcriptomic and proteomic datasets has grown enormously, accelerated by continuous advances in DNA-sequencing technology. Initially intended to provide annotation for the reference human genome, we have extended our framework to support the genomes of all species as well as richer assembly models. Cross-referenced links to other informatics resources facilitate searching our database with a variety of popular identifiers such as UniProt and RefSeq. Our comprehensive and robust framework storing a large diversity of genome annotations in one location serves as a platform for other groups to generate and maintain their own tailored annotation. We welcome reuse and contributions: our databases and APIs are publicly available, all of our source code is released with a permissive Apache v2.0 licence at and we have an active developer mailing list ( ).

    Database url:

    Database : the journal of biological databases and curation 2017;2017;1

  • The essential genomic landscape of the commensal Bifidobacterium breve UCC2003.

    Ruiz L, Bottacini F, Boinett CJ, Cain AK, O'Connell-Motherway M, Lawley TD and van Sinderen D

    School of Microbiology and APC Microbiome Institute, National University of Ireland, Cork, Western Road, Ireland.

    Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.

    Scientific reports 2017;7;1;5648

  • Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae.

    Runcharoen C, Moradigaravand D, Blane B, Paksanont S, Thammachote J, Anun S, Parkhill J, Chantratita N and Peacock SJ

    Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.

    Background: Klebsiella pneumoniae is a gram-negative bacterial species capable of occupying a broad range of environmental and clinical habitats. Known as an opportunistic pathogen, it has recently become a major causative agent of clinical infections worldwide. Despite growing knowledge about the highly diverse population of K. pneumoniae, the evolution and clinical significance of environmental K. pneumoniae, as well as the relationship between clinical and environmental K. pneumoniae, are poorly defined.

    Methods: We isolated and sequenced K. pneumoniae from in-patients in a single hospital in Thailand, as well as hospital sewage, and surrounding canals and farms within a 20-km radius.

    Results: Phylogenetic analysis of 77 K. pneumoniae (48 clinical and 29 non-clinical isolates) demonstrated that the two groups were intermixed throughout the tree and in some cases resided in the same clade, suggesting recent divergence from a common ancestor. Phylogenetic comparison of the 77 Thai genomes with 286 K. pneumoniae from a global collection showed that Thai isolates were closely related to the clinical sub-population of the global collection, indicating that Thai clinical isolates belonged to globally circulating lineages. Dating of four Thai K. pneumoniae clades indicated that they emerged between 50 and 150 years ago. Despite their phylogenetic relatedness, virulence factors and β-lactamase resistance genes were more numerous in clinical than in environmental isolates. Our results indicate that clinical and environmental K. pneumoniae are closely related, but that hospitals may select for isolates with a more resistant and virulent genotype.

    Conclusions: These findings highlight the clinical relevance of environmental K. pneumoniae isolates.

    Genome medicine 2017;9;1;6

  • Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand.

    Runcharoen C, Raven KE, Reuter S, Kallonen T, Paksanont S, Thammachote J, Anun S, Blane B, Parkhill J, Peacock SJ and Chantratita N

    Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand.

    Background: Tackling multidrug-resistant Escherichia coli requires evidence from One Health studies that capture numerous potential reservoirs in circumscribed geographic areas.

    Methods: We conducted a survey of extended β-lactamase (ESBL)-producing E. coli isolated from patients, canals and livestock wastewater in eastern Thailand between 2014 and 2015, and analyzed isolates using whole genome sequencing.

    Results: The bacterial collection of 149 isolates consisted of 84 isolates from a single hospital and 65 from the hospital sewer, canals and farm wastewater within a 20 km radius. E. coli ST131 predominated the clinical collection (28.6%), but was uncommon in the environment. Genome-based comparison of E. coli from infected patients and their immediate environment indicated low genetic similarity overall between the two, although three clinical-environmental isolate pairs differed by ≤ 5 single nucleotide polymorphisms. Thai E. coli isolates were dispersed throughout a phylogenetic tree containing a global E. coli collection. All Thai ESBL-positive E. coli isolates were multidrug resistant, including high rates of resistance to tobramycin (77.2%), gentamicin (77.2%), ciprofloxacin (67.8%) and trimethoprim (68.5%). ESBL was encoded by six different CTX-M elements and SHV-12. Three isolates from clinical samples (n = 2) or a hospital sewer (n = 1) were resistant to the carbapenem drugs (encoded by NDM-1, NDM-5 or GES-5), and three isolates (clinical (n = 1) and canal water (n = 2)) were resistant to colistin (encoded by mcr-1); no isolates were resistant to both carbapenems and colistin.

    Conclusions: Tackling ESBL-producing E. coli in this setting will be challenging based on widespread distribution, but the low prevalence of resistance to carbapenems and colistin suggests that efforts are now required to prevent these from becoming ubiquitous.

    Genome medicine 2017;9;1;81

  • Finding the needle in the haystack.

    Rutledge GG and Ariani CV

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

    Nature reviews. Microbiology 2017;15;3;136

  • Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

    Rutledge GG, Böhme U, Sanders M, Reid AJ, Cotton JA, Maiga-Ascofare O, Djimdé AA, Apinjoh TO, Amenga-Etego L, Manske M, Barnwell JW, Renaud F, Ollomo B, Prugnolle F, Anstey NM, Auburn S, Price RN, McCarthy JS, Kwiatkowski DP, Newbold CI, Berriman M and Otto TD

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

    Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

    Funded by: Wellcome Trust: 091625

    Nature 2017;542;7639;101-104

  • Genomic Characterization of Recrudescent Plasmodium malariae after Treatment with Artemether/Lumefantrine.

    Rutledge GG, Marr I, Huang GKL, Auburn S, Marfurt J, Sanders M, White NJ, Berriman M, Newbold CI, Anstey NM, Otto TD and Price RN

    Plasmodium malariae is the only human malaria parasite species with a 72-hour intraerythrocytic cycle and the ability to persist in the host for life. We present a case of a P. malariae infection with clinical recrudescence after directly observed administration of artemether/lumefantrine. By using whole-genome sequencing, we show that the initial infection was polyclonal and the recrudescent isolate was a single clone present at low density in the initial infection. Haplotypic analysis of the clones in the initial infection revealed that they were all closely related and were presumably recombinant progeny originating from the same infective mosquito bite. We review possible explanations for the P. malariae treatment failure and conclude that a 3-day artemether/lumefantrine regimen is suboptimal for this species because of its long asexual lifecycle.

    Emerging infectious diseases 2017;23;8

  • Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a mendelian randomisation analysis.

    Saleheen D, Haycock PC, Zhao W, Rasheed A, Taleb A, Imran A, Abbas S, Majeed F, Akhtar S, Qamar N, Zaman KS, Yaqoob Z, Saghir T, Rizvi SN, Memon A, Mallick NH, Ishaq M, Rasheed SZ, Memon FU, Mahmood K, Ahmed N, Frossard P, Tsimikas S, Witztum JL, Marcovina S, Sandhu M, Rader DJ and Danesh J

    Department of Biostatistics and Epidemiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA; Centre for Non-Communicable Diseases, Karachi, Pakistan. Electronic address:

    Background: The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease.

    Methods: In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease.

    Findings: The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90-0·97; p<0·0001) per 1-SD increment in LPA KIV2 repeats after adjustment for lipoprotein(a) concentration and conventional lipid concentrations. The OR for myocardial infarction was 1·10 (1·05-1·14; p<0·0001) per 1-SD increment in lipoprotein(a) concentration, after adjustment for LPA KIV2 repeats and conventional lipids. Genome-wide analysis identified rs2457564 as a variant associated with smaller apolipoprotein(a) isoform size, but not lipoprotein(a) concentration, and rs3777392 as a variant associated with lipoprotein(a) concentration, but not apolipoprotein(a) isoform size. In 60 801 patients with coronary heart disease and 123 504 controls, OR for myocardial infarction was 0·96 (0·94-0·98; p<0·0001) per 1-SD increment in apolipoprotein(a) protein isoform size in serum due to rs2457564, which was directionally concordant with the OR observed in PROMIS for a similar change. The OR for myocardial infarction was 1·27 (1·07-1·50; p=0·007) per 1-SD increment in lipoprotein(a) concentration due to rs3777392, which was directionally concordant with the OR observed for a similar change in PROMIS.

    Interpretation: Human genetic data suggest that both smaller apolipoprotein(a) isoform size and increased lipoprotein(a) concentration are independent and causal risk factors for coronary heart disease. Lipoprotein(a)-lowering interventions could be preferentially effective in reducing the risk of coronary heart disease in individuals with smaller apolipoprotein(a) isoforms.

    Funding: British Heart Foundation, US National Institutes of Health, Fogarty International Center, Wellcome Trust, UK Medical Research Council, UK National Institute for Health Research, and Pfizer.

    The lancet. Diabetes & endocrinology 2017

  • Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

    Saleheen D, Natarajan P, Armean IM, Zhao W, Rasheed A, Khetarpal SA, Won HH, Karczewski KJ, O'Donnell-Luria AH, Samocha KE, Weisburd B, Gupta N, Zaidi M, Samuel M, Imran A, Abbas S, Majeed F, Ishaq M, Akhtar S, Trindade K, Mucksavage M, Qamar N, Zaman KS, Yaqoob Z, Saghir T, Rizvi SNH, Memon A, Hayyat Mallick N, Ishaq M, Rasheed SZ, Memon FU, Mahmood K, Ahmed N, Do R, Krauss RM, MacArthur DG, Gabriel S, Lander ES, Daly MJ, Frossard P, Danesh J, Rader DJ and Kathiresan S

    Department of Biostatistics and Epidemiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

    Funded by: NHLBI NIH HHS: R01 HL107816; NIGMS NIH HHS: R01 GM104371

    Nature 2017;544;7649;235-239

  • Societal challenges of precision medicine: Bringing order to chaos.

    Salgado R, Moore H, Martens JWM, Lively T, Malik S, McDermott U, Michiels S, Moscow JA, Tejpar S, McKee T, Lacombe D and IBCD-Faculty

    EORTC Pathobiology Group, Belgium; Department of Pathology GZA, Antwerp, Belgium; Breast Cancer Translational Research Laboratory, Jules Bordet Institute, Belgium.

    The increasing number of drugs targeting specific proteins implicated in tumourigenesis and the commercial promotion of relatively affordable genome-wide analyses has led to an increasing expectation among patients with cancer that they can now receive effective personalised treatment based on the often complex genomic signature of their tumour. For such approaches to work in routine practice, the development of correspondingly complex biomarker assays through an appropriate and rigorous regulatory framework will be required. It is becoming increasingly evident that a re-engineering of clinical research is necessary so that regulatory considerations and procedures facilitate the efficient translation of these required biomarker assays from the discovery setting through to clinical application. This article discusses the practical requirements and challenges of developing such new precision medicine strategies, based on leveraging complex genomic profiles, as discussed at the Innovation and Biomarkers in Cancer Drug Development meeting (8th-9th September 2016, Brussels, Belgium).

    European journal of cancer (Oxford, England : 1990) 2017;84;325-334

  • A longitudinal study of the infant nasopharyngeal microbiota: The effects of age, illness and antibiotic use in a cohort of South East Asian children.

    Salter SJ, Turner C, Watthanaworawit W, de Goffau MC, Wagner J, Parkhill J, Bentley SD, Goldblatt D, Nosten F and Turner P

    Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    A longitudinal study was undertaken in infants living in the Maela refugee camp on the Thailand-Myanmar border between 2007 and 2010. Nasopharyngeal swabs were collected monthly, from birth to 24 months of age, with additional swabs taken if the infant was diagnosed with pneumonia according to WHO clinical criteria. At the time of collection, swabs were cultured for Streptococcus pneumoniae and multiple serotype carriage was assessed. The bacterial 16S rRNA gene profiles of 544 swabs from 21 infants were analysed to see how the microbiota changes with age, respiratory infection, antibiotic consumption and pneumococcal acquisition. The nasopharyngeal microbiota is a somewhat homogenous community compared to that of other body sites. In this cohort it is dominated by five taxa: Moraxella, Streptococcus, Haemophilus, Corynebacterium and an uncharacterized Flavobacteriaceae taxon of 93% nucleotide similarity to Ornithobacterium. Infant age correlates with certain changes in the microbiota across the cohort: Staphylococcus and Corynebacterium are associated with the first few months of life while Moraxella and the uncharacterised Flavobacteriaceae increase in proportional abundance with age. Respiratory illness and antibiotic use often coincide with an unpredictable perturbation of the microbiota that differs from infant to infant and in different illness episodes. The previously described interaction between Dolosigranulum and Streptococcus was observed in these data. Monthly sampling demonstrates that the nasopharyngeal microbiota is in flux throughout the first two years of life, and that in this refugee camp population the pool of potential bacterial colonisers may be limited.

    PLoS neglected tropical diseases 2017;11;10;e0005975

  • Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells.

    Sampaziotis F, de Brito MC, Geti I, Bertero A, Hannan NR and Vallier L

    Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge, UK.

    The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality. Compared with alternative protocols for biliary differentiation of hPSCs, our system does not require coculture with other cell types and relies on chemically defined conditions up to and including the generation of CPs. A complex extracellular matrix is used for the maturation of CLCs; therefore, experience in hPSC culture and 3D organoid systems may be necessary for optimal results. Finally, the capacity of our platform for generating large amounts of disease-specific functional cholangiocytes will have broad applications for cholangiopathies, in disease modeling and for screening of therapeutic compounds.

    Nature protocols 2017;12;4;814-827

  • Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

    Sampaziotis F, Justin AW, Tysoe OC, Sawiak S, Godfrey EM, Upponi SS, Gieseck RL, de Brito MC, Berntsen NL, Gómez-Vázquez MJ, Ortmann D, Yiangou L, Ross A, Bargehr J, Bertero A, Zonneveld MCF, Pedersen MT, Pawlowski M, Valestrand L, Madrigal P, Georgakopoulos N, Pirmadjid N, Skeldon GM, Casey J, Shu W, Materek PM, Snijders KE, Brown SE, Rimland CA, Simonic I, Davies SE, Jensen KB, Zilbauer M, Gelson WTH, Alexander GJ, Sinha S, Hannan NRF, Wynn TA, Karlsen TH, Melum E, Markaki AE, Saeb-Parsy K and Vallier L

    Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK.

    The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

    Nature medicine 2017;23;8;954-963

  • Whole-exome sequencing of 228 patients with sporadic Parkinson's disease.

    Sandor C, Honti F, Haerty W, Szewczyk-Krolikowski K, Tomlinson P, Evetts S, Millin S, Keane T, McCarthy SA, Durbin R, Talbot K, Hu M, Webber C, Ponting CP and Wade-Martins R

    Oxford Parkinson's Disease Centre, University of Oxford, Oxford, United Kingdom.

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting 1% of the population over 65 years characterized clinically by both motor and non-motor symptoms accompanied by the preferential loss of dopamine neurons in the substantia nigra pars compacta. Here, we sequenced the exomes of 244 Parkinson's patients selected from the Oxford Parkinson's Disease Centre Discovery Cohort and, after quality control, 228 exomes were available for analyses. The PD patient exomes were compared to 884 control exomes selected from the UK10K datasets. No single non-synonymous (NS) single nucleotide variant (SNV) nor any gene carrying a higher burden of NS SNVs was significantly associated with PD status after multiple-testing correction. However, significant enrichments of genes whose proteins have roles in the extracellular matrix were amongst the top 300 genes with the most significantly associated NS SNVs, while regions associated with PD by a recent Genome Wide Association (GWA) study were enriched in genes containing PD-associated NS SNVs. By examining genes within GWA regions possessing rare PD-associated SNVs, we identified RAD51B. The protein-product of RAD51B interacts with that of its paralogue RAD51, which is associated with congenital mirror movements phenotypes, a phenotype also comorbid with PD.

    Scientific reports 2017;7;41188

  • Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish.

    Scahill CM, Digby Z, Sealy IM, Wojciechowska S, White RJ, Collins JE, Stemple DL, Bartke T, Mathers ME, Patton EE and Busch-Nentwich EM

    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

    KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.

    PLoS genetics 2017;13;8;e1006959

  • Genomic investigation of a suspected outbreak of Legionella pneumophila ST82 reveals undetected heterogeneity by the present gold-standard methods, Denmark, July to November 2014.

    Schjørring S, Stegger M, Kjelsø C, Lilje B, Bangsborg JM, Petersen RF, David S, Uldum SA and ESCMID Study Group for Legionella Infections (ESGLI)

    Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhagen, Denmark.

    Between July and November 2014, 15 community-acquired cases of Legionnaires´ disease (LD), including four with Legionella pneumophila serogroup 1 sequence type (ST) 82, were diagnosed in Northern Zealand, Denmark. An outbreak was suspected. No ST82 isolates were found in environmental samples and no external source was established. Four putative-outbreak ST82 isolates were retrospectively subjected to whole genome sequencing (WGS) followed by phylogenetic analyses with epidemiologically unrelated ST82 sequences. The four putative-outbreak ST82 sequences fell into two clades, the two clades were separated by ca 1,700 single nt polymorphisms (SNP)s when recombination regions were included but only by 12 to 21 SNPs when these were removed. A single putative-outbreak ST82 isolate sequence segregated in the first clade. The other three clustered in the second clade, where all included sequences had < 5 SNP differences between them. Intriguingly, this clade also comprised epidemiologically unrelated isolate sequences from the UK and Denmark dating back as early as 2011. The study confirms that recombination plays a major role in L. pneumophila evolution. On the other hand, strains belonging to the same ST can have only few SNP differences despite being sampled over both large timespans and geographic distances. These are two important factors to consider in outbreak investigations.

    Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 2017;22;25

  • Fast, Quantitative and Variant Enabled Mapping of Peptides to Genomes.

    Schlaffner CN, Pirklbauer GJ, Bender A and Choudhary JS

    Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK; Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Cambridge, Cambridgeshire CB2 1EW, UK. Electronic address:

    Current tools for visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies and capture only basic sequence identity information. Furthermore, the frequent reformatting of annotations for reference genomes required by these tools is known to be highly error prone. We developed PoGo for mapping peptides identified through mass spectrometry to overcome these limitations. PoGo reduced runtime and memory usage by 85% and 20%, respectively, and exhibited overall superior performance over other tools on benchmarking with large-scale human tissue and cancer phosphoproteome datasets comprising ∼3 million peptides. In addition, extended functionality enables representation of single-nucleotide variants, post-translational modifications, and quantitative features. PoGo has been integrated in established frameworks such as the PRIDE tool suite and OpenMS, as well as a standalone tool with user-friendly graphical interface. With the rapid increase of quantitative high-resolution datasets capturing proteomes and global modifications to complement orthogonal genomics platforms, PoGo provides a central utility enabling large-scale visualization and interpretation of transomics datasets.

    Cell systems 2017;5;2;152-156.e4

  • Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

    Schneider VA, Graves-Lindsay T, Howe K, Bouk N, Chen HC, Kitts PA, Murphy TD, Pruitt KD, Thibaud-Nissen F, Albracht D, Fulton RS, Kremitzki M, Magrini V, Markovic C, McGrath S, Steinberg KM, Auger K, Chow W, Collins J, Harden G, Hubbard T, Pelan S, Simpson JT, Threadgold G, Torrance J, Wood JM, Clarke L, Koren S, Boitano M, Peluso P, Li H, Chin CS, Phillippy AM, Durbin R, Wilson RK, Flicek P, Eichler EE and Church DM

    National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA.

    The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

    Funded by: NHGRI NIH HHS: R01 HG002385, U41 HG007635, U54 HG003079; Wellcome Trust

    Genome research 2017;27;5;849-864

  • Malaria host candidate genes validated by association with current, recent and historic measures of transmission intensity.

    Sepúlveda N, Manjurano A, Campino SG, Lemnge M, Lusingu J, Olomi R, Rockett KA, Hubbart C, Jeffreys A, Rowlands K, MalariaGen consortium, Clark TG, Riley EM and Drakeley CJ

    London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

    Background: Human malaria susceptibility is determined by multiple genetic factors. It is however unclear which genetic variants remain important over time.

    Methods: Genetic associations of 175 high quality polymorphisms within several malaria candidate genes were examined in a sample of 8,096 individuals from northeast Tanzania using altitude, seroconversion rates and parasite rates as proxies of historical, recent and current malaria transmission intensity. A principal component (PC) analysis was used to derive two alternative measures of overall malaria propensity of a location across different time scales.

    Results: Common red blood cell polymorphisms (i.e., HbS, G6PD and α-thalassaemia) were the only ones to be associated with all three measures of transmission intensity and the first PC. Moderate associations were found between some immune response genes (i.e., IL3 and IL13) and parasite rates, but these could not be reproduced using the alternative measures of malaria propensity.

    Conclusions: We have demonstrated the potential of using altitude and seroconversion rate as measures of malaria transmission capturing medium to long term time scales to detect genetic associations that are likely to persist over time. These measures also have the advantage of minimizing the deleterious effects of random factors affecting parasite rates on the respective association signals.

    The Journal of infectious diseases 2017

  • Concern regarding the alleged spread of hypervirulent lymphogranuloma venereum Chlamydia trachomatis strain in Europe.

    Seth-Smith HM, Galán JC, Goldenberger D, Lewis DA, Peuchant O, Bébéar C, de Barbeyrac B, Bénard A, Carter I, Kok J, Bruisten SM, Versteeg B, Morré SA, Thomson NR, Egli A and de Vries HJ

    Clinical Microbiology, University Hospital Basel, Basel, Switzerland.

    Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 2017;22;15

  • European Chlamydia abortus livestock isolate genomes reveal unusual stability and limited diversity, reflected in geographical signatures.

    Seth-Smith HMB, Busó LS, Livingstone M, Sait M, Harris SR, Aitchison KD, Vretou E, Siarkou VI, Laroucau K, Sachse K, Longbottom D and Thomson NR

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

    Background: Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics.

    Results: Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied.

    Conclusions: The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable.

    BMC genomics 2017;18;1;344

  • Regulation of bone mass through pineal-derived melatonin-MT2 pathway.

    Sharan K, Lewis K, Furukawa T and Yadav VK

    Systems biology of bone, Department of Mouse and Zebrafish Genetics, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, United Kingdom.

    Tryptophan, an essential amino acid through a series of enzymatic reactions gives rise to various metabolites viz., serotonin and melatonin that regulate distinct biological functions. We show here that tryptophan metabolism in the pineal gland favors bone mass accrual through production of melatonin, a pineal-derived neurohormone. Pineal gland-specific deletion of Tph1, the enzyme that catalyzes the first step in the melatonin biosynthesis lead to a decrease in melatonin levels and a low bone mass due to an isolated decrease in bone formation while bone resorption parameters remained unaffected. Skeletal analysis of the mice deficient in MT1 or MT2 melatonin receptors showed a low bone mass in MT2-/- mice while MT1-/- mice had a normal bone mass compared to the WT mice. This low bone mass in the MT2-/- mice was due to an isolated decrease in osteoblasts numbers and bone formation. In vitro assays of the osteoblast cultures derived from the MT1-/- and MT2-/- mice showed a cell intrinsic defect in the proliferation, differentiation and mineralization abilities of MT2-/- osteoblasts compared to WT counterparts and the mutant cells did not respond to melatonin addition. Finally, we demonstrate that daily oral administration of melatonin can increase bone accrual during growth and can cure ovariectomy-induced structural and functional degeneration of bone by specifically increasing bone formation. By identifying pineal-derived melatonin as a regulator of bone mass through MT2 receptors, this study expands the role played by tryptophan derivatives in the regulation of bone mass and underscores its therapeutic relevance in postmenopausal osteoporosis. This article is protected by copyright. All rights reserved.

    Journal of pineal research 2017

  • Recurrent Rearrangements of Human Amylase Genes Create Multiple Independent CNV Series.

    Shwan NA, Louzada S, Yang F and Armour JA

    School of Life Sciences, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.

    The human amylase gene cluster includes the human salivary (AMY1) and pancreatic amylase genes (AMY2A and AMY2B), and is a highly variable and dynamic region of the genome. Copy number variation of AMY1 has been implicated in human dietary adaptation, and in population association with obesity, but neither of these findings has been independently replicated. Despite these functional implications, the structural genomic basis of copy number variation (CNV) has only been defined in detail very recently. In this work we use high-resolution analysis of copy number, and analysis of segregation in trios, to define new, independent allelic series of amylase CNVs in sub-Saharan Africans, including a series of higher-order expansions of a unit consisting of one copy each of AMY1, AMY2A and AMY2B. We use fibre-FISH (fluorescence in situ hybridization) to define unexpected complexity in the accompanying rearrangements. These findings demonstrate recurrent involvement of the amylase gene region in genomic instability, involving at least five independent rearrangements of the pancreatic amylase genes (AMY2A and AMY2B). Structural features shared by fundamentally distinct lineages strongly suggest that the common ancestral state for the human amylase cluster contained more than one, and probably three, copies of AMY1. This article is protected by copyright. All rights reserved.

    Human mutation 2017

  • Ancient genomes show social and reproductive behavior of early Upper Paleolithic foragers.

    Sikora M, Seguin-Orlando A, Sousa VC, Albrechtsen A, Korneliussen T, Ko A, Rasmussen S, Dupanloup I, Nigst PR, Bosch MD, Renaud G, Allentoft ME, Margaryan A, Vasilyev SV, Veselovskaya EV, Borutskaya SB, Deviese T, Comeskey D, Higham T, Manica A, Foley R, Meltzer DJ, Nielsen R, Excoffier L, Lahr MM, Orlando L and Willerslev E

    Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350 Copenhagen, Denmark.

    Present-day hunter-gatherers (HGs) live in multilevel social groups essential to sustain a population structure characterized by limited levels of within-band relatedness and inbreeding. When these wider social networks evolved among HGs is unknown. Here, we investigate whether the contemporary HG strategy was already present in the Upper Paleolithic (UP), using complete genome sequences from Sunghir, a site dated to ~34 thousand years BP (kya) containing multiple anatomically modern human (AMH) individuals. We demonstrate that individuals at Sunghir derive from a population of small effective size, with limited kinship and levels of inbreeding similar to HG populations. Our findings suggest that UP social organization was similar to that of living HGs, with limited relatedness within residential groups embedded in a larger mating network.

    Science (New York, N.Y.) 2017

  • Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations.

    Silva Martins WF, Wilding CS, Steen K, Mawejje H, Antão TR and Donnelly MJ

    Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

    Background: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species.

    Methodology/principal findings: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci).

    Conclusions/significance: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex.

    PLoS neglected tropical diseases 2017;11;10;e0005917

  • Family aggregation of cardiovascular disease mortality: a register-based prospective study of pooled Nordic twin cohorts.

    Silventoinen K, Hjelmborg J, Möller S, Ripatti S, Skythe A, Tikkanen E, Pedersen NL, Magnusson PK, Christensen K and Kaprio J

    Population Research Unit, Department of Social Research, University of Helsinki, Helsinki, Finland.

    Background: Familial factors play an important role in the variation of risk factors of cardiovascular diseases (CVD), but less is known about how they affect the risk of death from CVD. We estimated familial aggregation of CVD mortality for twins offering the maximum level of risk due to genetic and other familial factors.

    Methods: Altogether, 132 771 twin individuals, including 65 196 complete pairs from Denmark, Finland and Sweden born in 1958 or earlier, participated in this study. During the register-based follow-up, 11 641 deaths occurred from coronary heart disease (CHD), including 6280 deaths from myocardial infarct and 4855 deaths occurred from stroke, with 1092 deaths from ischaemic stroke and 1159 deaths from haemorrhagic stroke. Relative recurrence risk ratios (RRRs) with 95% confidence intervals (95% CIs) for monozygotic and dizygotic twins were calculated.

    Results: In the analyses pooling men and women, the RRR for monozygotic twins was 1.49 (95% CI 1.40-1.57) for CHD and 1.81 for any stroke (95% CI 1.54-2.09). The highest RRR was found for haemorrhagic stroke (3.53 95% CI 2.01-5.04). For dizygotic twins, the RRRs were generally lower.

    Conclusions: Family aggregation was found for CHD and haemorrhagic stroke. Clustering of risk factors in families increases the risk of CVD.

    International journal of epidemiology 2017

  • The contribution of rare variants to risk of schizophrenia in individuals with and without intellectual disability.

    Singh T, Walters JTR, Johnstone M, Curtis D, Suvisaari J, Torniainen M, Rees E, Iyegbe C, Blackwood D, McIntosh AM, Kirov G, Geschwind D, Murray RM, Di Forti M, Bramon E, Gandal M, Hultman CM, Sklar P, INTERVAL Study, UK10K Consortium, Palotie A, Sullivan PF, O'Donovan MC, Owen MJ and Barrett JC

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

    By performing a meta-analysis of rare coding variants in whole-exome sequences from 4,133 schizophrenia cases and 9,274 controls, de novo mutations in 1,077 family trios, and copy number variants from 6,882 cases and 11,255 controls, we show that individuals with schizophrenia carry a significant burden of rare, damaging variants in 3,488 genes previously identified as having a near-complete depletion of loss-of-function variants. In patients with schizophrenia who also have intellectual disability, this burden is concentrated in risk genes associated with neurodevelopmental disorders. After excluding known risk genes for neurodevelopmental disorders, a significant rare variant burden persists in other genes intolerant of loss-of-function variants; although this effect is notably stronger in patients with both schizophrenia and intellectual disability, it is also seen in patients with schizophrenia who do not have intellectual disability. Together, our results show that rare, damaging variants contribute to the risk of schizophrenia both with and without intellectual disability and support an overlap of genetic risk between schizophrenia and other neurodevelopmental disorders.

    Nature genetics 2017

  • WDR26 Haploinsufficiency Causes a Recognizable Syndrome of Intellectual Disability, Seizures, Abnormal Gait, and Distinctive Facial Features.

    Skraban CM, Wells CF, Markose P, Cho MT, Nesbitt AI, Au PYB, Begtrup A, Bernat JA, Bird LM, Cao K, de Brouwer APM, Denenberg EH, Douglas G, Gibson KM, Grand K, Goldenberg A, Innes AM, Juusola J, Kempers M, Kinning E, Markie DM, Owens MM, Payne K, Person R, Pfundt R, Stocco A, Turner CLS, Verbeek NE, Walsh LE, Warner TC, Wheeler PG, Wieczorek D, Wilkens AB, Zonneveld-Huijssoon E, Deciphering Developmental Disorders Study, Kleefstra T, Robertson SP, Santani A, van Gassen KLI and Deardorff MA

    Division of Genetics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

    We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

    Funded by: Wellcome Trust

    American journal of human genetics 2017;101;1;139-148

  • Interacting networks of resistance, virulence and core machinery genes identified by genome-wide epistasis analysis.

    Skwark MJ, Croucher NJ, Puranen S, Chewapreecha C, Pesonen M, Xu YY, Turner P, Harris SR, Beres SB, Musser JM, Parkhill J, Bentley SD, Aurell E and Corander J

    Department of Chemistry, Vanderbilt University, Nashville, TN, United States of America.

    Recent advances in the scale and diversity of population genomic datasets for bacteria now provide the potential for genome-wide patterns of co-evolution to be studied at the resolution of individual bases. Here we describe a new statistical method, genomeDCA, which uses recent advances in computational structural biology to identify the polymorphic loci under the strongest co-evolutionary pressures. We apply genomeDCA to two large population data sets representing the major human pathogens Streptococcus pneumoniae (pneumococcus) and Streptococcus pyogenes (group A Streptococcus). For pneumococcus we identified 5,199 putative epistatic interactions between 1,936 sites. Over three-quarters of the links were between sites within the pbp2x, pbp1a and pbp2b genes, the sequences of which are critical in determining non-susceptibility to beta-lactam antibiotics. A network-based analysis found these genes were also coupled to that encoding dihydrofolate reductase, changes to which underlie trimethoprim resistance. Distinct from these antibiotic resistance genes, a large network component of 384 protein coding sequences encompassed many genes critical in basic cellular functions, while another distinct component included genes associated with virulence. The group A Streptococcus (GAS) data set population represents a clonal population with relatively little genetic variation and a high level of linkage disequilibrium across the genome. Despite this, we were able to pinpoint two RNA pseudouridine synthases, which were each strongly linked to a separate set of loci across the chromosome, representing biologically plausible targets of co-selection. The population genomic analysis method applied here identifies statistically significantly co-evolving locus pairs, potentially arising from fitness selection interdependence reflecting underlying protein-protein interactions, or genes whose product activities contribute to the same phenotype. This discovery approach greatly enhances the future potential of epistasis analysis for systems biology, and can complement genome-wide association studies as a means of formulating hypotheses for targeted experimental work.

    PLoS genetics 2017;13;2;e1006508

  • Dataset of Sgo1 expression in cardiac, gastrointestinal, hepatic and neuronal tissue in mouse.

    Song AT, Galli A, Leclerc S, Nattel S, Mandato C and Andelfinger G

    Cardiovascular Genetics, Department of Pediatrics, CHU Sainte-Justine, Canada.

    The data shown in this article are related to the research article entitled "Characterization of Sgo1 expression pattern in developing and adult mouse" (Song et al., 2017) [3]. The article provides novel data on Sgo1 gene expression pattern utilizing Sgo1_LacZ_Knock in mouse line and immunohistochemistry in wild type mice. The data presents Sgo1 expression pattern during development, and in post-developmental proliferative and quiescent tissue. The article describes following tissues: developing heart, neural tube, adult colon, cerebellum, cerebral cortex, liver, and testis.

    Data in brief 2017;13;731-737

  • Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

    Song L, Jenner M, Masschelein J, Jones C, Bull MJ, Harris SR, Hartkoorn RC, Vocat A, Romero-Canelon I, Coupland P, Webster G, Dunn M, Weiser R, Paisey C, Cole ST, Parkhill J, Mahenthiralingam E and Challis GL

    Department of Chemistry, University of Warwick , Coventry CV4 7AL, United Kingdom.

    An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

    Journal of the American Chemical Society 2017;139;23;7974-7981

  • Whole genome sequencing and imputation in isolated populations identify genetic associations with medically-relevant complex traits.

    Southam L, Gilly A, Süveges D, Farmaki AE, Schwartzentruber J, Tachmazidou I, Matchan A, Rayner NW, Tsafantakis E, Karaleftheri M, Xue Y, Dedoussis G and Zeggini E

    Wellcome Trust Sanger Institute, Human Genetics, Hinxton CB10 1SA, UK.

    Next-generation association studies can be empowered by sequence-based imputation and by studying founder populations. Here we report ∼9.5 million variants from whole-genome sequencing (WGS) of a Cretan-isolated population, and show enrichment of rare and low-frequency variants with predicted functional consequences. We use a WGS-based imputation approach utilizing 10,422 reference haplotypes to perform genome-wide association analyses and observe 17 genome-wide significant, independent signals, including replicating evidence for association at eight novel low-frequency variant signals. Two novel cardiometabolic associations are at lead variants unique to the founder population sequences: chr16:70790626 (high-density lipoprotein levels beta -1.71 (SE 0.25), P=1.57 × 10(-11), effect allele frequency (EAF) 0.006); and rs145556679 (triglycerides levels beta -1.13 (SE 0.17), P=2.53 × 10(-11), EAF 0.013). Our findings add empirical support to the contribution of low-frequency variants in complex traits, demonstrate the advantage of including population-specific sequences in imputation panels and exemplify the power gains afforded by population isolates.

    Nature communications 2017;8;15606

  • NBEAL2 is required for neutrophil and NK cell function and pathogen defense.

    Sowerby JM, Thomas DC, Clare S, Espéli M, Guerrero JA, Hoenderdos K, Harcourt K, Marsden M, Abdul-Karim J, Clement M, Antrobus R, Umrania Y, Barton PR, Flint SM, Juss JK, Condliffe AM, Lyons PA, Humphreys IR, Chilvers ER, Ouwehand WH, Dougan G and Smith KG

    Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom.

    Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.

    The Journal of clinical investigation 2017;127;9;3521-3526

  • Reevaluation of SNP heritability in complex human traits.

    Speed D, Cai N, UCLEB Consortium, Johnson MR, Nejentsev S and Balding DJ

    UCL Genetics Institute, University College London, London, UK.

    SNP heritability, the proportion of phenotypic variance explained by SNPs, has been reported for many hundreds of traits. Its estimation requires strong prior assumptions about the distribution of heritability across the genome, but current assumptions have not been thoroughly tested. By analyzing imputed data for a large number of human traits, we empirically derive a model that more accurately describes how heritability varies with minor allele frequency (MAF), linkage disequilibrium (LD) and genotype certainty. Across 19 traits, our improved model leads to estimates of common SNP heritability on average 43% (s.d. 3%) higher than those obtained from the widely used software GCTA and 25% (s.d. 2%) higher than those from the recently proposed extension GCTA-LDMS. Previously, DNase I hypersensitivity sites were reported to explain 79% of SNP heritability; using our improved heritability model, their estimated contribution is only 24%.

    Nature genetics 2017

  • The antiviral restriction factor IFN-induced transmembrane protein 3 prevents cytokine-driven CMV pathogenesis.

    Stacey MA, Clare S, Clement M, Marsden M, Abdul-Karim J, Kane L, Harcourt K, Brandt C, Fielding CA, Smith SE, Wash RS, Brias SG, Stack G, Notley G, Cambridge EL, Isherwood C, Speak AO, Johnson Z, Ferlin W, Jones SA, Kellam P and Humphreys IR

    The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3-/- mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3-/- mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

    The Journal of clinical investigation 2017

  • Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

    Steinberg J, Ritchie GRS, Roumeliotis TI, Jayasuriya RL, Clark MJ, Brooks RA, Binch ALA, Shah KM, Coyle R, Pardo M, Le Maitre CL, Ramos YFM, Nelissen RGHH, Meulenbelt I, McCaskie AW, Choudhary JS, Wilkinson JM and Zeggini E

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

    Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two -omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three -omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

    Scientific reports 2017;7;1;8935

  • Genome annotation for clinical genomic diagnostics: strengths and weaknesses.

    Steward CA, Parker APJ, Minassian BA, Sisodiya SM, Frankish A and Harrow J

    Congenica Ltd, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1DR, UK.

    The Human Genome Project and advances in DNA sequencing technologies have revolutionized the identification of genetic disorders through the use of clinical exome sequencing. However, in a considerable number of patients, the genetic basis remains unclear. As clinicians begin to consider whole-genome sequencing, an understanding of the processes and tools involved and the factors to consider in the annotation of the structure and function of genomic elements that might influence variant identification is crucial. Here, we discuss and illustrate the strengths and weaknesses of approaches for the annotation and classification of important elements of protein-coding genes, other genomic elements such as pseudogenes and the non-coding genome, comparative-genomic approaches for inferring gene function, and new technologies for aiding genome annotation, as a practical guide for clinicians when considering pathogenic sequence variation. Complete and accurate annotation of structure and function of genome features has the potential to reduce both false-negative (from missing annotation) and false-positive (from incorrect annotation) errors in causal variant identification in exome and genome sequences. Re-analysis of unsolved cases will be necessary as newer technology improves genome annotation, potentially improving the rate of diagnosis.

    Genome medicine 2017;9;1;49

  • ANGPTL3 Deficiency and Protection Against Coronary Artery Disease.

    Stitziel NO, Khera AV, Wang X, Bierhals AJ, Vourakis AC, Sperry AE, Natarajan P, Klarin D, Emdin CA, Zekavat SM, Nomura A, Erdmann J, Schunkert H, Samani NJ, Kraus WE, Shah SH, Yu B, Boerwinkle E, Rader DJ, Gupta N, Frossard PM, Rasheed A, Danesh J, Lander ES, Gabriel S, Saleheen D, Musunuru K, Kathiresan S and PROMIS and Myocardial Infarction Genetics Consortium Investigators

    Cardiovascular Division, Department of Medicine, Department of Genetics, and McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

    Background: Familial combined hypolipidemia, a Mendelian condition characterized by substantial reductions in all 3 major lipid fractions, is caused by mutations that inactivate the gene angiopoietin-like 3 (ANGPTL3). Whether ANGPTL3 deficiency reduces risk of coronary artery disease (CAD) is unknown.

    Objectives: The study goal was to leverage 3 distinct lines of evidence-a family that included individuals with complete (compound heterozygote) ANGPTL3 deficiency, a population based-study of humans with partial (heterozygote) ANGPTL3 deficiency, and biomarker levels in patients with myocardial infarction (MI)-to test whether ANGPTL3 deficiency is associated with lower risk for CAD.

    Methods: We assessed coronary atherosclerotic burden in 3 individuals with complete ANGPTL3 deficiency and 3 wild-type first-degree relatives using computed tomography angiography. In the population, ANGPTL3 loss-of-function (LOF) mutations were ascertained in up to 21,980 people with CAD and 158,200 control subjects. LOF mutations were defined as nonsense, frameshift, and splice-site variants, along with missense variants resulting in <25% of wild-type ANGPTL3 activity in a mouse model. In a biomarker study, circulating ANGPTL3 concentration was measured in 1,493 people who presented with MI and 3,232 control subjects.

    Results: The 3 individuals with complete ANGPTL3 deficiency showed no evidence of coronary atherosclerotic plaque. ANGPTL3 gene sequencing demonstrated that approximately 1 in 309 people was a heterozygous carrier for an LOF mutation. Compared with those without mutation, heterozygous carriers of ANGPTL3 LOF mutations demonstrated a 17% reduction in circulating triglycerides and a 12% reduction in low-density lipoprotein cholesterol. Carrier status was associated with a 34% reduction in odds of CAD (odds ratio: 0.66; 95% confidence interval: 0.44 to 0.98; p = 0.04). Individuals in the lowest tertile of circulating ANGPTL3 concentrations, compared with the highest, had reduced odds of MI (adjusted odds ratio: 0.65; 95% confidence interval: 0.55 to 0.77; p < 0.001).

    Conclusions: ANGPTL3 deficiency is associated with protection from CAD.

    Journal of the American College of Cardiology 2017

  • Single-cell transcriptomics to explore the immune system in health and disease.

    Stubbington MJT, Rozenblatt-Rosen O, Regev A and Teichmann SA

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

    The immune system varies in cell types, states, and locations. The complex networks, interactions, and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity, and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as massively parallel single-cell RNA sequencing and sophisticated computational methods are catalyzing a revolution in our understanding of immunology. Here we provide an overview of the state of single-cell genomics methods and an outlook on the use of single-cell techniques to decipher the adaptive and innate components of immunity.

    Science (New York, N.Y.) 2017;358;6359;58-63

  • Multi-tissue DNA methylation age predictor in mouse.

    Stubbs TM, Bonder MJ, Stark AK, Krueger F, BI Ageing Clock Team, von Meyenn F, Stegle O and Reik W

    Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK.

    Background: DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse.

    Results: We have generated a comprehensive set of genome-scale base-resolution methylation maps from multiple mouse tissues spanning a wide range of ages. Many CpG sites show significant tissue-independent correlations with age which allowed us to develop a multi-tissue predictor of age in the mouse. Our model, which estimates age based on DNA methylation at 329 unique CpG sites, has a median absolute error of 3.33 weeks and has similar properties to the recently described human epigenetic clock. Using publicly available datasets, we find that the mouse clock is accurate enough to measure effects on biological age, including in the context of interventions. While females and males show no significant differences in predicted DNA methylation age, ovariectomy results in significant age acceleration in females. Furthermore, we identify significant differences in age-acceleration dependent on the lipid content of the diet.

    Conclusions: Here we identify and characterise an epigenetic predictor of age in mice, the mouse epigenetic clock. This clock will be instrumental for understanding the biology of ageing and will allow modulation of its ticking rate and resetting the clock in vivo to study the impact on biological age.

    Genome biology 2017;18;1;68

  • Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution.

    Suárez-Esquivel M, Baker KS, Ruiz-Villalobos N, Hernández-Mora G, Barquero-Calvo E, González-Barrientos R, Castillo-Zeledón A, Jiménez-Rojas C, Chacón-Díaz C, Cloeckaert A, Chaves-Olarte E, Thomson NR, Moreno E and Guzmán-Verri C

    Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica.

    Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97-99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication.

    Genome biology and evolution 2017;9;7;1901-1912

  • Brucella neotomae Infection in Humans, Costa Rica.

    Suárez-Esquivel M, Ruiz-Villalobos N, Jiménez-Rojas C, Barquero-Calvo E, Chacón-Díaz C, Víquez-Ruiz E, Rojas-Campos N, Baker KS, Oviedo-Sánchez G, Amuy E, Chaves-Olarte E, Thomson NR, Moreno E and Guzmán-Verri C

    Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed.

    Funded by: Wellcome Trust

    Emerging infectious diseases 2017;23;6;997-1000

  • Protein structure and phenotypic analysis of pathogenic and population missense variants in STXBP1.

    Suri M, Evers JMG, Laskowski RA, O'Brien S, Baker K, Clayton-Smith J, Dabir T, Josifova D, Joss S, Kerr B, Kraus A, McEntagart M, Morton J, Smith A, Splitt M, Thornton JM, DDD Study and Wright CF

    Nottingham Regional Genetics ServiceNottingham University Hospitals NHS TrustCity Hospital Campus, The Gables, Hucknall RoadNottinghamNG5 1PBUK.

    Background: Syntaxin-binding protein 1, encoded by STXBP1, is highly expressed in the brain and involved in fusing synaptic vesicles with the plasma membrane. Studies have shown that pathogenic loss-of-function variants in this gene result in various types of epilepsies, mostly beginning early in life. We were interested to model pathogenic missense variants on the protein structure to investigate the mechanism of pathogenicity and genotype-phenotype correlations.

    Methods: We report 11 patients with pathogenic de novo mutations in STXBP1 identified in the first 4293 trios of the Deciphering Developmental Disorder (DDD) study, including six missense variants. We analyzed the structural locations of the pathogenic missense variants from this study and the literature, as well as population missense variants extracted from Exome Aggregation Consortium (ExAC).

    Results: Pathogenic variants are significantly more likely to occur at highly conserved locations than population variants, and be buried inside the protein domain. Pathogenic mutations are also more likely to destabilize the domain structure compared with population variants, increasing the proportion of (partially) unfolded domains that are prone to aggregation or degradation. We were unable to detect any genotype-phenotype correlation, but unlike previously reported cases, most of the DDD patients with STXBP1 pathogenic variants did not present with very early-onset or severe epilepsy and encephalopathy, though all have developmental delay with intellectual disability and most display behavioral problems and suffered seizures in later childhood.

    Conclusion: Variants across STXBP1 that cause loss of function can result in severe intellectual disability with or without seizures, consistent with a haploinsufficiency mechanism. Pathogenic missense mutations act through destabilization of the protein domain, making it prone to aggregation or degradation. The presence or absence of early seizures may reflect ascertainment bias in the literature as well as the broad recruitment strategy of the DDD study.

    Molecular genetics & genomic medicine 2017;5;5;495-507

  • Pfk13-independent treatment failure in four imported cases of Plasmodium falciparum malaria given artemether-lumefantrine in the UK.

    Sutherland CJ, Lansdell P, Sanders M, Muwanguzi J, van Schalkwyk DA, Kaur H, Nolder D, Tucker J, Bennett HM, Otto TD, Berriman M, Patel TA, Lynn R, Gkrania-Klotsas E and Chiodini PL

    Public Health England Malaria Reference Laboratory, London School of Hygiene & Tropical Medicine, London, UK.

    We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015-16. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitaemia within 6 weeks of treatment with no intervening travel to malarious countries. Parasite isolates, all of African origin, harboured variants at some candidate resistance loci. No evidence of pfk13-mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended.

    Antimicrobial agents and chemotherapy 2017

  • Power analysis of single-cell RNA-sequencing experiments.

    Svensson V, Natarajan KN, Ly LH, Miragaia RJ, Labalette C, Macaulay IC, Cvejic A and Teichmann SA

    European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, UK.

    Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers ( We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.

    Nature methods 2017

  • Detection of Atherosclerotic Inflammation by (68)Ga-DOTATATE PET Compared to [(18)F]FDG PET Imaging.

    Tarkin JM, Joshi FR, Evans NR, Chowdhury MM, Figg NL, Shah AV, Starks LT, Martin-Garrido A, Manavaki R, Yu E, Kuc RE, Grassi L, Kreuzhuber R, Kostadima MA, Frontini M, Kirkpatrick PJ, Coughlin PA, Gopalan D, Fryer TD, Buscombe JR, Groves AM, Ouwehand WH, Bennett MR, Warburton EA, Davenport AP and Rudd JH

    Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom.

    Background: Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([(18)F]FDG PET), [(18)F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover.

    Objectives: This study tested the efficacy of gallium-68-labeled DOTATATE ((68)Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation.

    Methods: We confirmed (68)Ga-DOTATATE binding in macrophages and excised carotid plaques. (68)Ga-DOTATATE PET imaging was compared to [(18)F]FDG PET imaging in 42 patients with atherosclerosis.

    Results: Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific (68)Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo (68)Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). (68)Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). (68)Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [(18)F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [(18)F]FDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [(18)F]FDG spillover rendered coronary [(18)F]FDG scans uninterpretable in 27 patients (64%). Coronary (68)Ga-DOTATATE PET scans were readable in all patients.

    Conclusions: We validated (68)Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that (68)Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [(18)F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188).

    Journal of the American College of Cardiology 2017;69;14;1774-1791

  • Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib.

    Taylor SJ, Duyvestyn JM, Dagger SA, Dishington EJ, Rinaldi CA, Dovey OM, Vassiliou GS, Grove CS and Langdon WY

    School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009, Australia.

    We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

    Science translational medicine 2017;9;402

  • Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2.

    Tchasovnikarova IA, Timms RT, Douse CH, Roberts RC, Dougan G, Kingston RE, Modis Y and Lehner PJ

    Department of Medicine, Cambridge Institute for Medical Research, Cambridge, UK.

    Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.

    Nature genetics 2017

  • Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity.

    Thomas DC, Clare S, Sowerby JM, Pardo M, Juss JK, Goulding DA, van der Weyden L, Storisteanu D, Prakash A, Espéli M, Flint S, Lee JC, Hoenderdos K, Kane L, Harcourt K, Mukhopadhyay S, Umrania Y, Antrobus R, Nathan JA, Adams DJ, Bateman A, Choudhary JS, Lyons PA, Condliffe AM, Chilvers ER, Dougan G and Smith KG

    Department of Medicine, University of Cambridge, University of Cambridge School of Clinical Medicine, Cambridge CB2 0QQ, England, UK.

    The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

    The Journal of experimental medicine 2017;214;4;1111-1128

  • Faster Average Case Low Memory Semi-external Construction of the Burrows–Wheeler Transform

    Tischlerm G,

    Mathematics in Computer Science 2017;11;2;159-76

  • Alternative Splicing May Not Be the Key to Proteome Complexity.

    Tress ML, Abascal F and Valencia A

    Structural Biology and Bioinformatics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro, 3, 28029 Madrid, Spain.

    Alternative splicing is commonly believed to be a major source of cellular protein diversity. However, although many thousands of alternatively spliced transcripts are routinely detected in RNA-seq studies, reliable large-scale mass spectrometry-based proteomics analyses identify only a small fraction of annotated alternative isoforms. The clearest finding from proteomics experiments is that most human genes have a single main protein isoform, while those alternative isoforms that are identified tend to be the most biologically plausible: those with the most cross-species conservation and those that do not compromise functional domains. Indeed, most alternative exons do not seem to be under selective pressure, suggesting that a large majority of predicted alternative transcripts may not even be translated into proteins.

    Trends in biochemical sciences 2017;42;2;98-110

  • Most Alternative Isoforms Are Not Functionally Important.

    Tress ML, Abascal F and Valencia A

    Department of Structural and Computational Biology, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. Electronic address:

    Trends in biochemical sciences 2017

  • Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes.

    Unnikrishnan A, Papaemmanuil E, Beck D, Deshpande NP, Verma A, Kumari A, Woll PS, Richards LA, Knezevic K, Chandrakanthan V, Thoms JAI, Tursky ML, Huang Y, Ali Z, Olivier J, Galbraith S, Kulasekararaj AG, Tobiasson M, Karimi M, Pellagatti A, Wilson SR, Lindeman R, Young B, Ramakrishna R, Arthur C, Stark R, Crispin P, Curnow J, Warburton P, Roncolato F, Boultwood J, Lynch K, Jacobsen SEW, Mufti GJ, Hellstrom-Lindberg E, Wilkins MR, MacKenzie KL, Wong JWH, Campbell PJ and Pimanda JE

    Adult Cancer Program, Lowy Cancer Research Centre, UNSW, Sydney, NSW 2052, Australia; Prince of Wales Clinical School, UNSW, Sydney, NSW 2052, Australia. Electronic address:

    Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.

    Cell reports 2017;20;3;572-585

  • Normalizing single-cell RNA sequencing data: challenges and opportunities.

    Vallejos CA, Risso D, Scialdone A, Dudoit S and Marioni JC

    MRC Biostatistics Unit, Cambridge Institute of Public Health, Cambridge, UK.

    Single-cell transcriptomics is becoming an important component of the molecular biologist's toolkit. A critical step when analyzing data generated using this technology is normalization. However, normalization is typically performed using methods developed for bulk RNA sequencing or even microarray data, and the suitability of these methods for single-cell transcriptomics has not been assessed. We here discuss commonly used normalization approaches and illustrate how these can produce misleading results. Finally, we present alternative approaches and provide recommendations for single-cell RNA sequencing users.

    Nature methods 2017

  • The Profile and Dynamics of RNA Modifications in Animals.

    van Delft P, Akay A, Huber SM, Bueschl C, Rudolph KLM, Di Domenico T, Schuhmacher R, Miska EA and Balasubramanian S

    Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

    More than a hundred distinct modified nucleosides have been identified in RNA, but little is known about their distribution across different organisms, their dynamic nature and their response to cellular and environmental stress. Mass-spectrometry-based methods have been at the forefront of identifying and quantifying modified nucleosides. However, they often require synthetic reference standards, which do not exist in the case of many modified nucleosides, and this therefore impedes their analysis. Here we use a metabolic labelling approach to achieve rapid generation of bio-isotopologues of the complete Caenorhabditis elegans transcriptome and its modifications and use them as reference standards to characterise the RNA modification profile in this multicellular organism through an untargeted liquid-chromatography tandem high-resolution mass spectrometry (LC-HRMS) approach. We furthermore show that several of these RNA modifications have a dynamic response to environmental stress and that, in particular, changes in the tRNA wobble base modification 5-methoxycarbonylmethyl-2-thiouridine (mcm(5) s(2) U) lead to codon-biased gene-expression changes in starved animals.

    Chembiochem : a European journal of chemical biology 2017

  • Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

    van der Weyden L, Arends MJ, Campbell AD, Bald T, Wardle-Jones H, Griggs N, Velasco-Herrera MD, Tüting T, Sansom OJ, Karp NA, Clare S, Gleeson D, Ryder E, Galli A, Tuck E, Cambridge EL, Voet T, Macaulay IC, Wong K, Sanger Mouse Genetics Project, Spiegel S, Speak AO and Adams DJ

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

    Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

    Funded by: Cancer Research UK: 13031; NCI NIH HHS: P30 CA016059; NHGRI NIH HHS: U54 HG004028; Wellcome Trust: WT098051

    Nature 2017;541;7636;233-236

  • Genome wide in vivo mouse screen data from studies to assess host regulation of metastatic colonisation.

    van der Weyden L, Karp NA, Swiatkowska A, Adams DJ and Speak AO

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

    The process of metastasis is a multi-stage cascade with prior studies suggesting that the colonisation of the secondary site is the rate limiting step. This process involves contributions from the tumour cells and also non-tumour intrinsic factors such as the stroma and the haematopoietic system. In this study, we present data from screening 810 genetically-modified mouse lines with the experimental metastasis assay where intravenous delivery of murine metastatic melanoma B16-F10 cells was used to assess the formation of pulmonary metastasic foci. To date, these data have been studied with a two-step process cumulating in an integrative data analysis to identify genes controlling metastatic colonisation. We present the raw data, and a description to support fresh analyses where researchers can look both within and across gene sets to further elucidate process that regulate metastatic colonisation.

    Scientific data 2017;4;170129

  • A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming.

    Vasiliauskaitė L, Vitsios D, Berrens RV, Carrieri C, Reik W, Enright AJ and O'Carroll D

    European Molecular Biology Laboratory (EMBL), Monterotondo, Italy.

    In mice, the pathway involving PIWI and PIWI-interacting RNA (PIWI-piRNA) is essential to re-establish transposon silencing during male-germline reprogramming. The cytoplasmic PIWI protein MILI mediates piRNA-guided transposon RNA cleavage as well as piRNA amplification. MIWI2's binding to piRNA and its nuclear localization are proposed to be dependent upon MILI function. Here, we demonstrate the existence of a piRNA biogenesis pathway that sustains partial MIWI2 function and reprogramming activity in the absence of MILI.

    Nature structural & molecular biology 2017

  • Therapeutics: Click and discover.

    Vassiliou GS and Balasubramanian S

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK, and in the Department of Haematology, University of Cambridge.

    Nature 2017;548;7666;162-164

  • Phenotypic spectrum associated with de novo mutations in QRICH1 gene.

    Ververi A, Splitt M, Dean J, DDD study and Brady A

    North West Thames Regional Genetics Service, London North West Healthcare NHS Trust, Harrow, UK.

    Rare de novo mutations represent a significant cause of idiopathic developmental delay. The use of NGS has boosted the identification of de novo mutations in an increasing number of novel genes. Here we present three unrelated children with de novo LoF mutations in QRICH1, diagnosed through trio exome sequencing. QRICH1 encodes the glutamine-rich protein 1, which contains one Caspase Activation Recruitment Domain and is likely to be involved in apoptosis and inflammation. All three children had speech delay, learning difficulties, a prominent nose and a thin upper lip. In addition, two of them had mildly raised CK and one of them had autism. Despite their small number, the patients had a relatively consistent pattern of clinical features suggesting the presence of a QRICH1-associated phenotype. LoF mutations in QRICH1 are suggested as a novel cause of developmental delay.

    Clinical genetics 2017

  • Massive introgression drives species radiation at the range limit of Anopheles gambiae.

    Vicente JL, Clarkson CS, Caputo B, Gomes B, Pombi M, Sousa CA, Antao T, Dinis J, Bottà G, Mancini E, Petrarca V, Mead D, Drury E, Stalker J, Miles A, Kwiatkowski DP, Donnelly MJ, Rodrigues A, Torre AD, Weetman D and Pinto J

    Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon, Portugal.

    Impacts of introgressive hybridisation may range from genomic erosion and species collapse to rapid adaptation and speciation but opportunities to study these dynamics are rare. We investigated the extent, causes and consequences of a hybrid zone between Anopheles coluzzii and Anopheles gambiae in Guinea-Bissau, where high hybridisation rates appear to be stable at least since the 1990s. Anopheles gambiae was genetically partitioned into inland and coastal subpopulations, separated by a central region dominated by A. coluzzii. Surprisingly, whole genome sequencing revealed that the coastal region harbours a hybrid form characterised by an A. gambiae-like sex chromosome and massive introgression of A. coluzzii autosomal alleles. Local selection on chromosomal inversions may play a role in this process, suggesting potential for spatiotemporal stability of the coastal hybrid form and providing resilience against introgression of medically-important loci and traits, found to be more prevalent in inland A. gambiae.

    Scientific reports 2017;7;46451

  • Prevalence of M75 Streptococcus pyogenes Strains Harboring slaA Gene in Patients Affected by Pediatric Obstructive Sleep Apnea Syndrome in Central Italy.

    Viciani E, Montagnani F, Tordini G, Romano A, Salerni L, De Luca A, Ruggiero P and Manetti AG

    GSK Vaccines S.r.l.Siena, Italy; Host-Microbiota Interaction Team, Wellcome Trust Sanger InstituteCambridge, UK.

    Recently we reported an association between pediatric obstructive sleep apnea syndrome (OSAS) and Group A streptococcus (GAS) sub-acute chronic tonsil colonization. We showed that GAS may contribute to tonsil hyperplasia via a streptolysin O (SLO)-dependent cysteinyl leukotrienes (CysLTs) production, which can trigger T and B cell proliferation. In the present study, we characterized the GAS strains isolated from pediatric OSAS patients in comparison with a panel of age and sex matched GAS strains unrelated to OSAS, but isolated in the same area and during the same period ranging from 2009 to 2013. We found that slaA gene, previously reported to be associated to CysLTs production pathway, was significantly associated to GAS OSAS strains. Moreover, the most numerous group (32%) of the GAS OSAS strains belonged to M75 type, and 6 out of 7 of these strains harbored the slaA gene. Multilocus Sequence Typing (MLST) experiments demonstrated that the clone emm75/ST49/ smeZ, slaA was associated to OSAS cases. In conclusion, we found an association between slaA gene and the GAS OSAS strains, and we showed that the clone emm75/ST49 harboring genes smeZ and slaA was exclusively isolated from patients affected by OSAS, thus suggesting that this genotype might be associated to the pathogenesis of OSAS, although further studies are needed to elucidate the possible role of SlaA in tonsil hypertrophy development.

    Frontiers in microbiology 2017;8;294

  • Are cells from a snowman realistic? Cryopreserved tissues as a source for single-cell RNA-sequencing experiments.

    Vieira Braga FA, Teichmann SA and Stubbington MJ

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

    A recently published study in Genome Biology shows that cells isolated from cryopreserved tissues are a reliable source of genetic material for single-cell RNA-sequencing experiments.Please see related Method article:

    Genome biology 2017;18;1;54

  • Molecular diagnoses of century-old childhood tumours.

    Virasami A, Farndon SJ, McDermott U, Sebire N and Behjati S

    Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK; UCL Great Ormond Street Institute of Child Health, London, UK.

    The Lancet. Oncology 2017;18;5;e237

  • Mutational Processes Shaping the Genome in Early Human Embryos.

    Voet T and Vermeesch JR

    Department of Human Genetics, University of Leuven, KU Leuven, Herestraat 49, 3000 Leuven, Belgium; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. Electronic address:

    In-vitro-fertilized human embryos often acquire large structural and numerical chromosomal abnormalities. Liu et al. now show that multiple smaller copy number variations may also arise in in-vivo-conceived embryos. Analysis of these variations provides insight into the DNA mutational processes occurring in early embryos and the mechanisms underlying them.

    Cell 2017;168;5;751-753

  • Research benefits of storing genitourinary samples: 16S rRNA sequencing to evaluate vaginal bacterial communities.

    Wagner J, Kerry-Barnard S, Sadiq ST and Oakeshott P

    1 Wellcome Trust Sanger Institute, Cambridge, UK.

    International journal of STD & AIDS 2017;28;3;315-317

  • Novel Blood Pressure Locus and Gene Discovery Using Genome-Wide Association Study and Expression Data Sets From Blood and the Kidney.

    Wain LV, Vaez A, Jansen R, Joehanes R, van der Most PJ, Erzurumluoglu AM, O'Reilly PF, Cabrera CP, Warren HR, Rose LM, Verwoert GC, Hottenga JJ, Strawbridge RJ, Esko T, Arking DE, Hwang SJ, Guo X, Kutalik Z, Trompet S, Shrine N, Teumer A, Ried JS, Bis JC, Smith AV, Amin N, Nolte IM, Lyytikäinen LP, Mahajan A, Wareham NJ, Hofer E, Joshi PK, Kristiansson K, Traglia M, Havulinna AS, Goel A, Nalls MA, Sõber S, Vuckovic D, Luan J, Del Greco M F, Ayers KL, Marrugat J, Ruggiero D, Lopez LM, Niiranen T, Enroth S, Jackson AU, Nelson CP, Huffman JE, Zhang W, Marten J, Gandin I, Harris SE, Zemunik T, Lu Y, Evangelou E, Shah N, de Borst MH, Mangino M, Prins BP, Campbell A, Li-Gao R, Chauhan G, Oldmeadow C, Abecasis G, Abedi M, Barbieri CM, Barnes MR, Batini C, Beilby J, Blake T, Boehnke M, Bottinger EP, Braund PS, Brown M, Brumat M, Campbell H, Chambers JC, Cocca M, Collins F, Connell J, Cordell HJ, Damman JJ, Davies G, de Geus EJ, de Mutsert R, Deelen J, Demirkale Y, Doney ASF, Dörr M, Farrall M, Ferreira T, Frånberg M, Gao H, Giedraitis V, Gieger C, Giulianini F, Gow AJ, Hamsten A, Harris TB, Hofman A, Holliday EG, Hui J, Jarvelin MR, Johansson Å, Johnson AD, Jousilahti P, Jula A, Kähönen M, Kathiresan S, Khaw KT, Kolcic I, Koskinen S, Langenberg C, Larson M, Launer LJ, Lehne B, Liewald DCM, Lin L, Lind L, Mach F, Mamasoula C, Menni C, Mifsud B, Milaneschi Y, Morgan A, Morris AD, Morrison AC, Munson PJ, Nandakumar P, Nguyen QT, Nutile T, Oldehinkel AJ, Oostra BA, Org E, Padmanabhan S, Palotie A, Paré G, Pattie A, Penninx BWJH, Poulter N, Pramstaller PP, Raitakari OT, Ren M, Rice K, Ridker PM, Riese H, Ripatti S, Robino A, Rotter JI, Rudan I, Saba Y, Saint Pierre A, Sala CF, Sarin AP, Schmidt R, Scott R, Seelen MA, Shields DC, Siscovick D, Sorice R, Stanton A, Stott DJ, Sundström J, Swertz M, Taylor KD, Thom S, Tzoulaki I, Tzourio C, Uitterlinden AG, Völker U, Vollenweider P, Wild S, Willemsen G, Wright AF, Yao J, Thériault S, Conen D, Attia J, Sever P, Debette S, Mook-Kanamori DO, Zeggini E, Spector TD, van der Harst P, Palmer CNA, Vergnaud AC, Loos RJF, Polasek O, Starr JM, Girotto G, Hayward C, Kooner JS, Lindgren CM, Vitart V, Samani NJ, Tuomilehto J, Gyllensten U, Knekt P, Deary IJ, Ciullo M, Elosua R, Keavney BD, Hicks AA, Scott RA, Gasparini P, Laan M, Liu Y, Watkins H, Hartman CA, Salomaa V, Toniolo D, Perola M, Wilson JF, Schmidt H, Zhao JH, Lehtimäki T, van Duijn CM, Gudnason V, Psaty BM, Peters A, Rettig R, James A, Jukema JW, Strachan DP, Palmas W, Metspalu A, Ingelsson E, Boomsma DI, Franco OH, Bochud M, Newton-Cheh C, Munroe PB, Elliott P, Chasman DI, Chakravarti A, Knight J, Morris AP, Levy D, Tobin MD, Snieder H, Caulfield MJ and Ehret GB

    From the Department of Health Sciences (L.V.W., A.M.E., N. Shrine, C.B., T.B., M.D.T.), and Department of Cardiovascular Sciences and NIHR Leicester Biomedical Research Centre (C.P.N., P.S.B., N.J.S.), University of Leicester, United Kingdom; Department of Epidemiology (A.V., P.J.v.d.M., I.M.N., H. Snieder), Division of Nephrology, Department of Internal Medicine (M.H.d.B., M.A.S.), Interdisciplinary Center Psychopathology and Emotion Regulation (IPCE) (A.J.O., H.R., C.A.H.), Department of Genetics, (M.S.), and Department of Cardiology (P.v.d.H.), University of Groningen, University Medical Center Groningen, The Netherlands; Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences, Iran (A.V.); Department of Psychiatry, VU University Medical Center, Neuroscience Campus Amsterdam, The Netherlands (R. Jansen); Hebrew SeniorLife, Harvard Medical School, Boston, MA (R. Joehanes); National Heart, Lung and Blood Institute's Framingham Heart Study, MA (R. Joehanes, A.D.J., M. Larson); Institute of Psychiatry, Psychology and Neuroscience (P.F.O.), and Department of Twin Research and Genetic Epidemiology (M.M., C. Menni, T.D.S.), King's College London, United Kingdom; Clinical Pharmacology, William Harvey Research Institute (C.P.C., H.R.W., M.R.B., M. Brown, B.M., M.R., P.B.M., M.J.C.) and NIHR Barts Cardiovascular Biomedical Research Unit (C.P.C., H.R.W., M.R.B., M. Brown, P.B.M., M.J.C.), Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom; Division of Preventive Medicine, Brigham and Women's Hospital, Boston, MA (L.M.R., F.G., P.M.R., D.I.C.); Department of Epidemiology (G.C.V., A. Hofman, A.G.U., O.H.F.), Genetic Epidemiology Unit, Department of Epidemiology (N.A., B.A.O., C.M.v.D.), and Department of Internal Medicine (A.G.U.), Erasmus MC, Rotterdam, The Netherlands; Department of Biological Psychology, Vrije Universiteit, Amsterdam, EMGO+ Institute, VU University Medical Center, The Netherlands (J.-J.H., E.J.d.G., G.W., D.I.B.); Cardiovascular Medicine Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden (R.J.S., M. Frånberg, A. Hamsten); Centre for Molecular Medicine, Karolinska Universitetsjukhuset, Solna, Sweden (R.J.S., M. Frånberg, A. Hamsten); Estonian Genome Center (T.E., E.O., A. Metspalu), Institute of Biomedicine and Translational Medicine (S.S., M. Laan), and Estonian Genome Center (M.P.), University of Tartu, Estonia; Divisions of Endocrinology/Children's Hospital, Boston, MA (T.E.); Broad Institute of Harvard and MIT, Cambridge, MA (T.E., C.M.L., C.N.-C.); Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD (D.E.A., P.N., A. Chakravarti, G.B.E.); The Population Science Branch, Division of Intramural Research, National Heart Lung and Blood Institute (S.-J.H., D.L.), Laboratory of Neurogenetics, National Institute on Aging (M.A.N.), Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute (F.C.), and Center for Information Technology (Y.D., P.J.M., Q.T.N.), National Institutes of Health, Bethesda, MD; The Framingham Heart Study, Framingham, MA (S.-J.H., D.L.); The Institute for Translational Genomics and Population Sciences, Department of Pediatrics (X.G., J.Y.), and The Institute for Translational Genomics and Population Sciences, Departments of Pediatrics and Medicine (J.I.R.), LABioMed at Harbor-UCLA Medical Center, Torrance, CA; Institute of Social and Preventive Medicine, Lausanne University Hospital, Lausanne, Switzerland (Z.K., M. Bochud); Swiss Institute of Bioinformatics, Lausanne, Switzerland (Z.K.); Department of Cardiology (S. Trompet, J.W.J.) Department of Gerontology and Geriatrics (S. Trompet), Department of Clinical Epidemiology (R.L.-G., R.d.M., D.O.M.-K.), Department of Molecular Epidemiology (J.D.), and Department of Public Health and Primary Care (D.O.M.-K.), Leiden University Medical Center, The Netherlands; Institute for Community Medicine (A.T.), Department of Internal Medicine B (M.D.), and Interfaculty Institute for Genetics and Functional Genomics (U.V.), University Medicine Greifswald, Germany; DZHK (German Centre for Cardiovascular Research), partner site Greifswald, Germany (A.T., M.D., U.V.); Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany (J.S.R., A. Peters); Cardiovascular Health Research Unit, Department of Medicine (J.C.B., B.M.P.) and Departments of Biostatistics (K.R.), Epidemiology (B.M.P.), and Health Services (B.M.P.), University of Washington, Seattle; Icelandic Heart Association, Kopavogur, Iceland (A.V.S., V. Gudnason); Faculty of Medicine, University of Iceland, Reykjavik, Iceland (A.V.S., V. Gudnason); Department of Clinical Chemistry, Fimlab Laboratories, Tampere, Finland (L.-P.L., T.L.); Department of Clinical Chemistry, Faculty of Medicine and Life Sciences, University of Tampere, Finland (L.-P.L., T.L.); Wellcome Trust Centre for Human Genetics (A. Mahajan, A.G., M. Farrall, T.F., C.M.L., H.W., A.P.M.), and Division of Cardiovascular Medicine, Radcliffe Department of Medicine (A.G., M. Farrall, H.W.), University of Oxford, United Kingdom; MRC Epidemiology Unit, University of Cambridge School of Clinical Medicine, Institute of Metabolic Science, United Kingdom (N.J.W., J.L., C.L., R.J.F.L., R.A.S., J.H.Z.); Clinical Division of Neurogeriatrics, Department of Neurology (E.H., R. Schmidt), Institute of Medical Informatics, Statistics and Documentation (E.H.), and Department of Neurology (H. Schmidt), Medical University Graz, Austria; Centre for Global Health Research, Usher Institute of Population Health Sciences and Informatics (P.K.J., H.C., I.R., S.W., J.F.W.), Centre for Cognitive Ageing and Cognitive Epidemiology (L.M.L., S.E.H., G.D., A.J.G., D.C.M.L., J.M.S., I.J.D.), Medical Genetics Section, Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine (A. Campbell), Generation Scotland, Centre for Genomic and Experimental Medicine (A. Campbell, S.P., C.H.), Department of Psychology (G.D., D.C.M.L., A. Pattie, I.J.D.), Alzheimer Scotland Dementia Research Centre (J.M.S.), and Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine (C.H.), University of Edinburgh, Scotland, United Kingdom; Department of Health (K.K., A.S.H., T. Niiranen, P.J., A.J., S. Koskinen, P.K., V.S., M.P.), and Chronic Disease Prevention Unit (J.T.), National Institute for Health and Welfare (THL), Helsinki, Finland; Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milano, Italy (M.T., C.M.B., C.F.S., D.T.); Data Tecnica International, Glen Echo, MD (M.A.N.); Medical Genetics, IRCCS-Burlo Garofolo Children Hospital, Trieste, Italy (D.V., G.G., P.G.); Department of Medical, Surgical and Health Sciences, University of Trieste, Italy (D.V., I.G., M. Brumat, M. Cocca, A. Morgan, G.G., P.G.); Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck, Bolzano, Italy (F.D.G.M., P.P.P., A.S.P., A.A.H.); Department of Genetics and Genomic Sciences (K.L.A.), The Charles Bronfman Institute for Personalized Medicine (Y.L., E.P.B., R.J.F.L.), and Mindich Child health Development Institute (R.J.F.L.), Icahn School of Medicine at Mount Sinai, New York; Cardiovascular Epidemiology and Genetics, IMIM, and CIBERCV, Barcelona, Spain (J. Marrugat, R.E.); Institute of Genetics and Biophysics A. Buzzati-Traverso, CNR, Napoli, Italy (D.R., T. Nutile, R. Sorice, M. Ciullo); Department of Psychiatry, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin (L.M.L.); UCD Conway Institute, Centre for Proteome Research (L.M.L.), and School of Medicine, Conway Institute (D.C.S.), University College Dublin, Belfield, Ireland; Department of Immunology, Genetics and Pathology, Uppsala Universitet, Science for Life Laboratory, Sweden (S.E., Å. Johansson, U.G.); Department of Biostatistics and Center for Statistical Genetics, University of Michigan, Ann Arbor (A.U.J., M. Boehnke); NIHR Leicester Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester United Kingdom (C.P.N., P.S.B., N.J.S.); MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine (J.E.H., V.V., J. Marten, A.F.W., J.F.W.), and Medical Genetics Section, Centre for Genomic and Experimental Medicine and MRC Institute of Genetics and Molecular Medicine (S.E.H.), University of Edinburgh, Western General Hospital, Scotland, United Kingdom; Department of Epidemiology and Biostatistics, School of Public Health (W.Z., E.E., J.C.C., H.G., B.L., I.T., A.-C.V.), MRC-PHE Centre for Environment and Health, Department of Epidemiology and Biostatistics, School of Public Health (M.-R.J., P.E.), School of Public Health (N.P.), International Centre for Circulatory Health (S. Thom), and National Heart and Lung Institute (P.S.), Imperial College London, United Kingdom; Department of Cardiology, Ealing Hospital, London North West Healthcare NHS Trust, Southall, United Kingdom (W.Z., J.C.C., J.S.K.); Department of Medical Biology, Faculty of Medicine, University of Split, Croatia (T.Z.); Department of Hygiene and Epidemiology, University of Ioannina Medical School, Greece (E.E.); Medical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Scotland, United Kingdom (N. Shah, A.S.F.D., C.N.A.P.); Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan (N. Shah); National Institute for Health Research Biomedical Research Centre, London, United Kingdom (M.M.); Department of Human Genetics, Wellcome Trust Sanger Institute, United Kingdom (B.P.P., E.Z.); INSERM U 1219, Bordeaux Population Health Center, France (G.C., C.T., S.D.); Bordeaux University, France (G.C., C.T., S.D.); Hunter Medical Research Institute, New Lambton, NSW, Australia (C.O., E.G.H., R. Scott, J.A.); Center for Statistical Genetics, Department of Biostatistics, Ann Arbor, MI (G.A.); Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Iran (M.A.); Busselton Population Medical Research Institute, Western Australia (J.B., J.H.); PathWest Laboratory Medicine of Western Australia, Nedlands (J.B., J.H.); School of Pathology and Laboratory Medicine (J.B., J.H.), School of Population and Global Health (J.H.), and School of Medicine and Pharmacology (A. James), The University of Western Australia, Nedlands; Imperial College Healthcare NHS Trust, London, United Kingdom (J.C.C., J.S.K.); University of Dundee, Ninewells Hospital & Medical School, United Kingdom (J.C.); Institute of Genetic Medicine (H.J.C.), and Institute of Health and Society (C. Mamasoula), Newcastle University, Newcastle upon Tyne, United Kingdom; Department of Pathology, Amsterdam Medical Center, The Netherlands (J.J.D.); Department of Numerical Analysis and Computer Science, Stockholm University, Sweden (M. Frånberg); Department of Public Health and Caring Sciences, Geriatrics, Uppsala, Sweden (V. Giedraitis); Helmholtz Zentrum Muenchen, Deutsches Forschungszentrum fuer Gesundheit und Umwelt (GmbH), Neuherberg, Germany (C.G.); Department of Psychology, School of Social Sciences, Heriot-Watt University, Edinburgh, United Kingdom (A.J.G.); Intramural Research Program, Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging (T.B.H., L.J.L.); Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA (A. Hofman); Center For Life-Course Health Research (M.-R.J.), and Biocenter Oulu (M.-R.J.), University of Oulu, Finland; Unit of Primary Care, Oulu University Hospital, Finland (M.-R.J.); National Heart, Lung and Blood Institute, Cardiovascular Epidemiology and Human Genomics Branch, Bethesda, MD (A.D.J.); Department of Clinical Physiology, Tampere University Hospital, Finland (M.K.); Department of Clinical Physiology, Faculty of Medicine and Life Sciences, University of Tampere, Finland (M.K.); Cardiovascular Research Center (S. Kathiresan, C.N.-C.); Center for Human Genetics (S. Kathiresan), and Center for Human Genetic Research (C.N.-C.), Massachusetts General Hospital, Boston; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA (S. Kathiresan, C.N.-C.); Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge, United Kingdom (K.-T.K.); Department of Public Health, Faculty of Medicine, University of Split, Croatia (I.K., O.P.); Cardiology, Department of Specialties of Medicine, Geneva University Hospital, Switzerland (L. Lin, F.M., G.B.E.); Department of Medical Sciences, Cardiovascular Epidemiology (L. Lind, J.S.), and Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory (E.I.), Uppsala University, Sweden; Department of Psychiatry, EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam, The Netherlands (Y.M., B.W.J.H.P.); School of Molecular, Genetic and Population Health Sciences, University of Edinburgh, Medical School, Teviot Place, Scotland, United Kingdom (A.D.M.); Department of Epidemiology, Human Genetics and Environmental Sciences, School of Public Health, University of Texas Health Science Center at Houston (A.C.M.); British Heart Foundation Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences (S.P.), and Institute of Cardiovascular and Medical Sciences, Faculty of Medicine (D.J.S.), University of Glasgow, United Kingdom; Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland (A. Palotie, S.R., A.-P.S., M.P.); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada (G.P., S. Thériault); Department of Neurology, General Central Hospital, Bolzano, Italy (P.P.P.); Department of Neurology, University of Lübeck, Germany (P.P.P.); Department of Clinical Physiology and Nuclear Medicine, Turku University Hospital, Finland (O.T.R.); Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Finland (O.T.R.); Department of Cardiology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou, China (M.R.); Harvard Medical School, Boston, MA (P.M.R., D.I.C.); Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy (A.R.); Institute of Molecular Biology and Biochemistry, Centre for Molecular Medicine, Medical University of Graz, Austria (Y.S., H. Schmidt); INSERM U1078, Etablissement Français du Sang, Brest Cedex, France (A.S.P.); Faculty of Health, University of Newcastle, Callaghan, NSW, Australia (R. Scott, J.A.); John Hunter Hospital, New Lambton, NSW, Australia (R. Scott, J.A.); The New York Academy of Medicine, New York (D.S.); IRCCS Neuromed, Pozzilli, Isernia, Italy (R. Sorice, M. Ciullo); Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland (A.S.); Institute for Translational Genomics and Population Sciences, Los Angeles BioMedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA (K.D.T.); Division of Genetic Outcomes, Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA (K.D.T.); Department of Public Health (C.T.), and Department of Neurology (S.D.), Bordeaux University Hospital, France; Department of Internal Medicine, Lausanne University Hospital, CHUV, Switzerland (P.V.); Population Health Research Institute, McMaster University, Hamilton Ontario, Canada (D.C.); National Heart and Lung Institute, Imperial College London, Hammersmith Hospital Campus, United Kingdom (J.S.K.); Dasman Diabetes Institute, Kuwait (J.T.); Diabetes Research Group, King Abdulaziz University, Jeddah, Saudi Arabia (J.T.); Department of Neurosciences and Preventive Medicine, Danube-University Krems, Austria (J.T.); Division of Cardiovascular Sciences, The University of Manchester and Central Manchester University Hospitals NHS Foundation Trust, United Kingdom (B.D.K.); Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem (Y.M.L.); Kaiser Permanent Washington Health Research Institute, Seattle, WA (B.M.P.); Institute of Physiology, University Medicine Greifswald, Karlsburg, Germany (R.R); Department of Pulmonary Physiology and Sleep, Sir Charles Gairdner Hospital, Nedlands, Western Australia (A. James); Population Health Research Institute, St George's, University of London, United Kingdom (D.P.S.); Department of Medicine, Columbia University Medical Center, New York (W.P.); Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, CA (E.I.); Data Science Institute and Lancaster Medical School, Lancaster University, United Kingdom (J.K.); and Department of Biostatistics, University of Liverpool, United Kingdom (A.P.M.).

    Elevated blood pressure is a major risk factor for cardiovascular disease and has a substantial genetic contribution. Genetic variation influencing blood pressure has the potential to identify new pharmacological targets for the treatment of hypertension. To discover additional novel blood pressure loci, we used 1000 Genomes Project-based imputation in 150 134 European ancestry individuals and sought significant evidence for independent replication in a further 228 245 individuals. We report 6 new signals of association in or near HSPB7, TNXB, LRP12, LOC283335, SEPT9, and AKT2, and provide new replication evidence for a further 2 signals in EBF2 and NFKBIA Combining large whole-blood gene expression resources totaling 12 607 individuals, we investigated all novel and previously reported signals and identified 48 genes with evidence for involvement in blood pressure regulation that are significant in multiple resources. Three novel kidney-specific signals were also detected. These robustly implicated genes may provide new leads for therapeutic innovation.

    Hypertension (Dallas, Tex. : 1979) 2017

  • Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

    Webster SJ, Brode S, Ellis L, Fitzmaurice TJ, Elder MJ, Gekara NO, Tourlomousis P, Bryant C, Clare S, Chee R, Gaston HJS and Goodall JC

    Rheumatology Research Group, Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

    The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

    PLoS pathogens 2017;13;6;e1006383

  • CRISPR/Cas9-Mediated Deletion of Foxn1 in NOD/SCID/IL2rg(-/-) Mice Results in Severe Immunodeficiency.

    Wei X, Lai Y, Li B, Qin L, Xu Y, Lin S, Wang S, Wu Q, Liang Q, Huang G, Deng Q, Liu P, Wu D, Lai L, Yao Y and Li P

    School of Life Sciences, University of Science and Technology of China, Hefei, 230027, China.

    Immunodeficient mice engrafted with either normal or cancerous human cells are widely used in basic and translational research. In particular, NOD/SCID/IL2rg(-/-) mice can support the growth of various types of human cancer cells. However, the hairs of these mice interfere with the observation and imaging of engrafted tissues. Therefore, novel hairless strains exhibiting comparable immunodeficiency would be beneficial. Recently, the CRISPR/Cas9 system has been used for efficient multiplexed genome editing. In the present study, we generated a novel strain of nude NOD/SCID/IL2rg(-/-) (NSIN) mice by knocking out Foxn1 from NOD/SCID/IL2rg(-/-) (NSI) mice using the CRISPR/Cas9 system. The NSIN mice were deficient in B, T, and NK cells and not only showed impaired T cell reconstitution and thymus regeneration after allogeneic bone marrow nucleated cell transplantation but also exhibited improved capacity to graft both leukemic and solid tumor cells compared with NSI, NOG, and NDG mice. Moreover, the NSIN mice facilitated the monitoring and in vivo imaging of both leukemia and solid tumors. Therefore, our NSIN mice provide a new platform for xenograft mouse models in basic and translational research.

    Scientific reports 2017;7;1;7720

  • PSCA and MUC1 in non-small-cell lung cancer as targets of chimeric antigen receptor T cells.

    Wei X, Lai Y, Li J, Qin L, Xu Y, Zhao R, Li B, Lin S, Wang S, Wu Q, Liang Q, Peng M, Yu F, Li Y, Zhang X, Wu Y, Liu P, Pei D, Yao Y and Li P

    Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

    In recent years, immunotherapies, such as those involving chimeric antigen receptor (CAR) T cells, have become increasingly promising approaches to non-small-cell lung cancer (NSCLC) treatment. In this study, we explored the antitumor potential of prostate stem cell antigen (PSCA)-redirected CAR T and mucin 1 (MUC1)-redirected CAR T cells in tumor models of NSCLC. First, we generated patient-derived xenograft (PDX) mouse models of human NSCLC that maintained the antigenic profiles of primary tumors. Next, we demonstrated the expression of PSCA and MUC1 in NSCLC, followed by the generation and confirmation of the specificity and efficacy of PSCA- and MUC1-targeting CAR T cells against NSCLC cell lines in vitro. Finally, we demonstrated that PSCA-targeting CAR T cells could efficiently suppress NSCLC tumor growth in PDX mice and synergistically eliminate PSCA(+)MUC1(+) tumors when combined with MUC1-targeting CAR T cells. Taken together, our studies demonstrate that PSCA and MUC1 are both promising CAR T cell targets in NSCLC and that the combinatorial targeting of these antigens could further enhance the antitumor efficacy of CAR T cells.

    Oncoimmunology 2017;6;3;e1284722

  • Haploinsufficiency of ZNF462 is associated with craniofacial anomalies, corpus callosum dysgenesis, ptosis, and developmental delay.

    Weiss K, Wigby K, Fannemel M, Henderson LB, Beck N, Ghali N, Study DDD, Anderlid BM, Lundin J, Hamosh A, Jones MC, Ghedia S, Muenke M and Kruszka P

    Department of Medical Genetics, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.

    The introduction of whole-exome sequencing into the Pediatric Genetics clinic has increased the identification of novel genes associated with neurodevelopmental disorders and congenital anomalies. This agnostic approach has shed light on multiple proteins and pathways not previously known to be associated with disease. Here we report eight subjects from six families with predicted loss of function variants in ZNF462, a zinc-finger protein of unknown function. These individuals have overlapping phenotypes that include ptosis, metopic ridging, craniosynostosis, dysgenesis of the corpus callosum, and developmental delay. We propose that ZNF462 plays an important role in embryonic development, and is associated with craniofacial and neurodevelopmental abnormalities.

    European journal of human genetics : EJHG 2017;25;8;946-951

  • Targeted feature detection for data-dependent shotgun proteomics.

    Weisser H and Choudhary JS

    Label-free quantification of shotgun LC-MS/MS data is the prevailing approach in quantitative proteomics, but remains computationally non-trivial. The central data analysis step is the detection of peptide-specific signal patterns, called features. Peptide quantification is facilitated by associating signal intensities in features with peptide sequences derived from MS2 spectra. However, missing values due to imperfect feature detection are a common problem. A feature detection approach that directly targets identified peptides (minimizing missing values), but also offers robustness against false-positive features (by assigning meaningful confidence scores), would thus be highly desirable. We developed a new feature detection algorithm within the OpenMS software framework, leveraging ideas and algorithms from the OpenSWATH toolset for DIA/SRM data analysis. Our software, FeatureFinderIdentification ("FFId"), implements a targeted approach to feature detection based on information from identified peptides. This information is encoded in an MS1 assay library, based on which ion chromatogram extraction and detection of feature candidates are carried out. Significantly, when analysing data from experiments comprising multiple samples, our approach distinguishes between "internal" and "external" (inferred) peptide identifications (IDs) for each sample. Based on internal IDs, two sets of positive (true) and negative (decoy) feature candidates are defined. A support vector machine (SVM) classifier is then trained to discriminate between the sets, and is subsequently applied to the "uncertain" feature candidates from external IDs, facilitating selection and confidence scoring of the best feature candidate for each peptide. We validated FFId based on a public benchmark dataset, comprising a yeast cell lysate spiked with protein standards that provide a known ground-truth. The algorithm reached almost complete (>99%) quantification coverage for the full set of peptides identified at 1% FDR. Compared to other software solutions for label-free quantification, this is an outstanding result, which was achieved at competitive quantification accuracy and reproducibility across replicates. The FFId software is open-source and freely available as part of OpenMS (

    Journal of proteome research 2017

  • Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

    Westbury SK, Canault M, Greene D, Bermejo E, Hanlon K, Lambert MP, Millar CM, Nurden P, Obaji SG, Revel-Vilk S, Van Geet C, Downes K, Papadia S, Tuna S, Watt C, Consortium NBD, Freson K, Laffan MA, Ouwehand WH, Alessi MC, Turro E and Mumford AD

    School of Clinical Sciences, University of Bristol, United Kingdom.

    Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterised. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in three pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2,042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5,422 controls. Eleven cases harboured 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included five high-impact variants predicted to prevent CalDAG-GEFI expression and six missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signalling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical and dental bleeding from childhood, requiring at least one blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to ADP and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a non-syndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.

    Blood 2017

  • Genetic aetiology of glycaemic traits: approaches and insights.

    Wheeler E, Marenne G and Barroso I

    Department of Human Genetics, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    Glycaemic traits such as fasting and post-challenge glucose and insulin measures, as well as glycated haemoglobin (HbA1c), are used to diagnose and monitor diabetes. These traits are risk factors for cardiovascular disease even below the diabetic threshold, and their study can additionally yield insights into the pathophysiology of type 2 diabetes. To date, a diverse set of genetic approaches have led to the discovery of over 97 loci influencing glycaemic traits. In this review, we will focus on recent advances in the genetic aetiology of glycaemic traits, and the resulting biological insights. We will provide a brief overview of results ranging from common, to low- and rare-frequency variant-trait association studies, studies leveraging the diversity across populations, and studies harnessing the power of genetic and genomic approaches to gain insights into the biological underpinnings of these traits.

    Human molecular genetics 2017;26;R2;R172-R184

  • The Pursuit of Therapeutic Biomarkers with High-Throughput Cancer Cell Drug Screens.