Publications by authors named "Richard R Copley"

61 Publications

The Spindle Assembly Checkpoint Functions during Early Development in Non-Chordate Embryos.

Cells 2020 04 28;9(5). Epub 2020 Apr 28.

Sorbonne Universités, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), 06234 Villefranche-sur-mer, France.

In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.
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http://dx.doi.org/10.3390/cells9051087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290841PMC
April 2020

Molecular characterisation of a cellular conveyor belt in Clytia medusae.

Dev Biol 2019 12 8;456(2):212-225. Epub 2019 Sep 8.

Sorbonne Université, MNHN, CNRS, EPHE, Institut de Systématique, Evolution, Biodiversité (ISYEB UMR 7205), Paris, France. Electronic address:

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a "cellular conveyor belt". Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/β-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.
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http://dx.doi.org/10.1016/j.ydbio.2019.09.001DOI Listing
December 2019

Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria.

Curr Biol 2019 06 16;29(11):1818-1826.e6. Epub 2019 May 16.

Centre for Life's Origins and Evolution, Department of Genetics, Evolution and Environment, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK; Department of Computer Science, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK. Electronic address:

Xenoturbella and the acoelomorph worms (Xenacoelomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a clade called the Xenambulacraria [3-5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.
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http://dx.doi.org/10.1016/j.cub.2019.04.009DOI Listing
June 2019

The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle.

Nat Ecol Evol 2019 05 11;3(5):801-810. Epub 2019 Mar 11.

Laboratoire de Biologie du Développement de Villefranche-sur-mer, Sorbonne Université, CNRS, Villefranche-sur-mer, France.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterized by distinct transcriptome signatures reflecting abrupt life-cycle transitions and all deploy a mixture of phylogenetically old and new genes. Medusa-specific transcription factors, including many with bilaterian orthologues, associate with diverse neurosensory structures. Compared to Clytia, the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content evolution and sequence evolution. Absence of expression and gene loss among Clytia orthologues of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys the ancestral cnidarian-bilaterian transcription factor gene complement.
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http://dx.doi.org/10.1038/s41559-019-0833-2DOI Listing
May 2019

Kif2 localizes to a subdomain of cortical endoplasmic reticulum that drives asymmetric spindle position.

Nat Commun 2017 10 13;8(1):917. Epub 2017 Oct 13.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanologique, Villefranche sur-mer, 06230, France.

Asymmetric positioning of the mitotic spindle is a fundamental process responsible for creating sibling cell size asymmetry; however, how the cortex causes the depolymerization of astral microtubules during asymmetric spindle positioning has remained elusive. Early ascidian embryos possess a large cortical subdomain of endoplasmic reticulum (ER) that causes asymmetric spindle positioning driving unequal cell division. Here we show that the microtubule depolymerase Kif2 localizes to this subdomain of cortical ER. Rapid live-cell imaging reveals that microtubules are less abundant in the subdomain of cortical ER. Inhibition of Kif2 function prevents the development of mitotic aster asymmetry and spindle pole movement towards the subdomain of cortical ER, whereas locally increasing microtubule depolymerization causes exaggerated asymmetric spindle positioning. This study shows that the microtubule depolymerase Kif2 is localized to a cortical subdomain of endoplasmic reticulum that is involved in asymmetric spindle positioning during unequal cell division.Early ascidian embryos have a cortical subdomain of endoplasmic reticulum (ER) that controls asymmetric spindle positioning driving unequal cell division. Here the authors show that the microtubule depolymerase Kif2 is localized to a cortical subdomain of the ER that is involved in asymmetric spindle positioning.
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http://dx.doi.org/10.1038/s41467-017-01048-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640700PMC
October 2017

MicroRNAs That Contribute to Coordinating the Immune Response in .

Genetics 2017 09 13;207(1):163-178. Epub 2017 Jul 13.

Laboratory of Cell Biology, Development and Genetics, Department of Biochemistry, University of Oxford, OX1 3QU, United Kingdom

Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defenses. Here, we have used to explore the contribution of individual or clusters of miRNAs in countering systemic infection. From a total of 72 tested, we identify 6 miRNA allelic mutant backgrounds that modulate the survival response to infection and the ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts () and (). These are characteristics of defects in Toll signaling, and consistent with this, we demonstrate dependency for one of the miRNA mutants on the NF-κΒ homolog Dif. We also quantify changes in the miRNA expression profile over time in response to three pathogen types, and identify 13 mature miRNA forms affected by pathogens that stimulate Toll signaling. To complement this, we provide a genome-wide map of potential NF-κB sites in proximity to miRNA genes. Finally, we demonstrate that systemic infection contributes to a reduction in the total amount of branch-chained amino acids, which is miRNA-regulated. Overall, our data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection.
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http://dx.doi.org/10.1534/genetics.116.196584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586370PMC
September 2017

Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations.

Haematologica 2016 11 20;101(11):1306-1318. Epub 2016 Sep 20.

National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre, Oxford, UK.

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.
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http://dx.doi.org/10.3324/haematol.2016.144063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394871PMC
November 2016

Hydrozoan insights in animal development and evolution.

Curr Opin Genet Dev 2016 08 29;39:157-167. Epub 2016 Jul 29.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), 181 chemin du Lazaret, 06230, Villefranche-sur-mer, France.

The fresh water polyp Hydra provides textbook experimental demonstration of positional information gradients and regeneration processes. Developmental biologists are thus familiar with Hydra, but may not appreciate that it is a relatively simple member of the Hydrozoa, a group of mostly marine cnidarians with complex and diverse life cycles, exhibiting extensive phenotypic plasticity and regenerative capabilities. Hydrozoan species offer extensive opportunities to address many developmental mechanisms relevant across the animal kingdom. Here we review recent work from non-Hydra hydrozoans - hydromedusae, hydroids and siphonophores - shedding light on mechanisms of oogenesis, embryonic patterning, allorecognition, stem cell regulation and regeneration. We also highlight potential research directions in which hydrozoan diversity can illuminate the evolution of developmental processes at micro- and macro-evolutionary time scales.
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http://dx.doi.org/10.1016/j.gde.2016.07.006DOI Listing
August 2016

Identification of differentially expressed genes from multipotent epithelia at the onset of an asexual development.

Sci Rep 2016 06 6;6:27357. Epub 2016 Jun 6.

CNRS, Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-mer, Observatoire Océanographique, 06230, Villefranche-sur-mer, France.

Organisms that have evolved alternative modes of reproduction, complementary to the sexual mode, are found across metazoans. The chordate Botryllus schlosseri is an emerging model for asexual development studies. Botryllus can rebuild its entire body from a portion of adult epithelia in a continuous and stereotyped process called blastogenesis. Anatomy and ontogenies of blastogenesis are well described, however molecular signatures triggering this developmental process are entirely unknown. We isolated tissues at the site of blastogenesis onset and from the same epithelia where this process is never triggered. We linearly amplified an ultra-low amount of mRNA (<10ng) and generated three transcriptome datasets. To provide a conservative landscape of transcripts differentially expressed between blastogenic vs. non-blastogenic epithelia we compared three different mapping and analysis strategies with a de novo assembled transcriptome and partially assembled genome as references, additionally a self-mapping strategy on the dataset. A subset of differentially expressed genes were analyzed and validated by in situ hybridization. The comparison of different analyses allowed us to isolate stringent sets of target genes, including transcripts with potential involvement in the onset of a non-embryonic developmental pathway. The results provide a good entry point to approach regenerative event in a basal chordate.
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http://dx.doi.org/10.1038/srep27357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893630PMC
June 2016

The Unicellular Ancestry of Groucho-Mediated Repression and the Origins of Metazoan Transcription Factors.

Authors:
Richard R Copley

Genome Biol Evol 2016 06 27;8(6):1859-67. Epub 2016 Jun 27.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), 181 chemin du Lazaret, 06230 Villefranche-sur-mer, France

Groucho is a co-repressor that interacts with many transcription factors playing a crucial role in animal development. The evolutionary origins of Groucho are not clear. It is generally regarded as being a distinct animal-specific protein, although with similarities to the yeast Tup-like proteins. Here, it is shown that Groucho has true orthologs in unicellular relatives of animals. Based on their phylogenetic distribution, and an analysis of ligand-binding residues, these genes are unlikely to be orthologs of the fungal Tup-like genes. By identifying conserved candidate Groucho interaction motifs (GIMs) in nonmetazoan transcription factors, it is demonstrated that the details of molecular interactions between Groucho and transcription factors are likely to have been established prior to the origin of animals, but that the association of GIMs with many transcription factor types can be regarded as a metazoan innovation.
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http://dx.doi.org/10.1093/gbe/evw118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943189PMC
June 2016

Zoology: War of the Worms.

Curr Biol 2016 04;26(8):R335-7

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), 181 chemin du Lazaret, 06230 Villefranche-sur-mer, France.

The phylogenetic affinities of Xenacoelomorpha - the phylum comprising Xenoturbella bocki and acoelomorph worms - are debated. Two recent studies conclude they represent the earliest branching bilaterally symmetrical animals, but additional tests may be needed to confirm this notion.
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http://dx.doi.org/10.1016/j.cub.2016.03.015DOI Listing
April 2016

Factors influencing success of clinical genome sequencing across a broad spectrum of disorders.

Authors:
Jenny C Taylor Hilary C Martin Stefano Lise John Broxholme Jean-Baptiste Cazier Andy Rimmer Alexander Kanapin Gerton Lunter Simon Fiddy Chris Allan A Radu Aricescu Moustafa Attar Christian Babbs Jennifer Becq David Beeson Celeste Bento Patricia Bignell Edward Blair Veronica J Buckle Katherine Bull Ondrej Cais Holger Cario Helen Chapel Richard R Copley Richard Cornall Jude Craft Karin Dahan Emma E Davenport Calliope Dendrou Olivier Devuyst Aimée L Fenwick Jonathan Flint Lars Fugger Rodney D Gilbert Anne Goriely Angie Green Ingo H Greger Russell Grocock Anja V Gruszczyk Robert Hastings Edouard Hatton Doug Higgs Adrian Hill Chris Holmes Malcolm Howard Linda Hughes Peter Humburg David Johnson Fredrik Karpe Zoya Kingsbury Usha Kini Julian C Knight Jonathan Krohn Sarah Lamble Craig Langman Lorne Lonie Joshua Luck Davis McCarthy Simon J McGowan Mary Frances McMullin Kerry A Miller Lisa Murray Andrea H Németh M Andrew Nesbit David Nutt Elizabeth Ormondroyd Annette Bang Oturai Alistair Pagnamenta Smita Y Patel Melanie Percy Nayia Petousi Paolo Piazza Sian E Piret Guadalupe Polanco-Echeverry Niko Popitsch Fiona Powrie Chris Pugh Lynn Quek Peter A Robbins Kathryn Robson Alexandra Russo Natasha Sahgal Pauline A van Schouwenburg Anna Schuh Earl Silverman Alison Simmons Per Soelberg Sørensen Elizabeth Sweeney John Taylor Rajesh V Thakker Ian Tomlinson Amy Trebes Stephen Rf Twigg Holm H Uhlig Paresh Vyas Tim Vyse Steven A Wall Hugh Watkins Michael P Whyte Lorna Witty Ben Wright Chris Yau David Buck Sean Humphray Peter J Ratcliffe John I Bell Andrew Om Wilkie David Bentley Peter Donnelly Gilean McVean

Nat Genet 2015 Jul 18;47(7):717-726. Epub 2015 May 18.

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.
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http://dx.doi.org/10.1038/ng.3304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601524PMC
July 2015

Erythrocytosis associated with a novel missense mutation in the BPGM gene.

Haematologica 2014 Oct 11;99(10):e201-4. Epub 2014 Jul 11.

Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK

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http://dx.doi.org/10.3324/haematol.2014.109306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181271PMC
October 2014

Phylogenomic analysis of echinoderm class relationships supports Asterozoa.

Proc Biol Sci 2014 Jul;281(1786)

CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanographique, Villefranche-sur-mer 06230, France Sorbonne Universites, UPMC Univ Paris 06, Laboratoire de Biologie du Developpement de Villefranche-sur-mer, Observatoire Oceanographique, Villefranche-sur-mer 06230, France

While some aspects of the phylogeny of the five living echinoderm classes are clear, the position of the ophiuroids (brittlestars) relative to asteroids (starfish), echinoids (sea urchins) and holothurians (sea cucumbers) is controversial. Ophiuroids have a pluteus-type larva in common with echinoids giving some support to an ophiuroid/echinoid/holothurian clade named Cryptosyringida. Most molecular phylogenetic studies, however, support an ophiuroid/asteroid clade (Asterozoa) implying either convergent evolution of the pluteus or reversals to an auricularia-type larva in asteroids and holothurians. A recent study of 10 genes from four of the five echinoderm classes used 'phylogenetic signal dissection' to separate alignment positions into subsets of (i) suboptimal, heterogeneously evolving sites (invariant plus rapidly changing) and (ii) the remaining optimal, homogeneously evolving sites. Along with most previous molecular phylogenetic studies, their set of heterogeneous sites, expected to be more prone to systematic error, support Asterozoa. The homogeneous sites, in contrast, support an ophiuroid/echinoid grouping, consistent with the cryptosyringid clade, leading them to posit homology of the ophiopluteus and echinopluteus. Our new dataset comprises 219 genes from all echinoderm classes; analyses using probabilistic Bayesian phylogenetic methods strongly support Asterozoa. The most reliable, slowly evolving quartile of genes also gives highest support for Asterozoa; this support diminishes in second and third quartiles and the fastest changing quartile places the ophiuroids close to the root. Using phylogenetic signal dissection, we find heterogenous sites support an unlikely grouping of Ophiuroidea + Holothuria while homogeneous sites again strongly support Asterozoa. Our large and taxonomically complete dataset finds no support for the cryptosyringid hypothesis; in showing strong support for the Asterozoa, our preferred topology leaves the question of homology of pluteus larvae open.
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http://dx.doi.org/10.1098/rspb.2014.0479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046411PMC
July 2014

Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis.

Hum Mol Genet 2014 Jun 25;23(12):3200-11. Epub 2014 Jan 25.

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, NIHR Biomedical Research Centre, Oxford, UK,

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.
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http://dx.doi.org/10.1093/hmg/ddu030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030775PMC
June 2014

Causes and consequences of chromatin variation between inbred mice.

PLoS Genet 2013 Jun 13;9(6):e1003570. Epub 2013 Jun 13.

Wellcome Trust Centre for Human Genetics, Oxford, UK.

Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS). Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10%) of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.
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http://dx.doi.org/10.1371/journal.pgen.1003570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681629PMC
June 2013

Scaffolding low quality genomes using orthologous protein sequences.

Bioinformatics 2013 Jan 18;29(2):160-5. Epub 2012 Nov 18.

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

Motivation: The ready availability of next-generation sequencing has led to a situation where it is easy to produce very fragmentary genome assemblies. We present a pipeline, SWiPS (Scaffolding With Protein Sequences), that uses orthologous proteins to improve low quality genome assemblies. The protein sequences are used as guides to scaffold existing contigs, while simultaneously allowing the gene structure to be predicted by homology.

Results: To perform, SWiPS does not depend on a high N50 or whole proteins being encoded on a single contig. We tested our algorithm on simulated next-generation data from Ciona intestinalis, real next-generation data from Drosophila melanogaster, a complex genome assembly of Homo sapiens and the low coverage Sanger sequence assembly of Callorhinchus milii. The improvements in N50 are of the order of ∼20% for the C.intestinalis and H.sapiens assemblies, which is significant, considering the large size of intergenic regions in these eukaryotes. Using the CEGMA pipeline to assess the gene space represented in the genome assemblies, the number of genes retrieved increased by >110% for C.milii and from 20 to 40% for C.intestinalis. The scaffold error rates are low: 85-90% of scaffolds are fully correct, and >95% of local contig joins are correct.

Availability: SWiPS is available freely for download at http://www.well.ox.ac.uk/∼yli142/swips.html.

Contact: yang.li@well.ox.ac.uk or copley@well.ox.ac.uk
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http://dx.doi.org/10.1093/bioinformatics/bts661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546802PMC
January 2013

Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease.

Eur J Hum Genet 2013 Mar 12;21(3):274-80. Epub 2012 Sep 12.

Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.
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http://dx.doi.org/10.1038/ejhg.2012.172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573204PMC
March 2013

Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin.

Genet Med 2012 Nov 12;14(11):891-9. Epub 2012 Jul 12.

Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology, John Radcliffe Hospital, University of Oxford, UK.

Purpose: The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa.

Methods: Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance.

Results: Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic.

Conclusion: Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.
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http://dx.doi.org/10.1038/gim.2012.73DOI Listing
November 2012

Improving animal phylogenies with genomic data.

Trends Genet 2011 May 15;27(5):186-95. Epub 2011 Mar 15.

Department of Genetics, Evolution and Environment, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK.

Since the first animal genomes were completely sequenced ten years ago, evolutionary biologists have attempted to use the encoded information to reconstruct different aspects of the earliest stages of animal evolution. One of the most important uses of genome sequences is to understand relationships between animal phyla. Despite the wealth of data available, ranging from primary sequence data to gene and genome structures, our lack of understanding of the modes of evolution of genomic characters means that using these data is fraught with potential difficulties, leading to errors in phylogeny reconstruction. Improved understanding of how different character types evolve, the use of this knowledge to develop more accurate models of evolution, and denser taxonomic sampling, are now minimizing the sources of error. The wealth of genomic data now being produced promises that a well-resolved tree of the animal phyla will be available in the near future.
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http://dx.doi.org/10.1016/j.tig.2011.02.003DOI Listing
May 2011

Acoelomorph flatworms are deuterostomes related to Xenoturbella.

Nature 2011 Feb;470(7333):255-8

Centre Robert-Cedergren, Département de Biochimie, Université de Montréal, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.

Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data have revised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes and positioning the Acoelomorpha as the most basally branching bilaterian group(s). Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria. Here we assemble three independent data sets-mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements-and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.
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http://dx.doi.org/10.1038/nature09676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025995PMC
February 2011

Beyond the enhanceosome: cluster of novel κB sites downstream of the human IFN-β gene is essential for lipopolysaccharide-induced gene activation.

Blood 2010 Dec 20;116(25):5580-8. Epub 2010 Sep 20.

Imperial College of Science, Technology and Medicine, London, UK.

The expression of interferon-β (IFN-β) in virus-infected HeLa cells established a paradigm of multifactorial gene regulation, in which cooperative assembly of transcription factors (TFs) at the composite DNA element (enhanceosome), is central for amplification of weak activating signals provided by individual TFs. However, whether the same TFs and the same DNA element are essential for IFN-β induction in response to bacterial stimuli are less well understood. Here we report that rapid and transient transcription of IFN-β in response to TLR4 stimulation with bacterial lipopolysaccharide (LPS) follows nuclear factor-κB (NF-κB) RelA activation and recruitment to the IFN-β genomic locus at multiple spatially separated regulatory regions. We demonstrate that the IFN-β enhanceosome region is not sufficient for maximal gene induction in response to LPS and identify an essential cluster of homotypic κB sites in the 3' downstream of the gene. The cluster is characterized by elevated levels of histone 3 lysine 4 mono-methylation, a chromatin signature of enhancers, and efficiently binds RelA-containing NF-κB complexes in vitro and in vivo. These findings demonstrate that IFN-β gene activation via multifactorial enhanceosome assembly is potentiated in LPS-stimulated cells by NF-κB interactions with all functional κB sites in the locus.
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http://dx.doi.org/10.1182/blood-2010-05-282285DOI Listing
December 2010

Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts.

J Biol Chem 2009 Jun 21;284(25):16767-75. Epub 2009 Apr 21.

Henry Wellcome Building of Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom.

Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-alpha subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1alpha or HIF-2alpha at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1alpha and HIF-2alpha (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1alpha- versus HIF-2alpha-binding sites revealed that whereas some loci bound HIF-1alpha in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2alpha contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.
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http://dx.doi.org/10.1074/jbc.M901790200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719312PMC
June 2009

A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: the NIH heterogeneous stock.

Genome Res 2009 Jan 29;19(1):150-8. Epub 2008 Oct 29.

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

The laboratory rat (Rattus norvegicus) is a key tool for the study of medicine and pharmacology for human health. A large database of phenotypes for integrated fields such as cardiovascular, neuroscience, and exercise physiology exists in the literature. However, the molecular characterization of the genetic loci that give rise to variation in these traits has proven to be difficult. Here we show how one obstacle to progress, the fine-mapping of quantitative trait loci (QTL), can be overcome by using an outbred population of rats. By use of a genetically heterogeneous stock of rats, we map a locus contributing to variation in a fear-related measure (two-way active avoidance in the shuttle box) to a region on chromosome 5 containing nine genes. By establishing a protocol measuring multiple phenotypes including immunology, neuroinflammation, and hematology, as well as cardiovascular, metabolic, and behavioral traits, we establish the rat HS as a new resource for the fine-mapping of QTLs contributing to variation in complex traits of biomedical relevance.
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http://dx.doi.org/10.1101/gr.081497.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612958PMC
January 2009

POPE--a tool to aid high-throughput phylogenetic analysis.

Bioinformatics 2008 Dec 10;24(23):2778-9. Epub 2008 Oct 10.

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

Unlabelled: POPE (Phylogeny, Ortholog and Paralog Extractor) provides an integrated platform for automatic ortholog identification. Intermediate steps can be visualized, modified and analyzed in order to assess and improve the underlying quality of orthology and paralogy assignments.

Availability: POPE is available for download from the website: http://www.well.ox.ac.uk/~tota/pope.
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http://dx.doi.org/10.1093/bioinformatics/btn533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2639271PMC
December 2008

The animal in the genome: comparative genomics and evolution.

Authors:
Richard R Copley

Philos Trans R Soc Lond B Biol Sci 2008 Apr;363(1496):1453-61

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

Comparisons between completely sequenced metazoan genomes have generally emphasized how similar their encoded protein content is, even when the comparison is between phyla. Given the manifest differences between phyla and, in particular, intuitive notions that some animals are more complex than others, this creates something of a paradox. Simplistic explanations have included arguments such as increased numbers of genes; greater numbers of protein products produced through alternative splicing; increased numbers of regulatory non-coding RNAs and increased complexity of the cis-regulatory code. An obvious value of complete genome sequences lies in their ability to provide us with inventories of such components. I examine progress being made in linking genome content to the pattern of animal evolution, and argue that the gap between genomic and phenotypic complexity can only be understood through the totality of interacting components.
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http://dx.doi.org/10.1098/rstb.2007.2235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2614226PMC
April 2008

Human-mouse quantitative trait locus concordance and the dissection of a human neuroticism locus.

Biol Psychiatry 2008 May 20;63(9):874-83. Epub 2008 Feb 20.

Wellcome Trust Centre for Human Genetics, Headington, Oxford, United Kingdom.

Background: Exploiting synteny between mouse and human disease loci has been proposed as a cost-effective method for the identification of human susceptibility genes. Here we explore its utility in an analysis of a human personality trait, neuroticism, which can be modeled in mice by tests of emotionality. We investigated a mouse emotionality locus on chromosome 1 that contains no annotated genes but abuts four regulators of G protein signaling, one of which (rgs2) has been previously identified as a quantitative trait gene for emotionality. This locus is syntenic with a human region that has been consistently implicated in the genetic aetiology of neuroticism.

Methods: The functional candidacy of 29 murine sequence variants was tested by a combination of gel shift and transient transfection assays. Murine sequences that contained functional variants and exhibited significant cross-species conservation were prioritized for investigation in humans. Genetic association with neuroticism was tested in 1869 high and 2032 low unrelated individuals scored for neuroticism, selected from the extremes of 88,141 people from southwest England.

Results: Fifteen sequence variants contributed to variation in the expression of rgs18, the gene lying at the edge of the quantitative trait loci (QTL) interval. There was no evidence of association between neuroticism and single nucleotide polymorphisms (SNPs) lying in the human regions homologous to those of mouse functional variants. One SNP, rs6428058, in a region of sequence conservation 644 kb upstream of RGS18, showed significant association (p = .000631).

Conclusions: It is unlikely that a single variant is responsible for the mouse emotionality locus on chromosome 1. This level of underlying genetic complexity means that although cross-species QTL concordance may be invaluable for the identification of human disease loci, it is unlikely to be as informative in the identification of human disease-causing variants.
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http://dx.doi.org/10.1016/j.biopsych.2007.10.019DOI Listing
May 2008

Functional conservation of Rel binding sites in drosophilid genomes.

Genome Res 2007 Sep;17(9):1327-35

Wellcome Trust Centre for Human Genetics, Oxford University, Oxford OX3 7BN, United Kingdom.

Evolutionary constraints on gene regulatory elements are poorly understood: Little is known about how the strength of transcription factor binding correlates with DNA sequence conservation, and whether transcription factor binding sites can evolve rapidly while retaining their function. Here we use the model of the NFKB/Rel-dependent gene regulation in divergent Drosophila species to examine the hypothesis that the functional properties of authentic transcription factor binding sites are under stronger evolutionary constraints than the genomic background. Using molecular modeling we compare tertiary structures of the Drosophila Rel family proteins Dorsal, Dif, and Relish and demonstrate that their DNA-binding and protein dimerization domains undergo distinct rates of evolution. The accumulated amino acid changes, however, are unlikely to affect DNA sequence recognition and affinity. We employ our recently developed microarray-based experimental platform and principal coordinates statistical analysis to quantitatively and systematically profile DNA binding affinities of three Drosophila Rel proteins to 10,368 variants of the NFKB recognition sequences. We then correlate the evolutionary divergence of gene regulatory regions with differences in DNA binding affinities. Genome-wide analyses reveal a significant increase in the number of conserved Rel binding sites in promoters of developmental and immune genes. Significantly, the affinity of Rel proteins to these sites was higher than to less conserved sites and was maintained by the conservation of the DNA binding site sequence (static conservation) or in some cases despite significantly diverged sequences (dynamic conservation). We discuss how two types of conservation may contribute to the stabilization and optimization of a functional gene regulatory code in evolution.
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http://dx.doi.org/10.1101/gr.6490707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950901PMC
September 2007

Identification of common genetic variation that modulates alternative splicing.

PLoS Genet 2007 Jun;3(6):e99

University Department of Paediatrics, John Radcliffe Hospital, Oxford, United Kingdom.

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.
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http://dx.doi.org/10.1371/journal.pgen.0030099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904363PMC
June 2007

A high-resolution single nucleotide polymorphism genetic map of the mouse genome.

PLoS Biol 2006 Nov;4(12):e395

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

High-resolution genetic maps are required for mapping complex traits and for the study of recombination. We report the highest density genetic map yet created for any organism, except humans. Using more than 10,000 single nucleotide polymorphisms evenly spaced across the mouse genome, we have constructed genetic maps for both outbred and inbred mice, and separately for males and females. Recombination rates are highly correlated in outbred and inbred mice, but show relatively low correlation between males and females. Differences between male and female recombination maps and the sequence features associated with recombination are strikingly similar to those observed in humans. Genetic maps are available from http://gscan.well.ox.ac.uk/#genetic_map and as supporting information to this publication.
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http://dx.doi.org/10.1371/journal.pbio.0040395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635748PMC
November 2006