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WP6.2 Genomics and microbiology

WP6.2 Genomics and microbiology. Overall Objective: To demonstrate how the integration of pathway biology and host/ pathogen genomics can contribute to clinical diagnosis and treatment of infected patients. 7th Consortium Meeting, Amsterdam, 8 th and 9 th May 2006.

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WP6.2 Genomics and microbiology

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  1. WP6.2 Genomics and microbiology Overall Objective: To demonstrate how the integration of pathway biology and host/ pathogen genomics can contribute to clinical diagnosis and treatment of infected patients 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  2. WP6.2 Genomics and microbiology Work Package Focus: Host Interferon Signalling pathway Family of related proteins bind outside cell to specific receptors Interferon Homodimer binding receptor ‘Signalling’ Activation of the Interferon signalling pathwaysresults in changes in expression of 100 to 1000 genes depending on co-stimuli, cell type, environment etc ‘EFFECT’ 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  3. WP6.2 Genomics and microbiology What is the Effect? Development of an inflammatory response Inhibition of cellular proliferation Increased presentation of non-self to immune system An anti-infective state 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  4. WP6.2 Genomics and microbiology Interferon is a key Inflammatory mediator with a number of notable therapeutic applications Treatment of: Chronic Hepatitis C Viral Infection Melanoma Multiple Sclerosis Idiopathic Pulmonary Fibrosis An understanding of intracellular events following interferon receptor binding will enable rational development of therapeutic intervention in the future 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  5. WP6.2 Genomics and microbiology Medical Informatics Bioinformatics Viral Immuno- modulatory Interactions Clinical Host/ Viral Interactions Outcomes: Secretome 1. Further insight into the virus-host interaction 2. Identification of biomarkers for validation and potential therapeutic exploitation 3. Methods for storage, manipulationand exchange of phenotype data Patient-specific Clinical data Protein Interaction Network Host gene expression data microRNA Viral genotying Information Microarray/ Proteomic Profiling 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  6. WP6.2 Genomics and microbiology A clinical context for WP6.2 Treatment of Hepatitis C infected patients with Interferon alpha 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  7. WP6.2 Genomics and microbiology Hepatitis C • Up to 400 million people infected worldwide (1 – 3% world pop) • Common cause of cirrhosis and hepatocellular carcinoma • Transmitted by blood to blood contact (e.g. IV drug use) • 6 Major Genotypes with varying sensitivity to therapy 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  8. WP6.2 Genomics and microbiology Current therapeutic regime for HCV type1 12 weeks before response to therapy can be assessed [Viral Load] 1 year Outpatient Clinical Tracking; Full blood count (every 4 weeks) Liver function (Every 12 weeks) Respond Cure Patient Sampling HCV Genotyping 50% Weekly Ribavirin & IFN therapy 50% £200 £200 £200 Patient Management Adverse therapeutic effects: Flu-like symptoms, Neutropenia, Thrombocytopenia, Haemolysis, Depression DO NOT respond - Epo supplement - Transplant Treatment Stopped - Emerging protease inhibitor (Roche) 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  9. WP6.2 Genomics and microbiology Future therapeutic regime: A personalised approach Early sampling (<24hrs after 1st treatment) will allow: Clinical decision 8 to 10 weeks ahead of current standard 1 year Respond Cure Patient Sampling HCV Genotyping 50% Weekly Ribavirin & IFN therapy 50% DO NOT respond Benefits: Personal: Early patient treatment regime alteration, Individual avoids side-effects Financial: Saving of ~ £2K in treatments Treatment Stopped 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  10. WP6.2 Genomics and microbiology Pilot to establish precedent for: High-throughput analysis combined with a detailed knowledge of pathway biology enabling a patient-specific therapeutic regime Collaboration with: Professor Peter Hayes Patient Sampling HCV Genotyping Complete genome transcriptional analysis of each patient at 3 time points (48 arrays) Day -1 16 Hrs 12 Wks Strategy integrates Host and Pathogen Genomics with Clinical Parameters to define Early Responder (8 patients) Vs Non Responder (8 patients) response to treatment 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  11. WP6.2 Genomics and microbiology Integration of Pathway Biology and Clinical Data Scottish HCV clinical db project ~8000 patients GPX Microarray DB Local Clinical DB [400 patients] Integration by August 2006 Pathway DB Output Integration Patient Info, HCV PCR status and genotype, treatments [regime, side-effects, response], viral load, Treatment, History, Risk Category, methadone use, smoking, virology, complications, haematology, thyroid function, diabetes, heart disease, depression, outcome [cirrhotic etc] 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  12. WP6.2 Genomics and microbiology Integration of Pathway Biology and Clinical Data Progress: Ethical Approval Granted Laboratory personnel confirmed Sample extraction protocol in validation First patients now attending clinic Sampling to begin ASAP 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  13. WP6.2 Genomics and microbiology Personalised approach requires foundation: Comprehensive knowledge of Host-Pathogen pathway biology Pathway Notation, Recuration, Ontologies and DB Pathway Curation Novel Sequence Analysis Virus-Host Interaction Pathway DB DB Integration, Algorithm dev Protein Interaction Network Prediction pipeline Algorithm dev miRNA Control of pathways Laboratory Validation 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  14. WP6.2 Genomics and microbiology Recuration of the Interferon Pathways: • Implementation of new Edinburgh Pathway Notation [EPN] Systems Biology Graphical Notation http://sbgn.org/Workshops (SBGN-1) • Provide a compact view of a signalling pathway • Provide a logical view of the interactions in the pathway • Show the subcellular localisation of the interactions in the pathway • Allow for incomplete knowledge • Enable quantitative analysis of the pathway • Map to an unambiguous computational representation (including SBML) • Be understandable by biologists 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  15. WP6.2 Genomics and microbiology Recuration of the Interferon Pathways: 2. User testing of Edinburgh Pathway Editor [EPE] (Prof I. Goryanin) http://www.bioinformatics.ed.ac.uk/epe/ • Visual pathway editor • Designed for annotation, visualisation and presentation of wide variety of biological networks, including metabolic, genetic and signal transduction pathways. 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  16. WP6.2 Genomics and microbiology Recuration of the Interferon Pathways: Output Several biologist interpretations of pathways from which an improved consensus derived and stored in pathway DB Form basis for further protein interaction/ miRNA work relating to the Host/ Pathogen interaction 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  17. WP6.2 Genomics and microbiology Potential WP6.1 – 6.2 activity Pathway Exchange Formats • Current pathway curation activities: • Known or novel pathway components identified by literature review experimentation, text mining etc • Incorporated into evolving diagrams (typically with non-standard notation) • Supported by unstructured annotational information • Typically: • Curation not supported by an ontology • Result: explorative analysis and deductive reasoning are laborious • Exchange of pathway information is difficult and prone to error and ambiguity 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  18. WP6.2 Genomics and microbiology Potential WP6.1 – 6.2 activity Pathway Exchange Formats A variety of attempts have been made/ or are underway to develop pathway ontology/ exchange formats Examples: Systems Biology Markup Language Protein Standards Initiative – Molecular Interaction XML format Biological Pathway Exchange (BioPAX) project Cell signalling Networks Ontology Event Ontology (INOH) No Current Standard Initial scoping exercise suggests this is not a trivial task – Options to be discussed 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  19. WP6.2 Genomics and microbiology Pathway Database: Now under development • Aims for pathway database environment • Curate current core information and then incorporate • further signalling pathway information (type 1 interferon, • TLR, TNF-alpha) selected via objective criteria. • 2) Curate data generated in course of Infobiomed project Output by June 2006: (Interferon) Pathway Database Output by End 2006: Publicly accessible Interferon pathway database 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  20. WP6.2 Genomics and microbiology 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  21. WP6.2 Genomics and microbiology Datasets Interferon Gamma: JAK/STAT Apoptosis MHC/ Ag IRF subunit Published Data Williams et al etc TNF TLR P38 Kinase Interferon /  Clinical Data Gene lists Gene lists Gene and Edge lists Gene and Edge lists Output ‘Integration’ V0.1 [June] PIANA Validation Gene lists PIANA Gene network expansion Experimental Data Interferon Treated Macrophage time course Infobiomed Data 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  22. WP6.2 Genomics and microbiology Personalised approach requires foundation: Comprehensive knowledge of Host-Pathogen pathway biology Text Mining, Ontologies and Notation development Pathway Curation Novel Sequence Analysis Virus-Host Interaction Pathway DB DB Integration, Algorithm dev Protein Interaction Network Prediction pipeline Algorithm dev miRNA Control of pathways Laboratory Validation 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  23. WP6.2 Genomics and microbiology Virus-Host Interaction Mar Alba – IMIM, Barcelona Ana Angulo/ Pablo Engel - UB Objective: To investigate and characterise Host and Virus protein homologies Outputs: Provides insight into methods pathogen uses to survive and modulate host responses Provides potential targets for clinical intervention 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  24. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog • Member of CD150 Ig superfamily • Expressed on T and B lymphocytes • Binds homophilically to itself • Localises to contact area of cells during Ag dependent ‘immune synapse’ formation • Related to SLAM receptor family • regulation of lymphocyte activation and cytokine secretion • CD229 may attenuate T cell signalling 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  25. MASDVSSHLLTVTQSRWTIHHMYNKLLILALFTPVILESIIYVSGPQGGNVTLVSNFTSN ISARWFRWDGNDSHLICFYKRGEGLSTPYVGLSLSCAANQITIFNLTLNDSGRYGAEGFT RSGENETFLWYNLTVKPKPLETTTASNVTTIVTTTPTVIGTKSNVTGNASLAPQLRAVAG FLNQTPRENNTHLALVGVIVFIALIVVCIMGWWKLLCSKPKL Leader peptide; Ig-like domain;Transmembrane WP6.2 Genomics and microbiology UL7, a homolog of the lymphocyte cell surface protein CD229 in HCMV UL7 and CD229 V-like domains (31% identity, 49% homology) UL7: VSGPQGGNVTLVSNFTSNISARWFRWDGNDSHLICFYKRGEGLST---PYVG-LSLSCAA VSGGG+VTL N + + W G + + F + E ++Y+GL ++ + CD229: VSGILGGSVTLPLNISVDTEIENVIWIG-PKNALAFARPKENVTIMVKSYLGRLDITKWS UL7: NQITIFNLTLNDSGRYGAEGFTRSGENETFLWYNLTVKP +INLTLND+GYA+ R+ E T+L V CD229: YSLCISNLTLNDAGSYKAQINQRNFEVTTEEEFTLFVYE Preserved residues between members of the SLAM family 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  26. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog Highest homology in ligand binding region (almost all CD229 conserved residues present) HCMV UL7 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  27. WP6.2 Genomics and microbiology How might this viral product subvert the host immune response? Viral Homolog CD229 Signalling Human Virus Infected Macrophage MHC TCR T lymphocyte Inhibition of cytotoxicity 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  28. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog Validation in progress (Ana Angulo/ Pablo Engel - Barcelona) • Fluorescent Viral UL7 • anti-UL7 mAb • HCMV UL7 KO COS cells transfected with UL7-EGFP showing membrane expression of construct Current work: Molecular tool development -> functional assays to follow Output by summer 2006: Preliminary data on role of UL7 in viral infection Output by End 2006: Publication on functional role of UL7 in modulation of host during infection 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  29. WP6.2 Genomics and microbiology The personalised approach requires comprehensive knowledge of Host-Pathogen pathway biology Text Mining, Ontologies and Notation development Pathway Curation Novel Sequence Analysis Virus-Host Interaction Pathway DB DB Integration, Algorithm dev Protein Interaction Network Prediction pipeline Algorithm dev miRNA Control of pathways Laboratory Validation 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  30. WP6.2 Genomics and microbiology PIANA pilot work HCMV UL72 [dUTPase] 7 predicted interactions with host proteins [inc components of IFN pathway] UL72 shown not to be a dUTPase Homolog in closely related virus is immune modulator in host • Progress: • Recently appointed PhD student currently undertaking validation • Martin Messerle has produce a UL72KO virus – key reagent in validation process 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  31. WP6.2 Genomics and microbiology Future Plans based around 2 publications 1. ‘Validation of PIANA using interferon protein interaction network’ i. Recreation of network nodes and edges from a seed of components ii. Recreation of network edges using PIANA 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  32. WP6.2 Genomics and microbiology Future Plans based around 2 publications 2. ‘Expansion of Interferon protein network using PIANA’ • Existing interferon pathway components used to initiate expansion and identification of • i. New Nodes [Biomarkers] • ii. New Edges [Pathways] • Expanded network then compared with existing microarray data generated after cellular treatment with interferon • Work to be undertaken by Joan Planas during mobility exchange June/ July 2006 Output by end summer 2006:Manuscript in preparation Output by End 2006:Manuscript submitted 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  33. WP6.2 Genomics and microbiology The personalised approach requires comprehensive knowledge of Host-Pathogen pathway biology Text Mining, Ontologies and Notation development Pathway Curation Novel Sequence Analysis Virus-Host Interaction Pathway DB DB Integration, Algorithm dev Protein Interaction Network Prediction pipeline Algorithm dev miRNA Control of pathways Laboratory Validation 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  34. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus: Novel biomarkers for clinical intervention? Abundant class of tiny regulatory RNAs (21nt) Function post-transcriptionally to prevent protein production via: mRNA cleavage (‘High’ complementarity) &/or Translational repression (‘Low’ complementarity) Control and influence many aspects of cellular physiology 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  35. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus Focussed on answering question: How might a clinically relevant pathogens be subverting host immune response via miRNA expression? 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  36. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus 10 key components of interferon pathway found to contain miRNA target sites miRNA-ID HUGO Ensembl-ID miranda- Score ------------------------------------------------ Single targets: miRUL22A-1_3p HSPA5 ENSG00000044574 119 miRUL22A-1_3p CTSS ENSG00000163131 127 miRUL112-1_3p CBFB ENSG00000067955 113 miRUL112-1_3p DAP ENSG00000112977 132 miRUL148D-1_3p SOCS3 ENSG00000184557 113 miR-US5-1_3p CASP8 ENSG00000064012 124 miR-US5-2_3p PSMD12 ENSG00000197170 111 miRUS33-1_5p PRKCA ENSG00000154229 112 miRUL36-1_5p BCL6 ENSG00000113916 123 Double target: miRUL36-1_5p CAMK2A ENSG00000070808 115 miRUS25-2_3p CAMK2A ENSG00000070808 114 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  37. WP6.2 Genomics and microbiology First: understand the host • superimposition of predicted miRNA target networks for entire genome onto the interferon network • To understand how miRNAs may act to control the expression of interferon-related proteins in vivo. Prediction pipeline All potential miRNAs Map onto interferon genes Novel understanding of system wide control 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  38. WP6.2 Genomics and microbiology Next: understand the pathogen 2. Application of a new algorithm to identification of miRNAs in HCMV, MCMV, KSHV and HCV. > Implemented and retrained existing pipeline (called mirnafind) for precursor prediction from (unconserved) sequences only. 10 miRNA candidate sequences identified in mCMV – None with homology to human virus > Implemented and used existing pipeline for precursor prediction from sequences with homology to Human virus. 5 miRNA candidate sequences identified in mCMV with homology to human virus 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  39. WP6.2 Genomics and microbiology Finally: understand the host and pathogen Dr Amy Buck (UEDIN) working to verify expression and effect of miRNA expression on Interferon pathway Prediction of host target sites for viral miRNA binding Prediction of viral target sites for host miRNA binding Prediction of viral target sites for viral miRNA binding Prediction of host interferon-related target sites for predicted host miRNA binding 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

  40. WP6.2 Genomics and microbiology Milestones and Objectives Year 2 Year 3 Extension Gothenburg Aveiro Amsterdam Data acquisition and curation Pathway Biomarker Validation Data Interpretation - Preparation/ submission of Publications - Pathway DB development - Cross work package collaboration UEDIN/ FORTH Meet WP6-2/ UPM Meet UEDIN/ IMIM Meet WP6-2 Meet WP6-2 Meeting- Edinburgh WP6-2 Meeting- Barcelona Clinical Project Definition Sample collection and profiling Sample analysis and data integration Project Plan Reporting 7th Consortium Meeting, Amsterdam, 8th and 9th May 2006

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