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Our Objective: To demonstrate how the integration of [ Interferon ] Pathway

WP6.2 Genomics and microbiology. Our Objective: To demonstrate how the integration of [ Interferon ] Pathway Biology and host/ pathogen genomics can contribute to clinical diagnosis and treatment of infected patients. We define Pathway Biology as….

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Our Objective: To demonstrate how the integration of [ Interferon ] Pathway

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  1. WP6.2 Genomics and microbiology Our Objective: To demonstrate how the integration of [Interferon] Pathway Biology and host/ pathogen genomics can contribute to clinical diagnosis and treatment of infected patients We define Pathway Biology as… An approach for understanding a biological process through the functional association of multiple genes and gene products and metabolites defining networks of cause-effect relationships. 10th Consortium Meeting, Girona, 12th and 13th February 2007

  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’ 10th Consortium Meeting, Girona, 12th and 13th February 2007

  3. 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 10th Consortium Meeting, Girona, 12th and 13th February 2007

  4. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  5. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  6. WP6.2 Genomics and microbiology A Framework to support Host-Pathogen Pathway Medicine • Objectives: • To produce portal for pathway related data collected to-date. • Integration of pathway data and information with clinical records and laboratory output and resources • Provide standardised functionality [e.g. PSI-ML, BioPAX export] • Provide API for exploitation by external analysis/ visualisation resources Deliverable for June 2007 10th Consortium Meeting, Girona, 12th and 13th February 2007

  7. WP6.2 Genomics and microbiology A Framework to support Host-Pathogen Pathway Medicine Scottish HCV clinical db project ~8000 patients GPX Microarray DB Local Clinical DB [400 patients] Integration by August 2006 Pathway DB 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] Output Integration 10th Consortium Meeting, Girona, 12th and 13th February 2007

  8. WP6.2 Genomics and microbiology A Framework to support Host-Pathogen Pathway Medicine Requirements analysis undertaken - Current DB provides capability: • Publications • All human and mouse Entrez Gene • Gene to Uniprot mapping • Synonym mapping • Homolog Mapping • Predicted miRNAs • siRNA library annotation • Microarray probe mapping • Pathogen [Virus] ORF annotation Host-Pathogen Interactions Application of PSI-ML/ EBI IntAct Ontology/ Controlled Vocabulary [1092+ terms] Introduction of Biosource, Technique Modelling of complexes Infobiomed predicted interactions stored Curation application Web GUI 10th Consortium Meeting, Girona, 12th and 13th February 2007

  9. WP6.2 Genomics and microbiology A Framework to support Host-Pathogen Pathway Medicine 10th Consortium Meeting, Girona, 12th and 13th February 2007

  10. WP6.2 Genomics and microbiology 10th Consortium Meeting, Girona, 12th and 13th February 2007

  11. WP6.2 Genomics and microbiology 10th Consortium Meeting, Girona, 12th and 13th February 2007

  12. WP6.2 Genomics and microbiology 10th Consortium Meeting, Girona, 12th and 13th February 2007

  13. WP6.2 Genomics and microbiology Explorative tool allowing direct visualisation of pathways, disease associations, host-pathogen interactions etc. ‘Biolayout Express3D’ Freeman et al. 2007 10th Consortium Meeting, Girona, 12th and 13th February 2007

  14. WP6.2 Genomics and microbiology Framework Statistics by June 2007 Metabolic Pathways [Kegg] Metabolomic Interactions HostPathogen MicroRNA Interactions Host-Pathogen Protein Interactions Number of BioGrid P to P Interactions Protein Interactions and Disease associations [HPRD] Infobiomed Curated Pathways 1 10 100 1000 10000 100000 10th Consortium Meeting, Girona, 12th and 13th February 2007

  15. WP6.2 Genomics and microbiology Next Steps: Integration of Pathway, Microarray and Clinical Data GPX Database Clinical DB Project and Pathway Data request Pathway DB Application (Diagnosis/ treatment) Project and Anonymised Clinical Data request User selects or inputs pathway data and Clinical Project Integrated patient-specific pathway component data and clinical parameters Analysis: Classification 10th Consortium Meeting, Girona, 12th and 13th February 2007

  16. Feb Mar Apr May June WP6.2 Genomics and microbiology Timescales DB release Consolidation of Data Curation of Data gui refinement and development gui internal review begins Integration with array data and visualisation tool Clinical data integration requirements Clinical data integration specification Manuscript Preparation 10th Consortium Meeting, Girona, 12th and 13th February 2007

  17. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  18. WP6.2 Genomics and microbiology A clinical context for WP6.2 Treatment of Hepatitis C infected patients with Interferon alpha 10th Consortium Meeting, Girona, 12th and 13th February 2007

  19. 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 • In Edinburgh: Types 1 (50% response • and 3 (70+% response) prevalent 10th Consortium Meeting, Girona, 12th and 13th February 2007

  20. WP6.2 Genomics and microbiology Current therapeutic regime for HCV type1 12 weeks before response to therapy can be assessed [Viral Load] Cure orRelapse (Up to 50% depending on starting viral load and regime) 1 year Outpatient Clinical Tracking; Full blood count (every 4 weeks) Liver function (Every 12 weeks) Respond 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) 10th Consortium Meeting, Girona, 12th and 13th February 2007

  21. 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 10th Consortium Meeting, Girona, 12th and 13th February 2007

  22. 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 10th Consortium Meeting, Girona, 12th and 13th February 2007

  23. WP6.2 Genomics and microbiology • Current Phase: • Sample collection • Ethical approval • Delivered SOP, collection tubes etc to nurses • Patients currently undergoing evaluation • Actively recruiting • Existing Data – Development of analysis approaches Encouraging Publication He XS, Ji X, Hale MB, Cheung R, Ahmed A, Guo Y, Nolan GP, Pfeffer LM, Wright TL, Risch N, Tibshirani R, Greenberg HB. Global transcriptional response to interferon is a determinant of HCV treatment outcome and is modified by race. Hepatology. 2006 Aug;44(2):352-9. 10th Consortium Meeting, Girona, 12th and 13th February 2007

  24. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  25. 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) &/or Control and influence many aspects of cellular physiology *Possibly via the deadenylation of target mRNAs -> Processing bodies -> decapping and degradation Giraldez et al. Science (2006) 312 p75-79 10th Consortium Meeting, Girona, 12th and 13th February 2007

  26. WP6.2 Genomics and microbiology • Focussed on answering 2 questions: • How does host use miRNAs to control interferon pathway? • 2. How might a clinically relevant pathogens be subverting host immune response via miRNA expression? 10th Consortium Meeting, Girona, 12th and 13th February 2007

  27. WP6.2 Genomics and microbiology First: understand the pathogen miRNA prediction [M Reczko] miRNA cloning [A Buck] Viral genome Infected cells Small RNAs cloned and sequenced FORTH sequence prediction pipeline retrained Sequence analysis to identify virus-specific mature sequences Candidate precursor sequences with reliability scores Tens of sequences Thousands of sequences Compare Refine algorithm to reproduce biological results Lab Validation Target Prediction, Phenotype analysis and Biomarker identification New predictions 10th Consortium Meeting, Girona, 12th and 13th February 2007

  28. WP6.2 Genomics and microbiology Then: understand the host Human Target prediction [mirBase] miRNA sequences: Current screen (cloned RNAs) identified 10-15 Mouse Target prediction [M Reczko - UPENN] Temporal miRNA expression/ target analysis [Amy Buck] Functional pathway analysis Host-Pathogen Interaction Defined Lab Validation and Publication 10th Consortium Meeting, Girona, 12th and 13th February 2007

  29. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  30. WP6.2 Genomics and microbiology Protein-Protein Interactions • 2 key themes: • Informatics-based: Given our current knowledge of the interferon pathways, can we ‘tune’ PIANA to predict known pathways and networks? • 2. Biologically novel: Can we use PIANA to identify novel protein interactions within host and between host and pathogen? Work carried out by Joan Planas (IMIM, Barcelona) June – August 2006 10th Consortium Meeting, Girona, 12th and 13th February 2007

  31. Protein-Protein Interactions PIANA: Protein Interactions And Network Analysis • A software framework and tool which: • Integrates protein-related data from multiple sources • Provides a library which handles graph-related tasks • Automates the analysis of protein-protein interaction networks • Predicts novel protein interactions • Finds interaction distances between proteins • Match spots from electrophoresis experiments • Clusters proteins by their GO classification Aragues, R., D. Jaeggi, et al. (2006). "PIANA: protein interactions and network analysis." Bioinformatics 22(8): 1015-7. Espana, L., B. Martin, et al. (2005). Am J Pathol167(4): 1125-37. Mendez, O., B. Martin, et al. (2006). Carcinogenesis27(6): 1169-1179. 10th Consortium Meeting, Girona, 12th and 13th February 2007

  32. Protein-Protein Interactions Project Strategy Pathway DB Interferon/ apoptosis pathway components Uniprot IDs Compare to ‘real’ biological network Complete Network 5 x randomly selected 20% of curated network PIANA • Assess ability of 3 different data sources to recreate complete network: • All experimental DB [BIND, DIP, HPRD, MIPS] • STRING • ORI High confidence subset of interactions Network generation by all methods Threshold confidence values produced for each method: Applied to produce hi confidence subset Specificity/ Sensitivity plots for each of 3 approaches and merge with COG expansion data [enhanced by BLAST analysis] 10th Consortium Meeting, Girona, 12th and 13th February 2007

  33. Protein-Protein Interactions Pathway > Interferon Apoptosis BIND DIP MIPS HPRD Sensitivity = 0 Specificity = 0 Sensitivity = 0.808 Specificity = 0.163 Edges Prediction BIND DIP MIPS HPRD STRING Ori Sensitivity = 0.197 Specificity = 0.192 Sensitivity = 0.129 Specificity = 0.121 STRING: KEGG Reactome Biocarta Sensitivity = 0.75 Specificity = 0.88 Node Prediction Disparity between experimental protein interaction resources and accepted ‘canonical’ pathway representations 10th Consortium Meeting, Girona, 12th and 13th February 2007

  34. Protein-Protein Interactions • 1. Implement and assess the effect of orthology expansion incorporation into the 5 fold validation process. • 2. Incorporate microarray data into PIANA predictions in order to improve recovery of pathway interactions. • 3. Define data sources and thresholds for optimal pathway data recovery. • 4. Apply PIANA to the expansion of the known interferon pathways and analyse and interpret function of novel proteins identified. • 5. Apply PIANA to the identification and/ or prediction of novel host-pathogen interactions. • By end Infobiomed – • Manuscript submitted on application of PIANA to enlargement of interferon pathways 10th Consortium Meeting, Girona, 12th and 13th February 2007

  35. WP6.2 Genomics and microbiology Overall Project Structure Interferon Pathway Biology Clinical data and Patient Information Clinical Data Patient Response to treatment Molecular Profiling Pathway Curation and integration Protein-Protein Interaction Virus-Host Interactions miRNA regulation of Host and Pathogen Bioinformatics predictions, basic science laboratory validation and data integration Interferon treatment of HCV type 1 infected patients: Early prediction of success 10th Consortium Meeting, Girona, 12th and 13th February 2007

  36. WP6.2 Genomics and microbiology Transcriptional Control of Interferon Pathway Objective: To investigate and characterise transcriptional control of Interferon and related pathway members Outputs: Provides insight into host regulation of interferon pathways, Provides potential targets for clinical intervention and Provides insights into viral control of pathway. 10th Consortium Meeting, Girona, 12th and 13th February 2007

  37. WP6.2 Genomics and microbiology Interferons Induce a large number of genes through activating the janus tyrosine kinase (JAK)–signal transducers and activators of transcription proteins (STAT) pathway, Leading to… binding of transcription factors to upstream regions of the inducible genes (interferon-stimulated gene, ISG) at specific DNA regulatory elements known as interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS). 10th Consortium Meeting, Girona, 12th and 13th February 2007

  38. WP6.2 Genomics and microbiology Virus-Host Interactions Our aim: To characterise ISRE and GAS elements in curated and experimentally defined (and novel) genes Investigate hypothesis that genes belonging to the same pathway are share common gene expression regulatory mechanisms 10th Consortium Meeting, Girona, 12th and 13th February 2007

  39. WP6.2 Genomics and microbiology PEAKS • Uses a novel algorithm to identify and analyse regulatory motifs which cluster into high abundance ‘peaks’. • Identification of peaks can be used to: • - Increase the specificity of motif predictions • Provide information on promoter structure and • Help discover regulatory motifs specifically involved in the regulation of genes with similar expression or function • Termed ‘positional footprinting’. Positional footprinting has been applied to the identification of novel regulatory motifs in genes related to the interferon pathway Bellora N, Farre D, Alba MM. 2006. PEAKS: identification of regulatory motifs by their position in DNA sequences. Bioinformatics 10th Consortium Meeting, Girona, 12th and 13th February 2007

  40. WP6.2 Genomics and microbiology PEAKS Motifs mapped in genes: TATA, SP1, CAAT, TANTIGEN_B, ISRE GAS* Latest Build of IFN, apoptosis pathway + genes identified experimentally as controlled by interferon [702 Mouse, 650 Human] Focus on ISRE analysis in human genes: Found positional bias around -115bp Bellora N, Farre D, Alba MM. 2006. PEAKS: identification of regulatory motifs by their position in DNA sequences. Bioinformatics 10th Consortium Meeting, Girona, 12th and 13th February 2007

  41. WP6.2 Genomics and microbiology PEAKS • Whole genome characterisation of ISRE and GAS sequences [Novel ‘atlas’ of potential transcriptional control] • Identification of new genes directly controlled by interferon stimulation • - Does virus affect this control? • Ultimately compare with ChIP data from patients • Future - Begin to relate polymorphisms (host genomics) to lack of response to infection IFN proteins direct? indirect? ISRE &/or GAS +/- Virus Bellora N, Farre D, Alba MM. 2006. PEAKS: identification of regulatory motifs by their position in DNA sequences. Bioinformatics 10th Consortium Meeting, Girona, 12th and 13th February 2007

  42. Outcomes Pathway Biology Pathway Medicine Clinical Records and Data GPX DB Biomarker Discovery e.g. Patient Specific Molecular Signature Clinical DB Pathway DB BMI tools & Approaches 10th Consortium Meeting, Girona, 12th and 13th February 2007

  43. Outcomes Recent Publication: March 2007 Promoting Coherent Minimum Reporting Requirements for Biological and Biomedical Investigations: The MIBBI Project Taylor, CF. et al. Nature Biotechnology, 2007 Infobiomed presented as a project that cuts across domain boundaries and working towards resources that can be applied across biological, medical and technological domains. Publications (direct and indirect): Host-Virus miRNA control – Interferon pathway (Amy Buck) Re-annotation of CMV and publication of array (Andrew Livingston) A Framework for Pathway Medicine [DB work] Practical application of PIANA and novel insights into IFN pathway UL72 of HCMV functional significance (Tali Petchnick) UL7 (HCMV) modulation of host immune response ‘Exemplar’ clinical study on interferon and infection – publication/ grant? 10th Consortium Meeting, Girona, 12th and 13th February 2007

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