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Using Gene Ontology Annotations to Interpret DNA Array Data Stefan Pierrou PhD, AstraZeneca Spotfire Users Conference 2001-05-03 Department Author © 2001, AstraZeneca, Inc. - All Rights Reserved. COPD Genomics In collaboration with Southampton University Department Author AstraZeneca-Southampton Collaboration AZ R&D Lund and AZ R&D Charnwood Stephen Holgate, Donna Davies & Ratko Djukanovic et al., Univ. of Southampton, U.K. Analysis of Epithelial Gene Expression in COPD Hypotheses: COPD - caused by smoke and exacerbated by infections COPD - characterised by altered epithelial genotypes and phenotypes In the absence of epithelial activation there is no development of chronic bronchitis, and no progression of airways remodeling which ultimately leads to irreversible obstruction Epithelial responses to stress (smoke) determine the pathological and clinical presentations of COPD Molecular Sciences R&D Lund Stefan Pierrou 3 Objective To identify candidate genes associated with disease to provide opportunities for development of novel treatments of COPD. Molecular Sciences R&D Lund Stefan Pierrou Cycles of Tissue Damage in Pathogenesis of COPD Chronic irritation (Smoking, infections, etc) Genetic predisposition? Mucociliary Dysfunction Epithelial activation, injury & remodeling Disease progression Mucus hypersecretion Bacterial products Inflammatory Response • Proteases • Chemokines • Cytokines • Oxidants Molecular Sciences R&D Lund Stefan Pierrou Bacterial colonization COPD Pathology Chronic airflow obstruction due to chronic bronchitis and/or emphysema Molecular Sciences R&D Lund Stefan Pierrou Analysis of Epithelial Gene Expression in COPD “Critical Path” Smokers with/without COPD Tissue source Brushings Bronchial biopsies Lung resection Primary cell-based model Non-smokers Microarrays Identify differentially expressed genes Bioinformatics Tissue expression pattern RT-PCR IHC/in situ Functional assays Cytokine production Differentiation Proliferation Secretion Motility Molecular Sciences R&D Lund Stefan Pierrou Candidate Targets 7 Stress related to COPD Smoke Oxidants GFs Define the biochemical pathways initiated by COPD related stresses Analysis of Epithelial Gene Expression in COPD Study Design Day 0: Clinical assessment Day 14: Reversibility testing (salbutamol) Day 21: Sputum induction to characterize inflamm. cells & mediators Day 28: Bronchoscopy to obtain samples of bronchial epithelium Day 70: Bronchoscopy to obtain bronchial biopsies Molecular Sciences R&D Lund Stefan Pierrou 8 Analysis of Epithelial Gene Expression in COPD General Exclusion Criteria (1) Atopy (positive skin prick tests and history) (2) Asthma (reversibility to salbutamol <12%) (3) Respiratory diseases other than COPD (4) Other conditions which might compromise bronchoscopy (5) Recent respiratory or other infections (6 weeks) (6) Recent treatment with oral or inhaled corticosteroids Molecular Sciences R&D Lund Stefan Pierrou 9 Tests Performed Clinical screening MRC scale St.Georges´questionnarie Allergy testing Histamine challenge Diary card and peak flow Serum, DNA Sputum induction Blood gases Full Lung function Salbutamol reversibility CT Thorax Bronchoscopies Brushings Biopsies-IHC, ISH Molecular Sciences R&D Lund Stefan Pierrou 10 Data Analysis Sort and Select p-value P call E-lab Excel Results Sheet Molecular Sciences R&D Lund Stefan Pierrou Brushing+control+stimulated (Sammon) incl. ALI Molecular Sciences R&D Lund Stefan Pierrou 12 Clinical Data Overlay Molecular Sciences R&D Lund Stefan Pierrou GOAC - The Gene Ontology Annotation Campaign - some background Department Author Analysis and clustering of gene expression data generates most often lists of incomprehensible gene names Molecular Sciences R&D Lund Stefan Pierrou 15 Clustering of gene expression data according to protein function classification • Classification is currently done manually • Need for automatisation • Gene Ontology is a tool to make this happen Molecular Sciences R&D Lund Stefan Pierrou 16 History - why GO? • Need for data reduction based on biological information • Data overlay tool discussions with Spotfire • Spotfire created a plug in for GO • GO has since then started to become the de facto standard for annotation of genes. Molecular Sciences R&D Lund Stefan Pierrou 17 GO Consortium - www.geneontology.org • Drosophila (fruitfly) - FlyBase • Saccharomyces (budding yeast) - Saccharomyces Genome Database (SGD) • Mus (mouse) - Mouse Genome Database (MGD) & Gene Expression Database (GXD) • Arabidopsis (brassica or mustard family) - The Arabidopsis Information Resource (TAIR) • Caenorhabditis (nematode) - WormBase Molecular Sciences R&D Lund Stefan Pierrou 18 GOC Collaborators • Academic • SwissProt - annotations ongoing • Interpro - annotations ongoing - currently 1/2 done • Corporate • Celera - uses GO for Drosophila • Incyte - sponsor to Stanford group • GOC Sponsor - AstraZeneca Molecular Sciences R&D Lund Stefan Pierrou 19 The Gene Ontology • Molecular function describes the tasks performed by individual gene products; examples are transcription factor and DNA helicase. • Biological process describes broad biological goals, such as mitosis or purine metabolism, that are accomplished by ordered assemblies of molecular functions. • Cellular component encompasses subcellular structures, locations, and macromolecular complexes; examples include nucleus, telomere, and origin recognition complex Molecular Sciences R&D Lund Stefan Pierrou 20 GOC Annotation status - as of April 29 2001 SGD FlyBase MGI • Biological Process 5,684 1,306 3,461 • Molecular Function 5,780 5,290 4,574 • Cellular Component 2,350 1,347 3,545 • Total gene prod. Annot. 6,373 5,628 5,603 Molecular Sciences R&D Lund Stefan Pierrou 21 The GOAC development project How to make use of Gene Ontology annotations a reality Department Author Starting point for the GOAC development project Overlay expression data & visualise Gene ontology DB & browser Molecular Sciences R&D Lund Stefan Pierrou 23 GOAC components Excel Support file Gene ontology DB & browser GO mySQL DB Molecular Sciences R&D Lund Stefan Pierrou Annotation database with GO terms Oracle DB 24 Overlay expression data & visualise Molecular Sciences R&D Lund Stefan Pierrou Molecular Sciences R&D Lund Stefan Pierrou Summary • DNA micro array data can be analysed for a selected set of genes or complete profiles • We suggest the use of controled vocabulary such as GO for profile analysis • The possibility of overlaying expression data on a structure such as GO, will be essential • The Spotfire plugin developed in Göteborg will fill this need Molecular Sciences R&D Lund Stefan Pierrou Acknowledgement • AstraZeneca • Bo Servenius • Robert Virtala • Krzysztof Pawlowski • Jacob Sjöberg • Dan Gustavsson • Spotfire • Tobias Fändriks Molecular Sciences R&D Lund Stefan Pierrou