1. GeneGo. Your GPS in pathway analysis. Confidential © GeneGo Inc. Parkinson’s Disease GWAS and enrichment with MetaCore™ Ewan Hunter, PhD January 18, 2010

2. Confidential © GeneGo Inc.2 GeneGo Services Knowledge base Management Pathway Analysis Platforms Develop Ontologies Develop Disease platforms Annotation and Curation Data Repository Project analysis (on/off site) Wet Lab Services Data parsers Maps, interactions, interface skins Customization/ Integration Compound Assessment analysis

3. Confidential © GeneGo Inc.3 MetaMiner Initiatives Eli Lilly Astra Zeneca Chicago Children’s Hospital University of Glasgow Sheffield Queensland USC Research and DiscoveryClinical CNS Oncology Skin Stem Cells TSRI Cardiac Jackie Immunology Metabolic Knowledge Management Information Technology Vertex IOP Pharma TBA Eli Lilly Tgen Van Andel Harvard Johns Hopkins Exclusive PC contract with top Pharma GSK Top Pharma Nutrition TBA ToxicologyDrug Metabolism/ Positioning Dry Eye Exclusive Project 3 cosmetic co’s FDA ISB University of Amsterdam Respiratory TSRI Infectious Disease TBA Cystic Fibrosis Complete

4. Confidential © GeneGo Inc.4 MetaBase Content Overview Database  Human genes45.471  Human SwissProt proteins20.328  Mouse genes61.311  Mouse SwissProt proteins16.209  Rat genes36.870  Rat SwissProt proteins7.459  Chemical compounds691.896  Compounds with structures673.207  Drugs5.594 - FDA approved drugs1.085 - EU approved drugs169 - Drugs approved by other committees692 - US withdrawn drugs140 - EU withdrawn drugs54 - Clinical trial drugs2.258 - Discontinued drugs1.205 - Preclinical drugs134 - Biologics618  Endogenous compounds4.776  Nutritional compounds93  Metabolite of xenobiotic8.199 Networkable Objects  Human genes in network19.910  Mouse genes in network18.381  Rat genes in network16.228  Chemical compounds221.076  Drugs2.737  Endogenous compounds2.449  Metabolic Reactions8.899  Pubmed journals2.664  Pubmed articles (total)758.139  Pubmed articles (single citation)130.058 Total amount of interactions941.946  Protein-Protein365.129  Compound – Protein502.572  Compound – Compound 9.618  Protein -Reaction19.449  Substrate, Product-Reaction29.366  RNA – Protein15.812

5. Confidential © GeneGo Inc.5 GeneGo Ontologies Ontologies with graphic content GeneGo maps: 808 (regulatory processes maps Disease maps Metabolic maps) GeneGo networks 942 UNIQUE for GeneGo (regulatory networks drug target networks toxicity networks metabolic networks disease biomarker networks) Groups and Complexes: 2247 UNIQUE for GeneGo Schemas for human, mouse or rat ACM2 and ACM4 activation of ERK Inflammation_IL-6 signaling NF-kB_HUMAN

6. Confidential © GeneGo Inc.6 GeneGo Pathway Formats GeneGo Canonical MapsGeneGo Networks Insulin Receptor Signaling PathwaySignal transduction_Insulin signaling Total number to date: 808 Subset of a bigger-global network Static, pre-built Interactive Multistep Well accepted in the field “Review-article” style- mainstream signaling Total number to date: 942 Dynamic Interactive Pre-built and build your own Bigger-global network Include single or multistep Include newer findings, more than mainstream signaling

7. Confidential © GeneGo Inc.7 How to Approach MetaCore Input list Data Mining Functional Representation What do the items on the list collectively represent? Connectivity How do the items on the list connect?

8. Confidential © GeneGo Inc.8 GWAS- Pakinson’s Disease Data: SNP identifiers, allelic frequency and odds ratio The odds ratio = as the ratio of the odds of an event occurring in one group (PD siblings) to the odds of it occurring in another group (non-PD siblings). odds ratio =1  condition or event under study is equally likely in both groups. odds ratio > 1  condition or event is more likely in the first group odds ratio <1  condition or event is less likely in the second group.

9. Confidential © GeneGo Inc.9 Data Format and Data Loading

10. Confidential © GeneGo Inc.10 Define Thresholds

11. Confidential © GeneGo Inc.11 Single Experiment Workflow: Enrichment Histograms for Canonical Pathway Maps

12. Confidential © GeneGo Inc.12 Alzheimer's disease: extracellular Amyloid beta toxicity.

13. Confidential © GeneGo Inc.13 Disease Biomarker Map Overlay

14. Confidential © GeneGo Inc.14 Alzheimer's disease: extracellular Amyloid beta toxicity Ca++ Channels

15. Confidential © GeneGo Inc.15 From this table we can also appreciate that another gene of interest from this canonical pathway map is not only the Amyloid beta A4 precursor protein but that 3 subunits of the Ca2 channel have a significant association to Parkinson’s. This is of interest as there are studies that suggest the disruption in Ca2+ homeostasis within the dopamine neurons of the substantia nigra lead to loss of these SN neurons Sulzer and Schmitz (2007) Parkinson’s Disease: Return of an Old Prime Suspect. Neuron, 55, 8-10 Interesting SNPs and Associated Genes for Parkinson’s

16. Confidential © GeneGo Inc.16 A recent review in Trends in Neurosciences, “Calcium homeostasis, selective vulnerability and Parkinson's disease” ( TINS, 23 March 2009 doi:10.1016/) Has pointed that a very good theory of neuronal degeration/death of the dopamine neurons in the substantia nigra pas compacta (SNc) Is to do with the homeostatic control of Ca2+ within these neurons. The neurons of the SNc are selectively vulnerable to homeostatic Ca2+ stress as particularly Ca2+ ion channels are responsible for maintaining an autonomous pacemaking within these neurons. This Function is unlike other adult neurons in the brain, the pacemaking activity is believed to be important in maintaining dopamine levels in regions that are innervated by these neurons, particularly the striatum. The L-type Ca2+ channels are responsible for this pacemaker activity within the SNc neurons and the combined GWAS and Enrichment analysis within MetaCore, we have indentified 3 subunits of the Ca2+ channel Recent Review in the Parkinson’s Disease Field

17. Confidential © GeneGo Inc.17 A recent review in Trends in Neurosciences, “Calcium homeostasis, selective vulnerability and Parkinson's disease” ( TINS, 23 March 2009 doi:10.1016/) Has pointed that a very good theory of neuronal degeration/death of the dopamine neurons in the substantia nigra pas compacta (SNc) Is to do with the homeostatic control of Ca2+ within these neurons. The neurons of the SNc are selectively vulnerable to homeostatic Ca2+ stress as particularly Ca2+ ion channels are responsible for maintaining an autonomous pacemaking within these neurons. This Function is unlike other adult neurons in the brain, the pacemaking activity is believed to be important in maintaining dopamine levels in regions that are innervated by these neurons, particularly the striatum. The L-type Ca2+ channels are responsible for this pacemaker activity within the SNc neurons and the combined GWAS and Enrichment analysis within MetaCore, we have indentified 3 subunits of the Ca2+ channel Recent Review in the Parkinson’s Disease Field

18. Confidential © GeneGo Inc.18 The L-type Ca2+ channels are responsible for this pacemaker activity within the SNc neurons and the combined GWAS and Enrichment analysis within MetaCore, we indentified 3 subunits of the Ca2+ channel The beta-subunit of the L-type Ca2+ has a large Odds Ratio (1.781) And relatively significant association p-value 0.0094 for association To Parkinson’s Disease Recent findings in relation to results found with MetaCore™

19. Confidential © GeneGo Inc.19 Therefore Is PD simply a reflection of accelerated aging in neurons that rely too heavily upon Ca2+ channels to do theirbusiness? Age is undoubtedly the single strongest risk factor for PD. The ‘wear and tear’ theory of PD does not require a pathogenic agent. Moreover, it argues that there is no disease ‘onset’ other than the one created by the emergence of symptoms when the surviving SNc DA neurons Are incapable of fully compensating for the loss of neighboring neurons (1). It also predicts that we will all develop symptoms if we live long enough. Why then do some people become symptomatic in their 50 s, others in their 60 s or 70 s or not at all? Genetic and environmental factors certainly could account for this variation One of these genetic factors could be mutations within the Ca2+ L-type channel, which through malfunction, the Homeostasis of Ca2+ is not regulated and accelerate the ageing process within these neurons. (1) Zigmond, M.J. et al. (1990) Compensations after lesions of central dopaminergic neurons: some clinical and basic implications. Trends Neurosci. 13, 290–296 Conclusion of GWAS and MetaCore

20. Confidential © GeneGo Inc.20 Can PD be prevented? If PD is a consequence of Ca2+-accelerated aging in SNc DA neurons (and in those neurons with a similar phenotype) then reducing Ca2+ flux should delay the onset of PD symptoms and slow its progression. A recent case-control study of hypertensive patients found a significant reduction in the observed risk of PD with Calcium channel Antagonists( CCA) use, but not with medications that reduce blood pressure in other ways