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Investigation for Novel Schizophrenia Candidate

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Investigation for Novel Schizophrenia Candidate INVESTIGATION OF NOVEL SCHIZOPHRENIA CANDIDATE GENES THROUGH BIOCHEMICAL AND COMPUTATIONAL METHODS by CARRI-LYN REBECCA MEAD Bachelor of Science, University of Waterloo, 1999 (Science and Business) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2010 © Carri-Lyn Rebecca Mead, 2010 Abstract Schizophrenia is a complex highly heritable psychiatric disorder affecting ~1% of the human population. Complex disease research must consider the wide variety of confounding factors that contribute to disease pathology. Underlying genetic contributions to disease are often heterogeneous among the disease population and individual gene linkage and association signals may be weak and inconsistent within affected populations. The disease phenotype may actually result from multiple defects within one or more related functional pathways. Understanding the physical interactions that known susceptibility genes engage in provides insight into the functions and pathways contributing to disease, and also implicates the interacting genes and proteins as potential schizophrenia candidate genes. While many candidate schizophrenia genes have been proposed, findings for only a few genes have been sufficiently replicated for them to be considered schizophrenia susceptibility genes, including neuregulin-1 and dysbindin. The first aim of this thesis was to identify novel candidate schizophrenia genes through investigation of the interactions and pathways that known susceptibility genes neuregulin- 1 and dysbindin participate in. The second aim of this thesis was the generation of a novel method for whole genome linkage meta- analysis. Numerous genome-wide linkage studies have been performed on a wide variety of schizophrenia cohorts, however highly significant genome-wide linkage signals have not been prevalent and there has been little replication between studies. It is possible that individual studies contain weak linkage signals that are consistent across multiple studies, but due to their lack of significance in any one study, have not been identified. The Marker Footprint Linkage Meta-analysis method was developed to allow for refinement of candidate schizophrenia linkage regions from existing studies and identification of novel regions that show broadly consistent, but perhaps weak, linkage signals across multiple studies. Through these analyses a common protein interaction network that encompasses three of the current best schizophrenia susceptibility genes (neuregulin-1, dysbindin, and disrupted-In-schizophrenia-1) was identified. These findings greatly expand current knowledge of interactions with these important schizophrenia susceptibility genes. A novel method for performing genome-wide linkage meta-analyses was developed that incorporates recombination to refine existing linkage regions and identify novel linkage regions that have not previously been identified. ii Table of Contents Abstract .........................................................................................................................................................ii Table of Contents ......................................................................................................................................... iii List of Tables .................................................................................................................................................v List of Figures...............................................................................................................................................vi Abbreviations & Gene Definitions ............................................................................................................... vii Acknowledgements .................................................................................................................................... xiv Dedication....................................................................................................................................................xv Co-Authorship Statement........................................................................................................................... xvi 1 Introduction.............................................................................................................................1 1.1 Thesis Overview .....................................................................................................................1 1.2 Complex Disease ...................................................................................................................2 1.3 Schizophrenia.........................................................................................................................3 1.4 Schizophrenia Susceptibility Genes .......................................................................................6 1.4.1 Dysbindin (DTNBP1) ..............................................................................................................6 1.4.2 Neuregulin-1 (NRG1)..............................................................................................................6 1.4.3 Disrupted-In-Schizophrenia-1 (DISC1)...................................................................................7 1.5 Traditional Disease Gene Finding Methods ...........................................................................7 1.6 Recombination......................................................................................................................10 1.7 Non-Traditional Disease Gene Finding Tools ......................................................................11 1.7.1 Protein-protein interactions...................................................................................................11 1.7.1.1 High-throughput protein-protein interaction detection methods....................................12 1.7.1.2 Protein-protein interaction validation ............................................................................14 1.7.2 Protein-DNA interactions......................................................................................................14 1.7.3 Gene function .......................................................................................................................16 1.7.4 Visualization of biological information using networks .........................................................16 1.8 Statistical Analyses...............................................................................................................17 1.9 Thesis Chapters Summary...................................................................................................18 1.10 References ...........................................................................................................................21 2 Cytosolic Protein Interactions of the Schizophrenia Susceptibility Gene Dysbindin............31 2.1 Introduction...........................................................................................................................31 2.2 Materials and Methods .........................................................................................................32 2.2.1 Cloning..................................................................................................................................32 2.2.2 Antibodies.............................................................................................................................33 2.2.3 Cell culture............................................................................................................................33 2.2.4 Immunoprecipitation, protein complex preparation, and mass spectrometry.......................33 2.2.5 Identification of candidate interacting proteins .....................................................................34 2.2.6 Validation of protein interactions through immunoprecipitation-western analysis................35 2.2.7 Linkage and association with schizophrenia analysis ..........................................................35 2.3 Results..................................................................................................................................36 2.3.1 DTNBP1 protein interactions................................................................................................36 2.3.2 Exocyst and dynactin protein interactions............................................................................37 2.3.3 Verification of DISC1 interactions with exocyst and dynactin complexes ............................37 2.3.4 Gene ontology analysis of interacting proteins.....................................................................37 2.3.5 DTNBP1 and dynactin interacting proteins linked to schizophrenia ....................................38 2.4 Discussion ............................................................................................................................38 2.5 Chapter 2 Tables ..................................................................................................................42 2.6 Chapter 2 Figures.................................................................................................................76 2.7 References ...........................................................................................................................91 3 Protein and DNA Interactions of the Intracellular Domain
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