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Genetic Variation and Functional Analysis of the Cardiomedin Gene

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Genetic Variation and Functional Analysis of the Cardiomedin Gene TECHNISCHE UNIVERSITÄT MÜNCHEN LEHRSTUHL FÜR EXPERIMENTELLE GENETIK Genetic Variation and Functional Analysis of the Cardiomedin Gene Zasie Susanne Schäfer Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzende: Univ.-Prof. A. Schnieke, Ph.D. Prüfer der Dissertation: 1. apl. Prof. Dr. J. Adamski 2. Univ.-Prof. Dr. Dr. H.-R. Fries 3. Univ.-Prof. Dr. Th. Meitinger Die Dissertation wurde am. 31.05.2011 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 02.04.2012 angenommen. Table of Contents Table of contents Abbreviations ........................................................................................................................ 7 1. Summary ..........................................................................................................................10 Zusammenfassung ...............................................................................................................11 2. Introduction ......................................................................................................................12 2.1 Genome-wide association studies (GWAS) and post-GWAS functional genomics ......12 2.2 Genetic influences on cardiac repolarization and sudden cardiac death syndrome in GWAS and the chromosome 1q23.1 gene region .............................................................16 2.2.1 Genome-wide association study of cardiovascular disease ......................................17 2.2.2 Sudden cardiac death (SCD) ...................................................................................20 2.3 Sudden unexpected death syndrome (SUDS) and sudden infant death syndrome (SIDS) ...............................................................................................................................22 2.4 Illustration of relevant proteins in this study .................................................................25 2.4.1 Nitric oxid synthase 1 adaptor protein (NOS1AP) .....................................................25 2.4.2 Cardiomedin ............................................................................................................26 2.4.3 Potassium channel KCNH2 ......................................................................................30 2.5 Aims and structure of the work ....................................................................................33 3. Materials and Methods .....................................................................................................34 3.1 Material .......................................................................................................................34 3.1.1 Chemicals ................................................................................................................34 3.1.2 General equipment ..................................................................................................34 3.1.3 Protein chemistry .....................................................................................................34 3.1.4 Mass spectrometry ...................................................................................................35 3.1.5 Mammalian cell culture ............................................................................................35 3.1.6 Molecular biology .....................................................................................................35 3.1.7 Antibodies ................................................................................................................38 3.1.8 Software and databases ..........................................................................................39 3.2 Methods ......................................................................................................................40 3.2.1 Molecular biology .....................................................................................................40 3.2.2 Mammalian cell culture ............................................................................................44 3.2.3 Protein chemistry .....................................................................................................46 3.2.4 Protein complex purification .....................................................................................51 3.2.5 Mass spectrometry ...................................................................................................53 4 Table of Contents 3.2.6 Zebrafish morpholino experiments ...........................................................................55 3.2.7 Modeling and molecular dynamic simulations ..........................................................55 3.2.8 Oocyte preparation and voltage clamp .....................................................................56 4. Results .............................................................................................................................58 4.1 Work performed by cooperation partners in the course of this thesis ..........................58 4.1.1 Knock-down of NOS1AP and cardiomedin in zebrafish ............................................58 4.1.2 Identification of rare variants in human NOS1AP and cardiomedin ..........................60 4.1.3 Cardiomedin influences KR of potassium channel KCNH2 .......................................62 4.2 Characterization of cardiomedin ..................................................................................63 4.2.1 Illustration of cardiomedin in Western blot experiments ............................................63 4.2.2 Validation of monoclonal antibodies against cardiomedin ........................................64 4.2.3 Expression of endogenous cardiomedin on mRNA and protein level .......................66 4.3 Investigation of twelve mutations found in patients ......................................................67 4.3.1 Annotation of cardiomedin mutations with secretion phenotype ...............................69 4.3.2 Dominant negative effect of P504L, G515E and Y557H ...........................................71 4.4 ER – and Golgi stress .................................................................................................72 4.4.1 Examination of ER stress .........................................................................................72 4.4.2 Examination of Golgi stress......................................................................................75 4.5 Cardiomedin as a part of the extracellular matrix (ECM) .............................................77 4.6 Interaction partners of cardiomedin .............................................................................80 4.6.1 Purification of cardiomedin .......................................................................................80 4.6.2 Mass spectrometry results .......................................................................................84 4.7. Ion channel regulation by the cardiomedin protein .....................................................90 4.7.1 Interaction of cardiomedin and KCNH2 ....................................................................90 5. Discussion ........................................................................................................................94 5.1 Zebrafish knockdown experiments ..............................................................................94 5.2 Mutations and secretion phenotype ............................................................................94 5.3 Influences of cardiomedin on KCNH2 current .............................................................96 5.4 Interaction partners of cardiomedin .............................................................................97 5.5 GWAS gets functional ............................................................................................... 101 6. Bibliography ................................................................................................................... 104 Annex ................................................................................................................................. 110 1. Figure index ................................................................................................................ 110 2. Table index ................................................................................................................. 111 5 Table of Contents Acknowledgements ............................................................................................................ 113 Supplemental Tables .......................................................................................................... 114 6 Abbreviations ABBREVIATIONS APS Ammoniumperoxodisulfate AMD Age-related macular degeneration ANS Autonomic nervous system ARIC Atherosclerosis Risk in Communities BCA Bicinchoninic acid bp Base pair BrS Brugada Syndrome BSA Bovine serum albumin CAPON C-terminal PDZ domain ligand of neuronal nitric oxide synthase Chr. Chromosome CPVT Cetcholaminergic polymorphic ventricular Tachycardia C-Terminus Carboxy-terminus CD/CV Common disease common variant ddH2O Double distilled water ddNTPs Dideoxynucleotides DMEM Dulbecco’s modified eagle medium DNA Deoxyribonucleic acid dNTPs Deoxynucleotides DOC Sodium
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