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Genetic Factors Regulating Expression of Dopaminergic Genes

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Genetic Factors Regulating Expression of Dopaminergic Genes DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Elizabeth Stofko Barrie Graduate Program in Integrated Biomedical Science Program The Ohio State University 2014 Dissertation Committee: Professor Wolfgang Sadee, Advisor Professor R. Thomas Boyd Professor Howard Gu Professor Amanda Toland Copyright by Elizabeth Stofko Barrie 2014 Abstract Genetic differences are one of the main contributors to diversity in clinical phenotypes. This project identifies and defines genetic factors affecting dopamine dysregulation focusing on three genes in dopamine signaling: DBH, COMT and SNCA. To study this I measure allelic expression, selecting RNA samples from post-mortem human tissue that are heterozygous for a marker SNP, and quantitate the expression of each allele. Dopamine β-hydroxylase (DBH) encodes an enzyme which converts dopamine to norepinephrine. A promoter SNP, rs1611115 has been associated with low DBH and high dopamine plasma levels; however, underlying mechanisms remain uncertain. I found a tissue-specific effect of rs1611115 in liver, with up to 11 fold differences in allelic and overall mRNA expression (p<0.0004 to 2x10-7 and p<0.0001 respectively), indicating decreased transcription. Interestingly, locus coeruleus and adrenal gland, the main sources of DBH in the body, did not demonstrate this robust effect; only small AEI ratios were detected in these tissues. More frequent than rs1611115 and in linkage disequilibrium with it, a second SNP, rs1108580 was associated with reduced allelic mRNA expression in all tissues tested. This dual mechanism accounts for the previously described genotype effect on DBH plasma levels, with a novel role for liver as an important source of variability in DBH levels. In combination, rs161115 and rs1108580 contribute to strongly reduced mRNA expression in the liver, reducing transcription in a ii tissue selective manner. In mice, Dbh mRNA levels in the liver correlated with cardiovascular risk phenotypes. Using a PheWAS (phenome-wide association study) analysis, the minor alleles of rs1611115 and rs1108580 were associated with sympathetic phenotypes including angina pectoris. Testing the combined effects of rs1611115 and rs1108580 indicated robust protection against myocardial infarction in three clinical cohorts. These results demonstrate profound effects of common DBH variants on expression in sympathetically innervated organs, modulating clinical phenotypes responsive to peripheral sympathetic tone. In a pathway parallel to DBH, catechol-O- methyltransferase (COMT) converts dopamine to an inactive metabolite. Almost half of the African-American samples tested demonstrate a significant mRNA fold change, while only one Caucasian sample demonstrates AEI, indicating the presence of a regulatory variant which has not yet been described. Extensive sequencing of regions predicted to harbor this regulatory variant, up to 500 kb from the gene, did not reveal a variant associated with these instances of AEI. There is a factor, acting predominantly in African-Americans, regulating allelic expression and I have ruled out hundreds of SNPs as being the cause. Alpha-synuclein (SNCA) is involved in dopamine regulation, and implicated in degeneration of dopaminergic neurons. Allelic mRNA analysis indicates the presence of a regulatory variant. I have found an association with rs17016074, likely affecting, or marking differential 3′UTR usage. This approach has revealed the presence of frequent regulatory variants in all three genes studied. Functional SNPs contributing to dysregulation of dopamine can be tested for association with clinical phenotypes using large publicly available genome-wide association datasets. iii Dedication This document is dedicated to my family. Thanks to my mother for her many years of editing, my father for moral support, my brother for his sense of humor and my in-laws for being my home away from home. I’d especially like to thank my husband Mike for being my rock; his encouragement and patience have been vital to my success. Finally, I’m excited to meet our daughter who has helped me keep everything in perspective. iv Acknowledgments I am grateful to my advisor, Dr. Wolfgang Sadee, for helping me develop and define my research goals while maintaining a forward thinking view on my work and the field. He has challenged me to think critically about my research questions and work through issues that arise. He has undoubtedly shaped my graduate experience for the better and prepared me for a rewarding future scientific career. I appreciate the time and guidance from my committee members: Drs. R. Thomas Boyd, Howard Gu, and Amanda Toland. I am thankful to our laboratory manager, Audrey Papp as well as past and present members of the Sadee laboratory for their advice and helpful discussions: Amanda Curtis, Diane Delobel, Katherine Hartmann, Hannah Komar, Sebastiano Porcu, Jonathan Sanford, Gloria Smith, Adam Suhy, Danielle Sullivan, and Drs. Sam Handelman, Robert Moyer, Leslie Newman, Julia Pinsonneault, Ryan Smith, Danxin Wang, and Amy Webb. Pharmacology Department staff members Sherry Ring and Gina Pace were instrumental in managing paperwork and helping navigate the system. Collaborators from outside institutions, Drs. Deborah Mash, Sarah Pendergrass and David Weinshenker, also made vital contributions to this work. v Vita 2004................................................................Amherst Central High School 2008................................................................B.S. Biology, Case Western Reserve University 2008 to present ..............................................Graduate Research Associate, Department of Pharmacology, The Ohio State University Publications Barrie ES, Smith RM, Sanford JC, and Sadee W. (2012) mRNA Transcript Diversity Creates New Opportunities for Pharmacological Intervention, Molecular Pharmacology Barrie ES, Weinshenker D, Pendergrass S, Lange L, Ritchie M, Wilson J, Kuivaniemi H, Tromp G, Carey D, Gerhard G, Cubells J Sadee W. Regulatory polymorphisms in DBH affect peripheral gene expression and sympathetic phenotypes. Circulation Research. In Revision. vi Barrie ES, Lodder M, Weinreb P, Buss J, Rajab A, Adin C, Mi QS, Hadley GA. Role of CD103 in the development of autoimmune diabetes in NOD mice. Journal of Endocrinology. In Revision. Fields of Study Major Field: Integrated Biomedical Science Program vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Publications ........................................................................................................................ vi Fields of Study .................................................................................................................. vii Table of Contents ............................................................................................................. viii List of Tables ..................................................................................................................... xi List of Figures .................................................................................................................. xiii Chapter 1: Introduction ...................................................................................................... 1 1.1 Evolution of the Field of Genetics ............................................................................ 1 1.2 Mechanisms of Regulation and Evidence for cis-acting Polymorphisms ................. 3 1.3 Detecting Sequence Variation ................................................................................... 5 1.4 Targets of Interest: Dopamine and Norepinephrine .................................................. 6 1.5 Methods Common to All Projects ............................................................................. 7 viii 1.6 Summary of Studies ................................................................................................ 10 Chapter 2: Regulatory Genetics of Dopamine β-Hydroxylase (DBH) and Effect on Clinical Phenotypes .......................................................................................................... 11 2.1 Introduction and Background .................................................................................. 11 2.2 Known DBH SNPs and Clinical Correlations......................................................... 12 2.3 Myocardial Infarction and Coronary Heart Disease ............................................... 16 2.4 Study Overview ....................................................................................................... 18 2.5 Materials and Methods ............................................................................................ 19 2.6 Results ..................................................................................................................... 27 2.7 Discussion ..............................................................................................................
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