SEARCH FOR FUNCTIONAL ALLELES IN THE HUMAN GENOME WITH FOCUS ON CARDIOVASCULAR DISEASE CANDIDATE 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 Andrew Danner Johnson, B.S. ***** The Ohio State University 2007 Dissertation Committee: Approved by Professor Wolfgang Sadée, Advisor Professor Daniel A. Janies _ _______________ ________________ _ Professor Kirk Mykytyn Advisor Integrated Biomedical Sciences Graduate Program Copyright by Andrew Danner Johnson 2007 ii ABSTRACT The genetic investigation of human disorders largely through linkage mapping has led to the discovery of candidate genes and mutations as risk factors for those disorders where there is a high degree of penetrance. While twin studies have provided evidence that there are major genetic contributions to multifactorial diseases like coronary artery disease, it has proven difficult to find and replicate significant genetic associations for such diseases. Recent advances in technology, throughput and understanding of widespread human genetic variation at the genomic level (e.g., the HapMap project) have allowed the application of more genetic markers in larger sample studies, but we are still lacking a complete picture of genetic contributions to major multifactorial diseases. Searching for genetic variants with evidence of a direct molecular impact on the expression and function of genes vital to disease development and progression is one valid approach to this problem. There is a growing appreciation that one major class of variation acts at the level of mRNA expression. Traditional tools for studying this class of variation (e.g., reporter gene assays) in the laboratory have severe limitations, mainly in that they lack the in vivo context where the alleles are hypothesized to have a functional impact. This dissertation relies heavily on the application of a relatively novel technique, the measurement of allelic expression imbalances (AEI) between chromosomes in primary human tissues. Using these measurements as phenotypic traits, ii we demonstrate that cis-acting alleles exerting molecular affects on mRNA expression can often be readily mapped. In the largest survey to date of AEI in primary human tissues we find that AEI in disease candidate genes is quite common, and that the functional contributors to these expression phenotypes are often not regulatory polymorphisms, but polymorphisms found directly within the mRNAs and affecting mRNA processing and functions. Computational analysis of mRNA structures and genetic variation within human genomes indicates that modulation of mRNA structural plasticity to polymorphism is likely one contributor to human phenotypic variability. Focusing on a number of cardiovascular disease candidate genes I make a number of novel findings: 1) a strong ACE AEI phenotype common in the African-American population is mapped to specific upstream regulatory alleles and is significantly associated with relevant clinical phenotypes, 2) SOD2 is subject to extremely common and extensive AEI in the human population suggesting potential positive selection, and 3) our results call into question the strength of many previous association studies based on polymorphisms in ACE, CCL2 and NOS3 where there is weak evidence supporting putative functional alleles. iii Dedicated: First, to Meg, my partner in all; what a long, strange and wonderful trip we are on Second, to my family who brought me up curious Last, to colleagues and advisors past, present and future who keep me both inspired and on track iv ACKNOWLEDGMENTS I thank my adviser, Wolfgang Sadée, for his inspiration, time and direction. He showed me many facets of asking meaningful questions and becoming a successful scientist, and he gave me the space I needed to grow while demanding that I do it; I thank Dan Janies for frank talks from a different career perspective, collaborations both successful and published and those done out of interest, and for sharing computing resources and expertise, and teaching and travel opportunities; I thank Kirk Mykytyn for some interesting bioinformatics questions along the way and his some darn good chili at the annual Department party; I thank the whole lab crew: Audrey Papp, Danxin Wang, Zunyan Dai, Julia Pinsonneault, Ying Zhang and Gloria Smith for assistance with so many tiny details, and sharing moments and meetings, lunches and laughter, tea and talks and wells on 3730 plates. I fear I may never again work with such a nice group of people; I thank Jonathan Day who gave me my start in science at Penn State after I had knocked on other doors that were closed; I thank Helen Chamberlin who saw potential in me and acknowledged this by giving me my first start with publications; v I thank Ben Givens who laid the groundwork for me to take leadership roles in a lab setting, inadvertently set me off on computer programming and launched me into graduate school; I thank my research colloborators outside the lab whose hard work is also presented here: David Saffen and Jeong-Eun Lim, Philip Binkley and Amanda Lesinski, Glen Cooke, Clay Marsh, Chris Baran, Julie Johnson, Yan Gong, and Taimour Langaee; I am thankful for the support of IBGP program staff (Allan Yates, Christine Kerr, Angie Thomas, Darlene Johns) and Pharmacology staff (Sherry Ring, Gina Pace) whom never faltered in helping me navigate the system and always gave me the correct answers; I thank the Jest Jugglers of Columbus who provided stress relief on many Thursdays and may have helped spawn some critical neural circuitry; I thank, again, my wife Meg who lived through it and loved me all the way. I thank the anonymous tissue donors and their families without whom this work would not have been possible. I am also thankful for research support that contributed to this doctoral work: a predoctoral fellowship award from the American Heart Association (0515157B) and a predoctoral Distinguished University fellowship award from The Ohio State University. Travel grants from the OSU Ray Award, the OSU Medical Center Research Day and the Pharmaceutical Sciences World Congress supported conference presentation of my research. vi VITA November 15, 1975. .Born – Bryn Mawr, PA 1998. ………… . B.S. Biology (Vertebrate Physiology), The Pennsylvania State University 1999 – 2003. .. Researcher The Ohio State University. 2003 - 2004 . ………. Distinguished University Fellow The Ohio State University 2005 - 2007 . ………. American Heart Association Fellow The Ohio State University 2007. ………. ………. Distinguished University Fellow The Ohio State University PUBLICATIONS Research Publications 1. F. Habib, A.D. Johnson, R. Bandschuh and D. Janies, “Large scale genotype- phenotype correlation analysis based on phylogenetic trees.” Bioinformatics, 23, 785, (2007). 2. T. Kurc, D. Janies, A.D. Johnson, S. Langella, S. Oster, S. Hastings, F. Habib, T. Camerlengo, D. Ervin, U.V. Catalyurek and J. Saltz, “An XML-based system for synthesis of data from disparate databases.” J. Am. Med. Inform. Assoc., 13, 289, (2006). 3. Y. Zhang, D. Wang, A.D. Johnson, A.C. Papp and W. Sadée, “Allelic expression imbalance of human mu-opoid receptor (OPRM1) caused by variant A118G.” J. Biol. Chem., 280, 32618, (2005). vii 4. D. Wang, A.D. Johnson, A. Papp, D.L. Kroetz and W. Sadée, “Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability.” Pharmacogen. Gen., 15, 693, (2005). 5. A.D. Johnson, D. Wang and W. Sadée, “Polymorphisms affecting gene regulation and mRNA processing: Broad implications for pharmacogenetics.” Pharm. Ther., 106, 19, (2005). 6. H. McCartney, A.D. Johnson, Z. Weil and B. Givens, “Theta reset produces optimal conditions for long-term potentiation in the dentate gyrus.” Hippocampus, 14, 684, (2004). 7. A.D. Johnson, D. Fitzsimmons, J. Hagman, H.M. Chamberlin, “EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development.” Development, 128, 2857, (2001). FIELDS OF STUDY Major Field: Integrated Biomedical Sciences viii TABLE OF CONTENTS P a g e Abstract. ii Dedication. .iv Acknowledgments . .. ....v Vita . .. .. vii List of Tables. ... xi List of Figures . .. xiii List of Abbreviations . ...xv Chapters: 1. Introduction. …… .1 2. cis-acting genetic variation in the human genome…………………………….. ..10 2.1 Evidence for cis-acting effects on genes of clinical relevance………..……..10 2.2 Modes of cis-acting polymorphisms and methods for discovery...……….…16 2.2.1 Experimental methods for discovering cis-acting polymorphisms...16 2.2.2 In silico methods for discovering cis-acting polymorphisms…...…19 2.3. Review of evidence for cis-acting variation in genes of clinical relevance.. .22 2.3.1 Drug metabolizing enzymes…………………………………….....22 2.3.1.1 CYP1 family…………………………………………..…23 2.3.1.2 CYP2 family…………………………………………..…25 2.3.1.3 CYP3 family………………………………………….…30 2.3.1.4 Other CYPs……………..……………………………..…32 2.3.1.5 Other classes of drug metabolizing enzymes………….…33 2.3.2 Drug transporters……………………………………………….….37 ix 2.3.3 Drug targets and receptors………………………………………....39 2.3.4 cis-acting polymorphisms in relevant trans factors………………..42 2.4 Summary…………………………………………………………………..…44 3. Survey of allelic expression in human target tissues…………………………….46 3.1 Survey of allelic expression in human target tissues………………………...46 3.2 Method for allelic expression survey in human target tissues..……………...49 3.2.1 Tissue sources and processing……………………………………..49 3.2.2 Design, assay and analysis of allelic