View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ETD - Electronic Theses & Dissertations GENE EXPRESSION AND COPY NUMBER VARIATION IN COMMON COMPLEX DISEASES By Britney L. Grayson Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Microbiology and Immunology December, 2010 Nashville, Tennessee Approved: Thomas M. Aune Luc Van Kaer James W. Thomas Nancy J. Brown Marylyn D. Ritchie To my mom, Ruthie, your battle with lupus inspired every experiment on these pages. To my dad, Richard, a short pencil IS better than a long memory. To my sisters, Brandy and Brienne, I am but one-third of a whole. ii ACKNOWLEDGEMENTS I am thankful to my thesis committee, especially my mentor Tom Aune for his many hours of "interaction" and countless "pearls of wisdom." I am thankful to Luc Van Kaer for being an organized and timely chair and to all members, Drs. Aune, Van Kaer, Thomas, Ritchie and Brown, for insightful comments, critiques and advice that propelled both me and these projects forward. I am thankful for my lab members past and present- Kelly, Patrick, Zach, Erica, Sarah, Nyk, Chase, our honorary member, David and especially Mary Ellen, who performed many of the experiments you see on these pages, John, who helps to maintain our patient samples biobank and Mel, who had a solution to all of my lab problems. I am thankful to all of the physicians and clinics who gave of their time to recruit patients for these studies. This includes Drs. James W. Thomas, Howard Fuchs, Nancy J. Brown, Joe Huston, Bill Russell and Steve Davis, Margo Black and the Eskind Diabetes Clinic, and the nurses at the Clinical Research Center at Vanderbilt. I am also thankful to Dr. George Eisenbarth, Joy Jeffrey, Pam Fain and Priyaanka Nanduri at the Barbara Davis Center for Childhood Diabetes at the University of Colorado for collaborating with me on the work presented in Chapter IV. I would also like to acknowledge my many collaborators outside of the clinics: The Vanderbilt Functional Genomics Shared Resource, specifically Vicky Amann, Braden Boone and Phil Dexheimer for technical support with arrays both in experimental procedures and statistical analysis, and Latha Raju for her technical expertise in RT-PCR. My work would not be as "significant" without the help of our wonderful statistician, iii Lily Wang, and the Vanderbilt Department of Biostatistics. Every aspect of my work was made stronger by her tireless work. Thank you to my most important collaborators- the hundreds of patients who made this work possible. I am thankful to the MSTP, our director Terry Dermody and the rest of the team- Larry, Jim, Michelle, Susan, Jena, Gisel and Lindsay for unending support since my interview weekend nearly 7 years ago. Thanks to the Department of Microbiology and Immunology, chair Jacek Hawiger, administrator extraordinaire Jean Tidwell, director of graduate studies Chris Aiken, and the numerous others who have supported my graduate training for the last 4 years. On a personal note, I am so thankful to my family and friends. To all my MSTP colleagues, especially Aubrey and Katy, for understanding the highs and lows of graduate school life. Thanks to my Nashville friends, who are like family, for supporting every day of my crazy life- whether I could reciprocate or not. Finally, thank you to my family. In addition to those for whom this work is dedicated, I would like to thank Alecia, Angel, Nicole, Grandpa, Pop, Gram and the rest of the Noble gang for their unconditional love and support of my work. Financially, this work was supported by NIH grants R42 AI053984, T32 GM07347 (Medical Scientist Training Program), T32 DK07563 (Molecular Endocrinology Training Grant), and TL1 RR024978 (Clinical and Translational Research Award). iv TABLE OF CONTENTS Page DEDICATION .......................................................................................................................ii ACKNOWLEDGEMENTS ...................................................................................................iii LIST OF TABLES ................................................................................................................vii LIST OF FIGURES ..............................................................................................................viii LIST OF ABBREVIATIONS ...............................................................................................ix Chapter I. INTRODUCTION .............................................................................................................1 Common Complex Disease …………………………………………………..2 Autoimmune disease ……………………………………………………..2 Metabolic disorders ………………………………………………………7 Gene Expression Profiling …………………………………………………..10 Gene expression profiling in cancer …………………………………….12 Gene expression profiling in autoimmune disease ……………………...13 Peripheral blood gene expression in metabolic disorders ……………….18 Genetics and gene expression …………………………………………...18 Genetics ……………………………………………………………………..19 Single nucleotide polymorphisms ……………………………………….20 Copy number variation ………………………………………………….21 Copy number variation in the human genome …………………………..25 Copy number variation in type 1 diabetes ………………………………28 II. PERIPHERAL BLOOD GENE EXPRESSION PROFILES IN METABOLIC SYNDROME, CORONARY ARTERY DISEASE AND TYPE 2 DIABETES ………………………………………………………………30 Abstract ……………………………………………………………………...30 Introduction ………………………………………………………………… 31 Materials and Methods ………………………………………………………34 Patient recruitment ………………………………………………………34 Microarray gene expression experiments ……………………………….35 Microarray data analysis ………………………………………………...36 RT-PCR …………………………………………………………………37 Results ……………………………………………………………………….38 v Rheumatoid arthritis ……………………………………………………..46 Metabolic syndrome ……………………………………………………..48 Coronary artery disease ………………………………………………….49 Type 2 diabetes ………………………………………………………….50 Correlation among disease states ………………………………………..51 PCR validation …………………………………………………………..55 Discussion …………………………………………………………………...57 III. A COMPARISON OF GENOMIC COPY NUMBER CALLS BY PARTEK GENOMICS SUITE, GENOTYPING CONSOLE AND BIRDSUITE ALGORITHMS TO QUANTITATIVE PCR …………………….60 Abstract ……………………………………………………………………...60 Introduction ………………………………………………………………….61 Materials and Methods ………………………………………………………63 Patient recruitment ………………………………………………………63 Affymetrix SNP 6.0 Arrays ……………………………………………..63 Copy number analysis …………………………………………………..64 Quantitative PCR experiments ………………………………………….64 Results ……………………………………………………………………….65 Discussion …………………………………………………………………...73 IV. GENOME-WIDE ANALYSIS OF COPY NUMBER VARIATION IN TYPE 1 DIABETES …………………………………………………………76 Abstract ……………………………………………………………………..76 Introduction …………………………………………………………………77 Materials and Methods ………………………………………………………80 Ethics statement …………………………………………………………80 Patient recruitment ………………………………………………………81 Affymetrix copy number variation experiments….……………………...82 Copy number analysis …………………………………………………...82 Quantitative PCR experiments …………………………………………..83 Results ……………………………………………………………………….84 Discussion …………………………………………………………………...95 V. GENERAL DISCUSSION AND CONCLUSION……………………………...99 Appendix A. SUPPLEMENTAL DATA …………………………………………………………106 REFERENCES ………………………………………………………………………..107 vi LIST OF TABLES Table Page 2-1. SNPs associated with RA and T2D show differential gene expression .........................45 2-2. Differentially expressed gene sets .................................................................................47 2-3. RT-PCR determined ratios of differentially expressed genes .......................................56 3-1. Copy number calls at invariant regions of the genome .................................................66 3-2. Comparison of copy number calls at variant regions ....................................................67 3-3. Birdsuite agreement with qPCR calls in 18 genomic regions .......................................72 4-1. CNVs enriched in T1D and Twin cohorts, relative to CTRL ........................................89 4-2. CNVs depleted in T1D and Twin cohorts, relative to CTRL ........................................89 vii LIST OF FIGURES Figure Page 1-1. Comparison of the immune and autoimmune classes by cluster analysis .....................13 1-2. Hierarchical clustering using core autoimmune genes ..................................................15 1-3. Schematic model of a molecular mechanism for meiotic NAHR between low copy repeats ...........................................................................................................22 1-4. Genomic distributions of CNVRs ..................................................................................26 2-1. Hierarchical clustering of individual disease cohorts versus CTRL .............................40 2-2. Hierarchical clustering of all disease cohorts versus CTRL ..........................................43 2-3. Correlative relationships among disease cohort gene expression .................................53 3-1. Agreement of CN calls made by Partek, GTC, Birdsuite and qPCR ...........................69 3-2. Agreement between CN calls made by Birdsuite and qPCR .........................................73 4-1. Percent agreement between Birdsuite copy number calls and qPCR ............................85 4-2. Individual breakpoints of CNVR A588 .........................................................................91