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Epigenetics of Crohn's Disease, Ulcerative Colitis, And

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Epigenetics of Crohn's Disease, Ulcerative Colitis, And Epigenetics of Crohn’s Disease, Ulcerative Colitis, and Phenotypically Normal Individuals By © 2017 Delisa L Phillips M.S., University of New Mexico, 2009 M.A., University of Arkansas, 2005 B.A., University of Arkansas, 2003 Submitted to the graduate degree program in Anthropology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Chair: Michael Crawford Justin Blumenstiel Kristin Young Jennifer Raff Bartholomew Dean Date Defended: 2 May 2017 The dissertation committee for Delisa L Phillips certifies that this is the approved version of the following dissertation: Epigenetics of Crohn’s Disease, Ulcerative Colitis, and Phenotypically Normal Individuals Chair: Michael Crawford Date Approved: 2 May 2017 ii ABSTRACT Background It is difficult to distinguish between Crohn’s disease and ulcerative colitis, as they share many symptoms and many susceptibility genetic loci. Due to the similarities in phenotype between Crohn’s disease and ulcerative colitis, many patients are misdiagnosed for years. For this project, I collected genetic material, medical histories, and environmental variables from individuals with Crohn’s disease, individuals with ulcerative colitis, and individuals that are phenotypically normal. The purpose of this project is to determine the differential epigenetic methylation of genes in individuals with Crohn’s disease and ulcerative colitis compared to phenotypically normal controls. Materials and Methods Participants were recruited for this project at the Kansas Medical Center. Twenty of these individuals had Crohn’s disease, ten had ulcerative colitis, and thirty-one were phenotypically normal. Buccal swabs and information relevant to this project (via a questionnaire) was collected from the individuals. DNA was extracted then bisulfite-converted so that the unmethylated cytosines would be converted to uracils. Real-time PCR was performed on the promoter regions of three genes both prior to and post-bisulfite conversion, as well as on the regions of the genes containing known associated SNPs. The three genes of interest in this project are the NOD2, ATG16L1, and PTPN2 genes. Each of these genes has been found to be associated with Crohn’s disease. These three genes work in concert by identifying pathogenic bacteria, forming a response, and ultimately clearing out the bacteria. iii Results The statistical tests used in this project showed a few significant differences between Crohn’s disease, ulcerative colitis, and phenotypically normal samples from the questionnaire. From the bisulfite-converted samples, however, there were no obvious differences between the three groups. iv ACKNOWLEDGEMENTS I would like to thank the patients from the Kansas City Medical Center who volunteered for this study, as well as the wonderful doctors who allowed me to collect samples from their patients: Dr. John Bonino, Dr. Rajeswari Anaparthy, and Dr. Daniel Buckles. I would like to thank the members of my committee who have all worked so hard to insure that I succeed: Dr. Michael Crawford, Dr. Kristin Young, Dr. Justin Blumenstiel, Dr. Jennifer Raff, and Dr. Bartholomew Dean. I would also like to thank my husband and son for putting up with me through all of my moodiness and stress during the writing of this dissertation. I would like to dedicate this to my parents, Dale and Dee Phillips. The two of you have been my rock throughout my entire academic career. Over the years, you have provided me with a place to live, food to eat, financial support as needed, and most importantly, the emotional support that I needed to complete my Ph.D. You have made me the woman I am today, and I am thankful for the two of you every day of my life. This project was partially funded by the Carroll Clark Award. v Table of Contents ABSTRACT ................................................................................................................................................ III ACKNOWLEDGEMENTS ............................................................................................................................. V TABLE OF FIGURES ................................................................................................................................ VII CHAPTER 1: INTRODUCTION .......................................................................... 1 CHAPTER 2: LITERATURE REVIEW ............................................................. 3 HISTORY OF CROHN’S DISEASE ................................................................................................................3 AUTOIMMUNE DISEASES ...........................................................................................................................7 WHY STUDY AUTOIMMUNE DISEASES? ....................................................................................................9 GENETIC CONTRIBUTION TO AUTOIMMUNE DISEASES .........................................................................11 NORMAL FUNCTIONING OF THE IMMUNE SYSTEM ................................................................................20 BACTERIAL/VIRAL THEORY OF CROHN’S DISEASE ................................................................................41 GENETICS OF CROHN’S DISEASE ............................................................................................................48 HUMAN EPIGENETICS .............................................................................................................................82 CHAPTER 3: MATERIALS & METHODS .................................................... 128 SUBJECT SELECTION CRITERIA AND SAMPLE SIZE JUSTIFICATION ...................................................130 DNA EXTRACTION FROM BUCCAL SWABS ...........................................................................................132 PRIMER DESIGN ....................................................................................................................................133 METHODS FOR DETECTING DNA METHYLATION ................................................................................135 POLYMERASE CHAIN REACTION (PCR) ................................................................................................139 DATABASE ..............................................................................................................................................141 STATISTICAL TESTS ...............................................................................................................................142 CHAPTER 4: RESULTS ................................................................................... 145 PCR RESULTS ........................................................................................................................................145 STATISTICAL RESULTS ..........................................................................................................................146 MEANS AND STANDARD DEVIATIONS ...................................................................................................146 T-TESTS .................................................................................................................................................148 CORRELATIONS AND FISHER’S EXACT TESTS ......................................................................................150 CHAPTER 5: DISCUSSION AND CONCLUSIONS ..................................... 154 REFERENCES .................................................................................................... 158 APPENDICES ..................................................................................................... 175 APPENDIX 1 – IRB CONSENT FORM ...................................................................................................175 APPENDIX 2 – DATA COLLECTION WORKSHEET ...............................................................................183 APPENDIX 3 – COUNT OF EACH VARIABLE FROM QUESTIONNAIRE ..................................................186 APPENDIX 4 – AMPLIFICATION PLOTS AND MELT CURVES ..............................................................188 APPENDIX 5 – INTERVAL PLOTS POST-PCR .......................................................................................197 APPENDIX 6: CONTINGENCY TABLES WITH ODDS RATIOS, RELATIVE RISKS, AND CONFIDENCE INTERVALS .............................................................................................................................................200 vi TABLE OF FIGURES FIGURE 1 INCIDENCE OF IMMUNE DISORDERS FOR FOUR AUTOIMMUNE DISEASES. (BACH). THE X-AXIS DENOTES THE YEARS 1950-2000. THE Y-AXIS DENOTES THE PERCENT INCREASE OF IMMUNE DISORDERS. CROHN’S DISEASE HAS THE LARGEST INCREASE. THE DATA FOR THIS GRAPH WAS OBTAINED FROM FOUR SEPARATE ARTICLES (DUBOIS ET AL. 1998; PUGLIATTI ET AL. 2001; SWARBRICK ET AL. 2001; TUOMILEHTO ET AL. 1999). ...............10 TABLE 1 AUTOIMMUNE CONCORDANCE RATES IN TWINS ..............................................................................................14 FIGURE 2 FEMALE TO MALE RATIO OF SELECTED AUTOIMMUNE DISEASE EXPRESSED AS PERCENTAGE (X AXIS) AND CALCULATED AS AVERAGE (INVERNIZZI ET AL. 2009). THE POPULATIONS STUDIED WERE IN THE UNITED STATES AND DENMARK. INFLAMMATORY BOWEL DISEASES INCLUDE CROHN’S DISEASE AND ULCERATIVE COLITIS. .....17 TABLE 2 MEAN AGE OF DIAGNOSIS FOR SIX AUTOIMMUNE DISEASES. ...........................................................................19 FIGURE 3 THE COMPLEMENT
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