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Evaluation of Mismatch Repair Gene Polymorphisms and Their EVALUATION OF MISMATCH REPAIR GENE POLYMORPHISMS AND THEIR CONTRIBUTION TO COLORECTAL CANCER AND ITS SUBSETS By Miralem Mrkonjic A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Miralem Mrkonjic (2009) ABSTRACT Evaluation of Mismatch Repair Polymorphisms and Their Contribution to Colorectal Cancer and its Subsets Doctor of Philosophy, 2009 Miralem Mrkonjic Department of Laboratory Medicine and Pathobiology, University of Toronto Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair ii chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease. iii ACKNOWLEDGEMENTS I would like to extend my heartfelt gratitude to my supervisor, Dr. Bharati Bapat, for all her guidance and support over the years and for giving me an opportunity to pursue my interest in cancer research. I have gained much experience and knowledge and was able to grow both as a person and a researcher during my tenure in her laboratory. I would also like to thank my thesis advisory members, Dr. Julia Knight, Dr. Hilmi Ozcelik, and Dr. John McLaughlin, for their guidance, support, and inspiration over the last five years. A special thank you goes to Dr. Julia Knight who co-supervised my initial training. Her contributions to my projects have been substantial and very appreciated. I am grateful to our collaborators, Nicole Roslin, Dr. Andrew Paterson, and Dr. Celia Greenwood, from the Hospital for Sick Children, for their guidance and contribution to my final data chapter and beyond. I am also grateful to many individuals from the Mount Sinai Hospital/SLRI, OFCCR, NFCCR, FHCRC, and UCSC that contributed to my thesis project over the past five years. I would like to thank Neerav Monga, for his statistical support in my first data chapter, and to Michael Manno, for the statistical analyses in my many side projects. Many thanks to staff and administrators of the Laboratory Medicine and Pathobiology department, especially Dr. Harry Elsholtz, for all the support and friendly environment they have provided and to many CLAMPS members with whom I have had the privilege of working with over the years. I have had the pleasure of working with many exceptional people in the lab who have made my graduate experience truly memorable. Special thanks go to Roula for her patience and mentorship in the early stages of my project, and for correcting all the “thes” that I tended to neglect. To Sheron, with whom I have started my graduate journey with, thank you for all your support and for always lending me a helping hand when I needed it the most. You have been a great friend and a confidante. Many thanks to George who always willingly shared his experiences and offered guidance. Kenneth (Gyro James and countless other nicknames that I gave you), thank you for making my time in the lab an entertaining one – keep corrupting young minds and I promise to do the same. To Vaiju and the newbies, James and Liyang (Kiki), I have had an extraordinary time working with you. Additional thanks go to lab alumni, Susmita, Pinella, and Ted, with whom I share very fond memories. I have thoroughly enjoyed the time I spent with all of you, many thanks for all the support you have given me, and most importantly, your friendships, which I hope will continue well into the future. Finally and most importantly, countless thanks go to my wonderful family – my mother, my father, and my sister, Mirela. You have always supported me and encouraged me to do my utmost best. Thank you very much for many sacrifices you have made so that I can pursue my dreams. I love you very much. iv TABLE OF CONTENTS ABSTRACT .............................................................................................................ii ACKNOWLEDGEMENTS...................................................................................iv TABLE OF CONTENTS........................................................................................v LIST OF TABLES .............................................................................................. viii LIST OF FIGURES ...............................................................................................xi LIST OF ABBREVIATIONS............................................................................. xiii CHAPTER 1 GENERAL INTRODUCTION 1.1 COLORECTAL CANCER....................................................................................................... 1 1.1.1 PHYSIOLOGY AND ANATOMY OF THE COLORECTUM........................................................... 1 1.1.2 HISTORY AND EPIDEMIOLOGY OF COLORECTAL CANCER ................................................... 2 1.1.3 RISK FACTORS .................................................................................................................... 4 1.1.4 SCREENING, STAGING, AND PROGNOSIS.............................................................................. 6 1.1.5 MOLECULAR PATHWAYS IN CRC (CIN/MSS, MSI/MIN, CIMP) ...................................... 8 1.1.6 HEREDITARY SYNDROMES, PATHOLOGY AND PRESENTATIONS ........................................23 1.2 MISMATCH REPAIR (MMR)..............................................................................................30 1.2.1 SOURCES OF DNA NUCLEOTIDE MISMATCHES.................................................................30 1.2.2 MMR SYSTEM OVERVIEW AND HISTORY.........................................................................32 1.2.3 REPLICATION ERROR REPAIR............................................................................................32 1.2.4 MMR IN DNA DAMAGE SIGNALLING, CELL CYCLE ARREST, AND APOPTOSIS................41 1.2.5 OTHER ROLES OF MMR ...................................................................................................43 1.2.6 ROLE OF MMR SYSTEM IN CANCER.................................................................................47 1.3 GENETIC VARIATION........................................................................................................49 1.3.1 POLYMORPHISMS (DEFINITIONS AND INTRODUCTION)......................................................49 1.3.2 UTILITY AND ROLES OF SNPS IN GENETIC EPIDEMIOLOGY ..............................................51 1.3.3 IDENTIFICATION OF LOW-PENETRANCE ALLELES IN CRC AND FOUNDER POPULATIONS .53 1.3.4 SNPS IN MMR GENES ......................................................................................................55 1.4 HYPOTHESIS AND OBJECTIVES......................................................................................56 v CHAPTER 2 IDENTIFICATION OF MMR MODIFIER ALLELES IN CRC IN TWO CANADIAN POPULATIONS 2.1 SUMMARY............................................................................................................................60 2.2 INTRODUCTION..................................................................................................................61 2.3 MATERIALS AND METHODS ...........................................................................................64 2.3.1 SNP SELECTION CRITERIA................................................................................................64 2.3.2 STUDY SUBJECTS ..............................................................................................................64
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