Association of 19q, 8q24 regions and Mismatch Repair (MMR) Genes with Prostate Cancer in Caucasians A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of Environmental Health of the College of Medicine June, 2008 by Prodipto Pal M.B.B.S., University of Calcutta, Calcutta, India, 2000 Committee: Dr. Ranjan Deka, Ph.D. (Chair) Dr.Ranajit Chakraborty, Ph.D. Dr. Charles Ralph Buncher D.Sc. Dr. Daniel Woo, M.S., M.D. Dr. Divakar Choubey, Ph.D. ABSTRACT Prostate cancer (PCa) is the most commonly diagnosed visceral malignancy with over one-third of all incident cancers and the second leading cause of cancer deaths in men in the United States. Age, ethnicity and family history are among the classical epidemiological risk factors. Both environmental and genetic factors contribute to the etiopathogenesis of PCa. The region on 19q11-13, identified through linkage and association studies, was previously shown to harbor genes for PCa susceptibility as well as for biologic aggressiveness. Recently, admixture mapping and a number of independently performed genome-wide association studies (GWAS) further identified strong association signals on chromosome 8q24. The only biologically plausible candidate gene for cancers on 8q24 is the myc oncogene, located ~500 kb downstream to the identified region. The myc protein is a key regulator of cell cycle, growth and differentiation, apoptosis, genomic instability and angiogenesis. At the cellular level, myc synergistically interacts with mutL homolog 1 (MLH1), one of the key genes in mismatch repair (MMR) of DNA. Mutations in MMR pathway is implicated in the development of familial hereditary non-polyposis coli (HNPCC). Moreover, recently performed GWAS studies identified risk alleles for colorectal cancers on the same 8q24 region. It is possible that both prostate and colorectal adenocarcinomas share a common genetic origin, through interactions with myc and the MMR pathway. We have performed a comprehensive association analysis with PCa susceptibility primarily using tagging variants on 19q, 8q24 and the MMR pathway among men of European descent. Approximately two hundred single nulcleotide polymorphisms (SNPs) were analyzed in a sample of 590 histologically verified PCa cases and 567 ethnically matched controls. iii Multiple variants on chromosomes 19q and 8q24 show significant association with PCa susceptibility and/or aggressiveness. We further observe significant genetic contribution with tagging variants on MLH1 and mut S homolog 3 (MSH3), in the MMR repair pathway. Analyses reveal no population substructuring in our samples that could have confounded association results. These findings suggest a plausible biologic role for genetic variants on 19q, 8q24 and MMR pathway genes in the etiology of PCa among men of European ancestry. iv This page is intentionally left black v Acknowledgements The following work towards this dissertation is the culmination of several years of work, during which I received help, support and guidance from many persons. It is my pleasure to express my most sincere gratitude to all who have helped me achieve this goal. I am grateful to Dr. Ranjan Deka for his persistent and whole-hearted support at the time of need. Dr Deka has been the truest of advisors, with insightful scientific knowledge, passion for research and enthusiastic approach. I thank Dr Deka for helping me in my transition from a clinician to a researcher. I am thankful to Dr. Ranajit Chakraborty, Dr. Charles Ralph Buncher, Dr Daniel Woo and Dr. Divakar Choubey for kindly accepting to serve in my dissertation committee. Their valuable suggestions during committee meetings, and also while preparing manuscripts, have helped me gain new insights towards research. I am also thankful to Dr William Catalona at Northwestern University, Chicago who have graciously provided the samples for this study. I would like to express my thanks to all my co-workers at the Center for Genome Information, Complex Disease Genomics Lab, without whose help this work would not have been accomplished. My heart-felt thanks are due to my colleagues Dr Gyungun Sun and Dr Subbarao Indugula, who taught me everything that I know about laboratory work. I am especially grateful to two of my colleagues and friend, Dr Saurav Guha, post- doctoral fellow at Dr Chakraborty’s lab and my fellow graduate student Huifeng Xi, both of whom have helped me tremendously in the data analysis for this project. vi I wish to thank the faculty, staff and students of the Department of Environmental Health for being with me during one of the most important phases of my life and career. Lastly, and by no means the least, I wish to dedicate this dissertation to the dearest thing in my life, my family, my mother and father and my sister. They have guided me through the most difficult of times. I would not have been the person who I am today without them, and I owe them everything. vii Role in the study This research was developed as a part of a larger ongoing research at the Complex Disease Genetic lab at Center for Genome Inforamation, Department of Environmental Health, University of Cincinnati, of which Dr. Ranjan Deka is the principal investigator. The details of the research were planned by Prodipto Pal under the guidance of Dr. Ranjan Deka. Subjects for the study were recruited from out-patient clinics of the Washington University, St Louis by Dr. William Catalona’s group. Study design, hypothesis and specific aim development, database creation and maintenance, SNP-selection, Genotyping and statistical analysis were performed by primarily Prodipto Pal and in part by Huifeng Xi. Prodipto Pal has written this dissertation under the guidance of Dr. Ranjan Deka. Revisions and suggestions from Dr. Ralph Buncher, Dr. Ranajit Chakraborty, Dr. Daniel Woo and Dr. Divakar Choubey have been incorporated in this work. viii TABLE OF CONTENTS ABSTRACT .......................................................................................................... iii ACKNOWLEDGEMENTS ....................................................................................vi ROLE IN THE STUDY........................................................................................ viii TABLE OF CONTENTS .......................................................................................ix ABBREVIATIONS .............................................................................................. xiii CHAPTER 1 Introduction ....................................................................................1 1.1 HYPOTHESIS AND SPECIFIC AIMS .............................................................1 1.2 BACKGROUND AND SIGNIFICANCE............................................................3 1.3 MATERIALS AND METHODS.........................................................................8 1.3.1 Subjects – cases and controls..........................................................8 1.3.2 SNP selection strategy ...................................................................10 1.3.3 DNA analysis and Genotyping........................................................12 1.3.4 Statistical analysis ..........................................................................13 1.4 REFERENCES..............................................................................................18 CHAPTER 2 Association of Genetic Variants on 19q11-13 with Prostate Cancer in Men of European Origin....................................................................................31 2.1 ASSOCIATION WITH HPN ...........................................................................31 2.1.1 INTRODUCTION ...................................................................................31 2.1.2 MATERIALS AND METHODS ...............................................................32 2.1.2a Subjects........................................................................................32 ix 2.1.2b SNP selection and DNA analysis .................................................33 2.1.2c Statistical analysis ........................................................................34 2.1.3 RESULTS ..............................................................................................35 2.1.4 DISCUSSION.........................................................................................36 2.1.5 TABLES AND FIGURES........................................................................40 2.1.6 REFERENCES ......................................................................................43 2.2 ASSOCIATION WITH KLK3 and KLK2 .........................................................48 2.2.1 INTRODUCTION ...................................................................................48 2.2.2 METHODS .............................................................................................49 2.2.2a Subjects........................................................................................49 2.2.2b SNP selection and DNA analysis .................................................50 2.2.2c Statistical analysis ........................................................................52 2.2.3 RESULTS ..............................................................................................53 2.2.4 DISCUSSION.........................................................................................55 2.2.5 TABLES AND FIGURES........................................................................61