GENETIC VARIATION AND COMPLEX DISEASE: THE EXAMINATION OF AN X-LINKED DISORDER AND A MULTIFACTORIAL DISEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Catherine Elise Cottrell ***** The Ohio State University 2007 Dissertation Committee: Approved by Dr. Julie M. Gastier-Foster, Advisor Dr. John Bauer _________________________________ Dr. Gail Herman Advisor Graduate Program in Dr. Susan R. Mallery Integrated Biomedical Sciences ABSTRACT Two genetic studies are presented in this dissertation. The studies share a common theme of the influence of genetic variability in complex disease. The first study is entitled, “The Association of Antioxidant-Related Gene Polymorphisms and Second Primary Malignant Neoplasms in Pediatric Hodgkin Lymphoma.” Improved treatment strategies in pediatric Hodgkin Lymphoma (HL) have resulted in a cure rate approaching 95%, yet the development of a second primary malignant neoplasm (SMN) is a risk for long-term survivors. In a pediatric HL population, between 6-26% of patients will develop a SMN within 30 years following treatment. The etiology of HL is still largely unknown, as is the cause of certain types of SMNs. Oxidative stress has been linked to the development of cancer due to the damaging effects of reactive oxygen species (ROS) on DNA, lipids, and proteins. During periods of extended oxidative stress, the damaging effects of ROS are likely to increase which in turn raises the risk of genetic change. In a multi-stage model of tumorigenesis, genetic change is considered to be the initiating event of cancer development. Under normal circumstances, antioxidant enzymes within the cell mitigate the effects of ROS by converting reactive species into non-toxic molecules. Mechanisms within the cell maintain a steady state balance between ROS and antioxidant enzyme levels. We ii hypothesize that individuals predisposed to lower levels of antioxidant enzyme activity due to polymorphic variants within those genes may be at risk for increased damage caused by ROS. Although decreased amounts of antioxidant enzymes have been found in a variety of cancers, a correlation between polymorphisms in antioxidant genes and predisposition to secondary cancer has not been studied. The purpose this study was to assess the association of antioxidant gene alleles with the risk of developing a SMN in HL survivors. DNA samples were obtained from 768 HL patients enrolled in the Childhood Cancer Survivor Study (CCSS). The samples were genotyped for 90 polymorphisms in antioxidant related genes including SOD, GPX, NOS, CAT, and CYP2C9. Statistical analysis methods to determine risk of developing a SMN included association, haplotype, and multiple regression models. In our HL cohort, 131 patients developed a SMN. An additional 117 patients developed nonmelanoma skin cancer, and 34 patients developed 2 SMNs. Out of the 36 SNPs that were included in the final analysis, 4 SNPs in the GPX1, GPX3, GPX4, and SOD2 genes, were potentially suggestive (p<0.05) of an association between genotype and the development of a SMN. A general trend observed in the genotyping data suggested that an increased risk of SMN was conferred by the presence of the minor allele, while a decreased risk of SMN was conferred by the presence of the major allele in the population. Replication studies are necessary, though it is notable that polymorphisms within the GPX family may be associated with the development SMN in our cohort. Additionally, hazard ratios were only moderately increased in this population suggesting that the development of a SMN in a pediatric HL is likely multi-factorial in nature. The elucidation of critical genes involved in SMN development could influence iii patient follow-up and intervention strategies, and potentially aid in the prevention of SMN. The second study in this thesis is entitled, “Atypical X-Inactivation in an X;1 Translocation Patient Diagnosed with Otopalatodigital Syndrome.” X-chromosome inactivation (XCI) is an epigenetic process used to regulate gene dosage in mammalian females by silencing one X-chromosome. While the pattern of XCI is typically random in normal females, abnormalities of the X-chromosome may result in skewing due to disadvantaged cell growth. We describe a female patient with an X;1 translocation [46,X, t(X;1)(q28;q21)dn] and unusual pattern of XCI who was clinically diagnosed with Otopalatodigital syndrome (OPD) type 1. There was complete skewing of XCI in the patient, along with the atypical findings of an active normal X-chromosome and an inactive derivative X. An X-linked disorder, OPD1 is characterized by multiple congenital anomalies including skeletal abnormalities, craniofacial defects, and hearing loss. Mutations within the FLNA gene (Xq28) are known to cause OPD, though none were detected in our patient. Additionally, no abnormalities in FLNA mRNA or protein were detected in our patient. Characterization of the translocation revealed that the patient’s Xq28 breakpoint interrupts the DKC1 gene, located 400kB distal to FLNA. Molecular analysis of the breakpoint region revealed functional disomy of Xq28 genes distal to DKC1. No monosomy of 1q genes was detected. Possible explanations for the patient’s phenotype include a position effect due to the translocation breakpoint, an undetected FLNA-related mutation, or altered gene dosage due to consequences of atypical XCI. iv Dedicated to Andy Thank you for your love, constant support, and words of encouragement. & To my parents, Charles and Denise Thank you for inspiring me, fostering a sense of creativity, and for a lifetime of love and support. v ACKNOWLEDGMENTS It is with deepest gratitude that I would like to thank my advisor, Dr. Julie Gastier-Foster for her dedication and commitment to me on both a personal and professional level. You are a constant source of inspiration and an exceptional mentor. Thank you for your patience, guidance, and support over the years. Most of all, I thank you for your time, for the countless hours spent teaching me, and for the direction you provided so that I could mature on both a scientific and personal level. I would like to thank my committee members, Dr. Gail Herman, Dr. John Bauer, and Dr. Susan Mallery for your commitment to teaching, dedication to students, and exceptional guidance. I would like to express my sincere appreciation for Dr. Steve Qualman. Thank you for guiding me to choose a wonderful lab in which to complete my dissertation. Thank you for introducing me to Julie, because without her none of this would be possible. Finally, thank you most of all for your dedication, your commitment to research and teaching, and your passion in supporting Children’s Hospital. I would like to thank Dr. Allan Yates and Christine Kerr for your support of the IBGP program, for your devotion to the students, and for your sincere desire to see each of us succeed. vi I would especially like to thank all of the technologists and staff within the Molecular and Cytogenetics Laboratories at Children’s. Thank you for accepting me into the lab, and for sharing your time and resources with me. Thank you especially to Jessica who made me feel welcome when I first began and for your help along the way. Thanks to Beth and Morgan for teaching me the basics of FISH, cell harvests and for your expertise in taking wonderful photos on the scope. A special thank you goes to Eileen for your unwavering support, your friendship, and your sense of humor. I don’t think I could have made it through this without you. Finally, I would like to thank my family and friends for your support throughout this process. Thank you to Jen for understanding what I was going though when no one else did. Thank you for graduating ahead of me, so then I could ask you all sorts of questions on finishing up. Thank you for extended chats over coffee and for going to Buckeye football games with me. Thank you to Colleen for being a great sister. Thank you for your support and kind words, and for feeding the cats when I was working late or away on weekends. Thank you especially to Andy for your love and encouragement. Thank you for driving long hours to see me, for cheering me up during hard times, and for always making me laugh, especially when this day seemed very far away. Thank you to my parents for sparking my interest in science early on. Thank you for those kitchen science experiments, grade school science fair ideas, and for taking me to your lab when I was very young. Most of all, thank you for your love and guidance over the years. vii VITA August 3, 1979. Born – Columbus, OH June 2001. B.S. Molecular Genetics The Ohio State University 2001 – 2007. Graduate Fellow The Ohio State University. AWARDS 2004 . .Graduate Student Research Award Columbus Children’s Research Institute Annual Research Conference FELLOWSHIPS 2001 . .University Fellowship The Ohio State University 2001-2005 . Molecular Life Sciences Fellowship The Ohio State University 2002-2007 . General Mason Fellowship Columbus Children’s Hospital FIELD OF STUDY Major Field: Integrated Biomedical Sciences Specialization: Genetics viii TABLE OF CONTENTS P a g e Abstract. ii Dedication. v Acknowledgments . .vi Vita . viii List of Tables. xiv List of Figures . .xv Introduction . .1 Section I: The Association of Antioxidant Related Gene Polymorphisms and Second Primary Malignant Neoplasm in a Pediatric Hodgkin’s Lymphoma Population Chapters: 1. Hodgkin Lymphoma and the Childhood Cancer Survivor Study . 5 1.1 Introduction to Hodgkin Lymphoma . 5 1.2 Classification of Hodgkin Lymphoma . 6 1.3 Risk Factors and Causative Agents in Hodgkin Lymphoma . 6 1.4 Treatment of Hodgkin Lymphoma: Maximizing Efficacy while Minimizing Risk . .8 ix 1.5 The Childhood Cancer Survivor Study .