GENETIC, EVOLUTIONARY, AND GENOMIC ANALYSIS OF HOMOCYSTEINE AND FOLATE PATHWAY REGULATION By TOSHIMORI KITAMI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Joseph H. Nadeau Department of Genetics CASE WESTERN RESERVE UNIVERSITY January, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF TABLES 3 LIST OF FIGURES 4 ACKNOWLEDGEMENTS 5 ABSTRACT 6 CHAPTER 1: INTRODUCTION AND OBJECTIVES 8 Homocysteine, Folate, and Human Disease Risks 9 Introduction 9 Box 1: Different types of clinical studies 10 Non-genetic factors that affect homocysteine level 13 Effect of folate on homocysteine level 14 Cardiovascular disease 15 Neural tube defect 18 Cancer 20 Alzheimer’s disease and dementia 24 Genetic Mutations in Homocysteine and Folate Metabolism 25 Rare mutations 25 Common mutation (Mthfr C677T mutation) 26 Mthfr C677T mutation and disease risks 27 Mouse Models and Disease Mechanisms 32 Basic Biochemistry of Homocysteine and Folate Pathways 36 Regulation of Homocysteine and Folate Pathways 39 Positive and negative feedback 39 Polyglutamation of folate 40 Tissue specific regulation 40 Folate transport 41 Transcriptional regulation 42 Global perspective on homocysteine and folate pathway regulation 43 Research Objective 45 CHAPTER 2: GENETIC AND PHENOTYPIC ANALYSIS OF MTHFR ENZYME ACTIVITY, TAIL KINKS, AND SEIZURE SUSCEPTIBILITY IN PL/J MICE 48 Abstract 49 Introduction 50 Materials and Methods 52 Results 54 Discussion 60 1 CHAPTER 3: GENE DUPLICATION, METABOLIC NETWORKING, AND GENETIC BUFFERING IN PHYSIOLOGICAL PATHWAYS OF HUMAN AND MICE 65 Abstract 66 Introduction 67 Materials and Methods 70 Results 76 Discussion 84 CHAPTER 4: GLOBAL GENE EXPRESSION AND METABOLITE PROFILING OF RESPONSES TO DIETARY FOLATE PERTURBATIONS IN TWO GENETICALLY DISTINCT INBRED MOUSE STRAINS 88 Abstract 89 Introduction 90 Materials and Methods 93 Results 100 Discussion 112 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS 117 Summary 118 Future Directions 120 APPENDIX 127 Appendix 1: Nomenclature of Homocysteine and Folate Enzymes 128 Appendix 2: Nutrient Composition of Folate Deficient and Control Diets 129 BIBLIOGRAPHY 130 2 LIST OF TABLES CHAPTER 1 1.1 Summary of cohort studies on colon cancer risk and homocysteine and folate levels 23 1.2 Summary of association studies between Mthfr C677T and colon cancer risk 29 1.3 Summary of association studies between Mhtfr C677T and neural tube defect risk 31 CHAPTER 3 3.1 Average Ka/Ks values for orthologous genes in various metabolic pathways in humans and mice 78 CHAPTER 4 4.1 Significant over-representation of Gene Ontology terms 103 4.2 Effect of ApoE knockout and dietary choline supplements on metabolite and gene expression profiles 107 3 LIST OF FIGURES CHAPTER 1 1.1 Homocysteine and folate pathways 38 CHAPTER 2 2.1 Amino acid sequence alignment of Mthfr in five model organisms 56 2.2 X-ray photograph of a kinky tail in a PL/J mouse 56 2.3 Number of tests with seizures in parental, F1, and F2 mice 59 CHAPTER 3 3.1 Schematic for defining networks based on the structure of metabolic pathways 75 3.2 Distribution of Ka/Ks values for 241 genes in various metabolic pathways 79 3.3 Scatter plot of the Ka/Ks values and the number of tissues with gene expression from mouse and human gene expression data 83 CHAPTER 4 4.1 Dietary folate perturbation protocols 94 4.2 Metabolite profiles of serum homocysteine and folate, and total cholesterol in serum and liver 101 4.3 2-dimensional plots of metabolite profiles for A/J and C57BL/6J 101 4.4 Average linkage hierarchical clustering of liver gene expression profile 103 4.5 Gene expression profile of choline kinase 111 4 ACKNOWLEDGEMENTS My thesis is a collaborative work of many people who have impacted me both on educational and personal level throughout my life. I like to thank my adviser Dr. Joseph Nadeau for teaching me to think independently, write and speak effectively, and to approach science with curiosity and passion. I also like to thank my committee members for providing variety of views on my thesis and expanding my breadth of scientific thinking. I also thank the members of the Nadeau Lab for inputs and supports throughout my graduate career. I like to thank my family for providing generous financial support throughout my life, always providing the best environment for my education and personal growth, and providing me freedom to pursue any career choice of my own. I also like to thank my Cleveland family, the Tomciks and the Linnevers, for helping me situated in this city as well as inviting me to their holiday dinners. I also like to thank my tutor from my days in Hawaii, Mrs. Murata, who taught me English as well as many valuable lessons in life. Without her work, none of my academic success would have been possible. I also like to thank her family for their kindness and generosity, delicious meals, and fun memories. I also like to thank my friends in graduate school, Josephine and Ricky (a.k.a. Mr. & Mrs. Chan), Kirsten, Sheila, Lesil, Martha, Matt, Keith, Karen and Mike, Lisa and Brian, for making my long six years enjoyable, providing me with lots of fun memories, and motivating each other to survive the pains of graduate school. 5 Genetic, Evolutionary, and Genomic Analysis of Homocysteine and Folate Pathway Regulation Abstract By TOSHIMORI KITAMI Abnormalities in homocysteine and folate metabolism are associated with increased risk for several common human diseases. Both elevated serum homocysteine and low dietary folate intake increase the risk for cardiovascular diseases, neural tube defects, cancers, and neurodegeneration. Although common mutations in these pathways have been identified, they do not fully account for the variety of disease types that are associated with increased homocysteine or low folate levels. Mouse models allow us to control many of the genetic and environmental variables inherent in human studies, allowing us to dissect genes, pathways, and nutrients important for disease pathogenesis. Previous mouse studies showed that mutations in other pathways can significantly modulate functions of homocysteine and folate metabolism and modify disease phenotypes suggesting that these pathway interactions and their regulations are crucial to understanding the role of homocysteine and folate metabolism in complex diseases. To study these pathway interactions and their regulation, I used genetic, evolutionary, and genomic approaches. I first used a unique mouse strain PL/J to dissect the genetic control of key enzyme methylenetetrahydrofolate reductase (MTHFR) and its potential role in disease phenotypes. I found that seizure and kinky tail phenotype in these mice were 6 polygenic and showed strong environmental contributions. Next, I used evolutionary approaches to address whether biochemical interactions in metabolic pathways buffer deleterious mutations. I found that redundant metabolic paths do not provide genetic buffering as inferred from estimates of variation in gene evolution rates. However, higher level interactions explained some of the evolutionary rate variability. Lastly, I used genomic approaches to identify pathways that modulate homeostatic responses to dietary folate perturbations in two genetically distinct inbred strains. I found striking strain differences in homeostatic responses on folate retention and global gene expression profiles. I also found that cholesterol and choline metabolisms are involved in the folate perturbation response, which may play a more general role in complex disease mechanisms. Overall, these analyses revealed pathway interactions that are important for functionality of homocysteine and folate metabolism and highlighted some future strategies for dissecting the role of these pathways in complex diseases. 7 CHAPTER 1 Introduction and Objectives 8 Homocysteine, Folate, and Human Disease Risks Introduction Abnormalities in homocysteine and folate metabolism have been associated with a striking variety of complex human diseases. An elevated level of serum homocysteine is associated with increased risk for cardiovascular disease and Alzheimer’s disease. Low serum folate level or low dietary folate intake has also been associated with increased risk for mothers having children with neural tube defects and for colon and breast cancer. Genetic mutations in these metabolic pathways also elevate risks for these complex diseases (see Genetic Mutations in Homocysteine and Folate Metabolism). Serum homocysteine level in this thesis refers to the total serum homocysteine, which includes protein-bound homocysteine and soluble homocysteine. Protein-bound homocysteine accounts for nearly 80% and soluble homocysteine accounts for 20% of total homocysteine (Ueland 1995). The normal range of homocysteine is 5 to 15µM (Ueland et al. 1993, Graham et al. 1997). Elevated serum homocysteine beyond the normal range (>15uM) is traditionally