GENETIC AND MOLECULAR DISSECTION OF HOMOCYSTEINEMIA IN MICE by SHEILA ERNEST Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Joseph H. Nadeau Department of Genetics CASE WESTERN RESERVE UNIVERSITY August, 2004 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. À mes parents très chers 1 TABLE OF CONTENTS 2 TABLE OF CONTENTS .......................................................................................1 LIST OF TABLES.................................................................................................4 LIST OF FIGURES ...............................................................................................7 ACKNOWLEDGEMENTS...................................................................................11 ABSTRACT.........................................................................................................12 CHAPTER I INTRODUCTION AND RESEARCH AIMS.........................................................14 HOMOCYSTEINEMIA .....................................................................................15 Definitions of homocysteinemia ....................................................................15 Environmental determinants of homocysteinemia ........................................16 Genetic determinants of homocysteinemia...................................................19 HOMOCYSTEINEMIA AND HUMAN DISEASES............................................22 Neural Tube Defects.....................................................................................23 Vascular disease ..........................................................................................26 Cancer ..........................................................................................................28 Neurodegenerative disorders........................................................................30 Down Syndrome ...........................................................................................31 HOMOCYSTEINEMIA AND ANIMAL MODELS ..............................................32 Environmental determinants of homocysteinemia ........................................32 Mouse models with deficiencies of genes involved in HCY-folate metabolism ......................................................................................................................33 Mouse models with phenotypes associated with hyperhomocysteinemia in humans.........................................................................................................34 MECHANISM OF HYPERHOMOCYSTEINEMIA IN DISEASE .......................42 Neural tube defects.......................................................................................42 Vascular disease ..........................................................................................42 Cancer ..........................................................................................................44 Neurodegenerative disorders........................................................................45 Down Syndrome ...........................................................................................46 HOMOCYSTEINE............................................................................................47 Chemistry......................................................................................................47 Regulation of HCY metabolism.....................................................................48 FOLATE...........................................................................................................53 Chemistry......................................................................................................53 Regulation of folate metabolism....................................................................53 HOMOCYSTEINE AND FOLATE ....................................................................57 RESEARCH AIMS ...........................................................................................59 3 CHAPTER II GENETIC AND MOLECULAR CONTROL OF FOLATE-HOMOCYSTEINE METABOLISM IN MUTANT MICE .....................................................................63 ABSTRACT......................................................................................................64 INTRODUCTION .............................................................................................65 RESULTS ........................................................................................................68 DISCUSSION ..................................................................................................79 MATERIAL and METHODS.............................................................................87 CHAPTER III PARALLEL CHANGES IN METABOLITE LEVELS AND EXPRESSION PROFILES IN FOLATE-DEFICIENT AND CROOKED TAIL MUTANT MICE...96 ABSTRACT......................................................................................................97 INTRODUCTION .............................................................................................98 RESULTS ......................................................................................................101 DISCUSSION ................................................................................................113 MATERIAL AND METHODS .........................................................................115 CHAPTER IV NUTRIGENES, FUNCTIONAL GENOMICS AND SYSTEMS BIOLOGY ........119 ABSTRACT....................................................................................................120 INTRODUCTION ...........................................................................................121 RESULTS AND DISCUSSION ......................................................................122 CHAPTER V INHERITANCE OF HOMOCYSTEINEMIA IN A/J AND C57BL/6J MICE: DIET, GENDER, PARENTAL AND GENETIC EFFECTS..........................................124 ABSTRACT....................................................................................................125 INTRODUCTION ...........................................................................................126 RESULTS ......................................................................................................133 DISCUSSION ................................................................................................148 MATERIAL AND METHODS .........................................................................152 CHAPTER VI QTL ANALYSIS OF HOMOCYSTEINEMIA IN A/J AND C57BL/6J MICE USING C57BL/6J-Chr(i)A/J CHROMOSOME SUBSTITUTION STRAINS....................157 ABSTRACT.......................................................................................................158 INTRODUCTION ..............................................................................................159 RESULTS .........................................................................................................162 DISCUSSION ...................................................................................................179 MATERIAL AND METHODS ............................................................................184 CHAPTER VII SUMMARY AND FUTURE DIRECTIONS........................................................190 APPENDIX 1 ....................................................................................................198 APPENDIX 2 ....................................................................................................200 BIBLIOGRAPHY ..............................................................................................202 4 LIST OF TABLES 5 CHAPTER ONE I-1. Nutrition correction of NTDs in seven mouse models………………………..37 BOX 1: Homocysteine metabolism……………………………………………….....50 BOX 2: Folate metabolism……………………………………………………………54 CHAPTER TWO II-1. List of genes used on the arrays……………………………………………….75 II-2. Summary of RNA abundance results………………………………………….77 II-3. Significant differences in RNA abundance in mutants ……………………...86 CHAPTER THREE III-1. Effect of folic acid treatment on craniofacial and neural tube defects in Ski homozygous mutant mice…………………………………………………………...112 CHAPTER FIVE V-1. Gender and diet effects on mean homocysteine levels……………………138 V-2. Gender and diet effects on homocysteine variance………………………..139 V-3. Variation in mean homocysteine levels and in variance for females that were raised on the ‘lower folate’ diet……………………………………………….144 V-4. Variation in mean HCY levels and in variance for males that were raised on the ‘lower folate’ diet……………………………………………….145 6 CHAPTER FIVE (continued) V-5. Variation in mean HCY levels and in variance for females that were raised on the ‘higher folate’ diet……………………………………………...146 V-6. Variation in mean HCY levels and in variance for males that were raised on the ‘higher folate’ diet……………………………………………...147 V-7. Heritability of homocysteinemia……………………………………………….147 CHAPTER