Genetic and Molecular Dissection of Homocysteinemia in Mice

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

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