GENETIC AND PHENOTYPIC RESPONSE OF NEURAL TUBE DEFECT MOUSE MUTANTS TO FOLIC ACID By GHUNWA A. NAKOUZI 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, 2009 CASE WESTERN RESREVER UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of GHUNWA A. NAKOUZI _________________________________________________________ candidate for the PhD degree*. Shawn McCandless (signed)___________________________________________________ (chair of the committee) Joseph Nadeau ____________________________________________________ Mark Adams ____________________________________________________ Donald Jacobsen ____________________________________________________ Radhika Atit ____________________________________________________ June 30, 2009 (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 ............................................................................................................ 5 ACKNOWLEDGEMENTS AND DEDICATIONS .......................................................... 6 Abstract ............................................................................................................................... 8 CHAPTER 1 ..................................................................................................................... 10 NTDs in Humans .......................................................................................................... 11 Clinical significance.................................................................................................. 11 Genetic and environmental risk factors. ................................................................... 13 Folate-Homocysteine association. ............................................................................ 14 Prevention measures. ................................................................................................ 16 Adverse effects of high levels FA. ............................................................................ 18 NTDs in Mice ............................................................................................................... 20 Mice as models for human NTDs. ............................................................................ 20 Supplementation studies and metabolic pathways. ................................................... 24 Previous response predictive profiles. ...................................................................... 29 Summary of mouse mutants selected for modeling human NTDs. .......................... 30 Research Objectives ...................................................................................................... 35 CHAPTER 2 ..................................................................................................................... 40 Abstract ......................................................................................................................... 41 Introduction ................................................................................................................... 42 Materials and Methods .................................................................................................. 45 Results ........................................................................................................................... 50 Discussion ..................................................................................................................... 58 CHAPTER 3 ..................................................................................................................... 62 Abstract ......................................................................................................................... 64 Introduction ................................................................................................................... 65 Materials and Methods .................................................................................................. 69 Results ........................................................................................................................... 76 Discussion ..................................................................................................................... 84 CHAPTER 4 ..................................................................................................................... 88 Abstract ......................................................................................................................... 90 1 Introduction ................................................................................................................... 92 Materials and Methods .................................................................................................. 94 Results ......................................................................................................................... 102 Discussion ................................................................................................................... 117 CHAPTER 5 ................................................................................................................... 121 Summary ..................................................................................................................... 122 Future Directions ........................................................................................................ 126 APPENDIX ..................................................................................................................... 136 APPENDIX 1 .............................................................................................................. 137 APPENDIX 2 .............................................................................................................. 138 APPENDIX 3 .............................................................................................................. 139 APPENDIX 4 .............................................................................................................. 139 APPENDIX 5 .............................................................................................................. 139 APPENDIX 6 .............................................................................................................. 139 APPENDIX 7 .............................................................................................................. 140 APPENDIX 8 .............................................................................................................. 143 BIBLIOGRAPHY ........................................................................................................... 144 2 LIST OF TABLES CHAPTER 1 Table 1.1 Mouse NTD responses to different nutritional supplementations or treatments ....................................................................................................................... 28 Table 1.2 NTD mutants chosen for study ...................................................................... 34 CHAPTER 2 Table 2.1 Apob: effect of maternal FA supplementation on occurrence of resorptions 51 Table 2.2 Effect of maternal FA supplementation on incidence of exencephaly in Apob-/- embryos .............................................................................................. 51 Table 2.3 Lp: effect of maternal FA supplementation on occurrence of resorptions .... 53 Table 2.4 Effect of maternal FA supplementation on incidence of craniorachischisis in Lp-/- embryos ................................................................................................. 53 Table 2.5 Effect of maternal FA supplementation on incidence of looped tail in Lp+/- embryos ......................................................................................................... 53 Table 2.6 Genotype distribution of observed Apob embryos ........................................ 55 Table 2.7 Genotype distribution of observed Lp embryos ............................................. 55 Table 2.8 Gender specific distribution of exencephaly frequency and embryonic loss in Apob............................................................................................................... 56 Table 2.9 Gender specific distribution of craniorachischisis and looped tail frequency and embryonic loss in Lp............................................................................... 57 CHAPTER 3 Table 3.1 Test and control mice used for experiments .................................................. 71 Table 3.2 Comparison of metabolite levels in each mutant to its corresponding control (unpaired t-tests with Welch’s correction) .................................................... 82 3 CHAPTER 4 Table 4.1 Early lethality of FA-supplemented Lrp6-/- embryos ................................... 104 Table 4.2 Effect of maternal FA supplementation on incidence of NTDs in Lrp6-/- embryos ....................................................................................................... 105 Table 4.3 Genotype distribution of observed embryos from Lrp6+/- x