Investigations into the Early Steps of Cobalamin Metabolism Isabelle Racine Miousse Doctorate of Philosophy Department of Human Genetics McGill University Montreal, Quebec, Canada June 6, 2011 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctorate of Philosophy Copyright 2011 All rights reserved. DEDICATION This work is dedicated to the persons living with cobalamin disorders and their families. ACKNOWLEDGMENTS I would like to thank my supervisors David Rosenblatt and James Coulton for their support and inspiration during the years I have spent in their company. I am grateful for all the opportunities they have opened for me, for their time and their patience. Thank you to my committee members, Dr. Eric Shoubridge and Dr. Rima Slim, for their advice and guidance. Special thanks also to Drs. David Watkins and Maria Plesa for their technical guidance and their great depth of knowledge. I would also like to thank them for proofreading work. Thank you to the students, particularly Woranontee Werarpachai for teaching me microcell-mediated chromosome transfer, Abigail Gradinger and Amanda Duval-Loewy for getting me started with PCR and cell culture, and all the other students I had the chance to work with. iii TABLE OF CONTENTS DEDICATION .......................................................................................................................ii ACKNOWLEDGMENTS...................................................................................................... iii TABLE OF CONTENTS ......................................................................................................iv LIST OF TABLES ................................................................................................................xi LIST OF FIGURES ............................................................................................................. xii CLAIMS TO ORIGINALITY ............................................................................................... xiii LIST OF ABBREVIATIONS .............................................................................................. xiv CHAPTER 1: LITERATURE REVIEW ................................................................................ 1 1.1 Historical perspectives ......................................................................................... 2 1.1.1 First descriptions of pernicious anemia ............................................................. 2 1.1.2 A cure for pernicious anemia ............................................................................. 3 1.1.3 Cobalamin absorption ....................................................................................... 4 1.1.4 Pernicious anemia as an immune disorder ....................................................... 5 1.1.5 Cobalamin-binding proteins ............................................................................... 6 1.1.6 Cobalamin malabsorption .................................................................................. 6 1.1.7 Isolation of cobalamin ........................................................................................ 7 1.1.8 Structure and synthesis of cobalamin ............................................................... 8 1.1.9 Discovery of complementation groups .............................................................. 9 1.1.10 Genetic era .................................................................................................... 10 1.2 The cobalamin molecule .................................................................................... 11 1.2.1 Coordination chemistry .................................................................................... 11 1.2.2 Cobalamin structure ........................................................................................ 12 1.2.3 Oxidation states of cobalamin ......................................................................... 16 1.2.4 Cobalt-carbon bond ......................................................................................... 16 1.2.5 Cobalamin-related compounds ....................................................................... 17 1.2.6 Cobalamin synthesis in bacteria ...................................................................... 18 iv 1.2.7 Cobalamin import in bacteria ........................................................................... 19 1.3 Cobalamin absorption in humans ....................................................................... 20 1.3.1 Cobalamin binding in the GI tract .................................................................... 20 1.3.2 Crossing of the enteric epithelium ................................................................... 21 1.3.3 Cobalamin in the circulation ............................................................................ 26 1.3.4 Entry into the cells ........................................................................................... 27 1.3.5 Cobalamin-dependant enzymes in human ...................................................... 28 1.4 Homocysteine: an overview ............................................................................... 29 1.4.1 Inborn errors of homocysteine metabolism ..................................................... 32 1.4.2 Folic acid and cobalamin ................................................................................. 33 1.4.3 Cardiovascular disease in adults..................................................................... 34 1.4.4 Neural tube defects- Folic acid ........................................................................ 35 1.4.5 Neural tube defects- Cobalamin ...................................................................... 36 1.4.6 Cardiac malformations .................................................................................... 37 1.5 Methylmalonic acid: an overview ........................................................................ 38 1.5.1 Metabolic pathways leading to methylmalonic aciduria .................................. 41 1.5.2 Genetic causes of methylmalonic aciduria ...................................................... 41 1.5.3 Presentation .................................................................................................... 43 1.5.4 Suspected etiologies ....................................................................................... 44 1.5.5 Treatment ........................................................................................................ 45 1.6 Intracellular defects of cobalamin ....................................................................... 46 1.6.1 Biochemical signs of cobalamin disorders ...................................................... 46 1.6.2 Newborn screening ......................................................................................... 47 1.6.3 Biochemical characterization ........................................................................... 48 1.6.4 Somatic cell complementation ......................................................................... 49 v 1.7 Complementation Groups .................................................................................. 53 1.7.1 Disorders affecting methionine synthase ........................................................ 53 cblG (MIM #250940) ............................................................................................ 53 cblE (MIM #236270) ............................................................................................. 54 1.7.2 Disorders affecting methylmalonyl-CoA mutase ............................................. 55 cblB (MIM #251110) ............................................................................................. 55 cblA (MIM #251100) ............................................................................................. 57 mut (MIM #251000) .............................................................................................. 59 1.7.3 Disorders affecting methionine synthase and methylmalonyl-CoA mutase .... 61 cblF (MIM #277380) ............................................................................................. 61 cblC (MIM #277400)............................................................................................. 64 cblD (MIM #277410)............................................................................................. 67 1.8 Gene discovery in Mendelian disorders ............................................................. 70 1.8.1 Homology with known genes ........................................................................... 70 1.8.2 Linkage analysis .............................................................................................. 71 1.8.3 Exome capture ................................................................................................ 72 1.8.4 Microcell-mediated chromosome transfer ....................................................... 73 Introduction .......................................................................................................... 73 Principle ............................................................................................................... 74 Application ............................................................................................................ 77 1.9 Protein-protein interactions ................................................................................ 78 1.9.1 Protein interactions in cobalamin metabolism ................................................