Spectrum of Mutations in MMAB Identified by High Resolution Melting Analysis Margaret Lamb Illson Department of Human Genetics McGill University Montréal, Québec, Canada August 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Masters of Science Copyright 2012 Margaret L. Illson DEDICATION To all of my family and friends, but especially John, for their encouragement, support and understanding. ACKNOWLEDGMENTS I would like to express my appreciation to my mentor, David Rosenblatt, for providing me the opportunity, not only once but twice, to be actively engaged in a field of personal interest. Being a member of the Rosenblatt team provides the friendship, support and unquestioning assistance of a very special group of colleagues headed by Dr.David Watkins. He points us in the right direction, gives valuable information and offers only the kindest critiques when asked. Memorable days have been passed in the company of Alison Brebner, Jaesung Kim and Laura Dempsey Nunez, and former lab mates who occasionally make welcome appearances. They have always been available for science discussions and troubleshooting, IT assistance and quirky bits of fun. The successful completion of this project was due to the training, mentoring and support of a large cast of people. My co-supervisor, Dr. Brian Gilfix, and committee members, Dr. Ron Agatep and Dr. George Chong helped us wade through all challenges with very practical advice. Many members of Dr. Carl Wittwer’s Lab (Department of Pathology , University of Utah, Salt Lake City, UT) put aside time to first train us, and then offer continued support. Thank you Carl, Dr. Bob Palais, Quiying Huang, Jana Kent, Luming Zhou and Zach Dwight. I would also like to thank Gail Dunbar, Maria Galvez, Junhui Liu, Leah Ladorez and Jocelyne Lavallé, Kandance Springer, Thomas Leslie, Ross MacKay and Laura Benner for help in their areas of expertise, and smiling faces even when they saw me coming. I also appreciate the willingness of Francis Petrella, Kush Prithipaul, Dylan Tanzer and Tracy Wang to do countless, but not thankless tasks. However, the biggest contributer in helping me achieve my personal goal was Laura Dempsey Nunez who never tired of joining forces and brains to work our way through this project together. Thank you all! iii TABLE OF CONTENTS DEDICATION ................................................................................................................................. ii ACKNOWLEDGMENTS............................................................................................................... iii TABLE OF CONTENTS ................................................................................................................ iv LIST OF TABLES .......................................................................................................................... vi LIST OF FIGURES........................................................................................................................ vii LIST OF ABBREVIATIONS ....................................................................................................... viii ORIGINAL CONTRIBUTIONS TO SCIENCE............................................................................. ix ABSTRACT......................................................................................................................................x ABRÉGÉ........................................................................................................................................ xii Introduction and Objectives of Study..............................................................................................14 CHAPTER 1: Cobalamin .............................................................................................................15 1.1 Significance..........................................................................................................................15 1.2 Identification of cobalamin..................................................................................................15 1.3 The cobalamin molecule.......................................................................................................16 1.4 Clinical Impact ....................................................................................................................17 1.5 Cobalamin Metabolism ........................................................................................................17 1.5.1 Extracellular Metabolism..............................................................................................18 1.5.2 Intracellular Metabolism...............................................................................................19 1.6 Inherited Cobalamin Disorders (Complementation groups)................................................21 1.6.1 Combined methylmalonic aciduria and homocystinuria...............................................23 1.6.2 Isolated homocystinuria ...............................................................................................26 1.6.3 Isolated Methylmalonic Aciduria (MMA) ...................................................................28 CHAPTER 2: Isolated Methylmalonic Aciduria........................................................................30 2.1 Organic Aciduria (acidemia): an overview...........................................................................31 2.2 Etiology of Methylmalonic Aciduria...................................................................................31 2.3 Clinical spectrum – Isolated MMA .....................................................................................33 2.4 Vitamin B12 Responsiveness ...............................................................................................35 CHAPTER 3: The MMAB gene ...................................................................................................36 3.1 Historical Overview..............................................................................................................36 3.2 Identification of gene responsible for cblB disorders ..........................................................36 3.3 Location and Structure ........................................................................................................37 3.4 Function of ATR..................................................................................................................38 3.5 Spectrum of Variants...........................................................................................................39 3.6 Genotype / Phenotype correlation ......................................................................................40 CHAPTER 4: Mutation detection for cblB type disorders........................................................42 4.1 Overview of screening methods .........................................................................................42 4.2 Screening by functional assays............................................................................................42 4.3 New advances in sequencing technology ............................................................................43 4.4 High Resolution Melting Analysis ......................................................................................44 4.4.1 Simultaneous gene scanning and genotyping for common polymorphisms.............46 iv 4.4.2 HRMA Analysis...........................................................................................................46 4.4.3 Interpretation of HRMA results ....................................................................................48 4.5 Considerations in choosing a screening method..................................................................48 CHAPTER 5: Rationale for evaluating HRMA as a cblB screening tool................................50 5.1 Gene scanning to detect unidentified variants..................................................................51 5.2 Genotyping of common polymorphisms.........................................................................52 CHAPTER 6: Methods and Materials.........................................................................................53 6.1 Optimization........................................................................................................................53 6.1.1 Assay design .................................................................................................................53 6.1.2 PCR optimization..........................................................................................................55 6.2 Cell line selection ................................................................................................................56 6.2.1 DNA quality control and preparation...........................................................................57 6.3 Assay protocol with mixing..................................................................................................59 6.4 Protocol for genotyping with snapback primers ...................................................................59 6.5 Analysis................................................................................................................................60 6.6 Confirmation of results........................................................................................................61 CHAPTER 7: Results....................................................................................................................62