GENETIC ANALYSIS OF NOVEL MODELS OF THROMBOCYTOPENIA AND LEUCOPENIA by ERNEST RICKY CHAN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Mark D. Adams, Ph.D. Department of Genetics CASE WESTERN RESERVE UNIVERSITY August 2009 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of Ernest Ricky Chan candidate for the Doctor of Philosophy degree.* Ron Conlon (Chair of the Committee) Mark Adams (Mentor) Brian Bai Kevin Bunting Keith McCrae Joseph Nadeau June 23, 2009 *We also certify that written approval has been obtained for any proprietary material contained therein. Contents LIST OF TABLES ........................................................................................................................................ III LIST OF FIGURES ...................................................................................................................................... IV ACKNOWLEDGEMENTS AND DEDICATION ................................................................................................ V THESIS ABSTRACT .................................................................................................................................... VI CHAPTER 1: INTRODUCTION..................................................................................................................... 1 HEMATOPOIESIS ....................................................................................................................................... 2 THROMBOPOIESIS ..................................................................................................................................... 3 Platelets ........................................................................................................................................... 3 TPO, MPL, and major pathways of platelet development- ................................................................. 6 Thrombocytopenia and Platelet Disorders ....................................................................................... 11 Models of thrombocytopenia .......................................................................................................... 21 ENU MUTAGENESIS AND THE HLBS STRAINS .................................................................................................. 22 CHAPTER 2: THROMBOCYTOPENIC STRAIN HLB219 ............................................................................... 25 ABSTRACT ............................................................................................................................................. 26 INTRODUCTION ....................................................................................................................................... 27 MATERIALS AND METHODS ....................................................................................................................... 30 Mapping ........................................................................................................................................ 30 Platelet lifespan assay .................................................................................................................... 30 Sequencing ..................................................................................................................................... 31 Circulating TPO concentration ........................................................................................................ 31 Assay for colony forming units of hematopoietic progenitors .......................................................... 32 Competitive repopulation assay ...................................................................................................... 32 hlb219 Construction of Mpl or Mpl vectors .......................................................................................... 33 Stable transformation of Ba/F3 cells with Mpl, Mplhlb219, or MGIN ............................................... 33 Proliferation assay .......................................................................................................................... 34 Downstream signaling pathways .................................................................................................... 35 RESULTS ............................................................................................................................................... 36 Phenotypic Characterization of the HLB219 Strain ........................................................................... 36 Identifying the mutation ................................................................................................................. 38 Supporting evidence for a mutation in Mpl ..................................................................................... 40 hlb219 Mpl supports TPO-independent proliferation in Ba/F3 cells ...................................................... 43 hlb219 Mpl downstream signaling ...................................................................................................... 45 DISCUSSION ........................................................................................................................................... 48 ACKNOWLEDGEMENTS.............................................................................................................................. 52 CHAPTER 3: THROMBOCYTOPENIC STRAIN HLB381 ............................................................................... 53 ABSTRACT ............................................................................................................................................. 54 INTRODUCTION ....................................................................................................................................... 55 MATERIALS AND METHODS ....................................................................................................................... 58 Intercross, whole genome scan, and interval mapping .................................................................... 58 Candidate-gene and interval sequencing ........................................................................................ 59 Blood phenotype and general characterization ............................................................................... 61 Circulating TPO concentration ........................................................................................................ 61 Bleeding time ................................................................................................................................. 62 CFU assays ..................................................................................................................................... 62 Competitive repopulation and bone marrow transplant .................................................................. 63 i Platelet lifespan assay .................................................................................................................... 64 Splenectomy ................................................................................................................................... 64 Serum Injection .............................................................................................................................. 65 IVIg Treatment ............................................................................................................................... 65 Western blot of platelet proteins .................................................................................................... 66 Rasa3 mutation confirmation and expression ................................................................................. 66 RESULTS ............................................................................................................................................... 68 General characterization ................................................................................................................ 68 Genetic mapping and sequencing ................................................................................................... 70 Examining hematopoiesis ............................................................................................................... 79 Examining platelet turnover ........................................................................................................... 82 DISCUSSION ........................................................................................................................................... 87 HLB381 hematopoietic development .............................................................................................. 87 HLB381 platelet survival ................................................................................................................. 88 Searching for the mutation ............................................................................................................. 91 Compare and contrast HLB381 and scat.......................................................................................... 94 CHAPTER 4: LEUCOPENIC STRAIN HLB156 .............................................................................................. 99 ABSTRACT ........................................................................................................................................... 100 INTRODUCTION ....................................................................................................................................