GERMLINE MUTATIONS IN ETV6 RESULT IN AUTOSOMAL DOMINANT THROMBOCYTOPENIA By LEILA J. NOETZLI B.S., University of California San Diego, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Human Medical Genetics and Genomics Program 2017 This thesis for the Doctor of Philosophy degree by Leila J. Noetzli has been approved for the Human Medical Genetics and Genomics Program by Robert Sclafani, Chair Jorge Di Paola, Advisor Jay Hesselberth Richard Spritz Rytis Prekeris Date: May 19, 2017 ii Noetzli, Leila J. (Ph.D., Human Medical Genetics and Genomics) Germline mutations in ETV6 result in autosomal dominant thrombocytopenia Thesis directed by Associate Professor Jorge Di Paola ABSTRACT Whole exome sequencing on a family with autosomal dominant thrombocytopenia and two occurrences of acute lymphoblastic leukemia (ALL) of unknown cause identified a heterozygous single nucleotide change in the gene ETV6, encoding a p.Pro214Leu amino acid substitution. We screened families with the same phenotype and found mutations in ETV6 in two additional families: one with the identical p.Pro214Leu mutation and one with a p.Arg418Gly substitution. ETV6 is a transcriptional repressor that functions through self-oligomerization and is essential for hematopoietic stem cell proliferation and platelet production in mice. Our report was among the first to describe pathogenic germline mutations in ETV6. Functional characterization of the corresponding ETV6 protein mutations showed aberrant cytoplasmic localization, impaired transcriptional repression, and delayed megakaryocyte maturation. Furthermore, we found that mutant ETV6 protein oligomerizes with WT ETV6 and sequesters it from the nucleus suggesting a dominant negative mechanism. Platelet transcriptome analysis of patients with the p.P214L mutation revealed significant differential expression of 189 transcripts between patients and controls. Downregulated transcripts in patients with the P214L mutation were significantly enriched in transcripts encoding extracellular matrix and cytoskeletal proteins. Interestingly, maintenance of the extracellular matrix is essential for megakaryocyte migration in the bone marrow and plays a role in cancer iii progression. Furthermore, microtubule and cytoskeletal organization are essential for platelet production. Mouse models of Etv6 disruption, including one expressing the human P214L mutation, exhibit thrombocytopenia and defects in megakaryocyte maturation, similar to humans. My thesis project describes germline mutations in ETV6 that are associated with megakaryocyte differentiation and platelet production as well as ALL predisposition. The form and content of this abstract are approved. I recommend its publication. Approved: Jorge Di Paola iv DEDICATION My dissertation is dedicated to my dog, Cali, who has been my constant companion throughout graduate school and who patiently watched me write this thesis. v ACKNOWLEDGEMENTS This work would not have been possible without the contributions from individuals at multiple institutions. These people include, but are not limited to, Walter Kahr and Richard Lo from The Hospital for Sick Kids in Toronto, Michael Callaghan and Madhvi Rajpurkar from Children’s Hospital Michigan, Ken Jones and Katherine Gowan from the University of Colorado Anschutz Medical Campus, Andrew Weyrich and Jesse Rowley from the University of Utah, and Anna Savoia from the University of Trieste. I would like to thank members of the Di Paola lab, my committee, and the students and faculty in the Human Medical Genetics and Genomics Program for their support and expertise. Finally, I sincerely acknowledge Dr. Jorge Di Paola, my PhD advisor, for his mentorship and guidance and for providing me with countless opportunities for intellectual and professional growth. vi TABLE OF CONTENTS CHAPTER I. HEMATOPOIESIS, MEGAKARYOPOIESIS, AND THROMBOPOIESIS ..... 1 Hematopoiesis and Megakaryopoiesis ................................................. 1 Megakaryocyte Maturation and Thrombopoiesis .................................. 7 ETS Transcription Factors .................................................................. 14 ETV6 .................................................................................................. 16 II. HETEROZYGOUS GERMLINE MUTATIONS IN ETV6 ARE ASSOCIATED WITH THROMBOCYTOPENIA, HIGH MEAN CORPUSCULAR VOLUME OF RED BLOOD CELLS, AND PREDISPOSITION TO ACUTE LYMPHOBLASTIC LEUKEMIA ..................................................................... 24 Introduction ......................................................................................... 24 Methods .............................................................................................. 26 Patient Tissue Acquisition ............................................................ 26 Exome Sequencing ...................................................................... 27 Bioinformatics of Exome Sequencing .......................................... 27 Plasmids and Mutagenesis .......................................................... 28 Luciferase Reporter Assays ......................................................... 29 Immuno Pull Down Assay ............................................................ 29 Lentivirus CD34+ Cell Transduction ............................................. 30 vii Immunofluorescence and Confocal Microscopy ........................... 30 Transmission Electron Microscopy and Immunoblots .................. 31 RNA Sequencing (Bone Marrow) ................................................. 32 Bioinformatics of RNA Sequencing .............................................. 32 Results ............................................................................................... 33 Phenotypic Characterization of the Families ................................ 33 Whole Exome Sequencing and Identification of ETV6 Mutations 37 Germline Mutations in ETV6 Impair Transcriptional Repression .. 42 Germline Mutations in ETV6 do not Alter Oligomerization in vitro 44 Germline Mutations in ETV6 Alter Cellular Localization in vitro ... 46 Germline Mutations in ETV6 Affect Megakaryocyte Maturation in vitro .............................................................................................. 52 Leukemia Sample Shows Second Hit Translocations Associated with Leukemia .............................................................................. 56 Discussion .......................................................................................... 58 Function of the P214L and R418G Mutations .............................. 59 Analysis of Leukemia Mutations ................................................... 61 Analysis of Germline Mutations in ETV6 ...................................... 62 III. TRANSCRIPTIONAL CHANGES IN PATIENTS WITH ETV6-P214L MUTATION ................................................................................................... 71 viii Introduction ......................................................................................... 71 Methods .............................................................................................. 73 DAVID, Gene Ontology, and Gene Set Enrichment Analysis ....... 74 Hierarchical Clustering ................................................................. 74 Comparison of Chip Data to our Transcriptome Data .................. 75 Expression of ETV6 in K562 Cells ............................................... 75 Flow Cytometry of K562 Cells ...................................................... 76 Results ............................................................................................... 76 Hierarchical Clustering Shows Distinct Clusters between Patients and Controls ................................................................................. 76 Analysis of Significantly Different Transcripts .............................. 78 ETV6 Predicted Targets ............................................................... 81 Comparison of P214L Modulated Transcripts with ChIP Seq Data ..................................................................................................... 84 Interesting Differentially Expressed Genes .................................. 89 Expression of ETV6 in K562 Cells ............................................... 90 Discussion .......................................................................................... 93 Hierarchical Clustering ................................................................. 93 Extracellular Matrix and Cell Adhesion Transcripts ...................... 94 Microtubule and Cytoskeleton Transcripts ................................... 95 ix Platelet and Erythrocyte Transcripts ............................................ 97 Combinatorial Action with Other Transcription Factors ................ 99 Interesting Differentially Expressed Genes ................................ 100 IV. MOUSE MODELS OF ETV6 DISRUPTION ........................................... 104 Introduction ....................................................................................... 104 Methods ............................................................................................ 107 Generation of Mouse Models ..................................................... 107 Genotyping of Mouse Models .................................................... 107 Complete Blood Counts and Flow Cytometry of Peripheral Blood ..................................................................................................