ELUCIDATION OF THE GENETIC ETIOLOGY OF HEMOSTASIS AND THROMBOSIS THROUGH FAMILY-BASED STUDIES By JESSE DARRELL HINCKLEY B.S., Brigham Young University, 2005 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 Program 2013 This thesis for the Doctor of Philosophy degree by Jesse Darrell Hinckley has been approved for the Human Medical Genetics Program by Tasha Fingerlin, Chair Jorge Di Paola, Advisor Douglas Graham Elaine Spector Richard Spritz Date __04/15/13__ ii Hinckley, Jesse Darrell (Ph.D., Human Medical Genetics) Elucidation of the Genetic Etiology of Hemostasis and Thrombosis through Family- Based Studies Thesis directed by Associate Professor Jorge A. Di Paola ABSTRACT Von Willebrand Factor (VWF) plays a key role in primary hemostasis by initiating platelet recruitment and adhesion and an indirect role in secondary hemostasis as the noncovalent chaperone of coagulation factor VIII (FVIII) in circulation. Larger blood components such as erythrocytes and leukocytes contribute to the etiology of hemostasis and thrombosis by displacing platelets and VWF toward the endothelium. I first characterized an Amish pedigree with VWD type 2. Genome-wide linkage analyses identified candidate quantitative trait loci (QTL) of VWF and FVIII levels on chromosomes 6p22, 9q34 (ABO), and 16q11. I also replicated previously reported associations confirming STXBP5, SCARA5, STAB2, and TC2N as candidate QTL. Secondly, I evaluated VWF single nucleotide polymorphisms (SNPs) as sources of genetic variation of VWF levels in the Amish pedigree. I replicated association with two SNPs in three replication cohorts (n=3,316). I also found significant differences of the minor allele frequency of five SNPs between VWD type 1 index cases (n=310) and a control cohort. Thirdly, I mapped candidate QTL of complete blood count traits by genome-wide quantitative trait linkage analysis in the Amish pedigree. I identified four candidate loci with LOD scores above 2.0: 6q25, 9q33, 10p12, and 20q13. I also demonstrated that the most common causal variant of hereditary hemochromatosis was associated with common variation of complex erythrocyte traits. iii Fourthly, I characterized the novel VWF c.3911T>G mutation in a family that demonstrated phenotypic variability. Results demonstrated the extent of mutant VWF peptide incorporation into multimers may explain phenotypic variability in VWD patients. Fifthly, I studied two families with inherited thrombocytopenias. Homozygosity mapping and analysis of platelet mRNA in a large Native American kindred identified NBEAL2 as the most likely cause of gray platelet syndrome. Whole genome sequence analysis in a family with autosomal dominant thrombocytopenia (THC2) identified MASTL and ANKRD26 as the most likely causal genes. Taken together, these findings demonstrate the importance of multiple family- based study strategies in elucidating the genetic etiology of hemostasis and thrombosis. The genes and candidate QTL reported herein provide the foundation for molecular biology experiments to further characterize the complex pathways that mediate hemostasis and thrombosis. The form and content of this abstract are approved. I recommend its publication. Approved: Jorge A. Di Paola iv DEDICATION This thesis is dedicated to Frank Frerman and MJ Stewart, two valuable mentors and friends whose lives and friendship motivate me to excel in my academic pursuits in order to be able make a meaningful contribution to the field of hematology and oncology. They grounded me and gave my training purpose. This thesis is also dedicated to my wife who followed me to Colorado and has supported me in decisions that have taken us away from family and friends on more than one occasion. v ACKNOWLEDGMENTS This work would not have been possible without my thesis advisor, Jorge Di Paola. He provided me the space and resources to grow as a scientist by allowing me to lead out on collaborative projects and to attend conferences and meetings. I greatly appreciate all that has been provided during my dissertation. This work also would not have been possible without all of the amazing families that were willing to give of their time and blood (literally). My experience was enriched by participating in patient enrollment and meeting so many selfless patients. I would not be here without the many others that made my transfer possible, including the Human Medical Genetics and Genomics Program director Rich Spritz. Rich has also been an extremely valuable mentor and committee member. He challenged me, improving the depth of my training and the quality of my science. During my time in the program, I had two of the best committee chairs: Frank Frerman and Tasha Fingerlin. Frank was a close friend and mentor who treated students as equals. He was an anchor through the turbulence of academic politics and a true advocate as my committee chair. Tasha is no second to Frank as a committee chair. I thank her for agreeing to serve as my chair this past year. Her expertise and support were invaluable. She has also been an advocate and a voice of reason. I am also grateful for the mentorship I have received from Doug Graham and Elaine Spector as committee members. Their friendship and insight enriched my training experience. Doug also served as my first clinical mentor in the Medical Scientist Training Program at Colorado, and I have learned much from him as a physician scientist. I also owe a debt of gratitude to Stephen Hunger, section chief for Hematology and Oncology at Children’s Hospital Colorado, for his efforts to make my transfer possible. I am thankful to those that entered the trenches of science, helped me find my footing, and contributed their own time and efforts to the work presented herein. Kai vi Wang and Trudy Burns at the University of Iowa spent countless hours conducting statistical genetics analyses and editing manuscripts. Amy Shapiro and Meadow Heimann at the Indiana Hemophilia and Thrombosis Center made it possible to enroll so many members of the Amish pedigree. Sandy Haberichter and Pam Christopherson at the Blood Center of Wisconsin were invaluable in providing expertise and resources. Many other scientists have made their labs and resources available including Andy Weyrich at the University of Utah; Walter Kahr at the Hospital for Sick Kids in Toronto; Karl Desch, Jordan Shavit, and David Ginsburg at the University of Michigan; Bob Montgomery at the Blood Center of Wisconsin; Jose Lopez at the Puget Sound Blood Center; and Jeff Murray at the University of Iowa. Friends and coworkers at the University of Colorado have also assisted with this research including Di Paola Lab alumni Shay Fabbro, Shawn Jobe, and Brie Nixon and our current lab manager Gary Brodsky. Randy Wong has been invaluable. I am also grateful for resources provided by Uma Pugazhenthi, Anna Peljto, and many others. In addition to those who have assisted at the bench, I am thankful for all our amazing administrators who make science happen including Tina Green, Cammie Kennedy, Judie Lehman, and MJ Stewart. My research was also supported by the Hemophilia and Thrombosis Center, and I owe a debt of gratitude to Marilyn Manco- Johnson and Judy Primeaux for their support. I also appreciate the support of Tamim Shaikh, Arthur Gutierrez-Hartmann, Angie Ribera, and Jodi Cropper. Most importantly, I am thankful for good friends and family that have grounded me during my research experience including fellow genetics students, Taylor Blades, Alex Blasky, Cassidy Punt, and Erin Bashal. My wife, Latha, has tolerated me spending nights and weekends sequestered at the computer. She has been very understanding and facilitating. I am also grateful to my parents and immediate family members who have been encouraging and understood our absence during mandatory training events. vii CONTENTS CHAPTER I. HEMOSTASIS AND THROMBOSIS......................................................................1 Significance............................................................................................................1 Background............................................................................................................2 Thrombus formation: VWF, FVIII, and platelets.....................................................4 Von Willebrand factor.............................................................................................6 FVIII and the coagulation cascade.......................................................................12 Environmental and genetic etiology of VWF and FVIII........................................16 Platelets...............................................................................................................20 Objective..............................................................................................................24 II. ANALYSIS IN A MULTIGENERATIONAL AMISH PEDIGREE IDENTIFIES NOVEL CANDIDATE QTL THAT AFFECT VWF AND FVIII LEVELS AND REPLICATES GWAS QTL ASSOCIATIONS.......................................................26 Abstract................................................................................................................26 Introduction..........................................................................................................27 Methods...............................................................................................................29 Participants..............................................................................................29