IDENTIFICATION OF GENETIC PREDICTORS OF RESISTANT HYPERTENSION THROUGH GENOME WIDE ASSOCIATION ANALYSIS COUPLED WITH INDUCED PLURIPOTENT STEM CELLS By NIHAL EL ROUBY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Nihal El Rouby To my family, My mother Camilia, My father Mohammed El Rouby, My husband Ihab and sons Yaseen and Yousef, My brother Ameer, My sisters Iman, Sara and Heba, My friends Amira Khalil, Maha El badry and Amira Sirag ACKNOWLEDGMENTS I would like to give my sincere gratitude to my mentor, Dr. Julie Johnson, for her mentorship, guidance, and continuous support throughout my PhD training. I cannot thank her enough for providing me the opportunity to work in her lab and under her supervision. Her continuous support and mentoring helped me grow and become a better researcher. I learned from her how to think critically of all the details in the research and how to criticize my own work. I believe that any success in my career will be attributed to her continuous teaching, encouragement and support. I can say that I owe her a debt of gratitude for all she has done to help me succeed in my PhD and thereafter. I would like to thank all my committee members for their continuous guidance and extended support along the way. I am especially grateful to Dr. Caitrin McDonough who taught me everything about genetic analysis. She has been supportive since the very first day I joined the Johnson’s lab. She was always there whenever I wanted to consult and communicate any difficulties that I encounter during my analysis. Her feedback and valuable input have certainly helped me accomplish my project successfully. I would like to thank Dr. Yan Gong, for her tremendous help and support to my study and specifically the statistical aspects of the project. Special thanks Dr. Naohiro Terada who welcomed me in his lab and provided all the support and help me to learn and grasp a very new field to me. I would also like to thank Dr. Jefferey Harrison for his advices and insights on the CX3CR1 project. Dr. Harrison has always made time to discuss and support different aspects of my project. Special thanks to Dr. Matthew Gitzendanner for all his help, guidance and support, especially in the bioinformatics analysis. He always provided help whenever I needed and was always 4 willing to help me troubleshoot some of my codes that never worked. I would like to thank Dr. Jatinder lamba for her valuable advice, input and support for my work during my PhD training. I would like to also thank Dr. Mark Wallet for his advice and input on the monocytes differentiation and CX3CR1. My sincerest gratitude goes to Dr. Taimour Langaee, Dr. Rhonda Copper- DeHoff, Dr. Larissa Cavallari, and Dr. Reggie Frye for their support, valuable advice, and help during my PhD. Special thanks to Dr. Carl Pepine for giving me the opportunity to participate in the resistant hypertension clinic during my training, and for his continuous support. My utmost gratitude also goes to Dr. Katherine Santostefano who taught me everything about iPSC and wet lab experiments. Thank you for your gracious help and support throughout my project. I would also like to thank Dr. Nikolette Biel, Dr. Sonal Singh, Dr. Natalie Fredette, Dr. Noriko Watanabe, Dr. Chul Han, and Bayli DiVita for all their help with the iPSC project. They have been supportive to my experience in the Terada’s lab. I would like to thank Dr. Charles Wood in the department of Physiology and Functional Genomics for his continuous support throughout my PhD. I must acknowledge NIH grant T32HL083810 and the Department of Physiology and Functional Genomics for support of my PhD training. I would like to extend a special thanks to all present and former graduate students and postdocs in the Department of Pharmacotherapy and Translational Research who have been a family for me. Special thanks to Dr. Mohamed Mohamed Eslam who has graciously helped me when I first came her to UF. Dr. Mohamed and his 5 wife, Rana have been a family to me. Special thanks to Dr. Mohamed Hossam Shahin who has been always a brother to me. Dr. Mohamed Shahin has never hesitated in helping me and answering any questions I have. Special thanks to my colleague, Carol Sa, who started her PhD journey at the same time. Carol has been always there to support me during my journey. Special thanks to Dr. Issam Hamadeh, Dr. Shin-wen Chang, Dr. Mohamed Solayman, Dr. Sonal Singh for their kindness, support and friendship. I would also like to extend my thanks to Ben Burkley, Cheryl Galloway, Lynda Stauffer, who facilitated part of the research in this dissertation. I would like to deeply thank my wonderful husband – Ihab– for his tremendous support, love, caring, patience and encouragement; without his support, the completion of my PhD would have never been possible. I would like to thank my sons, Yaseen and Yousef, for the joy they have created in my life. They always kept me going as I have wanted be a role model for them when they grow up. I would like to dedicate my great and sincere thanks to my best friend Amira Khalil, who has been always a motivation force for me. Special thanks to my friends Maha El Badry and Amira Sirag who have always supported me and made this journey pleasant. Special thanks to my friends, Yasmeen Magdy, Amal Bakry, Razan Alfakir, Nadia Salloom and Souad Benchemsi for their kindness and support. Last, but not least, I would like to thank my family members, my father, Dr. El Rouby, who has been a role model for me. He taught me, by example, the importance of hard work, humbleness and selflessness. My mother has always showered me with unconditional love, support and encouragement. My brother and sisters have 6 surrounded me with care and support throughout every stage of my life; without your support, I would have never accomplished any of my goals. 7 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 12 LIST OF FIGURES ........................................................................................................ 13 ABSTRACT ................................................................................................................... 15 CHAPTER 1 GENETICS OF RESISTANT HYPERTENSION – A NOVEL PHARMACOGENOMICS PHENOTYPE ................................................................. 18 Introduction ............................................................................................................. 18 Recent RHTN Pharmacogenetics Studies........................................................ 20 Genetic Variation in Sodium and Water Handling Pathways ............................ 21 1) Epithelial Sodium Channel (ENAC) ....................................................... 21 2) Aldosterone synthase (Cytochrome P45011B2, CYP11B2) .................. 22 3) Cytochrome P450, family 4, subfamily A, polypeptide 11, CYP4A11 .... 23 Genetic Variation Affecting Response to Dietary Salt Reduction and Diuretics ........................................................................................................ 24 -Adducin ................................................................................................. 24 Guanine nucleotide-binding protein -polypeptide 3 (GNB3) ..................... 25 Neural precursor cell expressed developmentally down – regulated 4- like (NEDD4L) ......................................................................................... 25 Recent Genetic Studies in RHTN ..................................................................... 26 Genes related to vascular function ............................................................. 26 eNos variants and phosphodiesterase type 5 inhibitors ............................. 28 RHTN Associations in Genes of Unrelated Pathways ...................................... 29 Need for Expansive Genetic Approaches in RHTN Pharmacogenomics.......... 32 Conclusions ............................................................................................................ 37 Significance ............................................................................................................ 38 2 QUALITY CONTROL AND IMPUTATION PROCEDURES FOR THE SECONDARY PREVENTION OF SUBCORTICAL STROKES DATASET ............. 42 Introduction ............................................................................................................. 42 Methods .................................................................................................................. 45 Tools and software used for QC and imputation .............................................. 45 Study participants ............................................................................................. 45 Secondary Prevention of Subcortical Strokes (SPS3) ...................................... 45 Genotyping ....................................................................................................... 45 Per marker QC procedures .............................................................................. 45 8 SNP genotyping call rate and removing SNPs below the missingness call rate ................................................................................................................ 46 SNP deviating from Hardy