Role of modifier gene SLC6A14 in Cystic Fibrosis and the path to personalized medicine by Saumel Bashir Ahmadi A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Saumel Bashir Ahmadi (2018) Role of modifier gene SLC6A14 in Cystic Fibrosis and the path to personalized medicine Saumel Bashir Ahmadi Doctor of Philosophy Department of Physiology University of Toronto 2018 Abstract Cystic Fibrosis (CF) is the most common fatal genetic disorder in Canada. It is a multi-system disorder caused by mutations in the CFTR gene, expressed in epithelial tissues. Decrease in lung function over time is the most common cause of morbidity and mortality in CF. However, affliction to other organs systems like the gastro-intestinal system, also contributes to significant disease burden. Variation in disease severity among CF patients is well established, attributable to CFTR gene and modifier gene mutations. There are over 2,000 disease causing mutations in the CFTR gene. F508del is the most common CF causing mutation present on at least one allele in 90% of the CF population. However, patients bearing the same F508del mutation on both alleles also exhibit a tremendous variation in disease severity, which has been attributed to modifier gene mutations. Two FDA approved drugs that work directly on CFTR protein – Ivacaftor and Lumacaftor, are shown to have a heterogenous response in CF patients. The heterogeneity in response has also been attributed to modifier genes. With a greater understanding of modifier genes and CFTR genetics, the variation in patient responses to currently available CF therapies could be explained. This has led to efforts for in vitro phenotypic profiling of individual patient derived tissues, in the context of CF. Towards this we developed the apical CFTR conductance assay, to measure CFTR function in vitro using cultured ii airway epithelia from individual patients. Later we applied this technology to murine intestinal tissue to understand the mechanism of a genetic modifier of Cystic Fibrosis – SLC6A14, which was a top hit in a recent genome wide association study. Using a murine CF model and a SLC6A14 knockout mouse, we discovered that SLC6A14 modifies the intestinal phenotype of CF by regulating the fluid secretory capacity of the CF affected epithelium, via the nitric-oxide pathway. Thus, we explored the biologic basis of SLC6A14 as a modifier of CF. Taken together, the studies described in this thesis will facilitate the path towards personalized medicine in CF. iii Acknowledgments Foremost, I would like to thank my supervisors Dr. Christine Bear and Dr. Johanna Rommens for having me in their laboratories and for being excellent mentors and caring individuals. Their hard work and conduciveness to new ideas have always been very inspiring. They have helped me grow as a scientist to tackle the next generation of challenges in Biomedical research. Dr. Bear’s question of “what would be the best experiment?” helped me push the limit of innovation. The collegial and collaborative environment at The Hospital for Sick Children was a cornerstone to the success of everyone involved in the projects of this thesis. I am thankful to my committee members Dr. Scott Heximer and Dr. Reinhart Reithmeier for their helpful discussions, insights and keeping my progress on track. I am very thankful to my dear friend and colleague Dr. Stan Pasyk, who helped me grow professionally as well as socially. I am grateful for his insight, critical arguments and for inspiring me by sharing his passion for science. Over the years I have been fortunate to have many students and collaborators, without whose help this thesis would not be possible. I am thankful to Timothy Chung for discussions in physical chemistry and for maintaining a mutual inspiration for long experiments, and to Rupinder Mangat for her help. I am also thankful to Wilson Wu and Randolph Kissoon for the fun times in the animal facility, productive discussions and for their hard work with the mice. All of you have bright futures ahead of you. The people with whom I worked the most in the lab – my dear friends Sunny Xia and Michelle Di Paola, are really special to me. Their hard work, passion for science and their support was indispensable to this thesis. They are success bound with bright futures. I am grateful to Dr. Mohabir Ramjeesingh for supporting me, believing me and nurturing me as a scientist. I am thankful to my friend and colleague Dr. Steven Molinski for helpful discussions, help with molecular biology, and for a mutually productive life-long collaboration. I am also very thankful to Dr. Canhui (Danny) Li, who normalized my crazy ideas and helped them become a reality. His vision and passion for science has always been motivational. I have had helpful discussions with my colleagues and friends Stephanie Chin and Maurita Hung and I see bright futures for them. I am thankful to Ling Jun Huan, for her help in the lab all along the way, and maintaining a collegial environment. I am grateful to all past and present members of the Bear Lab including Elyse Watkins, Ida Szarics, Dr. Paul Eckford, Dr. Leigh Wellhauser, Dr. Kai Du, Wilson Yu, Janet Jiang, Onofrio Laselva, Donghe Yang, Angela Skoutakis, Anick Auger and Roman Pekhletski for their help at various stages of my project, and for sharing interesting conversations. iv I would like to give my sincere gratitude to Catherine Luk, whose help was critical in maintaining the mouse facility for this project. Her care, devotion and tireless efforts kept the project smooth and fun for everyone. I am thankful to all members of Dr. Johanna Rommens’ laboratory including Dr. Stéphane Gagnon, Rikesh Gandhi, Dr. Marina Tourlakis, Dr. Holly Liu, Rashmi Parekh and Fan Lin, who helped me all along especially in the area of molecular genetics. I am grateful for collaborating with Dr. Julie Forman-Kay’s laboratory. I made a life-long friend Dr. Zoltan Bozoky, whose insight into computational biology and big data handling was unmatched. I had a very productive professional and social relationship with him. I am also very grateful to collaborate with Dr. Theo Moraes’ and Dr. Tanja Gonska’s laboratories, and all the present and past members of their labs including Andrew Lloyd-Kuzik, Wan Ip, Claire Bartlett, Hong Ouyang and Wenming Duan. I was also fortunate to have a productive collaboration with Dr. Amy Wong in Dr. Janet Rossant’s lab, and with Dr. Felix Ratjen, Julie Avolio and Dr. Jeremy Hirota. I am also thankful to all my medical school mentors, and also my medical school friends Dr. Parth Vaishnav, Dr. Vidhi Patel and Dr. Mihir Chauhan for their support in my endeavors. Finally, I would also like to thank my parents Dr. Bashir Ahmadi and Nafisa Ahmadi for their constant support and trust. I am also very grateful to my grandparents Dr. Abdulmuttalib Ahmadi and Zubeda Ahmadi for constantly inspiring me to work hard, and to my sister Swela Bashir for her support. This study would not have been possible without the help and support of the Cystic Fibrosis patients and their families, and the inspiration they provided to find a cure. I hope this thesis will be a small step towards reaching a cure for everyone. v Table of Contents Contents Acknowledgments .......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. xi Abbreviations ................................................................................................................................ xii List of Figures .............................................................................................................................. xvi List of Appendices ....................................................................................................................... xix Chapter 1 Introduction .................................................................................................................... 1 1.1 Cystic Fibrosis (CF) .................................................................................................................. 1 1.2 Cystic Fibrosis Transmembrane conductance Regulator (CFTR) ............................................ 2 1.2.1 Introduction ................................................................................................................... 2 1.2.2 CFTR Genetics and regulation of gene expression ....................................................... 4 1.2.3 CFTR protein and regulation ........................................................................................ 6 1.2.4 CFTR anion conductance and epithelial pH regulation .............................................. 11 1.2.5 Cystic Fibrosis causing mutation F508del CFTR ....................................................... 11 1.3 Mechanisms underlying CF pathogenesis ............................................................................. 11 1.4 CF animal models .................................................................................................................. 14 1.4.1 Murine CF intestinal phenotype ............................................................................... 14