MULTI-OMIC DATA PROVIDE A MORE COMPLETE UNDERSTANDING OF THE AUTISTIC BRAIN by Shannon E. Ellis A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March, 2016 © 2016 Shannon E. Ellis All Rights Reserved ABSTRACT Autism is a complex neurodevelopmental disorder characterized by persistent social deficits and restricted or repetitive patterns of behavior. Despite an established genetic basis of the disorder, efforts to elucidate the genetic underpinnings of the disorder and our understanding of its etiology remains incomplete. As such, we set out to study the effects downstream of genetic variation by studying alterations in both gene expression and DNA methylation (DNAm) in post-mortem brain samples collected from individuals affected with autism and controls. This work highlights that even when there is no primary genetic lesion detected, the autistic brain shows a characteristic pattern of upregulation at M2-activation state microglia genes, a state potentially driven by Type I interferon responses. Additionally, by combining transcriptomic data across autism and two related neuropsychiatric disorders, schizophrenia and bipolar disorder, we have garnered a better understanding of the relationship between these disorders, where genes differentially expressed in autism are concordantly differentially expressed in schizophrenia, but not in bipolar disorder. Finally, as gene expression is regulated, at least in part, by DNAm, we have characterized DNAm at cytosines across the genome and have detected hypermethylation at cytosines outside the commonly-studied CpG context, suggesting that autistic brains have slight increases at many CpH sites (where H=A,T, or C) throughout their genome. These sites are enriched in repetitive regions of the genome and regions containing human-specific CpGs, offering an insight into how this hypermethylation may be functioning mechanistically. Taken together, by studying the downstream effects of genetic variation, at the levels of DNAm and gene expression, we have moved toward a more complete understanding of the autistic brain. Advisor: Dan E. Arking, Ph.D. Reader: Jeff Leek, Ph.D. ii PREFACE I can only begin to express how thankful I am for the opportunity I have had to pursue my Ph.D. in the McKusick-Nathans Institute of Genetic Medicine. I will do my very best to express my gratitude here, but it will undoubtedly fall short of expressing just how thankful I truly am. First and foremost, I want to thank my advisor, Dr. Dan Arking, for his unwavering support and incredible mentorship throughout graduate school. Having never coded before graduate school, I am thankful to Dan for taking a chance on me and spending the time to help make me a successful student. Dan has taught me how to generate, clean, and analyze data critically, creatively and thoroughly, for which I will be forever grateful. Additionally, Dan was always there to answer any questions I had and to help push me past each and every mental roadblock – whether that meant encouraging me to work through it on my own or supplying me with the tools to move past it. Last, I would be remiss not to mention Dan’s ability to think critically and quickly, to act ethically, to manage effectively, and to present data clearly – all skills that I will try my very best to emulate throughout my own career. Additionally, my thesis work was only accomplished thanks to contributions from a number of other people in Dan’s lab. Dr. Simone Gupta, a former postdoc in the lab, was immeasurably helpful my first few years in the lab. Her patience when I joined the lab is worth its weight in gold. I cannot even begin to explain how much Simone taught me. On my first day she sat and explained what a server was to me, demonstrated how to transfer files, and taught me how to log on to a node without ever making me feel like I didn’t know anything (which, in fact, I didn’t). Since that was my level of computational understanding at the start of my PhD, I think it’s fair to say that at least half of what I now know can be directly attributed to her teaching. Additionally, I was so lucky to gain Anna Moes, our longtime lab manager, as a friend and coworker when I joined the lab. Her impeccable organizational skills, attention to detail, and wonderful personality are rarely found in one individual. My work was also immensely influenced by Foram Ashar. She played a pivotal role in every single thing I accomplished in Dan’s lab. From discussing problems I was struggling with to editing both code and everything I’ve ever written and everything in between, Foram was there to support, help, listen, and be the best labmate and friend for which I could have ever asked. iii Finally, every member of the Arking lab during my time played a role in making me the scientist I am today and truly made coming into work an enjoyable experience day in and day out. Thank you for always being there to bounce ideas off of, to get ice cream when things got tough, and for being incredible scientists and even more amazing people. Outside of the Arking lab, there are many people who have been essential along the way. First, I am incredibly thankful to each and every collaborator with whom I’ve had the pleasure of working. Specifically, the scientific input Andy West has had throughout all of my work is impossible to measure. Additionally, he has not only read every draft of every paper, offering helpful comments every single time, but also responded with speedy answers to every e-mail I’ve ever written to him, for which I am so thankful. Additionally, I hit the jackpot with the members of my thesis committee: Drs. Joel Bader, Dani Fallin, Jeff Leek, and Juan Troncoso. The reasons I am indebted to this group are manifold, but I’ll highlight two. First, and foremost, each person offered a different skillset to jointly help me work through each and every issue I ran into along the way; however, secondly, they were all wonderful people to work with. Their criticism was always constructive and their feedback relevant. It was so nice to feel as though we were working as a team toward a common goal each time I scheduled a meeting. Finally, the human genetics program has provided me with a second family throughout graduate school. The supportive environment of the program is unique and indispensable. Thank you to Dr. Dave Valle, Dr. Kirby Smith, and Sandy Muscelli for the countless and tireless hours you all invest so that our program continues to thrive and so that us students always feel supported. And, thank you to all of the faculty members within the IGM who have played a role in my life these past six years. As a graduate student, I was also fortunate to go through the Human Genetics program with the most amazing classmates. I never expected to become close friends with my classmates, but I am so glad my expectation was wrong. Working as a team made getting through our coursework together so much less painful. And, I am so thankful we had one another as we studied for our comprehensive exams. However, I am most thankful for the friendships we formed beyond class and lab. Memories of our trip to Florida, annual white water rafting trips, and seemingly never-ending debates will be memories that I cherish long past our time together at Hopkins. iv I would also like to thank the mentors that helped me get to Hopkins. I struggle to find the words to explain what a huge impact Dr. Jeramia Ory, my undergraduate research mentor, has had on my career. He is a tough but incredible instructor, a supportive mentor, and a wonderful scientist. Not only did he offer me my first position in a research lab, but he helped ensure that I was given every opportunity going forward. He encouraged me to pursue summer research interests at other institutions, gave me opportunities to present at numerous conferences as an undergrad from a tiny liberal arts college, and made sure that I applied to top graduate programs. Through Jeramia’s encouragement, I also had the great fortune of being mentored by Dr. Joseph Ayoob during a summer at Carnegie Mellon University. Joe is not only responsible for any pipetting chops I have (even if I may not use them very much these days) but is also the reason I applied to Hopkins in the first place, as he received his PhD from Hopkins and strongly encouraged that I apply. Joe is a wonderful mentor, a superb scientist, and a great friend. I certainly lucked out getting the chance to work with him and am thankful for all the support and advice he’s given me since my summer at Carnegie Mellon. Beyond science, I have no idea how to thank the amazing friends who helped me make it to Baltimore – a city that I now absolutely love – and those I have made since making the move. I feel so lucky to have friends that have known me since I was five years old, whom know me better than I know myself, whom know where I come from, and who completely understand how I became the person I am today. Additionally, college brought me friends that are so different from myself in so many ways but who share the same importance I place on family, who taught me how to truly enjoy life, and showed me just how big others’ hearts can be, and for all of that I am so very grateful.