INSIGHTS INTO EARLY DEVELOPMENT FROM GENETIC AND EPIGENETIC DATA ACROSS TISSUE TYPES By Shan V. Andrews A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland April, 2018 © 2018 Shan V. Andrews All Rights Reserved ABSTRACT The etiology of autism spectrum disorder (ASD) remains largely undiscovered. Common genetic variants are increasingly being realized, but the functional biology implicated by these SNPs has yet to be elucidated. Integrating epigenetic data, particularly DNA methylation (DNAm), with ASD SNP data is warranted, given its success in schizophrenia and bipolar disorder and the evidence for an epigenetic basis to ASD. Given that DNAm is tissue-specific, it is of interest to consider DNAm from brain, the main tissue of interest for ASD, and peripheral tissues simultaneously so that SNP and CpG-based insights can be compared across tissues. One peripheral tissue, the placenta, is understudied in an ASD context, despite being the critical organ regulating the gestational period, a key window of interest for ASD etiology. Given this role, the placenta also “programs” early and later life phenotypes, and sex-biased ratios in many phenotypes, including ASD, may be driven by sex-specific placenta functions. In Chapter 1, we first describe the current literature on ASD etiology, and then review placenta structure and function, role in fetal brain development, and known differences by fetal sex. Next we use DNAm data across tissues in conjunction with paired genotype data to find that, across blood and brain, ASD-associated SNPs are enriched for methylation quantitative trait loci (meQTLs), or SNPs that are associated with DNAm, and that the DNAm sites controlled by ASD-related SNPs are engaged in immune response pathways (Chapter 2). We then focus on studies of the placenta, first demonstrating the additional enrichment of ASD-associated SNPs with placenta meQTLs (Chapter 3), and conducting queries for differentially methylated regions (DMRs) and global methylation differences related to ASD (Chapter 4). Finally, we discover a genome-wide significant DMR for fetal sex in placenta near ZNF300. DNAm levels in this region are related to placenta morphology, demonstrating a mechanism by which sex-specific placenta structure, and subsequent sex-biased vulnerability to disease, may occur (Chapter 5). This dissertation ii contributes to our understanding of how epigenetics mechanisms, particularly acting in conjunction with genetic variation, contributes to understanding of ASD etiology across tissues, and govern placental functions to create sex-specific gestational environments. Thesis Readers: Christine Ladd-Acosta, PhD (Advisor) M. Daniele Fallin, PhD (Advisor) Dan Arking, PhD Debashree Ray, PhD Alternates: Alden Gross, PhD and Xiaobin Wang, ScD iii ACKNOWLEDGEMENTS First I would like to thank my advisors, Chris Ladd-Acosta and Dani Fallin. I am extremely grateful for your mentorship for the past 6 years during my master’s degree and then of course my PhD. Thank you for always being open to new ideas, no matter at what stage in development you heard them, and for teaching me how to blend epidemiology, genomics, and bioinformatics in the right ways to lead to meaningful work. I know I am the researcher I am today due to both of your guidance. I am proud of the work we have accomplished together and I will miss working with you both. Next I would like to thank Kasper Hansen, who has been a great additional mentor to me. I always appreciated your frank criticism and I always felt that I walked out of your office with a very tangible improvement in my ability as a scientist. I would like to also thank you specifically for improving my ability to write R code; I greatly appreciated your insights into how to best write concise, user-friendly functions and I know you have given my knowledge that I will carry with me for years to come. I have additional mentors I would like to thank as well. First, to Dan Arking, thank you for all of your advice over the years. I always marveled at your ability to be able to give great insights and helpful next steps after looking at my results for only a few minutes. Next, to Andy Feinberg, thank you for all your insights, I always felt very lucky to gain your perspective on my work. To Kelly Bakulski, thank you for your friendship, support and guidance over the years. It is often said in academia that it is important to have someone at the stage ahead of you who you can look up to and who can serve as a role model, and you were undoubtedly that person for me. Finally I would like to thank Kelly Benke and Brion Maher, who I started my first research project with in iv the fall of 2012. Thank you setting me off on the right foot and easing me in to the research process. To Shannon Ellis – thank you being a wonderful collaborator and friend. I learned so much in our meetings together figuring out how to do our various meQTL analyses and I will always be very grateful for that time we spent working together. I need to give a special thanks to all the study staff and participants for the Study to Explore Early Development and the Early Autism Risk Longitudinal Investigation. In particular I would like to thank Jamie Kaczaniuk and Michelle Landrum, who always made navigating study requirements and logistics very easy and who are some of the friendliest faces at the School of Public Health. Thank you to the Maryland Genetics, Epidemiology, and Medicine Training program and all of the faculty behind it, in particular Priya Duggal, Dave Valle, Dani Fallin, and Jennifer Deal. I have gained numerous experiences I would not have had otherwise through this program and I know I am a more well-rounded researcher because of it. Thank you to the JHSPH Epidemiology department, in particular the genetic epidemiology faculty who have taught me and who I have TA’d for, including Priya Duggal, Terri Beaty, Robert Wojciechowski, and Linda Kao. To Ali Abraham and Bryan Lau, thank you for giving me the opportunity to TA for you for several years. I learned so much about the best ways to teach students and I always enjoyed your company. Thank you to all of my friends for their support during this process, in particular John, Robby, and Chet, and all of my roommates during my time in Baltimore. Many of you could relate to my v experiences first hand, and even if you didn’t, I always felt privileged to have you in my life as an outlet during the more difficult times. Thank you to my sisters for your love and support throughout my life and especially in this PhD process. You two have always been wonderful role models for me. I respect you both so much and am proud of everything you have accomplished. I must say a very deep thank you to my parents, to whom I don’t say that nearly enough. Thank you for immigrating to this country and giving me opportunities and experiences I would not have had otherwise. Thank you for all of your hard work during my childhood and for fostering a supporting environment. I am grateful for all you have given me and I hope to now start repaying the favor. And finally thank you to my dearest Brooke, my future wife and my best friend. I can’t describe what a joy it is to have you in my life and how lucky I was throughout this process to know that no matter what happened during the day, I could always come home to you. Thank you for all of your support and, of course, for bringing Leonard into my life. As I’ve always said, I’m not sure if I’m a dog person now, but I’m definitely a Leonard person. vi TABLE OF CONTENTS Abstract……………………………………………………………………………………………ii Acknowledgements……………………………………………………………………………….iv Table of contents………………………………………………………………………………....vii List of Tables……………………………………………………………………………………..ix List of Figures…………………………………………………………………………………......x Chapter 1: Introduction…………………………………………………………………………....1 1.1 Autism spectrum disorder………………………………………………………..……1 1.1.1 Definition………………………………………………………….………1 1.1.2 ASD heritability estimates………………………………………………...1 1.1.3 Environmental risk factors associated with ASD…………………………2 1.1.4 Genetic risk factors associated with ASD ………………………………...4 1.2 Epigenetics and ASD…………………………………………..……………………...6 1.2.1 Description of epigenetics………………………………………………....6 1.2.2 The relevance of epigenetics in ASD etiology……………………………7 1.2.3 Framework for understanding epigenetic involvement in ASD………..…9 1.2.4 Challenges with studying epigenetics in autism: tissue availability………9 1.2.5 Challenges with studying epigenetics in autism: measurement platforms…………………………………………………………………10 1.2.6 Importance of integrating genetic and epigenetic data in ASD………….11 1.3 The placenta……………………………………………………………….…………13 1.3.1 General structure and function………………………………...................13 1.3.2 The role of the placenta in fetal brain development…………………..…14 1.3.3 Differences in disease prevalence by sex and sexual dimorphism in the placenta …………………………………………………...……………..16 1.3.4 Placenta epigenetics……………………………………………………...18 1.4 Focus of this dissertation………………………………………………………….....21 1.5 References……………………………………………………………………………23 Chapter 2: Cross-tissue integration of genetic and epigenetic data offers insight into autism spectrum disorder………………………………………………………………………………...38 2.1 Abstract………………………………………………………………………………40 2.2 Introduction………………………………………………………………………..…41 2.3 Methods………………………………………………………………………………43 2.4 Results………………………………………………………………………………..55 2.5 Discussion……………………………………………………………………………59