The Genetic Architecture of Alopecia Areata Lynn Petukhova Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 ©2013 Lynn Petukhova All rights reserved ABSTRACT The Genetic Architecture of Alopecia Areata Lynn Petukhova Alopecia areata (AA) is the most prevalent autoimmune disease in the US. With An estimated lifetime risk of 1.7%, it affects both genders with similar frequencies and people of all ages. AA affects more individuals than most other autoimmune diseases combined, and yet despite its prevalence, there is an enormous unmet medical need, in part due to the dearth of information about the underlying pathogenesis. In AA, autoimmunity arises against the hair follicles in the skin, which causes hair loss associated with an aberrant accumulation of immune-response cells around the affected hair follicles. Evidence supporting a genetic basis for AA stems from multiple lines of research, including increased risk of disease in first degree relatives, twin studies, and more recently, our initial family-based linkage study and genome wide association study (GWAS) in a cohort of unrelated individuals. Importantly, our GWAS identified a set of 16 statistically independent risk haplotypes across 8 loci, implicating specific genes that increase risk of AA, all of which have been validated. Genome wide genetic studies can provide critical insight into disease mechanisms, especially when little is known about the underlying causes of disease. In this study, I use complementary gene mapping methods, performing one study in a cohort of families and a second study in a cohort of unrelated cases and controls. Using these two approaches, I obtain new evidence about the genetic influences on AA. Our family cohort contains statistically significant evidence for linkage at a new locus, on chromosome 2q36.1-q37.3 (LOD=4.17) and family-based tests of association implicate 47 genes. I then conducted a GWAS that expanded our initial cohort with the addition of 800 cases and obtained statistically significant evidence for a new locus at chromosome 16p13.13 (p=4.6x10-7). This region has been implicated in several other autoimmune diseases and contains several genes that are known to be involved with immune processes. Taken together, these two studies demonstrate the presence of both rare and common variants are contributing to AA etiology and support emerging evidence that suggests the genetic architecture of common complex diseases involves both rare and common variants. Table of Contents Page Paper 1 1 Introduction 2 Clinical Manifestations of Alopecia Areata 5 Treatment 7 Epidemiological Studies in Alopecia Areata 9 Etiology 13 Future Directions 23 Figures and Legends 26 References 28 Paper 2 39 Abstract 40 Introduction 41 Materials and Methods 44 Results 46 Discussion 50 Figures and Legends 54 Tables 62 References 157 Paper 3 161 Abstract 162 Introduction 162 Materials and Methods 164 Results 166 Discussion 167 Figures and Legends 170 Tables 173 References 174 Conclusions and Future Directions 176 Introduction 177 Summary of Findings 179 Implications of Findings 180 Future Research 181 References 182 i Lists of Charts, Graphs, Illustrations Page Paper 1 Figure 1. Trajectory of Disease. 26 Figure 2. Trajectory for the discovery of Crohn’s disease risk genes. 27 Paper 2 Figure 1. Pedigrees. 55 Figure 2. Whole genome plot of nonparametric linkage analysis. 56 Figure 3. Whole genome plot of nonparametric linkage analysis for seven 57 families. Figure 4. Pedigree-based association tests identified significant associations 58 within eight linkage intervals. Figure 5. Pseudomarker identified significant associations within three 59 linkage intervals. Figure 6. Pseudomarker Results across all regions tested. 60 Figure 7. A comparison of SNP linkage scan with microsatellite linkage scan. 61 Table 1. Nonparametric linkage results. 62 Table 2. Linkage evidence within families. 63 Table 3. Results of PBAT|Linkage. 63 Table 4. Psuedomarker results. 68 Table 5. Genome-wide PBAT results. 79 Table 6. Integrated association results. 153 Table 7. Summary of genes implicated in this study. 154 Table 8. Distribution of GWAS risk alleles in our family cohort. 156 Paper 3 Figure 1. Manhattan plot for genome-wide tests of association 170 Figure 2. Detailed map of associated SNPs and gene locations for 171 chromosome 16p13.13. Figure 3. Manhattan plot for a case only genome-wide tests of association. 172 Table 1. Case-only results. 173 ii Acknowledgments This dissertation is the culmination of many hours of academic training and informal mentoring, encouragement and support that that have been generously bestowed upon me by numerous members of the Columbia University Medical Center community. I thank all of the members of my dissertation committee. I am enormously grateful and forever indebted to Professor Angela M. Christiano for the pivotal role that she has played in this process. She has inspired me from the beginning, encouraging me to apply to the doctoral program in Epidemiology, serving as my benefactor, training me as a scientist and an academic, teaching me perseverance, navigating me through the many challenges of graduate school, and showing me how to light the world on fire by blazing trails. I thank Professor Ruth Ottman for serving as my academic advisor and transitioning into my dissertation sponsor. I am greatly appreciative of our weekly meetings, her patience in both listening and responding to my evolving thoughts, and the critical feedback that she gave me on earlier versions of my dissertation. I thank Professor Sharon Schwartz who inspired my love of causal inference and provided invaluable guidance and encouragement as I sought (and continue to seek) to apply this knowledge to the field of human genetics. I thank Professor Susan E. Hodge for teaching me to think and write with precision and clarity, and for her advice on biostatistics, parenting, and finding balance in life. Finally, I thank Professor Neil W. Schluger for serving as the Chair of my dissertation committee. I thank Drs. Karestan C. Koenen and Joseph H. Lee for serving as readers on my dissertation proposal. I additionally thank Dr. Joseph H. Lee, who along with Drs. Joseph Terwilliger, Krzysztof Kiryluk, Prakash Gorroochurn, and William C. Stewart, and Professors Peter K. Gregersen, David Greenberg, and Martina Durner, provided guidance and advice about the design and analysis of human genetic studies. I thank Professors Leslie Davidson iii and Sandro Galea for their wisdom and support. I am grateful to the many students whose comradery was inspirational and in particular to Drs. Ann Madsen and Ettie Lipner. I thank the members of Team Christiano, especially Drs. Yutaka Shimomura, Katherine Fantauzzo, Hyunmi Kim, Alison Barber, Courtney Luke, Claire A. Higgins, and Sivan Harel. Finally, I thank Liliane Zaretsky for always having an open door and a warm smile. I thank Professor Jurg Ott from Rockefeller University and the Chinese Academy of Sciences for encouraging me to pursue academic training and introducing me to the Columbia University Medical Center. There are an army of people who will forever hold my gratitude for helping me to equilibrate parenting with my academic aspirations, including the administration, faculty, and fellow parents at St. Ignatius Loyola School and the Convent of the Sacred Heart, 91st Street. This accomplishment could not have been achieved without the support of my family, including most significantly from my husband, Dr. Sergei A. Petukhov, and my parents, Dr. and Mrs. Lawrence D. Orvieto. iv Dedication My dissertation is dedicated to my children, Kira Petukhova and Aleksandr Petukhov, for several reasons. First, they are the unsung heroes of this endeavor; enduring the ups and downs of graduate school with me, compromising schedules, and chipping in with chores so that we, as a family, could carve out time for my doctoral program. Second, because they were, and continue to be, an eternal source of inspiration; in an obvious way with their loud cheerleading and quiet prayers; and in a subtle way, the way children do, by providing an entirely new perspective on life’s decisions. Finally, because there is an often iterated trepidation in science that children will be an impediment to success in research. While there is clearly a need for improved institutional infrastructure to support women in science, in dedicating this work to my children, I hope to highlight benefits of balancing motherhood with research. Children remind you to be curious and find awe in nature, they train you to be exceptionally organized, and they inspire you to be great. My children are the foundation to the success that I have achieved in my doctoral program. v 1 Paper 1 Towards Defining the Causal Structure of Alopecia Areata 2 Introduction Alopecia areata is an autoimmune disease that targets the hair follicles in the skin, causing disfiguring hair loss marked histologically by an accumulation of immune-response cells around the affected hair follicles. AA is the most prevalent autoimmune disease in the United States.1,2 With a lifetime risk of 1.7%,3 it targets both men and women and affects people of all ages.4-6 AA affects more individuals than most other autoimmune diseases combined, yet despite its prevalence, there is an enormous unmet medical need7 that arises primarily from the dearth of information about its underlying pathogenesis and is compounded by a lack of studies on its natural history, which presents challenges in designing robust and efficient clinical trials. As an autoimmune disease, AA falls within a broader category of related disorders that are characterized by an aberrant targeted immune attack on cells, tissues or organs. Collectively autoimmune diseases carry a tremendous public health burden in the US and globally.8-11 In the US, 5-8% of the population is affected by an autoimmune disease,1 which results in annual direct health care costs of at least $100 billion,2 and additional economic burden due to lost earnings.