FROM VARIANTS TO PATHWAYS: INTERROGATING THE GENETIC ARCHITECTURE OF AGE-RELATED MACULAR DEGENERATION by ANDREA ROSE WAKSMUNSKI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve of the dissertation of Andrea Rose Waksmunski candidate for the degree of Doctor of Philosophy*. Committee Chair Dana C. Crawford, Ph.D. Thesis Advisor Jonathan L. Haines, Ph.D. Committee Member Thomas LaFramboise, Ph.D. Committee Member Mark Cameron, Ph.D. Committee Member Alexander Miron, Ph.D. Date of Defense March 30, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 DEDICATION To my Mom and Dad. Your love and faith shaped me into the person I am today. Thank you for your endless encouragement and teaching me to value learning, hard work, and problem-solving. To my siblings, Alexis and Derek. Being triplets has given us a special bond. I am so honored to be your sister and eternally grateful for your unwavering love and support. To my grandparents, Andrew and Rose Ondecko. You became angels during the first year of my Ph.D. program and have been watching over me ever since. May you continue to be my guardian angels in everything I do. 3 Table of Contents LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ACKNOWLEDGEMENTS ........................................................................................... 10 LIST OF ABBREVIATIONS ........................................................................................ 12 Abstract ............................................................................................................................ 16 CHAPTER 1 .................................................................................................................... 18 Age-Related Macular Degeneration as a Public Health Concern........................... 18 Age-Related Macular Degeneration: Symptoms and Treatment Options ............. 19 Age-Related Macular Degeneration: Known Pathology ......................................... 22 Age-Related Macular Degeneration: Risk Factors .................................................. 24 Genetic Epidemiology of Age-Related Macular Degeneration: Methods and Populations ................................................................................................................... 25 The Amish as an Isolated, Founder Population ....................................................... 29 The Amish in Genetics Research ............................................................................... 31 Summary and Unanswered Questions ...................................................................... 34 CHAPTER 2 .................................................................................................................... 36 Abstract ........................................................................................................................ 37 Introduction ................................................................................................................. 38 Methods ........................................................................................................................ 40 Study Demographics .................................................................................................. 40 Blood Collection ........................................................................................................ 40 Nucleic Acid Extraction ............................................................................................. 41 P503A Genotyping ..................................................................................................... 41 Amish Pedigrees ........................................................................................................ 42 Age at Exam ............................................................................................................... 42 CFH RNA Quantification and Analysis ..................................................................... 42 Protein Modeling ....................................................................................................... 43 Western Blots and Quantitative Analysis .................................................................. 44 ELISA ......................................................................................................................... 45 Genetic Risk Score Analysis ...................................................................................... 46 4 Results .......................................................................................................................... 47 Identification of Additional CFH P503A Carriers in the Amish ............................... 47 Age at Diagnosis by P503A Carrier Status ............................................................... 49 CFH RNA Quantification and Analysis ..................................................................... 50 Protein Modeling ....................................................................................................... 52 CFH Protein Expression ........................................................................................... 54 Genetic Risk Scores ................................................................................................... 57 Discussion ..................................................................................................................... 59 CHAPTER 3 .................................................................................................................... 65 Abstract ........................................................................................................................ 66 Introduction ................................................................................................................. 67 Materials and Methods ............................................................................................... 68 Study Participants...................................................................................................... 68 Genotyping and Quality Control ............................................................................... 71 Association Analysis .................................................................................................. 73 Linkage Analysis ........................................................................................................ 74 In Silico Functional Analysis of Genes from 1-HLOD Support Intervals of Linkage Regions ...................................................................................................................... 76 Results .......................................................................................................................... 76 Association Analysis .................................................................................................. 76 Linkage Analysis ........................................................................................................ 80 In Silico Functional Analysis of Genes from 1-HLOD Support Intervals of Linkage Regions ...................................................................................................................... 85 Discussion ..................................................................................................................... 87 CHAPTER 4 .................................................................................................................... 95 Abstract ........................................................................................................................ 96 Introduction ................................................................................................................. 97 Methods ........................................................................................................................ 98 Study Subjects and GWAS Summary Statistics .......................................................... 98 PARIS: Knowledge-Driven Pathway Analysis of GWAS Data ................................. 99 Identification of Statistical Pathway Driver Genes ................................................. 100 Protein-Protein Interaction Network for Statistical Pathway Driver Genes .......... 100 Motif Analysis for Statistical Pathway Driver Genes .............................................. 101 5 Results ........................................................................................................................ 101 In Silico Pathway Analysis ...................................................................................... 101 Statistical driver genes among advanced AMD-associated pathways .................... 102 Discussion ................................................................................................................... 111 CHAPTER 5 .................................................................................................................. 117 Abstract ...................................................................................................................... 118 Background ...............................................................................................................