EXPLORING MOLECULAR PATHOGENESIS TO STREAMLINE FUTURE THERAPEUTICS IN RARE DISEASES USING GSD1A AS A MODEL By KATHLEEN LYNN PLONA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Mitchell L. Drumm, PhD Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY August 2021 Case Western Reserve University School of Graduate Studies We hereby approve the thesis of Kathleen Lynn Plona Candidate for the degree of Doctor of Philosophy* Committee Chair Paul Tesar Committee Member Anna Mitchell Committee Member Colleen Croniger Committee Member Mitchell L. Drumm Date of Defense May 27, 2021 * We also certify that written approval has been obtained for any proprietary material contained therein 2 Dedication This work is dedicated to everyone who has ever experienced the confusion and struggle of finding a diagnosis. 3 Table of Contents List of Tables ...................................................................................................................... 6 List of Figures .................................................................................................................... 7 AcKnowledgements ........................................................................................................... 8 Abstract ............................................................................................................................ 11 Chapter 1: BacKground and Significance ...................................................................... 13 1.1 Rare diseases ................................................................................................... 13 1.2 Role of sequencing in diagnostics ................................................................... 14 1.3 Variant interpretation ....................................................................................... 15 1.4 G6PC Family .................................................................................................. 16 1.5 Slc37a4 (G6PT) .............................................................................................. 19 1.6 Glycogen storage disease type 1 ...................................................................... 19 1.7 Glycogen storage disease type 1a ................................................................... 20 1.8 Significance ..................................................................................................... 22 1.9 Figures for Chapter 1 ...................................................................................... 27 1.10 Tables for Chapter 1 ................................................................................. 30 Chapter 2: Classifying molecular phenotypes of G6PC variants for pathogenic properties and to guide therapeutic development ........................................................ 31 2.1 Abstract ........................................................................................................... 31 2.2 Synopsis .......................................................................................................... 32 2.3 Introduction ..................................................................................................... 32 2.4 Materials and Methods .................................................................................... 35 2.5 Results ............................................................................................................. 39 4 2.6 Discussion ....................................................................................................... 42 2.7 Figures for Chapter 2 ...................................................................................... 45 2.8 Tables for Chapter 2 ....................................................................................... 50 Chapter 3: Investigating localization and movement of G6PC family members and G6PC1 variants ............................................................................................................... 51 3.1 Abstract ........................................................................................................... 51 3.2 Synopsis .......................................................................................................... 52 3.3 Introduction ..................................................................................................... 52 3.4 Materials and Methods .................................................................................... 54 3.5 Results ............................................................................................................. 56 3.6 Discussion ....................................................................................................... 59 3.7 Figures for Chapter 3 ...................................................................................... 63 Chapter 4: Discussion ..................................................................................................... 74 4.1 Figures for Chapter 4 ...................................................................................... 89 References ........................................................................................................................ 91 5 List of Tables Table 1-1 Associated diseases and phenotype overlay for G6PC family members and G6PT ................................................................................................................................. 30 Table 2-1 Summary of G6PC variant molecular phenotype results ................................. 50 Table 3-1 Plasmids used in Localization and Movement Analysis .................................. 55 6 List of Figures Figure 1-1. Functional relationship of G6PC and G6PT in G6P metabolism .................. .27 Figure 1-2. Tissue expression and cellular localization of G6PC family and G6PT ......... 28 Figure 1-3. Potential molecular consequences of DNA variants ....................................... 29 Figure 2-1 Selection of G6PC variants ............................................................................. 45 Figure 2-2 Protein levels are significantly reduced for a subset of G6PC variants ..........46 Figure 2-3 G6PC N-linked glycosylation is altered in several G6PC variants .................47 Figure 2-4 Several G6PC variants have altered protein localization................................ 48 Figure 2-5 C109Y, a Variant of Uncertain Significance, shows features consistent with pathogenicity .................................................................................................................... .49 Figure 3-1 Localization and movement of G6PC family members...................................63 Figure 3-2 Movement and colocalization of G6PC family members with G6PT ............64 Figure 3-3 Movement of G6PC1 variants ........................................................................ .67 Figure 3-4 Abnormal movement in G6PC1 variants ........................................................ 72 Figure 4-1. Protein levels of G6PC1 active site variants in HepG2 cells .......................... 89 Figure 4-2. Protein levels of G6PC1 missense variants in HepG2 cells ........................... 90 7 AcKnowledgements I thank the expertise and assistance from Yuriy Fedorov and equipment from the Small Molecule and Drug Development (SMDD) core facility. I would like to acknowledge Biorender for creation of figures and Snapgene for plasmid map generation. This work was supported by grants from the Children’s Fund for Glycogen Storage Disease, the Research Institute for Children’s Health, and NIH grant T32 GM007250. I thank my advisor, Dr. Mitchell Drumm, for welcoming me into his lab and giving me the opportunity to work on a project I was passionate about. For caring about rare diseases and finding new ways to investigate them and give the attention they deserve. I am thankful to my thesis committee for their insights and support along the way, and especially to Dr. Anna Mitchell for supporting me in my clinical interests and mentoring me in both the clinical and research worlds. I am grateful to all of my colleagues in the lab and program who helped support me through this journey sharing reagents, expertise, and kindness whenever needed. So many have helped me through hard times and celebrated my successes and the community of support helped me along the way. I want to thank all of the scientists and friends who graced my couch with their presence and shared science and support along the way. The cross-discipline discussions helped build the science into its final form. I especially thank Dr. Tristan de Jesús, Sarah Holden, and Dr. Zach Stanfield, for their many years of friendship, patience, and ideas, and Dr. Doug Oswald for being the best glycobiologist I have ever met. 8 I thank Anjali and the entire Prabhakaran family for sharing their experiences with me and teaching me the most valuable lessons about living with GSD1a. And to Anjali for being a stellar young scientist and helping shape this project inside and outside of the lab. I thank Katherine Halloran, Little Katie, for starting as my little shadow and ending as my colleague and friend. I am so grateful you stayed with me the whole way adding ideas, editing writing, and keeping the energy alive in the lab. I thank the rest of the summer dream team students- Henry Stitzel and Noah Taylor, for tirelessly sequencing, cloning, and putting mCherry where it belonged, and being the extra sets of hands and heads for everything