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Identification and Functional Analysis of Novel Genes Associated with Inherited Bone Marrow Failure Syndromes

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Identification and Functional Analysis of Novel Genes Associated with Inherited Bone Marrow Failure Syndromes Identification and Functional Analysis of Novel Genes Associated with Inherited Bone Marrow Failure Syndromes by Anna Matveev A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Anna Matveev 2020 Abstract Identification and Functional Analysis of Novel Genes Associated with Inherited Bone Marrow Failure Syndromes Anna Matveev Master of Science Institute of Medical Science University of Toronto 2020 Inherited bone marrow failure syndromes are multisystem-disorders that affect development of hematopoietic system. One of IBMFSs is Shwachman-Diamond-syndrome and about 80-90% of patients have mutations in the Shwachman-Bodian-Diamond-Syndrome gene. To unravel the genetic cause of the disease in the remaining 10-20% of patients, we performed WES as well as SNP-genotyping in families with SDS-phenotype and no mutations in SBDS. The results showed a region of homozygosity in chromosome 5p-arm DNAJC21 is in this region. Western blotting revealed reduced/null protein in patient. DNAJC21-homolog in yeast has been shown facilitating the release of the Arx1/Alb1 heterodimer from pre-60S.To investigate the cellular functions of DNAJC21 we knocked-down it in HEK293T-cells. We observed a high-level of ROS, which led to reduced cell proliferation. Our data indicate that mutations in DNAJC21 contribute to SDS. We hypothesize that DNAJC21 related ribosomal defects lead to increased levels of ROS therefore altering development and maturation of hematopoietic cells. ii Acknowledgments I would like to take this opportunity to extend my deepest gratitude to everyone who has helped me throughout my degree. First, I would like to thank my supervisor Dr. Yigal Dror for giving me the opportunity to study under his supervision, guiding me through the experimental design, supporting me through my degree, and without whom this project would not have been possible. I am grateful for all the time and attention that he always put into ensuring that I was successful every step of the way. I am also appreciative of my committee members, Dr. Herman Yeger and Dr. Sevan Hopyan for their precious discussion and constructive criticism. I would also like to thank and to give credit to the members of Dror lab, especially to our lab manager Hongbing Li who always has had time for me. He has a huge knowledge and always knows the answers to my questions, providing constructive criticism. I would like to thank our clinical research project manager Bozana Zlateska for acquiring patient samples with informed consent and for always providing us with all the knowledge she has. In addition, I want to express my gratitude to our former research associate Chetankumar Tailor for producing lentivirus, optimizing lentiviral transduction of hiPSCs, and assisting with the establishment of transduced hiPSCs. Also, I want to thank former M.Sc student Steph NG for teaching me and helping with hiPSCs and our former research fellow Stephanie Heidemann for assisting with PCR work. Moreover, I want to extend my deepest gratitude to PhD candidate Santhosh Dhanraj for his assistance in my experiments, for teaching me the bioinformatics tools required for my work, and especially for his constructive criticism. I would like to express my appreciation to Dr. Supanun Lauhasurayotin for her helpful contribution to my experiments and for providing her valuable expertise in medicine related aspects of the project. I would also like to thank M.Ss candidate Alejandra Lagos Monzon for always being there for me in hard times. Finally, I would iii like to thank our summer student Brian Bursic for helping me to finish experiments for a manuscript during my last summer in the lab. I would also like to express my appreciation to the facilities that helped us with our project: the Centre for Commercialization of Regenerative Medicine (CCRM) for creating reprogrammed hiPSC and their characterization. I would like to express my appreciation to the former Sick Kids Embryonic Stem Cell Facility for providing mouse embryonic fibroblasts (MEFs) for our experiments. I would like to thank The Centre for Applied Genomics (TCAG) for performing Sanger sequencing and for generating PCR primers. I would like to thank the Sick Kids- University Health Network Flow Cytometry Facility for fluorescence-activated cell sorting (FACS) and purity assessment. From a personal side, I would like to thank my husband for always being there and supporting me, for my parents that helped me a lot and gave me an opportunity to continue my studies. I express a deep and special gratitude to my daughter who is always there for me with a smile. Lastly and most importantly, I would like to thank all the families that participated in this study, and the funding agencies that supported my work, the Canadian Institute for Health Research and the Nicola’s Kids Triathlon. iv Statement of Contributions My supervisor Dr. Yigal Dror and my committee members Dr. Herman Yeger and Dr. Sevan Hopyan contributed to the experimental design, data interpretation, and thesis revision and approval. Hongbing Li, Santosh Dhanraj, Steph NG, and Dr. Chetankumar Tailor contributed to the experimental design and data interpretation. Bozana Zlateska and the Canadian Inherited Marrow Failure Registry acquired patient samples with informed consent. CCRM reprogrammed and characterized hiPSCs. TCAG performed the Sanger sequencing, whole genome sequencing, and PCR primer generation. The Sick Kids Embryonic Stem Cell Facility provided mouse embryonic fibroblasts. The Sick Kids-University Health Network Flow Cytometry Facility performed FACS and provided flow cytometers. I contributed to the experimental design; performed cell culture for different cell lines, hematopoietic differentiation, colony assays, RT-PCR, western blot, DNA/RNA/protein preparation, whole exome sequencing analysis and gene prioritization, lentiviral transduction, and FACS; acquired flow cytometry data; prepared DNA for Sanger sequencing; performed statistical analysis; interpreted data; and wrote the thesis. I designed and performed the experiments described in the thesis, unless indicated otherwise. This work was funded by Canadian Institute of Health Research. v Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments........................................................................................................................... ii Statement of Contributions ..............................................................................................................v Table of Contents ........................................................................................................................... vi List of Abbreviations .......................................................................................................................x List of Tables ............................................................................................................................... xiv List of Figures ................................................................................................................................xv Chapter 1 Introduction .....................................................................................................................1 1.1. Clinical characteristics of the inherited bone marrow failure syndromes................1 1.2. Genetic background of the inherited bone marrow failure syndromes ....................2 1.3. Shwachman-Diamond syndrome .............................................................................3 1.3.1. Hematological abnormalities .....................................................................5 1.3.2. Non-hematological abnormalities ...........................................................10 1.3.3. Treatment for SDS ...................................................................................17 1.3.4. Characterization of Shwachman-Bodian Diamond Syndrome gene SBDS. .......................................................................................................18 1.3.5. Ribosome Biogenesis and SBDS protein function ..................................20 1.3.6. Extra-ribosomal Consequences of SBDS Deficiency .............................22 1.3.7. SDS Patients without Biallelic SBDS Mutations ....................................27 1.3.8. Characterization of DnaJ Heat Shock Protein Family (Hsp40) Member C21 gene ...................................................................................28 Chapter 2 Research Aims and Hypothesis .....................................................................................31 2.1 Rationale. ...............................................................................................................31 2.2 Hypothesis..............................................................................................................31 2.3 AIM 1: To identify novel SDS-related genetic alterations and novel SDS genes through comprehensive Genome-wide genetic screen ................................32 vi 2.4 AIM 2: Studying the mechanism of how novel IBMFS-related gene DNAJC21 can cause Shwahman Diamond Syndrome ............................................................32 Chapter 3 Methods .........................................................................................................................33 3.1 Patients and Controls
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