RARE GERMLINE VARIANT CONTRIBUTIONS TO MYELOID MALIGNANCY SUSCEPTIBILITY by SAMUEL TZE KIN LI Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy Dissertation Advisor: Thomas LaFramboise, PhD Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Samuel Tze Kin Li Candidate for the degree of Doctor of Philosophy* Committee chair David Buchner Committee Member Thomas LaFramboise Committee Member David Wald Committee Member Sudha Iyengar Committee Member Dana Crawford Date of Defense May, 2020 2 *We also certify that written approval has been obtained for any proprietary material contained therein. Acknowledgements To start off, I would like to thank my advisor Thomas LaFramboise, or Tom. I still remember the day that I received his email 6 years ago saying that I could join his lab, which also marked the starting day of my dissertation. At that time, I knew nothing about programming, not to mention computational biology. Tom tried to teach me as much as he could and now I feel confident about computational biology. I am lucky enough that Tom always stands by me to provide all the assistance that I need, both in science and life. I want to thank my lab mates for their help and support, especially Meetha, Sneha, Janet and Jianghong. They always gave me help when I most needed. I want to thank my committee members, Dr. David Buchner, Dr. David Wald, and Dr. Sudha Iyengar for their help and input over the years, and for helping to guide this thesis to where it is today I want to thank my family, my mother, father, brother, sister-in-law, and of course my two smart and cute nephews, who always bring me a lot of joy. Their support is the key reason that I can finish the whole Ph.D. program 3 Lastly, I would like to acknowledge my friends in the department for providing encouragement and help with project ideas, which include Stevephen Hung, Chen Weng, Shiyi Yin, Shanshan Zhang, Dean Pontiue, Justine Ngo and Ian Bayles. Contents Acknowledgements .............................................................................................. 3 List of Figures: ...................................................................................................... 6 List of Tables: ....................................................................................................... 7 Abstract ................................................................................................................ 8 Chapter 1. Background and Significance ........................................................... 10 Chapter 1-1: Production of blood cells – Hematopoiesis .................................................. 10 Chapter 1-2: What if things go wrong in hematopoiesis? ................................................. 12 Chapter 1-3: Oncogenes ........................................................................................................ 12 Chapter 1-4: Tumor suppressor genes ................................................................................ 13 Chapter 1-5: The two-hit model applies to leukaemogenesis .......................................... 15 Chapter 1-6: The World Health Organization (WHO) classification of AML ................. 16 Chapter 1-7: Somatic mutations in myeloid malignancies ................................................ 17 Chapter 1-8: Myeloid Malignancies: Knowns and Unknowns .......................................... 19 Chapter 1-9: The importance of rare variants ..................................................................... 22 Chapter 1-10: Difficulties in conducting GWAS in myeloid malignancies ...................... 23 Chapter 1-11: Rationale for this study ................................................................................. 24 Chapter 2: Patient collections, variant calling and description of variants .......... 27 Chapter 2-1: Germline whole exome sequencing and ancillary data collection ............ 27 Chapter 2-2: Raw read processing, variant calling, and quality filtering ......................... 71 Chapter 2-3: Variant annotation and variant/gene/patient filtering .................................. 73 Chapter 2-4: Filtering putative compound heterozygotes and rare homozygotes ...... 103 Chapter 2-5: Statistical testing ............................................................................................ 104 Chapter 3: Results ............................................................................................ 105 4 Chapter 3-1: Rare variants in genes with known roles in germline myeloid malignancy susceptibility ........................................................................................................................... 105 Introduction ......................................................................................................................... 105 Result .................................................................................................................................. 106 Discussion .......................................................................................................................... 108 Chapter 3-2: Pathogenic rare variant burden and MPO as a candidate predisposition gene ......................................................................................................................................... 109 Introduction ......................................................................................................................... 109 Result .................................................................................................................................. 110 Discussion .......................................................................................................................... 113 Chapter 3-3: Exploring the overall germline rare variant burden and investigating two- hit hypothesis among germline rare variants and with somatic mutations. .................. 114 Introduction ......................................................................................................................... 114 Result .................................................................................................................................. 115 Discussion .......................................................................................................................... 123 Chapter 4: Discussion & Future Directions ....................................................... 124 The first population-based germline rare variants study of myeloid malignancy......... 124 Whole genome sequencing instead of whole exome sequencing ................................. 125 MPO as a novel myeloid malignancy predisposition gene ............................................. 126 The importance of FA genes in myeloid malignancies .................................................... 126 Crosstalk between germline rare variants and somatic mutations in the same patient ................................................................................................................................................. 127 Improvement in sample size ................................................................................................ 128 Limitations of our study ........................................................................................................ 129 Conclusion .............................................................................................................................. 129 Bibliography ...................................................................................................... 131 5 List of Figures: Figure 1: Hematopoietic stem cells differentiation pathway ................................................. 10 Figure 2: Description of pipeline to call, annotate, and filter variants from fastq files. ..... 72 Figure 3: Filtering suspected somatic contamination ............................................................ 73 Figure 4: Overview of patient and variant characteristics. .................................................. 102 Figure 5: Germline mutations in genes implicated in myeloid neoplasms with germline predisposition. ............................................................................................................................ 107 Figure 6: Myeloperoxidase (MPO) as a candidate susceptibility gene in myeloid malignancy. ................................................................................................................................ 112 Figure 7: Autosomal recessive genes and Fanconi anemia genes have higher overall and truncating rare germline variant burden. ........................................................................ 116 Figure 8: Genes with rare variants in both copies in the same patient. ............................ 119 Figure 9: Global and patient-specific concordance of rare germline variants and somatic mutations in each gene. ........................................................................................................... 121 6 List of Tables: Table 1: Tumor suppressor genes list ...................................................................................... 14 Table 2: Common cytogenetic abnormalities in AML ............................................................ 16 Table 3: Genes which are recurrently somatically mutated in myeloid malignancies grouped by their functional