Faculty of Medicine and Health Sciences (University of Antwerp) Molecular genetic dissection of disease in the era of next-generation sequencing Thesis submitted in order to obtain the degree of Doctor in Medical Sciences from the University of Antwerp Nikhita. Ajit Bolar Promotors: Prof. Dr. Bart Loeys Prof. Dr. Lut Van Laer Antwerp, 2017 MOLECULAR GENETIC DISSECTION OF DISEASE IN THE ERA OF NEXT-GENERATION SEQUENCING | Table of Contents Abbreviations ................................................................................................................................................................ 5 Abbreviations............................................................................................................................................................ 6 Summary ....................................................................................................................................................................... 9 Summary ................................................................................................................................................................ 10 Chapter 1 Introduction Molecular genetic dissection of disease in the era of next-generation sequencing ...................................................................................................................................................................................... 12 Introduction ........................................................................................................................................................... 13 Identifying inheritance patterns ......................................................................................................................... 13 Strategies for Disease gene identification ....................................................................................................... 18 Applications of NGS technology ......................................................................................................................... 28 Whole Exome Sequencing (WES) ....................................................................................................................... 28 Filtering Strategies ............................................................................................................................................... 34 Combinatorial gene identification strategies in conjunction with WES ....................................................... 37 Aims of this research ........................................................................................................................................... 41 Chapter 2 GFI1B mutations and the Gray Platelet Syndrome ........................................................................... 42 The hematopoietic system .................................................................................................................................. 43 Megakaryocyte development and Platelet formation .................................................................................... 46 Grey Platelet syndrome: Inheritance and molecular genetics ...................................................................... 49 Role of GFI1B in Megakaryocyte- Platelet development ................................................................................ 50 A Dominant–Negative GFI1B Mutation in the Gray Platelet Syndrome ....................................................... 53 Abstract .................................................................................................................................................................. 54 Introduction ........................................................................................................................................................... 54 Methods ................................................................................................................................................................. 55 Results .................................................................................................................................................................... 55 Discussion .............................................................................................................................................................. 61 Acknowledgements .............................................................................................................................................. 62 Supplementary Appendix .................................................................................................................................... 62 Supplementary Material ...................................................................................................................................... 63 Supplementary Figures ........................................................................................................................................ 66 Supplementary Tables ......................................................................................................................................... 74 Chapter 3 Autosomal Dominant Tubulo-Interstitial Kidney Disease and SEC61A1 ....................................... 76 Chronic kidney disease- An Introduction........................................................................................................... 77 Tubulo-interstitial kidney disease ...................................................................................................................... 77 Genetics of Autosomal Dominant tubulo-interstitial kidney disease ........................................................... 79 SEC61A1 - A novel gene for ADTKD ................................................................................................................... 82 Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia ................................................................................................. 84 Abstract .................................................................................................................................................................. 85 Introduction ........................................................................................................................................................... 85 2 Methods ................................................................................................................................................................. 86 Results .................................................................................................................................................................... 89 Discussion .............................................................................................................................................................. 98 Acknowledgements ............................................................................................................................................ 100 Conflict of Interest Statement .......................................................................................................................... 100 Web References .................................................................................................................................................. 100 Supplementary Appendix .................................................................................................................................. 101 Supplementary Figures ...................................................................................................................................... 102 Supplementary Tables ....................................................................................................................................... 107 Chapter 4 Mitochondrial function and OXPHOS Deficiency ............................................................................. 108 The Mitochondria and the OXPHOS system ................................................................................................... 109 OXPHOS deficiency – the genetics .................................................................................................................. 111 Iron Sulphur Biogenesis and Assembly ........................................................................................................... 118 S-adenosyl methionine transport and mitochondrial function .................................................................... 122 Chapter 4.1 .......................................................................................................................................................... 124 Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy ............................................................................................................................................................................... 124 Abstract ................................................................................................................................................................ 125 Introduction ......................................................................................................................................................... 125 Methods ..............................................................................................................................................................