Exploring the Genetics of Primary Open-angle Glaucoma with Next Generation Sequencing By Dr Tiger Zhou, BPhysio (Hons), BMBS Thesis Submitted to Flinders University for the degree of Doctor of Philosophy College of Medicine and Public Health Awarded 7th May 2021 Table of Contents Declaration and acknowledgments............................................................................................................ 6 Summary of thesis....................................................................................................................................... 8 Chapter 1: Introduction ............................................................................................................................ 10 1.1 Glaucoma ........................................................................................................................................ 10 1.1.1 Epidemiology and disease burden .......................................................................................... 11 1.1.2 Pathophysiology ...................................................................................................................... 13 1.1.3 Current therapies .................................................................................................................... 17 1.2 Endophenotypes and risk factors for primary open-angle glaucoma.......................................... 18 1.2.1 Intraocular pressure ................................................................................................................ 18 1.2.2 Optic disc parameters ............................................................................................................. 19 1.2.3 Central corneal thickness ........................................................................................................ 20 1.2.4 Myopia ..................................................................................................................................... 21 1.2.5 Systemic factors ....................................................................................................................... 23 1.2.6 Family history........................................................................................................................... 24 1.3 Genetic sequencing ........................................................................................................................ 25 1.3.1 Human genome and disease ................................................................................................... 25 1.3.2 First generation DNA sequencing ........................................................................................... 28 1.3.3 Next generation sequencing ................................................................................................... 28 1.3.4 Whole exome capture ............................................................................................................. 30 1.3.5 RNA sequencing ....................................................................................................................... 31 1.4 Gene discovery techniques in glaucoma ....................................................................................... 32 1.4.1 Linkage studies ........................................................................................................................ 32 1.4.2 Genome-wide association studies .......................................................................................... 34 1.5 Monogenic causes of glaucoma ..................................................................................................... 37 1.5.1 Glaucoma locus GLC1A – Myocilin ......................................................................................... 37 1.5.2 Glaucoma locus GLC1C – IL20RB ............................................................................................ 43 1.5.3 Glaucoma locus GLC1E – Optineurin ...................................................................................... 44 1.5.4 Glaucoma locus GLC1F – ASB10.............................................................................................. 48 1.5.5 Glaucoma locus GLC1G – WDR36 ........................................................................................... 49 1.5.6 Glaucoma locus GLC1O – NTF4 ............................................................................................... 51 1.5.7 Glaucoma locus GLC1P – TBK1 ............................................................................................... 52 1.5.8 Glaucoma locus GLC3A – CYP1B1 ........................................................................................... 53 1.5.9 Glaucoma locus GLC3D – LTBP2 ............................................................................................. 55 1.6 Rationale and hypotheses for research ......................................................................................... 56 1.6.1 Aims .......................................................................................................................................... 58 Chapter 2: Whole exome sequencing case-control study for rare potentially disease-causing monogenic variants in primary open-angle glaucoma ........................................................................... 59 2.1 Ethics ............................................................................................................................................... 59 2.2 Participant recruitment .................................................................................................................. 59 2.3 Selection of participants for whole exome sequencing ............................................................... 60 2.4 Whole exome sequencing .............................................................................................................. 61 2.5 Post sequencing bioinformatics analysis ....................................................................................... 62 2.5.1 Variant annotation .................................................................................................................. 62 2.5.2 Variant filtering ........................................................................................................................ 64 2.5.3 Counting minor alleles ............................................................................................................ 67 2.5.4 SKAT analysis............................................................................................................................ 73 2.6 Results ............................................................................................................................................. 75 2.6.1 Functional analysis of the top candidate - Neuroglobin ....................................................... 80 2.6.2 Generation of wildtype and mutant constructs .................................................................... 81 2.6.3 Reverse transcription .............................................................................................................. 81 2.6.4 Polymerase chain reaction for amplification of full length NGB .......................................... 82 2.6.5 Site directed mutagenesis ....................................................................................................... 83 2.6.6 Recombinant DNA cloning ...................................................................................................... 86 2.6.7 Differentiation of experimental human neurons .................................................................. 89 2.6.8 Transfection of NGB overexpressing plasmids ...................................................................... 94 2.7 Discussion ........................................................................................................................................ 99 Chapter 3: Contributions of known monogenic glaucoma genes to primary open-angle glaucoma. 102 3.1 Methods ........................................................................................................................................ 104 3.1.1 Participants ............................................................................................................................ 104 3.1.2 Data analysis .......................................................................................................................... 105 3.2 Results ........................................................................................................................................... 106 3.3 Discussion ...................................................................................................................................... 111 Chapter 4: Rare variants in known genome wide association genes ................................................... 119 4.1 Methods ........................................................................................................................................ 120 4.1.1 Participants ............................................................................................................................ 120 4.1.2 Data acquisition and analysis ................................................................................................ 121 4.1.3 Validation of variants ............................................................................................................ 121 4.2 Results ..........................................................................................................................................