Identifying Glaucoma Susceptibility Genes in Extended Families by Patricia Stacey Graham BSc(Hons), GradDipEd Menzies Institute for Medical Research | College of Health and Medicine Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (Medical Studies) University of Tasmania, March 2020 Declaration of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. 13/3/2020 i Authority of access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. 13/3/2020 ii Statement of ethical conduct The research associated with this thesis abides by the international and Australian codes on human experimentation and the guidelines and the rulings of the Ethics Committee of the University. Ethics Approval Numbers: University of Tasmania Human Research Ethics Committee H0014085 Oregon Health and Science University Institutional Review Board #1306 13/3/2020 iii Dedication To my parents Susie and Micky Lowry Who instilled in me the love of learning and who would have been so proud iv Acknowledgements What a rollercoaster the last four years have been …. it’s been an amazing ride. Rollercoasters are no fun alone, and I would sincerely like to thank the following people who kept the ride rolling and to those who sat with me in the carriage and didn’t let me fall out. To the Australian Government for my Research Training Program Scholarship and to the Menzies Institute for supporting me and providing the opportunity to get back into medical research again. To my supervisors Professor Kathryn Burdon and Dr Jac Charlesworth; the dynamic duo of supervisory teams. Kath, you are a “mentor extraordinaire”. Your intelligence, passion and kindness are the perfect combination. You’ve always had the confidence in me that sometimes I didn’t have in myself, and I truly appreciate that. Jac, thank you for trusting me with your POAG families and teaching me linkage. You are one of the most patient people I know and you have taught me so much. You’ve constantly challenged me, which has made me a better scientist. Thank you both for taking me on, teaching me so much and getting me to the end. To the participants of OGGS and GIST, without whom this study would not have been possible. To the researchers, clinicians and statisticians involved with this project and to those who kindly provided us with data: Professors John Blangero, Jamie Craig, Joanne Curran, Alex Hewitt, Stuart MacGregor, David Mackey, Mary Wirtz, and Dr Tiger Zhou. To Sionne and JoJo – my amigos. It’s been the best ride with you the past four years. You have both been super patient with my “old school genetics” and getting me up to speed. More importantly, I’ve loved our morning chats, procrasti-coffees, lunches in the roundabout, time spent at various sports and having the odd celebratory drink. Cheers! To the team of bioinformaticians who have taught me so much along the way. Juan, Nick and Bennet, I absolutely could not have done this project without you. An extra thank you to Juan for helping me troubleshoot my methods and get them up and running. To the CompGen team and my “office-kids” in 502 – Ming, Duran, Raj, Kelsie, Alex, Aparna, Emma and Van. You’ve kept me young! To my friends from outside Menzies. Although many of you never really understood why I would do this, you’ve been nothing but supportive. Special thanks to my coffee-buddy, Karen. Most importantly, to my family; Al, Jess and Nicki. I could not have done this without your support, encouragement and love. I promise – no more mid-life crises …. for a while at least. v Abstract Glaucoma encompasses a heterogenous group of eye diseases and is the leading cause of irreversible blindness worldwide. Death of retinal ganglion cells causes degeneration of the optic nerve with resultant loss in vision. Primary open-angle glaucoma (POAG) is the most common form of glaucoma. An increase in pressure inside the eye, the intraocular pressure (IOP), is the main risk factor for developing POAG. Pressure reducing medication and surgery are currently the only preventative measures, and means of slowing the progression of the disease, available to those at risk of POAG or already diagnosed with this disease. There is currently no cure for glaucoma. POAG is a highly heritable, complex disease. Genome-wide association studies (GWAS) have identified many common risk variants, each with small effect sizes, which may increase an individual’s susceptibility to developing this disease. However, these variants together only account for around 3% of the heritability of POAG. Rare variants have been proposed as a source of the missing heritability, and the most powerful way to enrich for rare variants is through family studies. Linkage studies have identified genes harbouring rare variants which cause the disease in around 10% of cases. It is proposed that other, unidentified rare variants with large effect sizes, may be important in POAG pathogenesis. The clinical intermediate traits of POAG that have been used for successful genetic studies include IOP, vertical cup to disc ratio (VCDR) and central corneal thickness (CCT). These highly heritable, quantitative traits are measurable in all individuals, regardless of their POAG disease status. IOP and VCDR are significantly genetically correlated with POAG disease status, making them ideal endophenotypes to use to identify variants and genes involved with POAG pathogenesis. CCT is not significantly genetically correlated with POAG, but is a well- recognised risk factor for the disease, with thinner corneas associated with an increased risk of developing POAG. Identifying genes important in corneal development still gives us the opportunity to better understand the risk of developing POAG. GWAS have been very successful in identifying variants associated with each of these clinical intermediate traits, however, only a small proportion of the overall heritability for each trait is accounted for by these variants. For this study, we hypothesised that rare genetic variants associated with heritable ocular clinical traits could be identified in extended pedigrees and would improve our understanding vi of the genetic susceptibility to POAG. The first aim of this study was to determine the contribution of published genetic loci associated with POAG and related traits, to the trait variance of the clinical phenotypes in five extended POAG enriched families. The next aim was to use linkage analysis to identify regions harbouring potential POAG related risk variants in these five families. The final aim was to use in silico tools to investigate genetic variants within the most significant peaks identified from the linkage analysis. This study utilised whole exome sequencing data from 249 members of five large POAG enriched families, with detailed clinical data including IOP, VCDR and CCT measurements, to perform family-based association and linkage analyses within a variance components framework. These five extended families provide the opportunity to identify rare variants involved in disease due to the potential for variants to be enriched by transmission from founder individuals to successive generations. To identify the previously reported POAG loci, Aim 1 of this study began with a comprehensive literature review. To determine whether these loci contributed to the variance of the clinical intermediate traits of the families in this study, a family-based association analysis was conducted on genetic variants within literature reported regions. Several variants within published linkage regions were significantly associated with each of the three traits; including a single rare variant within MST1 on chromosome 3 (p = 9.60x10-5) and a haplotype within FLRT3 on chromosome 20 (p = 1.17x10-4 to p = 3.18x10-4), which were associated with a large increase in IOP in the family members who carry them. FLRT3 lies in a previously mapped glaucoma linkage region, GLC1K, and is an ideal candidate gene for further study. To identify novel genes associated with POAG and its endophenotypes, Aim 2 focused on linkage analyses using whole exome sequencing data, which were conducted for the IOP and VCDR traits in the five families. These are both significantly heritable traits, with heritabilities calculated at 52% (p = 5.13x10-10) for IOP and 41% (p = 9.0x10-7) for VCDR in the families of this study. Identity by descent relationships between family members were calculated directly from the whole exome sequencing data using IBDLD, a program specifically designed for dense genotyping data. Variance components linkage analysis was conducted using SOLAR, including correction for both the ascertainment of the POAG enriched families used in this study, and non-normal trait distributions. Linkage peaks reaching significance were identified for IOP, but not VCDR. For IOP, peaks on chromosomes 9q34.3 and 15q11.2-13.2 reached full significance (maximum LOD = 4.25 and 3.40 respectively), and peaks on vii chromosomes 2q23.1-31.1, 3p13-12.1, 6q24.2-25.1 and 7p11.2-q11.23 reached suggestive significance (maximum LOD > 1.86). All but one of the peaks were novel, with the chromosome 15 peak completely overlapping the previously identified GLC1I locus. Aim 3 involved analysing the identified linkage peaks using in silico strategies. Variants within each region were prioritised based on their family-based association results, their effect on reducing the linkage peak when included in the linkage model, and predicted in silico functional effects.