IDENTIFYING GENETIC VARIATION CONTRIBUTING TO KERATOCONUS by Sionne Edie Marguerite Lucas BMedSci, BMedRes (Hons) 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, December, 2018 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. Sionne Lucas 18/12/2018 i AUTHORITY OF ACCESS STATEMENT The publishers of the papers comprising Chapter 3 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non-published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. Sionne Lucas 18/12/2018 ii STATEMENT OF ETHICAL CONDUCT The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. Sionne Lucas 18/12/2018 iii STATEMENT OF CO-AUTHORSHIP Sionne E. M. Lucas has incorporated versions of two first-author papers, Lucas et al. (2017)1 and Lucas et al. (2018),2 into Chapter 3. The contribution of co-authors for each paper is presented below. Lucas SEM, Zhou T, Blackburn NB, et al. Rare, potentially pathogenic variants in ZNF469 are not enriched in keratoconus in a large Australian cohort of European descent. Invest Ophthalmol Vis Sci 2017;58(14):6248-56. The co-authors and each author’s contribution are outlined as follows: Lucas, S. E. M. was involved the study design, data generation and data curation, she conducted data analysis and laboratory experiments and wrote the manuscript and prepared the tables and figures; Zhou, T. was involved in method development; Blackburn, N. B. was involved in method development, conducted preliminary data analysis and was involved in manuscript revision; Mills, R. A. conducted clinical examinations and was involved in patient recruitment; Ellis, J. was involved in preliminary data analysis; Leo, P. was involved in preliminary data analysis; Souzeau, E. was involved in patient recruitment, data curation and manuscript revision; Ridge, B. was involved in patient recruitment and data curation; Charlesworth, J. C. was involved in study design and manuscript revision; Brown, M. A. was involved in recruitment and data curation; Lindsay, R. conducted clinical examinations and was involved in patient recruitment; Craig, J. E. conducted clinical examinations and was involved in patient recruitment; Burdon, K. P. was involved in study design, funding acquisition, methodology development and manuscript revision. Lucas SEM, Zhou T, Blackburn NB, et al. Rare, potentially pathogenic variants in 21 keratoconus candidate genes are not enriched in cases in a large Australian cohort of European descent. PLoS One 2018;13(6):e0199178. The co-authors and each author’s contribution are outlined as follows: Lucas, S. E. M. was involved in data generation, data curation, and study design, conducted data analysis and laboratory experiments for variant validation, and wrote the manuscript and prepared the tables and figures; Zhou, T. was involved in method development; Blackburn, N. B. was involved in method development, conducted preliminary data analysis and was involved in manuscript revision; Mills, R. A. conducted clinical examinations and was involved in patient recruitment; Ellis, J. was involved in preliminary data analysis; Leo, P. was involved in preliminary data analysis; Souzeau, E. iv was involved in patient recruitment, data curation and manuscript revision; Ridge, B. was involved in patient recruitment and data curation; Charlesworth, J. C. was involved in study design and manuscript revision; Lindsay, R. conducted clinical examinations and was involved in patient recruitment; Craig, J. E. conducted clinical examinations and was involved in patient recruitment; Burdon, K. P. was involved in study design, funding acquisition, methodology development and manuscript revision. Prof. Kathryn Burdon Prof. Alison Venn Supervisor Director Menzies Institute for Medical Research Menzies Institute for Medical Research University of Tasmania University of Tasmania 18/12/2018 08/05/2019 v ACKNOWLEDGEMENTS As anticipated, an almost four-year project generates a long list of ‘thank yous’. To the Australian Government for my Research Training Program Scholarship and the Pennicott Foundation for the financial support over the last 3 and a half years. To all of our collaborators, the clinicians and the participants, without which this study would not be possible. A particularly big thank you to both Emmanuelle Souzeau and Sandra Staffieri for being the in-betweens and answering my many questions about the patients’ and their clinical data. To Kathryn Burdon, my primary supervisor, for being the most incredible mentor, critic, advocate and friend. Thank you for inspiring me every single day. To Jac Charlesworth, my co-supervisor, for always pushing me to be better and do better. And for reminding me, at potentially the most critical moment, that the most amazing things are made from crushing pressure and the passage of time. To Pat and JoJo, for our many ‘coffees’, lunches and early morning chats - your support over the past four years has been immeasurable. You’ve made me laugh on even the hardest of days and helped celebrate some of the best. Thank you being my sounding boards, my teachers, my students and such good friends. Don’t forget about me now that I don’t get to sit with the cool kids. To Bennet for your bioinformatic support, especially your R expertise, and for keeping Sunnydale running smoothly. To the rest of our CompGen team and 502 office buddies – Ming, Duran, Elise, Kelsie, Alex, Van, Emma and Aparna – for your support over the years, both professionally and socially. To James Marthick, ‘the boss’, for all of your sequencing expertise and support. To Nick, for teaching me all the bioinformatics that you could in the first year of my project. I would have been lost without you and such a strong foundation. To Sian, for our sporadic and always lengthy phone calls filled with commiserations and encouragement. You’ve got this too. To Hayley, for being the best cheerleader and friend anyone could ask for. Always. To my parents, for your never-ending support, advice (while it was sometimes unsolicited, in retrospect it was usually pretty accurate), and encouragement. To Jenna, my wife (I still have to get used to that!), for everything. For putting up with the stress, the non-existent weekends, and most importantly, the hours of dishes you endured towards the end of my PhD. I’m so glad I’ve had you by my side every step of the way. vi ABSTRACT Keratoconus (OMIM 148300) is a complex disease characterised by progressive stromal thinning and conical protrusion of the cornea. These abnormalities usually develop in the second decade of life and can lead to severe visual impairment as a result of high myopia and irregular astigmatism. Due to the early onset of disease, keratoconus affects individuals during their most productive years and quality of life estimates demonstrate a significant decline over time that is disproportional to visual acuity. The global incidence of keratoconus is approximately 1 in 50,000 and the prevalence in Caucasians is reported between 55 and 265 per 100,000 individuals. While keratoconus is a complex disease, it has a strong genetic basis. Familial examples display both autosomal dominant and recessive inheritance patterns and first-degree relatives have at least a 15-fold increased risk of developing keratoconus, compared to the general population. Current treatment options for keratoconus are suboptimal, require invasive surgical procedures and have limited applicability and effectiveness in early-onset and advanced disease. Currently, keratoconus is the second leading indication for corneal transplants in Australia and there is a great need to better understand keratoconus aetiology and pathophysiology to aid early diagnosis and the development of novel treatments to improve patient outcomes. Therefore, the overarching aim of this project was to identify genetic variants involved in keratoconus susceptibility through the assessment of individuals and families in our keratoconus DNA repository. This aim was achieved through three distinct studies: a candidate gene screen; novel variant discovery in families with multiple cases of severe and early-onset keratoconus; and association analysis in a large case-control cohort. The gene-screen aimed to determine if rare, protein-coding variants predicted to be pathogenic were enriched in in keratoconus-candidate genes in our cohort of keratoconus cases of European descent compared to ethnically matched controls. By combining whole exome-sequencing and targeted gene sequencing, a total of 22 keratoconus candidate genes were assessed in 385 unrelated cases, making this the most comprehensive study of its kind to date. Two control cohorts were used for comparison, including up to 396 population controls (unassessed for eye disease) and 230 individuals without keratoconus. The candidate genes were selected from the literature and included genes near associated loci, genes harbouring putative disease-causing variants in family studies, and genes proposed to play a role in keratoconus based on a known function in the cornea or involvement in a postulated disease process. Specifically, this study examined the frequency of potentially pathogenic variants in MPDZ, RXRA, RAB3GAP1, FOXO1, BANP, ZNF469, HGF, COL5A1, IMMP2L, FNDC3B, NFIB, ILRN, SLC4A11, CAST, COL4A3, COL4A4, TF, SOD1, VSX1, RAD51, IL1A and IL1B.