Disorders of sex development: Genetic analysis and development of a novel in vitro cell model Ingrid May Knarston B.Sc. (Hons.) Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2018 Department of Paediatrics The University of Melbourne 2 Abstract Disorders/Differences of Sex Development (DSDs) are conditions where the chromosomal, anatomical or gonadal sex is atypical. DSDs are caused by a breakdown in the molecular pathways controlling development of the reproductive organs, such as ovarian/testicular differentiation. These conditions can carry a number of clinical complications such as an increased risk of gonadal cancer, infertility and psychosocial consequences. Importantly, the underlying genetic cause is still unknown in 60% of DSD patients, meaning clinical care is severely compromised. In the first part of this thesis, I studied a cohort of 34 patients with 46,XX (ovo)testicular DSDs. In these individuals, the testicular differentiation pathway is activated in 46,XX genetic females, resulting in the formation of testes or ovotestes. The cohort was studied using massively parallel sequencing and PCR-based approaches. This identified diagnostic findings in nine patients in two known DSD genes (NR5A1 and SOX9), as well as variants in candidate DSD genes (EMX2, FOXL2, LGR5, RXFP2 and WNT9A). In vitro analysis of the NR5A1 variants showed how these variants repress ovarian signalling pathways and factors, sufficient to switch ovarian to testicular development. In vitro and in vivo analyses of three of the candidate genes (EMX2, LGR5 and RXFP2) indicated that they are likely benign variants that don’t contribute to the phenotype. Ongoing studies of two further candidate genes (FOXL2 and WNT9A) will establish their potential role in these DSD phenotypes. In the second part of the thesis, I aimed to develop an improved in vitro model for functionally analysing DSD gene variants. Several recent studies have differentiated human induced pluripotent stem cells (iPSCs) into many different tissues, which can be used as human- and tissue-specific disease models. I developed a protocol to differentiate human iPSCs into testis- like lineages. In this step-wise protocol, cells are directed through the developmental stages that give rise to the embryonic testis. Gene expression profiling has shown that at day 10-12 of iPSC differentiation, cells reach a bipotential gonad-like stage and by day 15 testis-like lineages are induced. This protocol will continue to be optimised, yet already I have shown its promising utility to study novel DSD genes. In summary, genetic analysis of a DSD cohort revealed diagnoses for a number of patients; these findings will likely improve their clinical management. It has also provided information on the most suitable genetic testing approach for 46,XX (ovo)testicular DSDs, a phenotypic group traditionally challenging to diagnose. Further, I showed functional insights into the molecular pathogenesis underlying NR5A1-mediated 46,XX (ovo)testicular DSD. Finally, development of a stem cell-based model of the human testis will help us to establish how novel DSD genes and variants affect human gonad development. ii Declaration This is to certify that: i. This thesis comprises only my original work towards the PhD except where indicated in the Preface, ii. Due acknowledgement has been made in the text to all other material used, iii. This thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices Ingrid Knarston: iii Preface Work carried out in collaboration with others as part of this thesis: Chapter 2 DNA and clinical information from all patients used in the studies presented here were collected from national and international collaborating clinicians. MLPA reactions and data analysis was performed in collaboration with Dr. Thomas Ohnesorg (Reproductive Development group, MCRI). Follow up CGH arrays were performed and analysed by Jocelyn van den Bergen (Reproductive Development group, MCRI). Preparation of DNA libraries for MPS was performed by Gorjana Robevska and Jocelyn van den Bergen (Reproductive Development group, MCRI) or at sequencing facilities (Australian Genomics Research Facility and Garvan Institute). The bioinformatic analysis of MPS data was undertaken in collaboration with Dr. Simon Sadedin (MCRI), Katrina Bell (MCRI) and Ben Lundie (Garvan Institute). The in vitro assay testing mutant RXFP2 function was performed by Prof. Ross Bathgate’s group (Florey Institute). The Emx2 and Wnt9a CRISPR mutant mice were generated by Dr. Liang Zhao (Institute of Molecular Bioscience, The University of Queensland) and characterised by Dr. Ella Thomson (Institute of Molecular Bioscience, The University of Queensland) and Dr. Anthony Bird (Hudson Institute for Medical Research, Monash University) respectively. Chapter 3 The in silico modelling of NR5A1 variant proteins and immunofluorescence staining for NR5A1 protein in COS-7 cells was performed by Gorjana Robevska (Reproductive Development group, MCRI). I conducted all other work, comprising 90% of the thesis. iv Publications Ingrid Knarston*, Katie L. Ayers*, Andrew H. Sinclair. Molecular mechanisms associated with 46, XX disorders of sex development. Clinical Science 2016 March 01; 130 (6): 421-432. Corresponding chapter in the thesis: Chapter 1 Contribution to manuscript: I was involved in all aspects of this manuscript. I conceptualised and designed the investigation with KA, conducted the literature analysis and co-wrote the paper with KA. Brittany Croft*, Thomas Ohnesorg*, Josephine Bowles, Katie Ayers, Jacky Hewitt, Jacqueline Tan, Vincent Corbin, Emanuele Pelosi, Jocelyn van den Bergen, Alexander Quinn, Rajini Sreenivisan, Ingrid Knarston, Gorjana Robevska, Dung Vu Chi, John Hutson, Vincent Harley, Peter Koopman and Andrew Sinclair. Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9. Nature Communications 2018 Dec 14; 9 (1): 5319. Corresponding chapter in the thesis: Chapter 2 Contribution to manuscript: I was involved in the identification of duplications in the upstream region of SOX9 in two 46,XX DSD patients. Ingrid M. Knarston*, Gorjana Robevska*, Jocelyn A. van den Bergen, Stefanie Eggers, Brittany Croft, Jason Yates, Remko Hersmus, Leendert H.J. Looijenga, Fergus J. Cameron, Klaus Monhike, Katie L. Ayers* and Andrew H. Sinclair*. NR5A1 gene variants repress the ovarian-specific WNT signalling pathway in 46,XX Disorders of Sex Development patients. Human Mutation 2019 Feb; 40 (2): 207-216. Corresponding chapter in thesis: Chapter 3 Contribution to manuscript: I was involved in all aspects of this manuscript. I conceptualised and designed the study with GR and KLA. Experimental work was performed in collaboration with GR and I and co-wrote the paper with GR and KLA. v Acknowledgements I would like to thank each of my supervisors for their support, Dr. Katie Ayers, Prof. Andrew Sinclair, Prof. Melissa Little, Dr. Alex Combes and Dr. Stefanie Eggers. My principal supervisors Katie Ayers and Andrew Sinclair have shaped a PhD project that has been incredibly exciting and challenging. Katie has been so generous with her time in developing my skills as a researcher and providing a constant source of advice and guidance. I am also very grateful for her encouragement to reach outside my comfort zone in so many instances. I am incredibly grateful for the opportunities and guidance that Andrew has provided me, particularly in shaping a PhD project that allowed me to gain experience in both the genomics and iPSC fields. I would like to thank both Melissa Little and Alex Combes for their support in my iPSC project; our discussions have taught me so much about developmental biology and their questions have been really valuable in directing this work. Finally, I would like to thank Stefanie Eggers for sharing her knowledge on DSD and genetic analyses. I would like to thank the entire Reproductive Development group for being such a welcoming and supportive group, Prof. Andrew Sinclair, Katie Griffin, Dr. Katie Ayers, Jocelyn van den Bergen, Gorjana Robevska, Dr. Elena Tucker, Dr. Rajini Sreenivasan, Brittany Croft, Dr. Aurore Bouty, Dr. Thomas Ohnesorg and Chloe Hanna. It has been a privilege to work alongside and learn from such a talented team of people. In particular I would like to thank Gorjana Robevska for helping me with so many aspects of this project and teaching me so many skills, as well as for her constant support and friendship over the years. I would also like to thank Jocelyn van den Bergen for being a constant source of helpful advice and such a big support. I would also like to thank the Kidney group at MCRI for allowing me to learn about iPSC and organoid modelling alongside them. I am very grateful to Irene Ghobrial and Pei Xuan Er for sharing so much of their knowledge and time training me in iPSC and organoid culture. I would also like to thank Dr. Santhosh Kumar for his valuable advice on organoid culture and Dr. Minoru Takasato for his guidance in the early stages of this project. I would like to thank my advisory committee, including Assoc. Prof. Shireen Lamande and Dr. Tiong Tan, for their input on the development of my project. Also at MCRI, I would like to thank vi the Animal house staff for their help with my mouse work and Matt Burton for sharing his knowledge on confocal imaging. Outside of MCRI, I would like to thank all members of the Australian DSD genetics program for the annual retreats that provided a fantastic forum to share ideas and get feedback on my project. Particularly I would like to thank Dr. Liang Zhao, Dr. Ella Thomson and Dr. Anthony Bird for their work on the mutant mouse models. I would also like to thank our collaborators Prof. Ross Bathgate (Florey Institute), Dr. Simon Sadedin (MCRI), Katrina Bell (MCRI) and Ben Lundie (Garvan Institute), for each of their contributions to this work. I would like to thank all of the patients and families who have been involved in this study, as well as all of the collaborating clinicians.