Monogenic Disorders of Glucose-Stimulated Insulin Secretion: Massively Parallel Sequencing Approaches Stephanie R. Johnson FRACP, MBBS, BSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Faculty of Medicine i Abstract Maturity-onset diabetes of the young (MODY) and congenital hyperinsulinism (CHI) are monogenic disorders of glucose-stimulated insulin secretion. MODY is the commonest form of monogenic diabetes and results from a heterozygous mutation in one of thirteen identified genes. It is estimated that up to 90% of cases of MODY are not diagnosed, possibly due to the insufficient awareness of or access to genetic screening. CHI is the commonest cause of persistent hypoglycemia in infancy and childhood, for which there are ten identified genes (mutation-positive disease found in 45% of all CHI, and 88% of diazoxide-unresponsive cases). Genetic confirmation of MODY and CHI traditionally involved Sanger sequencing of each exon of each gene individually; however, the development of massively parallel sequencing (MPS) allows simultaneous sequencing of multiple target regions efficiently and cost-effectively. This thesis assessed two forms of MPS - whole exome sequencing and targeted sequencing, in population and small family cohorts. An entire paediatric diabetes clinic underwent targeted MPS for MODY genes, which showed the prevalence of pathogenic/likely pathogenic MODY variants to be 2.1%. Based on these results, a cost-effectiveness analysis was performed, demonstrating that routine MPS screening for MODY in all paediatric cases with presumed type 1 diabetes was cost-saving. The utility of WES was evaluated in MODY and CHI in a proof-of-concept study. Subjects with known mutations in MODY and CHI genes were re-sequenced using two “off-the-shelf” WES capture technologies. All mutations were confirmed; however, the study showed the importance of careful selection of appropriate capture technology to ensure targeting of relevant genes and the use of bioinformatics tools appropriate to mutation type. Seven probands diagnosed with MODY but without a previously identified mutation were also assessed using WES. Three novel variants in known MODY genes were identified by WES, which variants segregated with diabetes in the families. A potential novel MODY gene was also identified through sequencing of one extended family. ii Six probands with CHI but without a known mutation were also sequenced. Two probands and their affected family members had the same variant in a potential novel CHI gene. MPS is an extremely useful addition to the diagnostic workup for paediatric cases of diabetes overall, in addition to its utility in screening clinically diagnosed MODY/CHI cases. Additionally, MPS can lead to novel gene identification for these conditions. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Conference presentations Stephanie R. Johnson, Elizabeth Davis, Louise S. Conwell, Mark Harris, Lisa Anderson, Paul Leo, Matthew Brown, Emma L. Duncan (2016) “Massively parallel sequencing to determine the prevalence of maturity onset diabetes of the young in an entire paediatric diabetes clinic, including antibody positive type 1 diabetes” . Australasian Paediatric Endocrine Group annual scientific meeting, Alice Springs, (oral presentation in Young Investigators) S.R. Johnson , E.A. Davis, L.S. Conwell, M. Harris, E.L. Duncan. Prevalence of maturity onset diabetes of the young in a Western Australian paediatric diabetes clinic using targeted massively parallel sequencing. [Abstract O2] Pediatric Diabetes 2015;16(Suppl 21):24-49. [Awarded APEG Young Investigator Award] Stephanie R. Johnson , Louise S Conwell , Mark Harris , Emma L. Duncan. Next generation Sequencing for Maturity Onset Diabetes of the Young. [Abstract P105] Pediatric Diabetes 2014;15(Suppl.19)1-137 Stephanie R Johnson , Ivan N. McGown, Louise Conwell, Mark Harris, Emma L. Duncan. Next Generation Sequencing (NGS) to identify a novel INS gene mutation in a family with Maturity Onset Diabetes of the Young (MODY). [Abstract P2-d1-857] Hormone Research 2013;80(Suppl 1):267 Stephanie R Johnson , Ivan McGown, Mark Williams, Joyce Wu, Mark Harris, Emma Duncan, David Cowley. (2012) “Genetic Testing in Patients with a Clinical Diagnosis of Maturity-Onset Diabetes of the Young (MODY) – The Australian Experience” Monogenic Disorders of Insulin Secretion: Congenital Hyperinsulinism and Neonatal Diabetes, Philadelphia v Publications included in this thesis No publications included. vi Contributions by others to the thesis Chapter 3: Professor Emma Duncan supervised the study design, along with Assoc. Professor Louise Conwell, Dr Mark Harris, Professor Matthew Brown and Assoc. Professor Elizabeth Davis. Ms Janelle MacFarlane, Assoc. Professor Elizabeth Davis and Ms Nirubasini Paramalingam assisted with ethics submission. Dr Uma Ganti, Dr Jacqueline Curran, Professor Timothy Jones and Assoc. Professor Elizabeth Davis assisted with sample acquisition. Ms Lisa Anderson, Ms Xiaoxia Song and Ms Jessica Harris performed library preparation for the massively parallel sequencing and Ms Jessica Harris performed Sanger sequencing. Dr Paul Leo and Dr Jonathan Ellis performed the bioinformatics analysis. Dr McInerney-Leo and Professor Emma Duncan assisted in analysis of the massively parallel sequencing data. Chapter 4 : Dr Hannah Carter, Professor Nicholas Graves, Dr Samantha Hollingworth, Professor Matthew Brown and Professor Emma Duncan had a role in study design. Dr Hannah Carter and Dr Samantha Hollignworth provided data from the literature for the model. Dr Hannah Carter created the Markov Model and Dr Hannah Carter and Dr Samantha Hollingworth ran the model supervised by Professor Nicholas Graves. Professor Timothy Jones and Assoc. Professor Elizabeth Davis provided additional data from the WACDD database. Chapter 5 : Mr Ivan McGown performed the massively parallel sequencing and analysis. Professor Udo Oppermann provided the in-silico modelling. Chapter 6 : Professor Emma Duncan assisted with study design. Ms Janelle MacFarlane, Dr Aideen McInerney-Leo and Professor Emma Duncan assisted with ethics submission. Mr Ivan McGown assisted with sample acquisition. Ms Xiaixia Song and Ms Lisa Anderson performed the whole exome sequencing. Dr Paul Leo, Ms Mhairi performed the bioinformatics analysis. Dr Felicity Newell analysed the data to detect deletions. Ms Mhairi Marshall also provided the R script to calculate target and capture regions vii Chapter 7 : Professor Emma Duncan assisted with study design. Ms Janelle MacFarlane and Professor Emma Duncan assisted with ethics submission. Mr Ivan McGown assisted with sample acquisition. Ms Xiaixia Song and Ms Lisa Anderson performed the whole exome sequencing. Dr Paul Leo, Ms Mhairi performed the bioinformatics analysis. Dr Felicity Newell analysed the data to detect deletions. Ms Jessica Harris assisted with Sanger sequencing Chapter 8 : Professor Emma Duncan assisted with study design. Ms Janelle MacFarlane and Professor Emma Duncan assisted with ethics submission. Mr Ivan McGown assisted with sample acquisition. Ms Xiaoxia Song and Ms Lisa Anderson performed the whole exome sequencing. Dr Paul Leo, Ms Mhairi performed the bioinformatics analysis. Ms Jessica Harris assisted with Sanger sequencing Professor Emma Duncan provided the majority of the assistance with conceptualising the studies and with study design, data analysis and performed the majority of the paper editing. Professor Matthew Brown assisted with study design as provided all the laboratory equipment and laboratory space as well as staff to assist me and teach me. viii Statement of parts of the thesis submitted to qualify for the award of another degree None ix Acknowledgements I would firstly like to thank my wonderful supervisors, Emma, Mark and Louise, who had the belief in me and in this project from the start. You allowed me enough free rein to re-design the thesis as new opportunities arose,