This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Expanding the genetics of microcephalic primordial dwarfism Jennie Elaine Murray Thesis submitted for the degree of Doctor of Philosophy The University of Edinburgh October 2014 This thesis is composed of original research undertaken by myself, and where the work of others is included their contributions have been duly acknowledged. This work has not been submitted for any other degree or professional qualification. Jennie Murray 2 Acknowledgements Firstly, I would like to say a big thank you to my supervisor, Professor Andrew Jackson, for his fantastic guidance and support over the last 3 years. Your integrity and dedication has been inspiring and, although I still have a long way to go, your input has been invaluable in helping me grow and develop as a clinical scientist. I am also in a huge debt of gratitude to Louise Bicknell for her superb day to day supervision, teaching, mentoring, encouragement, general life coaching and eternal optimism. You always kept me going when things got tough. I am also incredibly grateful for the rapid proof reading of this thesis enabling me to achieve my rather tight deadline. I also want to thank Professors David Fitzpatrick and Mary Porteous for their additional supervision and career guidance which helped me to obtain a clinical fellowship and complete this thesis. Next, I would like to thank Alison Meynert for the huge bioinformatic support she provided and always promptly answering my endless stream of programming questions which made such a large part of this thesis possible; Mike Halachev also for all things bioinformatic and particularly for help with the CNV analysis. I would also like to thank the members of the IGMM computing services; especially Graeme Grimes for getting me started with SQL and John Ireland, Ewan McDowall and Semir Kazazic for always quickly coming to my rescue on the many occasions my minimal computer skills failed me. I am also indebted to Carol-Anne Martin for inducting me into the field of cell biology and imparting some of her immense knowledge base to me as well as patiently teaching me many of the experimental techniques used in this thesis including cell culture, immunoblotting, immunofluorescence and microscopy. A big thank you also to Morad Ansari for all his technical help with performing array- CGH, generous donation of reagents and slides as well as support with AmpliSeq™. Additionally I would like to thank all the other members of my lab, past and present for all their helpful input and discussions during lab meetings and general lab 3 support, in particular Anna Klingseissen for introducing me to the world of zebrafish, Maggie Harley for help with various techniques including cell culture and immunoblotting, Paula Carroll for being ever willing to help at all times, Martin Reijns and James Ding for help with cloning, Rachel Rigby for all things RNA related, Andrea Robertson for being such a lovely desk buddy, providing much needed light relief as well as providing invaluable help in the lab and Karen MacKenzie for lifts to work and regularly letting me vent my frustrations. I would also like to thank Anne Seawright for all her assistance in and around the lab including ensuring there was always milk and coffee in the office! I would also like to express my gratitude to Stephen Brown, Agnes Gallacher and Stewart McKay in technical services for all their support in anything sequencing related; Lizzie Freyer for assistance with flow cytometry; Andrew Wood for insightful discussions and generous donation of antibodies; Craig Nicol for teaching me all things graphical for which I believe this thesis has greatly benefited and Matt Pearson for additional microscopy support. A huge thank you also to all the patients and their families whose selfless contributions to this research in the hope of providing help to future generations has been incredibly humbling and has been a big motivation for me over the last three years. Additionally I’d also like to thank Eilidh Guild and Angela Sandilands for their administrative support, keeping me organised with patient data, samples and reports and also our clinical counterparts in the USA, Mike Bober and Angela Duker for their ongoing collaboration, providing access to patient samples and expert clinical opinion. Finally, I would like to thank my family and friends for their eternal support, always believing in me and always attempting to be interested in what I was doing. And last but certainly not least, my incredible husband Andrew, for all your love, support and encouragement. You have been my absolute rock throughout as you always are in everything I do and therefore this thesis is dedicated to you along with our new beautiful baby daughter, Nina. 4 Abstract Body mass varies considerably between different mammals and this variation is largely accounted for by a difference in total cell number rather than individual cell size. Insights into mechanisms regulating growth can therefore be gained by understanding what governs total cell number at any one point. In addition, control of cell proliferation and programmed cell death is fundamental to other areas of research such as cancer and stem cell research. Microcephalic Primordial Dwarfism (MPD) is a group of rare Mendelian human disorders in which there is an extreme global failure of growth with affected individuals often only reaching a height of around one metre in adulthood. To date, all identified disease genes follow an autosomal recessive mode of inheritance and encode key regulators of the cell cycle, where mutations impact on overall cell number and result in a substantially reduced body size. MPD therefore provides a valuable model for examining genetic and cellular mechanisms that impact on growth. The overall aims of this thesis were to identify novel disease causing genes, as well as provide further characterisation of known disease causing genes, through the analysis of whole exome sequencing (WES) within a large cohort of MPD patients. Following the design and implementation of an analytical bioinformatics pipeline, deleterious mutations were identified in multiple disease genes including LIG4 and XRCC4. Both genes encode components of the non-homologous end joining machinery, a DNA repair mechanism not previously implicated in MPD. Additionally, the pathogenicity of novel mutations in subunits of a protein complex involved in chromosome segregation was assessed using patient-derived cells. These findings demonstrate WES can be successfully implemented to identify known and novel disease causing genes within a large heterogeneous cohort of patients, expanding the phenotype of known disorders and improving diagnosis as well as providing novel insights into intrinsic cellular mechanisms critical to growth. 5 Table of Contents Acknowledgements………………………………………………………………….3 Abstract……………………………………………………………………….……...5 Table of Contents…………………………………………………………………....6 List of Figures…………………………………………………………………........13 List of Tables………………………………………………………………….........15 Abbreviations…………………………………………………………………........13 Chapter 1: Introduction…………………………………………………………...19 1.1 Overview of microcephalic primordial dwarfism (MPD) .......................... 19 1.1.1 Definition .................................................................................................. 19 1.1.2 Diagnostic criteria ..................................................................................... 20 1.1.3 Aetiology ................................................................................................... 21 1.1.4 Spectrum of phenotypes ............................................................................ 21 1.1.4.1 Seckel syndrome (MIM 210600) ....................................................... 21 1.1.4.2 Microcephalic Osteodysplastic Primordial Dwarfism Type I (MOPDI, MIM 210710) ................................................................................................. 23 1.1.4.3 Microcephalic Osteodysplastic Primordial Dwarfism Type II (MOPDII, MIM 210720) ............................................................................... 24 1.1.4.4 Meier-Gorlin Syndrome ..................................................................... 24 1.1.4.5 Other disorders which overlap MPD ................................................. 25 1.1.4.5.1 Syndromic conditions affecting global growth ........................... 25 1.1.4.5.2 Autosomal recessive primary microcephaly (1° MCPH) ........... 25 1.2 Mechanisms governing body size .................................................................. 26 1.2.1 Signalling pathways promoting cell growth and proliferation .................. 27 1.2.1.1 Mitogen activated