Exome Sequencing and Human Disease The molecular characterisation of genetic disorders by Diana Maria Walsh A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Biomedical Research School of Clinical and Experimental Medicine College of Medical and Dental Sciences University of Birmingham September 2015 0 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Since the completion of the human genome project in 2001, the field of genomics has advanced exponentially, largely in part to the introduction of next generation sequencing (NGS); a technique that has revolutionised the ways in which genetic disease is investigated. NGS enables the simultaneous sequencing of multiple reads in parallel, which provides researchers with the opportunity to interrogate vast numbers of candidate genes in order to establish the genetic eitiology and key components of disease. Exome sequencing in particular offers an efficient method to investigate disease, as the exomic regions make up 1% of the whole genome, but can contain up to 85% of functional variants responsible for disease. Next generation sequencing has been employed to investigate and identify the genetic cause of Acrocallosal syndrome (a rare autosomal recessive disorder). Exome sequencing was then also applied to investigate the genetic associations with both familial and sporadic pheochromocytomas and paragangliomas (neuroendocrine tumours). This study describes s the various applications, challenges and potential benefits that can be achieved by using exome sequencing as a tool to investigate rare autosomal recessive disorders in addition to more complex disorders including familial and sporadic cancer. This study aims to employ cutting edge technology to investigate human disease, in order to enhance current understandings of disease biology and pathogenesis. Through this, it is hoped that these findings may help to contribute to on-going efforts to develop novel therapeutic strategies and improve the clinical management of these disorders. 1 Acknowledgements I would like to offer a sincere thank you to everyone from the molecular labs that have helped me over the years, especially Dean, Dewi and Malgosia for all of their scientific advice. I would also like to say thank you to my supervisor, Eamonn Maher for all of his guidance and for providing me with the amazing opportunity to carry out work in an exciting, cutting edge field. I would also like to say thank you to my second supervisor, Farida Latif, who offered me a lot of support throughout my time on this project. I would like to thank Jan, for putting up with the endless amount of pipette tips I used to generate, and for all of our lovely chats. I would also like to say a huge thank you my office, including Naomi Wake, for the incredible help she gave me throughout the project, Thoraia, Abdullah and Seley for our great conversations (also for the dates & Arabic coffee!). My partner in crime, and coffee bud, Amy- I enjoyed all of our chats in the office and will miss all of the fun and jokes we used to share (I still check my desk in the morning for pretend spiders!). I would like to dedicate this thesis and offer an incredibly special thank you to my mum and dad, who spent many Friday evenings in the Country Girl with me, listening to all of my genetics troubles over a glass of wine. To my dad for always having such a keen interest in everything that I do, I probably wouldn’t have made it this far without your encouragement. Finally, to my new husband, Jamie, thank you so much for all of your support during the tough times, and for sticking with me through them! Coming home to you and Nova (our dog) used to make all the troubles seem so distant. I can’t wait to finally spend some cosy evenings with you, without being surrounded by papers! You made it all worth it. 2 Table of Contents Chapter One: ..................................................................................................................... 11 Introduction ....................................................................................................................... 11 1.1 THE GENETIC EPIDEMIOLOGY OF INHERITED DISEASE .............................. 12 1.1a Mendelian Diseases: Clinical Aspects .................................................................. 12 1.1a.i Autosomal Recessive Disorders .................................................................. 13 1.1a.ii Autosomal Dominant Disorders ................................................................. 13 1.1a.iii Variable Penetrance and Variable Expressivity ........................................ 14 1.2 IDENTIFICATION OF THE GENETIC BASIS OF DISEASE ................................ 21 1.2a Cytogenetics .......................................................................................................... 22 1.2b Molecular Methods for the Investigation of Genetic Disease ............................... 23 1.2b.i Positional Cloning ....................................................................................... 23 1.2b.ii Candidate Gene Approach ......................................................................... 24 1.3 CANCER AS A GENETIC DISEASE ....................................................................... 26 1.3a Oncogenes ............................................................................................................. 27 1.3b Tumour Suppressor Genes and Knudson’s 2-hit Hypothesis ............................... 28 1.3c Intratumoural Heterogeneity ................................................................................. 30 1.4 FAMILIAL CANCER ................................................................................................ 32 1.5 SPORADIC CANCER ................................................................................................ 33 1.6 NEXT GENERATION SEQUENCING ..................................................................... 34 1.6a Main Principles of Exome Sequencing ................................................................. 37 1.6b Exome Sequencing Process .................................................................................. 38 1.6c Applications of Exome Sequencing to Investigate Disease .................................. 48 1.7 MAIN AIM OF PROJECT ......................................................................................... 51 Chapter Two: Materials and Methods ............................................................................... 53 2.1 MATERIALS .............................................................................................................. 54 2.1a Patient Material ..................................................................................................... 54 2.2 Chemicals, Reagents and Suppliers .................................................................. 56 2.3 EXOME SEQUENCING ............................................................................................ 57 2.4 POLYMERASE CHAIN REACTION (PCR) ............................................................ 58 3 2.4a Standard PCR amplification for Candidate Genes ................................................ 58 2.4b Touchdown PCR amplification ............................................................................. 59 2.4c Gradient PCR ........................................................................................................ 61 2.4d Analysis of Products from Standard PCR Cycle .................................................. 61 2.5 SEQUENCING OF PCR PRODUCTS ....................................................................... 62 2.5a PCR product clean-up ........................................................................................... 62 2.5b Sequencing Reactions ........................................................................................... 62 2.5c Sequencing Reactions Clean-up ........................................................................... 63 2.5d Whole Genome Amplification .............................................................................. 64 Chapter Three: Exome Sequencing and Autosomal Recessive Disease ........................... 66 3.1 INTRODUCTION TO ACROCALLOSAL SYNDROME ........................................ 67 3.1a Consanguinity........................................................................................................ 67 3.1a.i Prevalence of Consanguineous Unions ....................................................... 68 3.1a.ii Genetic Consequences of Consanguinity ................................................... 71 3.2 The Ciliopathies ..................................................................................................... 73 3.2a The Cilium............................................................................................................. 73 3.2b Oligogenic Inheritance and the Ciliopathies ........................................................