Investigation of the genetic cause and related phenotypes of rare early onset retinal dystrophies Sarah Hull Institute of Ophthalmology, University College London Submitted to the University College London for the degree of Doctor of Philosophy Supervisors: Prof Andrew R Webster Prof Anthony T Moore March 2016 1 Declaration I, Sarah Hull, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis 2 Abstract Early onset retinal dystrophies (EORD) are a group of disorders presenting in childhood with degenerative abnormalities in photoreceptor cells. They are one of the leading causes of sight impairment in the United Kingdom. Since the initial discovery of Rho causing dominant retinitis pigmentosa in 1990, more than 160 genes have been associated with retinal dystrophy. Many, including CRB1, CRX, and RPE65 exhibit phenotypic heterogeneity and have been associated with more than one retinal disorder. Increasingly, with the advent of next generation sequencing, the association of non-syndromic retinal dystrophy with mutations in syndromic genes has been reported including CEP290, CLN3, and BBS1. In this thesis, a large cohort of patients with EORD underwent both detailed phenotyping to characterise their condition and molecular genetic investigations to identify and investigate the underlying causative variants. Many areas of the presented research were driven by novel findings on whole-exome sequencing such as the association of IFT140 with non-syndromic retinal dystrophy or CRX with macular dystrophy. Other areas were driven by unusual groups of patients with limited published data on their condition such as COL18A1 and Knobloch syndrome, with novel phenotypic features of cone-rod dysfunction and pigmentary glaucoma. Sanger sequencing was performed for confirmation and segregation of identified variants but in addition, for investigation of phenotypically similar patient panels for unusual gene associations. This included systemically mild Hermansky-Pudlak syndrome due to HPS6, juvenile macular dystrophy and CDH3, macular dystrophy and CRX and microcephaly with familial exudative vitreoretinopathy due to LRP5. Functional investigation of missense variants in IFT140 related retinal dystrophy was performed with transient cell transfection. This thesis highlights the vast heterogeneity of rare forms of EORD, presents novel clinical and molecular data and describes the key features of conditions to aid diagnosis and opportunities for future research. 3 Acknowledgments I have been very fortunate to study within such a supportive, motivated and dynamic department. I am most grateful to my supervisors, Prof Tony Moore and Prof Andrew Webster, whose experience, expertise and enthusiasm have taught me so much. Within the laboratory, Dr Gavin Arno has been a great teacher of all molecular techniques and a great collaborator on papers. I am very grateful to Dr Nick Owen for his instruction and advice on cell studies. I would also like to thank Samantha Malka, research coordinator in the department for her essential administrative assistance and support as well as the genetic counsellor team. I am grateful for the invaluable help and advice from many different experts in particular Prof Graham Holder and Dr Anthony Robson in the electrophysiology department as well as Prof Mike Cheetham and Prof Mike Michaelides. This research would not be possible without the generous support of funding bodies specifically the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, Special Trustees of Moorfields Eye Hospital, and the Foundation Fighting Blindness, USA. To my incredible husband and family, thank you for your constant support and encouragement. Finally, it has been a privilege to be able to investigate and care for a large number of families affected by early onset retinal dystrophies; their willingness to help with research and their optimism despite such challenges is an inspiration. 4 Table of contents DECLARATION............................................................................................................ 2 ABSTRACT .................................................................................................................. 3 ACKNOWLEDGMENTS ............................................................................................... 4 TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ....................................................................................................... 8 LIST OF TABLES ......................................................................................................... 8 ABBREVIATIONS ...................................................................................................... 10 1 INTRODUCTION ................................................................................................ 11 1.1 The neurosensory retina ........................................................................... 12 1.1.1 Retinal development ................................................................................ 14 1.1.2 Phototransduction and the visual cycle .................................................... 14 1.2 Clinical assessment .................................................................................. 16 1.2.1 Visual function ......................................................................................... 16 1.2.2 Ophthalmoscopy and retinal imaging ....................................................... 18 1.3 Molecular genetics .................................................................................... 19 1.3.1 Mendelian inheritance ............................................................................. 20 1.4 Types of retinal dystrophy ........................................................................ 22 1.4.1 Leber congenital amaurosis ..................................................................... 22 1.4.2 Rod cone dystrophy ................................................................................. 25 1.4.3 Cone rod dystrophy ................................................................................. 26 1.4.4 Macular dystrophy ................................................................................... 26 1.4.5 Abnormal retinal vasculogenesis ............................................................. 26 1.4.6 Syndromic retinal dystrophies .................................................................. 27 1.5 Thesis aims ................................................................................................ 27 2 METHODS .......................................................................................................... 28 2.1 Clinical methods ........................................................................................ 28 2.1.1 History and examination .......................................................................... 28 2.1.2 Retinal imaging ........................................................................................ 29 2.1.3 Electrophysiology .................................................................................... 32 2.2 Molecular genetic methods ...................................................................... 35 2.2.1 DNA isolation and quantification .............................................................. 35 2.2.2 Polymerase chain reaction ...................................................................... 35 2.2.3 Sanger sequencing .................................................................................. 38 2.2.4 Next generation sequencing .................................................................... 39 2.2.4.1 Massively parallel sequencing ...................................................................... 39 2.2.4.2 Variant call alignment and analysis .............................................................. 40 2.2.4.3 Whole genome sequencing .......................................................................... 41 2.2.5 Other molecular investigations................................................................. 41 2.2.6 Variant analysis and functional predictive tools ........................................ 43 2.2.6.1 Determining novelty ...................................................................................... 43 2.2.6.2 Predicting pathogenicity................................................................................ 43 2.2.6.3 Protein modelling .......................................................................................... 44 2.3 Cell studies ................................................................................................ 44 2.3.1.1 Plasmid transformation of cells ..................................................................... 44 2.3.1.2 Culture of single colonies.............................................................................. 45 2.3.1.3 Sequencing of plasmid open reading frame ................................................. 46 2.3.1.4 Site directed mutagenesis ............................................................................ 46 2.3.1.5 Culture of hTERT-RPE1 cells ....................................................................... 49 2.3.1.6 Transient transfection of hTERT-RPE1 cells ................................................ 50 2.4 Zebrafish studies ......................................................................................