May 2013 The use of next generation sequencing technologies to dissect the aetiologies of neurodegenerative diseases Arianna Tucci, MD This thesis is submitted to the University College of London for the degree of Doctor of Philosophy 1 Declaration I, Arianna Tucci, hereby declare that the work presented in this thesis was performed by myself. Exceptions to this are indicated elsewhere in the thesis. 2 A Martino “Meglio aggiungere vita ai giorni che non giorni alla vita.” Rita Levi Montalcini 3 Abstract Advances in next generation sequencing technologies have brought a paradigm shift in how medical researchers investigate human disorders. Whole exome sequencing (WES) allows to comprehensively study, in a single experiment, coding variations of a human genome. My PhD thesis focuses on the use of WES to dissect genetic aetiologies of neurodegenerative diseases. First, I present a pilot study where I prove the feasibility of this technology and I test the methods used in the following projects. I then describe the use of WES in rare Mendelian disorders: i) in patients with Kohlschütter-Tönz Syndrome, where WES has failed to identify the disease gene, because the mutation is in a non-coding region; ii) in a family with Charcot-Marie- Tooth type 6, where coupled with linkage data, WES rapidly identified the mutation causing the disease. Last, I present two projects where next generation sequencing plays an important role in elucidating the role of variants present in two loci (PARK16 and EIF4G1) in Parkinson’s Disease. 4 Acknowledgements The work presented here was made possible by the people that surround, or have surrounded myself in the past. I am deeply grateful to my supervisor John Hardy, for his exceptional mentorship and constant support. Particular thanks to Prof. Henry Houlden for his support, Dr Vincent Plagnol for his advice, Dr Coro Paisán-Ruiz for her patience teaching me the basis of genetic research, Prof. Nick Wood for his support and advice, Dr Elia Stupka and Francesco Lescai for introducing myself to bioinformatics. Thanks to the members of the laboratory of neurogenetics at Institute of Neurology; Anna Sailer, Boniface Mok, Reema Paudel, Nuria Seto, Lucia Schottlaender, Eleanna Kara, Niccolo’ Mencacci, Una Sheerin, Alan Pittman, Mina Ryten, Daniah Trabzuni, Raquel Duran, Selina Wray, Lee Stanyer, June Smalley, Jana Vandrovcova, Dalia Kasperaviciute, Rohan de Silva and Roberto Simone. Thanks to the members of the neurogenetics lab at NIH: Dr Andy Singleton, Dena Hernandez, Dr Mike Nalls and especially Celeste Sassi. I am also very grateful to the people who first introduced myself to research, from the Laboratory of Genetics and Biochemistry in Milan: Stefania Corti, Monica Nizzardo and Giacomo Comi. Thanks to my lifetime friends sorelle Rugis Stefania e Ale, Giulia. Special thanks to Viola, Silvia and Licia; to my university fellows Alberto, Sara, Mimi’, Chiarina, SDM, Patoz, Luca, Leo, Tommi. Thanks to M’and the pizzica, Salvatore and Marta Wolek. Thanks to my family: my late grandma Nonna Giovanna, Zia Margherita. Thanks to the wonderful Emilia and Luigi, to Laura e Pietro and the little cousins Elena and Alessandro. Thanks to my parents for their encouragement, help and support. Thanks to the person that knows me better than I know myself, Fiora. Thanks to the astonishing beauty of Manarola and Tellaro. 5 Thanks to Martino and the brand new Riccardo, thanks for coming to this world, my little ones. And thanks to Pietro, for being always there – with a smile. 6 Table of contents 1 INTRODUCTION ................................................................................................... 17 1.1 NEURODEGENERATIVE DISEASES: IMPORTANCE OF GENETIC ANALYSIS .... 17 1.2 GENETIC RESEARCH OVERVIEW .................................................................... 18 1.2.1 Genetic analysis using high throughput SNP genotyping ...................... 21 1.3 WHOLE EXOME SEQUENCING ....................................................................... 28 1.3.1 Overview: next generation sequencing technologies ........................... 28 1.3.2 Whole exome sequencing: premises ..................................................... 30 1.3.3 The technology ....................................................................................... 31 1.3.4 How to identify the causal variants in Mendelian disorders ................. 35 1.3.5 How is WES changing neurogenetics ..................................................... 39 1.3.6 Limitations and challenges ..................................................................... 42 1.4 GENETICS OF SELECTED NEURODEGENERATIVE DISEASES .......................... 44 1.4.1 Genetics of Parkinson’s Disease ............................................................ 44 1.4.2 Genetics of Kohlschütter-Tönz syndrome ............................................. 51 1.4.3 Genetics of complex neuropathies ........................................................ 57 2 WHOLE EXOME SEQUENCING PILOT PROJECT ................................................... 61 2.1 STATEMENT OF CONTRIBUTION TO THIS RESEARCH ................................... 61 2.2 BACKGROUND ............................................................................................... 61 2.3 MATERIALS AND METHODS .......................................................................... 61 2.4 DNA samples selection .................................................................................. 61 2.4.1 Library preparation ................................................................................ 62 2.4.2 Sequencing ............................................................................................. 66 2.4.3 Bioinformatics ........................................................................................ 67 2.5 RESULTS ......................................................................................................... 77 2.5.1 Coupling NimbleGen SeqCap EZ to Illumina sequencing ....................... 77 2.5.2 Data analysis .......................................................................................... 85 2.6 CONCLUSIONS ............................................................................................... 91 7 3 KOHLSCHÜTTER-TÖNZ SYNDROME: ROGDI MUTATIONS AND GENETIC HETEROGENEITY ........................................................................................................ 92 3.1 STATEMENT OF CONTRIBUTION TO THIS RESEARCH ................................... 92 3.2 BACKGROUND ............................................................................................... 92 3.3 MATERIALS AND METHODS .......................................................................... 92 3.3.1 Samples .................................................................................................. 92 3.3.2 Genetic investigations ............................................................................ 98 3.4 RESULTS ....................................................................................................... 100 3.5 DISCUSSION ................................................................................................. 110 4 C12orf65 MUTATIONS CAUSE AXONAL NEUROPATHY WITH OPTIC ATROPHY 115 4.1 STATEMENT OF CONTRIBUTION TO THIS RESEARCH ................................. 115 4.2 BACKGROUND ............................................................................................. 115 4.3 METHODS .................................................................................................... 116 4.3.1 Samples ................................................................................................ 116 4.3.2 Nerve biopsy ........................................................................................ 116 4.3.3 SNP genotyping and autozygosity mapping ........................................ 117 4.3.4 Mutation validation and screening in the additional cohorts ............. 118 4.3.5 Lymphoblast cells cultures ................................................................... 118 4.3.6 Transcript analyses ............................................................................... 118 4.3.7 Blue native in-gel complex V assay ...................................................... 120 4.3.8 Oxygen Consumption ........................................................................... 121 4.4 RESULTS ....................................................................................................... 121 4.4.1 Clinical details ...................................................................................... 121 4.4.2 Genetic analyses .................................................................................. 127 4.4.3 Mitochondrial impairment ................................................................... 133 4.4.4 Oxygen consumption ........................................................................... 133 4.5 DISCUSSION ................................................................................................. 136 5 A GENOME-WIDE ASSOCIATION STUDY FOLLOW UP: THE PARK16 LOCUS .... 139 5.1 STATEMENT OF CONTRIBUTION TO THIS RESEARCH ................................. 139 5.2 BACKGROUND ............................................................................................. 139 5.3 MATERIALS AND METHODS ........................................................................ 140 8 5.3.1 Samples ................................................................................................ 140 5.3.2