Dissecting the Genetic Basis of Parkinson Disease, Dystonia and Chorea A thesis submitted to the University College London for the degree of Doctor of Philosophy June 2016 by Dr Niccolò Emanuele Mencacci 1 Declaration of Authorship I, Niccolò Emanuele Mencacci, 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 Incontenibile andare, di monte in monte, inquieti dietro un mistero che sempre ti seduce, da un’altra valle 3 Abstract In this thesis I used of a range of genetic methodologies and strategies to unravel the genetic bases of Parkinson disease (PD), myoclonus-dystonia (M-D), and chorea. First, I detail the work I performed in PD, including (1) the screening of GBA in a cohort of early-onset PD cases, which led to the identification of the allele E326K (p.Glu365Lys) as the single most frequent, clinically relevant, risk variant for PD; (2) a detailed genetic analysis in a large cohort of PD cases who underwent deep-brain stimulation treatment and a longitudinal comparison of the phenotypic features of carriers of mutations in different genes; (3) the observation that rare GCH1 coding variants, known to be responsible for the childhood-onset disorder DOPA-responsive dystonia, are a novel risk factor for PD. Then, I describe the work I performed to identify novel causes of M-D, including (1) the discovery of the missense p.Arg145His mutation in KCTD17 as a novel cause of autosomal dominant M-D; (2) the identification of tyrosine hydroxylase deficiency as a novel treatable cause of recessive M-D; and (3) the conclusive disproof of the pathogenic role of the p.Arg1389His variant in CACNA1B as a cause of M-D. Finally, I detail my work in the field of choreic syndromes, including (1) the genetic screening of NKX2-1 in the Queen Square cohort of benign hereditary chorea (BHC) cases; (2) the identification of ADCY5 mutations, the gene thought to be responsible for the condition familial dyskinesias with facial myokymia, as an important cause of BHC; and (3) the identification of de novo mutations in PDE10A as a novel genetic cause of chorea. These findings are discussed in light of the recent literature. Following my analysis, I suggest future directions for the identification of novel genetic causes of movement disorders, in light of my recent findings and ongoing research. 4 Table of Contents Declaration of Authorship .............................................................................................. 2 Abstract ............................................................................................................................ 3 Table of Contents ............................................................................................................. 5 List of Figures .................................................................................................................. 9 List of Tables .................................................................................................................. 12 Peer-Reviewed Publications ......................................................................................... 14 Acknowledgements ........................................................................................................ 18 Chapter 1. General Introduction ............................................................................ 20 1.1 Thesis Aim ...................................................................................................... 20 1.2 Summary of Relevant Techniques in Genetics ........................................... 21 1.2.1 Dideoxynucleotide Sequencing ................................................................... 21 1.2.2 Next Generation Sequencing....................................................................... 22 1.2.3 Linkage Analysis ......................................................................................... 24 1.3 Models and Strategies to Study the Genetic Architecture of Diseases ..... 26 1.4 Strategies for Gene Identification in Mendelian Diseases ......................... 28 1.5 Identifying Causal Alleles: How to Prioritise Variants? ............................ 31 1.5.1 Filtering Based on the Inheritance Mode and Pedigree Information .......... 32 1.5.2 Filtering Based on Rarity ............................................................................ 33 1.5.3 Filtering Based on Deleteriousness and Conservation ................................ 34 1.6 Limitations of Next-Generation Sequencing .............................................. 35 Chapter 2. Overview on Genetics of Selected Movement Disorders ................... 37 2.1 The Genetics of Parkinson disease .............................................................. 37 2.1.1 Introduction ................................................................................................. 37 2.1.2 Monogenic Causes of Parkinson Disease ................................................... 40 2.1.3 Rare Variants Predisposing to PD ............................................................... 50 2.1.4 Common Low-Risk Variants Predisposing to PD ....................................... 56 2.2 Overview on the Genetics of Dystonia ......................................................... 59 2.2.1 Introduction ................................................................................................. 59 2.2.2 Overview of Monogenic Forms of Dystonia .............................................. 63 2.3 Genetics of Choreas ....................................................................................... 77 5 2.3.1 Introduction ................................................................................................. 77 2.3.2 Huntington’s disease ................................................................................... 81 2.3.3 Huntington’s disease-like syndromes .......................................................... 82 2.3.4 Benign Hereditary Chorea........................................................................... 87 Chapter 3. Materials and Methods ......................................................................... 88 3.1 DNA Extraction ............................................................................................. 88 3.1.1 Extraction from Whole Blood ..................................................................... 88 3.1.2 Extraction from Saliva ................................................................................ 88 3.2 Polymerase-Chain Reaction ......................................................................... 89 3.3 Agarose Gel Electrophoresis ........................................................................ 89 3.4 PCR Purification ........................................................................................... 90 3.5 Sanger Sequencing ........................................................................................ 90 3.6 Multiplex Ligation-Dependent Probe Amplification ................................. 90 3.7 Analysis of Microsatellites ............................................................................ 92 3.8 DNA Array SNP Analysis ............................................................................. 92 3.9 Genome-Wide Parametric Multipoint Linkage Analysis .......................... 93 3.10 Whole-Exome Sequencing ............................................................................ 94 3.11 Bioinformatic Analysis of Whole-Exome Sequencing ................................ 96 3.12 Regional Gene Expression Profiling in Brain Tissue ................................. 98 Chapter 4. Exploring the Genetics of Parkinson Disease ................................... 100 4.1 Outline Of The Chapter .............................................................................. 100 4.2 GBA Mutational Analysis In Early Onset Parkinson Disease ................. 100 4.2.1 Statement Of Contribution ........................................................................ 100 4.2.2 Background ............................................................................................... 100 4.2.3 Subjects, Materials and Methods .............................................................. 101 4.2.4 Results ....................................................................................................... 103 4.2.5 Discussion ................................................................................................. 106 4.3 Genotype-Phenotype Correlation in A Cohort Of Parkinson Disease Patients Treated With Deep-Brain Stimulation.................................................... 109 4.3.1 Statement of Contribution ......................................................................... 109 4.3.2 Background ............................................................................................... 109 4.3.3 Subjects, Materials and Methods .............................................................. 110 4.3.4 Results ....................................................................................................... 112 4.3.5 Discussion ................................................................................................. 118 6 4.4 Mutational Analysis of the GTP cyclohydrolase-1 Gene in Parkinson Disease ...................................................................................................................... 121 4.4.1 Statement of Contribution ........................................................................