From the Institute of Neurogenetics of the University of Lübeck Director: Prof. Dr. med. Christine Klein Identification and characterization of novel genetic causes of dystonia Dissertation for Fulfilment of the Requirements for the Doctoral Degree at the University of Lübeck from the Department of Natural Sciences Submitted by Hauke Baumann from Celle Lübeck, 2020 First referee: Prof. Dr. rer. nat. Katja Lohmann Second referee: Prof. Dr. rer. nat. Henrik Oster Date of oral examination: 24.07.2020 Approved for printing, Lübeck, 28.07.2020 To Bernd Baumann LIST OF CONTENTS 1. ABSTRACT ................................................................................................................................................ 1 2. ZUSAMMENFASSUNG .............................................................................................................................. 3 3. INTRODUCTION........................................................................................................................................ 5 3.1 CLINICAL CHARACTERISTICS OF DYSTONIA ......................................................................................................... 5 3.2 GENETICS OF DYSTONIA................................................................................................................................ 6 3.2.1 DYT-KMT2B dystonia ......................................................................................................................... 7 3.2.2 GNB1-related disorders ..................................................................................................................... 8 3.2.3 VAC14-related disorders ................................................................................................................. 10 3.2.4 DYT-THAP1 dystonia ........................................................................................................................ 11 3.3 REDUCED PENETRANCE IN GENETIC DYSTONIAS ............................................................................................... 12 3.4 CONVERGING PATHWAYS OF GENETIC DYSTONIAS ............................................................................................ 13 3.5 AIMS ...................................................................................................................................................... 14 4. PATIENTS, MATERIALS, AND METHODS ................................................................................................. 16 4.1 PATIENTS ................................................................................................................................................ 16 4.2 MATERIALS ............................................................................................................................................. 18 4.2.1 Solutions .......................................................................................................................................... 18 4.2.2 Factors for differentiation ............................................................................................................... 19 4.2.3 Technical equipment ....................................................................................................................... 19 4.2.4 Kits ................................................................................................................................................... 20 4.2.5 Antibodies ....................................................................................................................................... 20 4.2.6 Laboratory supplies and reagents ................................................................................................... 21 4.2.7 Primer .............................................................................................................................................. 23 4.3 METHODS ............................................................................................................................................... 24 4.3.1 Mammalian cell culture ................................................................................................................... 25 4.3.1.1 Fibroblasts .............................................................................................................................................. 25 4.3.1.2 Reprogramming of skin fibroblasts into iPSCs ........................................................................................ 25 4.3.1.3 Coating ................................................................................................................................................... 26 4.3.1.4 iPSC culture ............................................................................................................................................. 27 4.3.1.5 Embryoid body formation ....................................................................................................................... 28 4.3.1.6 Differentiation of iPSCs into cortical neurons ......................................................................................... 28 4.3.1.7 Mycoplasma detection ........................................................................................................................... 29 4.3.2 Nucleic acid-based methods ........................................................................................................... 30 4.3.2.1 Extraction of nucleic acids ...................................................................................................................... 30 4.3.2.2 First-strand cDNA synthesis .................................................................................................................... 30 4.3.2.3 Quantitative real-time PCR (qRT-PCR) .................................................................................................... 31 4.3.2.4 Polymerase chain reaction (PCR) ............................................................................................................ 31 4.3.2.5 Sanger sequencing .................................................................................................................................. 32 4.3.2.6 Short tandem repeat (STR) analysis ........................................................................................................ 33 4.3.2.7 RNA integrity number (RIN) .................................................................................................................... 33 4.3.2.8 SNP karyotype analysis ........................................................................................................................... 33 4.3.2.9 Whole transcriptome analysis ................................................................................................................ 34 4.3.3 Molecular cloning ............................................................................................................................ 34 4.3.3.1 Insert generation and ligation ................................................................................................................ 35 4.3.3.2 Site-directed mutagenesis ...................................................................................................................... 36 4.3.3.3 Transformation ....................................................................................................................................... 36 4.3.3.4 Gel extraction ......................................................................................................................................... 36 4.3.3.5 Plasmid extraction .................................................................................................................................. 37 4.3.3.6 Subcloning of genomic DNA fragments .................................................................................................. 37 4.3.3.7 Lentiviral transduction ............................................................................................................................ 38 4.3.4 Immunofluorescence ...................................................................................................................... 38 4.3.5 Western blotting ............................................................................................................................. 38 4.3.6 Cytometry ........................................................................................................................................ 39 V 4.3.7 Systematic literature search ............................................................................................................ 39 4.3.8 Statistical analysis............................................................................................................................ 40 5. RESULTS ................................................................................................................................................. 41 5.1 KMT2B GENE EXPRESSION ANALYSIS ............................................................................................................ 41 5.2 FUNCTIONAL EVALUATION OF GNB1 MUTATIONS ........................................................................................... 42 5.3 MOLECULAR CHARACTERIZATION OF VAC14 ................................................................................................. 44 5.4 CLINICAL SPECTRUM AND GENETIC ANALYSIS OF DYT-THAP1 PATIENTS .............................................................. 47 5.4.1