FAKULTÄT FÜR MEDIZIN INSTITUT FÜR HUMANGENETIK Lehrstuhl: Prof. Dr. Thomas Meitinger Characterization of a Novel Monogenic Form of Liver Failure Marlies Gwendolyn Köpke Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Medizin (Dr. med.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Ernst J. Rummeny Prüfer der DissertAtion: 1. Prof. Dr. Thomas Meitinger 2. Prof. Dr. Percy A. Knolle Die Dissertation wurde am 07.08.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 04.07.2018 angenommen. Marlies G. Köpke ChArActerizAtion of A Novel Monogenic Form of Liver Failure Table of Contents Table of contents List of abbreviations ................................................................................................. V List of prefixes ........................................................................................................ VIII Introduction ............................................................................................................... 1 Acute liver failure in children ............................................................................................ 1 Definition .......................................................................................................................... 1 Epidemiology ................................................................................................................... 2 Pathophysiology ............................................................................................................... 2 Aetiology .......................................................................................................................... 3 ClinicAl phenotype ............................................................................................................ 8 DiAgnostics ...................................................................................................................... 8 TherApy ............................................................................................................................ 9 Prognosis ....................................................................................................................... 12 Chronic liver failure in children ...................................................................................... 14 Definition ........................................................................................................................ 14 Epidemiology ................................................................................................................. 14 Aetiology ........................................................................................................................ 14 ClinicAl presentAtion ....................................................................................................... 14 TherApy .......................................................................................................................... 15 Fever and its association with liver disease ................................................................. 16 Definition ........................................................................................................................ 16 Physiologic And pAthophysiologic effects ...................................................................... 16 TherApy .......................................................................................................................... 16 AssociAtion with liver diseAse ........................................................................................ 17 Recurrent acute liver failure syndrome ......................................................................... 18 NBAS ................................................................................................................................. 20 Discovery ....................................................................................................................... 20 Gene locAtion, gene structure And protein structure ...................................................... 20 Expression in different tissues ....................................................................................... 21 Physiologic function of NBAS ........................................................................................ 22 AssociAted diseAses ...................................................................................................... 22 Purpose of this work ............................................................................................... 24 I Marlies G. Köpke ChArActerizAtion of A Novel Monogenic Form of Liver Failure Table of Contents Materials and methods ........................................................................................... 25 Preamble ........................................................................................................................... 25 Reagents and media ........................................................................................................ 25 Buffers and solutions ...................................................................................................... 27 Antibodies, ladders and enzymes .................................................................................. 29 Kits .................................................................................................................................... 29 Equipment and software ................................................................................................. 30 Materials ........................................................................................................................... 31 Cell lines ........................................................................................................................... 31 Molecular biology methods ............................................................................................ 32 PlAsmid extrAction from bActeriA (Miniprep/Midiprep) .................................................... 32 DNA extrAction from humAn cells (AllPrep) ................................................................... 32 DeterminAtion of concentrAtion of DNA ......................................................................... 32 PolymerAse chAin reAction (PCR) .................................................................................. 32 Microbiological methods ................................................................................................. 37 CultivAtion of bActeriA .................................................................................................... 37 Cell biology methods ....................................................................................................... 38 CultivAtion of humAn cells .............................................................................................. 38 Collection of cells ........................................................................................................... 38 Splitting of cells .............................................................................................................. 38 GenerAtion of stocks ...................................................................................................... 38 GenerAtion of pellets for further AnAlysis ....................................................................... 38 ContAminAtion test for mycoplAsmA ............................................................................... 39 BlAsticidin killing curve ................................................................................................... 39 DeterminAtion of cell count using the Scepter™ Handheld Cell Counter ...................... 39 DeterminAtion of cell count using the CyQUANT® Cell ProliferAtion AssAy Kit ............. 39 Growth curve .................................................................................................................. 41 Knockdown of a gene in cell culture .............................................................................. 42 PreAmble ........................................................................................................................ 42 Design And hAndling of oligonucleotides for shRNA ...................................................... 42 TrAnsformAtion of chemicAlly competent E. coli ............................................................ 43 Cloning of entry construct .............................................................................................. 43 Cloning of expression construct ..................................................................................... 44 PrepArAtion of mediA for the lentivirAl trAnsduction ........................................................ 44 Production of lentivirus ................................................................................................... 44 II Marlies G. Köpke ChArActerizAtion of A Novel Monogenic Form of Liver Failure Table of Contents Lentiviral transduction .................................................................................................... 45 MaintenAnce of trAnsduced cells ................................................................................... 46 Proteomic analysis .......................................................................................................... 47 PreAmble .......................................................................................................................