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Markus Draaken Aus Krefeld Molekulargenetische Untersuchungen bei uro-rektalen Fehlbildungen Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Markus Draaken aus Krefeld Bonn (April 2013) Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Die vorliegende Arbeit wurde am Institut für Humangenetik der Rheinischen Friedrich-Wilhelms-Universität Bonn angefertigt. 1. Gutachter: Prof. Dr. Markus M. Nöthen 2. Gutachter: Prof. Dr. Michael Hoch Tag der Promotion: 17.09.2013 Erscheinungsjahr: 2014 Inhaltsverzeichnis I Inhaltsverzeichnis Inhaltsverzeichnis ............................................................................................................................................ I Abbildungs- und Tabellenverzeichnis ................................................................................................... VI Abkürzungsverzeichnis ............................................................................................................................... IX 1 Einleitung ................................................................................................................................................ 1 1.1 Untersuchte uro-rektale Fehlbildungen ...................................................................................... 1 1.1.1 Blasenekstrophie-Epispadie-Komplex (BEEK) ........................................................... 1 1.1.1.1 Isolierte Epispadie (E) .............................................................................................. 1 1.1.1.2 Klassische Blasenekstrophie (KBE) ..................................................................... 2 1.1.1.3 Kloakenekstrophie (KE) ........................................................................................... 3 1.1.1.4 Embryologie des BEEK ............................................................................................. 4 1.1.1.5 Epidemiologie des BEEK .......................................................................................... 5 1.1.1.6 Genetik des BEEK ........................................................................................................ 6 1.1.2 Anorektale Malformationen (ARM) .............................................................................. 11 1.1.2.1 VATER/VACTERL-Assoziation ........................................................................... 12 1.1.2.2 Prune-Belly-Syndrom (PBS) ................................................................................ 13 1.1.2.3 Embryologie der ARM ............................................................................................ 15 1.1.2.4 Epidemiologie der ARM ......................................................................................... 15 1.1.2.5 Genetik der ARM allgemein ................................................................................. 16 1.2 Systematische Identifizierung von kausalen Genen und Regionen bei seltenen angeborenen uro-rektalen Fehlbildungen .............................................................................. 17 1.2.1 Untersuchungen zu ursächlichen Kopienzahlveränderungen (CNV- Analysen) ................................................................................................................................ 18 1.2.2 Next Generation Sequencing (NGS) .............................................................................. 20 1.3 Netzwerk für kongenitale uro-rektale Fehlbildungen (CURE-Net) ............................... 22 2 Zielsetzung ........................................................................................................................................... 23 3 Material und Methoden................................................................................................................... 24 II Inhaltsverzeichnis 3.1 Verwendete Materialien ................................................................................................................. 24 3.1.1 Geräte ....................................................................................................................................... 24 3.1.2 Chemikalien und Enzyme ................................................................................................. 27 3.1.3 Lösungen ................................................................................................................................. 28 3.1.4 Kommerzielle Systeme (Kits) ......................................................................................... 28 3.1.5 Software und Datenbanken ............................................................................................. 29 3.2 Molekularbiologische Untersuchungen .................................................................................... 31 3.2.1 Isolierung genomischer DNA und RNA ....................................................................... 31 3.2.2 Konzentrations- und Reinheitsmessung .................................................................... 32 3.2.3 Fällung von verunreinigter DNA .................................................................................... 33 3.2.4 Genomweite SNP-Array Genotypisierung mit Illumina ........................................ 33 3.2.5 Multiplex ligations-abhängige Sondenamplifikation (MLPA) ............................ 35 3.2.6 Auswahl der Oligonukleotidsequenzen (Primer-Design) .................................... 36 3.2.7 DNA-Amplifikation (Polymerase-Kettenreaktion; PCR) ...................................... 37 3.2.7.1 PCR-Reaktionsansätze ........................................................................................... 37 3.2.7.2 PCR-Reaktionsbedingungen ................................................................................ 38 3.2.7.3 Agarose-Gelelektrophorese ................................................................................. 39 3.2.8 Quantitative PCR (qPCR) .................................................................................................. 40 3.2.8.1 qPCR-Reaktionsansatz und-bedingungen ...................................................... 40 3.2.8.2 Schmelzkurve ............................................................................................................ 41 3.2.8.3 -Methode ................................................................................. 41 3.2.9 AutomatisierteAuswertung Sanger ΔΔCt-Sequenzierung ....................................................................... 42 3.2.9.1 Probenvorbereitung und Primer für die Sanger-Sequenzierung .......... 43 3.2.9.2 Sanger-Sequenzierungsansatz und -Reaktionsbedingungen ................. 43 3.2.9.3 Vorbereitung, Auftrennung und Verarbeitung der Sequenzierfragmente ............................................................................................. 44 3.2.10 Next Generation Sequencing (NGS) .............................................................................. 44 3.2.10.1 Roche 454 GenomeSequencer FLX (GS-FLX) Titanium Instrument .... 45 3.2.10.2 Illumina (Solexa) Genome Analyzer (GA)....................................................... 46 3.2.10.3 Applied Biosystems (ABI) SOLiD System ....................................................... 47 3.2.11 Ganzpräparat in situ Hybridization (WISH) .............................................................. 47 Inhaltsverzeichnis III 3.2.12 Powerplex® 16-System zur Bestätigung der Elternschaft ................................... 48 3.2.13 Auswahl der Verifikationsmethoden ........................................................................... 48 3.3 Bioinformatische und statistische Methoden......................................................................... 49 3.3.1 GenomeStudio (GS) ............................................................................................................. 49 3.3.1.1 B-Allel Frequenz (BAF) .......................................................................................... 49 3.3.1.2 Log-R-Ratio (LRR) ................................................................................................... 50 3.3.1.3 CNV Detektion in GS ................................................................................................ 50 3.3.1.4 Ermittlung der Abstammung in GS ................................................................... 51 3.3.2 Genomweite Detektion von CNVs mittels QuantiSNP ........................................... 52 3.3.3 Einschätzung der biologischen Relevanz von CNVs ............................................... 53 3.3.3.1 Nicht öffentlich zugängliche interne Kontrollkollektive .......................... 53 3.3.3.2 Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER) ........................................................... 54 3.3.3.3 Database of Genomic Variants (DGV) .............................................................. 54 3.3.3.4 University of California Santa Cruz (UCSC) Genome Browser ............... 54 3.3.3.5 Mouse Genome Informatics (MGI) Datenbank ............................................. 55 3.3.4 Cartagenia Bench™ Sofware (Cartagenia) .................................................................. 55 3.3.5 Auswertung und computerbasierte Analyse der NGS Daten .............................. 56 3.3.6 Prädiktionsprogramm (PS)2-v2 zur Vorhersage der Proteinstruktur ............ 56 3.3.7 Prädiktionsprogramme zur Interpretation
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