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Molekulargenetische Charakterisierung Konstitutioneller Chromosomaler Translokationen Zur Positionsklonierung Krankheitsverursachender Gene

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Molekulargenetische Charakterisierung Konstitutioneller Chromosomaler Translokationen Zur Positionsklonierung Krankheitsverursachender Gene Molekulargenetische Charakterisierung konstitutioneller chromosomaler Translokationen zur Positionsklonierung krankheitsverursachender Gene Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Michael Kraft aus Forchheim Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 20.02.2013 Vorsitzender der Promotionskommission: Prof. Dr. J. Barth Erstberichterstatter: Prof. Dr. Reis Zweitberichterstatter: Prof. Dr. Slany Inhaltsverzeichnis 1 Einleitung ..................................................................................................... 1 1.1 Positionsklonierung krankheitsverursachender Gene ............................. 1 1.1.1 Methoden der Positionsklonierung ......................................................... 2 1.1.2 Positionsklonierung unter Verwendung Phänotyp-assoziierter, balancierter Translokationen ................................................................. 3 1.2 Zielsetzung ............................................................................................. 6 2 Material und Methoden ................................................................................. 7 2.1 Patienten ................................................................................................ 7 2.1.1 Patient 46,XY,t(10;13) – Noonan-Syndrome-Like .................................... 8 2.1.2 Patientin 46,XX,t(1;8) – Adipositas & Hypogonadismus ...........................11 2.1.3 Patientin 46,XX,t(5;14) – Schwere Mentale Retardierung ........................14 2.1.4 Patientin 47,XXX,t(7;11) – DiGeorge-Syndrome-Like ..............................16 2.2 Methoden ............................................................................................. 18 2.2.1 Biologisches Material ..........................................................................18 2.2.2 Amplifikation gesamtgenomischer DNA (Whole Genome Amplification) .....18 2.2.3 Gelelektrophorese ..............................................................................18 2.2.4 Polymerase-Kettenreaktion (PCR) ........................................................19 2.2.5 Sequenzierung ...................................................................................23 2.2.6 Bruchpunkt-Sequenzierung .................................................................24 2.2.7 Fluoreszenz-in-situ-Hybridisierung (FISH) .............................................25 2.2.8 Reverse Transkriptase-Polymerase-Kettenreaktion (RT-PCR) ...................29 2.2.9 Quantitative “Real-Time” PCR (qPCR) ...................................................30 2.2.10 Multiplex Ligation-dependent Probe Amplification (MLPA) ........................35 2.2.11 Expressions-Array ..............................................................................36 2.2.12 Array-basierte molekulare Karyoptypisierung .........................................37 2.2.13 Zellkultur ..........................................................................................37 2.2.14 Histonacetylierungs-Assay ...................................................................40 2.2.15 Western-Blot .....................................................................................40 2.2.16 ChIP-on-chip .....................................................................................42 2.2.17 Gene ontology- (GO) und Signalweg-Analysen .......................................44 2.3 Material ................................................................................................ 45 2.3.1 Verbrauchsmaterialien ........................................................................45 2.3.2 Chemikalien ......................................................................................45 2.3.3 Antikörper .........................................................................................47 2.3.4 Lösungen ..........................................................................................47 2.3.5 Kits ..................................................................................................51 I Inhaltsverzeichnis 2.3.6 TaqMan-Sonden (pre-designed) ...........................................................51 2.3.7 Selbstentworfene TaqMan-Sonden ........................................................52 2.3.8 Oligonukleotide ..................................................................................52 2.3.9 MLPA-Sonden-Oligonukleotide .............................................................59 2.3.10 BAC-Klone .........................................................................................60 2.3.11 Arrays ..............................................................................................60 2.3.12 Software und Datenbanken .................................................................61 2.3.13 Geräte ..............................................................................................62 3 Ergebnisse .................................................................................................. 64 3.1 Ergebnisse Patient 46,XY,t(10;13) – Noonan-Syndrome-like ............... 64 3.1.1 Ausgangslage ....................................................................................64 3.1.2 MYST4-Knockdown in Zellmodellsystemen .............................................64 3.1.3 Analyse der Histon H3- und H4-Acetylierung ..........................................65 3.1.4 Isformspezifisches Expressionsmuster von MYST4 ..................................66 3.1.5 Expressionsanalysen auf Proteinebene mittels Westernblot ......................68 3.1.6 Genomweite Expressionsanalysen ........................................................70 3.1.7 Chromatin-Immunopräzipitation (ChIP-on-chip) .....................................75 3.1.8 Kombinierte Analyse der Ergebnisse aus der genomweiten Expressionsanalyse mit denen aus der ChIP-on-chip ...............................77 3.1.9 Phosphorylierungsstatus wichtiger Komponenten des MAP-Kinase- Signalwegs ........................................................................................79 3.1.10 Mutations-Screening ...........................................................................81 3.2 Ergebnisse Patientin 46,XX,t(1;8) – Adipositas & Hypogonadismus ..... 82 3.2.1 Bruchpunktsequenzierung ...................................................................82 3.2.2 Analyse der Bruchpunkte und der umgebenden genomischen Region ........83 3.2.3 Expressionsanalysen mittels TaqMan-basierter qPCR ..............................87 3.2.4 Molekulare Karyotypisierung ................................................................88 3.3 Ergebnisse Patientin 46,XX,t(5;14) – Schwere mentale Retardierung .. 90 3.3.1 Bruchpunktsequenzierung ...................................................................90 3.3.2 Molekulare Karyotypisierung ................................................................91 3.3.3 Untersuchung der bruchpunktumgebenden genomischen Region und Identifikation des Kandidatengens FOXG1 .............................................91 3.3.4 Nachweis einer FOXG1-Defizienz ..........................................................93 3.4 Ergebnisse Patientin 47,XXX,t(7;11) – DiGeorge-Syndrome-Like ......... 94 3.4.1 Bruchpunkteingrenzung und Sequenzierung ..........................................94 3.4.2 Bruchpunktanalysen ...........................................................................96 3.4.3 Expressionsanalysen von Kandidatengenen im Bereich der Bruchpunktregionen ...........................................................................98 3.4.4 Molekulare Karyotypisierung ................................................................99 II Inhaltsverzeichnis 4 Diskussion ................................................................................................ 101 4.1 Diskussion Patient 46,XY,t(10;13) – Noonan-Syndrome-like ............. 101 4.1.1 Phänotyp des Patienten und Identifikation des Kandidatengens .............. 101 4.1.2 Die Histon-Acetyltransferase MYST4 ................................................... 101 4.1.3 Nachweis einer kausalen MYST4-Haploinsuffizienz ................................ 103 4.1.4 Isoformspezifische Expression von MYST4 korreliert mit dem murinen und humanen Phänotyp .................................................................... 104 4.1.5 MYST4-abhängige Expressionsregulation ............................................. 104 4.1.6 Identifizierung des MAP-Kinase-Signalwegs als ein Hauptziel MYST4- vermittelter Regulation ..................................................................... 105 4.1.7 MYST4-Haploinsuffizienz stellt eine seltene Ursache für Erkrankungen des Noonan-Syndrome-Like Spektrums dar ......................................... 106 4.2 Diskussion Patientin 46,XX,t(1;8) – Adipositas & Hypogonadismus ... 108 4.2.1 Phänotyp der Patientin ...................................................................... 108 4.2.2 Bruchpunktkartierung ....................................................................... 108 4.2.3 Kandidatengene in den Bruchpunktregionen ........................................ 109 4.2.4 Translokationsunabhängige genomische Imbalance .............................. 112 4.2.5 Zusammenfassende Genotyp-Phänotyp-Korrelation .............................. 113 4.3 Diskussion Patientin 46,XX,t(5;14) – Schwere mentale Retardierung 115 4.3.1 Phänotyp der Patientin .....................................................................
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