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Genetische Charakterisierung Chromosomaler Bruchpunkte

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Genetische Charakterisierung Chromosomaler Bruchpunkte Genetische Charakterisierung chromosomaler Bruchpunkte Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Dipl.-Biol. Anita Heller geboren am 07.05.1973 in Schmalkalden Inhaltsverzeichnis Inhaltsverzeichnis 1 EINLEITUNG ----------------------------------------------------------------------------------- 1 1.1 ZYTOGENETIK------------------------------------------------------------------------------------------ 2 1.1.1 Klassische Zytogenetik -----------------------------------------------------------------------------2 1.1.2 Bänderungs-Zytogenetik ---------------------------------------------------------------------------2 1.1.3 Molekulare Zytogenetik -----------------------------------------------------------------------------3 1.1.3.1 Prinzip der Fluoreszenz-in-situ-Hybridisierung (FISH)--------------------------------------3 1.1.3.2 Gebräuchliche Sonden in der FISH -------------------------------------------------------------4 1.1.3.3 Von der Ein- zur Vielfarben-FISH----------------------------------------------------------------5 1.1.3.4 FISH-Bänderungs-Methoden ---------------------------------------------------------------------6 1.1.3.5 Weitere Methoden der molekularen Zytogenetik---------------------------------------------7 1.2 BRUCHPUNKTE ---------------------------------------------------------------------------------------- 8 1.2.1 Chromosomale Bruchpunkte ---------------------------------------------------------------------8 1.2.2 Molekulargenetische Bruchpunkte--------------------------------------------------------------9 1.2.3 Mögliche Auswirkungen von Brüchen auf Proteinebene --------------------------------9 1.3 FRAGESTELLUNG ----------------------------------------------------------------------------------- 11 2 MATERIAL UND METHODEN -----------------------------------------------------------12 2.1 UNTERSUCHUNGSMATERIAL ---------------------------------------------------------------------- 12 2.1.1 Chromosomenpräparation ----------------------------------------------------------------------- 12 2.1.1.1 Chromosomenpräparation aus Blut ----------------------------------------------------------- 12 2.1.1.2 Chromosomenpräparation aus Knochenmark ---------------------------------------------- 13 2.1.1.3 Chromosomenpräparation aus Amnionzellen ---------------------------------------------- 13 2.1.1.4 Chromosomenpräparation der GGO- und HLA-Zellinien -------------------------------- 15 2.1.2 Herstellung von Objektträger-Präparaten für konventionelle zytogenetische Methoden und FISH -------------------------------------------------------------------------------- 15 2.2 VERWENDETE DNA-SONDEN--------------------------------------------------------------------- 15 2.2.1 YAC-, BAC-Sonden und Plasmide------------------------------------------------------------- 15 2.2.2 Durch Mikrosezierung hergestellte DNA-Sonden ---------------------------------------- 17 2.2.3 Zentromerspezifische Sonden------------------------------------------------------------------ 18 2.2.4 Weitere Lokusspezifische Sonden ------------------------------------------------------------ 18 2.2.5 Verwendete Sondenmixe ------------------------------------------------------------------------- 18 2.3 MIKROSEZIERUNG VON CHROMOSOMEN BZW. KERNEN (MICRO-CGH)------------------ 18 2.3.1 Verwendete Lösungen und Materialien ------------------------------------------------------ 18 2.3.2 Durchführung der Chromosomen-Mikrosezierung -------------------------------------- 20 2.3.3 Sammeln von Kernen für Micro-CGH--------------------------------------------------------- 21 2.4 MOLEKULARGENETISCHE TECHNIKEN ---------------------------------------------------------- 23 2.4.1 Präparation von Plasmid-DNA ------------------------------------------------------------------ 23 2.4.2 c-DNA Synthese ------------------------------------------------------------------------------------- 23 2.4.2.1 Isolierung von RNA aus fixierten Knochenmarkzellen------------------------------------ 23 2.4.2.2 Reverse-Transkriptase-Reaktion--------------------------------------------------------------- 23 2.4.3 Polymerasekettenreaktion (PCR) -------------------------------------------------------------- 24 2.4.3.1 DOP-PCR-------------------------------------------------------------------------------------------- 24 2.4.3.1.1 DNA-Amplifikation der mikrosezierten Chromosomenfragmente------------------------------25 2.4.3.1.2 DNA-Amplifikation der mikrosezierten Kerne-------------------------------------------------------27 2.4.3.1.3 DNA-Amplifikation der YAC- und Plasmid-Sonden -----------------------------------------------27 2.4.3.1.4 Reamplifizierungs-PCR ----------------------------------------------------------------------------------27 2.4.3.1.5 Markierungs-PCR------------------------------------------------------------------------------------------28 2.4.3.2 Spezifische PCR ----------------------------------------------------------------------------------- 29 2.4.4 Nick-Translation zum Einbringen von markierten Nukleotiden---------------------- 30 2.4.5 DNA-Sequenzierung-------------------------------------------------------------------------------- 30 2.5 KLASSISCHE METHODEN DER BÄNDERUNGS-ZYTOGENETIK------------------------------- 32 2.5.1 GTG-Bandentechnik-------------------------------------------------------------------------------- 32 2.5.2 CBG-Färbung----------------------------------------------------------------------------------------- 32 2.5.3 NOR-Färbung----------------------------------------------------------------------------------------- 33 Inhaltsverzeichnis 2.6 MOLEKULARZYTOGENETISCHE TECHNIKEN---------------------------------------------------- 33 2.6.1 DNA-Fällung der Sonden ------------------------------------------------------------------------- 33 2.6.2 Lösungen und verwendete Detektionssysteme------------------------------------------- 34 2.6.3 Hybridisierung --------------------------------------------------------------------------------------- 35 2.6.3.1 Objektträger-Vorbehandlung und Denaturierung ------------------------------------------ 35 2.6.3.2 Präparation und Denaturierung der DNA-Sonden ----------------------------------------- 36 2.6.3.3 Aufbringen der DNA-Sonden auf den Objektträger---------------------------------------- 38 2.6.4 Detektion----------------------------------------------------------------------------------------------- 38 2.6.5 Auswertung ------------------------------------------------------------------------------------------- 38 3 ERGEBNISSE --------------------------------------------------------------------------------40 3.1 ETABLIERUNG NEUER MOLEKULARZYTOGENETISCHER TECHNIKEN ---------------------- 40 3.1.1 Multicolor-Bänderung der Chromosomen 2, 13 und 22 mit einem auf YAC- und BAC-Banken basierendem Sondenmix (YAC/BAC-MCB) ------------------------------ 40 3.1.2 Der etablierte, auf YAC-Klonen basierende Chromosome-bar-code für Chromosom 13--------------------------------------------------------------------------------------- 43 3.1.3 Multicolor-Bänderung (MCB) für alle menschlichen Chromosomen basierend auf Mikrosezierungsbanken --------------------------------------------------------------------- 44 3.2 EVALUIERUNG DER MCB-METHODE ------------------------------------------------------------ 46 3.2.1 Charakterisierung der Bruchpunkte von konstitutionellen und tumorspezi- fischen Chromosomenaberrationen ---------------------------------------------------------- 46 3.2.2 Nachweis chromosomaler Umbauten in ZOO-FISH-Studien-------------------------- 54 3.2.2.1 Darstellung der Homologie der Gorilla gorilla (GGO)-Chromosomen im Vergleich zu Homo sapiens (HSA) ------------------------------------------------------------------------- 54 3.2.2.2 Darstellung der Homologie der Hylobates lar (HLA)-Chromosomen im Vergleich zu Homo sapiens (HSA) ------------------------------------------------------------------------- 58 3.3 CHARAKTERISIERUNG CHROMOSOMALER VERÄNDERUNGEN DURCH MCB- ANALYSEN-------------------------------------------------------------------------------------------- 61 3.3.1 MCB bestätigt Bruchpunkte der GTG-Bänderung---------------------------------------- 61 3.3.2 Bruchpunktkorrektur nach Anwendung von MCB --------------------------------------- 62 3.3.2.1 Konstitutionelle Aberrationen ------------------------------------------------------------------- 62 3.3.2.2 Tumorspezifische Aberrationen ---------------------------------------------------------------- 65 3.3.3 Fälle mit einer Trisomie 8 lassen mittels MCB für Chromosom 8 keine Aberration erkennen ------------------------------------------------------------------------------- 69 3.4 EINGRENZUNG DER BRUCHREGIONEN EINER T(16;21) MITTELS MCB UND NACHWEIS EINER FUS/ERG-GENFUSION -------------------------------------------------------------------- 70 3.5 GRENZEN DER MCB-METHODE ------------------------------------------------------------------ 72 3.5.1 Nicht eindeutig charakterisierbare Fälle----------------------------------------------------- 72 3.5.2 Robertsonsche Translokationen lassen durch FISH mit spezifischen Sonden der acro-p11-Region konservierte Bruchpunkte erkennen---------------------------- 73 3.6 DURCH MCB SICHTBARE UNTERSCHIEDE IN DER BRUCHPUNKTVERTEILUNG BEI KONSTITUTIONELLEN UND TUMORSPEZIFISCHEN ABERRATIONEN------------------------- 74 4 DISKUSSION ---------------------------------------------------------------------------------76
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