Identification of genes involved in progression of retinoblastoma [Elektronische Ressource] / by Sandrine Gratias

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Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2005
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Identification of genes involved in progression of
retinoblastoma



Inaugural Dissertation




to obtain the degree of
Dr. rer. nat.
Fachbereich Bio- und Geowissenschaften
at the
University Duisburg-Essen





by
Sandrine Gratias
From Figeac (France)


April 2005

A part of the present work will be published or has been submitted in the following
journals:



Sandrine Gratias, Andreas Schüler, Ludger Klein Hitpass, Harald Stephan, Harald
Rieder, Stephanie Schneider, Bernhard Horsthemke, Dietmar R. Lohmann. (2005)
Genomic gains on chromosome 1q in retinoblastoma: consequences on gene expression
and association with clinical manifestation (in press at the International Journal of
Cancer).


Boris Zielinski, Sandrine Gratias, Grischa Toedt, Frank Mendrzyk, Daniel E. Stange,
Bernhard Radlwimmer, Dietmar R. Lohmann, and Peter Lichter. Detection of
chromosomal imbalances in retinoblastoma by matrix-based comparative genomic
hybridization (under revision in Genes Chromosome and Cancer).

Corinna Grasemann, Sandrine Gratias, Harald Stephan, Andreas Schüler, Alexander
Schramm, Ludger Klein Hitpass, Harald Rieder, Stephanie Schneider, Ferdinand
Kappes, Angelika Eggert, Dietmar R. Lohmann. Gains and overexpression identify
DEK and E2F3 as targets of chromosome 6p gains in retinoblastoma (submitted to
Oncogene).






























Die der vorliegenden Arbeit zugrundeliegenden Experimente wurden am Institut für
Humangenetik der Universität-Duisburg-Essen durchgeführt.

1. Gutachter: Prof. Dr. Bernhard Horsthemke
2. Gutachter: PD Dr. Ludger Klein-Hitpass
3. Gutachter: /
Vorsitzender des Prüfungsausschusses: Prof. Dr. Peter Bayer
Tag der mündlichen Prüfung: 29.06.2005

1 INTRODUCTION .................................................................................................... 1
1.1 Tumorigenesis................................................................................................... 1
1.2 Cancer genes ..................................................................................................... 3
1.2.1 Oncogenes................................................................................................. 3
1.2.2 Tumour suppressor genes ......................................................................... 4
1.2.3 Stability genes........................................................................................... 5
1.3 Retinoblastoma 5
1.3.1 Genetics of retinoblastoma ....................................................................... 6
1.3.2 Histopathology of retinoblastoma............................................................. 7
1.3.3 The origin of retinoblastoma..................................................................... 7
1.3.4 The RB1 gene 9
1.3.5 Progression of retinoblastoma................................................................... 9
1.4 Aims................................................................................................................ 10
1.4.1 Chromosome 1q gains............................................................................. 11
1.4.2 osome 16q losses ......................................................................... 11
1.4.3 Chromosome 1p gains 11
2 MATERIAL AND METHODS.............................................................................. 12
2.1 Material........................................................................................................... 12
2.1.1 Buffers, reagents and solutions............................................................... 12
2.1.2 Enzymes.................................................................................................. 13
2.1.3 Oligonucleotide arrays............................................................................ 13
2.1.4 Primary tumours...................................................................................... 13
2.1.5 Cell lines ................................................................................................. 13
2.1.6 Clones ..................................................................................................... 13
2.1.7 Primers and Probes ................................................................................. 16
2.1.8 Equipment............................................................................................... 16
2.2 Methods........................................................................................................... 16
2.2.1 Cell culture.............................................................................................. 16
2.2.2 DNA extraction from blood samples ...................................................... 17
2.2.3 tumour samples.................................................... 17
2.2.4 BAC clones.......................................................... 18
2.2.5 Restriction cleavage of DNA.................................................................. 19
2.2.6 RNA extraction from tumour samples 19
2.2.7 DNA bisulfite treatment.......................................................................... 20
2.2.8 Reconcentration of the DNA/RNA eluates............................................. 20
2.2.9 Measurement of DNA and RNA concentration...................................... 21
2.2.10 PCR analyses (with and without labelled primers)................................. 21
2.2.11 Methylation Specific PCR (MS-PCR).................................................... 22
2.2.12 Quantitative Multiplex PCR (QMPCR).................................................. 22
2.2.13 tive Microsatellite Analysis Real Time PCR: QuMA............... 26
2.2.14 MSA for LOH study ............................................................................... 28
2.2.15 Electrophoresis and analysis................................................................... 29
2.2.15.1 Agarose Gel electrophoresis ............................................................... 29
2.2.15.2 Automatic Capillary Electrophoresis (Genescan and Sequencing) .... 29
2.2.15.3 DNA sequencing................................................................................. 30
2.2.15.4 Genotyping of STR loci for identification of LOH on chromosome 16
30
2.2.16 Pyrosequencing (PSQ)............................................................................ 31
2.2.17 Comparative Genomic Hybridization..................................................... 33
2.2.18 Matrix-CGH............................................................................................ 35

2.2.18.1 Chromosome 1 specific chip............................................................... 35
2.2.19 Expression analysis................................................................................. 36
2.2.20 Statistical analysis of genetic findings and clinical manifestation.......... 36
3 RESULTS ............................................................................................................... 37
3.1 Identification of target genes on the long arm of chromosome 1 ................... 37
3.1.1 Matrix CGH ............................................................................................ 37
3.1.1.1 Custom chip 37
3.1.1.2 Matrix CGH with DNA arrays representing the whole genome............. 39
3.1.2 Quantitative multiplex PCR (QMPCR) .................................................. 41
3.1.2.1 Establishing and validating QMPCR...................................................... 41
3.1.2.2 QMPCR of DNA from specimens of primary retinoblastoma ............... 42
3.1.2.3 fm retinoblastoma cell lines.................................... 42
3.1.3 Confirming the results of QMPCR by independent methods ................. 46
3.1.3.1 Genomic Real-Time PCR ....................................................................... 46
3.1.3.2 Pyrosequencing of SNP loci ................................................................... 47
3.1.3.3 Conventional comparative genomic hybridization (CGH) ..................... 48
3.1.4 Expression data ....................................................................................... 49
3.1.5 Genomic gains in 1q and clinical manifestation..................................... 52
3.2 Identification of an oncogene on chromosome 1p.......................................... 54
3.2.1 Matrix-CGH............................................................................................ 54
3.2.2 RNA expression analysis ........................................................................ 55
3.3 Identification of allelic loss on chromosome 16............................................. 57
3.3.1 Microsatellite analysis (MSA) ................................................................ 57
3.3.2 PSQ ......................................................................................................... 59
3.3.3 MSPCR ................................................................................................... 60
3.3.4