Genetic alterations in MENX-associated pheochromocytoma [Elektronische Ressource] / Alena Shyla

Bahar.Sanli

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Technische Universität München
Lehrstuhl für Entwicklungsgenetik





Genetic alterations in MENX-associated pheochromocytoma



Alena Shyla


Vollständiger Abdruck der von der Fakultät
Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
der Technischen Universität München zur Erlangung des akademischen
Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. S. Scherer
Prüfer der Dissertation: 1. apl. Prof. Dr. J. Graw
2. Univ.- Prof. Dr. A. Gierl
3. Univ.- Prof. Dr. M. J. Atkinson





Die Dissertation wurde am 18.02.2008 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 15.05.2008 angenommen.
Ich erkläre hiermit an Eides statt, dass ich die vorliegende Arbeit selbständig ohne unzulässige
fremde Hilfe angefertigt habe.

Die verwendeten Literaturquellen sind im Literaturverzeuchnis vollständig zitiert.




München, den



Alena Shyla
































TABLE OF CONTENTS

1. INTRODUCTION 1

1.1. Genetics and biology of pheochromocytoma (PC) 1
1.2. Familial forms of PC and mechanisms of their tumorigenesis 3
1.2.1. Multiple endocrine neoplasia type 2 syndrome (MEN2) 3
1.2.2. Neurofibromatosis type I (NF1) 6
1.2.3. von Hippel-Lindau disease (VHL) 8
1.2.4. PC-PGL syndromes characterized by germline mutations of the succinate
dehydrogenase gene (SDH) family 11
1.2.5. Inherited PC susceptibility genes and neuronal developmental
apoptosis pathway 14
1.3. Nonsyndromic sporadic PC 16
1.4. Involvement of other genes in PC development 18
1.4.1. Novel PC susceptibility loci 18
1.4.2. Somatic mutations in PC 18
1.5. Comparative genomic hybridization (CGH) and gene expression
array analyses of PC 20
22 1.6. Animal models of PC
1.7. The rat model of multiple endocrine neoplasia X (MENX) syndrome 24
1.8. Cyclin-dependent kinase inhibitor CDKN1B 26
1.8.1 CDKN1B and its role in human PC and other neuroendocrine tumors
formation 28
1.9. Aim of the work 29

2. MATERIALS 31

3. METHODS 36

3.1. Tumor and nontumor tissue samples 36
3.1.1. Preparation of tumor and nontumor tissue for molecular analysis 36
3.1.2. Genotyping on the Cdkn1b gene 36
3.2. Isolation of Nucleic Acids 36
3.2.1. Isolation of genomic DNA from fresh-frozen tissue 36
I3.2.2. Isolation of genomic DNA for CGH array 38
3.2.3. Isolation of RNA from snap - frozen tissue 38
3.2.4. Isolation of RNA from microdissected tissue 39
3.3. Quantification of Nucleic Acids 39
3.4. LOH analysis 39
3.4.1. Microsatellite markers 40
3.4.2. Amplification of DNA microsatellites 41
3.4.3. Electrophoresis of the DNA extraction products and PCR amplification
products 42
3.4.4. Quantification of the allelic ratio 44
3.5. Fragment analysis 44
3.6. TaqMan PCR 45
3.6.1. Reverse transcription 45
3.6.2. PCR amplification 46
3.6.3. Quantification 48
3.7. Automated fluorescent sequencing 49
3.7.1. PCR reaction 50
3.7.2. Purification of PCR products 50
3.7.3. Sequencing reaction 51
52 3.8. Fluorescence in situ hybridization analysis (FISH)
3.8.1. Probe preparation and labelling 53
3.8.2. Sample preparation 55
3.8.3. Denaturation of probe and sample 56
3.8.4. Hybridization of probe to sample (annealing) 57
3.8.5. Detection 57
3.8.6. Analysis of the data 59
3.9. CGH array 59

4. RESULTS 62

4.1. The group of experimental animals 62
4.1.1. Histological analysis of rat adrenal glands with PC 63
4.2. Microsatellite-based allelotyping of somatic genetic changes in PC
63 (LOH analysis)
4.2.1. Optimization of PCR conditions 65
II4.2.2. Allele dilution experiments 67
4.2.3. Genome-wide screening for LOH/AI 68
4.2.4. LOH/AI analysis for genes predisposing to PC development 73
4.3. Quantification of mRNA level of Cdkn2a, Cdkn2c, Sctr, Sdhd and
Rassf1 gene in MENX-affected rats 77
4.3.1. Validation of standard curves 77
4.3.2. mRNA expression level of Cdkn2a, Cdkn2c, Sctr genes in adrenal glands
with PC 79
4.3.3. mRNA expression level of Sdhd, Rassf1 genes in adrenal glands with PC 79
4.4. Sdhd and Rassf1 sequence analysis 83
4.5. FISH analysis of AI regions on chromosomes 8 and 19 84
4.5.1. Verification and validation of the BAC clones as FISH probes, metaphase
FISH analysis 84
4.5.2. Interphase FISH analysis 87
4.6. CGH array studies to identify genomic losses or gains in PC of
MENX rats 96
4.6.1. CGH array data analysis 98
4.6.2. Comparative analysis of data received after microsatellite analysis, FISH
and CGH array analysis for rat chromosomes 8 and 19 105

5. DISCUSSION 107

5.1. Candidate LOH/AI loci in MENX-PCs 107
5.1.1. LOH/AI loci on rat chromosome 8 and their syntenic regions in the
human genome 109
5.1.1.1. Rat chromosomal locus 8q31-q32 109
5.1.1.2. Rat chromosomal loci 8q11-q12 and 8q22-q24 112
5.1.1.3. Rat chromosomal locus 8q24 113
5.1.2. LOH/AI loci on rat chromosome 19 and their syntenic regions in the
113
human genome
5.1.2.1. Chromosomal loci 19p14-p12 and 19q12 (37.6-53.9 Mb) 113
5.1.2.2. Rat chromosomal locus 19q11 (24.7-26.3 Mb) 116
5.1.2.3. Rat chromosomal locus 19q12 (53.9-56.9 Mb) 116
5.1.2.4. Rat chromosomal locus 19q11 (26.3-34.4 Mb) 117
5.2. Copy number changes of candidate regions in MENX-PCs 117
III5.3. Analysis of rat homologs of genes predisposing to or involved in PC
development 119

6. SUMMARY 123
ZUSAMMENFASSUNG 127

131 7. REFERENCES

8. APPENDIX 150

PRESENTATIONS

ACKNOWLEDGEMENTS

LEBENSLAUF




















IVABBREVIATIONS

AI allelic imbalance
BAC clone bacterial artificial chromosome clone
Bcl-2 apoptosis regulator Bcl-2 protein, encoded by the
BCL2 gene
Biotin -16-dUTP biotin-16-2'- deoxyuridine -5'- triphosphate
BMPs bone morphogenetic proteins
BMP2 bone morphogenetic protein 2, encoded by the BMP2
gene
BMP4 bone morphogenetic protein 4, encoded by the BMP4
gene
CDK cyclin dependent kinase
CDKIs cyclin-dependent kinase inhibitors
Cdkn1a cyclin-dependent kinase inhibitor 1A (Rattus
norvegicus )
CDKN1B kinase inhibitor 1B
CDKN1C kinase inhibitor 1C
CDKN2B kinase inhibitor 2B
CDKN2A kinase inhibitor 2A
CDKN2C kinase inhibitor 2C
cDNA complementary DNA
CgA chromogranin A
CGH comparative genomic hybridization
CSR cysteine-rich region of neurofibromin
CT value threshold cycle value
DAPI 4',6-diamino-2-phenylindole dihydrochloride
dATP deoxyadenosin-5'-triphosphat
dCTP deoxycytidine-5'- triphosphate
ddNTP dideoxyribonucleotide
dGTP deoxyguanosine-5'
Digoxigenin -11- UTP digoxigenin-11-uridine-5'- triphosphate
dITP deoxyinosine-5'-triphosphate
DNA deoxyribonucleic acid
dNTP deoxynucleotide triphosphate
VdTTP deoxythymidine-5'-triphosphate
dUTP deoxyuridine-5'
Egl