Molecular genetics of abdominal aortic aneurysm

523 Pages·2017·17.99 MB·English

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Molecular genetics of abdominal aortic aneurysm
Chinien, Ganessen
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King's College London
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This electronic theses or dissertation has been
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Title:
Molecular genetics of abdominal aortic aneurysm

A uthor:Ganessen Chinien
The copyright of this thesis rests with the author and no quotation from it or information
derived from it may be published without proper acknowledgement.
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Molecular genetics of abdominal
aortic aneurysm
Ganessen Chinien
Thesis submitted for the degree of
Doctor of Medicine (Research)
2012
Academic Department of Surgery
Cardiovascular Division
King’s College London
1
Statement of originality
The work contained in this thesis is my own original work, except where
acknowledged in the text.
Acknowledgements
I would like to thank my supervisors, Prof Alberto Smith and Mr Matthew
Waltham for their support, guidance and patience throughout my MDRes. I would
also like to thank Prof Alberto Smith for his meticulous proof-reading of this thesis
and his patience. I am also grateful to Prof Kevin Burnand for his invaluable
guidance and support throughout my MDRes.
I would also like to thank Dr Matthew Arno and his team at the Genomics Centre
at Kings’ College London for their expert advice on microarray and real-time
PCR. Dr Julia Jenkins has been of great help with her expert advice on pathway
analysis.
I would also like to thank my colleagues at St Thomas’ Hospital for their help: Mr
Sanjay Patel, Mr Ashar Wadoodi, Mr James Orbell and Mr Prakash Saha for their
advice, support in specimen collection and for being good friends. Mrs Monica
Brennan has been instrumental to getting my thesis ferried from South Africa to
UK and back numerous times for the multiple corections.
Finally, I would like to thank my wife, my daughter and my parents for their
constant support and encouragement.
2
TABLE OF CONTENTS
LIST OF ABBREVIATIONS .................................................................................7
List of Figures...................................................................................................13
List of Tables.....................................................................................................18
ABSTRACT........................................................................................................21
Chapter 1...........................................................................................................24
OVERVIEW of AAA ...........................................................................................24
1.1 Definition............................................................................................................................25
1.2 Epidemiology......................................................................................................................26
1.3 Population screening..........................................................................................................28
1.4 Clinical presentation..........................................................................................................29
1.5 Risk Factors........................................................................................................................29
1.6 Management of AAA.........................................................................................................30
1.6.1 Historical.....................................................................................................................................30
1.6.2 Surgical management..................................................................................................................31
1.6.3 Medical management of AAA.....................................................................................................35
1.7 PATHOGENESIS OF AAA..............................................................................................39
1.7.1 Structure of the abdominal aortic wall.........................................................................................39
1.7.2 Extracellular matrix turnover.......................................................................................................40
1.7.3 Inflammation and auto-immune response....................................................................................41
1.7.3.1 TH1 TH2 responses.........................................................................................................43
1.7.3.2 Antigen activation in wall of AAA....................................................................................45
1.7.3.3 Autoantibodies and AAA..................................................................................................46
1.7.3.4 Putative self and non-self antigens involved in cellular and antibody responses in
AAA..................................................................................................................................................46
1.8 Proteolysis and extra-cellular matrix remodelling.........................................................47
1.8.1 Matrix metalloproteinases...........................................................................................................48
1.8.2 Cysteine proteases.......................................................................................................................52
1.8.3 Serine Proteases...........................................................................................................................53
1.9 Angiogenesis.......................................................................................................................55
1.10 Apoptosis..........................................................................................................................57
1.11 Atherosclerosis and AAA................................................................................................58
1.12 Oxidative Stress Theory..................................................................................................59
1.13 Haemodynamic factors and biomechanical wall stress................................................60
1.14 Genetics of AAA...............................................................................................................64
1.14.1 Candidate gene studies..............................................................................................................64
1.14.1.1 Genes in aortic wall structure.......................................................................................65
1.14.1.2 Genes involved in remodeling of aortic wall...............................................................66
1.14.1.3 Genes of the cardiovascular system...........................................................................68
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1.14.1.4 Genes involved in methionine metabolism.................................................................70
1.14.1.5 Genes involved in inflammation...................................................................................71
1.14.2 Linkage studies..........................................................................................................................73
1.14.3 Genomewide association studies...............................................................................................75
1.14.4 Gene expression studies.............................................................................................................77
1.15 Hypothesis and Aims.......................................................................................................79
1.15.1 Hypothesis.................................................................................................................................79
1.15.2 Aims..........................................................................................................................................80
Chapter 2...........................................................................................................81
RNA EXTRACTION AND QUALITY CONTROL................................................81
2.1 Introduction........................................................................................................................82
2.1.1 RNA and its extraction................................................................................................................82
2.1.1.1 Liquid phase extraction....................................................................................................84
2.1.1.2 Solid phase RNA extraction............................................................................................84
2.1.2 RNA QUALITY CONTROL......................................................................................................85
2.2 Material and Methods.......................................................................................................92
2.2.1 Samples used...............................................................................................................................92
2.3 RNA EXTRACTION.........................................................................................................93
2.3.1 Tissue preparation........................................................................................................................93
2.3.2 Tissue homogenisation and RNA extraction...............................................................................94
2.3.3 RNA purification.........................................................................................................................95
2.3.3.1 Precipitation with isopropanol.........................................................................................95
2.3.3.2 Purification with RNEasy kit (Qiagen)............................................................................95
2.4 RNA quantification and quality.......................................................................................97
2.5 RESULTS...........................................................................................................................97
2.5.1 Comparison of two methods of RNA isolation...........................................................................97
2.5.2 Quality of all RNA samples extracted.........................................................................................99
2.5.3 Patient demographics for microarray study...............................................................................103
2.5.4 RNA profiles of aortic tissues used in the microarray experiment............................................104
2.6 Discussion.........................................................................................................................109
Chapter 3.........................................................................................................111
Micro-array analysis.......................................................................................111
3.1 Introduction......................................................................................................................112
3.1.1 Principle of DNA microarray technology..................................................................................112
3.1.2 Fabrication of DNA microarray.................................................................................................113
3.1.3 General overview of target preparation and hybridisation.........................................................116
3.2 Methods............................................................................................................................118
3.2.1 Target preparation......................................................................................................................118
3.3 Data output quality control.276........................................................................................128
3.3.1 Probe array image inspection.....................................................................................................128
3.3.2 B2 Oligo performance...............................................................................................................128
3.3.3 Average background..................................................................................................................130
3.3.4 Noise..........................................................................................................................................130
3.3.5 Hybridisation controls: bioB, bioC, bioD and Cre....................................................................130
3.3.6 Internal control genes................................................................................................................131
3.3.7 Percent present...........................................................................................................................131
3.3.8 Scaling factor.............................................................................................................................131
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3.4 Results...............................................................................................................................133
3.4.1 cRNA synthesis.........................................................................................................................133
3.4.2 Fragmented cRNA.....................................................................................................................136
3.4.3 Background................................................................................................................................140
3.4.4 Noise..........................................................................................................................................142
3.4.5 Hybridisation controls: bioB, bioC, bioD and Cre....................................................................144
3.4.6 Internal control genes................................................................................................................146
3.4.7 Percent probe present.................................................................................................................148
3.4.8 Scaling factor.............................................................................................................................150
3.5 Discussion.........................................................................................................................152
Chapter 4.........................................................................................................153
Biostatistical analysis: differential expression............................................153
4.1 Introduction......................................................................................................................154
4.2 Methods............................................................................................................................155
4.2.1 Normalisation............................................................................................................................155
4.2.2 Quality control...........................................................................................................................156
4.2.3 Generating list of genes of interest............................................................................................157
4.2.4 Statistical analysis.....................................................................................................................158
4.2.5. Gene ontology..........................................................................................................................159
4.2.6 Identifying genes on chromosomes 19q13 and 4q31.................................................................159
4.3 Results...............................................................................................................................160
4.3.1 Normalisations...........................................................................................................................160
4.3.2 Principal component analysis....................................................................................................161
4.3.3 Hierarchal clustering..................................................................................................................164
4.3.4 Filtering for genes of interest.....................................................................................................167
4.3.5 Statistical analysis.....................................................................................................................168
4.3.6 Gene ontology...........................................................................................................................177
4.3.7 Genes differentiated on chromosome 19q13.............................................................................180
4.4 Discussion.........................................................................................................................181
Chapter 5.........................................................................................................183
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)................183
5.1 Introduction......................................................................................................................184
5.1.1 Overview of qRT-PCR..............................................................................................................184
5.1.2 Quantification strategies............................................................................................................184
5.1.3 Choice of housekeeping genes for relative quantification.........................................................185
5.1.4 Reverse transcription.................................................................................................................187
5.1.4 Priming consideration................................................................................................................188
5.1.5 qRT- PCR chemistry options....................................................................................................189
5.1.6 Real time amplification and analysis.........................................................................................194
5.2 Methods............................................................................................................................196
5.2.1 Experimental design..................................................................................................................196
5.2.2 Reverse transcription (RT)........................................................................................................198
5.2.3 qPCR Step.................................................................................................................................199
5.2.4 Statistical analysis of qRT-PCR data.........................................................................................202
5.3 Results...............................................................................................................................203
5.4 Discussion.........................................................................................................................212
Chapter 6.........................................................................................................213
5
Biostatistical analysis: pathways and networks..........................................213
6.1 Introduction......................................................................................................................214
6.2 Methods............................................................................................................................215
6.3 Results...............................................................................................................................217
6.3.1 Pathway analysis.......................................................................................................................217
6.3.2 Network analysis.......................................................................................................................221
6.4 Discussion.........................................................................................................................233
6.4.1 Pathway analysis.......................................................................................................................233
6.4.2 Network analysis.......................................................................................................................235
Chapter 7.........................................................................................................239
Discussion, future work and conclusions....................................................239
7.1 General Discussion...........................................................................................................240
7.1.1 Comparison with other microarray studies................................................................................240
7.1.2 Genes differentially expressed on chromosomes 4q31 and 19q13............................................241
7.1.3 Inflammation.............................................................................................................................243
7.1.4 Proteolysis.................................................................................................................................245
7.1.5 Apoptosis...................................................................................................................................246
7.1.6 Angiogenesis.............................................................................................................................247
7.1.7 Bone morphogenic protein 6.....................................................................................................247
7.2 Limitations of this study..................................................................................................248
7.3 Future work and conclusions..........................................................................................249
Reference List.................................................................................................251
Appendices .....................................................................................................284
6
LIST OF ABBREVIATIONS
A 260/280 Ratio of absorbance of RNA at 260 and 280 nm
AAA Abdominal aortic aneurysm
ACE Angiotensin converting enzyme
ADAM A disintegrin and metallopeptidase
AHCY Adenosylhomocysteine
ALK Anaplastic lymphoma receptor tyrosine kinase
Apo Apolipoprotein
AOD Aortic occlusive disease
BAI-1 Brain specific angiogenesis inhibitor 1
BTG-2 B – cell translocation gene - 2
DNA Deoxyribonucleic acid
CCR3 C – C chemokine receptor type 3
CD40L CD40 ligand
cDNA Complementary deoxyribonucleic acid
C/EBPalpha CCAAT/enhancer binding protein, alpha
cMyc myelocytomatosis viral oncogene homolog (avian)
COL3A1 Collagen type 3A1
COLEC12 Collectin subfamily member 12
7
COX-2 Cyclooxygenase 2
CREB1 cAMP responsive element binding protein 1
CSH Chorionic somatomammotropin hormone
CTLA4 Cytotoxic T – lymphocyte associated antigen – 4
D-PBS Dulbecco’s phosphate buffered saline
DTT Dithiothreitol
DMRT1 Doublesex and mab-3 related transcription factor 1
dNTP Deoxyribonucleotide triphosphate
EGR Early growth response
ELN Elastin
ER Endoplasmic recticulum
ESR Estrogen receptor
Ets- E-twenty six
FAM 6-carboxyfluorescin
FCGRT Fc fragment of IgG, receptor, transporter, alpha
FGF Fibroblast growth factor
FOLH1 Folate hydrolase
FOS FBJ murine osteosarcoma viral oncogene homolog
FOXM1 Forkhead box M1
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