Table Of ContentAn investigation into genetic polymorphisms and
abdominal aortic aneurysms
John Benjamin Wild BSc (Hons), MBChB, MRCS(Eng)
Thesis submitted for the degree of Doctor of
Medicine, University of Leicester
September 2015
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Abstract
Introduction
A wide variety of genetic polymorphisms have demonstrated a significant association to
the disease abdominal aortic aneurysm (AAA). Polymorphisms in rs217120 (Cathepsin C
gene CTSC) and rs1466535 (low density lipoprotein receptor related protein 1 gene, LRP1)
have both been associated with AAA, rs1466535 from a genome wide association study
(GWAS).
Materials & Methods
Associations were first confirmed then exons in close proximity to the polymorphisms
were re-sequenced in order to detect new mutations. Protein levels were assayed and
tissue was stained in order to determine the arterial layer that the LRP1 protein resides
within. TaqMan genotyping was undertaken in order to determine allele frequency of the
SNPs of interest in cohorts from Belfast (211 AAA, 262 controls), Viborg (473 AAA, 195
controls), Leeds (214 AAA and 249 controls) and Leicester (266 AAA, 143 controls).
Subsequently a list of linked SNPs was generated and the same samples were analysed in
order to fine map the gene(s) of interest.
Samples from Leicester (96 AAA, 96 controls) were sequenced. Serum protein
concentrations were measured with an ELISA (43 AAA, 26 controls). Immunohistochemical
analysis of aortic biopsies (n = 6) was also performed.
Results
rs217120 failed to associate with AAA in 3 of 4 the replication cohorts and meta-analysis
of all data was also not significant, odds ratio 1.17 (95% Confidence Interval 0.98-1.39), P
value = 0.08. rs1466535 did not significantly associate with AAA in the replication cohorts
however when the data was meta-analysed with the discovery phase of the GWAS (1866
AAA, 5435 controls) the association was significant, odds ratio 1.23 (95% Confidence
Interval 1.14-1.33), P value 1.25x10-7. A second SNP within LRP1, rs11172114, was found to
have a more significant association, odds ratio 1.23 (95% Confidence Interval 1.14 to 1.31),
P value 3.87x10-8. Re-sequencing of the 3 LRP1 exons did not detect any novel mutations.
Serum LRP1 levels between cases and controls were not different (mean concentration
583.4 v 631.3ng/ml, P value = 0.69). There was also no significant difference when samples
were analysed by SNP genotype, rs1466535 P value = 0.07. Analysis of aortic tissue
biopsies did not determine an association between LRP1 genotype and cellular location.
Conclusion
SNPs within LRP1 associate with AAA, with rs11172114 being most significant. However
the polymorphism is not associated with protein levels in serum and is not clearly
localizing to any given layer of the arterial wall. More investigation is required to
determine how LRP1 affects aneurysm development and growth.
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Acknowledgments
Firstly I would like to thank all of the individuals that contributed their samples to
my study, without then nothing would have been possible. Also I have to also
thank Heart Research UK for providing me valuable funding, without them I would
not have been able to achieve as much as I have.
I received excellent supervision from Mr M Bown and Prof. R Sayers, they guided
me through so much and I will never forget that. The people of the Department of
Vascular Surgery, Leicester Royal Infirmary, allowed me to access to their patients
and for this I am grateful, the teams from AAA surveillance and the Vascular
Technologists and the consultant surgeons, Mr McCarthy, Mr Nasim, Mr Dennis,
Prof. London and Prof. Naylor.
At the University of Leicester I have to thank the people from the RKCSB, Janet,
Kay, Jonathon and Julie, for putting up with me day after day. To Dr Nic Sylvius, for
lessons in genotyping and French. To Dr Mervin Thomas for his tutoring in DNA
analysis. Also to my fellow Clinical Research Fellows, Phil, Eyad, Jeremy, Metz,
Nikesh, Mo, David and Badri, you all helped me one way or another from coffee
making to hours of lab work.
Finally to my family, mum & dad, without you there is no me. To my wonderful
Aoife and Thomas, born at the start and finish of my research, you are the best
distraction anyone could have wished for and I love you to pieces. Finally to the
beautiful and long suffering Mrs Wild, Laura I love you with all my heart and you
know that this would never have come in to existence without all of your support,
you rock my world.
“Learn from yesterday, live for today, hope for tomorrow. The
important thing is not to stop questioning”
Albert Einstein.
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Statement of Originality
The work on which this thesis is based is my own independent work except where
acknowledged.
J B Wild
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Funding, Publications and Presentations arising from this
thesis
Funding:
Heart Research UK, Research Training Fellowship 2011, £49,947.
Publications:
Wild JB, Stather PW, Sylvius N, Choke E, Sayers RD, Bown MJ. Low density
lipoprotein receptor related protein 1 and abdominal aortic aneurysms. Eur J Vasc
Endovasc Surg. 2012 Aug;44(2):127-32
Presentations:
Rayt HS, Wild B, Scott J, Johnson A, Hughes A, Bradley D, Lindholt J, Samani N,
Thompson J, Sayers R, Bown MJ. Evaluation of the cathepsin gene and abdominal
aortic aneurysms. Vascular Society Annual Meeting 2011 Society for Academic
Surgery Session, Edinburgh.
Wild JB, Butt HZ, Sylvius N, Stather PW, Jones GT, Scott DJA, Lindholt J, Choke E,
Sayers RD, Bown MJ. Refining the association between low density lipoprotein
receptor related protein 1 and abdominal aortic aneurysm. Vascular Society
Annual Meeting 2012 BJS Prize Session, Manchester.
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Contents
CHAPTER 1: THE AORTA AND THE ANEURYSM ...................................................................................... 1
1.1 AORTA .......................................................................................................................................... 2
1.2.1 ANEURYSM ................................................................................................................................... 3
1.2.2 HISTORICAL PERSPECTIVE ................................................................................................................ 3
1.2.3 CLASSIFICATION OF ANEURYSMS ....................................................................................................... 4
1.2.3.1 LOCATION................................................................................................................................. 4
1.2.3.2 TRUE AND FALSE ANEURYSMS ....................................................................................................... 5
1.2.3.3 ANEURYSM MORPHOLOGY ........................................................................................................... 6
1.2.3.4 CAUSE ..................................................................................................................................... 7
1.2.4 ABDOMINAL AORTIC ANEURYSM ...................................................................................................... 7
1.2.4.1 AAA DEFINITION ....................................................................................................................... 7
1.2.5 DIAGNOSIS OF AAA ...................................................................................................................... 8
1.2.5.1 AAA SCREENING ..................................................................................................................... 10
1.2.6 SEQUELAE OF AAA ...................................................................................................................... 12
1.2.5.1 RUPTURE OF AAA .................................................................................................................... 12
1.2.5.2 PRIMARY AORTOENTERIC FISTULA ............................................................................................... 14
1.3.1 INCIDENCE OF AAA ..................................................................................................................... 15
1.3.2 PREVALENCE OF AAA .................................................................................................................. 16
1.4 PATHOLOGICAL PROCESSES IN AAA .............................................................................................. 18
1.4.1 INFILTRATION WITH INFLAMMATORY CELLS ....................................................................................... 18
1.4.2 DESTRUCTION OF THE EXTRACELLULAR MATRIX .................................................................................. 19
1.4.3 PATHOGENESIS OF AAA: SUMMARY ............................................................................................... 20
1.5 RISK FACTORS FOR AAA ................................................................................................................. 21
1.5.1 SMOKING .................................................................................................................................. 21
1.5.2 MALE GENDER ........................................................................................................................... 23
1.5.3 INCREASING AGE ......................................................................................................................... 24
1.5.4 DIABETES .................................................................................................................................. 25
1.5.5 HYPERTENSION ........................................................................................................................... 25
1.5.6 HEIGHT ..................................................................................................................................... 26
1.5.7 ETHNICITY ................................................................................................................................. 27
1.5.8 FAMILY HISTORY ......................................................................................................................... 28
1.5.9 MODE OF INHERITANCE ................................................................................................................ 29
1.6 MEDICAL TREATMENT OF AAA ...................................................................................................... 31
1.6.1 MEDICAL TREATMENT .................................................................................................................. 31
1.6.2 LIFESTYLE .................................................................................................................................. 31
1.6.3 BETA BLOCKERS .......................................................................................................................... 34
1.6.4 STATINS .................................................................................................................................... 34
1.6.5 ANGIOTENSIN CONVERTING ENZYME............................................................................................... 35
1.6.6 ADDITIONAL MEDICATIONS ........................................................................................................... 36
1.6.1.6 MEDICAL TREATMENT OF AAA CONCLUSION ................................................................................. 38
1.7 SURGICAL TREATMENT OF AAA ..................................................................................................... 39
1.7.1 HISTORICAL PERSPECTIVE .............................................................................................................. 39
1.7.2 PROFESSOR OSCAR CREECH ........................................................................................................... 40
1.7.2.1 COMPLICATIONS OF OPEN ANEURYSM REPAIR ............................................................................... 42
1.7.2.2 LAPAROSCOPIC AAA REPAIR ...................................................................................................... 42
1.7.3 ENDOVASCULAR ANEURYSM REPAIR ............................................................................................... 43
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1.7.3.1 FENESTRATED EVAR................................................................................................................. 47
1.7.3.2 BRANCHED EVAR .................................................................................................................... 47
1.7.3.3 COMPLICATIONS OF EVAR ......................................................................................................... 48
1.7.3.4 ENDOVASCULAR ANEURYSM SEALING........................................................................................... 50
1.7.4 SURGICAL TREATMENT CONCLUSION ............................................................................................... 50
1.8 CHAPTER 1 CONCLUSION ............................................................................................................... 52
CHAPTER 2: GENES AND ABDOMINAL AORTIC ANEURYSMS2.1 AAA GENETICS.................................. 53
2.1.1 SINGLE NUCLEOTIDE POLYMORPHISMS ............................................................................................ 54
2.2 EXTRACELLULAR MATRIX DEGRADATION ...................................................................................... 56
2.2.1 ELASTIN .................................................................................................................................... 56
2.2.2 COLLAGEN ................................................................................................................................. 58
2.2.3 ECM PROTEIN SUMMARY ............................................................................................................. 58
2.3 MATRIX METALLOPROTEINASES ................................................................................................... 60
2.3.1 MMP9 .................................................................................................................................... 60
2.3.3 MMP3 .................................................................................................................................... 63
2.3.4 MMP13................................................................................................................................... 64
2.3.5 MMP1 .................................................................................................................................... 65
2.3.6 MMP12................................................................................................................................... 66
2.3.7 MMP10................................................................................................................................... 67
2.3.8 1 TISSUE INHIBITORS OF MATRIX METALLOPROTEINASES .................................................................... 69
2.3.8.2 TIMP1 .................................................................................................................................. 69
2.3.8.3 TIMP2 .................................................................................................................................. 70
2.3.8.4 TIMP3 .................................................................................................................................. 71
2.3.9 OTHER MMP REGULATORS........................................................................................................... 72
2.3.10 MMP AND AAA SUMMARY ....................................................................................................... 73
2.4 OTHER PROTEOLYTIC ENZYMES THAT AFFECT THE ECM................................................................ 74
2.5 ECM DEGRADATION AND GENETIC POLYMORPHISMS: CONCLUSION ........................................... 76
2.6. INFLAMMATION AND AAA ........................................................................................................... 77
2.6.1 INTERLEUKINS............................................................................................................................. 77
2.6.1.1 INTERLEUKIN 1 ........................................................................................................................ 77
2.6.1.2 INTERLEUKIN 6 ........................................................................................................................ 78
2.6.1.3 INTERLEUKIN 10 ...................................................................................................................... 79
2.6.2.1 TUMOUR NECROSIS FACTOR ....................................................................................................... 80
2.6.2.2 C REACTIVE PROTEIN ................................................................................................................. 82
2.6.2.3 HUMAN LEUKOCYTE ANTIGEN .................................................................................................... 83
2.6.3 OTHER INFLAMMATORY MEDIATORS ............................................................................................... 85
2.6.4 INFLAMMATORY POLYMORPHISM CONCLUSION ................................................................................ 86
2.7 CARDIOVASCULAR SYSTEM ........................................................................................................... 87
2.7.1.1 ANGIOTENSIN CONVERTING ENZYME............................................................................................ 87
2.7.1.2 ANGIOTENSINOGEN .................................................................................................................. 88
2.7.1.3 RECEPTORS RELATED TO ANGIOTENSIN .......................................................................................... 89
2.7.2 HOMOCYSTEINE METABOLISM ....................................................................................................... 90
2.7.2.1 METHYLENETETRAHYDROFOLATE REDUCTASE................................................................................. 91
2.7.2.2 METHYLHYDROFOLATE ENZYMES ................................................................................................. 92
2.7.3 CARDIOVASCULAR SYSTEM GENES CONCLUSION ................................................................................ 94
2.8 AAA AND GENOME WIDE ASSOCIATION STUDIES ......................................................................... 95
2.8.1 HELGADOTTIR ET AL. .................................................................................................................... 96
2.8.2 ELMORE ET AL. ........................................................................................................................... 96
2.8.3 GRETARSDOTTIR ET AL. ................................................................................................................. 97
2.8.4 BOWN ET AL............................................................................................................................... 98
2.8.5 JONES ET AL. ............................................................................................................................ 100
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2.8.6 BRADLEY ET AL. ........................................................................................................................ 101
2.8.7 AAA GWAS CONCLUSION ......................................................................................................... 103
2.9 CHAPTER 2 CONCLUSION ............................................................................................................. 104
CHAPTER 3: LRP1 AND ABDOMINAL AORTIC ANEURYSMS ............................................................... 105
3.1 LRP1 AND AAA ............................................................................................................................. 106
3.2 LRP1 STRUCTURE ......................................................................................................................... 108
3.3 LRP1 AND EXTRACELLULAR MATRIX DEGRADATION ................................................................... 110
3.4 LRP1 AND VASCULAR SMOOTH MUSCLE CELL DEPLETION .......................................................... 112
3.5 LRP1 AND INFLAMMATION ......................................................................................................... 115
3.6 LRP1 REGULATION ....................................................................................................................... 116
3.7 IMPLICATIONS FOR THE TREATMENT OF SMALL AAA ................................................................. 117
3.8 CHAPTER 3 CONCLUSION ............................................................................................................. 118
CHAPTER 4: AIMS .............................................................................................................................. 120
4.1 AIMS OF THIS THESIS ................................................................................................................... 121
4.2 HYPOTHESES ................................................................................................................................ 122
CHAPTER 5: SAMPLE COLLECTION ..................................................................................................... 123
5.1 COLLECTION OF SAMPLES ............................................................................................................ 124
5.2 COLLECTION OF PLASMA AND BUFFY COAT ................................................................................ 126
5.3 DNA EXTRACTION FROM BUFFY COAT. ....................................................................................... 127
5.4 ASSESSMENT OF DNA QUALITY ................................................................................................... 129
5.5 CHAPTER 5 CONCLUSION ............................................................................................................. 131
CHAPTER 6: SNP GENOTYPING .......................................................................................................... 132
6.1 SNP GENOTYPING BACKGROUND ................................................................................................ 133
6.2 SNP GENOTYPING METHODS ....................................................................................................... 133
6.3 QUALITY CONTROL ...................................................................................................................... 137
6.4 VALIDATION OF THE ASSOCIATION OF RS217120 WITH AAA ...................................................... 138
6.4.1 RS217120 GENOTYPING RESULTS ................................................................................................ 140
6.4.2 DISCUSSION OF THE VALIDATION OF RS217120 ............................................................................... 142
6.4.3 META-ANALYSIS OF RS217120 DATA ............................................................................................ 143
6.5 ANALYSIS OF SNPS WITHIN LRP1 ................................................................................................. 144
6.5.1 IMPUTATION ............................................................................................................................ 146
6.5.2 DNASE HYPESENSITIVITY ............................................................................................................ 150
6.5.3 GENOTYPING OF SAMPLES FOR SNPS WITHIN LRP1 ......................................................................... 152
6.5.4 RS1466535 GENOTYPING RESULTS ............................................................................................... 154
6.5.5 RS11172114 GENOTYPING RESULTS ............................................................................................. 156
6.5.6 RS4257011 GENOTYPING RESULTS ............................................................................................... 158
6.5.7 RS11172113 GENOTYPING RESULTS ............................................................................................. 160
6.5.8 RS4759044 GENOTYPING RESULTS ............................................................................................... 162
6.5.9 RS715498 GENOTYPING RESULTS ................................................................................................. 164
6.5.10 SNPS ASSOCIATED WITH RS1466535 DISCUSSION ......................................................................... 166
6.6 HAPLOTYPE ANALYSIS ................................................................................................................. 168
6.7 SNP GENOTYPING: DISCUSSION .................................................................................................. 170
6.7.1 CHAPTER 6 NULL HYPOTHESIS ..................................................................................................... 172
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CHAPTER 7: RE-SEQUENCING OF LRP1 EXONS ................................................................................... 173
7.1 RE-SEQUENCING .......................................................................................................................... 174
7.2 PATIENTS ..................................................................................................................................... 178
7.3 PRIMER DESIGN ........................................................................................................................... 179
7.4 OPTIMIZATION ............................................................................................................................ 180
7.4.1 TEMPERATURE GRADIENT............................................................................................................ 181
7.4.2 MAGNESIUM CONCENTRATION .................................................................................................... 183
7.4.3 DMSO CONCENTRATION ........................................................................................................... 184
7.5 REACTION REAGENTS .................................................................................................................. 187
7.6 SEQUENCING ............................................................................................................................... 189
7.7 DNA ANALYSIS ............................................................................................................................. 190
7.8 RESULTS OF MUTATION ANALYSIS .............................................................................................. 192
7.9 CHAPTER 7 CONCLUSION ............................................................................................................. 194
7.9.1 CHAPTER 7 NULL HYPOTHESIS ..................................................................................................... 194
CHAPTER 8: DETECTION OF SOLUBLE LRP1 IN PLASMA ..................................................................... 195
8.1 INTRODUCTION ........................................................................................................................... 196
8.2 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) .................................................................... 197
8.3 PATIENT DEMOGRAPHICS............................................................................................................ 201
8.4 RESULTS ....................................................................................................................................... 202
8.4.1 EFFECT OF CASE/CONTROL STATUS UPON SLRP1 CONCENTRATION ..................................................... 203
8.4.2 THE INFLUENCE OF GENOTYPE UPON SLRP1 CONCENTRATION ............................................................ 204
8.5 CHAPTER 8 CONCLUSION ............................................................................................................. 207
8.5.1 CHAPTER 8 NULL HYPOTHESIS ..................................................................................................... 207
CHAPTER 9; IMMUNOHISTOCHEMICAL STAINING OF AORTIC BIOPSIES ........................................... 208
9.1 CHAPTER 9 INTRODUCTION ......................................................................................................... 209
9.2 IMMUNOHISTOCHEMISTRY OF AORTIC BIOPSIES ....................................................................... 210
9.2.1 SAMPLE PREPARATION ............................................................................................................... 210
9.2.2 HAEMATOXYLIN AND EOSIN STAINING ........................................................................................... 210
9.2.3 LRP1-ACTIN DOUBLE STAIN ....................................................................................................... 211
9.3 IMMUNOHISTOCHEMICAL STAINING RESULTS ............................................................................ 213
9.4 CHAPTER 9 CONCLUSION ............................................................................................................. 222
9.4.1 NULL HYPOTHESIS ..................................................................................................................... 223
CHAPTER 10: DISCUSSION ................................................................................................................. 224
10.1 INTRODUCTION TO DISCUSSION................................................................................................ 225
10.2 SUMMARY OF FINDINGS ........................................................................................................... 226
10.2.1 SNP GENOTYPING ................................................................................................................... 226
10.2.2 RE-SEQUENCING ..................................................................................................................... 226
10.2.3 DETECTION OF SOLUBLE LRP1 IN PLASMA .................................................................................... 228
10.2.4 IMMUNOHISTOCHEMICAL STAINING OF AORTIC BIOPSIES ................................................................. 228
10.3 GENERAL LIMITATIONS .............................................................................................................. 229
10.4 FUTURE DIRECTIONS .................................................................................................................. 230
10.5 PRACTICAL USES FOR SNPS ASSOCIATED WITH AAA ................................................................. 231
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10.6 CONCLUSION ............................................................................................................................. 232
10.7 CORRECTIONS ............................................................................................................................ 233
REFERENCES ...................................................................................................................................... 235
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Description:SNPs of interest in cohorts from Belfast (211 AAA, 262 controls), Viborg (473 AAA, 195 controls), Leeds .. 1.7.2.2 LAPAROSCOPIC AAA REPAIR .
