Genomic Research to Identify Novel Pathways in the Development of Abdominal Aortic Aneurysm

Abstract

Abdominal aortic aneurysm (AAA) is a common disease with a large heritable component. There is a need to improve our understanding of AAA pathogenesis in order to develop novel treatment paradigms. Genomewide association studies have revolutionized research into the genetic variants that underpin the development of many complex diseases including AAA. This article reviews the progress that has been made to date in this regard, including mechanisms by which loci identified by GWAS may contribute to the development of AAA. It also highlights potential post-GWAS analytical strategies to improve our understanding of the disease further.

Full text

Hindawi Publishing Corporation
Cardiology Research and Practice
Volume 2012, Article ID 852829, 8pages
doi:10.1155/2012/852829
Review Article
Genomic Research to Identify Novel Pathways in
the Development of Abdominal Aortic Aneurysm
Seamus C. Harrison,1Anastasia Z. Kalea,1Michael V. Holmes,2
Obi Agu,3and Steve E. Humphries1
1Centre for Cardiovascular Genetics, BHF Laboratories, the Rayne Building, Department of Medicine,
University College London (UCL), 5 University Street, London WC1E 6JF, UK
2Genetic Epidemiology Group, Department of Epidemiology and Public Health, University College London,
1-19 Torrington Place, London WC1E 7HB, UK
3Department of Vascular Surgery, University College London Hospital, London, NW1 2BU, UK
Correspondence should be addressed to Seamus C. Harrison, seamus.harrison@ucl.ac.uk
Received 29 July 2011; Accepted 27 October 2011
Academic Editor: Janice Tsui
Copyright © 2012 Seamus C. Harrison et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Abdominal aortic aneurysm (AAA) is a common disease with a large heritable component. There is a need to improve our
understanding of AAA pathogenesis in order to develop novel treatment paradigms. Genome wide association studies have
revolutionized research into the genetic variants that underpin the development of many complex diseases including AAA. This
article reviews the progress that has been made to date in this regard, including mechanisms by which loci identified by GWAS may
contribute to the development of AAA. It also highlights potential post-GWAS analytical strategies to improve our understanding
of the disease further.
1. Introduction
Abdominal aortic aneurysm (AAA) is a common, late onset
disease which, left untreated, can rupture with a high resul-
tant mortality. Approximately 5% of Caucasian males aged
65–74 will harbor a AAA [1] and the major risk factors for
the condition include male sex, cigarette smoking, a history
of cardiovascular disease, and a family history of AAA [2,3].
Currently, the best predictor of rupture is maximal aneurysm
diameter and surgical repair is indicated in AAA greater than
5.5 cm [4]. Population screening with abdominal ultrasound
scans (US) reduces the burden of aneurysm related death
[5,6], but there is a lack of evidence to support any
pharmacological therapies to attenuate AAA progression
and/or rupture. The advent of endovascular aneurysm repair
has reduced short-term perioperative mortality associated
with AAA repair [7] but nationwide audits indicate that
elective repair still carries a mortality risk in region of 1.5–
7% [8]. In patients deemed unfit for surgical repair ten-
year survival is less than 25% [9]. Understanding the genetic
architecture of the condition may provide a blueprint for
uncovering novel pathobiological pathways and targets for
nonsurgical treatments.
The role that genetic factors play in the development
of AAA has become increasingly prominent in recent
years following Clifton’s initial observation that the disease
appeared to run in families [10]. Family history of AAA is an
established risk factor for the disease, with male first-degree
relatives of probands at approximately fourfold greater risk
than the general population [11–13]. A twin-study of AAA
has estimated the heritability to be as high as 70% [14], and
familial studies have failed to demonstrate consistent modes
of inheritance, suggesting that it is likely to be a complex
disease [13,15], resulting from a complicated network of
environmental and genetic risk factors. There has been some
progress in discovery of rare monogenic cause of aneurysmal
disease in thoracic aorta (Ta ble 1 ) but in common with
other complex disorders, deciphering causal genetic variants
in AAA has proved a difficult task. Familial-based linkage
studies have identified areas of the genome that are strongly

2 Cardiology Research and Practice
Tab le 1: Monogenic causes of thoracic aortic diseases.
Phenotype/syndrome Gene Reference
Marfan syndrome FBN1 [17]
Loeys-Doetz—ascending
aortic aneurysm TGFBR1 and TGFBR2 [18]
Thoracic aortic aneurysm MYH11, ACTA2, SMAD3 [19–21]
associated with the disease, but attempts to refine the signal
have so far been unsuccessful [15,16].
2. Genetic Studies of AAA
2.1. Candidate Gene Approaches. The “common-disease
common-variant” hypothesis poses that common complex
diseases arise from the accumulation of genetic variants,
each with a modest effect on risk (low penetrance) and
environmental risk factors [22,23]. It is this hypothesis that
has underpinned the developments of genetic association
studies, whereby the frequency of indexed genetic variants is
compared between cases and controls.
A number of candidate gene association studies for AAA
have been published. Review of the literature, however,
reveals that many studies were underpowered and gave
inconsistent results, a problem shared by many other com-
plex disorders [24]. Small studies with a low Pvalue obtained
by chance have been more readily published than negative
findings (so-called publication bias), and the results are often
not replicated in larger studies with greater statistical power.
Despite this, meta-analysis of candidate gene studies suggests
that single nucleotide polymorphisms (SNPs) in genes of
the renin-angiotensin system and folate metabolism are
consistently associated with an increased risk of developing
AAA (Tabl e 2)[25,26]. There has been considerable interest
in the role of polymorphisms in the TGF-βsuperfamily and
risk of developing AAA as these genes have been causally
implicated in aneurysmal disease affecting the thoracic aorta.
Baas et al. found association between SNPs in TGF-βreceptor
1and2(TGFBR1 and TGFBR2) and risk of AAA in a Dutch
cohort [27], but these associations were not replicated in
two cohorts from New Zealand and Australia [28]. There
have also been studies demonstrating suggestive associations
between SNPs in Latent Transforming Growth Factor 4
(LTBP 4) and expansion of AAAs, but again, this finding has
not been replicated in independent follow-up studies [29].
Efforts by the Human HapMap consortium (http://
hapmap.ncbi.nlm.nih.gov/index.html.en/), the SNP consor-
tium (http://www.ncbi.nlm.nih.gov/SNP/), and more re-
cently the 1000 genome project (http://www.1000genomes
.org/) have uncovered much of the common variation seen
throughout the human genome. Linkage disequilibrium
(LD: the nonrandom association of alleles at two or more
loci) means that only a fraction of all possible SNPs require
genotyping, in order to impute information on nontyped
genetic variation, and chips that simultaneously genotype up
to 1 million variants at a time are commercially available.
In genomewide association studies (GWASs), a panel
of common SNPs (minor allele frequency >5%) capturing
common genetic variation across the entire genome is
compared in groups of cases and controls. This approach
is “hypothesis-free” and therefore not subject to potential
biases seen in candidate gene studies. Owing to the large
number of independent test in a single association study,
there are many factors to consider when designing a GWAS.
Most importantly, this multiple testing strategy results in
a large number of potentially false positive associations.
To adjust for this, stringent criteria for “genome wide
significance” are applied and replication of findings in
independent cohorts is required. A potential consequence
of this is that many true-positive associations may be lost
in the “statistical noise”. A second issue is that with only a
few exceptions, the effect size of common variants is small.
Carriers of risk alleles are generally at 10–30% increased odds
of disease compared to noncarriers. These characteristics
necessitate extremely large sample sizes in order to have suf-
ficient statistical power, with recent publications combining
multiple studying hundreds of thousands of subjects at a
time [14,30]. As of June 2011, 951 GWASs have now been
published in a wide range of disorders and traits (http://www
.genome.gov/gwastudies/).
2.2. GWASs and AAA. In 2007, the field of genomic research
was ignited by simultaneous publication of three GWASs
of cardiovascular disease [31–33]. Each of the studies
demonstrated a strong association between common SNPs
on Chromosome 9p21.3 [34], in a gene desert (an area of the
genome with no known protein-coding genes). These data
exemplified the power of GWAS, as this locus would not have
been given priority using a candidate gene approach. The
limitations were, however, also highlighted as the functional
significance of this locus was unclear. It has taken a further
3-4 years to understand the biological mechanisms by which
these variants act, and the translational benefit of these
discoveries is not likely to be realized in the near future. A
year following publication of these GWAS, it was reported
that SNPs at this locus were also strongly associated with
the presence of AAA [34].TheseSNPsarecommoninthe
population (risk allele frequency ∼45–50%), and individuals
carry 0, 1, or 2 risk alleles. The risk of developing AAA is
increased by ∼30% per allele carried. The association with
AAA has now been replicated in a number of well-powered
case-control studies (Tabl e 3)[35–37].
The first GWAS specifically of AAA was published in
2009 and identified association of on SNP on Chr3p12.3 with
AAA [38]. This association did not meet conventional levels
of genomewide significance and has not been replicated in
independent sample sets [39]. However, in 2010 a larger
GWAS with greater statistical power reported a novel asso-
ciation with sequence variant in DAB2IP on Chr9q33 [40].
The discovery phase included 1,292 individuals with AAA
(defined as an infrarenal aortic diameter >3 cm) and 30,530
unscreened controls (a small proportion of whom are likely
to harbor AAA), while follow-up replication studies included
3,297 cases and 7,451 controls (all cases and controls were

Cardiology Research and Practice 3
Tab le 2: SNPs associated with AAA after meta-analysis of candidate gene studies [25,26].
Gene/polymorphism Number of studies (total cases/controls) Effect size (OR and 95% CI)
Angiotensin type 1 Receptor/A116C (rs5186) 1 study, 3 populations (1226/1712) 1.386 (1.2–1.601)
Angiotensin converting Enzyme I/D (rs4646994) 4 (1657/2238) 1.238 (1.12–1.36)
Methlyenetetrahydrofolate reductase +677C>T 5 (1086/895) 1.234 (1.020–1.494)
Matrix metalloproteinase 9 (MMP9, 1562C>T ) 3 (848/802) 1.09 (1.01–1.18)
Tab le 3: Association with SNPs in the 9p21 locus with AAA.
Author Cases/Controls SNP OR (P-value)
Helgadottir et al. [34] 2836/16732 rs10757278 1.31 (1.2E−12)
Bown et al. [35] 899/815 rs1333049 1.22 (0.004)
Thompson et al. [36] 741/1366 rs10757278 1.38 (0.03)
of European ancestry). The variant conferred a per allele
odds ratio for AAA of 1.21, a smaller effect than that seen
with the 9p21 variant. Interestingly, the investigators also
found an association between this SNP and CHD, venous
thromboembolism, and peripheral arterial disease and the
association with CHD has now been replicated in further
independent cohorts [41]. Further GWASs are expected in
the future [30], and it is possible that meta-analyses of these
datasets will uncover further variants associated with the
disease.
2.3. Functional Analysis of GWAS Loci to Uncover Novel Pa-
thobiological Pathways. Initial excitement from three sepa-
rate GWAS reporting robust associations between common
risk variants on Chr9p21.3 and myocardial infarction was
tempered by the fact that the functional significance of
the locus was not immediately obvious. The lead SNP
(or any in close LD with it) does not lie in a protein
coding gene. It has, however, been identified that this risk
variant overlaps with the recently annotated noncoding RNA
(ncRNA), ANRIL. NcRNAs can alter expression of protein
coding genes by mechanisms such as gene silencing, DNA
methylation, chromatin remodeling, and RNA interference
[42]. Functional studies of this locus have demonstrated
that carriers of the risk variant have reduced expression of
ANRIL, along with other nearby genes such as CDKN2A and
CDKN2B [43] which are inhibitors of cellular senescence
involved on controlling cellular proliferation and apoptosis.
Jarinova et al. found that the risk locus has enhancer activity
in primary human aortic smooth muscle cells and that
pathways involved in cellular proliferation were upregulated
in risk allele carriers [44]. Visel et al. then demonstrated
that targeted deletion of this region in a mouse model leads
to increased expression of the CDKN2A and CDKN2B and
that aortic smooth muscle cells from these animals displayed
excessive proliferation and diminished senescence [45]. More
recently, it was shown that the region at 9p21 is densely
packed with enhancer sites that are capable of altering
expression of both neighboring and distant genes by physical
interaction. Specifically, variants associated with CHD (and
AAA) disrupt binding of the transcription factor STAT1,
which results in altered expression CDKN2A and CDKN2B,
MTAP (methylthioadenosine phosphorylase, an enzyme that
plays a major role in polyamine metabolism), and IFNA21
(interferon alpha-21).It was also demonstrated that the
transcriptional control of the 9p21 enhancers was remodeled
with interferon-γ, providing evidence that genetic variation
is one factor that determines the response to inflammatory
stimuli within the vasculature [46].
The SNP in DAB2IP discovered by GWAS also associates
with coronary artery disease, peripheral arterial disease,
venous thromboembolism, and pulmonary embolism but
shows no association with any classical CHD risk factors
[40,41]. DAB2IP, located on Chromosome 9q33, is a GTPase
activating protein thought to play an important role in
prostate cancer metastasis [47].ASNPinthisgenehas
been associated with aggressive prostate cancer [48], while
in vitro functional studies have demonstrated that loss of
the protein leads to enhanced cell proliferation and reduced
apoptosis, via the PI3-Akt pathway [49]. DAB2IP expression
is significantly reduced in AAA tissue compared to tissue
from healthy controls [50], and this SNP did correlate with
reduced expression of the protein in aortic tissue (though
this was not reproduced in mammary artery tissue) [40].
It is possible, therefore, that this variant also promotes
excessive vascular smooth muscle cell (VSMC) proliferation,
through reduced expression of DAB2IP in aortic tissue.
Interestingly, DAB2IP expression is modulated by EZH2,
a histone methyltransferase forms part of the polycomb
repressor complex, and has been proposed as a potential
drug target in prostate cancer [51,52]. If, at a molecular
level, the link between genetic variation at this locus, DAB2IP
expression, and vascular disease was uncovered, enzymes
such as EZH2 could also be potential novel targets in
pharmacological therapies to attenuate AAA formation.
Whilst it appears that the two SNPs discovered for AAA
may both be influencing a common disease pathway, there
was no evidence of epistatic interaction between the 9p21
and DAB2IP SNP, with simply additive effects on AAA risk
[40]. We have found the same with regard to risk of CHD;
approximately 40% of the population who carry 2 or more
risk alleles at these loci have a hazard ratio for myocardial
infarction of 1.7 compared to individuals carrying zero
risk alleles [41]. This suggests that accumulation of small

4 Cardiology Research and Practice
disturbances in different elements of the VSMC proliferation
pathway combines to increase the risk of both atherosclerosis
and AAA.
The small effect sizes seen with GWAS-identified variants
do not preclude potential biological importance, as they
may highlight important pathways in disease [53]. For
example, genes highlighted by GWAS of type 2 diabetes
mellitus (T2DM) are known targets for thiazolidinediones
and sulphonylureas [54], drugs commonly used in this
condition. For AAA, the genomewide data are pointing to
pathways involved in promoting excessive VSMC prolifera-
tion. Cigarette smoking, a major environmental risk factor
for both diseases, leads to increased levels of proliferation in
VSMCs [48,55], whilst a role for excessive VSMC prolifera-
tion in aneurysm formation elsewhere in the arterial tree has
been demonstrated—mutations in ACTA2 (smooth muscle
actin alpha 2) and TGFBR2 (transforming growth factor
beta receptor 2) promote excessive VSMC proliferation and
are causal for thoracic aneurismal disease [19,56]. Indeed,
evidence from candidate gene studies also suggests a role
for excessive VSMC proliferation. The Angiotensin II type
1 receptor 1166C polymorphism has been associated with
AAA in three independent cohorts [26] (per allele odds ratio
1.60, 95% CI 1.32–1.93, P=1.1×10−6), and it has been
shown that this polymorphism increased vascular response
to circulating Angiotensin II [57], a potent stimulator of
VSMC proliferation and migration [58].
3. Future Directions for Genomics and
Pathobiology of AAA
3.1. Study Design to Refine and Augment Signals. In genetic
studies a useful alternative to dichotomizing complex disor-
ders is to consider the clinical end-point as a combination
of different continuous traits [59]. Within the population,
infrarenal aortic diameter is a continuously distributed
phenotype (skewed to the right) [60], with AAA rupture
(the clinical end-point of interest) in aortas less than 4 cm
almost unheard of. Rather than dichotomizing into AAA
versus no AAA (cut-offthreshold 3 cm), another option as
suggested by Plomin [59] would be to study the trait across
the range of variation in the population. This strategy has
been used to great effect in other complex disorders; for
example, following discovery of loci for T2DM, a binary
outcome, signals have been refined by studying continuous
traits associated with the disease such as fasting glucose,
insulin secretion, and obesity. Population-based studies
provide greater freedom from biases, better definition of
environmental exposures before disease onset, and clearer
characterization of the evolution of traits over time [61].
Another area that has received limited attention in the
literature to date is the discovery of variants that associate
with rapid aneurysm expansion of small AAA. It is not clear
whether this phenotype has a large heritable component or
whether the genes that predispose to AAA are also those
that predispose to rapid expansion. For example, it does
not appear that the 9p21 SNP associates with expansion
rates [36]. It should be noted, however, that genetic studies
of expansion have often been small and underpowered,
with heterogeneity in the cohorts with regard to how the
phenotype is actually measured and modeled, which is a
major methodological concern. These problems will only
be overcome by a large-scale collaborative effort to produce
standardized methods for phenotype definitions and data-
collection.
3.2. Rare Variants/Exome Sequencing. Despite the discovery
of large numbers of variants associated with many complex
diseases by GWAS, the majority of observed heritability in
most of these diseases remains unexplained [62]. This has
prompted speculation that rare variants of large effect (which
are poorly covered on currently available gene chips) may
be important in development of common diseases [62].
Rare causative mutations have been identified by exome-
sequencing experiments in a handful of single genedisorders
[63,64], whilst deep resequencing efforts have identified
rare variants of large effect at loci implicated by GWAS
in traits such as triglyceride levels [65]. Despite these
successes, the whole genome/exome resequencing studies
for common complex diseases remain limited by expense,
statistical power, and computational capacity [66].
3.3. MicroRNAs and AAA. MicroRNAs (miRNAs) are a class
of endogenous noncoding single-stranded RNAs (19–24
nucleotides) that are important regulators of gene expres-
sion. miRNAs are transcribed as primary miRNAs (pri-
miRNAs), processed to precursor miRNAs (pre-miRNAs),
and then to mature miRNAs. It is estimated that more than
60% of protein-coding genes are regulated by these small
RNAs [67,68], composing a new complicated regulatory
network with a significant role in biological functions that
are frequently regulated cooperatively by large numbers of
genes.
The VSMC is crucial to the progression of almost all
vascular wall disorders including AAA [69,70]. Liu et
al. studied the expression of miRNAs in an experimental
animal model of AAA and discovered a group of miRNAs
differentially expressed in AAA versus normal Sprague
Dawley rat aortas [71]. Bioinformatics analyses for predicted
mRNA targets of differentially expressed miRNAs showed
enrichment for cell signaling pathways thought to play a role
in human AAA development, such as the mitogen-activated
protein kinase pathway. Recently Leeper et al. performed
an in vitro miRNA microarray analysis of human VSMC to
identify 28-upregulated and 3-downregulated miRNAs that
were significantly and sustainably altered during the differ-
entiation process [72]. Among the regulatory miRNAs for
VSMC,miRNA-26awasofparticularinterestasitappeared
to serve as an inhibitor of VSMC differentiation by inhibiting
the effect on the signaling pathways downstream of the
TGF-β/BMP superfamily of growth factors. Cells deficient in
miRNA-26a lost their migratory phenotype toward a growth
factor/serum gradient and displayed enhanced rates of
programmed cell death. These effects on the TGF-βpathway
and on VSMC proliferation, migration, and apoptosis in
particular suggested that miRNA-26a could be important in

Cardiology Research and Practice 5
Translational No translational
repression repression
1166 A 1166 C
C
T
T
A
5
55
5
3
3
3
3
Pre-miR-155
AGTR1
A
GTR1
miR-155 + RISC
Figure 1: Model of miR-155 binding to the 3UTR of AGTR1
and association with the 1166A>C polymorphism (rs5186).The
hairpin-shaped precursor of miR-155 (pre-miR-155) after being
exported in the cytoplasm is assembled into the RNA-induced
silencing (RISC) complex (mature miR-155), which transfers it to
recognise specific mRNA targets. miR-155 binds with complete
sequence complementarity to its target mRNA seed site and induces
posttranscriptional gene silencing. In the presence of the −1166 C
allele in the 3UTR of AGTR1, the compensatory base pairing is
affected, and miR-155 binding is inhibited leading to increased
AGTR1 expression.
AAA development. These results were confirmed using two
independent animal models of AAA disease, where miRNA-
26a expression coincides temporally with VSMC apoptosis
and cell loss.
Another miRNA-related mechanism of diseases suscepti-
bility is where SNPs alter miRNA target sites. For example,
the 1166A>C polymorphism (rs5186) in the angiotensin
receptor 1 (AGTR1) that has been associated with AAA
by candidate gene analysis appears to abolish miR-155-
mediated regulation of the AGTR1 gene [73,74](Figure 1).
Angiotensin II receptor type 1 (AT1) signaling stimulates
proliferation of VSMC and vascular fibrosis [75], while the
AT1 receptor blocker Losartan has been shown to reduce
experimental aneurysm formation in mouse models of
Marfan’s disease [76]. Daugherty et al. showed that selective
blockade of AT1 signaling with Losartan attenuated AAA
formation in the Angiotensin II-infused apoE−/−mouse
model, but blockade of AT2 signalling resulted in more severe
atherosclerosis and aneurysmal disease [77]. Taken together,
these data suggest that micro-RNAs are likely to play a role in
the remodeling process seen during aneurysm development,
and further research of genetic variation in both microRNAs
and their targets may uncover some novel insights.
4. Conclusions
Whilst GWASs are redefining our understanding of many
complex diseases including AAA, it is clear that they repre-
sent only an early step in the process of genetic discovery.
It is too early to define specific translational roles for
any of the loci identified so far that associate with AAA,
but light is being shed on pathobiological pathways such
as those involved in excessive VSMC proliferation, which
has potential implications for development of nonsurgical
therapies. Further discoveries will rely upon collaboration of
large research consortia as seen in other complex diseases and
careful consideration of how information from genomewide
data could be harnessed to develop specific therapies and
individualized preventative strategies.
Acknowledgments
S. C. Harrison is supported by a BHF Clinical Training
Fellowship (FS/11/16/28696). A. Z. Kalea is funded by the
BHF as a chair scholar. S. E. Hurphries is funded by the
British Heart Foundation RG2008/08. M. V. Holmes is
funded by a Population Health Scientist Fellowship from the
Medical Research Council (G0802432).
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