The contribution of six polymorphisms to cardiovascular risk in a Dutch high-risk primary care population: the HIPPOCRATES project.

Abstract

This study was designed to examine the contribution of six polymorphisms to the occurrence of cardiovascular disease (CVD) in a Dutch primary care population with a high prevalence of cardiovascular risk factors. In this cross-sectional case-control study, 232 patients with CVD and 571 event-free controls were studied. Patients were genotyped for the AGTR1 (A1166C), AGT (M235T), ACE (4656rpt), NOS3 (E298D), GNB3 (C825T) and ADD1 (G460W) polymorphisms. Univariate and multivariate odds ratios (ORs) were calculated to assess the relationship between genotypes and CVD. Receiver operating characteristic (ROC) analysis was used to quantify the contribution of the polymorphisms to the prediction of CVD. No differences in either genotype or allele frequencies were found between CVD cases and controls. Multivariate analyses, corrected for multiple testing according to Bonferroni, showed significant protective associations for the T-allele of AGT (OR=0.55 (0.34-0.84)) and for the T-allele of ADD1 (OR=0.52 (0.31-0.82)). ROC analysis showed only a very small improvement of CVD risk prediction by adding the six polymorphisms to a model with traditional risk factors. Our data suggest that a major attribution of the six polymorphisms to the cardiovascular risk prediction in a primary care population such as HIPPOCRATES is unlikely.

Full text

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Genetics and cardiovascular risk
in a primary care population
Studies from the HIPPOCRATES project

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© A.W. Plat, Maastricht 2009

ISBN: 978-90-5278-821-0


Layout: Tiny Wouters
samenwerking met Leen Liefsoens
Production: Datawyse | Universitaire Pers Maastricht

Scientific studies in this thesis were supported by unrestricted research grants from SOHO (Stichting
Ondersteuning Hypertensie Onderzoek) Amsterdam, the Netherlands, and the Maastricht University
Medical Center (Profileringsfonds grant no. PF194), Maastricht, the Netherlands.

Printing of this thesis was financially supported by Astellas Pharma B.V., AstraZeneca B.V., Pfizer B.V.,
Schering-Plough B.V.





Cover: Datawyse in

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PROEFSCHRIFT



ter verkrijging van de graad van doctor aan de Universiteit Maastricht,
op gezag van de Rector Magnificus, Prof. mr. G.P.M.F. Mols
volgens besluit van het College van Decanen in het openbaar te verdedigen
op vrijdag 29 mei 2009 om 14.00 uur


door


Adrianus Wilhelmus Plat
geboren op 1 maart 1975 te Steenwijk

Genetics and cardiovascular risk
in a primary care population
Studies from the HIPPOCRATES project
UNIVERSITAIRE
PERS MAASTRICHT
U
P
M

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Promotores

Prof. dr. C.P. van Schayck
Prof. dr. P.W. de Leeuw



Copromotores

dr. H.E.J.H. Stoffers
dr. A.A. Kroon



Beoordelingscommissie

Prof. dr. M.H. Prins, voorzitter
Prof. dr. M.C. Cornel, (VUMC Amsterdam)
Prof. dr. J.P.M. Geraerdts
Prof. dr. J.A. Knottnerus
Prof. dr. H.A.J. Struijker Boudier


















Financial support by the Netherlands Heart Foundation for the publication of this
thesis is gratefully acknowledged.

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de morgen breekt aan
de stormen van vannacht zijn
in sneeuw begraven
(Shiro)




























Voor Ma
Ter herinnering aan Pa
Aan Leen

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Contents


Chapter 1

Chapter 2


List of abbreviations 9
Introduction 11
Regional differences in cardiovascular risk factor profile
cannot fully explain differences in cardiovascular morbidity
in the Netherlands: a comparison of two urban areas
The Netherlands Journal of Medicine 2005;63:309-315
21

Chapter 3




Chapter 4




Chapter 5




Chapter 6


The contribution of six polymorphisms to cardiovascular risk
in a Dutch high risk primary care population:
The HIPPOCRATES project
Journal of Human Hypertension 2009; in press
35
The influence of six cardiovascular polymorphisms on a
first event of ischemic heart disease is modified by gender
and age
Submitted
51
Gender-specific effect of the α-adducin (G460W) and
AGTR1 (A1166C) polymorphism on carotid intima media
thickness
Submitted
67
The association between arterial stiffness and the
angiotensin II type 1 receptor (A1166C) polymorphism
is influenced by the use of cardiovascular medication
Journal of Hypertension 2009;27:69-75
85

Chapter 7



Chapter 8










Family history of common diseases as genetic tool in primary
care: what is the evidence? A systematic review
Submitted
101
General discussion 117
Summary 129
Samenvatting 135
List of publications 143
Dankwoord 147
Curriculum vitae 153

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Abbreviations ⏐9
List of abbreviations
ACE
AGT
ADD1
AGTR1
AngI
AngII
ANOVA
AUC
BMI
CBS
CIMT
CVD
DBP
DM
DNA
GNB3
GP
FHQ
HDL
HGP
HR
ICPC
I/D
IHD
LDL
mRNA
RNH
NOS3
OR
PCR
PP
PHARMO RLS
PWV
RAS
ROC
SBE
SBP
SD
SEM
SNP































angiotensin-converting enzyme
angiotensinogen
alpha adducin
angiotensin II type 1 receptor
angiotensin I
angiotensin II
analysis of variance
area under the curve
body mass index
statistics Netherlands
carotid intima media thickness
cardiovascular disease
diastolic blood pressure
diabetes mellitus
desoxy-ribonucleic acid
G-protein β3 subunit
general practitioner
family history questionnaire
high-density lipoprotein
human genome project
heart rate
international classification of primary care
insertion/deletion
ischemic heart disease
low-density lipoprotein
messenger ribonucleic acid
registration network general practices
endothelial nitric oxide synthase
odds ratio
polymerase chain reaction
pulse pressure
PHARmacoMOrbidity record linkage system
pulse wave velocity
renin-angiotensin system
receiver operating characteristic
multiplex single base extension
systolic blood pressure
standard deviation
standard error of the mean
single nucleotide polymorphism

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10⏐

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Chapter 1
Introduction

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12⏐Chapter 1

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Introduction ⏐13
Introduction
Cardiovascular genetics
Atherosclerotic cardiovascular disease (CVD) - involving the heart, brain and
peripheral arteries of the legs - is a major health problem worldwide.1,2 The aetiology
of CVD is multifactorial, with both genetic and environmental factors playing
important roles. Several monogenetic disorders contribute to CVD, such as familial
hypercholesterolemia and hypertrophic cardiomyopathy.3,4 Although most of these
monogenetic mutations are rare, they are associated with an elevated cardiovascular
risk. Moreover, the most common forms of CVD are believed to be multifactorial,
resulting from the interaction of several genes, each with a relatively small effect,
working alone or in combination with other modifiers, genes and/or environmental
factors. Little is currently known about which genes or mutations therein are involved
in atherosclerosis. However, there is accumulating evidence that a positive family
history for CVD in a first-grade family member is associated with an increased
cardiovascular risk in other (affected) family members.5–7 Therefore, there is active
research (predominantly epidemiologic studies) aimed at elucidating the combination
of genes or genetic factors that play active roles in the aetiology of CVD.

Genetic epidemiology involves the aetiology, distribution and control of disease in
groups of relatives and also in populations.8,9 Major topics considered in the genetic
epidemiology of CVD include cholesterol and lipid levels, blood coagulation, leukocyte
adhesion, endothelial cells of blood vessels, the renin-angiotensin system,
homocysteine and left ventricular hypertrophy.
Intermediate endpoints
The role of the renin-angiotensin system (RAS) in the development of CVD has being
studied for several years at the Department of Internal Medicine, Maastricht
University Medical Center. Figure 1.1 presents a schematic of the RAS, which is an
important system involved in blood pressure regulation, cardiovascular cellular
growth and remodelling, and thus also in the aetiology of atherosclerotic vascular
disease. Many association studies have connected specific genes of the RAS with
myocardial infarction, hypertrophic cardiomyopathy, hypertension, left ventricular
hypertrophy and stroke.10–12 Other association studies have found correlations
between certain polymorphisms of RAS genes and so-called intermediate vascular
endpoints, such as carotid intima media thickness (CIMT) and arterial stiffness13–15 as
assessed by pulse wave velocity (PWV) measurements. CIMT measurements provide a
non-invasive assessment of the (preclinical) atherosclerotic burden caused by
cardiovascular risk factors such as hypertension, diabetes mellitus and
hypercholesterolemia.16 Non-invasive PWV measurements have revealed increased
systolic and pulse pressures and, consequently, increased arterial stiffness.17 Both

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14⏐Chapter 1
increased CIMT and PWV have been indicated as independent predictors of future
CVD.16,17

























Figure 1.1 The renin-angiotensin system and several associated polymorphisms.
Polymorphisms
Polymorphisms are frequently occurring variations in the humane genome. By
definition, the frequency of the allele with the lowest prevalence is higher than 1%,
but the occurrence can vary between populations.9 The Department of Internal
Medicine of Maastricht University Medical Center has investigated the following
polymorphisms related to the RAS in hypertensive and normotensive patients:18–21
angiotensin II (AngII) type 1 receptor [AGTR1 (A1166C)], angiotensinogen [AGT
(M235T)], angiotensin-converting enzyme [ACE (4656rpt)], endothelial nitric oxide (NO)
synthase [NOS3 (E298D)], G-protein β3 subunit [GNB3 (C825T)] and α-adducin [ADD1
(G460W)].22 Figure 1.1 depicts the location of these polymorphisms, which function as
follows: Patients homozygous for the C allele of the AGTR1 polymorphism exhibit
increased sensitivity to AngII, which can - in combination with the DD genotype of the
ACE (I/D) polymorphism - lead to an increased risk of cardiovascular complications.23,24
KidneyKidney
AngiotensinogenAngiotensinogen
Renin Renin
Angiotensin I Angiotensin IAngiotensin IIAngiotensin II
Angiotensin converting
enzymeenzyme
AGTR1 receptor AGTR1 receptor
vasoconstrictionvasoconstriction
proliferationproliferation
Na+/wa ter
retention retention
sympathicus
activation activation
Adrenal glandAdrenal gland
Na+
Na+
excretion excretion
GNB3 (C825T)
Na+/H+-pump
Na+/H+-pump
ADD1 (G460W)
Na+/K+-pump
Na+/K+-pump
Blood
volumevolume
NOS3 (E298D) NOS3 (E298D)
AGT (M235T)AGT (M235T)
ACE (I/D)
ACE (4656rpt)
ACE (4656rpt)
AGTR1 (A1166C)AGTR1 (A1166C)
AldosteroneAldosterone
Heart Heart
Cardiac
outputoutput
Arterial
pressurepressure
Angiotensin converting
Na+/wa tersympathicus
GNB3 (C825T)
ADD1 (G460W)
Blood
ACE (I/D)
Cardiac Arterial

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Introduction ⏐15
The T allele of the AGT polymorphism is associated with high plasma levels of AGT and
increased responsiveness to AngII.11 The ACE (I/D) polymorphism was determined by
detecting ACE4656+CT. The ACE4656+CT polymorphism in the 3’-untranslated region of the
ACE gene consists of a repetition of two or three CT dinucleotides and is in complete
linkage disequilibrium with the ACE (I/D) polymorphism.25,26 Increased ACE levels have
been found In the presence of a 2 allele which is comparable to the D allele of the I/D
polymorphism, and they have been associated with an increased risk of cardiovascular
complications.27 The NOS3 (E298D) polymorphism is associated with reduced NO
production in endothelial cells of the arterial wall, inducing elevation of the arterial
pressure.19,28 The T allele of the GNB3 (C825T) polymorphism is associated with
increased intracellular signal transduction that stimulates the Na+/H+ exchanger29 to
increase Na+ transport across renal tubules and cause chronic volume expansion that
might lead to proliferation of vascular smooth muscle cells, vascular remodelling and
increased peripheral arterial resistance.30 Finally, the ADD1 (G460W) polymorphism is
of interest because the rare allele is also associated with increased Na+ transport,
though in this case the Na+/K+ exchanger is stimulated, which also causes chronic
volume expansion with related cardiovascular complications.31
Patient care
Collins et al. (1999) stated that genomics research will dramatically accelerate the
development of new strategies for the diagnosis, prevention and treatment of
disease, not just for single-gene disorders but for most of the more common complex
diseases, such as CVD.32 It was thought that the completion of the Human Genome
Project would lead naturally to the clinical application of medical genetics in both
hospital and general practice. The clinical meaning of both diagnostics and
therapeutics is strongly focused on the function of genes in both pathophysiological
(e.g. CVD) and healthy circumstances. As a consequence, the expectation is that
genetic knowledge will eventually have a major impact on daily clinical decision-
making by every doctor. Therefore, doctors need to know the genetic background of a
disease, not only for the treatment and prevention in an individual patient, but also as
a consequence in all first-degree family members.
Hypothesis
At the start of our studies, we reasoned that the above-mentioned polymorphisms
might be relevant individually or in combination as additional risk factors for CVD. We
hypothesized that these polymorphisms would lead to better cardiovascular risk
stratification, not only for an individual but probably also for all first-degree family
members. Furthermore, it might offer opportunities for genetically tailored
treatments. To explore this hypothesis, we studied the relationships between this set
of polymorphisms and biological (risk factors) and clinical (intermediate endpoints)