Published by Oxford University Press on behalf of the International Epidemiological Association
? The Author 2006; all rights reserved. Advance Access publication 24 March 2006
International Journal of Epidemiology 2006;35:922–931
Insight into the nature of the CRP–coronary
event association using Mendelian
Juan P Casas,1,2yTina Shah,1,3yJackie Cooper,3Emma Hawe,3Alex D McMahon,4Dairena Gaffney,5
Christopher J Packard,5Denis S O’Reilly,5Irene Juhan-Vague,6John S Yudkin,7Elena Tremoli,8,9
Maurizio Margaglione,10Giovanni Di Minno,10Anders Hamsten,11Teake Kooistra,12Jeffrey
W Stephens,3Steven J Hurel,13Shona Livingstone,14Helen M Colhoun,14George J Miller,15Leonelo
E Bautista,16Tom Meade,2Naveed Sattar,5Steve E Humphries3and Aroon D Hingorani1*
15 February 2006
It is unclear wheather the association between C-reactive protein (CRP) and
incident coronary events is free from bias and confounding. Individuals
homozygous for a 11444C.T polymorphism in the CRP gene have higher
circulating concentrations of CRP. Since the distribution of this polymorphism
occurs at random during gamete formation, its association with coronary events
should not be biased or confounded.
We calculated the weighted mean difference in CRP between individuals with
variants of the 11444C.T polymorphism in the CRP gene among 4659
European men from six studies (genotype-intermediate phenotype studies). We
used this difference together with data from previous observational studies to
compute an expected odds ratio (OR) for non-fatal myocardial infarction (MI)
among individuals homozygous for the T allele. We then performed four new
genetic association studies (6201 European men) to obtain a summary OR for
the association between the 11444C.T polymorphism and non-fatal MI
CRP was 0.68 mg/l [95% confidence interval (95% CI) 0.31–1.10; P 5 0.0001]
subjects for non-fatal MI corresponding to this difference in CRP was 1.20
(95% CI 1.07–1.38) using the Reykjavik Heart study data and 1.25 (1.09–1.43)
Laboratories at University College London (UCL), London, UK.
2Department of Epidemiology and Population Health, London School of
Hygiene and Tropical Medicine, London, UK.
3Centre for Cardiovascular Genetics, Department of Medicine, BHF
Laboratories at UCL, London, UK.
4Robertson Centre for Biostatistics, University of Glasgow, Glasgow,
5Department of Pathological Biochemistry, Glasgow Royal Infirmary,
Glasgow, Scotland, UK.
6Laboratoire d’He ´matologie, CHU Timone, Inserm Unit 626, Marseille,
7Diabetes and Cardiovascular Disease Academic Unit, Archway Campus,
Royal Free and UCL Medical School, London, UK.
8Department of Pharmacological Sciences, University of Milan, Milan, Italy.
9Monzino Cardiologic Center, Milan, Italy.
10Instituto di Ricovero e Cura a Carattere Scientifico, Ospedale Casa
Sollievo della Sofferenza, San Giovanni Rotondo, Italy.
11King Gustaf V Research Institute Department of Medicine, Karolinska
Hospital, Karolinska Institute, Stockholm, Sweden.
12The Gaubius Laboratory, The Netherlands Organization for Applied
Scientific Research-Prevention and Health, Leiden, The Netherlands.
13Department of Diabetes, Endocrinology and Metabolism, Middlesex
Hospital, London, UK.
14Department of Epidemiology at UCL, London, UK.
15Wolfson Institute of Preventive Medicine at the Medical College of
St Bartholomew’s Hospital, London, UK.
16Department of Population Health Sciences, University of Wisconsin
Medical School, Madison, WI, USA.
*Corresponding author. Centre for Clinical Pharmacology, BHF Laboratories
at UCL, Rayne Building, 5 University Street, London WC1E 6JJ, UK.
yBoth authors contributed equally to this article.
by guest on May 31, 2013
for all observational studies to 2004. The estimate for the observed adjusted-OR
for non-fatal MI among TT subjects was 1.01 (95% CI 0.74–1.38), lower than both
Conclusions A common CRP gene polymorphism is associated with important differences in
CRP concentrations, free from confounding. The null association of this variant
with coronary events suggests possible residual confounding (or reverse causation)
in the CRP–coronary event association in observational studies, though the
confidence limits are still compatible with a modest causal effect. Additional studies
of genotype (or haplotype) and coronary events would help clarify whether or not
the link between CRP and coronary events in observational studies is causal.
C-reactive protein, cardiovascular disease, genetics, polymorphism,
Prospective studies indicate a robust association, in healthy
individuals, between levels of the acute phase reactant
C-reactive protein (CRP) and later coronary events,1–3and it
has been proposed that measurement of CRP may be a useful
adjunct to coronary risk assessment and that CRP could be
causally involved in atheroslerosis.4However, CRP concentra-
tions are correlated with smoking status, blood pressure,
obesity, diabetes, physical activity, social class, low birth
weight, and other products of the inflammatory response and
are also higher in individuals with clinical cardiovascular
disease.5–8Despite statistical adjustment, this association may,
therefore, be subject to residual confounding or bias due to
reverse causation, where the disease even in its sub-clinical
state leads to elevation in the level of CRP.5–8
intervention to reduce CRP would provide an unbiased insight
into the nature of the association. Unfortunately, no such
selective intervention currently exists. Although inhibitors of
HMG-CoA reductase (statins) and glitazones reduce CRP,9,10
they also have major effects on lipid profile and glucose
metabolism. An alternative approach to control for confound-
ing and reverse causality is to identify a common polymorph-
ism in the CRP gene reliably associated with differences in
circulating CRP concentration. The inheritance of such a
variant should be subject to the random assortment of maternal
and paternal alleles at the time of gamete formation, according
to Mendel’s second law.11If CRP actually increases the risk of
coronary events then carriage of an allele that exposes indi-
viduals to a long-term elevation in CRP should confer an
increased risk of coronary events proportional to the difference
in CRP attributable to the allele. This relationship should be
largely unconfounded and free of reverse causality bias.12,13
Therefore, if non-genetic observational studies were unbiased,
the increase in risk estimated from these studies should be
consistent with the increase in risk conferred by carriage of the
allele.12,13This approach, known as ‘Mendelian randomiza-
tion’, has been used recently to investigate the link between
homocysteine, fibrinogen, and cardiovascular disease.14–17
CRP concentration is a heritable trait.18,19In three small
studies we found that homozygosity for the rarer T allele of a
11444C.T polymorphism in the 30-untranslated region of the
CRP gene was associated with higher basal and/or stimulated
CRP concentrations.20,21We have now genotyped a large
number of Caucasian men to test whether the increase in risk
of coronaryevents inindividuals
polymorphism is close to that predicted under the assumption
that the CRP–coronary event association from previous non-
genetic observational studies is free from residual confounding
and bias due to reverse causation.
After obtaining ethical approvals, genotyping for the CRP
polymorphism and measurement of plasma CRP concentration
were conducted in samples obtained from a number of cross-
sectional or prospective studies, or randomized controlled trials
summarized in Table 1 and in supplementary materials
available online.21–27To test the association of 11444C.T
genotype with CRP concentration, male subjects without
clinically evident cardiovascular disease from six studies
with availableDNA andplasma
(Table 1).21–23,25–27A second analysis was conducted to
evaluate the associationbetween genotype
myocardial infarction (MI) in male subjects from four studies
and risk of
Data on sex, mean age, systolic and diastolic blood pressure,
body mass index (BMI), smoking status, glucose, lipid profile,
alcohol consumption, fibrinogen, and plasma CRP values were
obtained from the original studies. Subjects were classified
using unified definitions of hypertension, hypercholestero-
guidelines on primary prevention of the American Heart
Association.28For the studies relating genotype and coronary
events,22–25non-fatal MI according to the WHO criteria29was
considered the primary outcome, as this end-point had been
uniformly used across all studies. Analyses of genotype and
CRP were limited to male subjects to preserve consistency with
available studies of genotype and coronary events, which were
all conducted in men.
and obesityfrom the
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