Exp Clin Cardiol Vol 15 No 3 2010e52
Lack of an association between connexin-37,
stromelysin-1, plasminogen activator-inhibitor type 1
and lymphotoxin-alpha genes and acute coronary
syndrome in Czech Caucasians
Jaroslav Alois Hubacek PhD1,2,3,4, Vladimír Stane ˇk MD PhD1, Marie Gebauerová MD1, Alexandra Pilipčincová PhD1,
Rudolf Poledne PhD1,5, Michal Aschermann MD PhD4, Hana Skalická MD4, Jana Matoušková MD6,
Andreas Kruger MD6, Martin Pe ˇnička MD6, Hana Hrabáková MD4, Josef Veselka MD5, Petr Hájek MD5,
Vera Lánská PhD1, Ve ˇra Adámková MD PhD1, Jan Pit’ha MD PhD1,2
1Institute for Clinical and Experimental Medicine; 2Centre for Cardiovascular Research, Prague; 3Faculty for Public Health and Social Studies,
South Bohemia University, Ceske Budejovice; 42nd Department of Internal Medicine, General Teaching Hospital; 5Cardiocenter, Department
of Cardiology, University Hospital Královské Vinohrady; 6Department of Cardiology, Homolka Hospital, Prague, Czech Republic
Correspondence: Dr Jaroslav Alois Hubacek, IKEM-DEM-LMG, Videnska 1958/9, 140 21 Prague 4, Czech Republic.
Telephone 420-261-363-367, fax 420-241-721-666, e-mail firstname.lastname@example.org
Received for publication March 24, 2010. Accepted May 10, 2010
risk factors of cardiovascular disease such as hypertension, dys-
lipidemia, obesity, smoking and diabetes. However, one-third
of cardiovascular disease cases cannot be characterized by these
generally accepted risk factor criteria and, despite intensive
research over the past decades, no major advances in the detec-
tion of additional risk factors have been made. Furthermore,
the current worldwide prevalence of CAD is increasing and is
expected to escalate even further in the future (1).
Molecular genetic methods offer an alternative to using the
classical risk factors for assessing CAD risk. Because of the high
heritability of CAD, it may be possible for this predisposition to
be detected by the identification of risk variants in the candi-
date genes believed to be associated with CAD. Further advan-
ces in the molecular genetic approach to CAD will contribute
to better understanding of the differences between individuals,
and may offer more effective treatment for those detected to be
The complexity of CAD, however, makes the application of
molecular methods somewhat more difficult, but two extreme
models of CAD have emerged (2). The ‘polygenic’ model pro-
poses that a large number of different alleles with small effects
at different genes interact with one another and with the
environment to cause cardiovascular disease. The ‘monogenic’
substantial portion of coronary artery disease (CAD)
events are explained by the commonly accepted ‘classical’
model proposes that rare alleles with substantial effects at a
large number of genes cause cardiovascular disease. In athero-
sclerosis, a combination of these two models with strong addi-
tional environmental influence (eg, smoking, high fat intake,
lack of physical activity and others) is believed to be involved.
Cardiovascular disease was calculated to have a significant
heritability component of approximately 60% in men and
approximately 40% in women (3), and that the risk of pre-
mature cardiac death was eightfold higher in men and 15-fold
higher in women whose siblings died as a result of heart attack
before 55 or 65 years of age, respectively (4).
Despite the known rare mutations, the majority of myocar-
dial infarction (MI) risk is under polygenic control, with many
variants in a few genes contributing to the total effect. The
estimated effect of each individual single nucleotide poly-
morphism (SNP) is estimated to be relatively low, yet signifi-
cant. Because atherosclerosis and ischemic heart disease are
very complex and nonhomogeneous diseases, recent estimates
suggest that as many as 500 to 800 genes influence the patho-
physiological processes from the onset of atherosclerosis to its
clinical manifestation. Consequently, thousands of articles in
the field of genetic predisposition to cardiovascular disease
have been published – the results of which are difficult to rep-
licate due to low statistical power, usually owing to a small
number of participants and ethnic differences.
CLiniCaL CardioLogy: originaL artiCLe
©2010 Pulsus Group Inc. All rights reserved
JA Hubacek, V Staněk, M Gebauerová, et al. Lack of an association
between connexin-37, stromelysin-1, plasminogen activator-inhibitor
type 1 and lymphotoxin-alpha genes and acute coronary syndrome in
Czech Caucasians. Exp Clin Cardiol 2010;15(3):e52-e56.
BACkGround: The majority of acute coronary syndrome (ACS) cases
cannot be explained by the analysis of commonly recognized risk factors;
thus, the analysis of possible genetic predispositions is of interest. The
genes for connexin-37, stromelysin-1, plasminogen activator-inhibitor
type 1 (PAI-1) and lymphotoxin-alpha are among many presently known
candidate genes that are associated with risk factors for ACS.
oBJECtiVE: To identify the potential impact of the functional variants
of connexin-37, stromelysin-1, PAI-1 and lymphotoxin-alpha on ACS in a
Caucasian Czech population.
MEtHodS: A total of 1399 consecutive patients (1016 men and 383 women)
with ACS from five coronary care units located in Prague (Czech Republic)
were analyzed; a representative sample of 2559 healthy individuals (1191 men
and 1368 women) were also genotyped and served as controls.
rESuLtS: The gene variants analyzed were not significantly associated
with the prevalence of ACS or the classical risk factors of ACS develop-
ment such as high plasma lipid levels, hypertension, diabetes, high body
mass index or smoking.
ConCLuSion: In a Caucasian Czech population sample, genetic vari-
ants of connexin-37, stromelysin-1, PAI-1 and lymphotoxin-alpha were
not significantly associated with a predisposition toward ACS.
key Words: Acute coronary syndrome; Connexin-37; Lymphotoxin-alpha;
Plasminogen activator-inhibitor type 1; Polymorphism; Stromelysin-1
Gene polymorphisms and ACS
Exp Clin Cardiol Vol 15 No 3 2010e53
Nevertheless, in past years, studies have been published in
which the authors examined sufficient numbers of patients
with acute coronary syndrome (ACS) (5-13).
Two interesting genetic studies (12,13) from Japan identi-
fied four genes of considerable interest that had a highly signifi-
cant effect on CAD development. These are the genes for
connexin-37, a gap junction protein involved in cell-to-cell
communication; stromelysin-1, a matrix metalloproteinase-3
involved in the destabilization of atherosclerotic plaques; plas-
minogen activator-inhibitor type 1 (PAI-1), a protein respon-
sible for prothrombogenic status of plasma; and the gene for
lymphotoxin-alpha – also known as tumour necrosis factor-
beta – a toxin produced by lymphocytes associated with inflam-
matory processes in artery walls leading to the initiation,
progression and destabilization of atherosclerotic plaques.
The significant differences in genetic predisposition toward
CAD, mainly among different ethnic groups, prompted us to
analyze the putative association between SNPs within these
four genes and ACS in a large group of Caucasian Czech
patients and healthy controls.
A total of 1686 patients younger than 65 years (men) or
75 years (women) of age hospitalized in five participating cor-
onary units (Clinic of Cardiology, Institute for Clinical and
Experimental Medicine; 2nd Department of Internal Medicine,
General Teaching Hospital; Department of Cardiology,
Teaching Hospital Motol; Department of Cardiology, Homolka
Hospital; and the Cardiocenter, Department of Cardiology,
University Hospital Královské Vinohrady) were included in
the present study (14). These facilities cover almost all areas of
health care in Prague (Czech Republic) for ACS (eg, acute MI,
minimal myocardial lesion and patients with subacute MI).
The only exclusion criteria were age of 65 years and older for
men, 75 years and older for women, and refusal to participate
in the study. Of the 1686 patients, detailed genetic association
analyses were performed for 1399 individuals (1016 men and
383 women) for which complete biochemical and anthropo-
metrical data were available.
The control groups (1191 men and 1368 women [response rate
84%]) were selected from 1% of a representative three-year
cohort sample of a Caucasian Czech population (from the
districts of Benesov, Prague-east, Cheb, Chrudim, Jindrichuv
Hradec, Pardubice, Kromeriz, Litomerice and Pilsen City). The
individuals were recruited in nine districts from 1997 to 1998
and re-invited in 2000/2001 according to the WHO protocol
(“MONICA Project”. Manual WHO/MNC 82.2, November
1983). The local ethics committee approved the study design
and written, informed consent was obtained from the partici-
pants. Individuals in the patient and control groups completed
a standard questionnaire regarding the presence of traditional
cardiovascular risk factors including family and personal his-
tory. The characteristics of the study participants are summar-
ized in Table 1.
Genetic and biochemical analysis
Patient and control DNA was isolated as previously described
(15). Individual SNPs (4G/5G polymorphism at –675 of the
PAI-1 promoter; A252→G of the lymphotoxin-alpha gene,
C1019→T in the connexin-37 gene; and the 5A/6A variant at
–1171 bp of the stromelysin-1 gene) were genotyped using a
high-throughput microplate array diagonal gel electrophoresis
methodology (16) (Table 2). The lipoprotein parameters were
measured at the WHO Regional Lipid Reference Centre,
IKEM (Prague) on a COBAS MIRA auto-analyzer (Roche,
The Hardy-Weinberg test was used to confirm the independent
segregation of the alleles of individual genotypes (www.tufts.
calculator.xls). The c2 test was used to test for differences
between the genotype frequencies of the participating groups
sh?nrow=2&ncolumn=3). ANOVA was used for additional
For genotype/allelic differences between the groups, P<0.05
was considered to be statistically significant. Because of the
high number of analyses performed, P<0.01 was considered to
be significant in the analysis of the putative association
between individual SNPs and the classical risk factors.
The basic characteristics of the study groups are summarized in
Table 1. As expected, patients (both men and women) were
older and had a higher prevalence of smoking, diabetes and
basic characteristics of the analyzed individuals
Control Acute coronary syndrome P Control Acute coronary syndrome P
Patients, n11911016– 1368 383–
Age, years49.0±10.855.2±7.50.001 48.6±10.662.6±8.50.001
Cholesterol, mmol/L5.76±1.065.22±1.150.0015.80±1.15 5.40±1.260.001
Body mass index, kg/m2
1.97±1.28 2.05±1.46 NS1.47±0.82 1.86±1.16 0.001
Never smokers, %67.3 12.90.001 78.564.50.001
Diabetes, % 8.9 34.7 0.0016.850.40.001
Data presented as mean ± SD unless otherwise indicated. NS Not significant
40.7 52.8 0.001 33.1 62.7 0.001
Hubacek et al
Exp Clin Cardiol Vol 15 No 3 2010 e54
hypertension than the control groups. However, the mean
body mass index of the male patients was similar to the mean
body mass index of the control group, and plasma cholesterol
levels were lower in ACS patients. This was due to the fact
that approximately 20% of the patients (in contrast to approxi-
mately 8% of the controls) were on lipid-lowering drugs
(almost all on statins) at the time of ACS.
Analyzed SnPs in controls and ACS patients
The distribution of all analyzed SNPs were similar to the frequen-
cies described in other Caucasian populations (Table 3). Overall,
the call rates for individual genotypes varied between 93.7%
(connexin-37 in male control groups) and 100% (PAI-1 variant
in female control groups). With exception of the PAI-1 variant
(borderline, P=0.03 and just for female controls), the distribu-
tions of individual genotypes were within the Hardy-Weinberg
equilibrium. The frequency of the genotypes and alleles con-
taining the analyzed SNPs were not significantly different
between MI patients and healthy controls (all P≥0.2) (Table 3).
Association between the analyzed SnPs and classical risk
The present study failed to identify associations between clas-
sical MI/CAD risk factors (total cholesterol, triglycerides, dia-
betes prevalence, blood pressure and glucose) and the four
analyzed gene variants, either in patients or controls, or in men
The present large study (investigating approximately 1500 patients
and 2500 controls) failed to replicate previous findings of an
association between four functional SNPs within the genes for
connexin-37, PAI-1, stromelysin-1 and lymphotoxin-alpha
with ACS. Furthermore, these variants were not significantly
associated with the ‘classical’ risk factors for ACS/CAD
Our results contrast with the original study (12) that found
the four genes to be important determinants of MI develop-
ment in a Japanese population; however, later studies (13) on
these variants were less clear.
The most controversial results have been published in associa-
tion with the C1019→T polymorphism in the connexin-37
gene – a variant that has been associated with cardiovascular risk;
however, it remains unclear as to which allele carries this risk.
Studies conducted in Japan (12,17) showed that the T allele
contributed to MI development, especially in high-risk individ-
uals; in contrast, however, studies on Caucasians (18,19) with the
same variant showed it to be protective. The present study – and
others (20) – did not detect an association between the C1019→T
variant and CAD/ACS risk.
The finding of an association between the lymphotoxin-
alpha variant and ACS suggests a strong ethnic-specific effect.
This variant was originally associated with CAD and later con-
firmed in Japanese populations (12,13), but not in German
(21) or Brazilian (22) populations.
The 4G/5G variant of the PAI-1 gene is among the most
analyzed hemostatic genetic polymorphisms in CAD. A large
meta-analysis (23) determined the per allele RR for CAD to be
significant, but very low (OR 1.06). Despite the large sample
size, the present study was inadequately powered to detect such
a small effect.
Finally, the available data regarding the stromelysin variant
are controversial. The largest study (24) performed to date did
not find an association between stromelysin and MI; however,
the same variant was associated with CAD.
Details of the analysis of four single-nucleotide polymorphisms
Gene (variant) Primer-pair sequences
enzymeSize, bp (allele)
Lymphotoxin-alpha gene (A252→G) 5′–AGA CCT CCC GCC CTG GGA GAC AGC ACC–3′
5′–TGG GCC TGG GCC TTG GTG GGT TTG G–3′
241 NcoI240 (T)
139 + 102 (C)
Stromelysin-1 gene (5A/6A)
5′–GAT TAC AGA CAT GGG TCA CA–3′
5′–TTT CAA TCA GGA CAA GAC GAA GTT T–3′
120, 121XmnI 120 (6G)
98 + 23 (5G)
Connexin-37 gene (C1019→T)
5′–CTG GAC CCA CCC CCT CAG AAT GGC CAA AGA–3′
5′–AGG AAG CCG TAG TGC CTG GTG G–3′
275 DdrI275 (T)
240 + 35 (C)
5′–ACC CAG CAC ACC TCC AAC CTC AGC CAG–3′
5′–CTC CGA TGA TAC ACG GCT GAC TCC CCC–3′
108, 109BbrI 108 (4G)
81 + 28 (5G)
PAI-1 Plasminogen activator-inhibitor type 1 gene; PCR Polymerase chain reaction
Frequencies of genotypes and alleles among healthy
controls and acute coronary syndrome (ACS) patients
Connexin-37 gene0.82 0.19
C/C543 (48.1) 471 (49.5)607 (45.1) 181 (49.6)
C/T482 (42.7) 397 (41.7)607 (45.1) 145 (39.7)
T/T 104 (9.2)84 (8.8)133 (9.9)39 (10.7)
Stromelysin-1 gene0.96 0.24
5A/5A 288 (24.9) 251 (25.4)337 (25.4)94 (24.9)
6A/5A 592 (51.2) 502 (50.9)666 (50.2) 176 (46.6)
6A/6A277 (23.9) 234 (23.7)323 (24.4) 108 (28.6)
4G/4G 347 (29.7) 324 (32.5)435 (31.8) 121 (31.8)
5G/4G 580 (49.7) 492 (49.3)638 (46.6) 188 (49.3)
5G/5G 241 (20.6) 182 (18.2) 295 (21.6) 72 (18.9)
Lymphotoxin-alpha gene0.39 0.24
A/A 570 (50.1) 513 (51.5)668 176 (46.6)
A/G458 (40.2) 381 (38.3) 539 171 (45.2)
Data presented as n (%) unless otherwise indicated. PAI-1 Plasminogen
activator-inhibitor type 1 gene
100 (8.8)102 (10.2)125 31 (8.2)
Gene polymorphisms and ACS
Exp Clin Cardiol Vol 15 No 3 2010e55
There are many reasons that could explain the differences
in the results of our study with previous findings. First, it is
known that the prevalence of MI is much higher in European
than in the Japanese populations, possibly due to a difference
in the gene effects in the two groups. This is supported by
recent findings analyzing thousands of SNPs. Briefly, if a large
number of SNPs are analyzed, different SNP patterns emerge,
resulting in many distinct ‘genetic fingerprints’ that may have
different effects in different populations (25). This may also
apply to some and, perhaps most, key individual SNPs. In fact,
not all of the thousands of SNPs analyzed are of functional
Second, gene-environment interactions definitely play an
important role. The negative gene effect seen in the Caucasian
Czech population may be partially masked by an unfavourable
environment. Generally speaking, a large proportion of the
Japanese diet consists of fish, which is highly regarded to be a
low-risk diet with respect to CAD. In contrast, a large propor-
tion of the typical Czech diet consists of red meat. Indeed, there
are published studies underlining the importance of individual
SNPs in population analyses that are placed in such a context.
For example, the effect of a common variant within the pro-
motor region of the hepatic lipase gene on plasma lipoprotein
levels was shown to be dependent on dietary fat intake (26).
Our negative results underline the importance of confirma-
tory analyses performed on a large number of appropriately
selected and characterized groups of patients and controls,
especially when considering populations with different ethnici-
ties and lifestyles.
Finally, it must be emphasized that the selection of the
genes analyzed in the present study was based on a candidate
gene approach, which is supported by functional data and
published results. Recently, genome-wide association stud-
ies investigating many thousands of SNPs (whose selection
was often based merely on the criterion ‘to equally cover the
genome’), mostly without known functionality, were analyzed.
Interestingly, of the significant SNPs detected and replicated
through genome-wide association studies, a substantial portion
were localized in new genes, mostly with unknown functions
and vice versa, and many candidate genes were not confirmed
(but definitely not excluded, and many other SNPs, especially
with lower frequency, were not covered by the genotyping
Both genetic and environmental factors contribute to ACS
risk in approximately equal proportions. However, with inten-
sive prescription of some medications (eg, lipid- and blood
pressure-lowering drugs), it is likely that future research will
reveal an even greater role of genetic predisposition.
We suspect that in the Czech-Slavonic population, genes other
than the connexin-37, PAI-1, stromelysin-1 and lymphotoxin-
alpha genes (eg, genes for apolipoproteins A5 and E) (27-29)
are likely to play a more important role in the detection and
development of ACS.
In the future, the detection of CAD risk alleles may lead, in
time, to the identification of individuals at risk. When clas-
sical risk factors for CAD/ACS (anthropometrical and/or
biochemical) are already present, it is clearly too late for pri-
mary prevention because the disease is, more or less, already in
ACknoWLEdGEMEntS: This study was supported by research
grant No. NR-9093-4/2006 (Internal Grant Agency of the
Ministry of Health, Czech Republic).
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