European Heart Journal (1999) 20, 584–592
Article No. euhj.1998.1340, available online at http://www.idealibrary.com on
The TT genotype of the methylenetetrahydrofolate
reductase C677T gene polymorphism is associated
with the extent of coronary atherosclerosis in
patients at high risk for coronary artery disease
A. Gardemann*, H. Weidemann*, M. Philipp*, N. Katz*, H. Tillmanns†,
F. Wilhelm Hehrlein‡ and W. Haberbosch§
*Institut für Klinische Chemie und Pathobiochemie, †Abteilung Kardiologie und Angiologie and †Klinik für
Herz- und Gefäßchirurgie der Justus-Liebig-Universität Giessen, Giessen; §Max-Planck-Institut für Experimentelle
und Klinische Forschung, Kerckhoff-Klinik, Bad Nauheim, Germany
Background There are conflicting results on the relationship of N5,N10-methylenetetrahydrofolate reductase C677T
gene variation in coronary artery disease and myocardial
infarction.
Methods and Results We analysed this gene variation
in 2453 male Caucasians whose coronary anatomy was
defined by coronary angiography. In the total sample, the
C677T gene polymorphism was not associated with the
presence or the extent of coronary artery disease (defined
by the degree of vessel disease or by the coronary heart
disease score according to Gensini). However, after
excluding individuals with low risk profiles, an association
between the C677T TT genotype and the Gensini score was
found. This observation applies only to individuals (i) with
high glucose levels, (ii) with low apolipoprotein Al/
apolipoprotein B ratios, (iii) with low apolipoprotein Al/
apolipoprotein B ratios and high lipoprotein (a) levels and
(iv) with low apolipoprotein Al/apolipoprotein B ratios and
high glucose concentrations. In patients with high glucose
levels, the paraoxonase 191 A/B gene variation presupposed
Introduction
Homocysteine is increasingly recognized as an
independent risk factor for coronary artery disease (for
review see[1–3]). Experimental evidence shows that
homocysteine-induced atherosclerosis is characterized
by endothelial dysfunction and injury followed by
Revision submitted 17 September 1998 and accepted 30 September
1998.
Correspondence: Andreas Gardemann, Institut für Klinische
Chemie und Pathobiochemie, Klinikum der Justus-LiebigUniversität Gießen, Gaffky-Strasse 11, 35392 Gießen, Germany.
0195-668X/99/080584+09 $18.00/0
whether differences in Gensini scores between C677T C
allele carriers and TT homozygotes became apparent, since
only in paraoxonase 191 AA homoxygotes, but not in
paraoxonase 191 B allele carriers, did C677 T TT homozygotes have clearly higher Gensini scores than C allele
carriers (two-way interaction; P=0·013). The MTHFR
C677T gene polymorphism was not associated with
non-fatal myocardial infarction.
Conclusion The present study extends previous observations by the finding that carriers of the N5,N10methylenetetrahydrofolate reductase C677T TT genotype
with various coronary high risk profiles had clearly higher
coronary heart disease scores than individuals with at least
one C677T C allele.
(Eur Heart J 1999; 20: 584–592)
Key Words: Homocysteine metabolism, coronary artery
disease, myocardial infarction, coronary risk factors.
See page 552 for the Editorial comment on this article
platelet activation and thrombus formation[4–6]. In
blood, homocysteine is rapidly auto-oxidized to form
homocystine, mixed disulfides, and homocysteine
thiolactone[7,8]. The potent reactive superoxide and
hydrogen peroxides, which are produced during this
process, are mainly responsible for the vascular toxicity
of homocysteine[9]. It has been proposed that endothelial
cell injury, caused by homocysteine and mediated by
reactive oxygen species, might expose the underlying matrix and promote activation of platelets and
leukocytes[4]. Homocysteine also alters the normal antithrombotic phenotype of endothelial cells (i) by enhancing the activities of factor V[10] and factor XII[11], (ii) by
1999 The European Society of Cardiology
Gene polymorphism and coronary atherosclerosis in CAD
depressing the activation of protein C[12], and (iii) by
inhibiting the expression of thrombomodulin[13] and of
endothelial heparan sulfate[14]. In addition, homocysteine (i) is a potent mitogen for vascular smooth
muscle cells[15], (ii) has been postulated to decrease the
bioavailability of nitric oxide by impairing its synthesis[9,16] and (iii) is able to increase the formation of
highly atherogenic oxycholesterols, the peroxidation of
lipids and the oxidation of low-density lipoprotein in
vitro[17,18].
The sulphur-containing amino acid homocysteine is
formed during the metabolism of methionine. It is
metabolized by either remethylation or transsulfuration.
In the remethylation cycle, homocysteine is salvaged by
the acquisition of a methyl group in a reaction catalyzed
by methionine synthase[19]. Vitamin B12 is an essential cofactor for methionine synthase, N5-methyltetrahydrofolate is the methyl donor in this
reaction, and N5,N10-methylenetetrahydrofolate reductase (MTHFR) functions as a catalyst in the remethylation process[19]. Increases in homocysteine
plasma levels are caused by nutritional deficiencies in
vitamin cofactors, or by genetic defects in the enzymes
which are involved in the metabolism of this amino acid
(for review see[1–3]). Cystathionine â-synthase deficiency
is the most common genetic cause of severe hyperhomocysteinaemia[2].
Besides other genetic defects, a thermolabile variant of
MTHFR has been described which is caused by a point
mutation (C677T) in the coding region for the MTHFR
binding site, leading to the substitution of valine for
alanine[20,21]. It has been shown that this gene variation
was associated with increased levels of homocysteine;
carriers of the C677T TT genotype had clearly higher
plasma levels than CT heterozygotes or CC
homozygotes[21–23]. In several studies, the potential link
between this gene polymorphism and ischaemic heart
disease has been investigated. Whereas some investigators identified the TT genotype as a risk factor of
cardiovascular disease[22,24–27], other scientists failed to
identify a link between the MTHFR C677T genotype and
this disease[28–39]. These discrepancies might be due to
differences in the study design. It is also conceivable that
gene variation other than MTHFR C677T gene polymorphism, which have also been postulated to be associated
with coronary heart disease, might contribute to the
discrepant results[22,24–39], by interfering with the link
between the MTHFR C677T gene variation and the
risk of coronary artery disease and myocardial infarction. Potential candidates are polymorphisms of
the renin–angiotensin system (angiotensin I-converting
enzyme insertion/deletion polymorphism[40], angiotensinogen T174M[41] and M235T gene variations[42],
angiotensin II type 1 receptor A1166C gene variation)[43]
and gene polymorphisms of the serum enzyme paraoxonase (54 L/M[44], 191 A/B[45]), which is entirely bound to
high density lipoprotein and is considered to play an
important role in limiting the accumulation of lipid
oxidation products in low density lipoprotein[46,47]. In
the present study population, the angiotensin I-
585
converting enzyme insertion/deletion polymorphism[48],
the angiotensinogen T174M and M235T gene variations
(unpublished observation) and the paraoxonase 54 L/M
gene polymorphism (unpublished observation), but
not the angiotensin II type 1 receptor A1166C gene
variation[49] and the paraoxonase 191 A/B gene polymorphism (unpublished observation) were associated
with the extent of coronary artery disease. It was the aim
of the present study to investigate in an overall group of
2453 male Caucasians, whose coronary anatomy was
defined by means of coronary angiography, whether the
MTHFR C677T gene polymorphism was related to the
presence and extent of coronary artery disease and to
myocardial infarction in the total population and among
subjects who were at lower or higher risk of these
diseases. In addition, we analysed potential interactions
between the MTHFR C677T polymorphism and gene
variations of the renin–angiotensin system, and of the
serum enzyme paraoxonase, on the risk of coronary
heart disease.
Methods
Detection of coronary artery disease and
myocardial infarction
The MTHFR C677T gene polymorphism was analysed in
2453 male Caucasian individuals. These were consecutive patients who underwent coronary angiography for
diagnostic purposes. About 80% of the participants
underwent coronary angiography on account of coronary heart disease as a verified illness or presumptive
diagnosis. The remainder of the group consisted almost
completely of patients who underwent coronary angiography for clarification of restricted left ventricular
function. In 90% of these patients, coronary artery
disease was the reason for this dysfunction. Only in 10%
of this subpopulation (=2% of the total sample), was
restricted left ventricular function caused by dilated
cardiomyopathy or longstanding arterial hypertension.
All patients who agreed to participate in the study
were evaluated with a detailed questionnaire which
provided information about coronary risk factors such
as smoking, diabetes mellitus and hypertension.
The retrospective study lasted 2·5 years and ended in
July 1997. Patients were initially recruited from the
departments of cardiovascular surgery and from the
department of cardiology of the university hospital in
Giessen, and — during the last year — from the department of cardiology of the Kerckhoff-Klinik in Bad
Nauheim. (W. H. who carried out or supervised the
majority of the coronary angiographies, moved from
Giessen to Bad Nauheim in July 1996.)
Coronary artery disease
Coronary angiography was performed by the Judkins
method. Coronary vessels with at least 50% stenosis
were defined as diseased. The severity of coronary heart
disease was also estimated by calculating the Gensini
Eur Heart J, Vol. 20, issue 8, April 1999
586
A. Gardemann et al.
score[50]; this score is subsequently designated the ‘CHD
score’. By means of coronary angiography, the study
population was divided into subjects without any
angiographically detectable coronary artery disease
or with coronary arterial stenoses less than 50% (no
vessel disease) and individuals with single-, double- or
triple-vessel disease.
Myocardial infarction
Angina pectoris and acute myocardial infarction were
diagnosed according to criteria established by the World
Health Organisation. Sixteen percent of patients without
vessel disease, 48% of individuals with single-vessel
disease, 55% of patients with double-vessel disease and
61% of patients with triple-vessel disease had suffered a
myocardial infarction before recruitment in the study.
Measurements of substrates, definition of
variables and detection of the MTHFR
C677T gene polymorphism
Triglycerides, total cholesterol, apolipoprotein B, apolipoprotein AI, lipoprotein (a), glucose and fibrinogen
were measured by conventional methods of clinical
chemistry[48]. Diabetes mellitus was defined as a binary
variable and not divided into subcategories. Hypertension (binary variable in the present study) was defined by
either treatment or diastolic blood pressure greater than
95 mmHg on two consecutive visits for those untreated.
Cigarette consumption was given as pack years (1 pack
year: e.g. 20 cigarettes per day for one year). The
MTHFR C677T gene polymorphism was analysed in all
study patients according to Nishinaga et al.[14]. Genotypes for angiotensin I-converting enzyme insertion/
deletion polymorphism[51,52], angiotensinogen T174M[53]
and M235T gene variations[54], angiotensin II type 1
receptor A1166C gene variation[55], paraoxonase 54
L/M[44] and paraoxonase 191 A/B gene polymorphisms[56] were determined in all study patients as
described.
Definition of low and high risk
subpopulations
The mean values of coronary risk factors from the whole
study population were used as cut points for the definition of low and high risk populations. With respect to
hypertension and diabetes, low and high risk groups
were defined by the absence or presence of these diseases. With respect to the plasma glucose level and the
apolipoprotein AI/apolipoprotein B ratio not only the
mean value but also the 10th, 25th, 50th, 75th and
90th percentiles were used to define low and high risk
populations.
Statistical analysis
Statistical analyses were performed using the SPSS
for Windows 95 software (Version 7·52). In order to
Eur Heart J, Vol. 20, issue 8, April 1999
compare established risk factors between MTHFR
C677T genotypes, the relationship of the MTHFR
C677T gene polymorphism to continuous variables
was tested by Kruskal–Wallis one-way ANOVA; the
relationship of the MTHFR C677T gene polymorphism
to diabetes and hypertension was checked by chisquare analysis. Established risk factors of coronary
artery disease and myocardial infarction were identified
by multiple regression analysis (extent of coronary
artery disease, CHD score) or multiple logistic regression (absence/presence of coronary artery disease or
myocardial infarction). The chi-square test was used to
test for deviation of genotype distribution from
Hardy–Weinberg equilibrium. The relationship of the
MTHFR C677T gene polymorphism to the extent of
coronary artery disease (coronary artery disease, CHD
score) was determined by multiple regression analysis;
coronary risk factors (age, cholesterol, apolipoprotein
AI, apolipoprotein B, lipoprotein (a), fibrinogen, body
mass index, smoking habit, diabetes, hypertension)
were introduced into the calculation. The relationship
of the MTHFR C677T gene polymorphism to the
presence of coronary artery disease and to myocardial
infarction was determined by multiple logistic regression with adjustment for the above-mentioned
coronary risk factors. The interactions between the
paraoxonase 191 A/B gene variation and the MTHFR
C677T gene polymorphism on the CHD score were
tested by a two-factor ANOVA procedure. A twosided probability value of less than 0·05 was considered
to indicate statistical significance.
Results
Distribution of MTHFR C677T genotypes
within the study population and effect of
MTHFR C677T gene polymorphism on
variables of clinical chemistry and clinics
The distributions of the MTHFR C677T genotypes in
controls (subjects without coronary artery disease and
myocardial infarction) were in Hardy–Weinberg’s equilibrium. Age, total cholesterol, triglycerides, apolipoprotein AI, apolipoprotein B, apolipoprotein AI/
apolipoprotein B ratio, lipoprotein (a), glucose,
fibrinogen, prevalence of diabetes and arterial hypertension, body mass index and cigarette consumption
were almost identical between MTHFR C677T genotypes (i) of the total study population, (ii) of patients
without or with single- or multi-vessel disease and (iii)
of subjects without and with myocardial infarction
(data not shown). The mean delay between the occurrence of myocardial infarction and recruitment in this
study (5·380·3 years for the total population) did
not differ between MTHFR C677T genotypes (data not
shown).
Gene polymorphism and coronary atherosclerosis in CAD
Table 1
587
Risk factors of coronary artery disease and myocardial infarction
Coronary artery disease
Risk factor
Age (years)
BMI (kg . m 2)
Smoking habit (pack years)
% Diabetes
% Hypertension
% treated hyperchol.
Glucose (mg/dl 2)
Cholesterol (mg/dl 2)
Triglycerides (mg/dl 2)
ApoAI (g . l 1)
ApoB (g . l 1)
ApoAI/ApoB ratio
Lp(a) (mg . dl 1)
Fibrinogen
Myocardial infarction
Mean value
CAD
(n=560)
+CAD
(n=1893)
2P
MI
(n=1301)
+MI
(n=1152)
2P
58·310·7
26·93·8
19·624
11
54
6
10940
20643
14494
1·470·30
1·210·32
1·290·46
2130
3·421·61
62·69·3
26·93·3
23·527
20
66
14
10949
21143
15694
1·420·29
1·300·35
1·160·44
3039
3·511·22
<0·0001
ns
<0·05
<0·01
<0·002
<0·001
ns
ns
ns
<0·005
<0·0001
<0·0001
<0·0001
ns
61·110·1
26·93·5
20·125
17
64
9
11043
21042
15398
1·450·29
1·260·34
1·220·41
26·937
3·441·31
62·29·5
26·93·3
25·427
19
62
15
11453
20943
15589
1·400·29
1·290·35
1·160·48
29·137
3·601·11
<0·01
ns
<0·0001
ns
ns
<0·005
<0·05
ns
ns
<0·005
<0·0001
—0·005
ns
<0·05
61·69·8
26·93·4
22·626
18
63
12
11248
21043
15494
1·430·29
1·280·34
1·190·45
28·237
3·511·22
Values are meansSD or % of a group. The relations of the coronary risk factors to the presence of coronary artery disease and to
myocardial infarction were analysed by multiple logistic regression. BMI=body mass index; hyperchol.=hypercholesterolaemia;
ApoAI=apolipoprotein AI; ApoB=apolipoprotein B; Lp(a)=lipoprotein (a); ns=not significant.
Table 2 Distribution of the MTHFR C677T genotypes in patients with or without coronary artery disease and with or
without myocardial infarction
Controls/Cases
CAD
CHD score=0 (no MI)
No vessel disease
Single-vessel disease
Double-vessel disease
Triple-vessel disease
Single, double or triple vessel disease
MI
No MI
At least 1 MI
Mean age (SD)
n
53·911·2
58·310·7
61·09·7
62·49·8
63·68·6
62·69·3
61·19·7
62·29·6
MTHFR C677T genotype
n (CC)
n (CT)
n (TT)
185
560
487
539
867
1893
88
242
220
256
415
891
82
254
211
237
357
805
15
64
56
46
95
197
1301
1152
606
527
544
515
151
110
C/T Alleles
C (95% CI)
0·66
0·66
0·67
0·69
0·69
0·68
(0·62–0·71)
(0·63–0·69)
(0·64–0·70)
(0·67–0·72)
(0·66–0·71)
(0·67–0·70)
0·675 (0·66–0·69)
0·68 (0·66–0·70)
T (95% CI)
0·34
0·34
0·33
0·31
0·31
0·32
(0·29–0·38)
(0·31–0·37)
(0·30–0·36)
(0·28–0·33)
(0·29–0·34)
(0·30–0·33)
0·325 (0·31–0·34)
0·32 (0·30–0·34)
CAD=coronary artery disease; MI=myocardial infarction; CHD=coronary heart disease.
Relation of established coronary risk factors
and of the MTHFR C677T genotypes to
coronary artery disease
Established coronary risk factors
Apolipoprotein B (P<0·0001), hypercholesterolaemia
(P<0·001), lipoprotein (a) (P<0·0001), diabetes mellitus
(P<0·01), hypertension (P<0·002), smoking habit
(P<0·05) and age (P<0·0001) could be demonstrated as
risk factors for coronary artery disease, and apolipoprotein AI (P<0·005) and consequently high apolipoprotein
AI/apolipoprotein B ratios (P<0·0001) as protective
factors against coronary artery disease (Table 1). The
mean values of coronary risk factors (Table 1) were
used as cut points for definition of low and high risk
populations.
MTHFR C677T gene polymorphism
In the total population, the MTHFR C677T gene polymorphism was not associated with the presence or the
extent of coronary artery disease (defined by the degree
of vessel disease) (Table 2). In addition, the mean CHD
score was similar between MTHFR C677T genotypes in
the total sample (Fig. 1). However, exclusion of individuals with ‘low risk profiles’ resulted in an association of
the TT genotype with the CHD score (Fig. 1). This
observation applies to restriction of individuals (i) with
high glucose levels (>112 mg . dl 1, mean value), (ii)
with low apolipoprotein AI/apolipoprotein B ratios
(<1·19, mean ratio), (iii) with low apolipoprotein AI/
apolipoprotein B ratios (<1·19) and high lipoprotein (a)
levels (>27·9 mg . dl 1, mean value) and (iv) with low
apolipoprotein AI/apolipoprotein B ratios (<1·19) and
Eur Heart J, Vol. 20, issue 8, April 1999
588
A. Gardemann et al.
100
apoAI/apoB < 1·19 +
–1
Glucose > 112 mg dl
apoAI/apoB < 1·19 +
Lp(a) > 27·9 mg dl–1
Gensini coronary heart disease score
90
Glucose > 112 mg dl–1
80
apoAI/apoB < 1·19
70
60
Total sample
TT
CC + CT
TT
CC + CT
TT
CC + CT
TT
CC + CT
TT
40
CC + CT
50
(2192)
(261)
(625)
(66)
(1181)
(126)
(404
(46)
(313)
(41)
P = 0·82
P = 0·018
P = 0·039
P = 0·040
P = 0·032
Figure 1 Comparison of mean Gensini scores between MTHFR C677T TT homozygotes and carriers of at
least one C allele. Values are meansSEM. The relationship of the MTHFR C677T gene polymorphism
(TT vs CT+CC subjects) to mean Gensini scores was checked by multiple regression analysis with
adjustment to coronary risk factors. Numbers of subjects are given in parenthesis. ApoAI=apolipoprotein
AI; apoB=apolipoprotein B; Lp(a)=lipoprotein (a).
high glucose plasma concentrations (>112 mg . dl 1)
(Fig. 1). Since the apolipoprotein B ratio, but not the
plasma glucose level was normally distributed in our
study sample, additional analyses were performed by
using the 10th, 25th, 50th, 75th and 90th percentiles
of the apolipoprotein AI/apolipoprotein B ratio and of
the glucose concentration as cut points for the inclusion of individuals in high risk populations. The
results of these analyses support our data, that within
subgroups of individuals with low apolipoprotein
AI/apolipoprotein B ratios or with high glucose levels,
TT homozygotes had clearly higher CHD scores
than C allele carriers (data not shown). This observation applies to the 50th (<1·12), 25th (<0·93)
and 10th (<0·78) percentile of the apolipoprotein
AI/apolipoprotein B ratio and to the 50th
(>98 mg . dl 1), 75th (>111 mg . dl 1) and 90th
(>151 mg . dl 1) percentile of the plasma glucose
level (data not shown). In other high risk and also
low risk subpopulations an association of the T
allele or of the TT genotype with the presence or
extent of coronary artery disease (degree of vessel
disease, CHD score) was not detected (data not
shown).
Eur Heart J, Vol. 20, issue 8, April 1999
Interactions between the MTHFR C677T gene variation
and other gene polymorphisms on the risk of coronary
artery disease
We analysed potential interactions between the
MTHFR C677T gene polymorphism and gene variations
of
the
renin–angiotensin
system
(angiotensin
I-converting enzyme insertion/deletion polymorphism[40], angiotensinogen T174M[41] and M235T gene
variations[42], angiotensin II type 1 receptor A1166C
gene variation[43]) and of the serum enzyme paraoxonase
(54 L/M[44], 191 A/B[45]) on the risk of coronary artery
disease. In the total sample, interactions between the
MTHFR gene variation and one of the abovementioned gene polymorphisms were not detected
(data not shown). In low and high risk populations,
this observation also applies to potential interactions
between the MTHFR C677T gene variation and one of
the polymorphisms of the renin–angiotensin system. In
contrast, in individuals with high glucose levels, the
paraoxonase 191 A/B gene variation pre-supposed
whether differences in CHD scores between MTHFR
C677T C allele carriers and TT homozygotes became
apparent; pronounced differences in CHD scores were
detected only in paraoxonase 191 AA homozygotes
Gene polymorphism and coronary atherosclerosis in CAD
589
110
Glucose > 112 mg dl–1 +
PON191 AA
90
80
Glucose > 112 mg dl–1
Glucose > 112 mg dl–1 +
PON191 AB + BB
70
(525)
(66)
P = 0·018
(264)
(39)
P = 0·801
(261)
TT
CC + CT
TT
40
CC + CT
50
TT
60
CC + CT
Gensini coronary heart disease score
100
(27)
P = 0·013
Figure 2 Comparison of mean Gensini scores between MTHFR C677T TT homozygotes and carriers of at
least one C allele according to paraoxonase PON191 A/B genotypes. Values are meansSEM. The
relationship of the MTHFR C677T gene polymorphism (TT vs CT+CC subjects) to mean Gensini scores
were checked by multiple regression analysis with adjustment to coronary risk factors. Numbers of
individuals are given in parentheses. PON=paraoxonase.
(two-way interaction between both gene variations on
CHD score; P=0·013), but not in paraoxonase 191 B
allele carriers (Fig. 2). Similar observations were made,
for example, in a subpopulation of individuals with high
glucose levels and low apolipoprotein AI/apolipoprotein
B ratios (data not shown). The paraoxonase 54 L/M
gene polymorphism[44] did not influence the association
of the MTHFR C677T gene polymorphism with the
CHD score (data not shown).
Relation of coronary risk factors and of the
MTHFR C677T gene polymorphism to
myocardial infarction
Established coronary risk factors
Age (P<0·01), ApoB (P<0·0001), hypercholesterolaemia
(P<0·005), glucose, (P<0·05), fibrinogen levels (P<0·05)
and cigarette consumption (P<0·002) were identified as
risk factors for myocardial infarction, and apolipoprotein AI (P<0·005) and consequently high apolipoprotein
AI/apolipoprotein B ratios as protective factors against
myocardial infarction (Table 1).
MTHFR C677T gene polymorphism
In the total population (Table 2), and in high and low
risk subpopulations (data not shown) an association of
the MTHFR C677T gene polymorphism with non-fatal
myocardial infarction was not detected. This observation applies to a comparison (i) of all three genotypes
(P=0·90), (ii) of the TT genotype vs CT+TT (odds ratio
0·76 (0·55–1·05); P=0·092), (iii) of the T allele (TT+TC)
vs CC homozygotes (odds ratio 1·09 (0·90–1·32);
P=0·39) and (iv) of TT genotypes vs CC homozygotes
(odds ratio 0·91 (0·77–1·08); P=0·28).
Interactions between the MTHFR C677T gene variation
and other gene polymorphisms on the risk of myocardial
infarction
Neither gene variations of the renin–angiotensin system
nor of paraoxonase had any significant influence on the
relationship of the MTHFR C677T gene polymorphism
to the risk of myocardial infarction (data not shown).
Discussion
Elevated levels of the thiol-containing amino acid
homocysteine are an independent, graded risk factor
for the development of atherosclerotic vascular disease
(for review see [1–3]). A thermolabile variant of the
enzyme N5, N10-methylenetetrahydrofolate reductase
(MTHFR) — involved in the remethylation pathway of
homocysteine to methionine — has been described and
Eur Heart J, Vol. 20, issue 8, April 1999
590
A. Gardemann et al.
is caused by a point mutation (C677T) in the coding
region for the MTHFR binding site[20,21]. TT homozygotes of this gene variation have been repeatedly
shown to have clearly higher homocysteine plasma values than CT heterozygotes or CC homozygotes[21–23].
However, conflicting results have been obtained with
respect to the potential association of the MTHFR
C677T gene variation with coronary artery disease and
myocardial infarction. Therefore, the present investigation was performed to analyse the relationship of the
MTHFR C677T gene polymorphism with coronary heart
disease in a large case-control study of 2453 participants.
The present findings allow the following conclusions.
Design of the present study
Established risk factors of coronary artery disease (age,
hypercholesterolaemia, apolipoprotein B, lipoprotein
(a), diabetes mellitus, hypertension, cigarette consumption) and of myocardial infarction (age, hypercholesterolaemia, apolipoprotein B, fibrinogen, smoking
habit) could be identified in the present study sample.
Also, known protective factors against coronary heart
disease, such as apolipoprotein AI and high apolipoprotein AI/apolipoprotein B ratios, were found in our
investigation. The present study did not show an association between cholesterol or fibrinogen and coronary
artery disease and between diabetes, hypertension, cholesterol or lipoprotein (a) and myocardial infarction.
However, when the group of patients without myocardial infarction was restricted to individuals without
coronary artery disease, lipoprotein (a) (P=0·0001),
diabetes (P=0·048) and hypertension (P=0·0006) could
be identified as risk factors of myocardial infarction
(data not shown). When this group was compared with
the subpopulation of patients with myocardial infarction, we obtained essentially the same results for the
relationship of the MTHFR C677T gene polymorphism
to myocardial infarction (data not shown). Therefore,
the present results strengthen the hypothesis that comparisons within our study group would lead to accurate
predictions of new coronary risk factors.
Distributions of the MTHFR C677T
genotypes
The frequencies of the C and T alleles of the present
study sample show great similarities to those of previous
investigations. For example, C allele frequencies of
0·61–0·70[22–39,57] were calculated in subpopulations of
control subjects.
Relation of the MTHFR C677T gene
polymorphism with coronary artery disease
Several studies have dealt with the question whether
the MTHFR C677T gene variation might be associated
Eur Heart J, Vol. 20, issue 8, April 1999
with an increased risk for the presence and/or the
extent of coronary artery disease[22,24–26,28,31,32,35,37–39].
In most studies, coronary artery disease has been
documented by means of coronary angiography. The
majority of investigations failed to detect an association of the MTHFR gene variation with the presence
of coronary artery disease[28,31,32,35,37–39]. Only in this
study and two others was the relationship of the
MTHFR gene polymorphism with the extent of coronary artery disease investigated (defined by the
degree of vessel disease)[25,37]. Whereas van Bockxmeer
et al.[37] and the present investigation found no association between the gene variation and the extent of
coronary artery disease, Morita et al.[25] observed that
the frequency of the MTHFR C677T TT genotype was
correlated with the severity of coronary artery disease.
However, we emphasize that we not only divided our
study sample into coronary heart disease-positive and
coronary heart disease-negative subjects or patients
with single-, double- or triple-vessel disease, but also
determined the precise extent of coronary heart disease
of all participants by calculating a CHD score[50].
These analyses enabled us to identify the MTHFR
C677T TT genotype as an independent risk factor of
coronary artery disease in various populations at high
risk for coronary artery disease. Since we did not
measure homocysteine levels of the 2453 study participants, we do not have direct evidence that the TT
genotype was also associated with elevated plasma
levels of homocysteine. Nevertheless, it has repeatedly
been shown that TT homozygotes have higher homocysteine levels than the other MTHFR C677T
genotypes[21–23]. Since Jacques et al.[58] demonstrated
that these differences disappeared when individuals had
folate levels greater than 15·4 nmol . l 1, they suggested that MTHFR C677T TT homozygotes may
require folate supplementation to regulate plasma
homocysteine concentrations. Further studies are
needed to clarify whether it is justified to perform
a PCR test and institute homocysteine-lowering
treatment instead of directly determining homocysteine
levels to guide homocysteine-lowering therapy.
Relation of the MTHFR C677T gene
polymorphism with myocardial infarction
In accordance with the majority of investigations[27,29–31,35], but in contrast to one study[24] we
failed to detect an association of the gene polymorphism
with non-fatal myocardial infarction. It appears inconsistent that in various high risk groups the TT genotype
was associated with the CHD score but not with
myocardial infarction. Nevertheless, in the present casecontrol study, it should be noted that survivors of
myocardial infarction and not patients with fatal outcome of this disease were included. This bias of selection
may explain, at least in part, the discrepancies.
Gene polymorphism and coronary atherosclerosis in CAD
Potential interactions between the MTHFR
C677T gene polymorphism and other gene
variations on the risk of coronary heart
disease
Regarding the divergent results obtained in recent
studies on the association of the MTHFR C677T gene
variation with coronary heart disease[22,24–39], we speculated that these discrepancies could, at least in part, be
explained by gene variations other than the MTHFR
gene polymorphism, which was also postulated to be
associated with coronary heart disease[40–47] and which
might interfere to a different degree with the link
between the MTHFR C677T gene variation and the risk
of coronary heart disease. No interactions could be
detected between one of the gene variations of the
renin–angiotensin system and the MTHFR gene polymorphism on the risk of coronary artery disease and
myocardial infarction. Only an interaction between
paraoxonase 191 A/B gene variation, and the MTHFR
C677T gene variation could be observed in a subpopulation of individuals with high plasma glucose levels.
Further investigations are required to examine whether
this interaction is also detectable in other populations
and to evaluate the clinical significance of this finding.
Conclusion
In the total study sample of 2453 participants, we could
not detect an association of the MTHFR C677T gene
polymorphism with the risk of coronary artery disease
and myocardial infarction. These observations are in
line with results obtained in the majority of other recent
investigations. However, the present study extends previous investigations by the finding that carriers of the
MTHFR C677T TT genotype with coronary high risk
profiles had clearly higher CHD scores than individuals
with at least one MTHFR C677T C allele.
References
[1] Mayer EL, Jacobsen DW, Robinson K. Homocysteine and
coronary atherosclerosis. J Am Coll Cardiol 1996; 27: 517–27.
[2] Welch GN, Loscalzo L. Homocysteine and atherothrombosis.
N Engl J Med 1998; 338: 1042–50.
[3] Miner SES, Evrovski J, Cole DEC. Clinical chemistry and
molecular biology of homocysteine metabolism: an update.
Clin Biochem 1997; 30: 189–201.
[4] Harker LA, Slichter SJ, Scott CR, Ross R. Homocysteinemia:
vascular injury and arterial thrombosis. N Engl J Med 1974;
291: 537–43.
[5] Harker LA, Ross R, Slichter SJ, Scott CR. Homocystineinduced arteriosclerosis: the role of endothelial cell injury and
platelet response in its genesis. J Clin Invest 1976; 58: 731–41.
[6] James TN. The spectrum of diseases of small arteries and
their physiologic consequences. J Am Coll Cardiol 1990; 15:
763–74.
[7] Stamler JS, Osborne JA, Jaraki O. Adverse vascular effects of
homocysteine are modulated by endothelium-derived relaxing
factor and related oxides of nitrogen. J Clin Invest 1993; 91:
308–18.
591
[8] Andersson A, Lindgren A, Hultberg B. Effect of thiol oxidation and thiol export from erythrocytes on determination of
redox status of homocysteine and other thiols in plasma from
healthy subjects and patients with cerebral infarction. Clin
Chem 1995; 41: 361–6.
[9] Welch GN, Upchurch Jr GR, Loscalzo J. Hyperhomocysteinaemia and atherothrombosis. Ann N Y Acad Sci 1997;
811: 48–58.
[10] Rodgers GM, Kane WH. Activation of endogenous factor V
by a homocysteine-induced vascular endothelial cell activator.
J Clin Invest 1986; 77: 1909–16.
[11] Ratnoff OD. Activation of Hageman factor by Lhomocysteine. Science 1968; 162: 1007–9.
[12] Rodgers GM, Conn MT. Homocysteine, an atherogenic
stimulus, reduces protein C activation by arterial and venous
endothelial cells. Blood 1990; 75: 895–901.
[13] Lentz SR, Sadler JE. Inhibition of thrombomodulin surface
expression and protein C activation by the thrombogenic
agent homocysteine. J Clin Invest 1991; 88: 1906–14.
[14] Nishinaga M, Ozawa T, Shimada K. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate
expression in cultured porcine aortic endothelial cells. J Clin
Invest 1993; 92: 1381–6.
[15] Tsai J-C, Perrella MA, Yoshizumi M. Promotion of vascular
smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci U.S.A. 1994; 91: 6369–73.
[16] Loscalzo J. The oxidant stress of hyperhomocysteinemia.
J Clin Invest 1996; 98: 5–7.
[17] Heinecke JW, Kawamura M, Suzuki L, Chait A. Oxidation of
low density lipoprotein by thiols: superoxide-dependent and
-independent mechanisms. J Lipid Res 1993; 34: 2051–61.
[18] Parthasarathy S. Oxidation of low-density lipoprotein by
thiol compounds leads to its recognition by the acetyl LDL
receptor. Biochem Biophys Acta 1987; 917: 337–40.
[19] Ueland PM, Refsum H, Stabler SP, Malinow MR, Andersson
A, Allen RH. Total homocysteine in plasma or serum: methods and clinical applications. Clin Biochem 1993; 93: 1764–79.
[20] Kang SS, Zhou J, Wong PWK, Kowalysin J, Strokosch G.
Intermediate homocysteinemia: a thermolabile variant of
methylentetrahydrofolate reductase. Am J Hum Genet 1988;
43: 414–21.
[21] Frosst P, Blom HJ, Milos R et al. A candidate genetic risk
factor for vascular disease: a common mutation in methylentetrahydrofolate reductase. Nature Genetics 1995; 10:
111–13.
[22] Ou T, Yamakawa Kobayashi K, Arinami T et al. Methylenetetrahydrofolate reductase and apolipoprotein E polymorphisms are independent risk factors for coronary heart disease
in Japanese: a case-control study. Atherosclerosis 1998; 137:
23–8.
[23] Gudnason V, Stansbie D, Scott J, Bowron A, Nicaud V,
Humphries S. C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): its
frequency and impact on plasma homocysteine concentration
in different European populations. Atherosclerosis 1998; 136:
347–54.
[24] Kluijtmans LAJ, Lambert PWJ, van den Heuvel LPWJ et al.
Molecular genetic analysis in mild hyperhomocysteinemia:
A common mutuation in the methylenetetrahydrofolate
reductase gene is a genetic risk factor for cardiovascular
disease. Am J Hum Genet 1996; 58: 35–41.
[25] Morita H, Taguchi J, Kurihara H et al. Genetic polymorphism of 5, 10-methylenetetrahydrofolate reductase
(MTHFR) as a risk factor for coronary artery disease.
Circulation 1997; 95: 2032–6.
[26] Brulhart MC, Dussoix P, Ruiz J, Passa P, Froguel P, James
RW. The (Ala-Val) mutation of methylenetetrahydrofolate
reductase as a genetic risk factor for vascular disease in
non-insulin-dependent diabetic patients. Am J Hum Genet
1997; 60: 228–9.
[27] Adams M, Smith , Martin D, Thompson JR, Samani NJ.
Genetic analysis of thermolabile methylenetetrahydrofolate
Eur Heart J, Vol. 20, issue 8, April 1999
592
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
A. Gardemann et al.
reductase as a risk factor for myocardial infarction. QJM
Monthly J Ass Physicians 1996; 89: 437–44.
Wilcken DEL, Wang XL, Sim AS, McCredie RM. Distribution in healthy and coronary populations of the methylenetetrahydrofolate reductase (MTHFR) C677T mutation. Art
Thromb Vasc Biol 1996; 16: 878–82.
Schmitz C, Lindpaintner K, Verhoef P, Gaziano JM, Buring
J. Genetic polymorphism of methylenetetrahydrofolate
reductase and myocardial infarction — a case-control study.
Circulation 1996; 94: 1812–14.
Ma J, Stampfer MJ, Hennekens CH et al. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US Physicians.
Circulation 1996; 94: 2410–16.
Anderson JL, King GJ, Thomson MJ et al. A mutation in the
methylenetetrahydrofolate reductase gene is not associated
with increased risk for coronary artery disease or myocardial
infarction. J Am Coll Cardiol 1997; 30: 1206–11.
Verhoef P, Kok FJ, Kluijtmans LAJ et al. The 677C->T
mutation in the methylenetetrahydrofolate reductase gene:
Associations with plasma total homocysteine levels and risk of
coronary atherosclerotic disease. Atherosclerosis 1997; 132:
105–13.
Schwartz SM, Siscovick DS, Malinow MR et al. Myocardial
infarction in young women in relation to plasma total homocysteine, folate, and a common variant in the methylenetetrahydrofolate reductase gene. Circulation 1997; 96: 412–417.
Christensen B, Frosst P, Lussier Cacan S et al. Correlation
of a common mutation in the methylenetetrahydrofolate
reductase gene with plasma homocysteine in patients with
premature coronary artery disease. Art Thromb Vasc Biol
1997; 17: 569–573.
Brugada R, Marian AJ. A common mutation in methylenetetrahydrofolate reductase gene is not a major risk of coronary artery disease or myocardial infarction. Atherosclerosis
1997; 128: 107–12.
Deloughery TG, Evans A, Sadeghi A et al. Common mutation
in methylenetetrahydrofolate reductase: Correlation with
homocysteine metabolism and late-onset vascular disease.
Circulation 1996; 94: 3074–8.
Van Bockxmeer FM, Mamotte CDS, Vasikaran SD, Taylor
RR. Methylenetetrahydrofolate reductase gene and coronary
artery disease. Circulation 1997; 95: 21–3.
Reinhardt D, Sigusch HH, Vogt SF, Farker K, Muller S,
Hoffmann A. Absence of association between a common
mutation in the methylenetetrahydrofolate reductase gene and
the risk of coronary artery disease. Eur J Clin Invest 1998; 28:
20–3.
Abbate R, Sardi I, Pepe G et al. The high prevalence of
thermolabile 5-10 methylenetetrahydro-folate reductase
(MTHFR) in Italians is not associated to an increased risk for
coronary artery disease (CAD). Thromb Haem 1998; 79:
727–30.
Cambien F, Poirier O, Lecerf L et al. Deletion polymorphism
in the gene for angiotensin-converting enzyme is a potent risk
factor for myocardial infarction. Nature 1992; 359: 641–4.
Ko YL, Ko YS, Wang SM et al. Angiotensinogen and
angiotensin I converting enzyme gene polymorphisms and the
risk of coronary artery disease in Chinese. Human Genetics
1997; 100: 210–14.
Katsuuya T, Koike G, Yee TW, Sharpe N, Jackson R, Norton
R. Association of angiotensinogen gene T235 variant with
increased risk of coronary artery disease. Lancet 1995; 345:
1600–3.
Eur Heart J, Vol. 20, issue 8, April 1999
[43] Tiret L, Bonnardeaux A, Poirier O et al. Synergistic effects
of angiotensin-converting enzyme and angiotensin-II type 1
receptor gene polymorphisms on risk of myocardial
infarction. Lancet 1994; 344: 910–13.
[44] Blatter Garin M-C, James RW, Dussoix P et al. Paraoxonase
polymorphism Met-Leu54 is associated with modified
serum concentrations of the enzyme. J Clin Invest 1997; 99:
62–6.
[45] Serrato N, Marian AJ. A variant of human paraoxonase/
arylesterase (HUMPONA) gene is a risk factor for coronary
heart disease. J Clin Invest 1995; 96: 3005–8.
[46] Mackness MI, Durrington PN. HDL, its enzymes and its
potential to influence lipid peroxidation. Atherosclerosis 1995;
115: 243–53.
[47] Watson AD, Berliner JA, Hama SY et al. Protective effect of
high density lipoprotein associated paraoxonase. Inhibition
of the biological activity of minimally oxidised low density
lipoprotein. J Clin Invest 1995; 96: 2882–91.
[48] Gardemann A, Fink M, Stricker J et al. ACE I/D gene
polymorphism: Presence of the ACE D allele increases the
risk of coronary artery disease in younger individuals. Atherosclerosis 1998; 139: 153–9.
[49] Gardemann A, Nguyen QD, Humme J et al. Angiotensin II
type 1 receptor A1166C gene polymorphism: Absence of an
association with the risk of coronary artery disease and
myocardial infarction and of a synergistic effect with the
angiotensin-converting enzyme gene polymorphism on the
risk of these diseases. Eur Heart J 1998; 19: 1657–65.
[50] Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol 1983;
51: 606.
[51] Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P,
Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the
variance of serum enzyme levels. J Clin Invest 1990; 86:
1343–6.
[52] Lindpaintner K, Pfeffer MA, Kreutz R et al. A prospective
evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J
Med 1995; 332: 706–11.
[53] Hegele RA, Brunt H, Connelly PW. A polymorphism
of the angiotensinogen gene associated with variation in
blood pressure in a genetic isolate. Circulation 1994; 90:
2207–12.
[54] Russ AP, Maerz W, Ruzicka V, Stein U, Gross W. Rapid
detection of the hypertension-associated Met235->Thr allele
of the human angiotensinogen gene. Hum Mol Genet 1993; 2:
609–10.
[55] Hingorani AD, Brown MJ. A simple molecular assay for the
C1166 variant of the angiotensin II type 1 receptor gene.
Biochem Biophys Res Commun 1995; 213: 725–9.
[56] Ruiz J, Blanche’ H, James RW et al. The polymorphism
(Gln-Arg192) of the high-density lipoprotein-bound enzyme
is an independent cardiovascular risk factor in non-insulin
dependent diabetic patients. Lancet 1995; 346: 869–72.
[57] Fodinger M, Mannhalter C, Wolfl G et al. Mutation (677 C to
T) in the methylenetetrahydrofolate reductase gene aggravates
hyperhomocysteinemia in hemodialysis patients. Kidney Int
1997; 52: 517–23.
[58] Jacques PF, Bostom AG, Williams RR et al. Relation between
folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations.
Circulation 1996; 93: 7–9.