European Journal of Internal Medicine 18 (2007) 474 – 483

www.elsevier.com/locate/ejim

Original article
Relation between plasma homocysteine, gene polymorphisms of
homocysteine metabolism-related enzymes, and angiographically proven
coronary artery disease
Abdelilah Laraqui a,b,c,⁎, Abdellatif Allami a,d , Alain Carrié c , Alain Raisonnier c ,
Anne-Sofie Coiffard c , Fatima Benkouka b , Abdenabi Bendriss d , Abdelaziz Benjouad b ,
N. Bennouar a , Nizar El Kadiri a , Anwar Benomar a , Seddik Fellat a , Mohamed Benomar e
a
Ligue Nationale de Lutte Contre les Maladies Cardiovasculaires, Unité d'Etudes des Facteurs Métaboliques et Polymorphismes Génétiques, Rabat, Morocco
b
UFR Biochimie Immunologie, Faculté des Sciences, Université Mohamed V. Rabat, Morocco
c
Laboratoire de Biochimie Médicale A, Unité Fonctionnelle Endocrinologie-Moléculaire-Oncologie, CHU Pitié-Salpêtrière, Paris, France
d
UFR Biologie Cellulaire et Moléculaire, Faculté des Sciences, Université Abdelmalek Es-Saadi, Tétouan, Morocco
e
Ligue Nationale de lutte contre les maladies cardiovasculaires, Service de Cardiologie A, CHU Ibn-Sina, Rabat, Morocco
Received 31 May 2006; received in revised form 12 November 2006; accepted 15 February 2007

Abstract

Background: Hyperhomocyteinemia (HHcy) is a risk factor for coronary artery disease (CAD), and methylenetetrahydrofolate reductase
(MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) polymorphisms may contribute to plasma total
homocysteine (tHcy) variation. We investigated the association of polymorphisms 1298A→C in the MTHFR gene, 2756A→G in the MTR
gene, and 66A→G in the MTRR gene with tHcy levels and with CAD in patients undergoing coronary angiography.
Methods: CAD patients (n = 151) and control subjects (n = 79) were compared regarding the prevalence of the polymorphisms, risk factors,
and biochemical parameters.
Results: The mean tHcy concentration was significantly higher in CAD patients than in control subjects (P b 0.001). HHcy (tHcy ≥ 15 μmol/l)
conferred an OR of CAD of 4.1 (95% CI 2.2–7.5, P b 0.001). In both cases and controls, smokers had a higher tHcy level than non-smokers
and demonstrated a markedly increased risk for CAD (OR = 2.5, 95% CI 1.7–3.3, P b 0.001). The allele frequencies of the MTHFR
1298A→C, MTR 2756A→G, and MTRR 66A→G mutations were 36.7%, 15.7%, and 36.6%, respectively. The 1298C allele frequency was
significantly higher in the CAD group than in controls (P b 0.05) and showed a significant association with CAD in heterozygote carriers.
There was no statistically significant difference between cases and controls in the frequencies of the A2756G alleles/genotypes in the MTR
gene and of the A66G alleles/genotypes in the MTRR gene. The contributions to tHcy levels of the three common mutations were statistically
significant. The heterozygosity of the MTHFR 1298AC genotype, MTR 2756G allele, and MTRR 66G allele yielded an OR of 3.4, 2.0, and
2.1, respectively, for having HHcy.
Conclusion: We suggest that HHcy confers a risk for CAD, and smokers with tHcy are at a greatly increased risk. Our finding supports an
important role of the MTHFR gene in CAD and provides evidence of polygenic regulation of tHcy.
© 2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Keywords: Coronary artery disease; Risk factors; Genes; Homocysteine; Metabolism

1. Introduction
⁎ Corresponding author. Unité d'Etudes des Facteurs Métaboliques et
Polymorphismes Génétiques, Ligue Nationale de Lutte Contre les Maladies
Cardiovasculaires, BP 1326-Rabat R.P. 10000, Morocco. Tel.: +212 63 14
Coronary artery disease (CAD) is a significant problem in
43 96; fax: +212 37 67 32 32. terms of morbidity as well as mortality. This condition is now
E-mail address: loranjad@yahoo.fr (A. Laraqui). becoming more frequent in people from developing countries
0953-6205/$ - see front matter © 2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ejim.2007.02.020
 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483
due to their unhealthy habits and behavior. Thus, any inter-
vention that can reduce CAD risks could have a tremendous
impact on public health. Several risk factors associated with
CAD have been identified which, more commonly in com-
bination than alone, interact to create a proatherogenic envi-
ronment. These risk factors are: advancing age, male sex,
smoking, hypertension, positive family history, hyperlipid-
emia, diabetes, and insulin resistance. Despite advances in
our knowledge, the established risk factors do not fully
explain its occurrence: about 30% of cardiovascular disease
cannot be explained by conventional risk factors. Plasma total
homocysteine (tHcy) has been identified as a risk factor for
CAD. Several case-control studies have demonstrated that
patients with CAD had fasting homocysteine concentrations
30% higher than those of controls [1–5]. In studies with a
prospective design, the association of hyperhomocysteinemia
(HHcy) with vascular disease is weaker; nevertheless, it is
statistically significant in most cases [6,7]. However, at
present, it is not known whether homocysteine is a primary
cause of arteriosclerosis [7,8] or whether it is only a secon-
dary marker of the vascular disease [9].
In contrast to metabolite levels that can change as a result
of vascular disease itself [10,11], allelic variants in enzymes
metabolizing homocysteine are fixed at conception and do
not change throughout life. Assuming “mendelian random-
ization”, the association of genetic variants with the studied
trait in case-control studies may suggest causality [12].
Therefore, an association of polymorphisms of the methio-
nine cycle with CAD, if observed, would suggest that gene-
tically determined alterations in metabolism of homocysteine
and related compounds are the cause or modifier of arte-
riosclerosis rather than its consequence.
Approximately ten common genetic variants in the en-
zymes of the methionine cycle have been reported [13,14].
Initial studies indicated that, in most cases, mild HHcy resulted
from heterozygosity for cystathionine β-synthase deficiency
[15,16]. These findings, however, could not be reproduced by
others studies [17,18], and a role for thermolabile methylene-
tetrahydrofolate reductase (MTHFR) in HHcy was proposed.
This MTHFR variant was reported by Kang et al., who
reported that 29% of CAD cases had less than half the MTHFR
activity of controls after 5 min of heat inactivation [19,20].
Later studies identified the 677C→T polymorphism in the
MTHFR gene as the genetic cause of thermolabile MTHFR
[21]. This variant, however, could not explain all cases of
HHcy and led to the search for other genetic determinants of
homocysteine, which is relevant because HHcy has a pre-
valence of 10–20% in the general population. The association
of common polymorphisms in other genes of the methionine
cycle with CAD was analyzed in only a limited number of
studies and remains to be determined.
In the current investigation, we measured tHcy levels and
determined three common mutations: the A1298C mutation
of the MTHFR gene, the A2756G mutation of the MTR
gene, and the A66G mutation of the MTRR gene with the
objective of (1) examining the relationships between plasma
475
tHcy levels and CAD and (2) determining the importance of
genetic influence on plasma tHcy levels and on CAD.
2. Methods
2.1. Subjects
The protocol was approved by the ethics committee of the
Ligue Nationale de Lutte Contre les Maladies Cardio-
Vasculaires, Rabat, Morocco. The study consisted of 230
individuals (160 males ranging in age from 28 to 76 years,
mean age 54 ± 10 years; and 70 females ranging in age from
20 to 70 years, mean age 52 ± 10 years) in whom coronary
angiography was clinically indicated. Reasons for angiogra-
phy were angina pectoris or high presumption of CAD.
Coronary angiography was performed using standard tech-
niques. Blinded assessment of coronary angiograms identi-
fied CAD as the presence of 50% or more stenosis in any
coronary vessel. According to this definition, 151 (65.7%) pa-
tients were found to have CAD (patient group), while 79
(34.3%) had a completely normal coronary anatomy or only
minor stenosis (b10% of lumen) and were, therefore, defined
as the control group. Angiograms were analyzed blinded to
risk factors and genetic studies, and were interpreted in-
dependently by two experienced cardiologists in order to
assess the available indication: coronary artery bypass graf-
ting or coronary angioplasty.
Patients with an acute illness, such as myocardial
infarction within the past three months, or a chronic disease,
such as chronic renal failure, were not included. None of the
subjects took vitamin supplements and all showed normal
hepatic function.
2.2. Definition of cardiovascular risk factor
Hypercholesterolemia was recorded if the subject had a
serum cholesterol above 6.1 mmol/l (235 mg/dl). Hyper-
tension was deemed to be present if the mean systolic
pressure was above 140 mm Hg and/or diastolic pressure
was above 90 mm Hg or if the subject was taking anti-
hypertensive medications. Cigarette smokers were catego-
rized as current smokers or non-smokers (the latter included
former smokers who had quit smoking for at least 6 months
before the study). A subject was defined as affected by
diabetes mellitus if this diagnosis was known to the patient
or if fasting glucose in the serum was 126 mg/dl or higher.
Weight and height were measured, and body mass index
(BMI) was calculated as total body weight (kg)/height
squared (m2).
2.3. Specimen collection and biochemical analyses
Fasting blood samples were obtained for measurement
of routine chemical variables and lipoprotein and apolipo-
protein levels and for isolation of DNA. Total cholesterol,
HDL-cholesterol, and triglyceride levels were measured by
476 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483

conventional methods of clinical chemistry. LDL-cholesterol
levels were calculated using the Friedewald formula.
2.4. Determination of homocysteine
Blood was drawn from fasting individuals into EDTA
vials. The samples were immediately ice packed and centri-
fuged within 30 min to avoid false increases in homocysteine
due to release from red blood cells. tHcy was determined with
an immunoassay (Axis Biochemicals, Oslo, Norway). The
assay method was based on enzymatic conversion of ho-
mocysteine to S-adenosyl-L-homocysteine (SAH), followed
by quantification of SAH by an enzyme-linked immunoassay
[22]. The CVs of variation within and between days for the
assays were 5% or less. Cross-reactivity with glutathione, L-
cysteine, adenosine, and L-cystathionine was below 0.1%, and
with S-adenosyl-L-methionine it was 12.5%. Plasma homo-
cysteine was recorded in units of μmol/l.
2.5. Genetic analysis
Genomic DNA was prepared from peripheral blood
leukocytes by the phenol/chloroform extraction procedure.
The MTHFR 1298A→C polymorphism was analyzed using a
modified method of Weisberg et al. [23]. Briefly, 500 ng
genomic DNA was amplified with 7 pmol each of the forward
primer 5′-CTT TGG GGA GCT GAA GGA CTA CTA C-3′
and the reverse primer 5′-CAC TTT GTG ACC ATT CCG
GTT TG-3′. PCR parameters were a 5-min denaturation cycle
at 94 °C and 38 cycles of the following: 96 °C for 1 min,
56 °C for 1 min, and 72 °C for 1 min. This was followed by a
10-min extension period at 72 °C. A 5-ìl aliquot of the 163-bp
PCR product was digested with 4 μl of 10x MboII buffer and
five units of MboII restriction enzyme at 37 °C overnight,
followed by electrophoresis on a 20% poly-acrylamide gel,
and silver-stained. The wild-type genotype (A1298A) pro-
duced five fragments of 56, 31, 30, 28, and 18 bp. Hete-
rozygotes (A1298C) produced six fragments of 84, 56, 31, 30,
28 and 18 bp. Homozygotes (C1298C) produced four frag-
ments of 84, 31, 30 and 18 bp.
The genotyping protocol for the detection of the MTR
2756A→G polymorphism was a modification of the method
of Chen et al. [25]. Briefly, 500 ng genomic DNA was ampli-
fied with 7 pmol each of the forward primer 5′-ATG GAA
GAA TAT GAA GAT ATT AGA C-3′ and the reverse primer
5′-GAA CTA GAA GAC AGA AAT TCT CTA-3′. PCR ther-
mal cycling conditions were a 2-min denaturation period at
95 °C and 35 cycles of the following: 94 °C for 40 s, 50 °C for
40 s, and 72 °C for 1 min. This was followed by a 10-min
extension period at 72 °C. HaeIII restriction digestion using
1 μl of 10x HaeIII buffer and five units of HaeIII restriction
endonuclease added to 8.5 μl of PCR product was incubated
at 37 °C overnight. Digestion products were silver-stained
after electrophoresis on a 3% agarose gel. The MTR PCR
product of 189 bp was cut into fragments of 159 and 30 bp in
the presence of the mutation.
The MTRR 66A→G polymorphism was analyzed using a
modified method of Jacques et al. [26]. Briefly, 500 ng
genomic DNA was amplified with 7 pmol each of the forward
primer 5′-CAG GCA AAG GCC ATC GCA GAA GAC AT-
3′ and the reverse primer 5′-CAC TTC CCA ACC AAA ATT
CTT CAA AG-3′. The reverse primer contains a mismatch
(underlined base in the primer sequence) that creates a res-
triction site for AflIII when the methionine-containing allele is
present. PCR parameters were a 6-min denaturation cycle at
95 °C and 35 cycles of the following: 94 °C for 40 s, 53 °C for
40 s, and 72 °C for 1 min. This was followed by a 10-min
extension period at 72 °C. The 7 μl of the 150-bp PCR
product was digested with 4 μl of 10x AflIII buffer and five
units of AflIII restriction enzyme at 37 °C overnight, followed
by electrophoresis on a 3% agarose gel. For the isoleucine-
containing allele, the 150 bp fragment remained undigested.
For the methionine-containing allele, the PCR products were
digested into fragments of 123 and 27 bp.

Table 1
Baseline clinical characteristics of controls and cases according to angiographic investigation
Controls Cases RR (95% CI) P
Number of subjects 79 151
Age (years ± SD) 51.62 ± 9.32 54.54 ± 9.77 1.03 (1–1.06) b0.05
Male gender, n (%) 41 (50.63) 120 (79.47) 3.77 (2.09–6.82) b0.05
History of hyperlipidemia, % 37.97 68.21 3.51 (1.98–6.91) b0.001
History of hypertension, % 30.38 70.20 5.40 (2.98–9.77) b0.001
History of smoking, % 26.58 73.51 7.66 (4.14–14.19) b0.001
History of diabetes, % 22.78 47.02 3.01 (1.63–5.56) b0.05
History of obesity, % 27.85 49.67 2.56 (1.42–4.60) b0.05
Cholesterol, mmol/l
Total 5.04 ± 0.85 5.72 ± 1.14 1.93 (1.41–2.66) b0.05
HDL 1.19 ± 0.78 1.05 ± 0.16 0.01 (0.001–0.06) b0.05
LDL 3.75 ± 0.94 4.58 ± 1.25 1.96 (1.45–2.64) b0.05
Triglycerides, mmol/l 1.48 ± 0.53 1.86 ± 0.58 3.88 (2.10–7.17) b0.05
CAD: coronary artery disease; SD: standard deviation; RR: relative risk; 95%CI: 95% confidence interval; P: degree of significant value. For comparison
between groups, the chi-square test was used to test prevalence differences of discontinuous variables; Student's t-test and analysis of variance (ANOVA) were
used for testing mean differences.
 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483 477

Table 2
Homocysteine and hyperhomocysteine distribution in the total population and according to angiographic investigation
Total population Case Control P
No of subjects 230 151 79
Homocysteinemia, μmol/l
Median 14.9 15.4 11.6 b0.001 a
Mean ± SD 14 ± 3.7 15.5 ± 2.8 11.2 ± 3.5 b0.001 b
(95% CI) (13.5–14.5) (15.1–15.9) (10.4–12.0)
Hyperhomocysteinemia, [n (%)] 105 (45.7) 89 (58.9) 16 (20.3) b0.001 c
95% CI: 95% confidence interval.
a
Comparison of homocysteine levels between cases and controls (median test).
b
Mean comparisons of homocysteine concentrations between cases and controls (Student's t-test).
c
Comparison of hyperhomocysteinemia distribution between cases and controls (chi-square test).

2.6. Statistical methods women) of the controls had HHcy. The corresponding RR
was 4.1 (95% CI 2.2–7.5, P b 0.001).
All tests were two-tailed and a P value below 0.05 was
deemed significant. All analyses considered only the 230 3.3. Homocysteine levels according to smoking status
subjects assessed by coronary angiography. Skewed variables
were natural log-transformed to normalize the distribution. To assess the interaction between smoking, tHcy, and CAD,
Between the two groups, a comparison was made using the subjects (n = 230) were grouped into current cigarette smokers
independent t-test and the chi-square test; the Kruskal–Wallis or non-smokers, and plasma homocysteine was defined as
test was applied to compare three or more groups. A one-way greater than 15 μmol/l. The tHcy concentrations were signif-
ANOVA was used to assess the effect of smoking status, BMI, icantly different between smokers and non-smokers (15.6 ± 3.1
diabetes, dyslipidemia, and genotype on homocysteine. versus 12.5 ± 3.8 μmol.l− 1; P b 0.05). In addition, smokers
Interactive effects of variables that were significantly corre- with HHcy demonstrated a markedly increased risk of CAD
lated were assessed using logistic regression analysis, which (OR = 2.5, 95% CI 1.7–3.3, P b 0.001) compared with a non-
was also applied to compute odds ratios (OR) as well as their smoker with normal homocysteine.
95% confidence intervals. To compute both allele and geno-
type frequencies and confidence intervals, SPSS (version
Table 3
10.0) for Windows was used. Genotype frequencies in controls and in cases
Gene All patients Controls Cases RR (95%CI)
3. Results
MTHFR 1298A→C, n (%)
AA 100 (43.5) 42 (53.2) 58 (38.4) 1.0
3.1. Mean differences between cases and control subjects
AC 93 (40.4) 23 (29.1) 70 (46.4) 2.2 (1.2–4.2)
CC 37 (16.1) 14 (17.7) 23 (15.2) 1.2 (0.6–2.6)
Table 1 shows the variables associated with case status. χ2 = 6.6 (P = 0.03)
Cases were older and more likely to be male. Their CAD risk
profile was unfavorable compared to control subjects. For MTR 2756→C, n (%)
AA 163 (70.9) 57 (72.2) 106 (70.2) 1.0
example, they had a higher blood pressure and total
AG 62 (27.0) 22 (27.8) 40 (26.5) 1.0 (0.53–1.80)
cholesterol level. Furthermore, their HDL cholesterol level GG 5 (22) – 5 (3.3) –
was lower and they were more likely to be smokers. The AG+GG 67 (29.3) 22 (27.8) 45 (29.8) 1.1 (0.6–2.0)
estimates of the relative risk (RR) of CAD according to χ2 = 0.88 (P = 0.120)
clinical and laboratory parameters examined in this study are
MTRR 66A→G, n (%)
shown in Table 1.
AA 83 (36.1) 31 (39.2) 52 (34.4) 1.00
AG 125 (54.4) 42 (53.2) 83 (55.0) 1.2 (0.7–2.1)
3.2. Levels of plasma total homocysteine and CAD GG 22 (9.6) 6 (6.7) 16 (10.6) 1.6 (0.6–4.5)
χ2 = 0.85 (P = 0.653)
The mean (±SD) homocysteine concentration in CAD P: degree of significant value. Significant differences in genotype
patients (15.5 ± 2.8 μmol/l; 95% CI 15.1–15.9 μmol/l) was distribution between cases and controls were tested by chi-square test.
significantly (P b 0.001) higher than the mean concentration Because GG genotype frequency was lower than 5, the chi-square test was
in controls (11.2 ± 3.5 μmol/l; 95% CI 10.4–12.0 μmol/l; used; after that, subjects were grouped into AG and GG. This made it
possible to estimate RR associated with the presence of the G allele.
Table 2). Using 15 μmol/l as a cut-off point to classify mild Chi-square test was used before subjects were grouped into AG and GG.
HHcy, 56.3% (45.0% in men and 11.3% in women) of the This grouping of genotype was used just to evaluate the RR associated with
CAD patients and 24.1% (38.0% in men and 11.4% in the presence of the G allele.
478 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483

Table 4
Homocysteine levels and hyperhomocysteinemia frequency distribution according to MTHFR, MTR, and MTRR genotypes and odds ratio (OR) of
hyperhomocysteinemia associated with each genotype
No of Homocysteinemia, μmol/l HHcy Odds ratio
subjects [n (%)]
Median Mean ± SD 95%CI Value 95% CI P
Total population 230 14.9 14.0 ± 3.7 13.5–14.5 106 (46.1) 4.3 2.3–7.9 0.000
MTHFR A1298C
AA 100 14.0 13.2 ± 3.5 12.5–13.9 35 (35.0) 1.0 –
AC 93 16.2 16.1 ± 3.7 15.3–16.8 60 (64.5) 3.4 1.9–6.1 0.001
CC 37 14.8 13.5 ± 4.8 12.0–15.1 15 (40.5) 1.3 0.6–6.1 0.55
P 0.000 a, b 0.000 c 0.000 d, b
MTR A2756G
AA 163 14.6 13.6 ± 3.6 13.1–14.2 67 (41.1) 1.0
AG 62 15.3 14.4 ± 3.4 13.5–15.3 35 (56.5) 1.9 1.0–3.4 0.040
GG 5 17.2 19.5 ± 4.6 13.8–25.1 4 (80.0) 5.7 0.6–51.6 0.123
AG + GG 67 15.3 14.8 ± 3.7 13.9–15.7 39 (58.2) 2.0 1.1–3.6 0.019
P 0.037 a, b 0.008 c 0.018 d, b
MTRR A66G
AA 83 14.1 13.1 ± 3.6 12.3–13.9 29 (34.9) 1.0
AG 125 15.1 14.3 ± 3.6 13.7–14.9 63 (50.4) 1.9 1.1–3.4 0.029
GG 22 15.4 15.5 ± 3.6 13.9–17.1 14 (63.6) 3.3 1.2–8.7 0.018
AG + GG 147 15.2 14.5 ± 3.6 13.9–15.1 77 (52.4) 2.1 1.2–3.6 0.011
P 0.015 a 0.007 c 0.020 d
a
Homocysteine comparison between genotypes (median test).
b
AG and GG genotypes were grouped if the expected counts were less than 5.
c
Comparison of homocysteine levels between genotypes (Kruskal–Wallis test).
d
Comparison of hyperhomocysteinemia frequency between genotypes (chi-square test).

3.4. Mutation frequencies and distribution in cases and
control subjects
The frequencies for the alleles were 36.7% (95% CI 32.0–
41.5) for the 1298C allele in the MTHFR, 15.7% (95% CI
12.3–19.0) for the 2756G allele in the MTR, and 36.6% (95%
CI 32.7–40.8) for the 66G allele in the MTRR, similar to
those previously reported in other populations [26]. Table 3
shows the allele and genotype frequencies for the MTHFR,
MTR, and MTRR genes in cases as well as in controls. The
results identified the MTHFR 1298CC genotype in 23
(15.2%) cases and 14 (17.7%) controls, the MTHFR 1298AC
genotype in 70 (46.4%) cases and 23 (29.1%) controls, and
the MTHFR 1298AA genotype in 58 (38.4%) cases and 42
(53.2%) controls. The occurrence of the MTHFR 1298AC
genotype was higher in cases than in the control group, sug-
gesting that the heterozygous genotype could have a role in
CAD (OR = 2.3, 95 % CI 1.2–4.2, P b 0.05).
There were no significant differences in the distribution of
MTR and MTRR genotypes between cases and controls. P
values of 0.120 and 0.653 were obtained for the comparative
frequencies of the MTR 2756A→G and MTR 66A→G va-
riants, respectively.
3.5. Association between genotypes and homocysteine levels
To further analyze the possible contribution of the MTHFR
1298A→C, MTR 2756A→G, and MTRR 66A→G poly-
morphisms to elevated tHcy levels, patients and controls were
grouped together (n = 230). As shown in Table 4, individuals
heterozygous for the MTHFR 1298A→C polymorphism had
significantly higher tHcy values (OR 3.4 CI 95% 1.9–6.1;
P b 0.001). The 2756G allele of the MTR gene tended to
have higher homocysteine levels (OR 2.0 CI 95% 1.1–3.6;
P b 0.05). Also, patients with the 66G allele tended to have
higher homocysteine levels than those with the 66AA
homozygous genotype of the MTRR gene (OR 2.1 CI 95%
1.2–3.6; P b 0.05). These quantitative differences were also
seen when we used the 15 μmol/l cut-off level for elevated
homocysteine. Among 1298AC heterozygotes of the MTHFR
gene, 64.5% had mild HHcy compared to 40.5% and 35.0%
for 1298CC and 1298AA, respectively. Among homozygous
carriers of the mutant 2756G allele for the MTR gene, 58.2%
had HHcy compared to 41.1% of the wild-type AA
homozygotes. In addition, 52.4% of the MTRR 66G
individuals had plasma homocysteine above 15 μmol/l,
whereas 34.9% of those with 66AA had levels above the
cut-off.
3.6. Multivariate analysis
The model of multiple linear regression was used to define
the independent correlates of tHcy concentration. Age, gender,
smoking, hypertension, diabetes, hypercholesterolemia and
MTHFR gene 1298A→C, MTR gene 2756A→G, and MTRR
gene 66A→G polymorphisms were included in the model.
The MTHFR gene 1298A→C, MTR gene 2756A→G, and
MTRR gene 66A→G polymorphisms were entered into the
 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483
model after being categorized as 1298AA versus 1298AC +
1298CC, 2756AA versus 2756AG + 2756GG, and 2756AA
versus 2756AG + 2756GG genotypes, respectively. The model
showed that smoking (P b 0.05), MTHFR 1298A→C
(P b 0.001), MTR 2756A→G (P b 0.001), and MTRR
66A→G (P b 0.001) polymorphisms were related to homocys-
teine concentration. A significant interaction between MTHFR
gene 1298A→C and MTR gene 2756A→G polymorphisms
(P b 0.05) or MTR gene 2756A→G and MTRR gene 66A→G
polymorphisms (P b 0.001) was observed to plasma homo-
cysteine levels.
Multiple logistic regression was used to test for indepen-
dent correlates of CAD. Included in the model were: age,
gender, smoking, hypertension, diabetes, hypercholesterol-
emia, tHcy, and MTHFR gene 1298A→C, MTR gene
2756A→G, and MTRR gene 66A→G polymorphisms. Age
(P b 0.001), smoking (P b 0.001), hypertension (P b 0.05),
diabetes (P b 0.05), tHcy (P b 0.001), and MTHFR gene
1298A→C polymorphism (P b 0.001) were independent
correlates to CAD. In addition, our results showed that tHcy
and smoking habits were related to CAD (P b 0.05).
4. Discussion
Several characteristics of the present study deserve to be
stressed. First, as in previous studies, we found that tHcy
concentrations above 15 μmol/l were a significant risk factor
for angiographically documented CAD, with a RR of 4.1
(95% CI 2.2–7.5, P b 0.001). Second, our study was
conducted as part of an initial ongoing study to test the
relative impact of environmental factors and of variation in
the MTHFR, MTR, and MTRR genes, which code for key
enzymes in the homocysteine metabolic pathways, in
determining tHcy levels. Third, by performing coronary
angiography in all patients and controls, our study provides a
very reliable phenotypic characterization of CAD.
4.1. Homocysteine and risk of CAD
The present study confirms the results of previous
findings [27] that an elevated plasma homocysteine level is
implicated as a risk factor for CAD. The etiologies of
elevated tHcy have been attributed to impairment of reme-
thylation of homocysteine, rather than impairment of
transsulfuration or increased dietary methionine [28]. Thus,
defects in the gene encoding MTHFR, MTR, or MTRR
enzymes involved in remethylation or inadequate status of
either folate or vitamin B12 will lead to a substantial increase
in plasma homocysteine concentrations under fasting con-
ditions [28,29]. The etiopathogenesis of HHcy in CAD may
be the proatherogenic and prothrombotic metabolic milieu
created by homocysteine. Probable causes are endothelial
cell injury due to patchy desquamation or production of
reactive oxygen species, increased platelet aggregation, oxi-
dation of LDL, and/or proliferation of vascular smooth
muscle cell [30,31].
479
4.2. Smoking
The role of environmental factors in determining homo-
cysteine levels was examined first. Cigarette smoking is
known to increase plasma homocysteine [32]. Our results
confirm this effect and suggest that smokers with high plasma
homocysteine above 15 μmol/l are at a greatly increased risk of
CAD (OR = 2.5, 95% CI 1.7–3.3). O'Callaghan et al. [33]
demonstrated that cigarette smokers with a tHcy above
12 μmol/l had a 12-fold increased risk of CVD (OR 12.4
95% CI 7.3–21.2) compared with non-smokers with a normal
tHcy [33]. Several mechanisms might explain the increased
risk in smokers with raised tHcy. Smoking affects the vascular
tree via several different interactive mechanisms [34]. Nicotine
and carbon monoxide separately produce tachycardia, hyper-
tension, and vasoconstriction, and both produce direct endo-
thelial damage. Smoking also affects vaso-occlussive factors
such as platelet aggregation, plasma viscosity, and fibrinogen
levels [34]. HHcy has been associated with impaired
endothelial function, and abnormal flow-mediated vasodilata-
tion has been demonstrated with mild HHcy [35,36]. It may
also damage the vascular tree via platelet activation, lipid
peroxidation, enhanced tissue factor activation, reduced Von
Willebrand factor, increased fibrinogen levels, and smooth
muscle proliferation [37–39]. The fact that both of these risk
factors can exert similar effects would suggest a strong
potential for interaction between them to produce vascular
damage.
4.3. MTHFR
Regarding the 1298A→C mutation, our results were in
agreement with those in the few published studies: the 1298C
allele frequency was 36.7%, demonstrating that the mutation
is also common in our population. To date, few reports are
available on the prevalence of the 1298A→C polymorphism
among different populations. The frequency among Cana-
dians [23] and Dutch [40] is approximately 9%, while it is
13.8%, 17%, 28.2%, and 41.1% for studies conducted on
populations from Germany [41], China [42], Portugal [43],
and Brazil [44], respectively.
The association of the 1298A→C mutation with decreased
MTHFR specific activity is confirmed, although its effect on
tHcy levels is not yet clear. Reports show either no effect of
this mutation on tHcy levels or an association with even lower
levels of tHcy in homozygous individuals [45]. We observed
a significant effect of the 1298A→C polymorphism on tHcy
levels. Subjects bearing the 1298AC genotype had signifi-
cantly higher tHcy levels than those bearing the 1298AA or
1298CC genotypes. In the first two studies that examine
homocysteine levels in individuals with the 1298A→C
change [23,40], homocysteine levels for heterozygotes and
homozygotes were not different from those with the wild-
type genotype (1298AA). However, Van der Put et al. [41]
suggested that combined heterozygosity for both polymorph-
isms resulted in reduced MTHFR specific activity, higher
480 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483
tHcy levels, and decreased plasma folate [40]. Other studies
[46,47] indicate that the MTHFR 1298A→C polymorphism
does not contribute significantly to HHcy, either by itself or in
combination with the 677C→T polymorphism. On the
contrary, the MTHFR 1298C allele was found to be asso-
ciated with a slight decrease in tHcy. Conceivably, this
association depends on the complete linkage disequilibrium
between the 1298A→C and 677C→T polymorphisms in
most populations. Individuals who are homozygous for the
677T allele, which predisposes to HHcy, carry with a few
exceptions the 1298AA genotype, and vice versa [48].
Distribution of the MTHFR 1298A→C polymorphism
has been studied in neural tube defects and acute leukemia,
but not yet in CAD. We presented a study in which the
A1298C polymorphism of the MTHFR gene was assessed in
patients with angiographically proven CAD. Allele 1298C
showed a significant association with CAD in heterozygous
carriers and predisposed individuals to CAD. In a case-
control study, Szczeklik et al. observed that the mutant
MTHFR 1298C allele was associated with early-onset CAD,
following a dominant mode of inheritance; the association
remained significant when adjusted for 677T homozygosity
or when compared to the general population sample [49].
This association is not related to HHcy, folic acid, or B12
deficiency. Other studies [50–52], conducted on individuals
from different populations, have concluded that there is a
strong association between the presence of the mutant
MTHFR A1298C and C677T variants and the occurrence of
CAD. Therefore, it seems that the association is influenced
by the ethnic origins of the examined subjects. The
association of MTHFR polymorphisms with CAD was
recently reviewed [53].
4.4. MTR
MTR, the enzyme involved in the vitamin B12-dependent
remethylation of homocysteine to methionine, plays an
important role in homocysteine metabolism. The gene
coding MTR has been cloned, sequenced and mapped. The
most prevalent mutation of the MTR gene is the A2756G
transition, which results in the substitution of aspartic acid by
glycine (D919G). The frequency of this mutant allele was
approximately 15–20%. In our study, approximately 15.7%
of the individuals were either heterozygous or homozygous
carriers of the 2756G allele. A previous study on the
relationship between the 2756A→G transition in the MTR
gene and tHcy showed that the 2756G allele was associated
with a lower tHcy; however, the difference in their study did
not reach statistical significance [54]. In the current study, we
were able to demonstrate that the 2756G allele, when present
in either the heterozygous or homozygous state, was related
to circulating homocysteine concentrations. Several other
studies, however, failed to note any association between the
MTR 2756A→G polymorphism and homocysteine concen-
trations [24,29,55]. A small number of reports even found
that both fasting and post-methionine load, homocysteine
concentration decreased with an increasing number of
2756G alleles. There are no features that clearly distinguish
the studies that failed to see any association from those that
observed an inverse association. Because of the low preva-
lence of the MTR GG genotype, statistical power could be an
issue for some of the smaller studies.
Relatively little is known about the effect of the MTR
gene on vascular disease. Increased cardiovascular disease
risk has been described among patients homozygous for this
polymorphism compared with subjects with wild-type alleles
[56]. This finding raises the possibility of an involvement of
MTR 2756A→G in CAD. In the present study, we did not
observe any association between this polymorphism and
CAD. Three out of four other case-control studies suggested
that the MTR 2756GG genotype has a protective, rather than
an adverse, effect on coronary heart disease [24,29,54].
However, these studies were small or were done in countries
with fortification of folic acid and other vitamins. Further-
more, an increase in formylated tetrahydrofolate plasma
levels has been observed in healthy subjects with the MTR
2756AG genotype in contrast to individuals with the AA
genotype [57]. Moreover, the MTR 2756AG genotype was
associated with longer event-free survival in patients with
CAD compared with patients with the AA genotype [57].
The discrepancy between our study and the other might
be explained by interaction between the MTR 2756A→G
polymorphism and vitamin B12, folate, or other cardiovas-
cular risk factors. For example, in a hospital-based case-
control study in Australia, the MTR D919G polymorphism
interacted with smoking to increase the risk of CAD [58]. An
interaction between the MTR 2756A→G polymorphism and
the polymorphisms of MTHFR on homocysteine concentra-
tion and homocysteine-related diseases has previously been
suggested. None of these studies showed a clear interaction
between these polymorphisms [29,57,59–61]. Unfortunate-
ly, case-control studies, in general, are much too small to
investigate possible effect modification.
4.5. MTRR
At the time of cloning and characterization of the MTRR
gene, two genetic polymorphisms were identified [62]
besides rare mutations, resulting in severe enzyme deficiency
[63]. Recently, more than ten SNPs have been found in exonic
regions [64], suggesting that the MTRR gene is highly
polymorphic. MTRR deficiency will decrease the amount of
active MTR enzyme. However, whether or not MTRR is the
only physiologically relevant pathway remains unknown.
The 66A→G polymorphism is situated at a site within the
sequence of the FMN-binding domain. Our present study
reported a significant association between this polymorphism
and homocysteine concentrations. This is consistent with
earlier studies that examined this relationship. Moreover, the
relative contribution of the MTRR 66GG genotype to the
variability of tHcy ranking was approximately half the effect
of the MTHFR 677TT genotype and twice the effect
 A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483
attributable to MTR 2756AA [65]. In contrast to this finding,
no association of the MTRR 66A→G genotype with either
fasting [51,66–69] or post-methionine loading [69] tHcy
levels has been reported by others in individuals known to be
at highest risk for CAD or with diagnosed premature CAD, in
patients with confirmed venous thromboembolism, or in
adults without a clinical history of cardio-or cerebrovascular
events and without current malignancy.
Mutation analysis of the MTRR gene has been identified in
patients with CAD in some studies. Our results are consistent
with an earlier report that failed to note any association
between the CAD and the 66A→G mutation. However, our
study does not exclude the potential for disease association
with the MTRR polymorphism. For example, Brown et al.
[67] reported a higher gender-adjusted risk for premature
CAD in MTRR GG patients, even though there was no
increase in the tHcy levels [70]. They postulated that the
MTRR 66A→G polymorphism could cause CAD through a
non-hyperhomocysteinemia mechanism. Also, Wilson et al.
described an association between MTRR 66A→G and spina
bifida, observing an increase in the number of affected
children of mothers with the mutant genotype and low coba-
lamin levels [62]. Hobbs et al. reported an association of
homozygosity for MTHFR 677C→T or MTRR 66A→G with
an increased risk of having a child with Down syndrome, with
the combined risk greater than either risk alone [71].
One limitation of our study is the relatively small number
of individuals included. Patients in this study were all
referred for coronary angiography, so our control group may
not be representative of the general population. However, the
use of patients with normal coronary arteries or minor
stenosis on angiography also has advantages over using
population-based controls, as described above.
Despite these limitations, tHcy concentration confers a
risk for CAD in the Moroccan population, and smokers with
tHcy are at a greatly increased risk of CAD. Our finding
supports an important role of the MTHFR gene in CAD and
provides evidence of polygenic regulation of tHcy. Further
studies of the genetic predisposition to hyperhomocysteine-
mia are needed to give us a better understanding of the etio-
logy of the relationships between hyperhomocysteinemia
and CAD.
5. Learning points
• Elevated tHcy levels may ultimately become an important
screening test for coronary artery disease in the Moroccan
population to determine cardiovascular risk.
• The polymorphisms related to homocysteine metabolism
(MTHFR 1298A→C, MTR 2756A→G, and MTRR
66A→G) contribute to the risk of hyperhomocysteine-
mia. Among the three common mutations, the MTHFR
1298A→C mutation is the major risk factor for the de-
velopment of CAD.
• An understanding of the genetic determinants of mild
hyperhomocysteinemia may be useful in pre-symptomat-
481
ic as well as post-symptomatic evaluation and for the
prevention and treatment of cardiovascular disease.
Acknowledgements
The authors would like to thank Pr. M-J Chapman and Pr.
Y. Touitou for their help and advice, and J.P. Lagarde for
technical assistance. We also thank the individuals who
voluntarily took part in this study.
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