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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) 474–483 475

due to their unhealthy habits and behavior. Thus, any inter- tHcy levels and CAD and (2) determining the importance of
vention that can reduce CAD risks could have a tremendous genetic influence on plasma tHcy levels and on CAD.
impact on public health. Several risk factors associated with
CAD have been identified which, more commonly in com- 2. Methods
bination than alone, interact to create a proatherogenic envi-
ronment. These risk factors are: advancing age, male sex, 2.1. Subjects
smoking, hypertension, positive family history, hyperlipid-
emia, diabetes, and insulin resistance. Despite advances in The protocol was approved by the ethics committee of the
our knowledge, the established risk factors do not fully Ligue Nationale de Lutte Contre les Maladies Cardio-
explain its occurrence: about 30% of cardiovascular disease Vasculaires, Rabat, Morocco. The study consisted of 230
cannot be explained by conventional risk factors. Plasma total individuals (160 males ranging in age from 28 to 76 years,
homocysteine (tHcy) has been identified as a risk factor for mean age 54 ± 10 years; and 70 females ranging in age from
CAD. Several case-control studies have demonstrated that 20 to 70 years, mean age 52 ± 10 years) in whom coronary
patients with CAD had fasting homocysteine concentrations angiography was clinically indicated. Reasons for angiogra-
30% higher than those of controls [1–5]. In studies with a phy were angina pectoris or high presumption of CAD.
prospective design, the association of hyperhomocysteinemia Coronary angiography was performed using standard tech-
(HHcy) with vascular disease is weaker; nevertheless, it is niques. Blinded assessment of coronary angiograms identi-
statistically significant in most cases [6,7]. However, at fied CAD as the presence of 50% or more stenosis in any
present, it is not known whether homocysteine is a primary coronary vessel. According to this definition, 151 (65.7%) pa-
cause of arteriosclerosis [7,8] or whether it is only a secon- tients were found to have CAD (patient group), while 79
dary marker of the vascular disease [9]. (34.3%) had a completely normal coronary anatomy or only
In contrast to metabolite levels that can change as a result minor stenosis (b10% of lumen) and were, therefore, defined
of vascular disease itself [10,11], allelic variants in enzymes as the control group. Angiograms were analyzed blinded to
metabolizing homocysteine are fixed at conception and do risk factors and genetic studies, and were interpreted in-
not change throughout life. Assuming “mendelian random- dependently by two experienced cardiologists in order to
ization”, the association of genetic variants with the studied assess the available indication: coronary artery bypass graf-
trait in case-control studies may suggest causality [12]. ting or coronary angioplasty.
Therefore, an association of polymorphisms of the methio- Patients with an acute illness, such as myocardial
nine cycle with CAD, if observed, would suggest that gene- infarction within the past three months, or a chronic disease,
tically determined alterations in metabolism of homocysteine such as chronic renal failure, were not included. None of the
and related compounds are the cause or modifier of arte- subjects took vitamin supplements and all showed normal
riosclerosis rather than its consequence. hepatic function.
Approximately ten common genetic variants in the en-
zymes of the methionine cycle have been reported [13,14]. 2.2. Definition of cardiovascular risk factor
Initial studies indicated that, in most cases, mild HHcy resulted
from heterozygosity for cystathionine β-synthase deficiency Hypercholesterolemia was recorded if the subject had a
[15,16]. These findings, however, could not be reproduced by serum cholesterol above 6.1 mmol/l (235 mg/dl). Hyper-
others studies [17,18], and a role for thermolabile methylene- tension was deemed to be present if the mean systolic
tetrahydrofolate reductase (MTHFR) in HHcy was proposed. pressure was above 140 mm Hg and/or diastolic pressure
This MTHFR variant was reported by Kang et al., who was above 90 mm Hg or if the subject was taking anti-
reported that 29% of CAD cases had less than half the MTHFR hypertensive medications. Cigarette smokers were catego-
activity of controls after 5 min of heat inactivation [19,20]. rized as current smokers or non-smokers (the latter included
Later studies identified the 677C→T polymorphism in the former smokers who had quit smoking for at least 6 months
MTHFR gene as the genetic cause of thermolabile MTHFR before the study). A subject was defined as affected by
[21]. This variant, however, could not explain all cases of diabetes mellitus if this diagnosis was known to the patient
HHcy and led to the search for other genetic determinants of or if fasting glucose in the serum was 126 mg/dl or higher.
homocysteine, which is relevant because HHcy has a pre- Weight and height were measured, and body mass index
valence of 10–20% in the general population. The association (BMI) was calculated as total body weight (kg)/height
of common polymorphisms in other genes of the methionine squared (m2).
cycle with CAD was analyzed in only a limited number of
studies and remains to be determined. 2.3. Specimen collection and biochemical analyses
In the current investigation, we measured tHcy levels and
determined three common mutations: the A1298C mutation Fasting blood samples were obtained for measurement
of the MTHFR gene, the A2756G mutation of the MTR of routine chemical variables and lipoprotein and apolipo-
gene, and the A66G mutation of the MTRR gene with the protein levels and for isolation of DNA. Total cholesterol,
objective of (1) examining the relationships between plasma 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 rozygotes (A1298C) produced six fragments of 84, 56, 31, 30,
levels were calculated using the Friedewald formula. 28 and 18 bp. Homozygotes (C1298C) produced four frag-
ments of 84, 31, 30 and 18 bp.
2.4. Determination of homocysteine The genotyping protocol for the detection of the MTR
2756A→G polymorphism was a modification of the method
Blood was drawn from fasting individuals into EDTA of Chen et al. [25]. Briefly, 500 ng genomic DNA was ampli-
vials. The samples were immediately ice packed and centri- fied with 7 pmol each of the forward primer 5′-ATG GAA
fuged within 30 min to avoid false increases in homocysteine GAA TAT GAA GAT ATT AGA C-3′ and the reverse primer
due to release from red blood cells. tHcy was determined with 5′-GAA CTA GAA GAC AGA AAT TCT CTA-3′. PCR ther-
an immunoassay (Axis Biochemicals, Oslo, Norway). The mal cycling conditions were a 2-min denaturation period at
assay method was based on enzymatic conversion of ho- 95 °C and 35 cycles of the following: 94 °C for 40 s, 50 °C for
mocysteine to S-adenosyl-L-homocysteine (SAH), followed 40 s, and 72 °C for 1 min. This was followed by a 10-min
by quantification of SAH by an enzyme-linked immunoassay extension period at 72 °C. HaeIII restriction digestion using
[22]. The CVs of variation within and between days for the 1 μl of 10x HaeIII buffer and five units of HaeIII restriction
assays were 5% or less. Cross-reactivity with glutathione, L- endonuclease added to 8.5 μl of PCR product was incubated
cysteine, adenosine, and L-cystathionine was below 0.1%, and at 37 °C overnight. Digestion products were silver-stained
with S-adenosyl-L-methionine it was 12.5%. Plasma homo- after electrophoresis on a 3% agarose gel. The MTR PCR
cysteine was recorded in units of μmol/l. product of 189 bp was cut into fragments of 159 and 30 bp in
the presence of the mutation.
2.5. Genetic analysis The MTRR 66A→G polymorphism was analyzed using a
modified method of Jacques et al. [26]. Briefly, 500 ng
Genomic DNA was prepared from peripheral blood genomic DNA was amplified with 7 pmol each of the forward
leukocytes by the phenol/chloroform extraction procedure. primer 5′-CAG GCA AAG GCC ATC GCA GAA GAC AT-
The MTHFR 1298A→C polymorphism was analyzed using a 3′ and the reverse primer 5′-CAC TTC CCA ACC AAA ATT
modified method of Weisberg et al. [23]. Briefly, 500 ng CTT CAA AG-3′. The reverse primer contains a mismatch
genomic DNA was amplified with 7 pmol each of the forward (underlined base in the primer sequence) that creates a res-
primer 5′-CTT TGG GGA GCT GAA GGA CTA CTA C-3′ triction site for AflIII when the methionine-containing allele is
and the reverse primer 5′-CAC TTT GTG ACC ATT CCG present. PCR parameters were a 6-min denaturation cycle at
GTT TG-3′. PCR parameters were a 5-min denaturation cycle 95 °C and 35 cycles of the following: 94 °C for 40 s, 53 °C for
at 94 °C and 38 cycles of the following: 96 °C for 1 min, 40 s, and 72 °C for 1 min. This was followed by a 10-min
56 °C for 1 min, and 72 °C for 1 min. This was followed by a extension period at 72 °C. The 7 μl of the 150-bp PCR
10-min extension period at 72 °C. A 5-ìl aliquot of the 163-bp product was digested with 4 μl of 10x AflIII buffer and five
PCR product was digested with 4 μl of 10x MboII buffer and units of AflIII restriction enzyme at 37 °C overnight, followed
five units of MboII restriction enzyme at 37 °C overnight, by electrophoresis on a 3% agarose gel. For the isoleucine-
followed by electrophoresis on a 20% poly-acrylamide gel, containing allele, the 150 bp fragment remained undigested.
and silver-stained. The wild-type genotype (A1298A) pro- For the methionine-containing allele, the PCR products were
duced five fragments of 56, 31, 30, 28, and 18 bp. Hete- 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 grouped together (n = 230). As shown in Table 4, individuals
control subjects heterozygous for the MTHFR 1298A→C polymorphism had
significantly higher tHcy values (OR 3.4 CI 95% 1.9–6.1;
The frequencies for the alleles were 36.7% (95% CI 32.0– P b 0.001). The 2756G allele of the MTR gene tended to
41.5) for the 1298C allele in the MTHFR, 15.7% (95% CI have higher homocysteine levels (OR 2.0 CI 95% 1.1–3.6;
12.3–19.0) for the 2756G allele in the MTR, and 36.6% (95% P b 0.05). Also, patients with the 66G allele tended to have
CI 32.7–40.8) for the 66G allele in the MTRR, similar to higher homocysteine levels than those with the 66AA
those previously reported in other populations [26]. Table 3 homozygous genotype of the MTRR gene (OR 2.1 CI 95%
shows the allele and genotype frequencies for the MTHFR, 1.2–3.6; P b 0.05). These quantitative differences were also
MTR, and MTRR genes in cases as well as in controls. The seen when we used the 15 μmol/l cut-off level for elevated
results identified the MTHFR 1298CC genotype in 23 homocysteine. Among 1298AC heterozygotes of the MTHFR
(15.2%) cases and 14 (17.7%) controls, the MTHFR 1298AC gene, 64.5% had mild HHcy compared to 40.5% and 35.0%
genotype in 70 (46.4%) cases and 23 (29.1%) controls, and for 1298CC and 1298AA, respectively. Among homozygous
the MTHFR 1298AA genotype in 58 (38.4%) cases and 42 carriers of the mutant 2756G allele for the MTR gene, 58.2%
(53.2%) controls. The occurrence of the MTHFR 1298AC had HHcy compared to 41.1% of the wild-type AA
genotype was higher in cases than in the control group, sug- homozygotes. In addition, 52.4% of the MTRR 66G
gesting that the heterozygous genotype could have a role in individuals had plasma homocysteine above 15 μmol/l,
CAD (OR = 2.3, 95 % CI 1.2–4.2, P b 0.05). whereas 34.9% of those with 66AA had levels above the
There were no significant differences in the distribution of cut-off.
MTR and MTRR genotypes between cases and controls. P
values of 0.120 and 0.653 were obtained for the comparative 3.6. Multivariate analysis
frequencies of the MTR 2756A→G and MTR 66A→G va-
riants, respectively. The model of multiple linear regression was used to define
the independent correlates of tHcy concentration. Age, gender,
3.5. Association between genotypes and homocysteine levels smoking, hypertension, diabetes, hypercholesterolemia and
MTHFR gene 1298A→C, MTR gene 2756A→G, and MTRR
To further analyze the possible contribution of the MTHFR gene 66A→G polymorphisms were included in the model.
1298A→C, MTR 2756A→G, and MTRR 66A→G poly- The MTHFR gene 1298A→C, MTR gene 2756A→G, and
morphisms to elevated tHcy levels, patients and controls were MTRR gene 66A→G polymorphisms were entered into the
A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483 479

model after being categorized as 1298AA versus 1298AC + 4.2. Smoking


1298CC, 2756AA versus 2756AG + 2756GG, and 2756AA
versus 2756AG + 2756GG genotypes, respectively. The model The role of environmental factors in determining homo-
showed that smoking (P b 0.05), MTHFR 1298A→C cysteine levels was examined first. Cigarette smoking is
(P b 0.001), MTR 2756A→G (P b 0.001), and MTRR known to increase plasma homocysteine [32]. Our results
66A→G (P b 0.001) polymorphisms were related to homocys- confirm this effect and suggest that smokers with high plasma
teine concentration. A significant interaction between MTHFR homocysteine above 15 μmol/l are at a greatly increased risk of
gene 1298A→C and MTR gene 2756A→G polymorphisms CAD (OR = 2.5, 95% CI 1.7–3.3). O'Callaghan et al. [33]
(P b 0.05) or MTR gene 2756A→G and MTRR gene 66A→G demonstrated that cigarette smokers with a tHcy above
polymorphisms (P b 0.001) was observed to plasma homo- 12 μmol/l had a 12-fold increased risk of CVD (OR 12.4
cysteine levels. 95% CI 7.3–21.2) compared with non-smokers with a normal
Multiple logistic regression was used to test for indepen- tHcy [33]. Several mechanisms might explain the increased
dent correlates of CAD. Included in the model were: age, risk in smokers with raised tHcy. Smoking affects the vascular
gender, smoking, hypertension, diabetes, hypercholesterol- tree via several different interactive mechanisms [34]. Nicotine
emia, tHcy, and MTHFR gene 1298A→C, MTR gene and carbon monoxide separately produce tachycardia, hyper-
2756A→G, and MTRR gene 66A→G polymorphisms. Age tension, and vasoconstriction, and both produce direct endo-
(P b 0.001), smoking (P b 0.001), hypertension (P b 0.05), thelial damage. Smoking also affects vaso-occlussive factors
diabetes (P b 0.05), tHcy (P b 0.001), and MTHFR gene such as platelet aggregation, plasma viscosity, and fibrinogen
1298A→C polymorphism (P b 0.001) were independent levels [34]. HHcy has been associated with impaired
correlates to CAD. In addition, our results showed that tHcy endothelial function, and abnormal flow-mediated vasodilata-
and smoking habits were related to CAD (P b 0.05). tion has been demonstrated with mild HHcy [35,36]. It may
also damage the vascular tree via platelet activation, lipid
4. Discussion peroxidation, enhanced tissue factor activation, reduced Von
Willebrand factor, increased fibrinogen levels, and smooth
Several characteristics of the present study deserve to be muscle proliferation [37–39]. The fact that both of these risk
stressed. First, as in previous studies, we found that tHcy factors can exert similar effects would suggest a strong
concentrations above 15 μmol/l were a significant risk factor potential for interaction between them to produce vascular
for angiographically documented CAD, with a RR of 4.1 damage.
(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 4.3. MTHFR
relative impact of environmental factors and of variation in
the MTHFR, MTR, and MTRR genes, which code for key Regarding the 1298A→C mutation, our results were in
enzymes in the homocysteine metabolic pathways, in agreement with those in the few published studies: the 1298C
determining tHcy levels. Third, by performing coronary allele frequency was 36.7%, demonstrating that the mutation
angiography in all patients and controls, our study provides a is also common in our population. To date, few reports are
very reliable phenotypic characterization of CAD. available on the prevalence of the 1298A→C polymorphism
among different populations. The frequency among Cana-
4.1. Homocysteine and risk of CAD dians [23] and Dutch [40] is approximately 9%, while it is
13.8%, 17%, 28.2%, and 41.1% for studies conducted on
The present study confirms the results of previous populations from Germany [41], China [42], Portugal [43],
findings [27] that an elevated plasma homocysteine level is and Brazil [44], respectively.
implicated as a risk factor for CAD. The etiologies of The association of the 1298A→C mutation with decreased
elevated tHcy have been attributed to impairment of reme- MTHFR specific activity is confirmed, although its effect on
thylation of homocysteine, rather than impairment of tHcy levels is not yet clear. Reports show either no effect of
transsulfuration or increased dietary methionine [28]. Thus, this mutation on tHcy levels or an association with even lower
defects in the gene encoding MTHFR, MTR, or MTRR levels of tHcy in homozygous individuals [45]. We observed
enzymes involved in remethylation or inadequate status of a significant effect of the 1298A→C polymorphism on tHcy
either folate or vitamin B12 will lead to a substantial increase levels. Subjects bearing the 1298AC genotype had signifi-
in plasma homocysteine concentrations under fasting con- cantly higher tHcy levels than those bearing the 1298AA or
ditions [28,29]. The etiopathogenesis of HHcy in CAD may 1298CC genotypes. In the first two studies that examine
be the proatherogenic and prothrombotic metabolic milieu homocysteine levels in individuals with the 1298A→C
created by homocysteine. Probable causes are endothelial change [23,40], homocysteine levels for heterozygotes and
cell injury due to patchy desquamation or production of homozygotes were not different from those with the wild-
reactive oxygen species, increased platelet aggregation, oxi- type genotype (1298AA). However, Van der Put et al. [41]
dation of LDL, and/or proliferation of vascular smooth suggested that combined heterozygosity for both polymorph-
muscle cell [30,31]. 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 concentration decreased with an increasing number of
[46,47] indicate that the MTHFR 1298A→C polymorphism 2756G alleles. There are no features that clearly distinguish
does not contribute significantly to HHcy, either by itself or in the studies that failed to see any association from those that
combination with the 677C→T polymorphism. On the observed an inverse association. Because of the low preva-
contrary, the MTHFR 1298C allele was found to be asso- lence of the MTR GG genotype, statistical power could be an
ciated with a slight decrease in tHcy. Conceivably, this issue for some of the smaller studies.
association depends on the complete linkage disequilibrium Relatively little is known about the effect of the MTR
between the 1298A→C and 677C→T polymorphisms in gene on vascular disease. Increased cardiovascular disease
most populations. Individuals who are homozygous for the risk has been described among patients homozygous for this
677T allele, which predisposes to HHcy, carry with a few polymorphism compared with subjects with wild-type alleles
exceptions the 1298AA genotype, and vice versa [48]. [56]. This finding raises the possibility of an involvement of
Distribution of the MTHFR 1298A→C polymorphism MTR 2756A→G in CAD. In the present study, we did not
has been studied in neural tube defects and acute leukemia, observe any association between this polymorphism and
but not yet in CAD. We presented a study in which the CAD. Three out of four other case-control studies suggested
A1298C polymorphism of the MTHFR gene was assessed in that the MTR 2756GG genotype has a protective, rather than
patients with angiographically proven CAD. Allele 1298C an adverse, effect on coronary heart disease [24,29,54].
showed a significant association with CAD in heterozygous However, these studies were small or were done in countries
carriers and predisposed individuals to CAD. In a case- with fortification of folic acid and other vitamins. Further-
control study, Szczeklik et al. observed that the mutant more, an increase in formylated tetrahydrofolate plasma
MTHFR 1298C allele was associated with early-onset CAD, levels has been observed in healthy subjects with the MTR
following a dominant mode of inheritance; the association 2756AG genotype in contrast to individuals with the AA
remained significant when adjusted for 677T homozygosity genotype [57]. Moreover, the MTR 2756AG genotype was
or when compared to the general population sample [49]. associated with longer event-free survival in patients with
This association is not related to HHcy, folic acid, or B12 CAD compared with patients with the AA genotype [57].
deficiency. Other studies [50–52], conducted on individuals The discrepancy between our study and the other might
from different populations, have concluded that there is a be explained by interaction between the MTR 2756A→G
strong association between the presence of the mutant polymorphism and vitamin B12, folate, or other cardiovas-
MTHFR A1298C and C677T variants and the occurrence of cular risk factors. For example, in a hospital-based case-
CAD. Therefore, it seems that the association is influenced control study in Australia, the MTR D919G polymorphism
by the ethnic origins of the examined subjects. The interacted with smoking to increase the risk of CAD [58]. An
association of MTHFR polymorphisms with CAD was interaction between the MTR 2756A→G polymorphism and
recently reviewed [53]. the polymorphisms of MTHFR on homocysteine concentra-
tion and homocysteine-related diseases has previously been
4.4. MTR suggested. None of these studies showed a clear interaction
between these polymorphisms [29,57,59–61]. Unfortunate-
MTR, the enzyme involved in the vitamin B12-dependent ly, case-control studies, in general, are much too small to
remethylation of homocysteine to methionine, plays an investigate possible effect modification.
important role in homocysteine metabolism. The gene
coding MTR has been cloned, sequenced and mapped. The 4.5. MTRR
most prevalent mutation of the MTR gene is the A2756G
transition, which results in the substitution of aspartic acid by At the time of cloning and characterization of the MTRR
glycine (D919G). The frequency of this mutant allele was gene, two genetic polymorphisms were identified [62]
approximately 15–20%. In our study, approximately 15.7% besides rare mutations, resulting in severe enzyme deficiency
of the individuals were either heterozygous or homozygous [63]. Recently, more than ten SNPs have been found in exonic
carriers of the 2756G allele. A previous study on the regions [64], suggesting that the MTRR gene is highly
relationship between the 2756A→G transition in the MTR polymorphic. MTRR deficiency will decrease the amount of
gene and tHcy showed that the 2756G allele was associated active MTR enzyme. However, whether or not MTRR is the
with a lower tHcy; however, the difference in their study did only physiologically relevant pathway remains unknown.
not reach statistical significance [54]. In the current study, we The 66A→G polymorphism is situated at a site within the
were able to demonstrate that the 2756G allele, when present sequence of the FMN-binding domain. Our present study
in either the heterozygous or homozygous state, was related reported a significant association between this polymorphism
to circulating homocysteine concentrations. Several other and homocysteine concentrations. This is consistent with
studies, however, failed to note any association between the earlier studies that examined this relationship. Moreover, the
MTR 2756A→G polymorphism and homocysteine concen- relative contribution of the MTRR 66GG genotype to the
trations [24,29,55]. A small number of reports even found variability of tHcy ranking was approximately half the effect
that both fasting and post-methionine load, homocysteine of the MTHFR 677TT genotype and twice the effect
A. Laraqui et al. / European Journal of Internal Medicine 18 (2007) 474–483 481

attributable to MTR 2756AA [65]. In contrast to this finding, ic as well as post-symptomatic evaluation and for the
no association of the MTRR 66A→G genotype with either prevention and treatment of cardiovascular disease.
fasting [51,66–69] or post-methionine loading [69] tHcy
levels has been reported by others in individuals known to be Acknowledgements
at highest risk for CAD or with diagnosed premature CAD, in
patients with confirmed venous thromboembolism, or in The authors would like to thank Pr. M-J Chapman and Pr.
adults without a clinical history of cardio-or cerebrovascular Y. Touitou for their help and advice, and J.P. Lagarde for
events and without current malignancy. technical assistance. We also thank the individuals who
Mutation analysis of the MTRR gene has been identified in voluntarily took part in this study.
patients with CAD in some studies. Our results are consistent
with an earlier report that failed to note any association
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