Relation between plasma homocysteine, gene polymorphisms of homocysteine metabolism-related enzymes, and angiographically proven coronary artery disease

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

⁎ 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 43 96; fax: +212 37 67 32 32. E-mail address: [email protected] (A. Laraqui).

1. Introduction Coronary artery disease (CAD) is a significant problem in terms of morbidity as well as mortality. This condition is now 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

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due to their unhealthy habits and behavior. Thus, any intervention 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 combination than alone, interact to create a proatherogenic environment. These risk factors are: advancing age, male sex, smoking, hypertension, positive family history, hyperlipidemia, 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 secondary 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 randomization”, the association of genetic variants with the studied trait in case-control studies may suggest causality [12]. Therefore, an association of polymorphisms of the methionine cycle with CAD, if observed, would suggest that genetically determined alterations in metabolism of homocysteine and related compounds are the cause or modifier of arteriosclerosis rather than its consequence. Approximately ten common genetic variants in the enzymes 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 methylenetetrahydrofolate 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 prevalence 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

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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 CardioVasculaires, 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 angiography were angina pectoris or high presumption of CAD. Coronary angiography was performed using standard techniques. Blinded assessment of coronary angiograms identified CAD as the presence of 50% or more stenosis in any coronary vessel. According to this definition, 151 (65.7%) patients 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 independently by two experienced cardiologists in order to assess the available indication: coronary artery bypass grafting 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). Hypertension 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 antihypertensive medications. Cigarette smokers were categorized 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 apolipoprotein levels and for isolation of DNA. Total cholesterol, HDL-cholesterol, and triglyceride levels were measured by

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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 centrifuged 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 homocysteine 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, Lcysteine, adenosine, and L-cystathionine was below 0.1%, and with S-adenosyl-L-methionine it was 12.5%. Plasma homocysteine 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) produced 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 fragments 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 amplified 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 thermal 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 AT3′ 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 restriction 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 isoleucinecontaining 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

Number of subjects Age (years ± SD) Male gender, n (%) History of hyperlipidemia, % History of hypertension, % History of smoking, % History of diabetes, % History of obesity, % Cholesterol, mmol/l Total HDL LDL Triglycerides, mmol/l

Controls

Cases

RR (95% CI)

P

79 51.62 ± 9.32 41 (50.63) 37.97 30.38 26.58 22.78 27.85

151 54.54 ± 9.77 120 (79.47) 68.21 70.20 73.51 47.02 49.67

1.03 (1–1.06) 3.77 (2.09–6.82) 3.51 (1.98–6.91) 5.40 (2.98–9.77) 7.66 (4.14–14.19) 3.01 (1.63–5.56) 2.56 (1.42–4.60)

b0.05 b0.05 b0.001 b0.001 b0.001 b0.05 b0.05

5.04 ± 0.85 1.19 ± 0.78 3.75 ± 0.94 1.48 ± 0.53

5.72 ± 1.14 1.05 ± 0.16 4.58 ± 1.25 1.86 ± 0.58

1.93 (1.41–2.66) 0.01 (0.001–0.06) 1.96 (1.45–2.64) 3.88 (2.10–7.17)

b0.05 b0.05 b0.05 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.

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Table 2 Homocysteine and hyperhomocysteine distribution in the total population and according to angiographic investigation

No of subjects Homocysteinemia, μmol/l Median Mean ± SD (95% CI) Hyperhomocysteinemia, [n (%)]

Total population

Case

Control

230

151

79

P

14.9 14 ± 3.7 (13.5–14.5) 105 (45.7)

15.4 15.5 ± 2.8 (15.1–15.9) 89 (58.9)

11.6 11.2 ± 3.5 (10.4–12.0) 16 (20.3)

b0.001 a b0.001 b 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 All tests were two-tailed and a P value below 0.05 was deemed significant. All analyses considered only the 230 subjects assessed by coronary angiography. Skewed variables were natural log-transformed to normalize the distribution. Between the two groups, a comparison was made using the independent t-test and the chi-square test; the Kruskal–Wallis test was applied to compare three or more groups. A one-way ANOVA was used to assess the effect of smoking status, BMI, diabetes, dyslipidemia, and genotype on homocysteine. Interactive effects of variables that were significantly correlated were assessed using logistic regression analysis, which was also applied to compute odds ratios (OR) as well as their 95% confidence intervals. To compute both allele and genotype frequencies and confidence intervals, SPSS (version 10.0) for Windows was used. 3. Results 3.1. Mean differences between cases and control subjects Table 1 shows the variables associated with case status. Cases were older and more likely to be male. Their CAD risk profile was unfavorable compared to control subjects. For example, they had a higher blood pressure and total cholesterol level. Furthermore, their HDL cholesterol level was lower and they were more likely to be smokers. The estimates of the relative risk (RR) of CAD according to clinical and laboratory parameters examined in this study are shown in Table 1.

women) of the controls had HHcy. The corresponding RR was 4.1 (95% CI 2.2–7.5, P b 0.001). 3.3. Homocysteine levels according to smoking status To assess the interaction between smoking, tHcy, and CAD, subjects (n = 230) were grouped into current cigarette smokers or non-smokers, and plasma homocysteine was defined as greater than 15 μmol/l. The tHcy concentrations were significantly different between smokers and non-smokers (15.6 ± 3.1 versus 12.5 ± 3.8 μmol.l− 1; P b 0.05). In addition, smokers with HHcy demonstrated a markedly increased risk of CAD (OR = 2.5, 95% CI 1.7–3.3, P b 0.001) compared with a nonsmoker with normal homocysteine. Table 3 Genotype frequencies in controls and in cases Gene

All patients

MTHFR 1298A→C, n (%) AA 100 (43.5) AC 93 (40.4) CC 37 (16.1)

MTR 2756→C, n (%) AA 163 (70.9) AG 62 (27.0) GG 5 (22) AG+GG 67 (29.3)

Controls

Cases

RR (95%CI)

42 (53.2) 58 (38.4) 23 (29.1) 70 (46.4) 14 (17.7) 23 (15.2) χ2 = 6.6 (P = 0.03)

1.0 2.2 (1.2–4.2) 1.2 (0.6–2.6)

57 (72.2) 106 (70.2) 22 (27.8) 40 (26.5) – 5 (3.3) 22 (27.8) 45 (29.8) χ2 = 0.88 (P = 0.120)

1.0 1.0 (0.53–1.80) – 1.1 (0.6–2.0)

31 (39.2) 52 (34.4) 42 (53.2) 83 (55.0) 6 (6.7) 16 (10.6) χ2 = 0.85 (P = 0.653)

1.00 1.2 (0.7–2.1) 1.6 (0.6–4.5)

3.2. Levels of plasma total homocysteine and CAD

MTRR 66A→G, n (%) AA 83 (36.1) AG 125 (54.4) GG 22 (9.6)

The mean (±SD) homocysteine concentration in CAD patients (15.5 ± 2.8 μmol/l; 95% CI 15.1–15.9 μmol/l) was significantly (P b 0.001) higher than the mean concentration in controls (11.2 ± 3.5 μmol/l; 95% CI 10.4–12.0 μmol/l; Table 2). Using 15 μmol/l as a cut-off point to classify mild HHcy, 56.3% (45.0% in men and 11.3% in women) of the CAD patients and 24.1% (38.0% in men and 11.4% in

P: degree of significant value. Significant differences in genotype distribution between cases and controls were tested by chi-square test. Because GG genotype frequency was lower than 5, the chi-square test was 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. Chi-square test was used before subjects were grouped into AG and GG. This grouping of genotype was used just to evaluate the RR associated with the presence of the G allele.

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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 subjects Total population MTHFR A1298C AA AC CC P MTR A2756G AA AG GG AG + GG P MTRR A66G AA AG GG AG + GG P a b c d

Homocysteinemia, μmol/l

HHcy [n (%)]

Odds ratio

Median

Mean ± SD

95%CI

Value

95% CI

P

230

14.9

14.0 ± 3.7

13.5–14.5

106 (46.1)

4.3

2.3–7.9

0.000

100 93 37

14.0 16.2 14.8 0.000 a, b

13.2 ± 3.5 16.1 ± 3.7 13.5 ± 4.8 0.000 c

12.5–13.9 15.3–16.8 12.0–15.1

35 (35.0) 60 (64.5) 15 (40.5) 0.000 d, b

1.0 3.4 1.3

– 1.9–6.1 0.6–6.1

0.001 0.55

163 62 5 67

14.6 15.3 17.2 15.3 0.037 a, b

13.6 ± 3.6 14.4 ± 3.4 19.5 ± 4.6 14.8 ± 3.7 0.008 c

13.1–14.2 13.5–15.3 13.8–25.1 13.9–15.7

67 (41.1) 35 (56.5) 4 (80.0) 39 (58.2) 0.018 d, b

1.0 1.9 5.7 2.0

1.0–3.4 0.6–51.6 1.1–3.6

0.040 0.123 0.019

83 125 22 147

14.1 15.1 15.4 15.2 0.015 a

13.1 ± 3.6 14.3 ± 3.6 15.5 ± 3.6 14.5 ± 3.6 0.007 c

12.3–13.9 13.7–14.9 13.9–17.1 13.9–15.1

29 (34.9) 63 (50.4) 14 (63.6) 77 (52.4) 0.020 d

1.0 1.9 3.3 2.1

1.1–3.4 1.2–8.7 1.2–3.6

0.029 0.018 0.011

Homocysteine comparison between genotypes (median test). AG and GG genotypes were grouped if the expected counts were less than 5. Comparison of homocysteine levels between genotypes (Kruskal–Wallis test). 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, suggesting 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 variants, 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 polymorphisms 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

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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 homocysteine 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 homocysteine levels. Multiple logistic regression was used to test for independent correlates of CAD. Included in the model were: age, gender, smoking, hypertension, diabetes, hypercholesterolemia, 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 remethylation 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 conditions [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, oxidation of LDL, and/or proliferation of vascular smooth muscle cell [30,31].

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4.2. Smoking The role of environmental factors in determining homocysteine 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, hypertension, and vasoconstriction, and both produce direct endothelial 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 vasodilatation 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 Canadians [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 significantly 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 wildtype genotype (1298AA). However, Van der Put et al. [41] suggested that combined heterozygosity for both polymorphisms resulted in reduced MTHFR specific activity, higher

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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 associated 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 casecontrol 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 concentrations [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 prevalence 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. Furthermore, 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 cardiovascular risk factors. For example, in a hospital-based casecontrol 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 concentration and homocysteine-related diseases has previously been suggested. None of these studies showed a clear interaction between these polymorphisms [29,57,59–61]. Unfortunately, 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

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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 cobalamin 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 hyperhomocysteinemia are needed to give us a better understanding of the etiology 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 hyperhomocysteinemia. Among the three common mutations, the MTHFR 1298A→C mutation is the major risk factor for the development of CAD. • An understanding of the genetic determinants of mild hyperhomocysteinemia may be useful in pre-symptomat-

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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. References [1] Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274: 1049–57. [2] Christensen B, Frosst P, Lussier-Cacan S, Selhub J, Goyette P, Rosenblatt DS, et al. Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. Arterioscler Thromb Vasc Biol 1997;17:569–73. [3] Graham IM, Daly LE, Refsum HM, Robinson K, Brattström LE, Ueland PM, et al. Plasma homocysteine as a risk factor for vascular disease: the European Concerted Action Project. JAMA 1997;277: 1775–81. [4] Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997;337:230–6. [5] Eikelboom JW, Lonn E, Genest J, Hankey G, Yusuf S. Homocysteine and cardiovascular disease: a critical review of the epidemiologic evidence. Ann Intern Med 1999;131:363–75. [6] Verhoef P, Stampfer M. Epidemiology of vascular and thrombotic associations. In: Carmel R, Jacobsen D, editors. Homocysteine in health and disease. Cambridge: Cambridge University Press; 2001. p. 357–70. [7] Ford ES, Smith SJ, Stroup DF, Steinberg KK, Mueller PW, Thacker SB. Homocysteine and cardiovascular disease: a systematic review of the evidence with special emphasis on case-control studies and nested case-control studies. Int J Epidemiol 2002;31:59–70. [8] Robinson K. Homocysteine and coronary artery disease. In: Carmel R, Jacobsen D, editors. Homocysteine in health and disease. Cambridge: Cambridge University Press; 2001. p. 371–83. [9] Ueland PM, Refsum H, Beresford SA, Vollset SE. The controversy over homocysteine and cardiovascular risk. Am J Clin Nutr 2000;72: 324–32. [10] Tsai MY, Bignell M, Yang F, Welge BG, Graham KJ, Hanson NQ. Polygenic influence on plasma homocysteine: association of two prevalent mutations, the 844ins68 of cystathionine β-synthase and A2756G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis 2000;149:131–7. [11] Dekou V, Gudnason V, Hawe E, Miller GJ, Stansbie D, Humphries SE. Gene–environment and gene–gene interaction in the determination of plasma homocysteine levels in healthy middle-aged men. Thromb Haemost 2001;85:67–74. [12] Clayton D, McKeigue PM. Epidemiological methods for studying genes and environmental factors in complex diseases. Lancet 2001;358: 1356–60. [13] Kraus JP, Oliveriusova J, Sokolova J, Kraus E, Vlcek C, de Franchis R, et al. The human cystathionine β-synthase (CβS) gene: complete sequence, alternative splicing, and polymorphisms. Genomics 1998;52: 312–24. [14] Rozen R. Polymorphisms of folate and cobalamin metabolism. In: Carmel R, Jacobsen D, editors. Homocysteine in health and disease. Cambridge: Cambridge University Press; 2001. p. 259–69.

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