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Plasma homocysteine, methylenetetrahydrofolate reductase mutation and carotid damage in elderly healthy women
Atherosclerosis 157 (2001) 175– 180 www.elsevier.com/locate/atherosclerosis
Plasma homocysteine, methylenetetrahydrofolate reductase mutation and carotid damage in elderly healthy women Angela Passaro a, Antonietta Vanini b, Fabio Calzoni a, Lorenzo Alberti a, Pier Francesco Zamboni a, Renato Fellin a, Anna Solini a,* a
Department of Clinical and Experimental Medicine, Section of Internal Medicine II, Uni6ersity of Ferrara, Via Sa6onarola, 9, I-44100 Ferrara, Italy b Section of Ultrasonography, S. Anna Hospital, Ferrara, Italy Received 24 May 2000; received in revised form 14 September 2000; accepted 5 October 2000
Abstract Plasma homocysteine (Hcy) is an independent vascular risk factor. Its remethylation to methionine is regulated by the activity of the enzyme 5,10-methylene tetrahydrofolate reductase (MTHFR). A C-to-T substitution at nucleotide 677 of the MTHFR gene is frequently associated to hyperhomocysteinemia. In this study, we evaluated the relationship among MTHFR C677T polymorphism, Hcy and some ultrasonographic parameters at the level of carotid arteries in 120 elderly women with normal ECG, normal blood pressure values, total cholesterol B250 mg/dl, normal glucose tolerance, normal albumin excretion rate. In all subjects, we measured Hcy by HPLC, MTHFR mutation by polymerase chain reaction followed by HinfI digestion and intima-media thickness (IMT), peak velocity of the systolic flow (SPV), end-diastolic velocity (EDV) and resistance and pulsatility indexes of intracranial circulation (RI and PI) by ultrasound imaging. Twenty-eight women were homozygotes for the wild type allele (Ala/Ala), 72 were heterozygotes (Ala/Val) and 20 were homozygotes for the mutation (Val/Val). Groups were comparable for age, blood pressure values and plasma lipid levels. Hcy was higher in Val/Val group; moreover, after adjustment for confounding factors, Val/Val had significantly greater IMT and EDV (P B0.001 and PB 0.05, respectively). Logistic analysis revealed that Val/Val genotype was the strongest risk factor for IMT (OR 30.8, 95% CI 2.82– 335.6). Our results show that, in elderly healthy women, Val/Val homozygosity for C677T mutation in MTHFR gene could identify subjects at risk for asymptomatic carotid atherosclerotic impairment. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Homocysteine; Methylentetrahydrofolate reductase; Folate; Intima-media thickness; Atherosclerosis; Ultrasonics
1. Introduction Homocysteine (Hcy) is a putatively atherothrombotic sulfur amino acid produced during methionine metabolism. It is catabolized to cystathionine and cysteine in a pathway in which cystathionine b-synthase is a rate-limiting enzyme. Alternatively, homocysteine undergoes a methylation process catalyzed by 5,10-methylene tetrahydrofolate reductase (MTHFR) [1]. Much evidence show that mild elevation in plasma Hcy concentration is an independent risk factor for cerebrovascular and cardiovascular disease in the gen* Corresponding author. Tel.: + 39-532-247409; fax: +39-532210884. E-mail address: [email protected] (A. Solini).
eral population [2,3]. The main determinants of mild hyperhomocysteinemia are folate and B12 intake, impaired renal function and genetic factors [4–6]. In contrast to the very low prevalence of homocystinuria (a hereditary disease caused by deficiency of this enzyme and characterized by severe hyperhomocysteinemia, homocystinuria and early atherosclerosis and thrombosis) [7], a relatively frequent thermolabile variant of MTHFR, defective in its enzymatic activity, is associated with increased levels of Hcy and coronary artery disease [8,9]. Its allele is polymorphic and a C-to-T substitution at nucleotide 677 of the MTHFR gene is responsible for the thermolability of the enzyme. The mutation is common and : 12% of all Caucasians are mutant homozygotes (TT genotype). Some authors have reported an association of the TT genotype with
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A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
CHD [10], while others have denied it [11,12]. Few data, mostly in patients with type 2 diabetes [13] or hypertension [14], are indeed available concerning the putative relation between this polymorphism and cerebrovascular disease or carotid impairment, particularly in the early stages of involvement. In order to offer a contribution to clarify this issue, we designed a study to evaluate the relation among MTHFR C677 T polymorphism, plasma homocysteine levels and some ultrasonographic parameters registered at the level of carotid arteries in a group of healthy post-menopausal women without other known cardiovascular risk factors.
2. Methods
2.1. Subjects A total of 120 healthy, non-smoking women aged 55 – 80 years, participated in the study. Inclusion criteria were normal glucose tolerance after an OGTT, normal blood pressure values (systolic blood pressure B140 and diastolic blood pressure B 85 mmHg in the absence of any antihypertensive treatment), no previous clinical history of angina, myocardial infarction, stroke or transitory ischemic attack, absence of signs of CHD in a resting ECG, plasma total cholesterol below 250 mg/dl not more that 6 months before the onset of the study, normal kidney function (plasma creatinine B 1.2 mg/dl), normal albumin excretion rate at least in two consecutive 24-h urine collections in the last 6 months. Postmenopausal status was defined as absence of menstrual cycle in the last 12 months preceding the enrolment and serum FHS \40 mUI/ml. None of the recruited women had assumed medications that may influence Hcy (estrogens, bile acid sequestrant, folate or multivitamins, anticonvulsant agents) within the last 6 months preceding the study.
2.2. Analytical procedures After an overnight fast, venous blood samples were drawn for the determination of fasting plasma glucose (by glucose oxidase method), total and HDL cholesterol and plasma triglycerides (by standard enzymatic methods), plasma B12 and folate concentrations (by chemiluminescent immunoassays). Plasma homocysteine concentration was measured by a modified procedure of Araki and Sako with high pressure liquid chromatography and fluorescence detection [15]. Intraassay variation of the method was 2.5%. An EDTA whole blood sample for DNA extraction was frozen and stored at − 20°C until analysis. MTHFR C677 T polymorphism was determined by polymerase chain reaction (PCR) amplification of DNA
samples. Restriction analysis was performed with the enzyme HinfI (Boehringer Manheim, Germany). Digestion fragments were separated by 2% agarose gel electrophoresis and visualized under ultraviolet light following ethidium bromide staining [9].
2.3. Carotid ultrasonography Within 1 week of the blood sampling, all participants underwent an ultrasonographic evaluation of carotid arteries. Examinations were performed at room temperature (23°C) on overnight fasting subjects kept in supine position with the head slightly extended, after 10-min resting. An electrocardiogram-triggered high resolution Echo Color Doppler ATL 3000, equipped with a 5–10 Mhz multifrequency linear probe was used. All measurements were registered in the common carotid arteries, 1–2 cm proximal to the bulb, upstream to the eventually present plaque. Plaque was defined as localized lesion \ 1.3 mm thick, with spectral broadening absent or present only during the deceleration phase of systole. IMT was quantified by an appropriate software. The maximum IMT was defined as the mean of the maximum IMT for near and far walls in both right and left sides. The average value of three consecutive measurements was taken for statistical analysis. The maximum spatial resolution in M-mode was 0.1 mm.
2.4. Blood flow measurements Blood flow and velocity measurements were performed by the same operator (AV) using a pulsed-wave Doppler by a method detailed elsewhere [16]. We registered peak velocity of the systolic flow (PSV); end diastolic velocity (EDV); resistance index of intracranial circulation (RI) measured as (PSV − EDV)/PSV and pulsatility index (PI), an index of parietal compliance, equal to (PSV − EDV)/MSV. The average of three measurements repeated for three consecutive heart cycles was taken for statistical analysis.
2.5. Statistical analysis For the distribution of MTHFR genotypes, Hardy– Weinberg equilibrium was assessed by 2 analysis. Data are expressed as mean9 S.D. unless otherwise specified. Comparison of mean values among groups was performed by ANOVA followed by Tukey test. Multivariate analysis was used to assess relationship among IMT and other variables of interest. Logistic regression analysis was used to test the relative risk to have IMT higher than 1 mm. A P value B 0.05 was considered statistically significant. Analysis was performed using SPSS 8.0 package for Windows (SPSS, Evanston, IL).
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180 Table 1 Clinical and biochemical characteristics of the whole study populationa Age (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Post-load plasma glucose (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Plasma triglycerides (mg/dl) AER (mg/min) Plasma homocysteine (mmol/l) a
62 9 4 27.39 3.2 132910 829 4 90 97 108 9 22 215916 128 9 22 50 912 130 9 25 5.2 (1–21) 10.69 2.4
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Table 3 Ultrasonographic parameters of the three study groupsa
IMT (mm) SPV (cm/s) EDV (cm/s) PI RI
Ala/Ala (n =28)
Ala/Val (n =72)
Val/Val (n = 20)
0.98 90.04 68.04 93.6 20.40 91.3 1.04 90.04 0.70 9 0.01
1.03 90.02 70.9 9 2.4 21.92 90.8 1.05 90.03 0.68 90.01
1.23 9 0.04* 74.99 94.9 25.84 9 1.7† 0.99 9 0.05 0.65 9 0.02
Data are expressed as mean 9S.E. and analyzed by ANOVA. Adjustment for age, HDL and LDL-cholesterol, systolic and diastolic blood pressure. * PB0.001 respect to Ala/Ala and Ala/Val. † PB0.05 respect to Ala/Ala. a
AER is expressed as median (range).
3. Results Clinical characteristics of the whole study group are shown in Table 1. All subjects had normal blood pressure values and normal fasting and post-OGTT plasma glucose by selection criteria. After MTHFR C677 T polymorphism determination, 28 women were wild type for the 677C allele (Ala/Ala), 72 were heterozygotes (Ala/Val) and 20 were homozygotes for the mutation (Val/Val). This allelic distribution was in Hardy– Weinberg equilibrium with a sample of 118 randomly selected individuals from the same geographical area of our study population. Table 2 shows clinical parameters of the three groups, that were well-matched for age, BMI, blood pressure values and plasma glucose concentrations. No significant differences were observed in the lipid pattern and in plasma vitamins among groups. Conversely, plasma Hcy concentration was significantly higher in Val/Val group with respect to Ala/Val and Ala/Ala
(12.79 2.7 vs. 10.59 2.2 in Ala/Val and 9.391.6 mmol/l in Ala/Ala, PB 0.001). When ultrasonographic parameters of the three groups were compared, after adjustment for age, HDL and LDL-cholesterol, systolic and diastolic blood pressure, plasma folate and B12 concentrations, IMT was significantly higher in women carrying the mutation (Table 3). This group also showed a significantly higher EDV compared to wild type and heterozygotes subjects (PB 0.05). No differences were observed for PSV, RI and PI among groups. Partial correlations, after adjustment for age, showed a weak but significant relationship between IMT and plasma Hcy (PB 0.05). Neither plasma B12 or folate was significantly related to IMT. In multivariate analysis, MTHFR genotype and, to a lesser extent, age and homocysteine levels were significant determinants of IMT (Table 4). After logistic regression analysis, the crude odds ratio representing the likelihood of the Val/Val genotype to have an IMT \ 1 mm with respect to Ala/Ala was 30.8 (95% CI,
Table 2 Clinical characteristics of the three study groupsa
Age (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Post-load plasma glucose (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) AER (mg/min) Vitamin B12 (pmol/l) Folate (nmol/l) Plasma HCy (mmol/l)
Ala/Ala (n =28)
Ala/Val (n = 72)
Val/Val (n = 20)
6394 27.19 3.0 1309 10 829 4 859 12 1119 21 210 915 1289 20 539 13 131928 5 (1–21) 6539 207 1694 9.39 1.6
63 9 3 26.9 9 3.1 131 9 9 81 9 5 87 9 13 107 9 23 218 9 13 132 9 17 49 9 10 137 9 20 5 (1–18) 679 9 216 18 96 10.5 9 2.2
61 93 27.2 9 2.9 129 98 81 93 90 916 107 922 216 912 125 925 46 915 130 926 4 (2–21) 673 9241 18 95 12.7 92.7*
Data are expressed as mean 9S.D. AER is expressed as median (range). * PB0.001 respect to both Ala/Ala and Ala/Val.
a
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
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Table 4 Multiple regression analysis of the relationship between IMT and other variables of interesta
Age Systolic blood pressure Diastolic blood pressure Total cholesterol HDL-cholesterol Plasma homocysteine Vitamin B12 Serum folate MTHFR polymorphism
Standardized b coefficient
P value*
0.203 −0.110 0.115
0.010 0.607 0.702
0.115 0.008 0.286 0.098 −0.101 0.425
0.267 0.970 0.010 0.936 0.508 0.006
a
MTHFR C677T genotype was entered as a categorical variable. * P= 0.03 for the entire model; R 2 = 0.547.
2.82 –335.6) (Table 5); other risk factors, however to a lesser extent than MTHFR genotype, were age, diastolic blood pressure and total cholesterol.
4. Discussion There is agreement in considering mild hyperhomocysteinemia as an independent risk factor for cardiovascular disease in the general population; it has also been associated with an increased prevalence of carotid intima-media thickness in asymptomatic subjects [17]. Conversely, the role of MTHFR gene mutation in influencing cardiovascular risk is still a matter for debate. Some authors have described a positive association of the MTHFR C677T polymorphism with CHD [18,19], but not with stroke [20]. CUDAS study, even describing associations of elevated homocysteine with carotid wall thickening in old populations, did not relate these observations to a particular allelic distribution in regulatory genes [21], while Bova et al. [22] have recently shown a significantly higher presence of severe carotid stenosis in subjects carrying the allele A677V of Table 5 Results of logistic regression analysisa OR Age (5 years) Diastolic blood pressure (10 mmHg) MTHFR Val/Val a
1.930 2.874 30.786
CI (95%)
P*
1.253–2.972 1.107–7.459
0.003 0.030
2.824–335.593
0.005
Variables introduced into the model: age (5 years increment), BMI, systolic blood pressure (10 mmHg increment), diastolic blood pressure (10 mmHg increment), total cholesterol (50 mg/dl increment), HDL-cholesterol (10 mg/dl increment), albumin excretion rate, plasma homocysteine (10 mmol/l increment), MTHFR Ala/Val, MTHFR Val/Val. * P = 0.001 for the entire model. Sensitivity of the model is 0.747.
MTHFR; the effect, however, was not more pronounced in homozygotes. Our data give further insight into the relationship between homocysteine metabolism and carotid atherosclerosis, showing that, in a selected sample of elderly healthy subjects substantially devoid of any other known cardiovascular risk factors, a strong role of MTHFR mutation in conferring this risk can be detected, because homozygosity identifies subjects at high risk to develop intima-media thickness. An important characteristic of our study, in our opinion, is that all subjects had the parameters commonly influencing cardiovascular risk (fasting and post-load plasma glucose, blood pressure values, lipid pattern, albumin excretion rate) within the normal range, outlining a very homogeneous population respect to other studies [22– 24]; it is possible, however, that the failure to detect differences could be due to the very few subjects studied by other authors [22]. A population in Hardy–Weinberg equilibrium for a particular polymorphism should display genotype frequencies that remain stable throughout life. The prevalence of homozygosity for the mutation in our study population was 16.5%, slightly higher than the frequency observed in the US and Australian white population, but virtually identical to that previously described in Italy by other authors [25]. Regarding the possible effect of age in allelic distribution, in a recent study in 90–100-year-old subjects, both allele T frequency and TT genotype were in Hardy–Weinberg equilibrium and similar to those in the background population [26]. This excludes a biased selection of allelic distribution in our study group. Another evaluation to be done is that many studies have shown that the impact was largely dependent on low serum folate; it has been also suggested that homozygotes for the mutant MTHFR allele may require a higher than usual folate intake to correct the associated hyperhomocysteinemia [26]. In our population, serum folate levels were normal in all the subjects studied, without significant difference among the three MTHFR genotypes; this is probably the reason why vitamin concentrations did not affect the impact of genotype on IMT. It is likely, however, that the link between vascular disease and the C677T mutation is partially mediated through folate effect on homocysteine metabolism — the lower the plasma folate concentration, the higher the impact of MTHFR mutation on vascular disease [2]. A large and consistent impact of the thermolabile MTHFR variant on plasma Hcy levels in different European populations has been shown [27]; the association is however more pronounced in Val/Val carriers [28]. Our study makes a contribution to assess the genotype influence on circulating Hcy levels in the general population, confirming these observations in aging healthy women.
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
In our study population, logistic regression analysis demonstrated that the odds ratio for having carotid intima media thickness \1 mm is 30 times higher in subjects carrying the mutation than in wild type subjects. We would like to comment that this result, probably due to the careful selection of the patients, allowed us to detect, within a very homogeneous study population, significantly different levels of IMT even B 1.3 mm. It is of particular interest to observe that heterozygosity does not confer any valuable relative risk of increased IMT; this indirectly confirms that homozygosity, inducing a defective enzymatic activity, via higher Hcy levels could promote an increase of IMT in healthy people. Interestingly, our results show that subjects carrying the mutation also had a higher EDV. This parameter is one of the most important in defining flux in internal carotid artery; it increases over 40 cm/s in the presence of a stenosis \ 50% of the artery diameter but usually does not modify for mild stenosis. Our observation of a significant relationship between this index and MTHFR mutation, still to be confirmed in a larger population, is however the first report showing a link between homocysteine metabolism and a measurement of blood flow velocity, suggesting a more careful evaluation of blood flow characteristics in these subjects. There is good agreement between in vitro ultrasound and histological measurements of intima-media thickness. In our patients we did not collect information on coronary conditions besides resting ECG. In vivo studies however suggest that extracranial carotid atherosclerosis measured by B-mode ultrasound, is associated with the extent of coronary atherosclerosis, measured by coronary angiography [29]; it is therefore likely that our findings on carotid artery thickening may also have implications for coronary atherosclerosis. Some hypotheses can be suggested to explain how hyperhomocysteinemia could promote the progression of intima-media thickness toward occlusive arterial disease. It has been proposed that an increased arterial wall thickness may reduce oxygen transmissibility and give rise to oxygen-derived free radicals with ensuing tissue damage [30]. Similarly, the oxidation of homocysteine might lead either to an increased production of free radicals and hydrogen peroxide, which may contribute to damage to endothelial cells, and to a modification of LDL, enhancing foam cells formation. Moreover, recent evidence suggests that homocysteine has the ability to stimulate DNA synthesis in human vascular smooth muscle [31]; this fact is considered an important component of the atherosclerotic process. In conclusion, this study demonstrates that in healthy individuals without other known vascular risk factors and with normal serum folate and B12 concentrations, the determination of MTHFR polymorphism and the identification of homozygotes for the defective allele (T)
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may be useful to identify subjects at risk of developing intima-media thickness.
Acknowledgements We are deeply indebted with Giuseppe Gilli for skilful statistical assistance.
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[17] Malinow MR, Nieto FJ, Szklo M, Chambless LE, Bond G. Carotid artery intimal-medial wall thickening and plasma homocysteine in asymptomatic adults. Circulation 1993;87:1107 – 13. [18] Ou T, Yamakawa-Kobayashi K, Arinami T, et al. Methylentetrahydrofolate reductase and apolipoprotein E polymorphisms are independent risk factors for coronary heart disease in Japanese: a case-control study. Atherosclerosis 1998;137:23 – 8. [19] van den Berg M, Stehouwer CDA, Bierdragen E, Rauwerda JA. Plasma homocysteine and severity of atherosclerosis in young patients with lower-limb atherosclerotic disease. Arterioscler Thromb Vasc Biol 1996;16:165 –71. [20] Markus HS, Ali N, Swaminathan R, Sankaralingam A, Molloy J, Powell J. A common polymorphism in the methylenetetrahydrofolate reductase gene, homocysteine, and ischemic cerebrovascular disease. Stroke 1997;28:1739 –43. [21] McQuillan BM, Beilby JP, Nidorf M, Thompson PL, Hung J. Hyperhomocysteinemia but not the C677T mutation of methylenetetrahydrofolate reductase is an independent risk determinant of carotid wall thickening. Circulation 1999;99:2383 – 8. [22] Bova I, Chapman J, Sylantiev C, Corczyn AD, Bornstein NM. The A677V methylenetetrahydrofolate reductase gene polymorphism and carotid atherosclerosis. Stroke 1999;30:2180 –2. [23] Kostulas K, Crisby M, Huang WX, Lannfelt L, Hagenfeldt L, Eggertsen G, Kostulas V, Hillert J. A methylenetetrahydrofolate reductase gene polymorphism in ischaemic stroke and in carotid artery stenosis. Eur J Clin Invest 1998;28:285 –9. [24] Spence JD, Malinow R, Barnett PA, Marian AJ, Freeman D, Hegele RA. Plasma homocysteine concentration, but not
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Plasma homocysteine, methylenetetrahydrofolate reductase mutation and carotid damage in elderly healthy women Angela Passaro a, Antonietta Vanini b, Fabio Calzoni a, Lorenzo Alberti a, Pier Francesco Zamboni a, Renato Fellin a, Anna Solini a,* a
Department of Clinical and Experimental Medicine, Section of Internal Medicine II, Uni6ersity of Ferrara, Via Sa6onarola, 9, I-44100 Ferrara, Italy b Section of Ultrasonography, S. Anna Hospital, Ferrara, Italy Received 24 May 2000; received in revised form 14 September 2000; accepted 5 October 2000
Abstract Plasma homocysteine (Hcy) is an independent vascular risk factor. Its remethylation to methionine is regulated by the activity of the enzyme 5,10-methylene tetrahydrofolate reductase (MTHFR). A C-to-T substitution at nucleotide 677 of the MTHFR gene is frequently associated to hyperhomocysteinemia. In this study, we evaluated the relationship among MTHFR C677T polymorphism, Hcy and some ultrasonographic parameters at the level of carotid arteries in 120 elderly women with normal ECG, normal blood pressure values, total cholesterol B250 mg/dl, normal glucose tolerance, normal albumin excretion rate. In all subjects, we measured Hcy by HPLC, MTHFR mutation by polymerase chain reaction followed by HinfI digestion and intima-media thickness (IMT), peak velocity of the systolic flow (SPV), end-diastolic velocity (EDV) and resistance and pulsatility indexes of intracranial circulation (RI and PI) by ultrasound imaging. Twenty-eight women were homozygotes for the wild type allele (Ala/Ala), 72 were heterozygotes (Ala/Val) and 20 were homozygotes for the mutation (Val/Val). Groups were comparable for age, blood pressure values and plasma lipid levels. Hcy was higher in Val/Val group; moreover, after adjustment for confounding factors, Val/Val had significantly greater IMT and EDV (P B0.001 and PB 0.05, respectively). Logistic analysis revealed that Val/Val genotype was the strongest risk factor for IMT (OR 30.8, 95% CI 2.82– 335.6). Our results show that, in elderly healthy women, Val/Val homozygosity for C677T mutation in MTHFR gene could identify subjects at risk for asymptomatic carotid atherosclerotic impairment. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Homocysteine; Methylentetrahydrofolate reductase; Folate; Intima-media thickness; Atherosclerosis; Ultrasonics
1. Introduction Homocysteine (Hcy) is a putatively atherothrombotic sulfur amino acid produced during methionine metabolism. It is catabolized to cystathionine and cysteine in a pathway in which cystathionine b-synthase is a rate-limiting enzyme. Alternatively, homocysteine undergoes a methylation process catalyzed by 5,10-methylene tetrahydrofolate reductase (MTHFR) [1]. Much evidence show that mild elevation in plasma Hcy concentration is an independent risk factor for cerebrovascular and cardiovascular disease in the gen* Corresponding author. Tel.: + 39-532-247409; fax: +39-532210884. E-mail address: [email protected] (A. Solini).
eral population [2,3]. The main determinants of mild hyperhomocysteinemia are folate and B12 intake, impaired renal function and genetic factors [4–6]. In contrast to the very low prevalence of homocystinuria (a hereditary disease caused by deficiency of this enzyme and characterized by severe hyperhomocysteinemia, homocystinuria and early atherosclerosis and thrombosis) [7], a relatively frequent thermolabile variant of MTHFR, defective in its enzymatic activity, is associated with increased levels of Hcy and coronary artery disease [8,9]. Its allele is polymorphic and a C-to-T substitution at nucleotide 677 of the MTHFR gene is responsible for the thermolability of the enzyme. The mutation is common and : 12% of all Caucasians are mutant homozygotes (TT genotype). Some authors have reported an association of the TT genotype with
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A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
CHD [10], while others have denied it [11,12]. Few data, mostly in patients with type 2 diabetes [13] or hypertension [14], are indeed available concerning the putative relation between this polymorphism and cerebrovascular disease or carotid impairment, particularly in the early stages of involvement. In order to offer a contribution to clarify this issue, we designed a study to evaluate the relation among MTHFR C677 T polymorphism, plasma homocysteine levels and some ultrasonographic parameters registered at the level of carotid arteries in a group of healthy post-menopausal women without other known cardiovascular risk factors.
2. Methods
2.1. Subjects A total of 120 healthy, non-smoking women aged 55 – 80 years, participated in the study. Inclusion criteria were normal glucose tolerance after an OGTT, normal blood pressure values (systolic blood pressure B140 and diastolic blood pressure B 85 mmHg in the absence of any antihypertensive treatment), no previous clinical history of angina, myocardial infarction, stroke or transitory ischemic attack, absence of signs of CHD in a resting ECG, plasma total cholesterol below 250 mg/dl not more that 6 months before the onset of the study, normal kidney function (plasma creatinine B 1.2 mg/dl), normal albumin excretion rate at least in two consecutive 24-h urine collections in the last 6 months. Postmenopausal status was defined as absence of menstrual cycle in the last 12 months preceding the enrolment and serum FHS \40 mUI/ml. None of the recruited women had assumed medications that may influence Hcy (estrogens, bile acid sequestrant, folate or multivitamins, anticonvulsant agents) within the last 6 months preceding the study.
2.2. Analytical procedures After an overnight fast, venous blood samples were drawn for the determination of fasting plasma glucose (by glucose oxidase method), total and HDL cholesterol and plasma triglycerides (by standard enzymatic methods), plasma B12 and folate concentrations (by chemiluminescent immunoassays). Plasma homocysteine concentration was measured by a modified procedure of Araki and Sako with high pressure liquid chromatography and fluorescence detection [15]. Intraassay variation of the method was 2.5%. An EDTA whole blood sample for DNA extraction was frozen and stored at − 20°C until analysis. MTHFR C677 T polymorphism was determined by polymerase chain reaction (PCR) amplification of DNA
samples. Restriction analysis was performed with the enzyme HinfI (Boehringer Manheim, Germany). Digestion fragments were separated by 2% agarose gel electrophoresis and visualized under ultraviolet light following ethidium bromide staining [9].
2.3. Carotid ultrasonography Within 1 week of the blood sampling, all participants underwent an ultrasonographic evaluation of carotid arteries. Examinations were performed at room temperature (23°C) on overnight fasting subjects kept in supine position with the head slightly extended, after 10-min resting. An electrocardiogram-triggered high resolution Echo Color Doppler ATL 3000, equipped with a 5–10 Mhz multifrequency linear probe was used. All measurements were registered in the common carotid arteries, 1–2 cm proximal to the bulb, upstream to the eventually present plaque. Plaque was defined as localized lesion \ 1.3 mm thick, with spectral broadening absent or present only during the deceleration phase of systole. IMT was quantified by an appropriate software. The maximum IMT was defined as the mean of the maximum IMT for near and far walls in both right and left sides. The average value of three consecutive measurements was taken for statistical analysis. The maximum spatial resolution in M-mode was 0.1 mm.
2.4. Blood flow measurements Blood flow and velocity measurements were performed by the same operator (AV) using a pulsed-wave Doppler by a method detailed elsewhere [16]. We registered peak velocity of the systolic flow (PSV); end diastolic velocity (EDV); resistance index of intracranial circulation (RI) measured as (PSV − EDV)/PSV and pulsatility index (PI), an index of parietal compliance, equal to (PSV − EDV)/MSV. The average of three measurements repeated for three consecutive heart cycles was taken for statistical analysis.
2.5. Statistical analysis For the distribution of MTHFR genotypes, Hardy– Weinberg equilibrium was assessed by 2 analysis. Data are expressed as mean9 S.D. unless otherwise specified. Comparison of mean values among groups was performed by ANOVA followed by Tukey test. Multivariate analysis was used to assess relationship among IMT and other variables of interest. Logistic regression analysis was used to test the relative risk to have IMT higher than 1 mm. A P value B 0.05 was considered statistically significant. Analysis was performed using SPSS 8.0 package for Windows (SPSS, Evanston, IL).
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180 Table 1 Clinical and biochemical characteristics of the whole study populationa Age (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Post-load plasma glucose (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Plasma triglycerides (mg/dl) AER (mg/min) Plasma homocysteine (mmol/l) a
62 9 4 27.39 3.2 132910 829 4 90 97 108 9 22 215916 128 9 22 50 912 130 9 25 5.2 (1–21) 10.69 2.4
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Table 3 Ultrasonographic parameters of the three study groupsa
IMT (mm) SPV (cm/s) EDV (cm/s) PI RI
Ala/Ala (n =28)
Ala/Val (n =72)
Val/Val (n = 20)
0.98 90.04 68.04 93.6 20.40 91.3 1.04 90.04 0.70 9 0.01
1.03 90.02 70.9 9 2.4 21.92 90.8 1.05 90.03 0.68 90.01
1.23 9 0.04* 74.99 94.9 25.84 9 1.7† 0.99 9 0.05 0.65 9 0.02
Data are expressed as mean 9S.E. and analyzed by ANOVA. Adjustment for age, HDL and LDL-cholesterol, systolic and diastolic blood pressure. * PB0.001 respect to Ala/Ala and Ala/Val. † PB0.05 respect to Ala/Ala. a
AER is expressed as median (range).
3. Results Clinical characteristics of the whole study group are shown in Table 1. All subjects had normal blood pressure values and normal fasting and post-OGTT plasma glucose by selection criteria. After MTHFR C677 T polymorphism determination, 28 women were wild type for the 677C allele (Ala/Ala), 72 were heterozygotes (Ala/Val) and 20 were homozygotes for the mutation (Val/Val). This allelic distribution was in Hardy– Weinberg equilibrium with a sample of 118 randomly selected individuals from the same geographical area of our study population. Table 2 shows clinical parameters of the three groups, that were well-matched for age, BMI, blood pressure values and plasma glucose concentrations. No significant differences were observed in the lipid pattern and in plasma vitamins among groups. Conversely, plasma Hcy concentration was significantly higher in Val/Val group with respect to Ala/Val and Ala/Ala
(12.79 2.7 vs. 10.59 2.2 in Ala/Val and 9.391.6 mmol/l in Ala/Ala, PB 0.001). When ultrasonographic parameters of the three groups were compared, after adjustment for age, HDL and LDL-cholesterol, systolic and diastolic blood pressure, plasma folate and B12 concentrations, IMT was significantly higher in women carrying the mutation (Table 3). This group also showed a significantly higher EDV compared to wild type and heterozygotes subjects (PB 0.05). No differences were observed for PSV, RI and PI among groups. Partial correlations, after adjustment for age, showed a weak but significant relationship between IMT and plasma Hcy (PB 0.05). Neither plasma B12 or folate was significantly related to IMT. In multivariate analysis, MTHFR genotype and, to a lesser extent, age and homocysteine levels were significant determinants of IMT (Table 4). After logistic regression analysis, the crude odds ratio representing the likelihood of the Val/Val genotype to have an IMT \ 1 mm with respect to Ala/Ala was 30.8 (95% CI,
Table 2 Clinical characteristics of the three study groupsa
Age (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Post-load plasma glucose (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) AER (mg/min) Vitamin B12 (pmol/l) Folate (nmol/l) Plasma HCy (mmol/l)
Ala/Ala (n =28)
Ala/Val (n = 72)
Val/Val (n = 20)
6394 27.19 3.0 1309 10 829 4 859 12 1119 21 210 915 1289 20 539 13 131928 5 (1–21) 6539 207 1694 9.39 1.6
63 9 3 26.9 9 3.1 131 9 9 81 9 5 87 9 13 107 9 23 218 9 13 132 9 17 49 9 10 137 9 20 5 (1–18) 679 9 216 18 96 10.5 9 2.2
61 93 27.2 9 2.9 129 98 81 93 90 916 107 922 216 912 125 925 46 915 130 926 4 (2–21) 673 9241 18 95 12.7 92.7*
Data are expressed as mean 9S.D. AER is expressed as median (range). * PB0.001 respect to both Ala/Ala and Ala/Val.
a
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
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Table 4 Multiple regression analysis of the relationship between IMT and other variables of interesta
Age Systolic blood pressure Diastolic blood pressure Total cholesterol HDL-cholesterol Plasma homocysteine Vitamin B12 Serum folate MTHFR polymorphism
Standardized b coefficient
P value*
0.203 −0.110 0.115
0.010 0.607 0.702
0.115 0.008 0.286 0.098 −0.101 0.425
0.267 0.970 0.010 0.936 0.508 0.006
a
MTHFR C677T genotype was entered as a categorical variable. * P= 0.03 for the entire model; R 2 = 0.547.
2.82 –335.6) (Table 5); other risk factors, however to a lesser extent than MTHFR genotype, were age, diastolic blood pressure and total cholesterol.
4. Discussion There is agreement in considering mild hyperhomocysteinemia as an independent risk factor for cardiovascular disease in the general population; it has also been associated with an increased prevalence of carotid intima-media thickness in asymptomatic subjects [17]. Conversely, the role of MTHFR gene mutation in influencing cardiovascular risk is still a matter for debate. Some authors have described a positive association of the MTHFR C677T polymorphism with CHD [18,19], but not with stroke [20]. CUDAS study, even describing associations of elevated homocysteine with carotid wall thickening in old populations, did not relate these observations to a particular allelic distribution in regulatory genes [21], while Bova et al. [22] have recently shown a significantly higher presence of severe carotid stenosis in subjects carrying the allele A677V of Table 5 Results of logistic regression analysisa OR Age (5 years) Diastolic blood pressure (10 mmHg) MTHFR Val/Val a
1.930 2.874 30.786
CI (95%)
P*
1.253–2.972 1.107–7.459
0.003 0.030
2.824–335.593
0.005
Variables introduced into the model: age (5 years increment), BMI, systolic blood pressure (10 mmHg increment), diastolic blood pressure (10 mmHg increment), total cholesterol (50 mg/dl increment), HDL-cholesterol (10 mg/dl increment), albumin excretion rate, plasma homocysteine (10 mmol/l increment), MTHFR Ala/Val, MTHFR Val/Val. * P = 0.001 for the entire model. Sensitivity of the model is 0.747.
MTHFR; the effect, however, was not more pronounced in homozygotes. Our data give further insight into the relationship between homocysteine metabolism and carotid atherosclerosis, showing that, in a selected sample of elderly healthy subjects substantially devoid of any other known cardiovascular risk factors, a strong role of MTHFR mutation in conferring this risk can be detected, because homozygosity identifies subjects at high risk to develop intima-media thickness. An important characteristic of our study, in our opinion, is that all subjects had the parameters commonly influencing cardiovascular risk (fasting and post-load plasma glucose, blood pressure values, lipid pattern, albumin excretion rate) within the normal range, outlining a very homogeneous population respect to other studies [22– 24]; it is possible, however, that the failure to detect differences could be due to the very few subjects studied by other authors [22]. A population in Hardy–Weinberg equilibrium for a particular polymorphism should display genotype frequencies that remain stable throughout life. The prevalence of homozygosity for the mutation in our study population was 16.5%, slightly higher than the frequency observed in the US and Australian white population, but virtually identical to that previously described in Italy by other authors [25]. Regarding the possible effect of age in allelic distribution, in a recent study in 90–100-year-old subjects, both allele T frequency and TT genotype were in Hardy–Weinberg equilibrium and similar to those in the background population [26]. This excludes a biased selection of allelic distribution in our study group. Another evaluation to be done is that many studies have shown that the impact was largely dependent on low serum folate; it has been also suggested that homozygotes for the mutant MTHFR allele may require a higher than usual folate intake to correct the associated hyperhomocysteinemia [26]. In our population, serum folate levels were normal in all the subjects studied, without significant difference among the three MTHFR genotypes; this is probably the reason why vitamin concentrations did not affect the impact of genotype on IMT. It is likely, however, that the link between vascular disease and the C677T mutation is partially mediated through folate effect on homocysteine metabolism — the lower the plasma folate concentration, the higher the impact of MTHFR mutation on vascular disease [2]. A large and consistent impact of the thermolabile MTHFR variant on plasma Hcy levels in different European populations has been shown [27]; the association is however more pronounced in Val/Val carriers [28]. Our study makes a contribution to assess the genotype influence on circulating Hcy levels in the general population, confirming these observations in aging healthy women.
A. Passaro et al. / Atherosclerosis 157 (2001) 175–180
In our study population, logistic regression analysis demonstrated that the odds ratio for having carotid intima media thickness \1 mm is 30 times higher in subjects carrying the mutation than in wild type subjects. We would like to comment that this result, probably due to the careful selection of the patients, allowed us to detect, within a very homogeneous study population, significantly different levels of IMT even B 1.3 mm. It is of particular interest to observe that heterozygosity does not confer any valuable relative risk of increased IMT; this indirectly confirms that homozygosity, inducing a defective enzymatic activity, via higher Hcy levels could promote an increase of IMT in healthy people. Interestingly, our results show that subjects carrying the mutation also had a higher EDV. This parameter is one of the most important in defining flux in internal carotid artery; it increases over 40 cm/s in the presence of a stenosis \ 50% of the artery diameter but usually does not modify for mild stenosis. Our observation of a significant relationship between this index and MTHFR mutation, still to be confirmed in a larger population, is however the first report showing a link between homocysteine metabolism and a measurement of blood flow velocity, suggesting a more careful evaluation of blood flow characteristics in these subjects. There is good agreement between in vitro ultrasound and histological measurements of intima-media thickness. In our patients we did not collect information on coronary conditions besides resting ECG. In vivo studies however suggest that extracranial carotid atherosclerosis measured by B-mode ultrasound, is associated with the extent of coronary atherosclerosis, measured by coronary angiography [29]; it is therefore likely that our findings on carotid artery thickening may also have implications for coronary atherosclerosis. Some hypotheses can be suggested to explain how hyperhomocysteinemia could promote the progression of intima-media thickness toward occlusive arterial disease. It has been proposed that an increased arterial wall thickness may reduce oxygen transmissibility and give rise to oxygen-derived free radicals with ensuing tissue damage [30]. Similarly, the oxidation of homocysteine might lead either to an increased production of free radicals and hydrogen peroxide, which may contribute to damage to endothelial cells, and to a modification of LDL, enhancing foam cells formation. Moreover, recent evidence suggests that homocysteine has the ability to stimulate DNA synthesis in human vascular smooth muscle [31]; this fact is considered an important component of the atherosclerotic process. In conclusion, this study demonstrates that in healthy individuals without other known vascular risk factors and with normal serum folate and B12 concentrations, the determination of MTHFR polymorphism and the identification of homozygotes for the defective allele (T)
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may be useful to identify subjects at risk of developing intima-media thickness.
Acknowledgements We are deeply indebted with Giuseppe Gilli for skilful statistical assistance.
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