Plasma homocysteine, methylenetetrahydrofolate reductase genotypes, and age at onset of symptoms of myocardial ischemia

Plasma Homocysteine, Methylenetetrahydrofolate Reductase Genotypes, and Age at Onset of Symptoms of Myocardial Ischemia Aviv Mager,

MD,

Alexander Battler, MD, Yochai Birnbaum, Mordechai Shohat, MD

MD,

Nurit Magal,

PhD,

and

Elevated fasting plasma homocysteine is a graded risk factor of coronary artery disease (CAD) and may accelerate onset of CAD. Homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is commonly but inconsistently associated with hyperhomocysteinemia. In the present study we examined the possible relation between levels of fasting plasma homocysteine and age at CAD onset in different MTHFR genotypes. We studied 182 patients with CAD, 74 patients with early onset CAD (aged <45 years), and 108 patients with later onset CAD (aged 46 to 65 years). Plasma homocysteine levels in 90 subjects without CAD were used for control. Fasting plasma homocysteine levels in T/T homozygotes with early onset CAD (20.2 ⴞ12.5 ␮mol/L) was markedly higher than in T/T homozygotes with later onset CAD (13.4 ⴞ 6.8 ␮mol/L) and in patients with early onset CAD who were not T/T homozygotes (11.9 ⴞ 3.7 ␮mol/L; p ⴝ 0.034 and p ⴝ 0.0001, respectively). CAD developed earlier in T/T ho-

mozygotes who were hyperhomocysteinemic (>15 ␮mol/L) than in the T/T homozygotes who were not (p ⴝ 0.036). Plasma homocysteine levels had no effect on age at onset of CAD in patients who were non-T/T genotypes. Homocysteine levels in control subjects and in patients who were non-T/T genotypes were comparable and were not influenced by age. The results reveal an inverse relation between the level of fasting plasma homocysteine and age at onset of CAD in T/T homozygotes as opposed to no association in patients who were non-T/T genotypes. Additionally, these results show that hyperhomocysteinemia and the T/T genotype have a stronger effect on the pathogenesis of CAD when they are combined, and that a marked increase (>15 ␮mol/L) in fasting plasma homocysteine in T/T homozygotes is a risk factor for early onset of CAD. 䊚2002 by Excerpta Medica, Inc. (Am J Cardiol 2002;89:919 –923)

asting plasma homocysteine is a graded and independent risk factor for coronary and other forms of F vascular disease, as well as a strong predictor of

tion between levels of fasting plasma homocysteine and age at onset of CAD in patients with CAD with different MTHFR genotypes.

1–3

4,5

6

mortality. Some investigators7,8 have found an association between plasma homocysteine and early onset of coronary artery disease (CAD), but this was not confirmed by others.9 Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in homocysteine metabolism. MTHFR deficiency results in homocysteinuria,10 a rare genetic disorder characterized by development of CAD very early in life. A common point mutation (C677T) in the MTHFR gene renders this enzyme thermolabile and less active.11 Homozygosity for this mutation is typically but inconsistently associated with hyperhomocysteinemia11–15 and with CAD.12–20 Interestingly, studies that reported an association between the C677T mutation and CAD often included younger patients.17,19,21 In the present study we examined the possible associaFrom the Departments of Cardiology and Medical Genetics, Rabin Medical Center-Beilinson Campus and Felsenstein Medical Research Center, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Manuscript received August 29, 2001; revised manuscript received and accepted December 27, 2001. Address for reprints: Aviv Mager, MD, Department of Cardiology, Rabin Medical Center-Beilinson Campus, Petah Tiqva 49100, Israel. E-mail: [email protected]. ©2002 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 89 April 15, 2002

METHODS The study candidates consisted of 210 consecutive patients with CAD from our Coronary Clinic. Inclusion criteria were onset of CAD symptoms at ⬍65 years of age and angiographically documented CAD (⬎50% stenosis of ⱖ1 epicardial coronary artery) or well-documented myocardial infarction diagnosed by clinical, electrocardiographic, and enzymatic criteria. All the patients underwent coronary angiography for purposes unrelated to this study. Patients with renal failure (serum creatinine ⱖ1.4 mg/dl), severe systemic disease, hypothyroidism, and known vitamin B12 deficiency, and patients taking medications or vitamins that could affect plasma homocysteine levels, were excluded. Ten additional patients were referred from other clinics. Of the original group, 2 died, 1 developed renal failure, and 35 could not be contacted or were not willing to participate. The final study population consisted of 182 patients. All were interviewed to collect data on smoking habits, body height and weight, use of medications including vitamins, age at onset of CAD symptoms, previous myocardial infarction, hypertension (blood pressure ⬎140/90 mm Hg or 0002-9149/02/$–see front matter PII S0002-9149(02)02239-7

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TABLE 1 Patient Characteristics* Variable Women Cigarette smoker Diabetes mellitus Dyslipidemia‡ Systemic㛳 hypertension§ Obesity

Early Onset (n ⫽ 74) 10 42 15 37 13 10

(13%) (64%) (23%) (56%) (20%) (15%)

Later Onset (n ⫽ 108) 24 45 19 58 29 21

(27%)† (51%) (21%) (65%) (33%) (24%)

*Data on risk factors are for 66 patients with early onset CAD and 89 with later onset CAD. † p ⬍0.05. ‡ Low-density lipoprotein ⬎130 mg/dl or triglycerides ⬎300 mg/dl, or on lipid-lowering medications. § Blood pressure ⬎140/90 mm Hg or on antihypertensive therapy. 㛳 Body mass index ⬎30 kg/m2.

TABLE 2 Plasma Homocysteine Levels (␮mol/L) in Patients and Age-matched Controls Patients

Controls

Early onset (age ⱕ45 yrs) 14.3 ⫾ 5.3 10.6 ⫾ 2.8 Later onset (age ⬎45 yrs) 11.8 ⫾ 3.3* 10.8 ⫾ 2.4†

p Value 0.008 0.16

*p ⫽ 0.018 for patients with early versus later onset; †p ⫽ 0.11 for older versus younger control subjects.

on antihypertensive treatment), diabetes mellitus, dyslipidemia (low-density lipoprotein ⬎130 mg/dl or triglycerides ⬎300 mg/dl, or on lipid-lowering medications), and co-morbidity. Obesity was defined as a body mass index ⬎30 kg/m2. All the patients were genotyped for the C677T mutation. After an 8-hour fast, blood was drawn for determination of plasma homocysteine, folate and vitamin B12 levels, a lipid profile, and creatinine. The patients were allocated into 2 groups according to age at onset of CAD symptoms21: ⱕ45 years (n ⫽ 74) and ⬎45 years (n ⫽ 108). Ninety subjects (aged 29 to 65 years) without a history of CAD or renal failure who underwent screening for plasma homocysteine for purposes not related to vascular disease served as a control group. The study was approved by the Ethics Committee of the Rabin Medical Center, and informed consent was obtained from each participant. Genetic and biochemical analyses: DNA was isolated from peripheral leukocytes with a DNA Isolation Kit for Mammalian Blood (Boehringer Mannheim, Mannheim, Germany). Screening for the 677C3 T substitution was performed by polymerase chain reaction of genomic DNA, followed by Hinf I digestion and agarose gel electrophoresis as described by Frosst et al.11 Plasma cobalamin (vitamin B12) and folate levels were measured using radioimmunoassay techniques. For determination of plasma homocysteine, the fresh blood samples were immediately cooled on ice and protected from light. The plasma was separated shortly thereafter by cold centifugation and stored at ⫺18°C until assay with the IMx analyzer (Abbott, Chicago, Illinois). A plasma homocysteine 920 THE AMERICAN JOURNAL OF CARDIOLOGY姞

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FIGURE 1. Fasting plasma homocysteine and age at onset of CAD in T/T homozygotes (top) and in the patients who were non-T/T genotypes (bottom).

level above the normal range (⬎15 ␮mol/L) was defined as hyperhomocysteinemia. Statistical analysis: Comparison between means was performed with Student’s t test and comparison between frequencies with the Pearson chi-square test. Comparison of biologic parameters according to age at onset of CAD (early vs late) and MTHFR genotype (T/T vs non-T/T) was performed with 2-way analysis of variance. Fisher’s exact test was performed for comparison of frequencies between small groups.

RESULTS Table 1 lists the clinical characteristics of the patients with early and later onset of CAD. The later onset group had a higher proportion of women. There were no differences in the prevalence of coronary risk factors between patients with early and later onset. Table 2 lists the mean plasma homocysteine levels in the control subjects by age and in the patients according to age at CAD onset. Patients with early onset CAD had higher plasma homocysteine levels than both the younger control subjects and the patients with later onset. The patients with later onset CAD had levels similar to the older control subjects. In the APRIL 15, 2002

TABLE 3 Plasma Homocysteine (Hcy) Levels in the Patients According to Age at Onset of Coronary Artery Disease and Methylenetetrahydrofolate Reductase Genotype Early Onset Genotype T/T C/T ⫹ C/C

Later Onset

TABLE 4 Age at Onset of Coronary Artery Disease According to Plasma Homocysteine (Hcy) Level and Methylenetetrahydrofolate Reductase Genotype Genotype

n

Hcy

n

Hcy

p Value

21 53

20.2 ⫾ 12.5* 11.9 ⫾ 3.7

22 86

13.4 ⫾ 6.8 11.6 ⫾ 3.6

0.034 0.36

*p ⫽ 0.0001 for T/T versus C/T ⫹ C/C with early onset.

control group, there was no difference in plasma homocysteine levels between younger and older subjects. Figure 1 shows the levels of fasting plasma homocysteine according to age at onset of CAD in T/T homozygotes and in patients with the C/T and C/C genotypes (non-T/T). In T/T homozygotes, there was an inverse relation between plasma homocysteine levels and age at CAD onset (r ⫽ ⫺0.351, p ⫽ 0.032). There was no association between plasma homocysteine level and the age at CAD onset in patients who were not T/T homozygotes (r ⫽ ⫺0.047, p ⫽ 0.58). Table 3 lists the levels of plasma homocysteine in the patients by MTHFR genotype and age at CAD onset (early vs later). T/T homozygotes with early onset had significantly higher plasma homocysteine levels than T/T homozygotes with later onset and patients with early onset CAD who were not T/T homozygotes. In non-homozygotes, there was no difference in plasma homocysteine between the patients with early and later onset or between those with the C/T and C/C genotypes in any age group. Plasma homocysteine levels in T/T homozygotes with early onset was markedly increased (69% higher than in non-homozygotes with early onset CAD and 51% higher than in T/T homozygotes with later onset). Plasma homocysteine levels in non-homozygotes was only 8% higher than in the control subjects (p ⫽ NS). There was no difference in plasma homocysteine levels between non-homozygotes and control subjects in any age group. To minimize the impact of the lower plasma homocysteine levels in non-homozygotes, we also compared the effect of elevated plasma homocysteine levels on age at onset in the T/T and non-T/T genotypes. Forty-two percent of the patients with the T/T genotype were hyperhomocysteinemic, as were 17% of the patients who were non-T/T genotypes. Hyperhomocysteinemia was more common in T/T homozygotes with early onset than in T/T homozygotes with later onset (57% vs 27%, p ⫽ 0.047). The prevalences of hyperhomocysteinemia in non-homozygotes with early and later onset CAD were similar (17% in each group). Table 4 lists the age at onset of CAD according to plasma homocysteine level (elevated vs nonelevated) and MTHFR genotype. In T/T homozygotes with elevated plasma homocysteine levels, CAD developed earlier than in those with plasma homocysteine within the normal range. In non-homozygotes, there was no difference in the age at CAD onset

Hcy ⬎15 ␮mol/L

Hcy ⱕ15 ␮mol/L

p Value

44.3 ⫾ 4.0 47.3 ⫾ 6.5

48.3 ⫾ 5.4 46.5 ⫾ 5.2

0.036 0.49

T/T C/T ⫹ C/C

TABLE 5 Coronary Risk Factors by Methylenetetrahydrofolate Reductase Genotype and Age at Onset of Coronary Artery Disease C/T ⫹ C/C

T/T

Risk Factor Cigarette smoker Dyslipidemia* Systemic hypertension† Diabetes mellitus Obesity‡

Early Onset (n ⫽ 21)

Later Onset (n ⫽ 20)

Early Onset (n ⫽ 45)

Later Onset (n ⫽ 69)

13 (62%) 10 (48%) 2 (10%)

10 (50%) 15 (75%) 5 (25%)

29 (64%) 26 (58%) 10 (22%)

35 (51%) 43 (62%) 25 (36%)

1 (5%)§ 3 (14%)

3 (15%) 4 (20%)

13 (29%) 7 (16%)

16 (23%) 17 (25%)

*Low-density lipoprotein ⬎130 mg/dl or triglycerides ⬎300 mg/dl, or on lipid-lowering medications. † Blood pressure ⬎140/90 mm Hg or on antihypertensive therapy. ‡ Body mass index ⬎30 kg/m2. § p ⫽ 0.038 for T/T versus C/C ⫹ C/T with early onset, Fisher’s exact test.

between the patients with and without hyperhomocysteinemia. The distribution of risk factors among the patients according to the MTHFR genotype is shown in Table 5. Diabetes mellitus was less frequent in T/T than in non-T/T patients with early onset. There was no difference in the distribution of the other risk factors among genotypes in both age groups and there was no association between plasma homocysteine levels and any of these risk factors. Log-transformed homocysteine values showed a weak inverse correlation with plasma folate (r ⫽ ⫺0.354 in T/T homozygotes and r ⫽ ⫺0.259 in nonhomozygotes; p ⫽ 0.037 and p ⫽ 0.008, respectively). Table 6 lists the mean levels of folate and cobalamin according to MTHFR genotype and age at CAD onset. T/T homozygotes tended to have lower plasma folate levels than non-homozygotes (p ⫽ 0.07). T/T homozygotes with early onset CAD tended to have lower plasma folate levels than both T/T homozygotes with later onset and non-homozygotes with early onset; however, these differences were not significant. There were no differences in plasma cobalamin levels between genotypes or between age groups.

DISCUSSION To the best of our knowledge, this is the first study to correlate the levels of plasma homocysteine and age at onset of CAD in different MTHFR genotypes. The results reveal a strong association between fasting plasma homocysteine levels and age at onset of CAD in T/T homozygotes, and no association in patients who are non-T/T genotypes. In patients with the T/T

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CAD (aged ⱕ45 years), but not with later onset.21 These results were supported by a recent study in male Japanese patients with familial hyperEarly Onset Later Onset cholesterolemia.22 In the present C/C ⫹ C/T T/T C/C ⫹ C/T T/T p Value* study, in Israeli Jews, we found that T/T homozygotes with early onset Folate (nmol/L) 19.2 ⫾ 9.1 15.8 ⫾ 9.3 21.0 ⫾ 9.9 18.9 ⫾ 6.9 0.70 had a marked increase in plasma hoB12 (pmol/L) 246 ⫾ 89 252 ⫾ 91 244 ⫾ 96 285 ⫾ 173 0.39 mocysteine, whereas in those with Values are mean ⫾ SD. later onset, plasma homocysteine *Two-way analysis of variance. levels were less elevated, indicating that the effect of the T/T genotype on the age at onset of CAD correlates genotype, we found an inverse relation between fast- with its effect on plasma homocysteine. Taken toing plasma homocysteine levels and age at onset of gether with the present findings, the data suggest that CAD; hyperhomocysteinemia (⬎15 ␮mol/L) in pa- the T/T genotype is associated with CAD only when it tients with this genotype was associated with a signif- is accompanied by a significant increase in fasting icantly earlier onset of CAD. In contrast, in patients plasma homocysteine, and that the difference among with non-T/T genotypes, even hyperhomocysteinemia studies in the risk associated with the T/T genotype had no effect on age at CAD onset. may be explained by the differences in the effect of Our findings of higher plasma homocysteine levels the T/T genotype on plasma homocysteine. in patients with early onset of CAD than in those with The levels of plasma homocysteine in our T/T later onset agree with the findings of Genest et al7 and homozygotes with early onset CAD are similar to Kawashiri et al.8 These investigators found higher those reported for T/T homozygotes in a study from plasma homocysteine levels in patients with prema- Japan that demonstrated an association between the ture CAD than in control subjects and concluded that T/T genotype and early onset of CAD in male patients elevated plasma homocysteine levels are an indepen- with heterozygous familial hypercholesterolemia. dent risk factor for this condition. However, we fur- Consequently, the association between elevated ther divided our patients with early onset of CAD plasma homocysteine and early onset of CAD in T/T according to the MTHFR genotype and found that homozygotes is not exclusive to our patients, and only those with the T/T genotype had higher plasma homozygosity for the C677T mutation with elevated homocysteine levels than the control subjects. Thus, in plasma homocysteine levels is probably a risk factor our patients, the T/T genotype accounted entirely for for early onset of CAD regardless of ethnic backthe difference in plasma homocysteine levels between ground. Moreover, in homozygotes for the C677T the patients with early onset CAD and the control mutation, there seems to be a dose-response relation subjects. Because the MTHFR genotype was not de- between the level of fasting plasma homocysteine and termined in the studies of Genest et al7 and Kawashiri the age at onset of CAD. This, and the lack of accelet al,8 the role of the C677T mutation in their patients erating effect of hyperhomocysteinemia on CAD in is unknown. the patients with non-T/T genotypes, suggest that hyOnly few of the case-control studies that examined perhomocysteinemia and the T/T genotype have a the possible association between the C677T mutation stronger effect on the pathogenesis of CAD when they and CAD in relatively younger patients provided infor- are combined. mation also on plasma homocysteine levels in the 3 MTHFR genotypes.12,16,17 Schwartz et al16 examined the risk of myocardial infarction in North American 1. Stampfer MJ, Malinow MR, Willet WC, Newcomer LM, Upson B, Ullman D, women aged ⬍45 years, and found no difference in Tishler PV, Hennekens CH. A prospective study of plasma homocyst(e)ine and of myocardial infarction in US physicians. JAMA 1992;268:877–881. nonfasting plasma homocysteine levels between patients risk 2. Arensen E, Refsum H, Bonaa KH, Ueland PM, Forde OH, Nordrehaug JE. with the T/T genotype and the other case patients (13.2 Serum total homocysteine and coronary artery disease. Int J Epidemiol 1995;24: vs 13.4 ␮mol/L, respectively). In another study, fasting 704 –709. 3. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative plasma homocysteine in patients with the T/T genotype assessment of plasma homocysteine as a risk factor for vascular disease: probable was only 23% higher than in those with the C/C geno- benefits of increasing folic acid intakes. JAMA 1995;274:1049 –1057. type (homozygous normal).12 Similar findings were re- 4. Verhoef P, Hennekens CH, Malinow MR, Kok FJ, Willet WC, Stampfer MJ. A prospective study of homocyst(e)ine and risk of ischemic stroke. Stroke ported in a study of United States physicians (aged 40 to 1994;25:1924 –1930. 13 84 years). In none of these 3 studies was there an 5. den Heijer M, Koster T, Blom HJ, Bos GM, Briet E, Reitsma PH, VandenJP, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep vein association between the MTHFR genotype and the risk brouke thrombosis. N Engl J Med 1996;334:759 –762. 17 of CAD. In contrast, in an Irish study, the T/T genotype 6. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollest SE. was associated both with an increased frequency of Plasma homocysteine levels and mortality in patients with coronary artery disN Engl J Med 1997;337:230 –236. elevated fasting and postload plasma homocysteine ease. 7. Genest JJ, McNamara JR, Salem DN, Wilson PW, Schaefer EJ, Malinow MR. and an increased risk of CAD. However, the levels of Plasma homocyst(e)ine levels in men with premature coronary artery disease. plasma homocysteine were not detailed. In a study J Am Coll Cardiol 1990:1114 –1119. Kawashiri M, Kajinami K, Nohara A, Yagi K, Inazu A, Koizumi J, Haraki T, that included Israeli Jewish patients, the T/T genotype 8. Takegoshi T, Mabuchi H. Plasma homocysteine level and development of corwas associated with an increased risk of early onset of onary artery disease. Coronary Artery Dis 1999;10:443–447. TABLE 6 Plasma Folate and Vitamin B12 Levels According to Methylenetetrahydrofolate Reductase Genotype and Age at Onset of Coronary Artery Disease

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