The deployment of endovascular stents offers a significant

Transcription

1 Homocysteine Status and Polymorphisms of Methylenetetrahydrofolate Reductase Are Not Associated With Restenosis After Stenting in Coronary Arteries Werner Koch, Gjin Ndrepepa, Julinda Mehilli, Siegmund Braun, Marc Burghartz, Harald Lengnick, Klaus Kölling, Albert Schömig, Adnan Kastrati Objective We investigated the influence of elevated homocysteine plasma levels and 2 polymorphisms, 677C/T and 1298A/C, of the methylenetetrahydrofolate reductase (MTHFR) gene on the risk of restenosis after stenting in patients with symptomatic coronary artery disease. Methods and Results Homocysteine levels and MTHFR genotypes were determined in 800 consecutive patients treated with coronary artery stenting. Angiographic restenosis ( 50% diameter stenosis at 6-month follow-up) was present in 25.8% of the patients with low homocysteine levels (at or below the median of 11.6 mol/l; n 400) and 24.1% of the patients with high homocysteine levels ( 11.6 mol/l; n 400; P 0.62). Rates of angiographic restenosis were 26.0%, 23.5%, and 26.9% in carriers of the 677CC, 677CT, and 677TT genotypes (P 0.75), respectively, and 24.4%, 25.9%, and 24.0% in patients with the 1298AA, 1298AC, and 1298CC genotypes (P 0.90), respectively. The need for restenosis-driven reintervention (clinical restenosis) was 18.8% in subjects with low homocysteine concentrations and 19.0% in subjects with high homocysteine concentrations during the first year after the intervention (P 0.93). Rates of clinical restenosis were 19.5%, 17.1%, and 23.3% in carriers of the 677CC, 677CT, and 677TT genotypes (P 0.37), respectively, and 17.6%, 18.6%, and 24.7% in patients with the 1298AA, 1298AC, and 1298CC genotypes (P 0.27), respectively. Conclusions Elevated levels of homocysteine and 2 polymorphisms of the MTHFR gene are not associated with restenosis after stenting in coronary arteries. (Arterioscler Thromb Vasc Biol. 2003;23: ) Key Words: homocysteine methylenetetrahydrofolate reductase stent restenosis genetics The deployment of endovascular stents offers a significant advance in the percutaneous treatment of atherosclerotic disease, but in-stent restenosis affects 30% of patients in the months after an initially successful intervention. 1 Elevated homocysteine plasma levels have been associated with the risk of coronary artery disease (CAD), myocardial infarction (MI), and adverse outcome after coronary balloon angioplasty. 2 6 Homocysteine levels are determined by environmental and genetic factors, including the single-nucleotide polymorphism (SNP) 677C/T of the gene encoding methylenetetrahydrofolate reductase (MTHFR). 7,8 MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is required as a methyl donor for conversion of homocysteine to methionine by methionine synthase. 9 The 677C/T SNP affects amino acid 222 (alanine or valine), located in the catalytic domain of MTHFR, and the MTHFR variant with valine 222 has decreased thermostability and reduced specific enzymatic activity. 7,10 Compared with subjects with the 677CC (Ala/Ala) or 677CT (Ala/Val) genotype, mild hyperhomocysteinemia and an increased risk of CAD were found to be associated with carriers of the 677TT (Val/Val) genotype, particularly in individuals with low plasma concentrations of folate. 11,12 In a limited series (n 62) of Japanese patients with CAD, an impact of the 677C/T SNP on the restenotic process after stenting was observed. 13 Another SNP of the MTHFR gene, 1298A/C, is associated with the presence of glutamate (1298A) or alanine (1298C) at amino acid position ,15 The polymorphic site is located in the putative regulatory domain of the polypeptide chain, and the MTHFR variant with alanine 429 has reduced enzymatic activity Inconsistent findings were reported on an association between the 1298A/C SNP and CAD, 17,18 and it is not known whether this SNP is related to restenosis after interventions in coronary arteries. Optimal procedures for blood sample collection and handling are critical when homocysteine is used for cardiovascular risk assessment. 2,3 Inadequate procedures potentially result in artificial elevation of homocysteine concentrations, which may be interpreted as increased risk. For this reason, Received September 16, 2003; revision accepted October 13, From the Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar, Technische Universität München, München, Germany. Correspondence to Werner Koch, PhD, Deutsches Herzzentrum München, Experimentelle Kardiologie, Lazarettstrasse 36, D München, Germany. wkoch@dhm.mhn.de 2003 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at DOI: /01.ATV

2 2230 Arterioscler Thromb Vasc Biol. December 2003 determination of patients MTHFR genotypes may provide valuable additional information about homocysteine status. In a relatively large number of patients, we investigated the influence of elevated homocysteine levels and polymorphisms (677C/T and 1298A/C) of the MTHFR gene on the rate of restenosis or other adverse events (death or nonfatal MI) after placement of stents in atherosclerotic coronary arteries. Methods Patients The study included 800 white patients with symptomatic CAD who underwent stent implantation in coronary arteries at Deutsches Herzzentrum München from September 2000 to September The protocols of stent placement and poststenting therapy have been described in detail elsewhere. 19,20 Postprocedural pharmacological therapy consisted of aspirin (100 mg twice daily, indefinitely) and clopidogrel (75 mg/d for at least 4 weeks). Patients who were considered at a higher risk for ischemic complications received additional therapy with the glycoprotein IIb/IIIa blocker abciximab, which was given as a bolus injection during the stent insertion procedure and as a 12-hour continuous infusion thereafter. All patients were scheduled for angiographic follow-up at 6 months. Written informed consent was obtained for the intervention itself, routine follow-up angiography at 6 months, and genotype determination. The study protocol conformed to the Declaration of Helsinki and was approved by the Institutional Ethics Committee. Coronary Angiography Lesion morphology was classified according to the modified American College of Cardiology/American Heart Association grading system as type A, B1, B2, or C, and lesions of types B2 and C were considered complex lesions. Angiograms were recorded just before and immediately after the intervention and at 6-month follow-up. Quantitative analysis of angiograms (CMS system; Medis Medical Imaging Systems) was performed by operators not involved in the stenting procedure and who were unaware of the laboratory or genetic data. Measurement of Homocysteine, Folate, and Vitamin B 12 Concentrations Blood samples were taken from individuals in the supine position before cardiac catheterization, collected into heparinized tubes, transported to the central laboratory, and immediately centrifuged at 1550g for 10 minutes. The separated plasma samples were stored at a temperature of 4 C and analyzed within 3 days. Plasma total homocysteine, folate, and vitamin B 12 concentrations were measured with AxSYM kits (Abbott Laboratories). Determination of MTHFR Genotypes Genotype analysis was performed with the TaqMan technique (Applied Biosystems). Primers 5 -GACCTGAAGCACTTGAAGGAGAAG-3 and 5 -CTTCACAAAGCGGAAGAATGTGT-3 were used to amplify a 96-bp portion of exon 4 that contained the polymorphic site 677C/T (dbsnp cluster ID rs ), and primers 5 -AGGAGGAGCTGC- TGAAGATGTG-3 and 5 -GTTCTCCCGAGAGGTAAAGAA- CAAA-3 were used to amplify an 87-bp portion of exon 7 that contained the polymorphic site 1298A/C (dbsnp cluster ID rs ). The sequences of the allele-specific probe oligonucleotides were FAM-5 - TGATGAAATCGGCTCC-3 (677C), VIC-5 -TGATGAAAT- CGACTCCC-3 (677T), FAM-5 -TCAAAGACACTTTCTTCACT-3 (1298A), and VIC-5 -AAAGACACTTGCTTCACT-3 (1298C). The fluorogenic dyes FAM, ie, 6-carboxy-fluorescein, or VIC (proprietary dye of Applied Biosystems) were attached to the 5 ends of the probe molecules to accomplish allelic discrimination. Minor groove binder groups were conjugated with the 3 ends of the probes. As a control, genotyping was repeated for 20% of the samples with DNA prepared separately from the original blood sample. Genotype determination was done by workers who had no knowledge of patients clinical, laboratory, or angiographic data. Definitions and Study End Points For assessment of the influence of elevated homocysteine concentrations on clinical and angiographic outcomes, patients were divided into 2 groups with the median value of total serum homocysteine levels used as a cutoff point. The 400 patients with homocysteine levels 11.6 mol/l constituted the group with a low homocysteine status, and the 400 patients with homocysteine levels 11.6 mol/l constituted the group with a high homocysteine status. To examine the impact of the MTHFR gene polymorphisms on outcome measures, patients were analyzed according to genotypes of the 677C/T SNP and the 1298A/C SNP, independent of their homocysteine status. The primary end point of the study was restenosis. Two definitions of restenosis were used: the incidence of a diameter stenosis of 50% at 6-month follow-up angiography (angiographic restenosis) and the need for target-vessel revascularization (TVR; percutaneous transluminal coronary balloon angioplasty or aortocoronary bypass grafting) because of symptoms or signs of ischemia in the presence of angiographic restenosis at the stented site during the first year after stent placement (clinical restenosis). Secondary end points of the study were the incidence of all-cause death and the rate of death or nonfatal MI at 1 year after stenting. The incidence of thrombotic events during the early 30-day period after stenting, resulting in death, acute MI, or urgent TVR, was assessed separately. The diagnosis of acute MI was based on the presence of a clinical episode of prolonged chest pain with either the appearance of 1 or more new pathological Q waves on the ECG or an increase in creatine kinase (or its MB isoenzyme) levels to at least twice the normal upper limit. Creatine kinase levels were determined systematically over 48 hours after the stenting procedure. The follow-up protocol included a phone contact or a medical visit at the outpatient clinic at 30 days and between 9 and 15 months after stent placement and a control angiography at 6 months. Clinical events were assessed on the basis of the information provided by hospital readmission records, the referring physician, or phone interview with the patient. For all patients who reported cardiac symptoms during the phone interview, at least 1 clinical and electrocardiographic evaluation was performed at the outpatient clinic or by the referring physician. Statistical Analysis Discrete variables are expressed as counts and percentages and were compared with 2 test or Fisher exact test, as appropriate. Continuous variables are expressed as mean SD and were compared by means of the unpaired, 2-sided t test or ANOVA for more than 2 groups. The independent role of homocysteine levels and the polymorphisms was evaluated in a multivariate model (multiple logistic regressions) for restenosis that included the baseline clinical, lesion-related, and procedural variables as potentially confounding factors. Survival free of MI was analyzed by a Cox regression model that allowed the calculation of hazard ratios and 95% CIs. Statistical analyses were performed with S-Plus software (Mathsoft Inc). A probability value of 0.05 was considered statistically significant. Results Patient Characteristics Table 1 shows a comparison of the baseline clinical, lesionrelated, and procedural characteristics between the 400 patients with low ( 11.6 mol/l) and the 400 patients with high ( 11.6 mol/l) total plasma homocysteine levels. Among the 800 patients, genotypes of the 677C/T SNP were distributed as 43.5% 677CC, 45.3% 677CT, and 11.3% 677TT, and genotypes of the 1298A/C SNP were distributed as 46.1% 1298AA, 41.8% 1298AC, and 12.1% 1298CC. As shown in Table 2, total homocysteine levels were significantly higher among carriers of the 677TT genotype than

3 Koch et al Homocysteine, MTHFR Polymorphisms, and Restenosis 2231 TABLE 1. Characteristics of Patients According to Total Plasma Homocysteine Status (n 800) Homocysteine 11.6 mol/l (n 400) Homocysteine 11.6 mol/l (n 400) P Homocysteine, mol/l 9.3 ( 1.6) 15.7 ( 4.4) Mean age, y 63.2 ( 10.1) 68.1 ( 10.8) Women 102 (25.5) 103 (25.8) 0.94 Arterial hypertension 312 (78.0) 324 (81.0) 0.29 Hypercholesterolemia 285 (71.3) 293 (73.3) 0.53 Current smoker 88 (22.0) 86 (21.5) 0.86 Diabetes mellitus 86 (21.5) 90 (22.5) 0.73 Unstable angina pectoris 79 (19.8) 80 (20.0) 0.93 Acute MI 72 (18.0) 42 (10.5) Previous MI 140 (35.0) 132 (33.0) 0.55 Previous aortocoronary bypass surgery 44 (11.0) 55 (13.8) 0.24 No. of narrowed coronary arteries (25.3) 102 (25.5) (32.5) 117 (29.3) (42.3) 181 (45.3) Left ventricular ejection fraction, % 55.7 ( 13.3) 53.6 ( 14.9) 0.04 Folate, ng/ml 10.6 ( 3.0) 8.4 ( 2.9) Vitamin B 12, pg/ml 447 ( 296) 361 ( 236) Creatinine, mg/dl 1.07 ( 0.19) 1.22 ( 0.36) Lipoprotein(a), mg/dl 40.7 ( 46.3) 41.6 ( 45.1) 0.79 Target coronary vessel 0.63 LMCA 7 (1.8) 8 (2.0) LAD 176 (44.0) 156 (39.0) LCx 81 (20.3) 88 (22.0) RCA 123 (30.8) 130 (32.5) Venous bypass graft 13 (3.3) 18 (4.5) Chronic occlusion 16 (4.0) 24 (6.0) 0.19 Restenotic lesion 36 (9.0) 31 (7.8) 0.52 Complex lesions (ACC/AHA type B2 or C) 291 (72.8) 316 (79.0) 0.04 Lesion length, mm 12.1 ( 6.8) 12.6 ( 6.8) 0.28 Reference diameter, mm 2.96 ( 0.60) 2.93 ( 0.57) 0.45 Diameter stenosis before stenting, % 61.6 ( 19.2) 61.7 ( 18.5) 0.90 Stented segment length, mm 21.4 ( 10.4) 22.6 ( 10.6) 0.12 Periprocedural therapy with abciximab 222 (55.5) 231 (57.8) 0.52 Diameter stenosis after stenting, % 4.5 ( 10.4) 5.3 ( 10.9) 0.24 Data are presented as mean ( SD) or number (%) of subjects. LMCA indicates left main coronary artery; LAD, left anterior descending coronary artery; LCx, left circumflex coronary artery; RCA, right coronary artery; and ACC/AHA, American College of Cardiology/American Heart Association. among patients with the 677CC or 677CT genotype (P 0.001). Inversely, significantly higher folate levels were associated with the 677CC genotype than with the 677CT or 677TT genotype (P 0.003; Table 2). Regarding the 1298A/C SNP, homocysteine and folate concentrations were not significantly different between genotypes. Carriers of the genotype combination 677CC/1298AA (CC/AA), CC/AC, CC/CC, CT/AA, CT/AC, or TT/AA were present among the study patients, but not subjects with the genotype combination CT/CC, TT/AC, or TT/CC. Early Clinical Events We assessed the occurrence of major adverse clinical events due to thrombosis during the early 30-day period after stenting. Comparison of patients with low homocysteine status and those with high homocysteine status did not indicate significant differences in the incidences of death, death or MI, or urgent TVR (P 0.43). Similarly, these events did not occur at significantly different frequencies among the genotype groups of the 677C/T SNP (P 0.29) or the 1298A/C SNP (P 0.07).

4 2232 Arterioscler Thromb Vasc Biol. December 2003 TABLE 2. Characteristics of Patients According to Genotypes of the MTHFR Gene 677C/T Polymorphism (n 800) Angiographic Restenosis Six-month angiography of coronary arteries was performed in 306 (76.5%) of patients with low homocysteine status and 295 (73.8%) of patients with high homocysteine status (P 0.37). The proportion of patients with angiographic restenosis was 25.8% in patients with low homocysteine levels and 24.1% in patients with high homocysteine levels (P 0.62). Among patients with angiographic restenosis (n 150), the mean homocysteine level was mol/l, and among subjects without angiographic restenosis (n 451), the mean homocysteine level was mol/l (P 0.19). Folate 677CC (n 348) 677CT (n 362) 677TT (n 90) P Mean age, y 64.7 ( 10.8) 66.2 ( 10.6) 67.1 ( 10.5) 0.07 Women 88 (25.3) 96 (26.5) 21 (23.3) 0.81 Arterial hypertension 277 (79.6) 286 (79.0) 73 (81.1) 0.90 Hypercholesterolemia 241 (69.3) 271 (74.9) 66 (73.3) 0.24 Current smoker 77 (22.1) 72 (19.9) 25 (27.8) 0.26 Diabetes mellitus 84 (24.1) 73 (20.2) 19 (21.1) 0.43 Unstable angina pectoris 70 (20.1) 72 (19.9) 17 (18.9) 0.97 Acute MI 45 (12.9) 60 (16.6) 9 (10.0) 0.18 Previous MI 111 (31.9) 135 (37.3) 26 (28.9) 0.18 Previous aortocoronary bypass surgery 46 (13.2) 38 (10.5) 15 (16.7) 0.23 No. of narrowed coronary arteries (24.1) 93 (25.7) 26 (28.9) (29.6) 117 (32.3) 27 (30.0) (46.3) 152 (42.0) 37 (41.1) Left ventricular ejection fraction, % 54.3 ( 14.9) 55.5 ( 13.7) 52.4 ( 12.7) 0.16 Homocysteine, mol/l 12.0 ( 3.9) 12.5 ( 4.5) 14.4 ( 6.5) Folate, ng/ml 10.0 ( 3.1) 9.3 ( 3.1) 9.0 ( 3.6) Vitamin B 12, pg/ml 405 ( 269) 392 ( 252) 451 ( 341) 0.18 Creatinine, mg/dl 1.13 ( 0.23) 1.16 ( 0.36) 1.12 ( 0.25) 0.31 Lipoprotein(a), mg/dl 39.7 ( 46.2) 41.8 ( 44.6) 44.2 ( 48.7) 0.70 Target coronary vessel 0.34 LMCA 9 (2.6) 5 (1.4) 1 (1.1) LAD 140 (40.2) 151 (41.7) 41 (45.6) LCx 72 (20.7) 83 (22.9) 14 (15.6) RCA 113 (32.5) 113 (31.2) 27 (30.0) Venous bypass graft 14 (4.0) 10 (2.8) 7 (7.8) Chronic occlusion 16 (4.6) 21 (5.8) 3 (3.3) 0.57 Restenotic lesion 31 (8.9) 31 (8.6) 5 (5.6) 0.58 Complex lesions (ACC/AHA type B2 or C) 252 (72.4) 282 (77.9) 73 (81.1) 0.11 Lesion length, mm 12.4 ( 7.1) 12.4 ( 6.6) 11.9 ( 6.4) 0.82 Reference diameter, mm 2.94 ( 0.58) 2.95 ( 0.59) 2.96 ( 0.58) 0.94 Diameter stenosis before stenting, % 61.7 ( 18.8) 62.1 ( 18.9) 59.7 ( 18.6) 0.57 Stented segment length, mm 21.7 ( 10.4) 22.1 ( 9.9) 22.6 ( 12.9) 0.78 Periprocedural therapy with abciximab 197 (56.6) 206 (56.9) 50 (55.6) 0.97 Diameter stenosis after stenting, % 5.1 ( 11.8) 4.7 ( 9.9) 4.7 ( 9.2) 0.88 Abbreviations as in Table 1. Data are presented as mean ( SD) or number (%) of subjects. concentration was ng/ml in patients with angiographic restenosis and ng/ml in those without angiographic restenosis (P 0.76). In addition, the relationship between homocysteine concentrations and angiographic restenosis was determined in quintiles of homocysteine. Within the quintiles, homocysteine concentrations were 3.6 to 9.1 mol/l, 9.1 to 10.8 mol/l, 10.8 to 12.5 mol/l, 12.5 to 15.0 mol/l, and 15.0 to 49.1 mol/l, and the restenosis rates in these groups were 28.8%, 20.5%, 25.0%, 24.8%, and 25.8%, respectively (P 0.68). Finally, in the uppermost decile (patients with homocysteine concentrations above the 90th percentile of 18.0 mol/l), the angiographic

5 Koch et al Homocysteine, MTHFR Polymorphisms, and Restenosis 2233 restenosis rate was 19.0%, and the clinical restenosis rate was 17.5%. Follow-up angiography was done in 262 (75.3%) of the patients with the 677CC genotype, 272 (75.1%) of the patients with the 677CT genotype, and 67 (74.4%) of the patients with the 677TT genotype (P 1). The angiographic restenosis rates were 26.0%, 23.5%, and 26.9% in carriers of the 677CC, 677CT, and 677TT genotype, respectively (P 0.75). Among patients with genotype 1298AA, 1298AC, and 1298CC, follow-up angiography was performed in 275 (74.5%), 251 (75.1%), and 75 (77.3%) subjects, respectively (P 0.98). The observed angiographic restenosis rates were 24.4%, 25.9%, and 24.0% in carriers of the 1298AA, 1298AC, and 1298CC genotype, respectively (P 0.90). A comparison of the group of patients without angiographic restenosis (n 451) with the group of patients with angiographic restenosis (n 150) showed that the proportion of each MTHFR genotype or genotype combination was not significantly different between the 2 groups (P 0.37). The independent role of homocysteine level and both polymorphisms was evaluated in a multivariate model of restenosis that included all baseline, lesion-related, and procedural characteristics displayed in Table 1 or Table 2. This model did not show any significant independent association for homocysteine (P 0.15), the 677C/T SNP (P 0.75), or the 1298A/C SNP (P 0.98). In addition, no significant interaction was observed between elevated homocysteine levels and low folate levels with respect to the risk of restenosis (probability value for interaction 0.93). Clinical Restenosis The need for restenosis-driven reintervention was 18.8% in the patient group with low homocysteine concentrations and 19.0% in the patient group with high homocysteine concentrations during the first year after stent placement (P 0.93). Patients with genotypes 677CC, 677CT, and 677TT had clinical restenosis rates of 19.5%, 17.1%, and 23.3% (P 0.37), respectively, and patients with genotypes 1298AA, 1298AC, and 1298CC had clinical restenosis rates of 17.6%, 18.6%, and 24.7% (P 0.27), respectively. One-Year Clinical Outcome The combined incidence of death and nonfatal MI was 2.5% in the group of patients with low homocysteine status and 4.5% in the group of patients with high homocysteine status (P 0.14, Cox model). Thus, a high homocysteine concentration was associated with a hazard ratio of 1.80 (95% CI 0.83 to 3.91) for the occurrence of death or MI. In the groups with the 677CC, 677CT, and 677TT genotypes, death or nonfatal MI occurred in 3.2%, 3.0%, and 6.7% of the patients, respectively (P 0.24, Cox model). The 677TT genotype was associated with a hazard ratio of 2.14 (95% CI 0.79 to 5.78) when compared with the 677CC genotype. Among patients with the 1298AA, 1298AC, and 1298CC genotypes, the rates of death or nonfatal MI were 3.5%, 3.9%, and 2.1%, respectively (P 0.71, Cox model). The 1298CC genotype was associated with a hazard ratio of 0.59 (95% CI 0.13 to 2.59) compared with the 1298AA genotype. Mortality rates were also not significantly associated with homocysteine levels or MTHFR genotypes. Discussion The results presented here strongly suggest that elevated plasma homocysteine concentrations are not associated with an increased risk of restenosis in patients treated with stenting in coronary arteries. In addition, the results provide evidence that the 677C/T SNP and the 1298A/C SNP of the MTHFR gene, either alone or in combination, are not related to in-stent restenosis. Homocysteine Levels and Restenosis After Coronary Interventions Experimental studies and clinical trials suggested a possible relationship between high homocysteine levels and restenosis after interventions in coronary arteries. 2 6,21,22 Homocysteine was found to stimulate processes critically involved in neointima formation and restenosis, including inflammatory reactions and growth of vascular smooth muscle cells. 21,22 Elevated total plasma homocysteine level was repeatedly reported to be a strong predictor of restenosis and major adverse clinical events after coronary balloon angioplasty. 2 6 In contrast to these results obtained in patients with balloon angioplasty, the present data do not suggest the existence of a relationship between homocysteine concentration and the risk of angiographic restenosis or the need for restenosisdriven reintervention after stenting in coronary arteries. In addition, the present results show that levels of total homocysteine were similar in patients with angiographic restenosis and those without angiographic restenosis. The latter finding is in line with a recent result obtained with stenting in Austrian patients and reports on patients from Canada and Israel treated with either stenting or balloon angioplasty Relationship Between MTHFR Gene Polymorphisms and Levels of Homocysteine and Folate In agreement with a number of reports, 7,11,12 we found that carriers of the 677TT genotype had significantly higher plasma homocysteine levels than carriers of the 677CC or 677CT genotype. Most likely, the association between the 677TT genotype and elevated homocysteine levels depends on the presence of a low plasma folate status. 11,12 Consistent with a previous report on patients with symptomatic atherosclerotic cerebrovascular or peripheral vascular disease, 26 the 677CC and 677TT genotype groups among the present study patients exhibited significant differences in plasma folate concentrations. With regard to the 1298A/C SNP, we observed no association with homocysteine or folate levels, which is in agreement with previous publications. 14,17 MTHFR Polymorphisms and Restenosis After Stenting A possible association between the 677C/T SNP and restenosis after stenting was suggested by the findings that the 677T allele or 677TT genotype was related to elevated homocysteine levels 7,11,12 and that increased homocysteine levels were associated with the stimulation of inflammatory

6 2234 Arterioscler Thromb Vasc Biol. December 2003 reactions and vascular smooth muscle cell proliferation. 21,22 However, although a relationship between the 677TT genotype and higher plasma homocysteine concentrations was evident in patients in the present study, a linkage between the 677C/T SNP and restenosis was not detected. In a series of 197 Australian subjects, the 677C/T SNP was not significantly related to the occurrence of angiographically documented restenosis after coronary balloon angioplasty. 27 Although this finding is in line with the present data on restenosis after stenting, the present results must not be interpreted merely as a confirmation, because fundamental differences underlie the mechanisms that lead to restenosis after balloon angioplasty and stenting. 28,29 To the best of our knowledge, the impact of the 1298A/C SNP on restenosis after coronary balloon angioplasty or stenting has not been examined previously. Study Limitations Although this is the largest study on the association between homocysteine concentration or MTHFR polymorphisms and restenosis after coronary stenting, it may not be sufficiently powered to detect subtle differences in restenosis. At a 2-sided -level of 0.05, the study has power values of 83%, 68%, 44%, and 22% to detect increases in the restenosis rate of 40%, 33%, 25%, and 20%, respectively, in the presence of a high homocysteine concentration or carriage of the 677T allele. Conclusions Elevated levels of total plasma homocysteine and 2 polymorphisms, 677C/T and 1298A/C, of the MTHFR gene were not associated with restenosis or the occurrence of other major adverse events in a series of 800 patients who underwent stenting in coronary arteries. References 1. Kastrati A, Schömig A, Elezi S, et al. Predictive factors of restenosis after coronary stent placement. J Am Coll Cardiol. 1997;30: Refsum H, Ueland PM, Nygård O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med. 1998;49: Eikelboom JW, Lonn E, Genest J Jr, Hankey G, Yusuf S. Homocyst(e)ine and cardiovascular disease: a critical review of epidemiological evidence. Ann Intern Med. 1999;131: The Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA. 2002;288: Schnyder G, Flammer Y, Roffi M, Pin R, Hess OM. Plasma homocysteine levels and late outcome after coronary angioplasty. J Am Coll Cardiol. 2002;40: Schnyder G, Roffi M, Flammer Y, Pin R, Hess OM. Association of plasma homocysteine with restenosis after percutaneous coronary angioplasty. Eur Heart J. 2002;23: Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10: Jee SH, Song KS, Shim WH, et al. Major gene evidence after MTHFRsegregation analysis of serum homocysteine in families of parents undergoing coronary arteriography. Hum Genet. 2002;111: Selhub J. Homocysteine metabolism. Annu Rev Nutr. 1999;19: Yamada K, Chen Z, Rozen R, Matthews RG. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci U S A. 2001;98: Jacques PF, Bostom AG, Williams RR, et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation. 1996;93: Klerk M, Verhoef P, Clarke R, Blom HJ, Kok FJ, Schouten EG, and the MTHFR Studies Collaboration Group. MTHFR 677C3T polymorphism and risk of coronary heart disease. JAMA. 2002;288: Kosokabe T, Okumura K, Sone T, et al. Relation of a common methylenetetrahydrofolate reductase mutation and plasma homocysteine with intimal hyperplasia after coronary stenting. Circulation. 2001;103: van der Put NMJ, Gabreëls F, Stevens EMB, et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet. 1998;62: Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998;64: Weisberg IS, Jacques PF, Selhub J, et al. The 1298A3C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis. 2001;156: Szczeklik A, Sanak M, Jankowski M, et al. Mutation A1298C of methylenetetrahydrofolate reductase: risk for early coronary disease not associated with hyperhomocysteinemia. Am J Med Genet. 2001;101: Rothenbacher D, Fischer HG, Hoffmeister A, et al. Homocysteine and methylenetetrahydrofolate reductase genotype: association with risk of coronary heart disease and relation to inflammatory, hemostatic, and lipid parameters. Atherosclerosis. 2002;162: Schömig A, Kastrati A, Mudra H, et al. Four-year experience with Palmaz-Schatz stenting in coronary angioplasty complicated by dissection with threatened or present vessel closure. Circulation. 1994;90: Schömig A, Neumann F-J, Kastrati A, et al. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronaryartery stents. N Engl J Med. 1996;334: Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med. 1998;338: Thambyrajah J, Townend JN. Homocysteine and atherothrombosis: mechanisms for injury. Eur Heart J. 2000;21: Genser D, Prachar H, Hauer R, Halbmayer W-M. Mlczoch J, Elmadfa I. Relation of homocysteine, vitamin B 12, and folate to coronary in-stent restenosis. Am J Cardiol. 2002;89: Miner SES, Hegele RA, Sparkes J, et al. Homocysteine, lipoprotein(a), and restenosis after percutaneous transluminal coronary angioplasty: a prospective study. Am Heart J. 2000;140: Hodish I, Matetzky S, Selah B-A, et al. Effect of elevated homocysteine levels on clinical restenosis following percutaneous coronary intervention. Cardiology. 2002;97: Deloughery TG, Evans A, Sadeghi A, et al. Common mutation in methylenetetrahydrofolate reductase: correlation with homocysteine metabolism and late-onset vascular disease. Circulation. 1996;94: van Bockxmeer FM, Mamotte CDS, Vasikaran SD, Taylor RR. Methylenetetrahydrofolate reductase gene and coronary artery disease. Circulation. 1997;95: Hoffmann R, Mintz GS, Dussaillant GR, et al. Patterns and mechanisms of in-stent restenosis: a serial intravascular ultrasound study. Circulation. 1996;94: Mintz GS, Popma JJ, Pichard AD, et al. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation. 1996;94:35 43.