Published online before print October 5, 2006, doi: 10.1161/01.STR.0000244779.30070.1a
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(Stroke. 2006;37:2667.)
© 2006 American Heart Association, Inc.
Original Contributions |
TGF-ß1 Polymorphisms and Risk of Myocardial Infarction and Stroke
The Rotterdam Study
From the Departments of Epidemiology and Biostatistics (M.P.S.S., A.G.U., M.J.B., M.M.B.B., H.A.P.P., A.H., C.M.v.D., J.C.M.W.), Internal Medicine (M.P.S.S., A.G.U., P.P.A., H.A.P.P.), and Neurology (M.J.B., P.J.K.), Erasmus Medical Center, Rotterdam, The Netherlands.
Correspondence to J.C.M. Witteman, PhD, Department of Epidemiology and Biostatistics, Erasmus Medical Center, Rotterdam, PO Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail j.witteman{at}erasmusmc.nl
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Abstract |
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Background and Purpose— Inflammation plays a pivotal role in the pathogenesis of atherosclerosis and of cardiovascular and cerebrovascular complications. Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic cytokine with a central role in inflammation. Little is known of the relation of variations within the gene and risk of cardiovascular and cerebrovascular disease. We therefore investigated 5 polymorphisms in the TGF-ß1 gene (–800 G/A, –509 C/T, codon 10 Leu/Pro, codon 25 Arg/Pro, and codon 263 Thr/Ile) in relation to the risk of myocardial infarction and stroke in a population-based study.
Methods— Participants (N=6456) of the Rotterdam Study were included in the current study. Analyses of the relations of genotypes with the risk of myocardial infarction and stroke were performed according to Cox proportional-hazards methods. All analyses were adjusted for age, sex, conventional cardiovascular risk factors, and medical history.
Results— We found no association with the risk of myocardial infarction. A significantly increased risk of stroke was found, associated with the T allele of the –509 C/T polymorphism (relative risk, 1.26; (95% CI, 1.06 to 1.49) and the Pro variant of the codon 10 polymorphism (relative risk, 1.24; 95% CI, 1.04 to 1.48).
Conclusions— No association between the TGF-ß1 polymorphisms and myocardial infarction was observed; however, the –509 C/T and codon 10 Leu/Pro polymorphisms were associated with the risk of stroke.
Key Words: myocardial infarction • polymorphisms • stroke • transforming growth factor-ß
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Introduction |
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Inflammation is an essential process in the pathogenesis of atherosclerosis and consequently, of coronary heart disease (CHD) and cerebrovascular disease.1,2 Inflammation is influenced by many different cytokines, such as transforming growth factor-ß1 (TGF-ß1), the most common variant of 3 isoforms.3 TGF-ß has many different functions, both proatherogenic and antiatherogenic. Some consider the overall effect of TGF-ß to be protective, by reducing the risk of cardiovascular and cerebrovascular diseases.4–10 Others describe TGF-ß as inducing or facilitating cardiovascular and cerebrovascular pathological states, such as vascular stenosis and thrombogenesis.11–16
The TGF-ß1 gene is located on chromosome 19q13.2. There are several commonly known (potentially) functional polymorphisms in this gene. Cambien et al17 described the –988 C/A, –800 G/A, and –509 C/T polymorphisms (all in the promoter region); a C insertion at position +72 (in the nontranslated region); and codons 10 Leu/Pro (c10) and 25 Arg/Pro (c25) (signal peptide sequence) and 263 Thr/Ile (c263) (in the precursor part of the protein). Strong linkage disequilibrium between the polymorphisms was described.17,18 The +72 form was in almost complete linkage disequilibrium with c25, whereas –988 C/A was extremely rare.17 Grainger et al19 described the –509 C/T polymorphism as associated with levels of TGF-ß1.
The c25 polymorphism has been associated with cardiovascular disease in several studies, as was the c10 polymorphism.17,20–22 However, other studies have reported no association with cardiovascular disease.17,18,20,23 To our knowledge, none of the polymorphisms was ever studied in relation to the risk of stroke in a general population. We therefore studied the TGF-ß1 –800 G/A, –509 C/T, c10, c25, and c263 polymorphisms in relation to the risk of myocardial infarction and stroke in a large population-based study.
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Subjects and Methods |
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Study Population
The Rotterdam Study is an ongoing, prospective, cohort study including 7983 participants aged 55 years and older. Its general aims are to investigate the determinants of chronic diseases.24 During the first phase (1990 to 1993), all inhabitants of the Rotterdam suburban area (Ommoord) aged 55 years and older were invited to participate. Baseline investigations included an interview and visits to the research center, where a varied number of measurements were performed. Approval of the medical ethics committee of Erasmus University Rotterdam was obtained for the Rotterdam Study. Written, informed consent was acquired from all participants. An in-depth description of the Rotterdam Study has already been published in an earlier report.24
Clinical Characteristics
Skilled investigators collected information with use of a computerized questionnaire. The information included current health status, medical history, and drug and smoking behavior. Blood samples were obtained, and established cardiovascular risk factors were measured as described elsewhere.25 Diabetes mellitus was defined as a nonfasting/postload serum glucose level of 
11.1 mmol/L and/or use of antidiabetic medications. A 12-lead ECG was recorded and analyzed with use of the modular ECG analysis system.26 A diagnosis of atrial fibrillation was based on ECG data and/or confirmation by a subject’s general practitioner (GP).
Follow-Up Procedures and Definition of Events
GPs in the research district, with whom 85% of the participants were enlisted, reported fatal/nonfatal cardiovascular events. Research assistants verified all information by checking medical records at the GPs’ offices. All medical records of the participants under the care of GPs outside the study area were checked annually. Letters and discharge reports from medical specialists were obtained. Information on vital status of the participants was obtained regularly from the municipal health authorities in Rotterdam. After notification, the cause and circumstances of death were established by a questionnaire from the GPs. Two research physicians independently coded all reported cardiovascular events, according to the International Classification of Diseases, 10th edition (World Health Organization, 1992).27 Codes on which the research physicians disagreed were discussed to reach consensus. Finally, a medical expert in cardiovascular disease, whose judgment was considered final, reviewed all events. Incident myocardial infarction (MI) was defined as the occurrence of a fatal or nonfatal MI (International Classification of Diseases-10 code I21) after the baseline examination. Medical records of subjects with a history of stroke were verified. For reported events, additional information was obtained from hospital records. Information on all potential strokes and transient ischemic attacks were reviewed by both a research physician and an experienced stroke neurologist (P.J.K.) to verify all diagnoses. Subarachnoid hemorrhages and retinal strokes were excluded. Stroke was subclassified as ischemic when a CT or MRI scan, made within 4 weeks after the stroke occurred, ruled out other diagnoses or when indirect evidence (deficit limited to 1 limb or completely resolved within 72 hours; atrial fibrillation in the absence of anticoagulants) suggested an ischemic nature of the stroke. A stroke was subclassified as hemorrhagic when a relevant hemorrhage was shown on the CT or MRI scan or when the subject lost consciousness permanently or died within hours after onset of focal signs. When a stroke could not be subclassified, it was called unspecified.
Genotyping
Genotyping of the TGF-ß1 polymorphisms (–800 G/A [rs1800468]; –509 C/T [rs1800469]; codon 10 Leu/Pro [T/C, rs1982073]; codon 25 Arg/Pro [G/C, rs1800471]; and codon 263 Thr/Ile [C/T, rs1800472]) was performed, regardless of disease status, on blood samples that had been acquired by venepuncture and stored at –80°C. DNA was isolated according to standard procedures. Genotypes were determined in 2-ng genomic DNA samples with the Taqman allelic discrimination assay (Applied Biosystems). Primer and probe sequences were optimized by using the single-nucleotide polymorphism assay-by-design service of Applied Biosystems (for details, see http://store.appliedbiosystems.com). Reactions were performed with the Taqman Prism 7900HT 384-well format in a 2-µL reaction volume. The polymorphisms were selected on the basis of (potential) functionality (–800 G/A and –509 C/T located in promotor and codons 10, 25, and 263, resulting in an amino acid change) and reports in the literature, thus making comparisons and replication feasible.
Measurement of IL-6 and CRP Plasma Levels
Levels of interleukin (IL)-6 and C-reactive protein (CRP) were determined in samples obtained at baseline. These methods have been described previously.28
Population for Analysis
The Rotterdam Study comprises 7983 subjects. The current study included participants on the basis of the largest, successfully genotyped group, ie, 6456 participants (for the –800 G/A polymorphism). Of these, 6392 subjects were successfully genotyped for the –509 C/T polymorphism, and 6187, for codons 10, 25 and 263.
Statistical Analyses
2 tests were performed to test for deviations from Hardy-Weinberg equilibrium. Missing data were imputed from expectation-maximization algorithms. Baseline characteristics were tested by ANOVA and logistic-regression analyses adjusted for age and sex. To correct for outliers in serum measurements, all values above the mean+3SD were excluded. Natural-log–transformed values of IL-6 and CRP levels were used to normalize the distribution of these variables. Cox proportional-hazards analyses were performed to obtain relative risks (RRs). All analyses were adjusted for age and sex and additionally for body mass index, systolic blood pressure, HDL and total cholesterol levels, baseline smoking, and diabetes mellitus. Additional adjustment was also made for a history of MI/stroke/atrial fibrillation or with the exclusion of subjects with a history of MI/stroke. A P value of 
0.05 was considered significant. Statistical analyses were performed with SPSS version 11.0.1 for MS Windows.
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Results |
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A total of 6456 participants were included. Baseline characteristics are described in Table 1. Few and small differences in clinical characteristics between genotypes were found (supplemental Table I
, available online at http://stroke.ahajournals.org). During a mean±SD follow-up of 9.2±3.0 years, 358 incident cases of MI and 540 incident cases of stroke, of which 312 strokes were ischemic strokes and 51 were hemorrhagic, were identified. At baseline, 756 (12%) subjects had a history of MI, and 191 (3%) had a history of stroke.
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Genotyping of the TGF-ß1 polymorphisms was performed for all 5 polymorphisms: –800 G/A (n=6456), –509 C/T (n=6392), c10 (n=6187), c25 (n=6187), and c263 (n=6187). All genotype and allele proportions were in Hardy-Weinberg equilibrium, except for c263 (P=0.00078), which was not included in the analyses. For any of the 4 analyzed polymorphisms, no significant associations with risk of MI were found when we compared individuals heterozygous and homozygous for the risk allele with those with the wild-type genotype (Table 2). Also, when we compared homozygotes versus nonhomozygotes and carriers versus noncarriers, no evidence for an association with the risk of MI was found.
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Subjects with the –509 CT genotype had a significantly increased risk of stroke compared with those with the wild-type genotype (CC; RR, 1.27; 95% CI, 1.06 to 1.51; P=0.01) (Table 3). Also, T-allele carriers had a significantly increased RR of 1.26 (95% CI, 1.06 to 1.49; P=0.01) compared with noncarriers. For ischemic stroke, we found an increased risk for subjects with the CT genotype compared with the wild-type CC (RR, 1.29; 95% CI, 1.02 to 1.63; P=0.03) and for T-allele carriers in comparison with noncarriers (RR, 1.29; 95% CI, 1.03 to 1.61; P=0.03) (Table 4).
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For the c10 polymorphism, a significantly increased risk of stroke of 1.24 (95% CI, 1.03 to 1.50; P=0.03) for subjects with the Leu/Pro genotype was found, compared with the wild-type genotype (Leu/Leu) (Table 3). Pro carriers also were at increased risk when compared with noncarriers: RR=1.24 (95% CI, 1.04 to 1.48; P=0.02). For ischemic stroke, a consistent but nonsignificantly increased risk was observed for subjects with the Leu/Pro genotype and the Pro/Pro genotype, as well as for Pro carriers (Table 4). The –800 G/A and c25 polymorphisms were not associated with the risk of (ischemic) stroke (Tables 3 and 4).
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All analyses were adjusted for age and sex. Further adjustments for body mass index, systolic blood pressure, HDL and total cholesterol levels, smoking, and diabetes mellitus did not essentially change our estimates (data not shown). Additional analyses, either with adjustment for a history of cardiovascular disease or with exclusion of subjects with a history of cardiovascular disease, yielded essentially similar results (data not shown). Separate analyses for men and women yielded no consistent results different from the overall analyses (data not shown).
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Discussion |
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In this prospective, population-based study, we found no significant association between 4 common TGF-ß1 polymorphisms and the risk of MI. We did find, however, an increased risk of stroke associated with the risk alleles of the –509 C/T and c10 Leu/Pro polymorphisms.
To date, few studies have been published on the association of the TGF-ß1 polymorphisms –800 G/A, –509 C/T, c10, and c25 and the risk of MI. To our knowledge, there are no previous studies on the association of these polymorphisms with risk of stroke in a general population. The TGF-ß polymorphisms have been studied before in relation to CHD (supplemental Table II, available online at http://stroke.ahajournals.org); the c25 polymorphism was associated with a risk of CHD in several larger and smaller studies, as was the c10 polymorphism.17,20–22 However, other studies reported no associations with risk of CHD.17,18,20,23 Overall, the sparse results available are inconsistent.
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TGF-ß is a pleiotropic cytokine with a diversity of effects, with both proatherogenic and antiatherogenic effects. It is not known yet what the overall effect of TGF-ß is on atherogenesis and its associated morbidity. Some consider an adequate level of TGF-ß to be protective, by reducing the risk of cardiovascular and cerebrovascular diseases.4–10 Indeed, Cipollone et al29 postulated a stabilizing effect of increased expression of TGF-ß on atherosclerotic plaques. Others assume that high levels are the cause of adverse events, inducing or facilitating cardiovascular and cerebrovascular pathological states, such as vascular stenosis and thrombogenesis.11–16
The –800 G/A and –509 C/T polymorphisms are located in the promoter region. Their precise effect is still unknown, but owing to their location, they are considered possible modulators of expression of the TGF-ß gene and levels.17,19 The c10 and c25 polymorphisms are located in the signal peptide sequence; this sequence is involved in the export of synthesized proteins across membranes of the endoplasmic reticulum.17 They are also located at potentially important positions that influence activation of the TGF-ß protein.18 We found an association with increased risk of (ischemic) stroke for the –509 C/T and c10 Leu/Pro polymorphisms. For c10 Leu/Pro, the findings in the overall stroke group were somewhat stronger than in the ischemic stroke group; we have no explanation for this result. Overall, the associations between the polymorphisms and (ischemic) stroke may very well be attributable to changes in the expression or activity of TGF-ß. It is unclear why similar effects on risk of MI were not observed.
Our study was based on a large, ongoing, population-based study in a relatively homogeneous population, because 98% of the participants are white and living in the same area. In contrast to case-control studies, the prospective nature of our study makes our results less prone to survival bias. We adjusted for common cardiovascular risk factors, including a history of cardiovascular disease. Because of high linkage disequilibrium between the 4 polymorphisms (D’0.97; data not shown), we did not use haplotypes or haplotype-based analyses. Unfortunately, no levels of TGF-ß were determined. Therefore, we were unable to elucidate the effect of the polymorphisms on TGF-ß levels. Despite the fact that the Rotterdam Study is a large study, small effects may have been missed because of the limited number of cases.
In conclusion, we found no association between the TGF-ß1 –800 G/A, –509 C/T, c10, and c25 polymorphisms and risk of MI. We observed, however, a significant but small association between the –509 C/T and c10 Leu/Pro polymorphisms and risk of stroke. These results warrant further studies, with larger numbers, to consolidate these findings, to investigate the functional effects of these polymorphisms, and to study the underlying pathophysiological mechanism. Only after replication in other studies can the implications of these findings be discussed.
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Acknowledgments |
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Sources of Funding
This study was supported by NWO (Netherlands Organization for Scientific Research) grant No. 904-61-196, the Center for Medical Systems Biology, and European Commission grant QLK6-CT-2002-02629 (GENOMOS).
Disclosures
None.
Received May 16, 2006; accepted July 18, 2006.
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