a Human Genetics Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, UK
b MRC Environmental Epidemiology Unit (University of Southampton), Southampton General Hospital, Southampton SO16 6YD, UK
* Corresponding author. Dr C. R. Gale, University of Southampton, MRC Environmental Epidemiology Unit, Southampton General Hospital, Southampton, Hants SO16 6YD, UK. Tel.: +44-23-80764080; fax: +44-23-80704021. Address for reprints: Dr S Ye, Human Genetics Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, UK.
E-mail address: crg{at}mrc.soton.ac.uk
Received 15 October 2002; revised 12 June 2003; accepted 2 July 2003
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Abstract |
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Methods and results We genotyped 471 Caucasian men and women, aged 6675 years, from Sheffield, UK, for the 1G/2G polymorphism in the MMP-1 gene and the 5A/6A polymorphism in the MMP-3 gene and ascertained the prevalence of coronary heart disease. People homozygous for the more transcriptionally active 2G allele of the MMP-1 gene had a reduced risk of coronary heart disease (OR 0.5, 95% CI 0.3 to 0.9) compared to people homozygous for the less transcriptionally active 1G allele. Heterozygotes had an intermediate risk (OR 0.7, 95% CI, 0.5 to 1.1). We found no association between the 5A/6A polymorphism in the MMP-3 gene and risk of coronary heart disease.
Conclusion Sequence variants at the MMP-1 genomic locus may influence risk of coronary heart disease in humans.
Key Words: Coronary heart disease Matrix metalloproteinase-1 Matrix metalloproteinase-3 Gene expression Polymorphism Epidemiology
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1. Introduction |
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Recent data from animal experiments suggest that MMP-1 can prevent or delay the progression of atherosclerotic plaques.2The apoE knockout mouse rapidly develops atherosclerotic lesions similar to those found in humans when fed a high cholesterol, Western-type diet. Insertion of a human MMP-1 transgene into such mice (wild-type mice do not possess a gene homologous to human MMP-1) reduces both the severity and extent of aortic atheromatous lesions.
A functional polymorphism is present in the promoter region of the human MMP-1 gene.3
The two alleles have either one (1G) or two (2G) guanine necleotides at position1607 relative to the transcriptional start site of the MMP-1 gene. It has been shown that the 2G allelic promoter of the MMP-1 gene has over 20 fold higher transcriptional activity than the 1G allelic promoter and is associated with elevated MMP-1 mRNA levels in ovarian carcinomas.4
We examined the MMP-1 gene polymorphism in a group of British Caucasian subjects and investigated whether they were related to the prevalence of coronary heart disease. In addition, we investigated whether the risk of coronary heart disease was associated with a MMP-3 gene variant, referred to as the 5A/6A polymorphism which had previously been reported to be associated with progression of coronary atherosclerosis.5,6
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2. Methods |
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Participants were interviewed by a fieldworker who administered the Rose/WHO Cardiovascular Questionnaire andenquired about history of cardiovascular disease, smoking habits and current medication. She also measured height, weight and blood pressure, recorded a 12-lead electrocardiogram and took a fasting venous blood sample for measurement of total cholesterol and MMP-1 and MMP-3 genotyping. Samples were stored at 80oC for later analysis. The methods used to determine genotypes for the MMP-1 1G/2G polymorphism and the MMP-3 5A/6A polymorphism have been described.7,8In brief, using subjects genomic DNA as template, PCR reactions were carried out to amplify the DNA sequence containing the polymorphism. The PCR products were then subjected to cleavage by restriction endonuclease Xmn I which cuts the 1G allele of the MMP-1 gene and the 5A allele of the MMP-3 gene. The digests were fractionated by non-denaturing polyacrylamide gel electrophoresis and subsequently the DNA bands were visualized by Vistra Green (Amersham) staining.
We tested that the allele frequencies conformed to Hardy-Weinberg equilibrium proportions by use of the 2test. We used ANOVA and the
2test to examine the relation between polymorphism of the MMP-1 gene and cardiovascular risk factors. Logistic regression was used to examine the relation between MMP-1 and MMP-3 genotype and the presence of coronary heart disease, defined as the presence of one or more of the following: angina according to the Rose/WHO Cardiovascular Questionnaire, Minnesota codes 1-1, 1-2 (Q and QS codes) on an electrocardiogram or a history of coronary-artery bypass grafting or coronary angioplasty. In the multivariate logistic regression analyses we adjusted for age, gender, body mass index, smoking habit, total cholesterol concentration, pulse pressure, and use of medication to treat hypertension, diabetes or hyperlipidaemia. We also examined whether there were any interactions between MMP-1 and MMP-3 polymorphisms and smoking habit, body mass index, total cholesterol concentration or pulse pressure.
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3. Results |
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Risk of coronary heart disease was reduced in men and women who were homozygous for the 2G allele of the MMP-1 gene compared to those who were homozygous for the 1G allele (OR 0.6, 95% CI 0.3 to 0.9 (Table 2). Heterozygotes had an intermediate risk (OR 0.7, 95% CI 0.5 to 1.1). This relation remained statistically significant after adjustment for age, gender and other cardiovascular risk factors. We examined whether the relation between coronary heart disease and MMP-1 polymorphisms varied according to smoking habit, blood pressure, body mass index or total cholesterol concentration, but there were no statistically significant interactions. We repeated the analysis using a more rigorous definition of coronary heart disease, i.e. Minnesota codes 1-1, 1-2 (Q and QS codes) on an electrocardiogram or a history of coronary-artery bypass grafting or coronary angioplasty. Risk estimates changed little: the odds ratio for coronary heart disease in men and women who were homozygous for the 2G allele of the MMP-1 gene compared to those who were homozygous for the 1G allele was 0.5, 95% CI 0.3 to 0.9, after multivariate adjustment.
We found no statistically significant association between the MMP-3 polymorphism and risk of coronary heart disease, either in univariate analysis or after adjustment for other risk factors (Table 2). Since a previous study showed an additive effect of the MMP-1 gene 2G allele and the MMP-3 6A allele on carotid atherosclerosis,9we tested whether there was an additive effect on these two alleles on risk of coronary heart disease. No such effect was detected in this study. In addition, as in vitro data show that the 5A allele of the MMP-3 gene and the 2G allele of the MMP-1 gene have higher promoter activity,3,5we examined whether homozygosity for both these alleles was associated with a lower risk of coronary heart disease. We found no evidence of a reduction in risk (OR 0.9, 95% CI 0.2 to 3.1), but only 10 participants had this combination of alleles.
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4. Discussion |
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Some studies have shown an association between progression of atherosclerosis and a functional polymorphism in the promoter of the gene encoding MMP-3, another important matrix-degrading enzyme, such that lesion progression is more rapid in individuals who are homozygous for the transcriptionally less active 6A allele than those who carry the more active 5A allele.5,6Genetic factors can be classed into disease susceptibility genes and disease modifying genes. The former may contribute to the initiation of the disease process, whereas the latter may influence the progression and outcome of the disease after it has been initiated.12The finding of an association between the MMP-3 gene 5A/6A polymorphism and progression of atherosclerosis in previous studies suggest that this genetic variant can exert a disease modifying effect. Whether this genetic variant can also influence disease susceptibility is not known and this was examined in the present study. In the sample studied, we did not find an association between this genetic variant and risk of coronary heart disease.
There is accumulating evidence that MMPs play diverse roles in atherosclerosis.13Although imbalanced MMP-1 activity in advanced atheroma may be one of the factors that contribute to plaque instability, we speculate that increased MMP-1 expression at the earlier stages of atherogenesis may be a beneficial response to collagen accumulation and help deter lesion progression.
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Acknowledgments |
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References |
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