Haplotype Effect of the Matrix Metalloproteinase-1 Gene on Risk of Myocardial Infarction
http://www.100md.com
循环研究杂志 2005年第11期
The Human Genetics Division (E.P., A.R.M., C.C., S.Y.), School of Medicine, University of Southampton, United Kingdom
INSERM U525 (D.-A.T), Facultee de Meedecine, Hpital Pitiee-Salpe簍rieere, Paris, France
Atherosclerosis Research Unit (A.S., A.H., P.E.), King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden.
Abstract
Myocardial infarction (MI) is commonly caused by atherosclerotic plaque rupture following excessive degradation of collagen fibers in the atherosclerotic lesion. We investigated whether interindividual variability in risk of MI was related to polymorphisms in the gene encoding matrix metalloproteinase (MMP)-1, a key fibrillar collageneCdegrading enzyme. Several single nucleotide polymorphisms in the MMP1 gene promoter were identified following sequencing DNA samples from 30 individuals. An analysis of the polymorphisms in a cohort of British whites with coronary atherosclerosis, including 639 patients with MI and 538 non-MI subjects, revealed a haplotype effect of the eC519A>G and eC340T>C polymorphisms on risk of MI, with the AeC519-CeC340 and GeC519-TeC340 haplotypes being protective (odds ratio=0.70 [0.57 to 0.86]; P=0.0007), whereas the GeC519-CeC340 haplotype increased MI risk (odds ratio=1.94 [1.15 to 3.28]; P=0.013). This finding was replicated in a subsequent analysis of 387 Swedish MI patients and 387 healthy controls (odds ratio=0.70 [0.55 to 0.89], P=0.003, for AeC519-CeC340 and GeC519-TeC340; odds ratio=1.54 [0.97 to 2.46], P=0.07, for GeC519-CeC340). In vitro assays showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes had lower promoter activity, whereas the GeC519-CeC340 haplotype had greater promoter strength, in driving gene expression in human macrophages. Haplotype-specific differences in MMP1 mRNA level in atherosclerotic tissues were also detected. The data indicate that MMP1 gene variation is a genetic factor contributing to interindividual differences in MI risk.
Key Words: matrix metalloproteinase genetics polymorphism haplotype analysis atherosclerosis
Introduction
Myocardial infarction (MI) is predominately caused by coronary atherosclerotic plaque rupture, a mechanical breakdown of the fibrous cap overlaying the lipid core of the plaque.1,2 Rupture of the plaque results in exposure of thrombogenic material (eg, tissue factor and lipids) located in the lipid core to the blood and leads to thrombus formation and, consequently, to the ischemic event.1,2 The biomechanical strength of the fibrous cap largely depends on its content of fibrillar collagens, type I and III in particular, which are cross-linked to provide structural support.3 Excessive degradation of these collagen fibers has been recognized as an important mechanism underlying atherosclerotic plaque rupture.4eC6
Matrix metalloproteinase (MMP)-1 (or interstitial collagenase) can degrade fibrillar collagens, especially types I and III, which are resistant to most proteinases.7,8 Pathological studies have shown that MMP1 is expressed by macrophages in atherosclerotic plaques and colocalizes with degraded fibrillar collagens in the shoulder region, which is prone to rupture.9eC11 MMP1 expression is significantly increased in vulnerable atherosclerotic plaques, characterized by having a thin fibrous cap overlaying a large lipid core, compared with stable atherosclerotic plaques, characterized by having a thick fibrous cap.11,12 Thus, it has been postulated that increased MMP1 expression promotes atherosclerotic plaque rupture.11
In this study, we investigated whether interindividual variability in risk of MI was related to MMP1 gene polymorphisms that might influence MMP1 expression or activity. We first performed a search for polymorphisms in this gene by sequencing DNA samples from a group of unrelated individuals. The polymorphisms were then studied in relation to MI in a cohort of coronary artery disease patients. The findings were verified in an independent sample from another population. We then performed functional analyses of the polymorphisms found to be associated with MI risk.
Subjects and Methods
Identification of MMP1 Gene Variants
The MMP1 gene promoter (1.9 kb), all 10 exons, and the introneCexon junctions were amplified by PCR using primers described in Table I in the online data supplement available at http://circres.ahajournals.org. The PCR amplicons were sequenced using ABI sequencers and BigDye terminator cycle sequencing reagents. The PCR and sequencing were performed using DNA templates from 30 unrelated whites, which provided >90% power to detect sequence polymorphisms with an allele frequency of >5%.13
Subjects in Studies of MMP1 Gene Polymorphisms and Haplotypes in Relation to MI Risk
Analyses of MMP1 polymorphisms and haplotypes in relation to MI risk were performed in 2 population samples. The first was a cohort of British whites with coronary artery disease, documented angiographically as having >50% diameter stenosis in at least 1 coronary artery, as described previously.14 It consisted of 2 groups, namely 639 patients who had experienced an MI and 538 non-MI subjects. The characteristics of the 2 groups are summarized in Table 1. The second sample consisted of 387 Swedish patients with an MI at the age of <60 years and 387 gender- and age-matched healthy Swedish individuals participating in a program aimed at identifying novel biomarkers and susceptibility genes for MI. The characteristics of the 2 groups are summarized in Table 1 and have been described in detail elsewhere.15 In both studies, MI was diagnosed according to standard clinical criteria, including electrocardiographic and/or enzymatic changes. Both studies were approved by the respective local research ethics committees, and all subjects gave informed consent.
Determination of Genotypes
For each polymorphism, a DNA sequence containing the polymorphic site was amplified by PCR using primers described in supplemental Table I. The amplicons were digested with an appropriate restriction endonuclease that specifically cleaved 1 of the 2 alleles. The restriction endonucleases used were XmnI (for eC1607GG>G), HindIII (for eC839G>A), MspI (for eC755G>T), KpnI (for eC519A>G), BanI (for eC422T>A), AflII (for eC340T>C), and HaeIII (for eC320T>C), respectively. Digests were subjected to gel electrophoresis, and DNA was detected by poststaining of the gel with Vistra Green and visualized using a fluorimager to determine genotypes.
Statistical Analyses of MMP1 Polymorphisms and Haplotypes in Relation to MI Risk
Allele and genotype frequencies were calculated by gene counting. Analyses of MMP1 polymorphisms in relation to MI were performed, first, by examining the polymorphisms individually and then, second, by studying haplotypes. Individual polymorphisms in relation to MI were examined by 2 analysis. In the haplotype analyses, a systematic analysis of all possible combinations of 1 to k polymorphisms to select the most informative and parsimonious haplotype configuration in terms of predicting disease status was performed using a previously described method that calculates the Akaike’s Information Criterion (AIC) values for each haplotype model and then subtracts the minimum AIC value obtained for each model over all models explored, giving a rescaled AIC value for each haplotype model.16 As described previously,16 the models with a rescaled AIC 2 are considered equivalent to the most informative model, and among these models, the most parsimonious model with the fewest polymorphisms is considered the best model. Because the choice of models is based on AIC values, it circumvents problems associated with methods based on null-hypothesis testing, such as the requirement of multiple testing correction. After identifying the best model, the haplotype effects of the polymorphisms in this model on MI risk were analyzed using the THESIAS program (www.genecanvas.org), which implements the stochastic-EM (ExpectationeCMaximization) algorithm.17 Haplotype effects on MI risk were adjusted for age, gender, body mass index, smoking, cholesterol levels, statin treatment, hypertension, and diabetes.
Transient Transfection and Reporter Assays
Transient transfection and reporter assays were performed to compare the strengths of MMP1 promoter of different haplotypes in driving gene expression. For each haplotype, the corresponding MMP1 promoter (from eC1870 bp to +42 bp relative to the transcriptional start site) was generated by PCR using genomic DNA as template and then cloned into a TOPO PCR cloning vector (Invitrogen). The cloned MMP1 promoter was sequenced to verify that there was no misincorporation during PCR and then subcloned into a plasmid (pGL3-Basic Vector; Promega) containing a firefly luciferase reporter gene. The resultant construct was mixed with a plasmid (pRL-TK, Promega) containing a renilla luciferase gene under the control of a thymidine kinase promoter and transferred into cultured THP-1 human monocytic cells using the lipofection method with FuGENE 6 transfection reagent. The transfected cells were treated with phorbol 12-myristate 13-acetate to induce differentiation of the cells into macrophages. At 36 hours after transfection, the cells were lysed and the activities of the firefly luciferase and renilla luciferase in the lysates measured with the use of a dual-luciferase assay kit (Promega). The ratio of firefly luciferase level to renilla luciferase level was used as a measure of the MMP1 gene promoter activity. Three independent experiments in duplicate were performed for each construct.
Electrophoretic Mobility-Shift Assays
For each allele, a double-stranded oligonucleotide corresponding to the sequence at and surrounding the polymorphic site was generated and used as a probe. The sequences of these probes are described in supplemental Table I. Each probe was labeled with 32P at the 5' terminus. The labeled probe was incubated with nuclear protein extracts from cultured human macrophages derived from THP-1 cells, followed by nondenaturing polyacrylamide gel electrophoresis and autoradiography, as described.18,19 Three independent experiments were performed for each polymorphism.
Real-Time RT-PCR
RNA was extracted from atherosclerotic plaques removed from patients (n=26) undergoing carotid endarterectomy and reverse transcribed to cDNA. Real-time PCR was performed in duplicates in an ABI Prism 7700 Sequence Detection System. PCR primers (described in supplemental Table I) were designed with the use of the Primer Express program (Applied Biosystems), with the forward and reserve primers placed in different exons. The MMP1 real-time RT-PCR results of different samples were standardized for the amounts of RNA template and efficiencies of reverse transcription using the housekeeping gene 36B4 (acidic ribosomal phosphoprotein PO subtype) as a reference20 and applying the 2eCCT method.21 The haplotype effects of the MMP1 eC519A>G and eC314C>T polymorphisms on MMP1 mRNA levels were analyzed using the THESIAS program, adjusting for statin treatment and atherosclerotic plaque types defined according to the revised American Heart Association classification system,22 which takes into account macrophage density, smooth muscle cell density, the size of lipid core, and the proportion of fibrous tissue.
Results
Identification of Polymorphisms in the MMP1 Gene
To search for naturally occurring, common sequence variants of the MMP1 gene, we sequenced the promoter, exons, and introneCexon junctions of the gene in 30 unrelated white individuals. In the promoter region, single-nucleotide polymorphisms were identified at positions eC1607 (GG/G, ie, G insertion/deletion), eC839 (G/A), eC755 (G/T), eC519 (A/G), eC422 (T/A), eC340 (T/C), and eC320 (T/C) (Figure 1A). Two of these 7 promoter polymorphisms, namely eC519A>G and eC340T>C, were found to be located in regions conserved in human, mouse, and rat (Figure 1B). In the coding region, only 2 polymorphisms were identified, neither of which changes the amino acid sequence. The subsequent studies were focused on the promoter polymorphisms.
MMP1 Haplotype Effects on Risk of MI
To investigate whether there was a genotypic or haplotypic effect of MMP1 on MI risk, we first studied the polymorphisms in a sample of British whites with hemodynamically significant coronary atherosclerosis, consisting of 639 patients who had experienced an MI and 538 non-MI subjects. Frequencies of alleles, genotypes, and haplotypes derived from the 7 polymorphisms are shown in supplemental Table II. No significant difference in genotype distribution of individual polymorphisms between the 2 groups was detected.
Data of a systematic analysis of all possible combinations of the polymorphisms in relation to MI are presented in supplemental Table III. The analysis showed that the best model in terms of predicting MI status was 1 that consisted of the eC519A>G and eC340T>C polymorphisms. An analysis of haplotypes based on these 2 polymorphisms showed that the haplotype frequencies were significantly different between the MI and non-MI groups (2=16.48 with 3 degrees of freedom, P<10eC3; Table 2). Compared with the AeC519-TeC340 haplotype, both the AeC519-CeC340 and GeC519-TeC340 haplotypes conferred a protective effect against MI (odds ratio [OR]=0.68 [0.52 to 0.88], P=0.004, for AeC519-CeC340; OR=0.71 [0.56 to 0.89], P=0.003, for GeC519-TeC340; the common OR associated with these 2 haplotypes being OR=0.70 [0.57 to 0.86], P=0.0007; Table 2), whereas the GeC519-CeC340 haplotype was associated with an increased risk of MI (OR=1.94 [1.15 to 3.28], P=0.013; Table 2).
To verify the findings above, a Swedish sample consisting of 387 MI patients and 387 healthy controls was genotyped for the polymorphisms. As in the British study, the Swedish study showed a protective effect of the AeC519-CeC340 and GeC519-TeC340 haplotypes (OR=0.72 [0.54 to 0.94], P=0.02, for AeC519-CeC340 versus AeC519-TeC340; OR=0.68 [0.52 to 0.89], P=0.005, for GeC519-TeC340 versus AeC519-TeC340; and OR=0.70 [0.55 to 0.89], P=0.003, for AeC519-CeC340+GeC519-TeC340 versus AeC519-TeC340; Table 2) and an increased risk for the GeC519-CeC340 haplotype (OR=1.54 [0.97 to 2.46], P=0.07, for GeC519-CeC340 versus AeC519-TeC340; Table 2).
Effects of MMP1 Polymorphisms on Promoter Activity in Driving Gene Expression
To investigate whether the eC519A>G and eC340T>C polymorphisms had a functional effect, we examined the promoter activity of MMP1 haplotypes in driving the expression of a luciferase reporter gene in transiently transfected THP-1 celleCderived human macrophages. Compared with the haplotypes encompassing both the AeC519 and TeC340 alleles, the haplotypes encompassing both the AeC519 and CeC340 alleles and those encompassing both the GeC519 and TeC340 alleles had lower promoter activity (P<0.01), whereas the haplotype containing both the GeC519 and CeC340 alleles had higher promoter activity (Table 3).
Effects of MMP1 Polymorphisms on Nuclear Protein Binding to the MMP1 Promoter
To investigate whether the eC519A>G and eC340T>C polymorphisms were located in DNA sequences recognized by nuclear proteins, and if so, whether the polymorphisms affected the DNA-protein interactions, we performed electrophoretic mobility shift assays with oligonucleotide probes corresponding to the different alleles and nuclear protein extracts from THP-1 human macrophages. For the eC519A>G polymorphism, a DNAeCprotein complex (indicated by an arrow in Figure 2A) was detected in assays using a probe corresponding to the AeC519 allele but not in assays using a probe corresponding to the GeC519 allele. For the eC340T>C polymorphism, a DNAeCprotein complex (indicated by an arrow in Figure 2B) was detected in assays using a probe corresponding to the CeC340 allele but not in assays using a probe corresponding to the TeC340 allele.
MMP1 Expression Levels in Atherosclerotic Plaques From Patients of Different MMP1 Haplotypes
We also examined MMP1 expression levels in atherosclerotic plaques from patients of different haplotypes using the real-time RT-PCR method. Compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with lower MMP1 expression in atherosclerotic plaques (P=0.033; Figure 3). The GeC519-CeC340 haplotype, which was rare, with a frequency of <6% in British whites, was not present in the patients (n=26) from whom the atherosclerotic plaques were obtained for MMP1 expression level measurement.
Discussion
The study provides evidence of differences in MI risk between individuals of different MMP1 gene haplotypes, which were observed initially in a sample from Southern England and then in an independent sample from the Swedish population. In both studies, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with a reduction in MI risk, whereas the GeC519-CeC340 haplotype was associated with an increase in MI risk. The data suggest a contribution of MMP1 gene variation to interindividual variability in risk of MI, which is known to have a multifactorial etiology.
The reporter assays showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes had lower promoter activity, whereas the GeC519-CeC340 haplotype had higher promoter activity, in driving gene expression in human macrophages. The mRNA assays in atherosclerotic tissues showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with reduced MMP1 expression in atherosclerosis plaques. These analyses suggest that differences in MMP1 gene promoter activity and expression might, perhaps in part, explain the differences in MI risk according to MMP1 haplotypes, as observed in the British and Swedish studies. Although the activity of MMPs is influenced not only by the level of MMP expression but also by MMP activators and inhibitors, previous studies have shown a strong relationship between MMP expression and MMP activity in atherosclerotic lesions.9,11
The electrophoretic mobility-shift assays showed that the eC519A>G and eC340T>C polymorphisms have an allele-specific effect on binding of nuclear proteins from human macrophages. This could potentially explain the haplotype effect of these 2 polymorphisms on MMP1 promoter activity detected in the reporter assays, but a complete explanation awaits analyses to characterize the nuclear proteins involved and their interactions. The eC519A>G and eC340T>C polymorphisms are located in conserved regions in the MMP1 promoter, as shown in Figure 1B. It has been suggested that conserved sequences often harbor transcription factoreCbinding sites.23 In silico examination showed that the eC340C allele (but not the eC340T allele) might contain a binding site (CACCT on the reverse strand) for transcription factor TCF8 (also known as AREB6). We performed supershift assays with a TCF8 antibody to examine whether TCF8 was present in the DNAeCprotein complexes detected in the electrophoretic mobility-shift assays. The supershift assays showed that none of the DNAeCprotein complexes was affected by the antibody, suggesting that TCF8 is probably not involved in these complexes. The in silico examination did not reveal other transcription factoreCbinding site in the DNA sequences surrounding eC519A>G or eC340T>C, suggesting that the nuclear proteins binding to these regions are not in the current transcription factor databases, which only include factors that have been characterized. Therefore, identifying the proteins binding to these polymorphic sites will likely require cloning techniques such as the 1-hybrid assay.
An increasing number of studies have shown that a disease phenotype can be associated with a haplotype made up of polymorphisms that are not individually associated with the phenotype.24eC29 The present study provides another such example. The study showed that MI risk was associated with haplotypes derived from the eC519A>G and eC340T>C polymorphisms, although there was no association between the disease and the polymorphisms individually. In a systematic analysis of all different combinations of the 7 MMP1 polymorphisms using an AIC-based method (described in Subjects and Methods) that circumvents problems associated with methods based on null-hypothesis testing, such as the requirement for multiple testing correction, the haplotype model consisting of the eC519A>G and eC340T>C polymorphisms was shown to be the best model in terms of predicting the disease status.
It appears that there are at least 2 reasons that might explain why a phenotype can be associated with a haplotype but not with the individual polymorphisms that make up the haplotype. First, a functional effect on gene expression can be dependent on the interaction between 2 or more polymorphisms, as demonstrated in a study by Terry et al, which showed a cooperative influence of several polymorphisms on interleukin-6 gene transcriptional regulation and that studying a single polymorphism in isolation would not uncover the overall functional effect of the polymorphism in combination with other functional polymorphisms.30 Second, haplotypes generally have a higher probability than individual polymorphisms of showing useful linkage disequilibrium with an unknown causal variant.31
It has been suggested that genetic factors may be classified into 2 broad categories: (1) susceptibility genes that affect certain initial events in the disease process and thus the susceptibility of an individual to the disease; and (2) disease modifying genes that affect the course of the disease process after it has been initiated and thus influence the disease outcome.32 The results of the British study suggest that the MMP1 gene can have a modifying effect on coronary artery disease and that subjects in this study had coronary atherosclerosis, and in these patients, there was an association between MMP1 haplotypes and MI risk. It is possible that atherosclerotic lesions in individuals carrying higher MMP1 expression haplotypes are more likely to develop into vulnerable plaques because of excessive degradation of fibrillar collagens.
In summary, this study has shown a haplotype effect of the MMP1 gene on risk of MI. The results are consistent with the notion that MMP1 plays an important role in the pathogenesis of atherosclerosis and its clinical complications1 and the notion that regulatory polymorphisms constitute an important type of genetic variant that influences susceptibility to complex diseases.33 The data might have utility in future development of approaches for identifying at-risk individuals and strategies to optimize treatment for this common, complex disease.
Acknowledgments
This work was supported by grants from the British Heart Foundation (PG/98183 and PG/2001105), the European Union Framework Programme 6 Degradome project (LSHC-CT-2003-503297), the Swedish Research Council (12660), the Swedish Heart-Lung Foundation, the Petrus and Augusta Hedlund Foundation, the Stockholm County Council, and AFA Insurance. We thank Barbro Burt for excellent technical assistance.
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INSERM U525 (D.-A.T), Facultee de Meedecine, Hpital Pitiee-Salpe簍rieere, Paris, France
Atherosclerosis Research Unit (A.S., A.H., P.E.), King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden.
Abstract
Myocardial infarction (MI) is commonly caused by atherosclerotic plaque rupture following excessive degradation of collagen fibers in the atherosclerotic lesion. We investigated whether interindividual variability in risk of MI was related to polymorphisms in the gene encoding matrix metalloproteinase (MMP)-1, a key fibrillar collageneCdegrading enzyme. Several single nucleotide polymorphisms in the MMP1 gene promoter were identified following sequencing DNA samples from 30 individuals. An analysis of the polymorphisms in a cohort of British whites with coronary atherosclerosis, including 639 patients with MI and 538 non-MI subjects, revealed a haplotype effect of the eC519A>G and eC340T>C polymorphisms on risk of MI, with the AeC519-CeC340 and GeC519-TeC340 haplotypes being protective (odds ratio=0.70 [0.57 to 0.86]; P=0.0007), whereas the GeC519-CeC340 haplotype increased MI risk (odds ratio=1.94 [1.15 to 3.28]; P=0.013). This finding was replicated in a subsequent analysis of 387 Swedish MI patients and 387 healthy controls (odds ratio=0.70 [0.55 to 0.89], P=0.003, for AeC519-CeC340 and GeC519-TeC340; odds ratio=1.54 [0.97 to 2.46], P=0.07, for GeC519-CeC340). In vitro assays showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes had lower promoter activity, whereas the GeC519-CeC340 haplotype had greater promoter strength, in driving gene expression in human macrophages. Haplotype-specific differences in MMP1 mRNA level in atherosclerotic tissues were also detected. The data indicate that MMP1 gene variation is a genetic factor contributing to interindividual differences in MI risk.
Key Words: matrix metalloproteinase genetics polymorphism haplotype analysis atherosclerosis
Introduction
Myocardial infarction (MI) is predominately caused by coronary atherosclerotic plaque rupture, a mechanical breakdown of the fibrous cap overlaying the lipid core of the plaque.1,2 Rupture of the plaque results in exposure of thrombogenic material (eg, tissue factor and lipids) located in the lipid core to the blood and leads to thrombus formation and, consequently, to the ischemic event.1,2 The biomechanical strength of the fibrous cap largely depends on its content of fibrillar collagens, type I and III in particular, which are cross-linked to provide structural support.3 Excessive degradation of these collagen fibers has been recognized as an important mechanism underlying atherosclerotic plaque rupture.4eC6
Matrix metalloproteinase (MMP)-1 (or interstitial collagenase) can degrade fibrillar collagens, especially types I and III, which are resistant to most proteinases.7,8 Pathological studies have shown that MMP1 is expressed by macrophages in atherosclerotic plaques and colocalizes with degraded fibrillar collagens in the shoulder region, which is prone to rupture.9eC11 MMP1 expression is significantly increased in vulnerable atherosclerotic plaques, characterized by having a thin fibrous cap overlaying a large lipid core, compared with stable atherosclerotic plaques, characterized by having a thick fibrous cap.11,12 Thus, it has been postulated that increased MMP1 expression promotes atherosclerotic plaque rupture.11
In this study, we investigated whether interindividual variability in risk of MI was related to MMP1 gene polymorphisms that might influence MMP1 expression or activity. We first performed a search for polymorphisms in this gene by sequencing DNA samples from a group of unrelated individuals. The polymorphisms were then studied in relation to MI in a cohort of coronary artery disease patients. The findings were verified in an independent sample from another population. We then performed functional analyses of the polymorphisms found to be associated with MI risk.
Subjects and Methods
Identification of MMP1 Gene Variants
The MMP1 gene promoter (1.9 kb), all 10 exons, and the introneCexon junctions were amplified by PCR using primers described in Table I in the online data supplement available at http://circres.ahajournals.org. The PCR amplicons were sequenced using ABI sequencers and BigDye terminator cycle sequencing reagents. The PCR and sequencing were performed using DNA templates from 30 unrelated whites, which provided >90% power to detect sequence polymorphisms with an allele frequency of >5%.13
Subjects in Studies of MMP1 Gene Polymorphisms and Haplotypes in Relation to MI Risk
Analyses of MMP1 polymorphisms and haplotypes in relation to MI risk were performed in 2 population samples. The first was a cohort of British whites with coronary artery disease, documented angiographically as having >50% diameter stenosis in at least 1 coronary artery, as described previously.14 It consisted of 2 groups, namely 639 patients who had experienced an MI and 538 non-MI subjects. The characteristics of the 2 groups are summarized in Table 1. The second sample consisted of 387 Swedish patients with an MI at the age of <60 years and 387 gender- and age-matched healthy Swedish individuals participating in a program aimed at identifying novel biomarkers and susceptibility genes for MI. The characteristics of the 2 groups are summarized in Table 1 and have been described in detail elsewhere.15 In both studies, MI was diagnosed according to standard clinical criteria, including electrocardiographic and/or enzymatic changes. Both studies were approved by the respective local research ethics committees, and all subjects gave informed consent.
Determination of Genotypes
For each polymorphism, a DNA sequence containing the polymorphic site was amplified by PCR using primers described in supplemental Table I. The amplicons were digested with an appropriate restriction endonuclease that specifically cleaved 1 of the 2 alleles. The restriction endonucleases used were XmnI (for eC1607GG>G), HindIII (for eC839G>A), MspI (for eC755G>T), KpnI (for eC519A>G), BanI (for eC422T>A), AflII (for eC340T>C), and HaeIII (for eC320T>C), respectively. Digests were subjected to gel electrophoresis, and DNA was detected by poststaining of the gel with Vistra Green and visualized using a fluorimager to determine genotypes.
Statistical Analyses of MMP1 Polymorphisms and Haplotypes in Relation to MI Risk
Allele and genotype frequencies were calculated by gene counting. Analyses of MMP1 polymorphisms in relation to MI were performed, first, by examining the polymorphisms individually and then, second, by studying haplotypes. Individual polymorphisms in relation to MI were examined by 2 analysis. In the haplotype analyses, a systematic analysis of all possible combinations of 1 to k polymorphisms to select the most informative and parsimonious haplotype configuration in terms of predicting disease status was performed using a previously described method that calculates the Akaike’s Information Criterion (AIC) values for each haplotype model and then subtracts the minimum AIC value obtained for each model over all models explored, giving a rescaled AIC value for each haplotype model.16 As described previously,16 the models with a rescaled AIC 2 are considered equivalent to the most informative model, and among these models, the most parsimonious model with the fewest polymorphisms is considered the best model. Because the choice of models is based on AIC values, it circumvents problems associated with methods based on null-hypothesis testing, such as the requirement of multiple testing correction. After identifying the best model, the haplotype effects of the polymorphisms in this model on MI risk were analyzed using the THESIAS program (www.genecanvas.org), which implements the stochastic-EM (ExpectationeCMaximization) algorithm.17 Haplotype effects on MI risk were adjusted for age, gender, body mass index, smoking, cholesterol levels, statin treatment, hypertension, and diabetes.
Transient Transfection and Reporter Assays
Transient transfection and reporter assays were performed to compare the strengths of MMP1 promoter of different haplotypes in driving gene expression. For each haplotype, the corresponding MMP1 promoter (from eC1870 bp to +42 bp relative to the transcriptional start site) was generated by PCR using genomic DNA as template and then cloned into a TOPO PCR cloning vector (Invitrogen). The cloned MMP1 promoter was sequenced to verify that there was no misincorporation during PCR and then subcloned into a plasmid (pGL3-Basic Vector; Promega) containing a firefly luciferase reporter gene. The resultant construct was mixed with a plasmid (pRL-TK, Promega) containing a renilla luciferase gene under the control of a thymidine kinase promoter and transferred into cultured THP-1 human monocytic cells using the lipofection method with FuGENE 6 transfection reagent. The transfected cells were treated with phorbol 12-myristate 13-acetate to induce differentiation of the cells into macrophages. At 36 hours after transfection, the cells were lysed and the activities of the firefly luciferase and renilla luciferase in the lysates measured with the use of a dual-luciferase assay kit (Promega). The ratio of firefly luciferase level to renilla luciferase level was used as a measure of the MMP1 gene promoter activity. Three independent experiments in duplicate were performed for each construct.
Electrophoretic Mobility-Shift Assays
For each allele, a double-stranded oligonucleotide corresponding to the sequence at and surrounding the polymorphic site was generated and used as a probe. The sequences of these probes are described in supplemental Table I. Each probe was labeled with 32P at the 5' terminus. The labeled probe was incubated with nuclear protein extracts from cultured human macrophages derived from THP-1 cells, followed by nondenaturing polyacrylamide gel electrophoresis and autoradiography, as described.18,19 Three independent experiments were performed for each polymorphism.
Real-Time RT-PCR
RNA was extracted from atherosclerotic plaques removed from patients (n=26) undergoing carotid endarterectomy and reverse transcribed to cDNA. Real-time PCR was performed in duplicates in an ABI Prism 7700 Sequence Detection System. PCR primers (described in supplemental Table I) were designed with the use of the Primer Express program (Applied Biosystems), with the forward and reserve primers placed in different exons. The MMP1 real-time RT-PCR results of different samples were standardized for the amounts of RNA template and efficiencies of reverse transcription using the housekeeping gene 36B4 (acidic ribosomal phosphoprotein PO subtype) as a reference20 and applying the 2eCCT method.21 The haplotype effects of the MMP1 eC519A>G and eC314C>T polymorphisms on MMP1 mRNA levels were analyzed using the THESIAS program, adjusting for statin treatment and atherosclerotic plaque types defined according to the revised American Heart Association classification system,22 which takes into account macrophage density, smooth muscle cell density, the size of lipid core, and the proportion of fibrous tissue.
Results
Identification of Polymorphisms in the MMP1 Gene
To search for naturally occurring, common sequence variants of the MMP1 gene, we sequenced the promoter, exons, and introneCexon junctions of the gene in 30 unrelated white individuals. In the promoter region, single-nucleotide polymorphisms were identified at positions eC1607 (GG/G, ie, G insertion/deletion), eC839 (G/A), eC755 (G/T), eC519 (A/G), eC422 (T/A), eC340 (T/C), and eC320 (T/C) (Figure 1A). Two of these 7 promoter polymorphisms, namely eC519A>G and eC340T>C, were found to be located in regions conserved in human, mouse, and rat (Figure 1B). In the coding region, only 2 polymorphisms were identified, neither of which changes the amino acid sequence. The subsequent studies were focused on the promoter polymorphisms.
MMP1 Haplotype Effects on Risk of MI
To investigate whether there was a genotypic or haplotypic effect of MMP1 on MI risk, we first studied the polymorphisms in a sample of British whites with hemodynamically significant coronary atherosclerosis, consisting of 639 patients who had experienced an MI and 538 non-MI subjects. Frequencies of alleles, genotypes, and haplotypes derived from the 7 polymorphisms are shown in supplemental Table II. No significant difference in genotype distribution of individual polymorphisms between the 2 groups was detected.
Data of a systematic analysis of all possible combinations of the polymorphisms in relation to MI are presented in supplemental Table III. The analysis showed that the best model in terms of predicting MI status was 1 that consisted of the eC519A>G and eC340T>C polymorphisms. An analysis of haplotypes based on these 2 polymorphisms showed that the haplotype frequencies were significantly different between the MI and non-MI groups (2=16.48 with 3 degrees of freedom, P<10eC3; Table 2). Compared with the AeC519-TeC340 haplotype, both the AeC519-CeC340 and GeC519-TeC340 haplotypes conferred a protective effect against MI (odds ratio [OR]=0.68 [0.52 to 0.88], P=0.004, for AeC519-CeC340; OR=0.71 [0.56 to 0.89], P=0.003, for GeC519-TeC340; the common OR associated with these 2 haplotypes being OR=0.70 [0.57 to 0.86], P=0.0007; Table 2), whereas the GeC519-CeC340 haplotype was associated with an increased risk of MI (OR=1.94 [1.15 to 3.28], P=0.013; Table 2).
To verify the findings above, a Swedish sample consisting of 387 MI patients and 387 healthy controls was genotyped for the polymorphisms. As in the British study, the Swedish study showed a protective effect of the AeC519-CeC340 and GeC519-TeC340 haplotypes (OR=0.72 [0.54 to 0.94], P=0.02, for AeC519-CeC340 versus AeC519-TeC340; OR=0.68 [0.52 to 0.89], P=0.005, for GeC519-TeC340 versus AeC519-TeC340; and OR=0.70 [0.55 to 0.89], P=0.003, for AeC519-CeC340+GeC519-TeC340 versus AeC519-TeC340; Table 2) and an increased risk for the GeC519-CeC340 haplotype (OR=1.54 [0.97 to 2.46], P=0.07, for GeC519-CeC340 versus AeC519-TeC340; Table 2).
Effects of MMP1 Polymorphisms on Promoter Activity in Driving Gene Expression
To investigate whether the eC519A>G and eC340T>C polymorphisms had a functional effect, we examined the promoter activity of MMP1 haplotypes in driving the expression of a luciferase reporter gene in transiently transfected THP-1 celleCderived human macrophages. Compared with the haplotypes encompassing both the AeC519 and TeC340 alleles, the haplotypes encompassing both the AeC519 and CeC340 alleles and those encompassing both the GeC519 and TeC340 alleles had lower promoter activity (P<0.01), whereas the haplotype containing both the GeC519 and CeC340 alleles had higher promoter activity (Table 3).
Effects of MMP1 Polymorphisms on Nuclear Protein Binding to the MMP1 Promoter
To investigate whether the eC519A>G and eC340T>C polymorphisms were located in DNA sequences recognized by nuclear proteins, and if so, whether the polymorphisms affected the DNA-protein interactions, we performed electrophoretic mobility shift assays with oligonucleotide probes corresponding to the different alleles and nuclear protein extracts from THP-1 human macrophages. For the eC519A>G polymorphism, a DNAeCprotein complex (indicated by an arrow in Figure 2A) was detected in assays using a probe corresponding to the AeC519 allele but not in assays using a probe corresponding to the GeC519 allele. For the eC340T>C polymorphism, a DNAeCprotein complex (indicated by an arrow in Figure 2B) was detected in assays using a probe corresponding to the CeC340 allele but not in assays using a probe corresponding to the TeC340 allele.
MMP1 Expression Levels in Atherosclerotic Plaques From Patients of Different MMP1 Haplotypes
We also examined MMP1 expression levels in atherosclerotic plaques from patients of different haplotypes using the real-time RT-PCR method. Compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with lower MMP1 expression in atherosclerotic plaques (P=0.033; Figure 3). The GeC519-CeC340 haplotype, which was rare, with a frequency of <6% in British whites, was not present in the patients (n=26) from whom the atherosclerotic plaques were obtained for MMP1 expression level measurement.
Discussion
The study provides evidence of differences in MI risk between individuals of different MMP1 gene haplotypes, which were observed initially in a sample from Southern England and then in an independent sample from the Swedish population. In both studies, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with a reduction in MI risk, whereas the GeC519-CeC340 haplotype was associated with an increase in MI risk. The data suggest a contribution of MMP1 gene variation to interindividual variability in risk of MI, which is known to have a multifactorial etiology.
The reporter assays showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes had lower promoter activity, whereas the GeC519-CeC340 haplotype had higher promoter activity, in driving gene expression in human macrophages. The mRNA assays in atherosclerotic tissues showed that compared with the AeC519-TeC340 haplotype, the AeC519-CeC340 and GeC519-TeC340 haplotypes were associated with reduced MMP1 expression in atherosclerosis plaques. These analyses suggest that differences in MMP1 gene promoter activity and expression might, perhaps in part, explain the differences in MI risk according to MMP1 haplotypes, as observed in the British and Swedish studies. Although the activity of MMPs is influenced not only by the level of MMP expression but also by MMP activators and inhibitors, previous studies have shown a strong relationship between MMP expression and MMP activity in atherosclerotic lesions.9,11
The electrophoretic mobility-shift assays showed that the eC519A>G and eC340T>C polymorphisms have an allele-specific effect on binding of nuclear proteins from human macrophages. This could potentially explain the haplotype effect of these 2 polymorphisms on MMP1 promoter activity detected in the reporter assays, but a complete explanation awaits analyses to characterize the nuclear proteins involved and their interactions. The eC519A>G and eC340T>C polymorphisms are located in conserved regions in the MMP1 promoter, as shown in Figure 1B. It has been suggested that conserved sequences often harbor transcription factoreCbinding sites.23 In silico examination showed that the eC340C allele (but not the eC340T allele) might contain a binding site (CACCT on the reverse strand) for transcription factor TCF8 (also known as AREB6). We performed supershift assays with a TCF8 antibody to examine whether TCF8 was present in the DNAeCprotein complexes detected in the electrophoretic mobility-shift assays. The supershift assays showed that none of the DNAeCprotein complexes was affected by the antibody, suggesting that TCF8 is probably not involved in these complexes. The in silico examination did not reveal other transcription factoreCbinding site in the DNA sequences surrounding eC519A>G or eC340T>C, suggesting that the nuclear proteins binding to these regions are not in the current transcription factor databases, which only include factors that have been characterized. Therefore, identifying the proteins binding to these polymorphic sites will likely require cloning techniques such as the 1-hybrid assay.
An increasing number of studies have shown that a disease phenotype can be associated with a haplotype made up of polymorphisms that are not individually associated with the phenotype.24eC29 The present study provides another such example. The study showed that MI risk was associated with haplotypes derived from the eC519A>G and eC340T>C polymorphisms, although there was no association between the disease and the polymorphisms individually. In a systematic analysis of all different combinations of the 7 MMP1 polymorphisms using an AIC-based method (described in Subjects and Methods) that circumvents problems associated with methods based on null-hypothesis testing, such as the requirement for multiple testing correction, the haplotype model consisting of the eC519A>G and eC340T>C polymorphisms was shown to be the best model in terms of predicting the disease status.
It appears that there are at least 2 reasons that might explain why a phenotype can be associated with a haplotype but not with the individual polymorphisms that make up the haplotype. First, a functional effect on gene expression can be dependent on the interaction between 2 or more polymorphisms, as demonstrated in a study by Terry et al, which showed a cooperative influence of several polymorphisms on interleukin-6 gene transcriptional regulation and that studying a single polymorphism in isolation would not uncover the overall functional effect of the polymorphism in combination with other functional polymorphisms.30 Second, haplotypes generally have a higher probability than individual polymorphisms of showing useful linkage disequilibrium with an unknown causal variant.31
It has been suggested that genetic factors may be classified into 2 broad categories: (1) susceptibility genes that affect certain initial events in the disease process and thus the susceptibility of an individual to the disease; and (2) disease modifying genes that affect the course of the disease process after it has been initiated and thus influence the disease outcome.32 The results of the British study suggest that the MMP1 gene can have a modifying effect on coronary artery disease and that subjects in this study had coronary atherosclerosis, and in these patients, there was an association between MMP1 haplotypes and MI risk. It is possible that atherosclerotic lesions in individuals carrying higher MMP1 expression haplotypes are more likely to develop into vulnerable plaques because of excessive degradation of fibrillar collagens.
In summary, this study has shown a haplotype effect of the MMP1 gene on risk of MI. The results are consistent with the notion that MMP1 plays an important role in the pathogenesis of atherosclerosis and its clinical complications1 and the notion that regulatory polymorphisms constitute an important type of genetic variant that influences susceptibility to complex diseases.33 The data might have utility in future development of approaches for identifying at-risk individuals and strategies to optimize treatment for this common, complex disease.
Acknowledgments
This work was supported by grants from the British Heart Foundation (PG/98183 and PG/2001105), the European Union Framework Programme 6 Degradome project (LSHC-CT-2003-503297), the Swedish Research Council (12660), the Swedish Heart-Lung Foundation, the Petrus and Augusta Hedlund Foundation, the Stockholm County Council, and AFA Insurance. We thank Barbro Burt for excellent technical assistance.
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