Influences of matrix metalloproteinase-3 gene variation on extent of coronary atherosclerosis and risk of myocardial infarction

Subjects

We recruited 1,240 consecutive Caucasian patients undergoing interventional or diagnostic coronary angiography in the Southampton General Hospital. Among the 1,240 subjects, 943 had significant stenosis (>50%) in at least one major epicardial coronary artery, and the remaining 297 had no significant stenosis (<50%). Coronary angiograms were assessed by one consultant cardiologist. We also recorded demographic and clinical data, including age, gender, weight, height, occupation, smoking habit, hyperlipidemia, hypertension, diabetes mellitus, previous MI, and coronary heart disease in first-degree relatives. The main characteristics of the subjects are summarized in Table 1. The group with MI had a higher percentage of smokers than did the group without MI (p = 0.016). There was no significant difference between patients with and those without MI in terms of age, gender, and prevalence of hypercholesterolemia, hypertension, diabetes, and family history of CAD. Patient recruitment was approved by the local Ethics Committee, and all subjects gave written, informed consent.

Sequence variant scanning

The bi-directional dideoxy fingerprinting (bi-ddF) method (19)was utilized to search for sequence variants in the MMP3 gene, including the promoter (∼2.3 kb), all 10 exons, and the intron–exon junctions. The nature and location of sequence variants identified by bi-ddF were determined by DNA sequencing. The assays were performed on genomic DNA samples from 20 unrelated Caucasian individuals (10 with early-onset MI [<45 years] and 10 healthy subjects). This sample size (40 chromosomes) provided ∼90% power to detect polymorphisms with a minor-allele frequency of >5% (20), on the basis that genetic variants contributing to a common disease in a large proportion of patients are likely to be common in the population, and thus the principal target of this work was common sequence variants.

Determination of genotypes

The subjects previously described were genotyped for the −1986 T>C, −1612 5A>6A, −1346 A>C, −709 A>C, −376 G>C, and +802 A>G polymorphisms. For each polymorphism, a sequence containing the polymorphic site was amplified by the polymerase chain reaction (PCR), and the amplicon was digested with an appropriate restriction endonuclease, which specifically cleaved one of the two alleles. Because of the limited amounts of DNA samples available to us, some of the polymorphisms could not be genotyped for some of the subjects.

Reporter assays

The MMP3 gene promoter region (from −2309 bp to +54 bp relative to the transcriptional start site) was amplified by PCR. The amplicon was inserted into a promoterless vector (pGL3-Basic Vector, Promega, Southampton, United Kingdom) containing a firefly luciferase reporter gene. The resultant construct was mixed with a plasmid (pRL-TK, Promega) containing a renillaluciferase gene under the control of a thymidine kinase promoter and transfected into cultured macrophages by electroporation. The transfectants were cultured with or without 1 μmol/l phorbol 12-myristate 13-acetate for 24 h. The cells were then lysed, and the activities of the firefly luciferase and renillaluciferase in the lysates were measured with the use of a Dual-Luciferase assay kit (Promega). The ratio of firefly luciferase level to renillaluciferase level was used as a measurement of the MMP3 gene promoter activity. At least four independent experiments in duplicates were carried out for each construct, and the mean values ± SEM are presented.

Electrophoretic mobility shift assays

With the use of a method by Alksnis et al. (21), nuclear protein extracts were prepared from cultured macrophages differentiated from human monocytic U937 cells with 1 μmol/l phorbol 12-myristate 13-acetate and from cultured human monocytoid MonoMac-6 cells. For each polymorphism, two double-stranded 26-mer oligonucleotides corresponding to the two alleles were used as probes and labeled with [γ³²-P]-adenosine triphosphate. The labeled probes were incubated with the aforementioned nuclear protein extracts, followed by nondenaturing polyacrylamide gel electrophoresis and autoradiography. Three independent experiments were carried out for each polymorphism.

Statistical analyses

The HWE program was used to test whether the observed genotype distributions deviated from the Hardy-Weinberg equilibrium. Linkage disequilibrium between the polymorphisms and the association metric D′ were analyzed with the use of the ASSOCIATE program and according to Devlin and Risch (22). Haplotype frequencies (Table 2) were estimated using the Haplotyper program (23), which employs a bayesian algorithm. To assess the effects of the genetic variants, the MMP3 gene polymorphisms were first examined individually in relation to the number of coronary arteries with >50% stenosis by ordinal logistic regression analysis (Table 3) and in relation to the risk of MI by binary logistic regression analyses (Table 4). Subsequently, stepwise logistic regression analysis with all genotypes and haplotypes, or with all haplotypes but not genotypes, inputted as independent variables, was performed to investigate which polymorphism(s) and haplotype(s) accounted for the association with MI susceptibility (Table 5).

Identification of novel variants in the MMP3 gene

Sequence variant scanning in the promoter region, coding region, and intron–exon junctions of the MMP3 gene identified a total of six novel sequence variants, in addition to the previously reported 5A/6A polymorphism. All newly identified variants were single nucleotide polymorphisms: four located in the promoter region and the remainder in the coding region (Fig. 1). The −1986C, −16126A, −1346C, −709G, −376C, +802G, and +814C alleles were detected in six of the 10 healthy individuals and five of the 10 MI patients subjected to the variant scanning. The frequencies of haplotypes in a group of individuals without significant coronary atherosclerosis are shown in Table 2. There was a substantial linkage disequilibrium between the polymorphisms, with the linkage disequilibrium metric D′ being over 0.9 for all polymorphisms, except for the −709 A>G polymorphism, which had a lower frequency of the minor allele (0.2 vs. >0.4 for the minor allele of the other polymorphisms).

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Figure 1

Schematic presentation of all polymorphisms identified in the matrix metalloproteinase-3 gene.

Association of MMP3 gene variation with extent of coronary atherosclerosis

Based on the finding from previous studies that progression of coronary atherosclerosis was more rapid in individuals with the MMP3 gene 6A/6A genotype (12–14), we hypothesized that variation in this gene could influence the extent of coronary atherosclerosis in CAD patients. To test this hypothesis, the polymorphisms in the MMP3 gene were analyzed in a cohort of Caucasian individuals undergoing coronary angiography. In those without a history of MI, the number of coronary arteries with >50% stenosis increased with increasing frequency of the 6A/6A genotype (odds ratio [OR] 1.52, p = 0.008) (Table 3). This association remained significant after adjustment for classic risk factors. Analyses in male and female subjects separately showed a consistent trend. No statistically significant association was detected between the other polymorphisms in the MMP3 gene and the extent of coronary atherosclerosis. There was no significant association between MMP3 genotype and the extent of coronary atherosclerosis in patients with MI.

Association of MMP3 gene variation with risk of MI in CAD patients

We then tested the hypothesis that variation in the MMP3 gene could influence the risk of MI in CAD patients. Logistic regression analyses showed that in patients with >50% stenosis in at least one coronary artery, there was an association between the −1612 5A/6A polymorphism and risk of MI, with the 5A/5A genotype conferring a twofold increase in MI risk (p = 0.016) (Table 4). Individuals who were heterozygous for the 5A/6A polymorphism had an intermediate risk (OR 1.78, p = 0.032). The association between this polymorphism and MI risk remained significant after adjustment for classic risk factors. A similar trend was observed when male and female subjects were analyzed separately.

In addition, there were approaching statistically significant differences in MI risk between the genotypes for the −1986 T>C polymorphism, such that individuals with the T/T genotype had 1.67-fold higher risk of MI compared with those with the C/C genotype (p = 0.089 after adjustment for covariates), whereas heterozygous individuals had an intermediate risk (OR 1.57, p = 0.096). There was no statistically significant association between MI risk and the other polymorphisms in the MMP3 gene (Table 2).

Stepwise logistic regression analysis

To estimate which variants and haplotypes largely accounted for the association between the MMP3 gene and MI susceptibility, we performed a stepwise logistic regression analysis with all genotypes (based on individual polymorphisms) and haplotypes (based on combinations of the alleles of the polymorphisms) inputted as independent variables. In this analysis, only the 5A/6A polymorphism was significantly associated with MI risk and remained in the equation (Table 5). We then carried out a stepwise logistic regression with all haplotypes, but not genotypes, inputted as independent variables. In this analysis without genotype terms, three haplotypes were associated with MI risk and remained in the equation (Table 5). These haplotypes—T-5A-A-A-G-A, T-5A-A-A-C-G, and C-5A-C-G-C-G—contained the 5A allele at the −1612 site but either the major or minor allele at the other polymorphic sites.

Differences in transcriptional activity between haplotypes

Our previous work showed that the 5A allele had a higher promoter activity than the 6A allele (11). In this study, we carried out transient transfection experiments and reporter assays to investigate whether the difference in promoter activity remained when the other polymorphisms were taken into account. In these experiments, the T-5A-A-A-G-A haplotype (which was the most common haplotype and was associated with increased MI risk) consistently showed a higher promoter activity than the C-6A-C-G-C-G haplotype. This difference was detected consistently in two different macrophage cell lines (RAW264.7 and MALU) and became more pronounced when these cells were stimulated with phorbol 12-myristate 13-acetate, a reagent that induces macrophage differentiation (Table 6).

Allele-specific binding of nuclear protein at polymorphic sites of the MMP3 promoter gene

We previously showed that the sequence surrounding the −1612 5A>6A polymorphic site was a transcription factor binding site and that compared with the 5A allele, the 6A allele had a higher affinity with the transcription factor (11). In the present study, we carried out electrophoretic mobility shift assays to investigate whether the other polymorphisms in the MMP3 gene promoter also showed an allele-specific interaction with transcription factors. These assays showed binding of a nuclear protein with a higher affinity to the −1986 T allele than to the −1986 C allele (Fig. 2). No differential binding of transcription factor was detected for the other promoter polymorphisms (−1346 A>C, −709 A>G, or −376 G>C; data not shown).

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Figure 2

Results of electrophoretic mobility shift assays. Two double-stranded oligonucleotide probes corresponding to the T and C alleles, respectively, of the −1986 T>C polymorphism were labeled with ³²P and incubated with nuclear extracts from macrophages (U937 [a]and MonoMac-6 [b]), followed by polyacrylamide gel electrophoresis and autoradiography. The arrowindicates a DNA–protein complex that is more readily detectable with the T probe (lanes 2 and 5)than the C probe (lanes 4 and 9). The DNA–protein complex is readily competed with unlabeled T probe (lanes 6 and 10), weakly competed with unlabeled C probe (lanes 7 and 11), but not affected by a nonspecific competitor (lanes 8 and 12).

Analysis of variants in the coding region

Both coding region polymorphisms identified are located in exon 2 of the MMP3 gene. Whereas the +814 A>G polymorphism is a synonymous substitution, the +659 A>G polymorphism results in a change from glutamic acid (Glu) to lysine (Lys) at residue 45, which is located just after the first alpha helix in the propeptide. The three-dimensional structure of the propeptide indicates that the region containing residue 45 is flexible. Thus, the change from glutamic acid (an acidic amino acid) to lysine (a basic amino acid) at this residue could potentially alter its interaction with other amino acids in this region and could have an effect on MMP3 activation. However, clustal alignment revealed that though most residues in this region were conserved across species, the Glu45 residue was not. The amino acid sequences in this region are YDL₄₄E₄₅KDVKQF in humans, YNL₄₄E₄₅KDVKQF in rabbits, YGL₄₄A₄₅KDVKQF in mice, and YGL₄₄E₄₅KDVKQF in rats. This suggests that the Lys45Glu polymorphism is unlikely to have a significant functional impact. We are currently seeking to address this question with in vitro studies.

Conclusions

The data in this study indicate that variation in the MMP3 gene influences the extent of coronary atherosclerosis and risk of MI in patients with CAD. These influences are largely attributable to the 5A/6A polymorphism, with the 6A/6A genotype being associated with the extent of atherosclerosis and the 5A allele-containing genotypes being associated with the risk of MI. These findings suggest that the 5A/6A polymorphism may contribute to the patient-to-patient variability in atherosclerotic plaque composition (3,5).