WO2002046462A2

WO2002046462A2 - Functional genetic variants of matrix metalloproteinases (nmps)

Info

Publication number: WO2002046462A2
Application number: PCT/GB2001/005365
Authority: WO
Inventors: David Robert Greaves; Simon Price; H. Watkins
Original assignee: Isis Innovation Limited
Priority date: 2000-12-07
Filing date: 2001-12-04
Publication date: 2002-06-13
Also published as: AU2002220886A1; WO2002046462A3

Abstract

A method of assessing an individual for predisposition to a disease in which MMP-2 plays a role comprises determining the nucleotide at a site of MMP-2 polymorphism associated with such disease. The site at -168 is especially important, providing an indication of coronary artery disease.

Description

Functional Genetic Variants

The present invention is concerned with the role played by gene polymorphisms and functional genetic variants.

There are links between genetic variation and the susceptibility to diseases such as atherosclerosis or cancer. The problem is first to find genes which relate in a significant manner to the development and clinical outcome of the diseases, and secondly to identify sites in such genes where genetic variation leads to consequences for the clinical outcome of the disease. The Common Disease-Common Variant hypothesis takes account of the observation that the human population has relatively limited genetic diversity, such that common variants may contribute significantly to genetic risk for common disease.

Atherosclerosis is a complex progressive disease of the large arteries characterised by cellular proliferation, lipid deposition and accumulation of fibrous elements. The extensive vascular remodelling is underpinned by multiple cell-cell and cell-matrix interactions principally between endothelial, inflammatory and smooth muscle cells of the vessel wall. The effectors of these remodelling processes include a family of enzymes called matrix metalloproteinases (MMPs).

The matrix metalloproteinases (MMPs) constitute a family of secreted and membrane associated structurally related zinc-dependent endopeptidases, that are capable of degrading a wide spectrum of both extracellular matrix (ECM) and non-matrix proteins. Currently upwards of 20 vertebrate MMPs have been reported which can be categorised, by substrate specificity, to give inter alia the collagenases, stromelysins, gelatinases and membrane-type MMPs. The broad range of substrates conveys a pivotal role for MMP involvement during both normal physiological processes {e.g. embryonic development, bone remodelling, angiogenesis, nerve growth, etc.) and pathological states {e.g. arthritis, cancer, atherosclerosis, liver fibrosis, etc.). Accordingly, MMP activity is tightly coordinated at several levels including tran scrip tional regulation, activation of latent zymogen and interaction with endogenous inhibitors. The pericellular activities of MMPs have been shown to facilitate inflammatory cell infiltration, vascular smooth muscle cell (SMC) migration and neointima formation, angiogenesis and plaque disruption.

The highly ordered nature of MMP regulation imparts a spatial and temporal distribution of enzymatic activity during lesional development, with certain family members being more prevalent than others. One such prominent enzyme is MMP-2, which has specific proteolytic activity against components of the basement membrane as well as other proteins known to be associated with both early and complicated plaques, such as fibrin and elastin. MMP-2 (gelatinase A, also termed 72Kd type IV collagenase) has type IV collagenolytic activity and is constitutively expressed by most connective tissue cells including endothelial cells, osteoblasts, fibroblasts and myoblasts. The membrane-bound activation of proMMP-2 ensures that proteolytic activity, predominantly against components of the basement membrane, is localised to discrete regions on the cell surface thereby potentiating ECM remodelling as well as uniquely acting to generate several different biologically active molecules including laminin, fibronectin and monocyte chemoattractant protein- 3.

The product of the human matrix metalloproteinase-2 gene (MMP-2) possesses proteolytic activity against type IV collagen, a major component of the basement membrane, and is therefore implicated in an extensive array of pathologies including atherogenesis, arthritis and tumour growth and metastasis. The majority of MMP-2 studies have focused on demonstrating the role in promoting cell invasiveness during tumour angiogenesis, arthritis and atherogenesis, as well as tumour metastasis where levels of MMP-2 expression can be correlated with tumour grade. The design of specific and selective inhibitors of MMP-2, for therapeutic or prophylactic intervention, remains an intense focus of research.

Active MMP-2 has been identified in human atheroma and MMP-2 expression increases after balloon catheter injury to pig, rabbit and rat vessels, correlating with VSMC migration to neointimal development. In the latter stages of plaque development, MMP-2 has been identified in fibrous and lipid- rich carotid plaques where expression is localised to vascular SMCs and macrophages. The accumulation of macrophages in the rupture-prone regions of plaques is known to correlate with loss of connective tissue and increased rupture vulnerability. Cultured macrophages expressing MMP-2 can digest collagen obtained from the human fibrous cap and promote plaque rupture suggesting that they may thereby participate in the pathogenesis of thrombus formation and consequent acute coronary syndromes such as unstable angina (UA) and myocardial infarction (MI).

Ohnishi et al, Hum. Genet. (2000) 16: 288-292, identified 187 single nucleotide polymorphisms (SNPs) among 41 candidate genes for ischemic heart disease in the Japanese population. They report the existence of 7 SNPs in MMP-2.

We reported in March 2001 a systematic survey of natural genetic variation across 28 kb of the human MMP-2 gene, encompassing the promoter and transcribed regions, see Price, S. J., Greaves, D. R., and Watkins, H. (2001) JBiol Chem 276, 7549-7558. We performed functional studies on the entire panel of seven promoter variants and identified polymorphisms which influence MMP-2 expression in an allele- specific manner. That article is incorporated in full by specific reference.

SUMMARY OF INVENTION

We have now demonstrated that matrix metalloproteinase-2, MMP-2, exhibits polymorphisms which have consequences for the development of disease.

The present invention identifies the naturally occurring genetic variation in the MMP-2 gene (the term 'gene' herein including the regulatory regions, untranslated regions, exons and introns, for example) to enable analysis of the functional effects of promoter variants on gene expression, and envisages tests for association with a variety of clinical phenotypes.

With the present invention, we have identified the nature and extent of genetic variation in the human matrix metalloproteinase-2 gene, including for example analysis of the 5' UTR, 3' UTR, regulatory regions including enhancers and promoters, and intron sequences.

Importantly we have undertaken functional analyses to identify polymorphisms within the MMP-2 gene that result in an altered transcriptional activity of the MMP-2 gene in a range of mammalian cell types. Moreover, we have determined the common haplotypes of the MMP-2 promoter in human populations.

With knowledge of the MMP-2 polymorphisms and haplotypes, it now becomes possible to evaluate the importance of such genetic variations and to apply an understanding of such relationships to the diagnosis and treatment of MMP-2-associated diseases.

Particular areas of interest include, inter alia, those where the specific predilection of MMP-2 for substrates important in basement membrane integrity indicate a role in a number of disease states including, for example, atherosclerosis, restenosis and metastasis. Common variants that alter the amount of protein expressed, for instance by affecting transcriptional regulation, protein processing, or that alter the activity of the protein, can be investigated to assess the extent to which they have a quantitative influence on disease activity.

PREFERRED EMBODIMENTS

Examples of polymorphic sites which affect transcriptional activity of the MMP-2 promoter and which may be indicators of disease include -168 (G/T), - 790 (T/G) +220 (G/C) and -1306 (C/T). We have now in particular found that the -168 polymorphism is a significant independent predictor of coronary atherosclerosis.

The invention provides in one aspect, a method of determining the sequence of the MMP-2 gene, which involves determining the nucleotide at one or more polymorphic sites in the MMP-2 gene. The MMP-2 gene includes the . promoter, coding and regulatory sequences, and detection of MMP-2 promoter polymorphisms is a feature of this invention. A method of sequencing can then form part of a method of diagnosis and/or prognosis of diseases or conditions associated with MMP-2 activity, where the sequenced nucleotide is evaluated against consequences of such polymorphisms. In a related aspect the invention provides a method of assessing for predisposition to diseases in which MMP-2 plays a role, comprising sequencing the MMP-2 gene and identification of polymorphisms within the MMP-2 gene linked to such diseases. Particularly preferred is sequencing to determine the nucleotide at position -168.

The invention can be applied to any disease or illness in which MMP-2 plays a role, including diseases involving tissue metalloproteinase remodelling, such as growth or destruction of tissue. For example, the present invention can play a significant part in assessment of predisposition to these diseases, prognosis and/ or prevention of such disease in individuals. Among MMP-2 related diseases and conditions we include myocardial infarction, thromboembolism, abdominal aortic aneurism, and other consequences of atherosclerosis; restenosis; arthritis; cancer caused by both metastatic and non metastatic tumours; and obstructive pulmonary disease, emphysema, hepatic fibrosis and other diseases involving fibrosis.

Furthermore, the invention finds application in the profiling and assessment of individuals for medical treatment including clinical trials. The sequencing of the MMP-2 gene in individuals enables screening for appropriate selection and allocation of individuals and, for instance, can help to avoid inappropriate treatments, or misleading findings in clinical trials. Thus, knowledge of functional MMP-2 polymorphisms allows a pharmacogenetic approach to treatment. Essentially, knowledge of the function of polymorphisms provided by the invention allows treatment and trials to be better targeted and understood. By identifying individuals with a predisposition to conditions associated with MMP-2 expression and activity (or lack of expression and inactivity) based on a polymorphism, they can be targeted for treatment with drugs, including MMP-2 inhibitors. Identification of individuals at risk because of MMP-2 polymorphism can be used in other situations, for example in deciding whether the coating of metal stents is warranted in balloon angioplasty to prevent restenosis or whether MMP2 inhibitors should be used as an adjunct to chemotherapy, for example.

One of the genetic variants which can be determined is -168 (G/T) which is found in the MMP-2 gene promoter at position - 168 bp from the major transcription start site, defined in Huhtala, P., Chow, L. T., and Tryggvason, K. (1990) J Biol Chem 265, 11077-82. The predominant polymorphism in the population has a guanidine nucleotide present at this position, the less common variant has a thymine nucleotide. Examples of other polymorphic sites include -790 (T/G) +220 (G/C) and -1306 (C/T). Our experiments have not shown that the -1306 polymorphism is important in cardiovascular disease for our test sample, but it might be important in other patients or diseases, or cancer metastases.

In a first embodiment, the method of the invention comprises the steps of subjecting a region of DNA containing the MMP-2 gene containing a polymorphic sequence to a restriction digestion using at least one restriction endonuclease, and analysing the resulting products to determine the presence or absence of a restriction site for the endonuclease as an indication of the nature of the genetic variant. Suitable analysis techniques include RFLP analysis, for example.

Preferably, the region containing the polymorphic sequence is amplified prior to restriction digestion, for example using a PCR technique. In an adaptation of the first embodiment of the method of the invention, the region of the MMP-2 gene containing a polymorphic sequence is amplified using one or more oligonucleotide primers designed to incorporate into the amplified DNA sequence a restriction endonuclease recognition site comprising a variant of the polymorphic sequence. This is useful where one or other of the variant forms of the polymorphic sequence does not itself contain a restriction site.

Analysis of the restriction digestion products in the method of the invention may be carried out by a size determination technique, for example, using a separation technique such as gel electrophoresis.

In an alternative approach the nature of the genetic variant may be identified without the need for restriction digest, for example, using PCR in combination with denaturing HPLC or SSCP analysis, which techniques are standard in the art.

In an alternative embodiment of the method of the invention the nature of the variant is determined by using at least one oligonucleotide capable of hybridising specifically to a variant of interest under suitably stringent conditions. The term Oligonucleotide' has no inherent length restriction, and generally refers herein to any suitable nucleotide sequence or polynucleotide, as appropriate. Such an oligonucleotide should be long enough to be specific for a particular variant of interest in the present embodiment. Suitably stringent hybridisation conditions need to be employed to enable an oligonucleotide specific for one variant of the polymorphic sequence to hybridise to that variant and not to any other variant. Such hybridisation conditions are known and/ or can be easily determined. Hybridisation may be detected by means of a detectable label on the specific oligonucleotides.

Specific oligonucleotides can be designed for each polymorphic site. For example, the identified polymorphisms at positions -1306 and -168 in the human MMP-2 promoter sequence are shown in the following four sequences, with the polymorphic base change in the DNA sequence being indicated in bold text. SEQ ID NO 1:

-1306T AUele 5'-ACCCAGCACTCTACCTCTTTAGCT SEQ ID NO 2:

-1306C Allele 5'-ACCCAGCACTCCACCTCTTTAGCT

SEQ ID NO 3:

-168G Allele 5'-GCAGGCGGCCGGGGAAAAGAGGTGG SEQ ID NO 4:

-168T Allele 5'-GCAGGCGGCCGGGTAAAAGAGGTGG

The specific oligonucleotide may be employed as a sequence specific probe. Methods wherein specific oligonucleotides are employed as primers in an amplification reaction are also provided by the invention. In one such method, the nature of the variant is determined by an amplification reaction which uses one or more sequence specific oligonucleotide primers. A region of the MMP-2 gene containing the polymorphic sequence may be subjected to PCR in the presence of at least one primer designed to hybridise with a particular variant of the polymorphism, at the 3'-terminal region of the primer, such as the 3'-terminal nucleotide, such that only fragments comprising that variant will be amplified. Two or more different primers having differing 3' -termini may be employed. Oligonucleotides which are complementary to a given DNA sequence except for a mis-match at their 3' -termini fail to function as primers in PCR. This technique is known as the amplification refractory mutation system (ARMS).

Other methods of variant sequence detection employing amplification reactions and other techniques are envisaged. For example, reference is made to the techniques described in WO 9957315. The invention also envisages a combination of tests, which combination includes determining the nature of one or more MMP-2 polymorphisms, for providing a prognosis for diseases involving changes in the extracellular matrix, including remodelling diseases for example. Such a combination can include for example a test for determining the nature of one or more polymorphisms in other genes such as MMP genes, for example the MMP-3 or MMP-9 gene.

In an alternative aspect, the invention provides an oligonucleotide, preferably between 8 and 50 base pairs, more preferably 15 to 30 base pairs in length, which under suitably stringent conditions specifically hybridises to a variant of a polymorphic sequence of this invention, or its reverse complement. This aspect of the invention is concerned in particular with such an oligonucleotide which hybridises to a polymorphic position, or its reverse complement, especially in a region containing position -168. The oligonucleotide may specifically hybridise to the polymorphic sequence containing the variant thymine at position -168.

Particularly preferred is an oligonucleotide which extends at least 7 nucleotides downstream or upstream of the site of polymorphism and includes a nucleotide known to occur in one of the polymorphisms, preferably at the polymorphic end, as well as oligonucleotides complementary thereto. Thus, for instance, in the case of the -168 polymorphism, preferred primers include the guanidine or the thymine appropriate for the polymorphism at - 168, together with at least a further 7 nucleotides extending downstream or upstream therefrom, or a complementary oligonucleotide.

The invention especially provides oligonucleotide primers having a sequence suitable for the preferential amplification of an MMP-2 gene from a nucleic acid DNA template molecule comprising a cytosine nucleotide at position -168 relative to the MMP-2 gene major transcription start site and oligonucleotide primers having a sequence suitable for the preferential amplification of an MMP-2 DNA template molecule comprising a thymine nucleotide at position -168.

The invention also relates to RNA deriveable from the polynucleotides of the present invention, both MMP-2 mRNA and to antisense RNA.

The invention also relates to proteins or peptides encoded by the variant sequences of the MMP-2 gene, and fragments thereof. Specific antibodies raised against such proteins or peptides are also envisaged. Proteins or peptides encoded by the variant sequences may be utilised for screening compounds that bind specifically to a molecule encoded by variants of a polymorphic sequence, in a search for compounds which modulate the activity of the enzyme. The structure of such variant proteins may be used for rational drug design. In addition, proteins or peptides encoded by the variant sequences may themselves potentially be used for prophylaxis or therapy, either alone or in combination with other pharmaceutically acceptable agents.

The invention also provides assay kits for use in the detection of MMP-2 gene variants such as those described herein. Such kits may comprise for example one or more oligonucleotides as described herein, and optionally a polymerase enzyme. Other suitable kits may comprise antibodies to the proteins encoded by the variant MMP-2 genes, optionally with suitable agents for detection of antibody binding.

The invention also relates to a gene chip comprising a polynucleotide according to the present invention. A gene chip suitably comprises a substrate onto which a polynucleotide such as DNA or RNA may be attached or synthesised. Examples of gene chips are well known in the art. Such chips can be used to screen for polymorphisms in DNA by hybridisation, for example, and allow screening for multiple polymorphisms to be carried out. In particular the invention relates to a gene chip comprising all or a part of the MMP-2 gene having the -168 (G/T) polymorphism, preferably a chip comprising polynucleotide sequences representing both the G and T variants.

To investigate whether the common functional variants of the MMP-2 gene confer risk for coronary artery disease (CAD), subjects with angiographic evidence of coronary atherosclerosis were compared with population controls for whom coronary disease was excluded on the basis of clinical history and angiographic data. The frequency of the C-1306/T genotypes and alleles were not significantly different between case and controls, signifying a lack of association between the C-1306/T allele and incidence of CAD. In contrast, a strong association existed between the G-168/T variant of the MMP-2 gene and risk of CAD that was independent of other coronary risk factors. The increased risk was assigned to individuals homozygous for the G allele, and homozygosity at this position resulted in a 2.7-fold increase risk for CAD compared with individuals who had at least one copy of the T allele.

We then investigated whether the distribution of genotypes could differ with differing coronary phenotypes, namely coronary atherosclerosis versus acute coronary events (i.e. unstable angina, UA; and, myocardial infarction, MD. We subdivided the patients based on clinical data with 384/782 (49%) subjects having MI and/ or UA versus those patients with neither acute coronary syndrome (i.e. 268/782; 34%). The distribution of genotypes and alleles were not significantly different between these two groups in this analysis.

Because MMP-2 has a potential role in vascular remodelling, the association with coronary atherosclerosis supports the hypothesis that genetic variation influencing transcriptional regulation might contribute to interindividual susceptibility of disease phenotype. Although the precise underlying mechanisms remain unclear, these data may indicate that the -168 variant has a different effect on pathways that mediate plaque evolution and progression, than on those which result in symptomatic plaque rupture. Differential responses to changes in MMP-2 expression would be consistent with the actions of a wide spectrum of upstream signalling molecules, such as cytokines and growth factors, influencing MMP-2 transcription profiles in a disease stage-specific manner.

Indeed, such differential responses would likely explain why an association is observed with only the G-168/T variant but not the C-1306/T polymorphism, the latter of which paradoxically displays increased differences in allelic expression when analysed in a series of in vitro assays. However, these functional studies are limited by their inability to duplicate the in vivo environment and were designed to examine basal levels of transcription. Consequently these assays are instructive for identifying elements and factors that mediate changes in transcriptional profiles within a defined cell culture model but are constrained by the absence of pathophysiological in vivo modulators. Such modulators would therefore be hypothesised to mediate increased differences in allelic expression of the G-168/T variant, rather than the C-1306/T variant, during the progression of atherosclerosis.

A parsimonious interpretation of these data is that individuals who are genetically predisposed to produce excess MMP-2 (i.e. -168 G homozygotes) have an increased ability to influence the architecture of the vessel wall in vivo in the early, rather than end-stages, of plaque evolution. However, we emphasise that although these findings suggest an important contribution of MMP-2 in CAD, it is not possible to infer whether the functional -168 polymorphism is a causative variant related to disease or a surrogate marker for an unidentified functional variant adjacent to this locus.

. In summary, we have identified a novel association between a functional polymorphism of the MMP-2 promoter and incidence of CAD. In particular, individuals who are homozygous for the -168 G allele are at an increased risk for the development of coronary artery stenosis, but not symptomatic plaque rupture. Our study design alleviates the burden of genotyping multiple candidate polymorphisms and therefore has implications for disease mapping methodologies. We believe that a systematic survey of qualitative and quantitative genetic variation at a locus allied to functional characterisation of variants leads to the identification of a robust panel of markers that are highly informative in genetic epidemiological studies. Such approaches will help identify subsets of variants that capture the disequilibrium structure of the genetic region being investigated, thereby increasing the sensitivity of subsequent candidate gene or genome-wide association studies.

DRAWINGS OF THE INVENTION

Figure 1 shows ethidium bromide-stained agarose gels used for restriction fragment length polymorphism analysis of the MMP-2 gene

Figure 2 shows genetic variants in the MMP-2 gene.

EXAMPLES OF THE INVENTION

MMP-2 spans 17kb and contains 13 exons encoding a 72kDa protein, see Huhtala, P., Chow, L. T., and Tryggvason, K. (1990) J Biol Chem 265, 11077-82. We scanned -3. Ikb of MMP-2 transcribed sequence, 1.9kb of promoter sequence and ~lkb of intronic sequence, see Price, S.P., Greaves, .D .R., and Watkins, H., [2001] J Biol Chem 276, 7549-7558.

Fifteen single base substitutions (SNPs, single-nucleotide polymorphisms) were identified as being distributed throughout the gene with six variants in the promoter, one in the 5'untranslated region, 5'UTR, six in the coding region, one in intronic sequence, and one in the 3'untranslated region, 3'UTR. Reference is made to Figure 2, genetic variants in the MMP-2 gene. It gives a summary of the location, nature and minor allele frequencies of single base substitutions identified in A, the coding region and B, the promoter of the human MMP-2 gene from a panel of 32 unrelated individuals. Nucleotide (nt) positions are referenced relative to the major transcription start-site and amino acids are designated by the standard one letter code.

Of most potential interest, we identified the SNPs at positions -1575 (G/A), -1306 (C/T), -955 (C/A), -790 (T/G), -787 (T/G), -168 (G/T), and +220 (G/C)

INITIAL CLINICAL DATA

We have obtained genotypic data from coronary artery disease (CAD) patients vs. non-CAD, which directly compares allele frequencies between patients (191) and controls (141), which allows for comparison of allele frequencies within the patient subgroup.

observed genotype numbers

-1306 patients controls -168 patients controls 220 patients controls ~CC~ 84 72 GG 171 116 GG 160 113 CT 89 55 GT 20 18 GC 30 27 TT 18 14 TT 0 7 CC 1 1 total 191 141 191 141 191 141

% observed genotype frequencies

expected genotype numbers

-1306 patients controls -168 patients controls 220 patients controls

CC 86 70 GG 172 111 GG 160 113

CT 84 59 GT 19 28 GT 29 26

TT 20 12 TT 1 2 TT 1 1

haplotypes

From this preliminary data, it can be seen that the observed genotype frequencies shows departures from the expected genotype frequencies. Thus, it can be concluded that the genetic variations may play a role in the predisposition to and severity of the disease.

FURTHER CLINICAL STUDIES

Study Population

The 782 unrelated subjects 599 men and 183 women; mean age 63.3 ± 10.1 years) were participants of the Southampton Atherosclerosis Study (SAS) which is an ongoing project designed to investigate genetic determinants of coronary artery disease. In this study, consecutive patients undergoing coronary angiography were invited to take part, and informed consent was obtained from all participants. Demographic (age, sex, body mass index, smoking habit, occupation), clinical (history of myocardial infarction or acute coronary syndrome, hypercholesterolaemia, hypertension, and diabetes mellitus, family history of coronary heart disease, cholesterol and triglyceride levels) and angiographic (location and degree of stenosis) data were recorded. All angiograms were independently evaluated by at least two cardiologists. The study was approved by the South and West Local Research Ethics Committee. All subjects were from a white British population in the Wessex region of Southern England. Control subjects were unrelated individuals in whom angiography did not detect a significant (i.e. >50%) stenosis in any coronary artery (n= 139; 69 men, 70 women; mean age 58.0 ± 11.7 years).

DNA Genotyping DNA was extracted from a 10 ml EDTA-blood sample using the standard salt precipitation method, see Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988) Nucleic Acids Res 16, 1215. Genotyping was performed by restriction fragment polymorphism analysis of engineered (C-1306/T) or naturally occurring (G-168/T) enzymatic sites. Briefly, genomic DNA (20 ng) was amplified in a 50 μl reaction volume containing 25 pmol primers, 200 gM dNTPs, 3 mM MgCb, 50 mM KCI and 2.5 units of Pfu Turbo polymerase (Roche). For C-1306/T, the restriction site cleaved by the enzyme BstXI was engineered using the following amplification primers, indicated thus: BG-1; 5'-TTCTCCAGTGCCTCTTGCTG-3' and BG-2, 5'-AGCTGAGACCTGAAGAGCCAAAG-3' (mismatch is underlined). For G-168/T, a naturally occurring BsaJI restriction site was amplified using the following PCR primers, indicated thus: Prl, 5'-GTTTCCCAGCAGGGGGTTCT-3' and LO-2, 5'-CCTACTCGCCCTCCTCCACTT-3'. A touchdown PCR programme was used for both variants; an initial denaturation at 95°C for 2 mm followed by 15 cycles of denaturation at 95°C for 30 sec, annealing at 62.5°C for 30 sec (minus 0.5°C/ cycle) and extension at 72°C for 30 sec, followed by 19 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec, and concluded with a final extension at 72°C for 7 mm.

For C-1306/T, the 113 bp amplicons (17 μl) were digested in 1 x Buffer 3 (NEB) and 10 units of BstXI at 55°C for 12 hours. The digests were size fractionated on a 5% SeaKem agarose gel at 100 V for 1 hour, and genotypes were independently scored using samples of known genotype as internal controls (-1306C, 93 bp and 20 bp; -1306T, 113 bp).

For G-168/T, the 168 bp amplicons (17 ~tl) were digested in 1 x Buffer 2 (NEB) and 2.5 units of BsaJJ at 60°C for 12 hours. The digests were visualised on a 4% SeaKem agarose gel and genotypes were independently scored using samples of known genotype as internal controls (-168G, 110 bp and 58 bp; -168T, 168 bp).

Statistical Analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc.), version 7.0. Statistically significant differences between groups for continuous or categorical variables were evaluated by Student's unpaired 2 -tailed t test or _² test, respectively. The frequencies of the genotypes and alleles in all subjects were determined by direct count and compared by _² analysis to test for Hardy- Weinberg equilibrium and to compare the case-control data for independence. The independent contribution of each variable to CAD was evaluated by multivariate logistic regression analysis. These analyses were performed in the total cohort as well as in the subgroup of CAD subjects categorised on the basis of acute coronary events (i.e. patients with history of unstable angina and/ or myocardial infarction versus patients with neither). Likelihood ratio statistics were used to compare models with different combinations of predictors. Odds ratios (ORs) and 95% confidence intervals (95% CIs) are reported.

RESULTS

Study Subjects

Demographic and clinical characteristics for the sample of 782 patients with angiographically proven coronary atherosclerosis, and 139 controls without, who participated in the SAS study are shown in Table 1. Table 1. Characteristics of the Study Population

Patients with Control

CAD Subjects

(N=782) (N=139) Risk Factors P values*

Age, y 63.3 + 10.1 58.0+11.7 <0.001

BMI, kg/rn² 27.4 ± 4.2 27.2 ± 4.7 NS

Male sex, n (%) 599 (76.5) 69 (49.6) <0.001

Current or former smoker, n (%) 574 (73.4) 86 (61.8) <0.01

Hypertension, n (%)** 340 (43.4) 50 (35.9) NS

Diabetes mellitus, n (%) 104 (13.2) 6 (4.3) <0.003

Age and BMI are given as mean ± SD. Other values represent number of individuals (n) with percentage (n/N) in parentheses.

* Continuous variables were compared by Student's t test whereas categorical variables were compared by _² test.

** Hypertension defined as systolic blood pressure -60 mm Hg.

As expected, the incidence of several conventional risk factors for CAD, including male sex, smoking, and diabetes mellitus, did significantly differ between CAD patients and controls (P<0.01). In contrast, the two groups were comparable for body mass index and hypertension.

MMP-2 Genotypes and Allele Frequencies of the Subjects

All subjects were genotyped for the C-1306/T and G-168/T promoter polymorphisms of the MMP-2 gene by restriction enzyme digestion, see Figure 1. Figure 1 shows ethidium bromide-stained agarose gels used for restriction fragment length polymorphism analysis of the MMP-2 gene. A, C-1306/T polymorphism of the MMP-2 promoter detected by BstXI restriction endonuclease digestion of the 113 bp PCR product. B, G-168/T polymorphism of the MMP-2 promoter detected by BsaJI restriction endonuclease digestion of the 168 bp PCR product. Inferred genotypes for both enzymatic digests are shown. M, standard 100 bp ladder.

The frequencies of the genotypes and alleles for both loci were determined by direct count and compared by the _² test with the values predicted assuming Hardy-Weinberg equilibrium (HWE). All data fitted the Hardy- einberg expectation with high fidelity (P>0.30). Genotype and allele frequencies of both promoter polymorphisms in CAD patients and controls are shown in Table 2.

Table 2. Distribution of MMP-2 Promoter Genotypes and Allele Frequencies in Patients With CAD and Controls.

Patients with CAD Controls

(N=782) (N=139)

Variables n/N % n/N %

Genotypes

-1306 CC 410/782 52.4 73/139 52.5

-1306 CT 313/782 40.0 52/139 37.4

-1306 TT 59/782 7.6 14/139 10.1 1.15 0.56

Alleles

C 1133/ 1564 72.4 198/278 71.2

T 431 / 1564 27.6 80/278 28.7 0.18 0.675

Genotypes

-168 GG 734/782 93.9 119/139 85.6 -168 GT 47/782 6.0 19/ 139 13.7

-168 T 1 /782 0.1 1/ 139 0.7 12.42 0.0019

Alleles

G 1515/ 1564 96.c 257/278 92.4

T 49/ 1564 3.1 21/278 7.6 12.62 0.0003

n indicates number of subjects (for genotype frequencies) or number of alleles (for allele frequencies). P values refer to the overall difference between groups.

In the total cohort, 365 (39.6%) of the 921 subjects were heterozygous for the C-1306/T variant, whereas 66 (7.1%) subjects were heterozygous for the G- 168/T polymorphism. The genotype and allele frequencies of control subjects were very similar to those previously reported in an independent white British population.

Coronary Atherosclerosis and MMP-2 Genotype

There was no significant difference in the frequencies of genotypes or alleles between patients and control subjects for the -1306 polymorphism, with the frequency of the T allele being 0.276 in patients and 0.287 in controls (Table 2). In contrast, the -168 G/T genotype was found to be under half as frequent in CAD patients than in control subjects (0.06 versus 0.137, respectively), and the difference was highly significant (P=0.0019). Also, allele frequencies were significantly different between the two groups, with the frequency of the T allele being 0.031 in patients and 0.076 in controls (P=0.0003).

Acute Coronary Events and MMP-2 Genotype

Clinical data on acute coronary events (i.e. myocardial infarction, MI; and, unstable angina, UA) were available for 670 CAD patients. To examine potential association with MMP-2 genotype the patient cohort was subdivided into subjects with MI and/or UA {308 men, 76 women; age 62.2 ± 10.2 years) versus patients with neither (213 men, 73 women; age 63.8 ± 10.1). The distributions of genotypes were consistent with HWE. There was no significant difference in the frequencies of genotypes or alleles between these subgroups (i.e. MI/UA vs. neither), with the frequency of the -1306 T allele being 0.268 and 0.28, and the frequency of the -168 T allele being 0.031 and 0.028, respectively, see Table 3.

Table 3. Distribution of MMP-2 Promoter Genotypes and Allele Frequencies in Subdivision of Patients Categorised By Acute Coronary Events

MI indicates rnyocardial infarction; UA, unstable angina

n indicates number of subjects (for genotype frequencies) or number of alleles (for allele frequencies).

P values refer to the overall difference between groups.

Multivariate Logistic Regression Analysis

The results of a logistic regression analysis showed that the association between the G-168/T polymorphism and CAD was independent of potentially confounding continuous (i.e. age and BMI) and binary (i.e. gender, smoking, hypertension, and diabetes mellitus) risk factors, see Table 4.

Table 4. Multivariate Logistic Regression Analysis of Predictors of CAD in Study Population

Y, yes; N, no. * Analysis included 921 subjects (782 with CAD and 139 controls).

** The relative risk associated with the MMP-2 ιβs variant was estimated between G-allele hornozygosity and at least one copy of the T allele.

Analysis showed that hornozygosity for the -168 G allele (seen in 94% of cases) was associated with a significantly higher risk of coronary artery stenosis with an OR of 2.76 (95% CI 1.50 to 5.07, PO.OOlj. A similar analysis was performed to test the potential association between the G-168/T variant and acute coronary events, by subdividing the patients according to history of MI and/ or UA, and no effect was observed, see Table 5.

Table 5. Multivariate Logistic Regression Analysis of Subdivision of Patients Categorised By Acute Coronary Events

Y, yes; N, no.

* Analysis included 670 patients (384 with MI and/ or UA and 286 with neither). ** The relative risk associated with the MMP-2 lββ variant was determined as described for Table 4.

Thus, matrix metalloproteinase-2 (MMP-2) is an enzyme with type IV collagenolytic activity that is implicated in vascular matrix remodelling during atherogenesis. The combinatorial nature of multiple cis-acting elements and trans-acting factors ensure that MMP-2 expression is regulated in a cell- and stimulus-specific manner. Two common promoter polymorphisms of the MMP-2 gene (C-1306/T and G-168/T) influence MMP-2 transcriptional profiles in an allele- specific manner. The invention considers the role of these functional variants in predisposing individuals to coronary artery disease (CAD). We determined the MMP-2 promoter genotypes of 782 subjects with angiographically proven coronary atherosclerosis and 139 control subjects without coronary stenoses by restriction enzyme digestion. The -1306 genotypes were not associated with CAD, whereas the frequency of -168 G/T heterozygotes was significantly lower in CAD patients than controls (6% versus 13.7% respectively; P<0.01 . Logistic regression analysis confirmed that hornozygosity for the -168 G allele (seen in the majority of the population) is associated with a significantly higher risk of CAD (odds ratio [OR] 2.76, 95% confidence interval [CI] 1.50 to 5.07; P<0.001). Among the 782 patients there was no significant relationship between genotype and risk of myocardial infarction and/or unstable angina, for either variant. We conclude that the functional G-168/T polymorphism of the MMP-2 gene is a significant independent predictor of coronary atherosclerosis but not acute coronary events.

Claims

Claim

1. A method of assessing an individual for predisposition to a disease in which MMP-2 plays a role, comprising determining the nucleotide at a site of MMP-2 polymorphism associated with such disease.

2. A method according to claim 1, when used in assessment of predisposition to the disease, or prognosis and /or prevention of such disease in the individual.

3. A method according to claim 1, when used in the profiling and assessment of individuals for medical treatment.

4. A method according to claim 3, wherein the medical treatment is a clinical trial.

5. A method according to any preceding claim, wherein the MMP-2 disease is myocardial infarction, thromboembolism, abdominal aortic aneurism, and other consequences of atherosclerosis; restenosis; arthritis; cancer caused by both metastatic and non metastatic tumours; and obstructive pulmonary disease, emphysema, hepatic fibrosis and other diseases involving fibrosis.

6. A method according to any preceding claim, wherein the polymorphic site is one or more of positions -168 (G/T), -790 (T/G) +220 (G/C) and -1306 (C/T). the position being defined as base pairs from the major transcription start site, itself defined in Huhtala, P., Chow, L. T., and Tiyggvason, K. (1990) J Biol Chem 265, 11077-82.

7. A method according to claim 6, which involves determining the nucleotide sequence at position -168.

8. A method according to any preceding claim, which comprises the steps of subjecting a region of DNA containing an MMP-2 polymorphic sequence to a restriction digestion using at least one restriction endonuclease, and analysing the resulting products to determine the presence or absence of a restriction site for the endonuclease as an indication of the nature of the genetic variant.

9. A method according to claim 8, in which the region containing the polymorphic sequence is amplified prior to restriction digestion.

10. A method according to claim 9, in which the region of the MMP-2 gene containing a polymorphic sequence is amplified using one or more oligonucleotide primers designed to incorporate into the amplified DNA sequence a restriction endonuclease recognition site comprising a variant of the polymorphic sequence.

11. A method according to any of claims 1 to 7, using PCR in combination with denaturing HPLC or SSCP analysis.

12. A method according to any of claims 1 to 7, wherein a given variant of the polymorphic sequence is determined by using at least one oligonucleotide capable of hybridising specifically to the variant of interest under suitably stringent conditions.

13. An oligonucleotide which under stringent conditions specifically hybridises to a variant of a polymorphic sequence of this invention, or its reverse complement.

14. An assay kit for use in the detection of MMP-2 gene variants, comprising one or more oligonucleotides of claim 13, and optionally a polymerase enzyme.