The present invention relates to a method of identifying a subject predisposed to ischemic stroke. The method includes the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

Description of the Invention

In one form the present invention provides a method of identifying a subject predisposed to ischemic stroke, the method including the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

The subject is any human subject of either gender for which the predisposition to ischemic stroke is to be determined. Preferably, the subject is a human of Caucasian origin.

The ischemic stroke is any thrombotic or embolic stoke that may occur in the subject, including a cardioembolic or atherothrombotic ischemic stroke. Preferably, the ischemic stroke is an atherothrombotic ischemic stroke.

Preferably, the ischemic stroke is a small vessel stroke (ie a lacunar stroke). Accordingly, in a preferred form, the present invention provides a method of identifying a subject predisposed to lacunar stroke, the method including the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

The mutation in the subject is any mutation in any gene or extragenic region that reduces the release rate of tPA from an endothelial cell. For example, the mutation may be in a gene or region not associated with the tPA locus, or be a mutation in the tPA locus, such as a mutation in a region upstream of the tPA coding region that affects transcription of the tPA gene, a mutation in an enhancer, a mutation in an exon of the tPA gene, a mutation in an intron that affects splicing, or a mutation in the 3' region of the tPA that affects translation or mRNA stability.

The mutation may be present in one or both alleles of the particular gene. Most preferably, the mutation is present in both alleles of the particular gene.

Preferably, the mutation is a mutation in the tPA locus. In a particularly preferred form, the mutation is in both alleles of the tPA locus.

In this regard, the structure of the human tPA locus is as described in Degen et aL (1986) J. Biol. Chem. 261(15):6972-6985. The tPA gene consists of 14 exons and 13 introns.

The upstream region includes a number of regions that act to regulate transcription of the gene. For example, transcription of the human tPA gene is regulated by a multi-hormonal responsive enhancer at -7 kb, and transient transfection studies have shown that tPA reporter constructs having the -7 kb enhancer require SP1 binding in the proximal promoter region allow induction of the promoter by retinoic acid mediated by the enhancer.

A C to T polymorphism at -7351 in the upstream region of tPA has been previously identified and this mutation reduces the release rate of tPA in vivo in individuals heterozygous and homozygous for the polymorphism (Ladenvall et al. (2000) Thromb. Haemost. 84: 150-155). This mutation is in an SP1 binding site.

The ability of a mutation to reduce the release rate of tissue plasminogen activator may be determined by a suitable method known in the art. For example, the ability of specific mutation to reduce the release rate of tPA from endothelial cells may be determined by culturing endothelial cells in vitro carrying the mutation and determining the rate of release of tPA from the endothelial cells, by washing the cells with fresh medium and determining the extent of rate of change of tPA released into the medium over time, as compared to wild type endothelial cells. An enzyme-linked immunosorbent assay (ELISA) may be employed for the quantitative determination of total t-PA antigen (eg TintElize.RTM. t-PA, Biopool AB). Such assays are based on the double-antibody principle. Free t-PA and t-PA in complex with inhibitors are detected with equal efficiency.

To induce release of tPA from the endothelial cells, agents known in the art that induce release of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method for inducing the release of tPA from endothelial cells in vitro is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc Biol 1 9(7):1 796-803.

Alternatively, the rate of release of tPA in a subject may be determined directly, for example by determination of forearm release rates as compared to a subject without the mutation. A suitable method for determining the rate of release of tPA in vivo is as described in Jern et al. (1999) Arterioscler. Thromb. Vasc. Biol 19(2): 454-459.

Preferably, the mutation is located in the tPA locus. More preferably, the mutation in the subject is a mutation in an upstream region of the tPA locus. More preferably, the mutation is a mutation in an enhancer element in the tPA locus. Most preferably, the mutation is a cytosine to thymine mutation at position -7351 of the upstream region of the tPA locus.

The -7351 cytosine to thymine mutation may be present in one or both alleles of the tPA locus. Most preferably, the mutation is present in both alleles of the tPA locus.

Accordingly, in a preferred form, the present invention provides a method of identifying a subject predisposed to ischemic stroke, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

As discussed previously, preferably the ischemic stroke is a lacunar stroke. Accordingly, in another preferred form, the present invention provides a method of identifying a subject predisposed to lacunar stroke, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

The identification of a mutation in the subject that reduces the release rate of tPA may be determined by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

Various methods may be used to identify the mutation. DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect the mutation. IL this case, identification of the mutation will usually involve amplification of the region containing the mutation from nucleic acid isolated from the subject (generally genomic DNA), although it is also possible to identify the mutation by sequencing a clone of the region derived from a particular subject, with or without amplification.

Another approach for identifying mutations is the single-stranded conformation polymorphism assay (SSCA) (as described in Orita et al. (1989) Genomics 5(4): 874-879. This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. Fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation.

Another approach is based on the detection of mismatches between two complementary DNA strands, including clamped denaturing gel electrophoresis (as described in Sheffield et al. (1991) Am. J. Hum. Genet. 49:699-706), heteroduplex analysis (as described in White et al. (1992) Genomics 12:301-303) and chemical mismatch cleavage (as described in Grompe et al. (1989) Proc. Natl. Acad. Sci. USA 86:5855-5892). Once a specific mutation is identified, an allele specific detection approach such as allele specific oligonucleotide hybridization can be utilized.

If DNA sequence analysis is used to identify a mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing, a region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. In the case of the mutation being the -7351 C to T mutation in the upstream region of the tPA locus, a preferred region for amplification. is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. If sequence specific primers are used to amplify the DNA, a consensus primer and one of two alternative primers will be used. Each of the alternative primers will have a 3' terminal nucleotide that either corresponds to the wild type sequence (a WT primer) or the polymorphic sequence (a SNP primer). In this case, amplification will only occur from the template having the correct complementary nucleotide.

Other methods to identify mutations involve hybridization of nucleic acid containing the mutation with other nucleic acids (ie a reporter nucleic acid) that allows discrimination between differences in nucleic acid sequences. For example, Southern analysis with an oligonucleotide may be used to detect mutations. Alternatively, methods are known in the art in which the oligonucleotide is attached to a solid substrate, such as chip, and the binding of a nucleic acid containing a mutation detected by binding (or lack thereof) to the oligonucleotide. In these cases, the identification of a mutation in a subject also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

In the case of the mutation being the -7351 C to T mutation, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Accordingly, in another form the present invention provides an isolated nucleic acid, the polynucleotide consisting of the sequence spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

The sequence of the region spanning nucleotides 1840 to 2245 of SEQ ID No.1 is as follows -- see Original Patent.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat ischemic stroke.

Accordingly, in another form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat ischemic stroke, the method including the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

In addition, in a preferred form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat ischemic stroke, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

The present invention also provides a method of identifying a subject predisposed to ischemic stroke, the method include the step of identifying a reduced rate of release of tissue plasminogen activator in the subject.

The ischemic stroke is any thrombotic or embolic stoke that may occur in the subject, including a cardioembolic or atherothrombotic ischemic stroke. Preferably, the ischemic stroke is an atherothrombotic ischemic stroke.

Preferably, the ischemic stroke is a small vessel stroke (ie a lacunar stroke). Accordingly, in a preferred form, the present invention provides a method of identifying a subject predisposed to lacunar stroke, the method include the step of identifying a reduced rate of release of tissue plasminogen activator in the subject.

The subject is any human subject of either gender for which the predisposition to ischemic stroke is to be determined. Preferably, the subject is a human of Caucasian origin.

Preferably, the subject has a mutation in the tPA locus that reduces the release rate of tPA in the subject. More preferably, the subject has a mutation in an upstream region of the tPA locus that reduces the release rate of tPA in the subject. More preferably, the subject has a mutation in an enhancer element in the tPA locus that reduces the release rate of tPA in the subject. Most preferably, the subject has a mutation in a cytosine to thymine at position -7351 of the upstream region of the tPA locus.

The -7351 cytosine to thymine mutation may be present in one or both alleles of the tPA locus. Most preferably, the mutation is present in both alleles of the tPA locus.

The identification of a reduced rate of release of tPA in the subject may by a suitable method known in the art. For example, the rate of release of tPA in a subject may be determined directly, by determination of forearm release rates. A suitable method for determining the rate of release of tPA in vivo is as described in Jern et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(2): 454-459.

This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat ischemic stroke.

Accordingly, in another form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat ischemic stroke, the method including the step of identifying a reduced release rate of tissue plasminogen activator in the subject.

The present invention also provides a method of identifying a subject predisposed to small vessel occlusion, the method including the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

The subject is any human subject of either gender for which the predisposition to small vessel occlusion is to be determined. Preferably, the subject is a human of Caucasian origin.

The small vessel occlusion in the various forms present invention is any thrombotic or embolic occlusion that may occur in a small vessel in a subject, including small vessel occlusion manifesting clinically as a lacunar stroke, dementia, ischemic heart disease (including ischemic cardiomyopathy), peripheral vascular disease, disseminated intravascular coagulation, small vessel vasculitis, ischemic neuropathy, ischemic retinopathy, ischemic gastropathy (including small and large bowel ischemia), diffuse pulmonary embolism and vascular impotence.

Preferably, the small vessel occlusion occurs in the brain, including a small vessel occlusion manifesting clinically as a lacunar stroke.

The mutation in the subject is any mutation in any gene or extragenic region that reduces the release rate of tPA from an endothelial cell. For example, the mutation may be a mutation in the tPA locus, such as a mutation in a region upstream of the tPA coding region, a mutation in an enhancer element, a mutation in an exon of the tPA gene, a mutation in an intron that affects splicing, or a mutation in the 3' region of the tPA locus that affects translation or mRNA stability.

The mutation may be present in one or both alleles of the particular gene. Most preferably, the mutation is present in both alleles of the particular gene.

As discussed previously, the identification of a mutation in the subject that reduces the release rate of tPA may be determined by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

The ability of a mutation to reduce the release rate of tissue plasminogen activator may be determined by a suitable method known in the art. For example, the ability of specific mutation to reduce the release rate of tPA from endothelial cells may be determined by culturing endothelial cells in vitro carrying the mutation and determining the rate of release of tPA from the endothelial cells, by washing the cells with fresh medium and determining the extent of rate of change of tPA released into the medium over time, as compared to wild type endothelial cells. An enzyme-linked immunosorbent assay (ELISA) may be employed for the quantitative determination of total t-PA antigen (eg TintElize.RTM. t-PA, Biopool AB). Such assays are based on the double-antibody principle. Free t-PA and t-PA in complex with inhibitors are detected with equal efficiency.

To induce release of tPA from the endothelial cells, agents known in the art that induce release of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method for inducing the release of tPA from endothelial cells in vitro is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc Biol. 19(7):1 796-803.

Preferably, the mutation in the subject is a mutation in the tPA locus. More preferably, the mutation is in an upstream region of the tPA locus. More preferably, the mutation is in an enhancer element in the tPA locus. Most preferably, the mutation is a cytosine to thymine mutation at position -7351 of the upstream region of the tPA locus.

The -7351 cytosine to thymine mutation may be present in one or both alleles of the tPA locus. Most preferably, the mutation is present in both alleles of the tPA locus. Accordingly, in a preferred form, the present invention provides a method of identifying a subject predisposed to small vessel occlusion, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

As discusses previously, the identification of a mutation in the subject that reduces the release rate of tPA may be determined by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

As discussed previously, various methods may be used to identify a mutation.

DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect a mutation. Another approach for identifying mutations is the single-stranded conformation polymorphism assay (SSCA) (as described in Orita et al. (1989) Genomics 5(4): 874-879. This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. Fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation.

Another approach is based on the detection of mismatches between two complementary DNA strands, including clamped denaturing gel electrophoresis (as described in Sheffield et al. (1991) Am. J. Hum. Genet. 49:699-706), heteroduplex analysis (as described in White et al. (1992) Genomics 12:301-303) and chemical mismatch cleavage (as described in Grompe et al. (1989) Proc. NatI. Acad. Sci. USA 86:5855-5892). Once a specific mutation is identified, an allele specific detection approach such as allele specific oligonucleotide hybridization can be utilized.

If DNA sequence analysis is used to identify a mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing, a region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. In the case of the mutation being the -7351 C to T mutation in the upstream region of the tPA locus, a preferred region for amplification is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. If sequence specific primers are used to amplify the DNA, a consensus primer and one of two alternative primers will be used. Each of the alternative primers will have a 3' terminal nucleotide that either corresponds to the wild type sequence (a WT primer) or the polymorphic sequence (a SNP primer). In this case, amplification will only occur from the template having the correct complementary nucleotide.

In the case of the mutation being the -7351 C to T mutation, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of the mutation also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat a disease or condition associated with small vessel occlusion.

Accordingly, in another form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat a disease or condition associated with small vessel occlusion, the method including the step of identifying a mutation in the subject that reduces the release rate of tissue plasminogen activator.

In addition, in a preferred form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat a disease or condition associated with small vessel occlusion, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

The present invention also provides a method of identifying a subject predisposed to small vessel occlusion, the method including the step of identifying a reduced rate of release of tissue plasminogen activator in the subject.

The subject is any human subject of either gender for which the predisposition to small vessel occlusion is to be determined. Preferably, the subject is a human of Caucasian origin.

The small vessel occlusion is any thrombotic or embolic occlusion that may occur in a small vessel in a subject, including small vessel occlusion manifesting clinically as a lacunar stroke, dementia, ischemic heart disease (including ischemic cardiomyopathy), peripheral vascular disease, disseminated intravascular coagulation, small vessel vasculitis, ischemic neuropathy, ischemic retinopathy, ischemic gastropathy (including small and large bowel ischemia), diffuse pulmonary embolism and vascular impotence.

Preferably, the small vessel occlusion occurs in the brain, including a small vessel occlusion manifesting clinically as a lacunar stroke.

Preferably, the subject has a mutation in the tPA locus that reduces the release rate of tPA in the subject. More preferably, the subject has a mutation in an upstream region of the tPA locus that reduces the release rate of tPA in the subject. More preferably, the subject has a mutation in an enhancer element in the tPA locus that reduces the release rate of tPA in the subject. Most preferably, the subject has a mutation in a cytosine to thymine at position -7351 of the upstream region of the tPA locus.

The -7351 cytosine to thymine mutation may be present in one or both alleles of the tPA locus. Most preferably, the mutation is present in both alleles of the tPA locus.

The identification of a reduced rate of release of tPA in the subject may by a suitable method known in the art. For example, the rate of release of tPA in a subject may be determined directly, by determination of forearm release rates.

A suitable method for determining the rate of release of tPA in vivo is as described in Jem et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(2): 454-459.

This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat a disease or condition associated with small vessel occlusion.

Accordingly, in another form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat a disease or condition associated with small vessel occlusion, the method including the step of identifying a reduced release rate of tissue plasminogen activator in the subject.

The present invention also provides a method of identifying a subject predisposed to a disease or condition associated with small vessel occlusion, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

The subject is any human subject of either gender for which the predisposition to developing a disease or condition associated with small vessel occlusion is to be determined. Preferably, the subject is a human of Caucasian origin.

Example of diseases or conditions associated with small vessel occlusion include lacunar stroke, dementia, ischemic heart disease (including ischemic cardiomyopathy), peripheral vascular disease, disseminated intravascular coagulation, small vessel vasculitis, ischemic neuropathy, ischemic retinopathy, ischemic gastropathy (including small and large bowel ischemia), diffuse pulmonary embolism and vascular impotence.

Preferably, the disease or condition associated with small vessel occlusion occurs in the brain, including a lacunar stroke.

The identification in the subject of the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus may be by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

To identify the cytosine to thymine mutation at position -7351 both alleles of the tissue plasminogen activator locus, DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect a mutation.

As discussed previously, various methods may be used to identify the mutation. If DNA sequence analysis is used to identify the mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides (C and T) at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing the mutation, a suitable region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. A preferred region for amplification is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation in both alleles may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. Once again, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of the mutation also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat a disease or condition associated with small vessel occlusion.

Accordingly, in another form the present invention also provides a method of identifying a subject suitable for intervention to prevent and/or treat a disease or condition associated with small vessel occlusion, the method including the step of identifying in the subject the presence of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus.

The present invention also provides a method of determining the risk of ischemic stroke in a subject, the method including the step of determining the presence in the subject of a cytosine to thymine mutation at position -7351 in one or both of the alleles of the tissue plasminogen activator locus.

The risk of ischemic stroke in a subject is the probability that a subject with a mutation in the tPA locus may suffer an ischemic stroke as compared to the probability that a subject in the general population may suffer an ischemic stroke, under circumstances where other risk factors (eg atrial fibrillation, history of smoking) for having an ischemic stroke between the subjects are the same.

In this regard, the presence of a cytosine to thymine mutation at position -7351 in one or both alleles of the tPA locus (ie an individual heterozygous for the polymorphism) indicates an elevated risk that the subject may suffer an ischemic stroke as compared to a subject in the general population, under circumstances where other risk factors (eg atrial fibrillation, history of smoking) for having an ischemic stroke or a small vessel occlusion between the subjects are the same. The presence of a mutation in both alleles indicates a further elevated risk over the presence of a mutation in one allele.

For example, the presence in the subject of a cytosine to thymine mutation at position -7351 in one allele of the tissue plasminogen activator locus indicates an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for a subject not having the mutation in either allele of the tissue plasminogen locus.

Alternatively, the presence in the subject of a cytosine to thymine mutation at position -7351 in one allele of the tissue plasminogen activator locus indicates an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for a subject in the general population, or an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for another subject with similar other risk factors for having an ischemic stroke.

In addition, the presence in the subject of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus indicates an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for a subject not having the mutation in one or both alleles of the tissue plasminogen locus.

Alternatively, the presence in the subject of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus indicates an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for a subject in the general population, or indicates an increased risk of ischemic stroke in the subject, as compared to the risk of ischemic stroke for a subject with similar other risk factors for having an ischemic stroke.

The subject is any human subject of either gender for which the risk of suffering an ischemic stroke is to be determined. Preferably, the subject is a human of Caucasian origin.

The ischemic stroke is any thrombotic or embolic stoke that may occur in the subject, including a cardioembolic or atherothrombotic ischemic stroke. Preferably, the ischemic stroke is an atherothrombotic ischemic stroke.

Preferably, the ischemic stroke is a small vessel stroke (ie a lacunar stroke). Accordingly, in a preferred form, the present invention provides a method of determining the risk of lacunar stroke in a subject, the method including the step of determining the presence in the subject of a cytosine to thymine mutation at position -7351 in one or both of the alleles of the tissue plasminogen activator locus.

As discussed previously, determination of the presence in the subject of a cytosine to thymine polymorphism at position -7351 in one or both of the alleles of the tissue plasminogen activator locus may be by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

As discussed previously, various methods can be use to determine the presence of the cytosine to thymine mutation at position -7351 in one or both alleles of the tissue plasminogen activator locus, such as DNA sequencing (either manual sequencing or automated fluorescent sequencing).

If DNA sequence analysis is used to identify the mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides (C and T) at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing the mutation, a suitable region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. A preferred region for amplification is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. Once again, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of the mutation also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

The present invention also provides a method of determining the risk of small vessel occlusion in a subject, or the risk of developing a disease or condition associated with small vessel occlusion in a subject, the method including the step of determining the presence in the subject of a cytosine to thymine mutation at position -7351 in one or both of the alleles of the tissue plasminogen activator locus.

The risk of small vessel occlusion in a subject is the probability that a subject with a mutation in the tPA locus may suffer small vessel occlusion as compared to the probability that a subject in the general population may suffer small vessel occlusion, under circumstances where other risk factors for having a small vessel occlusion between the subjects are the same.

In this regard, the presence of a cytosine to thymine mutation at position -7351 in one or both alleles of the tPA locus (ie an individual heterozygous for the polymorphism) indicates an elevated risk that the subject may suffer an a small vessel occlusion as compared to a subject in the general population, under circumstances where other risk factors for having a small vessel occlusion between the subjects are the same. The presence of a mutation in both alleles indicates a further elevated risk over the presence of a mutation in one allele.

For example, the presence in the subject of a cytosine to thymine mutation at position -7351 in one allele of the tissue plasminogen activator locus indicates an increased risk of small vessel occlusion or developing a disease associated with small vessel occlusion in the subject, as compared to the risk for a subject not having the mutation in either alleles of the tissue plasminogen locus.

Alternatively, the presence in the subject of a cytosine to thymine mutation at position -7351 in one allele of the tissue plasminogen activator locus indicates an increased risk of small vessel occlusion or developing a disease associated with small vessel occlusion in the subject, as compared to the risk for a subject in the general population, or an increased risk of small vessel occlusion or developing a disease associated with small vessel occlusion in the subject ischemic stroke in the subject, as compared to the risk for another subject with similar other risk factors for having a small vessel occlusion.

The presence in the subject of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus indicates an increased risk of srhall vessel occlusion or developing a disease associated with small vessel occlusion in the subject, as compared to the risk for a subject not having the mutation in one or both alleles of the tissue plasminogen locus.

Alternatively, the presence in the subject of a cytosine to thymine mutation at position -7351 in both alleles of the tissue plasminogen activator locus indicates an increased risk of small vessel occlusion or developing a disease associated with small vessel occlusion in the subject, as compared to the risk for a subject in the general population, or an increased risk of small vessel occlusion or developing a disease associated with small vessel occlusion in the subject, as compared to the risk for another subject with similar other risk factors for having a small vessel occlusion.

The subject is any human subject of either gender for which the risk of suffering a small vessel occlusion is to be determined. Preferably, the subject is a human of Caucasian origin.

The small vessel occlusion is any thrombotic or embolic occlusion that may occur in a small vessel in a subject, including small vessel occlusion manifesting clinically as lacunar stroke, dementia, ischemic heart disease (including ischemic cardiomyopathy), peripheral vascular disease, disseminated intravascular coagulation, small vessel vasculitis, ischemic neuropathy, ischemic retinopathy, ischemic gastropathy (including small and large bowel ischemia), diffuse pulmonary embolism and vascular impotence.

Preferably, the small vessel occlusion occurs in the brain, including a small vessel occlusion manifesting clinically as a lacunar stroke.

As discussed previously, determination of the presence in the subject of a cytosine to thymine polymorphism at position -7351 in one or both of the alleles. of the tissue plasminogen activator locus may be by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

To determine the presence of the cytosine to thymine mutation at position -7351 in one or both alleles of the tissue plasminogen activator locus, DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect a mutation.

If DNA sequence analysis is used to identify the mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides (C and T) at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing the mutation, a suitable region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. A preferred region for amplification is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. Once again, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCAAACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of the mutation also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

The present invention also provides an isolated nucleic acid consisting of the sequence according to SEQ. ID No. 3 or RNA equivalent thereof, or an isolated nucleic acid with one or more base substitutions in the sequence according to SEQ ID No. 3, wherein the nucleic acid hybridises with the complement of SEQ ID No. 3 under stringent hybridisation conditions and the stringent hybridisation conditions include hybridisation in 6.times. SSC at 42.degree. C. and washing in 2.times. SSC at 20.degree. C.

This form of the present invention contemplates an isolated nucleic acid consisting of the sequence according to SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), or a RNA equivalent thereof, or an isolated nucleic acid with one or more base substitutions of this sequence which hybridises with the complement of SEQ ID No. 3 under stringent hybridisation conditions, wherein the stringent reaction conditions include hybridisation in 6.times. SSC at 42.degree. C. and washing in 2.times. SSC at 20.degree. C.

The nucleic acid may be synthesized by a standard method known in the art. For example, phosphorothioate oligonucleotides may be synthesized by the method as described in Stein et al. (1988) Nucl. Acids Res. 16: 3209.

Stringent hybridisation conditions are conditions that allow complementary nucleic acids to bind to each other within a range from at or near the Tm (Tm is the melting temperature) to about 20.degree. C. below Tm.

Factors such as the length of the complementary regions, type and composition of the nucleic acids (DNA, RNA, base composition), and the concentration of the salts and other components (e.g. the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) must all be considered, essentially as described in in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).

For example, conditions that allowing the nucleic acid to hybridise with the complement of SEQ ID No. 3 under stringent conditions are as follows: prehybridization may be performed in a prehybridization solution (eg 6.times. SSC (1.times.=150 mM NaCI, 15 mM sodium citrate, pH 7.0), 5.times. Denhardt's reagent (1 g/l each of Ficoll, Polyvinyl-pyrrolidone, Bovine Serum Albumin), 1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA) for 2 to 12 hours. Hybridizition of the probe with the target (ie filter) may then be performed under conditions such as 6.times. SSC, 1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, at 42.degree. C. overnight. The filter may then be washed with 2.times. SSC and 0.5% SDS at room temperature for 15 min at 20.degree. C.

The present invention also provides an isolated nucleic acid consisting of the sequence according to SEQ. ID No. 4 or RNA equivalent thereof, or an isolated nucleic acid with one or more base substitutions in the sequence according to SEQ ID No. 4, wherein the nucleic acid hybridises with the complement of SEQ ID No. 4 under stringent hybridisation conditions and the stringent hybridisation conditions include hybridisation in 6.times. SSC at 42.degree. C. and washing in 2.times. SSC at 20.degree. C.

This form of the present invention contemplates an isolated nucleic acid consisting of the sequence according to SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3') or a RNA equivalent thereof, or an isolated nucleic acid with one or more base substitutions of this sequence which hybridises with the complement of SEQ ID No. 4 under stringent hybridisation conditions, wherein the stringent reaction conditions include hybridisation in 6.times. SSC at 42.degree. C. and washing in 2.times. SSC at 20.degree. C.

The nucleic acid may be synthesized by a standard method known in the art. For example, phosphorothioate oligonucleotides may be synthesized by the method as described in Stein et al. (1988) Nucl. Acids Res. 16: 3209.

Stringent hybridisation conditions are conditions that allow complementary nucleic acids to bind to each other within a range from at or near the Tm (Tm is the melting temperature) to about 20.degree. C. below Tm.

Factors such as the length of the complementary regions, type and composition of the nucleic acids (DNA, RNA, base composition), and the concentration of the salts and other components (e.g. the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) must all be considered, essentially as described in in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).

For example, conditions that allowing the nucleic acid to hybridise with the complement of SEQ ID No. 4 under stringent conditions are as follows: prehybridization may be performed in a prehybridization solution (eg 6.times. SSC (1.times.=150 mM NaCl, 15 mM sodium citrate, pH 7.0), 5.times.Denhardt's reagent (1 g/l each of Ficoll, Polyvinyl-pyrrolidone, Bovine Serum Albumin), 1.0% SDS, 10 ug/ml denatured, fragmented salmon sperm DNA) for 2 to 12 hours. Hybridizition of the probe with the target (ie filter) may then be performed under conditions such as 6.times. SSC, -1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, at 42.degree. C. overnight. The filter may then be washed with 2.times. SSC and 0.5% SDS at room temperature for 15 min at 20.degree. C.

The present invention also provides an isolated nucleic acid with one or more base substitutions in the sequence according to SEQ ID No. 3, wherein the nucleic acid has at least 80% homology to SEQ. ID No. 3 or RNA equivalent thereof.

This form of the present invention contemplates an isolated nucleic acid with one or more substitutions in the sequence of SEQ ID No. 3, the nucleic acid having at least 80% homology to SEQ ID No. 3 or RNA equivalent thereof.

Various algorithms exist for determining the degree of homology between any two nucleic acid sequences. For example, the BLAST algorithm can be used for determining the extent of sequence homology between two sequences. BLAST identifies local alignments between two sequences and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul etal., 1990, J. Mol. Biol. 215:403-410.

Preferably, the nucleic has at least 90% homology to SEQ. ID No. 3 or RNA equivalent thereof. Most preferably, the nucleic has at least 95% homology to SEQ. ID No. 3 or RNA equivalent thereof.

The nucleic acid may be synthesized by a standard method known in the art. For example, phosphorothioate oligonucleotides may be synthesized by the method as described in Stein et al. (1988) Nucl. Acids Res. 16: 3209.

The present invention also provides an isolated nucleic acid with one or more base substitutions in the sequence according to SEQ ID No. 4, wherein the nucleic acid has at least 80% homology to SEQ. ID No. 4 or RNA equivalent thereof.

This form of the present invention contemplates an isolated nucleic acid with one or more substitutions in the sequence of SEQ ID No. 4, the nucleic acid having at least 80% homology to SEQ ID No. 4 or RNA equivalent thereof.

Once again, various algorithms exist for determining the degree of homology between any two nucleic acid sequences. For example, the BLAST algorithm can be used for determining the extent of sequence homology between two sequences. BLAST identifies local alignments between two sequences and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al., 1990, J. Mol. Biol. 215:403-410.

Preferably, the nucleic has at least 90% homology to SEQ. ID No. 4 or RNA equivalent thereof. Most preferably, the nucleic has at least 95% homology to SEQ. ID No. 4 or RNA equivalent thereof.

The nucleic acid may be synthesized by a standard method known in the art. For example, phosphorothioate oligonucleotides may be synthesized by the method as described in Stein et al. (1988) Nucl. Acids Res. 16: 3209.

The present invention also provides a method of identifying an agent capable of increasing the release rate of tissue plasminogen activator from a cell, the method including the steps of: (a) exposing an agent to a cell including a mutation that decreases the release rate of tissue plasminogen activator from the cell; (b) determining the release rate of tissue plasminogen activator from the cell so exposed to the agent; and (c) identifying the agent as an agent capable of increasing the release rate of tissue plasminogen activator from the cell.

This form of the present invention is directed to the identification of agents that are capable of increasing the release rate of tissue plasminogen activator from a cell. Agents so identified are candidate compounds for administering to a subject to reduce the likelihood of the subject suffering an ischemic stroke or a small vessel occlusion, and in particular a lacunar stroke.

The agent is any agent for which the ability to increase the release rate of tissue plasminogen activator from the cell is to be determined.

The term "exposing" is be understood to include within its scope the external administration of the agent to a cell or the intracellular expression of the agent in the cell. Accordingly, the exposing of an agent to a cell may be by way of contacting the cell with the agent, or for example, by way of transforming the cell with a recombinant nucleic acid capable of directing the expression of the agent in the cell.

The cell is any cell having a mutation that decreases the release rate of tissue plasminogen activator (as compared to a similar cell not having the mutation).

Preferably the cell is an endothelial cell. Most preferably, the endothelial cell is a human endothelial cell. An example of a suitable endothelial cell is a human umbilical vein endothelial cell (HUVEC).

The mutation in the cell is any mutation that reduces the release rate of tPA from the cell. For example, the mutation may be in a gene or region not associated with the tPA locus, or be a mutation in the tPA locus, such as a mutation in a region upstream of the tPA coding region that affects transcription of the tPA gene, a mutation in an exon of the tPA gene, a mutation in an intron that affects splicing, or a mutation in the 3' region of the tPA that affects translation or mRNA stability.

The mutation may be present in one or both alleles of the particular gene.

Preferably, the mutation is a mutation in the tPA locus that reduces the release rate of tPA from an endothelial cell.

The ability of a mutation to reduce the release rate of tissue plasminogen activator may be confirmed by a suitable method known in the art. For example, the ability of specific mutation in the tPA locus to reduce the release rate of tPA from endothelial cells may be determined by culturing endothelial cells in vitro carrying the mutation and determining the rate of release of tPA from the endothelial cells, by washing the cells with fresh medium and determining the extent of rate of change of tPA released into the medium over time, as compared to wild type endothelial cells. To induce release of tPA from the endothelial cells, agents known in the art that induce secretion of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method for inducing the release of tPA from endothelial cells in vitro is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc Biol. 19(7):1796-803.

An enzyme-linked immunosorbent assay (ELISA) may be employed for the quantitative determination of total t-PA antigen (eg TintElize.RTM. t-PA, Biopool AB). Such assays are based on the double-antibody principle. Free t-PA and t-PA in complex with inhibitors are detected with equal efficiency.

Preferably, the mutation in the cell is a mutation in an upstream region of the tPA gene. More preferably, the mutation is a mutation in an enhancer element in the tPA gene. Most preferably, the mutation is a cytosine to thymine mutation at position -7351 of the upstream region of the tPA locus.

The -7351 cytosine to thymine mutation may be present in one or both alleles of the tPA locus.

Accordingly, in a preferred form, the present invention also provides a method of identifying an agent capable of increasing the release rate of tissue plasminogen activator from a cell, the method including the steps of: (a) exposing an agent to a cell including a cytosine to thymine polymorphism at position -7351 in one or both alleles of the tissue plasminogen activator locus; (b) determining the release rate of tissue plasminogen activator from the cell so exposed to the agent; and (c) identifying the agent as an agent capable of increasing the release rate of tissue plasminogen activator from the cell.

Most preferably, the -7351 cytosine to thymine mutation is present in both alleles of the tPA locus.

As will be appreciated, the ability of an agent to increase the release rate of tissue plasminogen activator from a cell will depend on the concentration of the agent exposed to the cell. Accordingly, the agent will be exposed to the cell at a suitable concentration for testing the ability of the agent at that concentration to increase the release rate of tissue plasminogen activator from the cell.

A known agent that increases rate of release of tPA from endothelial cells is monosodium [2-(6-hydroxynaphthalen-2-yl)-6-methyl-pyrimidin4-yloxy]acetate dihydrate (JTV-926) (Ueshima et al. (2002) "Function of tissue-type plasminogen activator releaser on vascular endothelial cells in thrombolysis in vivo" Thrombosis Haemostasis 87: 1069-74).

The determination of the release rate of tissue plasminogen activator from a cell may be performed by a suitable method known in the art. For example, the ability of an agent to alter the release rate of tPA from endothelial cells may be determined by culturing endothelial cells in vitro and determining the rate of release of tPA from the endothelial cells in the presence and absence of the agent. To determine release rates, the cells may be washed with fresh medium and the extent of rate of change of tPA released into the medium over time determined. To induce release of tPA from the endothelial cells, compounds known in the art that induce secretion of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method for inducing the release of tPA from endothelial cells in vitro is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc BioL 19(7):1796-803.

An enzyme-linked immunosorbent assay (ELISA) may be employed for the quantitative determination of total t-PA antigen (eg TintElize.RTM. t-PA, Biopool AB). Such assays are based on the double-antibody principle. Free t-PA and t-PA in complex with inhibitors are detected with equal efficiency.

Agents that increase the release rate of tPA may be so identified by comparison of the effect of the agents on tPA release rates, as compared to release rates in the absence of an agent, and as compared to known agents that increase the release rate of tPA (eg JTV-926). An agent capable of increasing the release rate of tissue plasminogen activator so identified is a candidate compound for administering to subject to reduce the likelihood of the subject suffering an ischemic stroke (in particular lacunar strokes) or a small vessel occlusion.

The present invention also provides a method of identifying an agent capable of increasing the release rate of tissue plasminogen activator from a cell, the method including the steps of: (a) exposing an agent to a cell transformed with all or part of the tissue plasminogen activator locus, wherein the transformed locus includes a cytosine to thymine mutation at position -7351 and the transformed locus regulates expression of a reporter gene; (b) determining the level of expression of the reporter gene in the cell so exposed to the agent; (c) identifying an agent capable of increasing the expression of the reporter gene; and (d) identifying the agent capable of increasing the expression of the reporter gene as an agent capable of increasing the release rate of tissue plasminogen activator from a cell.

This form of the present invention is also directed to the identification of agents that are capable of increasing the release rate of tissue plasminogen activator from a cell. Agents so identified are candidate compounds for administering to subject to reduce the likelihood of the subject suffering an ischemic stroke (in particular lacunar stroke) or a small vessel occlusion.

The cell is any cell transformed with all or part of the tissue plasminogen locus having a cytosine to thymine mutation at position -7351 and regulating the expression of a suitable reporter gene.

Examples of a suitable cell that may be transformed include endothelial cells (eg HUVECs, bovine aortic endothelial cells) and HeLa cells.

Preferably the transformed cell is an endothelial cell. More preferably, the transformed cell is a human endothelial cell. Most preferably, the endothelial cell is a HUVEC.

The DNA coding for all or part of the tPA locus which is to be used to drive the expression of a reporter gene may be derived from an appropriate human genomic clone, the clone produced by a suitable method known in the art.

For example, the tPA locus having the C to T mutation at position -7351 may be cloned from genomic DNA by performing a partial digestion of the DNA with a restriction enzyme (eg Sau 3A1) and shot gun cloning into a lambda or cosmid vector as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989). The library may be screened with an appropriate probe (eg a labelled oligonucleotide or a nick translated fragment from the upstream region of the tPA locus) to isolated an appropriate clone, as also described in Sambrook et al. (1998).

Alternatively, all or part of the tPA locus may be generated by PCR amplification from the genomic DNA, using appropriate primers, as described in as described in Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.

In the case where part of the tissue plasminogen locus is used to regulate expression of a reporter gene, the ability of the part of the tPA locus to regulate appropriate expression in endothelial cells will be determined. For example, the ability of the part of the tissue plasminogen locus to induce expression of the reporter gene in response to treatment with thrombin or calcium ionophore can be determined.

The DNA containing the region for cloning may then be fused to an appropriate reporter gene by standard cloning protocols, as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989)

Examples of suitable reporter genes include the chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (and variants thereof), and luciferase.

Transfection of endothelial cells with the reporter gene constructs may be performed by a suitable method known in the art, including the calcium phosphate precipitation method, incorporating a glycerol shock, as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989). After exposure, cells may be harvested and relative changes in reporter gene activity quantitated.

To induce expression driven from the tPA locus, an agent known in the art that induce release of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc Biol. 19(7):1796-803.

Transfection efficiency may also be evaluated by cotransfecting the cells with an appropriate control construct, such as the cytomegalovirus promoter and lac Z gene construct. .beta.-Galactosidase assay may then be performed as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), and may be taken as a direct index of the efficiency of transfection and used to normalize reporter gene activities among various experiments.

An agent that is capable of increasing the expression of the reporter gene may then be tested for its ability to increase the release rate of tPA from a cell. As will be appreciated, the ability of an agent to increase the expression of the reporter gene will depend on the concentration of the agent exposed to the cell. Accordingly, the agent will be exposed to the cell at a suitable concentration for testing the ability of the agent at that concentration to increase the expression of the reporter gene.

The ability of the agent to increase the release rate of tissue plasminogen activator from a cell may then be determined by a suitable method known in the art. For example, the ability of an agent to increase the release rate of tPA from endothelial cells (with or without the C to T mutation at position -7351 in the tPA locus) may be determined by culturing endothelial cells in vitro and determining the rate of release of tPA from the endothelial cells in the presence and absence of the agent. To determine release rates, the cells may be washed with fresh medium and the extent of rate of change of tPA released into the medium over time determined. To induce release of tPA from the endothelial cells, compounds known in the art that induce secretion of tPA may be utilised, such as thrombin or calcium ionophore. A suitable method for inducing the release of tPA from endothelial cells in vitro is as described in Rosnoblet et al. (1999) Arterioscler Thromb Vasc Biol. 19(7):1796-803.

An enzyme-linked immunosorbent assay (ELISA) may be employed for the quantitative determination of total t-PA antigen (eg TintElize.RTM. t-PA, Biopool AB). Such assays are based on the double-antibody principle. Free t-PA and t-PA in complex with inhibitors are detected with equal efficiency.

A known agent that increases rate of release of tPA from endothelial cells is monosodium [2-(6-hydroxynaphthalen-2-yl)-6-methyl-pyrimidin-4-yloxy]acetate dihydrate (JTV-926) (Ueshima et al. (2002) "Function of tissue-type plasminogen activator releaser on vascular endothelial cells in thrombolysis in vivo" Thrombosis Haemostasis 87: 1069-74).

The present invention also provides a method of identifying a subject suitable for treatment with an agent that increases the rate of release of tissue plasminogen activator, the method including the step of identifying in the subject. the presence of a cytosine to thymine polymorphism at position -7351 in one or both alleles of the tissue plasminogen locus.

The subject is any human subject of either gender for whom the treatment with an agent that increases the rate of release of tissue plasminogen activator may be beneficial. Preferably, the subject is a human of Caucasian origin.

In this regard, subjects that may benefit from the treatment with an agent that increase the rate of release of tissue plasminogen activator are subjects at increased risk of an ischemic stroke or a small vessel occlusion, and in particular an increased risk of lacunar stroke.

Preferably, the subject suitable for treatment has a cytosine to thymine polymorphism at position -7351 in both alleles of the tissue plasminogen locus.

Accordingly, in a preferred form, the present invention provides a method of identifying a subject suitable for treatment with an agent that increases the rate of release of tissue plasminogen activator, the method including the step of identifying in the subject the presence of a cytosine to thymine polymorphism at position -7351 in both alleles of the tissue plasminogen locus.

An example of an agent that increases the rate of release of tissue plasminogen activator is monosodium [2-(6-hydroxynaphthalen-2-yl)-6-methyl-pyrimidin-4-yloxy]acetate dihydrate (JTV-926) (Ueshima et al. (2002) "Function of tissue-type plasminogen activator releaser on vascular endothelial cells in thrombolysis in vivo" Thrombosis Haemostasis 87: 1069-74).

The identification in the subject of the presence of a cytosine to thymine mutation at position -7351 in one or both alleles of the tissue plasminogen activator locus may be by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3): 1215.

To identify the cytosine to thymine mutation at position -7351 in one or both alleles of the tissue plasminogen activator locus, DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect a mutation.

If DNA sequence analysis is used to identify the mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides (C and T) at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing the mutation, a suitable region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art. A preferred region for amplification is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. Once again, a preferred region of the tPA locus for amplification with WT and SNP primers is the region spanning nucleotides 1840 to 2245 of SEQ ID No. 1.

Preferably, the primers used to amplify the region of the tPA locus having the normal -7351 sequence are SEQ ID No. 2 (5'-ATTGGCGCAMCTCCTCACA-3') and SEQ ID No. 3 (5'-ATGGCTGTGTCTGGGGCG-3'), and the primers used to amplify the region of the tPA locus having the -7351 C to T polymorphic sequence are SEQ ID No. 2 (5'-ATTGGCGCMACTCCTCACA-3') and SEQ ID No. 4 (5'-ATGGCTGTGTCTGGGGCA-3'). Suitable reaction conditions to amplify the tPA locus with these primers include amplification using a PTC-200 Peltier Thermal Cycler (MJ Research) with the following PCR cycling parameters: 5 cycles of 96.degree. C. for 25 seconds, 70.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds; 21 cycles of 96.degree. C. for 25 seconds, 65.degree. C. for 50 seconds, and 72.degree. C. for 45 seconds; 4 cycles of 96.degree. C. for 25 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 125 seconds.

The amplification products may be detected by a suitable method known in the art. For example, the amplification products may be run on an agarose gel and stained with ethidium bromide for visualization.

Preferably, the identification of the mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of the mutation also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

The present invention also provides a method of treating a disease or condition associated with small vessel occlusion in a subject, the method including the step of administering to the subject an effective amount of an agent that increases the rate of release of tissue plasminogen activator in the subject.

The subject is any human subject susceptible to, or suffering from, a disease or condition associated with small vessel occlusion.

Accordingly, in another form, the present invention also provides a method of treating a subject susceptible to a disease or condition associated with small vessel occlusion, the method including the step of administering to the subject an effective amount of an agent that increases the rate of release of tissue plasminogen activator in the subject.

Example of diseases or conditions associated with small vessel occlusion include lacunar stroke, dementia, ischemic heart disease (including ischemic cardiomyopathy), peripheral vascular disease, disseminated intravascular coagulation, small vessel vasculitis, ischemic neuropathy, ischemic retinopathy, ischemic gastropathy (including small and large bowel ischemia), diffuse pulmonary embolism and vascular impotence.

Preferably, the disease or condition associated with small vessel occlusion occurs in the brain, including a lacunar stroke.

Accordingly, in a preferred form, the present invention also provides a method of treating a lacunar stroke in a subject, the method including the step of administering to the subject an effective amount of an agent that increases the rate of release of tissue plasminogen activator in the subject.

In another preferred form, the present invention provides a method of treating a subject susceptible to lacunar stroke, the method including the step of administering to the subject an effective amount of an agent that increases the rate of release of tissue plasminogen activator in the subject.

The agent is any agent that when administered to a subject has the capacity to increase the rate of release of tPA. An example of an agent that has the capacity to increase the release rate of tPA is monosodium [2-(6-hydroxynaphthalen-2-yl)-6-methyl-pyrimidin-4-yloxy]acetate dihydrate (JTV-926) (Ueshima et al. (2002) "Function of tissue-type plasminogen activator releaser on vascular endothelial cells in thrombolysis in vivo" Thrombosis Haemostasis 87: 1069-74).

The effective amount of the agent that increases the release rate of tPA to be administered to a subject is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful or therapeutic effect.

In this regard, an effective amount of the agent that increases the release rate of tPA may be appropriately chosen, depending upon the extent of increase in the rate of release of tPA to be achieved in the subject, the types of diseases or conditions associated with small vessel occlusion to be treated, the age and body weight of the subject, the frequency of administration, and the presence of other active agents.

The administration of the agent that increases the release rate of tPA may be within any time suitable to produce the desired effect of increasing the rate of release of tPA in the subject, and may be administered orally, parenterally or by any other suitable means, and therefore transit time of the drug must be taken into account.

The administration of the agent that increases the release rate of tPA may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the agent that increases the release rate of tPA to be administered.

For example, the agent that increases the release rate of tPA can be prepared into a variety of pharmaceutical preparations in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., and these preparations can be administered as intramuscular or subcutaneous injection or as injection to the organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions). Compositions containing the agent that increases the release rate of tPA may also contain a preservative, stabilizer, dispersing agent, pH controller or isotonic agent. Examples of suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilizers are dextran, gelatin, .alpha.-tocopherol acetate or alpha-thioglycerin. Examples of suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol.

The administration of the agent that increases the release rate of tPA may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant or sweetener, taking into account the physical and chemical properties of the agent that increases the release rate of tPA.

For these purposes, the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

When administered orally, the composition will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

A tablet may be made by compressing or molding the active ingredient optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

The administration of the agent that increases the release rate of tPA may also utilize controlled release technology. The agent that increases the release rate of tPA may also be administered as a sustained-release pharmaceutical. To further increase the sustained release effect, the composition may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2% aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1% aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2% aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1,200,000).

Alternatively, the agent that increases the release rate of tPA may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The composition of the invention may then be molded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agent that increases the release rate of tPA over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics. The composition may then be molded into a solid implant suitable for providing efficacious concentrations of the agent that increases the release rate of tPA over a prolonged period of time without the need for frequent re-dosing. The agent that increases the release rate of tPA can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be molded into a solid implant.
 

Claim 1 of 5 Claims

1. A method of identifying a subject predisposed to ischemic stroke, wherein said method comprises: determining the presence of a mutation in the subject that reduces the release rate from endothelial cells into the circulation of tissue plasminogen activator (t-PA), wherein said mutation is a cytosine to thymine mutation at position -7351 of the upstream region of the human tissue plasminogen activator locus, which is position 2228 of SEQ ID NO:1.

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