WO2002002812A2

WO2002002812A2 - Method for determining the susceptibility to stoke by analysing the ins gene

Info

Publication number: WO2002002812A2
Application number: PCT/GB2001/002891
Authority: WO
Inventors: Ian George Charles; Lizhi Liu; Weiming Xu
Original assignee: University College London
Priority date: 2000-06-30
Filing date: 2001-06-29
Publication date: 2002-01-10
Also published as: GB0016169D0; WO2002002812A3; AU2001266202A1

Abstract

A method for determining the susceptibility of a subject to stroke comprises determining the identity of the nucleotide 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1, thereby to determine the susceptibility of the subject to stroke. The identification of a subject susceptible to stroke can be used to reduce, prevent or delay the risk of stroke by administration of treatment of therapy before the onset of disease symptoms.

Description

DIAGNOSTIC METHOD

Field of The Invention

The invention relates to methods for identifying individuals who are at risk from stroke and to methods for treating individuals who have been identified as at risk from stroke.

Background to The Invention

Stroke is the single leading cause of severe disability and the third leading cause of death in the USA. Studies have identified sex, age, race, uncontrolled hypertension, diabetes, cigarette smoking and family history all to be significant risk factors in the causation of stroke.

Most strokes are caused by atherosclerotic plaques that develop on one or more of the arteries that carry blood to the brain. The plaque usually activates the clotting mechanism of the blood causing a clot to develop which then results in the artery being blocked or severely narrowed. This block causes deprivation of blood to the part of the brain supplied by the blocked artery leading to the acute loss of brain funciton in a localised area. A less common cause of stroke which occurs in about a quarter of people who develop strokes is as a result of high blood pressure. High blood pressure causes one or more of the blood vessels supplying the brain to burst resulting in haemorrhage. This compresses local brain tissue resulting in the loss of function of the affected tissue.

Genetic factors seem to play a part in the pathogenesis of practically every single human disease, by conferring some type of susceptibility to the diseased individual. Twin and family studies have demonstrated that there is a significant familial or genetic component underlying the occurrence of stroke (Boerwinkle et ah, 1999). Also, several studies have shown that a positive family history is a significant risk factor for stroke in future generations again indicating a genetic component. Genes known to be associated with stroke include the human Notch 3 gene on chromosome 19p 13, in which the autosomal dominant genotype contributes to a form of stroke called cerebral autosomal dominant ateriopathy with subcortical infarcts and leukoencephalopathy.

Summary of The Invention

This invention is based on our demonstration of an association between the iNOS gene and stroke. In particular, we have shown that polymorphism of a single nucleotide within intron 2 of the iNOS gene is associated with predisposition to stroke.

According to the invention there is thus provided a method for determining the susceptibility of a subject to stroke, which method comprises determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1, thereby to determine the susceptibility of the subject to stroke.

The invention also provides:

- a test kit suitable for use in a method for determining the susceptibility of a subject to stroke, which test kit comprises means for determining the sequence of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: l;

- an agent which reduces the risk of stroke for use in a method of treatment of a subject identified as susceptible to stroke by therapy; - use of an agent which reduces the risk of stroke in the manufacture of a medicament for use in a method of treatment of a subject identified as susceptible to stroke;

- a method for reducing the risk of stroke in a subject, which method comprises: (a) identifying whether the subject is susceptible to stroke; and

(b) administering to a subject identified in (a) as susceptible to stroke, a therapeutically effective amount of an agent which reduces the risk of stroke.

- products containing means for determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subj ect, which -J- position is defined by reference to SEQ ID NO: 1, and an agent which reduces the risk of stroke as a combined preparation for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy; and - a polynucleotide comprising the sequence set out in SEQ ID NO: 1 or a fragment or derivative thereof, wherein the polynucleotide encompasses position 1231 of SEQ ID NO: 1 and wherem the nucleotide at position 1231 of SEQ ID NO: 1 is G.

Brief Description of The Figures

Figure 1 shows the position of a single polynucleotide polymorphism (SNP) in intron 2 of the human iNOS (NOS2A) gene. The ATG start codon is in exon 2, and the numbering starts from the A of the ATG as position 1. PCR primer sequences are underlined and shown in capital letters. The polymorphism gtAc to gtGc is double underlined and shown at position 1231 with respect to the ATG start codon in exon 2. The SNP changes an Rsal restriction endonuclease site into a site that cannot be recognised by the enzyme.

Figure 2a shows a photograph of an agarose gel showing the PCR products prior to restriction enzyme digestion. Figure 2b shows a photograph of an agarose gel following restriction with

Rsal. The three arrows to the right of the photo point to the three bands caused by the three alleles. The upper band is caused by the "a" allele. The lower two bands are caused by digestion of the "b" allele at the polymorphic restriction site.

Figure 3 shows a graphical representation of the percentage of subjects in the stroke group and the control group presenting each of the three genotypes.

Figure 4 shows a graphical representation of the percentage of each allele present in the control group and the stroke group.

Description of the sequence listing

SEQ ID NO: 1 sets out part of the nucleotide sequence of exon 2 and intron 2 of the human iNOS (NOS2A) gene. SEQ ID NO: 2 sets out the nucleotide sequence of the human iNOS intron 2 primer F.

SEQ ID NO: 3 sets out the nucleotide sequence of the human iNOS intron 2 primer R.

Detailed Description of The Invention

We have shown that within intron 2 of the iNOS (NOS2A) gene, at nucleotide position 1231 as defined by reference to SEQ ID NO: 1, polymorphism occurs such that the adenine (A) residue may be replaced by any a guanidine (G) residue. We have also shown that the polymorphism we have identified in intron 2 is associated with susceptibility to stroke.

The position of the single nucleotide polymorphism (SNP) is defined above by reference to the sequence set out in SEQ ID NO: 1. If the subject carries an allelic variant of the sequence set out in SEQ ID NO: 1, the nucleotide of interest is the one in the allelic variant which corresponds to 1231 in SEQ ID NO: 1. Those skilled in the art will be able to determine the appropriate nucleotide. Comparison of an allelic variant with the sequence set out in SEQ ID NO: 1, using for example the PILEUP program referred to below, will allow the nucleotide corresponding to that at position 1231 to be identified.

An allele which has an A at nucleotide position 1231 of the iNOS gene intron 2, wherein the nucleotide position is defined by reference to SEQ ID NO: 1 is referred to as a "b" allele. An allele which has a G at position 1231 is referred to as an "a" allele.

Our results show that individuals of the "aa" genotype are much more likely to be stroke patients than normal control individuals, whereas individuals of the "ab" or "bb" genotypes are much more likely to be normal control individuals. The percentage of stroke patients with the genotypes aa, ab and bb were 55.3%, 36.5% and 8.2% respectively. Our results have further demonstrated that the "a" allele is presented more frequently in stroke patients, whereas the "b" allele is presented much more frequently in normal patients in comparison to stroke patients. The percentage occurrence of the "a" allele and "b" allele in stroke patients was 73.5% and 26.5% respectively. Thus, the "a" allele is marker highly suggestive of increased susceptibility to stroke.

Research to date has demonstrated that iNOS has both neurotoxic and neuroprotective roles within the body. Up-regulation of the gene could augment the neurotoxic effects of NO which could be a contributing factor to the causation of stroke. On the other hand , down-regulation of the gene could reduce the neuroprotective actions of NO contributing to the occurrence of stroke. Thus, it is possible that stroke could result from either the up-regulation of the gene or the down-regulation of the gene. It may be that the polymorphism we have identified in intron 2 of the NOS2A gene has an effect on the expression of the NOS2A gene and in consequence of that, the polymorphism affects susceptibility to stroke.

Alternatively, it may be that the polymorphism we have identified is simply a marker for susceptibility to stroke and the fact that the polymorphism is in the NOS2A gene may be coincidental. The invention provides methods for determining whether a subject is susceptible to stroke. Generally, the subject will be a human. The subject may be asymptomatic for stroke or alternatively may show clinical symptoms of predisposition to stroke, for example hypertension.

Subjects identified as at risk from stroke (i.e. subjects identified as susceptible or predisposed thereto) may be treated so as to reduce the risk of stroke.

Such treatment may take the form of, for example, a change of lifestyle or a pharmaceutical treatment.

The identity of the nucleotide at position 1231 of the iNOS gene intron 2 is determined for at least one of the alleles carried by a subject. Typically the identity of the nucleotide at position 1231 is determined for both alleles carried by a subject.

If the subject has an allelic variant sequence of that shown in SEQ ID NO: 1, the identity of the nucleotide corresponding to that shown at position 1231 as defined by reference to SEQ ID NO: 1 will be identified. The identity of that nucleotide may be determined for one or both alleles. A subject may be: homozygous for the "a" allele, i.e. have the genotype "aa"; heterozygous for the "a" allele, i.e. have the genotype "ab"; or homozygous for the "b" allele, i.e have the genotype "bb". A subject that has the genotype "aa" will have he nucleotide "G" at position 1231 for both alleles. A subject that has the genotype "ab" will have the nucleotide "G" at position 1231 for one allele and the nucleotide "A" at position 1231 for the other allele. A subject that has the genotype "bb" will have the nucleotide "A" at position 1231 for both alleles.

The identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject may be determined by any convenient method. Clearly, there are a large number of analytical techniques available to those skilled in the art for determining the identity of the nucleotide at position 1231. In general, however, a number of steps are common to whichever technique is used. Typically, a sample is obtained from the subject. Generally a nucleic acid preparation is prepared from the sample; and then the sequence of the nucleotide at position 1231 may be determined..

Typically, a sample is obtained from the subject. The test sample is conveniently, a sample of blood, bronchoalveolar lavage fluid, sputum or any other body fluid or tissue obtained from an individual. Generally, nucleic acid, is prepared from the sample according techniques well-known to those skilled in the art. Typically, a preparation of total nuclear DNA is prepared. The nucleic acid preparation may then be used to determine the sequence of the nucleotide at position 1231. Alternatively, it may not be necessary to include a nucleic acid preparation step and a polymorphism discrimination technique may be carried out directly on the sample.

In general the detection of allelic variation at position 1231 requires a polymorphism discrimination technique and a signal generation system. In addition, an optional amplification step is sometimes used. Suitable polymorphism discrimination techniques, some of which may include PCR, include:

General techniques, for example, DNA sequencing or sequencing by hybridisation;

Scanning techniques, for example protein truncation test (PTT) [not useful for the analysis of intron sequence polymorphism], single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel eletrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), cleavase, heteroduplex analysis, chemical mismatch analysis (CMC) or enzymatic mismatch cleavage;

Hybridisation techniques, for example solid phase hybridisation (for example, dot blots, multiple allele specific diagnostic assay [MASDA], reverse dot blots, oligonucleotide arrays [DNA chips]) or solution phase hybridisation (for example, Taqman™ [US-A-5210015 and US-A-5487972], molecular beacons [Tyagi et al, 1996, Nature Biotechnology,14, 303 and WO-A-95/13399]);

Extension based techniques, for example amplification refractory mutation system (ARMS™), amplification refractory mutation system linear extension (ALEX™) [EP-B-332435] or competitive oligonucleotide priming system (COPS) [Gibbs et al, 1989, Nucleic Acids Research, 17, 2347];

Incorporation based techniques, for example mini-sequencing or arrayed primer extension (APEX);

Restriction enzyme based techniques, for example restriction fragment length polymorphism (RFLP) or restriction site generating PCR;

Oligonucleotide based techniques: oligonucleotide ligation assay (OLA); and

Other types of assay, for example invader assay.

The above list provides examples of suitable polymorphism discrimination techniques and should not be construed as limiting. Suitable signal generation or detection systems include:

Fluorescence based techniques, for example fluorescence resonance energy transfer, fluorescence quenching or fluorescence polarisation (GB-B-2228998); and

Other techniques, for example chemiluminescence, eletrochemiluminescence, raman, radioactivity, colorimetric, hybridisation protection assay, mass spectrometry. Again, this list of signal generation or detection systems is merely illustrative and should not be construed as limiting.

A particular suitable technique for determining the identity of the nucleotide at position 1231 is a combination of a RFLP technique (the polymorphism discrimination technique) and agarose gel electrophoresis combined with ethidium bromide staining (the signal generation technique).

When the nucleotide at position 1231 is A, that base and those surrounding it give rise to a sequence, GTAC, which can be cut by the restriction enzyme Rsal. The site is cut between the T and the A residues. Polymorphism at this site may give rise to one of the following sequence, GTGC. That sequence can not be cut with Rsal and therefore "a" alleles can be conveniently identified by the lack of an Rsal site. PCR may be used to amplify a sequence comprising all or part (i.e. a fragment) of the sequence set out in SEQ ID NO: 1. Of course, if a fragment is amplified that fragment will comprise the nucleotide at position 1231 and at least two bases 5' thereto and at least one base 3' thereto.

The PCR product thus generated is digested with Rsal. The digestion products are then typically run on an agarose gel and the resulting DNA fragments stained with a suitable fluorescent molecule, for example ethidium bromide, and visualised under UV. The presence of a single band on the gel is indicative of a lack of Rsal sites and therefore the subject is homozygous for the "a" allele. The presence of two bands is indicative of an Rsal site at both alleles and therefore the subject is homozygous for the "b" allele. The presence of three bands is indicative of the presence of an Rsal site at one allele and the absence of an Rsal site at the other allele. Therefore, according to this combination of RFLP and gel electrophoresis analysis the genotype of a subject with respect to the nucleotide at position 1231 of the sequence set out in SEQ ID NO: 1 may be conveniently determined. The oligonucleotides for use in carrying out such RFLP analysis may be any suitable oligonucleotides. The design of suitable oligonucleotides will be apparent to the person skilled in the art. Suitable oligonucleotides will be of any convenient length, for example up to about 50 nucleotides in length, up to 40 nucleotides in length, or more conveniently up to 30 nucleotides in length, such as for example, from about 8 to about 25 nucleotides in length or from about 8 to about 15 nucleotides in length. In addition, the oligonucleotides will typically be designed such that the nucleotide at position 1231 in the amplified PCR product does not occur at the middle of that product. In this way, when and if the fragment is cut with Rsal, the two fragments that thus arise will be clearly distinguishable on the basis of size.

In general, suitable PCR primers will comprise sequences entirely complementary to the corresponding sequence to be amplified. However, if required, one or more, for example up to 3, up to 5 or up to 8 mismatches may be introduced, to introduce a convenient restriction enzyme site for example, provided that such mismatches do not unduly affect the ability of the primer to hybridize to its target sequence. Suitable primers may carry one or more labels to facilitate detection.

The sequences of two oligonucleotides which can be used in a method of the invention are give in SEQ ID NO: 2 (forward primer) and SEQ ID NO: 3 (reverse primer). Use of those sequences enables the generation of a polynucleotide of 441 base pairs in length. If the polynucleotide thus generated is an "b" allele it will be cleaved into two fragments of 264 and 177 base pairs in length on digestion with Rsal.

The presence of a G residue (an "a" allele) at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject is indicative of predisposition or susceptibility to stroke. Where both alleles have a G residue at position 1231, the indication is even stronger.

The invention also provides a test kit for use in a method for determining the susceptibility of a subject to stroke. A test kit of the invention comprises means for determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1. Any suitable means for determining the identity of the nucleotide at position

1231 may be included in a test kit of the invention. Typically, the means will comprise two oligonucleotides which can be used to amplify a polynucleotide from a subject which comprises the sequence set in SEQ ID NO: 1 or a fragment thereof or allelic variants thereof. If the oligonucleotides amplify a fragment, that fragment (or allelic variant thereof) will comprise at least the nucleotide at position 1231 and preferably the nucleotides at positions 1229 to 1232 of SEQ ID NO: 1. A test kit of the invention may comprise the restriction endonuclease Rsal.

A test kit of the invention may optionally comprise, appropriate buffer(s), enzymes, for example a thermostable polymerase such as Taq polymerase and/or control polynucleotides. A kit of the invention may also comprise appropriate packaging and instructions for use in a method for determining the susceptibility of a subject to stroke. A test kit of the invention may also comprise an agent which reduces the risk of stroke.

The invention allows diagnosis of subjects at risk from stroke before the onset of disease symptoms or before the onset of severe symptoms. The risk of stroke can thus be reduced, prevented or delayed by administration of treatment of therapy in advance of stroke appearance. Suitable treatments to reduce the risk of stroke include a change of lifestyle. Such changes of lifestyle include for example, behaviour to reduce obesity, reduction of alcohol intake, reduction of smoking, reduction of salt intake and an increase in exercise. Alternatively, the treatment can be pharmaceutical, in which case any suitable agent can be used which is known to reduce the risk of stroke.

Agents which reduce the risk of stroke may also be used in the manufacture of a medicament for use in a method of treatment of a subject identified as susceptible to stroke. Thus, the condition of a subject identified as susceptible to stroke can be improved by administration of an agent which reduces the risk of stroke. A therapeutically effective amount of an agent which reduces the risk of stroke may be given to a human patient identified as susceptible to stroke.

Examples of agents which can be used to reduce the risk of stroke include anti-hypertensive agents. Examples of particular anti-hypertensive agents include the beta-adrenoceptor blocking drugs, optionally in combination with a thiazide, calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors, vasodilators, alpha-blockers and centrally acting drugs such as prazosin, terazosin and doxazosin. Further agents which can be used to reduce the risk of stroke are modulators of iNOS. A modulator of iNOS is an antagonist or an agonist of iNOS. An antagonist of iNOS is an agent which causes reduction or even elimination of iNOS expression and/or activity. A modulator of iNOS is an agent which causes an increase of iNOS expression and/or activity.

Any pharmaceutically acceptable antagonist of iNOS can be used in the present invention. An antagonist may reduce the expression of the iNOS gene and/or reduce activity of the iNOS enzyme. Thus, competitive, non-competitive, reversible and irreversible inhibitors of the enzyme are suitable antagonists. The antagonist may antagonist eNOS and/or nNOS as well as iNOS. Preferably, an antagonist will achieve a higher antagonism of iNOS than of eNOS and/or nNOS. Preferred antagonists are thus selective iNOS antagonists. Typically, selective iNOS antagonists are those which antagonise iNOS at least twice as strongly, preferably at least five times as strongly, more preferably at least ten times as strongly, as eNOS and/or nNOS. Ideally, a selective iNOS antagonist substantially does not antagonise eNOS or nNOS.

Suitable antagonists include L-arginine analogues, thiocitrullines, indazole derivatives, imidazole derivatives, hydrazine derivatives, thioureas, tliiazoles, biotin derivatives and phenyl-substituted thiopene amidines.

Examples of suitable L-arginine analogues include methyl-L-arginine, N^G- nitro-L-arginine methyl esther (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), N^G-amino-L-arginine (L-NAA), N^w,N^w-dimethyl-L-arginine (ADMA), N^w,N^w2- dimethyl-L-arginine (SDMA), N^w-ethyl-L-arginine (L-NEA), N^w-methyl-L- homoarginine (L-NMHA), N^w-nitro-L-arginine (L-NOARG), N^δ-iminoethyl-L- ornithine (L-NIO), N^δ-iminoethyl-L-lysine (L-homo-NIO) and L-canavanine (L- CAN).

Examples of suitable thiocitrullines include S-methyl-L-thiocitrulline (SMTC), L-thiocitrulline (L-TC) and L-S-ethyl-thiocitrulline (Et-TC). Examples of suitable indazole derivatives include indazole and 7-substituted indazoles such as 7-nitro indazole and 3-bromo-7-nitroindazole.

Examples of suitable hydrazine derivatives include aminoguanidine.

Examples of suitable imidazole derivatives include phenyl substituted imidazoles such as 1-phenyl-imidazole. Examples of suitable thioureas include S-methylisothiourea sulphate, δ-(S- methylisothioureido)-L-norvaline (L-MIN), S-ethylisothiourea (SETU) and S- isopropylisothiourea (SIPT).

Examples of suitable thiazoles include 2-amino-thiazole and 2-amino-4,5- dimethyl tliiazole. Examples of suitable biotin derivatives include 2-iminobiotin.

Preferred iNOS antagonists are selective antagonists of iNOS. Selective antagonists include N-^δiminoethyl-L-ornithine (L-NIO) and aminoguanidine.

The above antagonists are commercially available, or may be made by analogy with known methods.

The antagonists may be a pharmaceutically acceptable salt of one the above compounds. Suitable salts include salts with pharmaceutically acceptable acids, both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Salts may also be formed with pharmaceutically acceptable bases such as alkali metal (eg sodium or potassium) and alkali earth metal (eg calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.

Antagonists of the iNOS enzyme can be identified by:

(a) contacting a candidate substance with iNOS and a substrate and co- factor therefor, under conditions under which iNOS activity, in the absence of an antagonist, would be expected to occur; and

(b) determining whether, or to what extent, NOS activity takes place.

A suitable such assay for identifying antagonists of NOS is a microtiter plate assay in which NOS activity is measured by determining the change in absorbance as NADPH is converted to NADP⁺. This assay comprises:

(a) adding a candidate compound, a known iNOS antagonist (for example L-NMMA) and a buffer solution to separate microtiter wells;

(b) adding to each well, iNOS enzyme, cofactors therefor, L-arginine and buffer; and (c) determining the change in absorbance in each well.

Typically, the buffer is a HEPES buffer capable of maintaining a pH of about 7, preferably about 7.4. The cofactors comprise oxyhemoglobin, NADPH and BH₄. They may also comprise CaCl₂, MgCl₂, FMN, FAD and/or CaM.

The iNOS may be a naturally occurring form of iNOS or may be a variant which retains NOS activity, for example variants produced by mutagenesis techniques. The iNOS used in the assay is preferably of mammalian origin, for example rodent (including rat and mouse) or primate (such as human). Preferably, the iNOS is of human origin.

The iNOS may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines. Preferably, iNOS used in the assay is recombinant. More preferably, it is obtained by expression in S/21 cells according to the methodology in Charles et al, Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press, Totowa), vol 100, pgs 51-60.

Step (c) of the assay may be carried out by reading the difference in absorbance between 420 and 405 nm. Typically, this is done by a spectrometer. Comparison of the well containing the candidate compound with the control wells containing a known iNOS antagonist (100% inhibition) and no inhibitor (0% inhibition) allows % inhibition achieved by the candidate substance to be calculated. A microtiter assay as set out above is described in detail in Dawson & Knowles, Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press, Totowa), vol 100, Chapt. 22, pgs 237-242.

Any substance which is identified as an iNOS antagonist using an assay as described above can be used in the present invention. The NOS antagonists used in the present invention typically achieve at least 25%, for example 50% iNOS antagonism, more preferably at least 80%, at least 90% or at least 95% NOS antagonism, or they achieve substantially complete iNOS antagonism at a concentration of the antagonist of lμg ml^"1, lOμg ml^"1, lOOμg ml"¹, 500μg ml^"1, lmg ml^"1' lOmg ml^"1, lOOmg ml^"1. The percentage antagonism represents the percentage increase in activity a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage antagonism and concentration of antagonist may be used to define an antagonist of the invention, with greater antagonism at lower concentrations being preferred. Selective antagonists of iNOS can be identified by conducting the above microtiter assay using different purified NOS isoforms. Any pharmaceutically acceptable agonist of iNOS can be used in the present invention. An agonist may, for example, increase the expression of the iNOS gene and/or increase activity of the iNOS enzyme. Thus, competitive, non-competitive, reversible and irreversible activators if the iNOS enzyme are suitable agonists. The agonist may agonise eNOS and or nNOS as well as iNOS. Preferably, an agonist will achieve a higher agonism of iNOS than of eNOS and/or nNOS. Preferred agonists are thus selective iNOS agonists. Typically, selective iNOS agonists are those which agonise iNOS at least twice as strongly, preferably at least five times as strongly, more preferably at least ten times as strongly, as eNOS and/or nNOS. Ideally, a selective iNOS agonist substantially does not agonise eNOS or nNOS. Suitable agonists include L-arginine and tetrahydrobiopterin (BH₄). The above agonists are commercially available, or may be made by analogy with known methods.

The agonist may be a pharmaceutically acceptable salt of one the above compounds. Suitable salts include salts with pharmaceutically acceptable acids, both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Salts may also be formed with pharmaceutically acceptable bases such as alkali metal (eg sodium or potassium) and alkali earth metal (eg calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.

Agonists of the iNOS enzyme can be identified by: (a) contacting a candidate substance with iNOS and a substrate and co- factor therefor, under conditions under which iNOS activity, in the absence of an agonist, would be expected to occur; and (b) determining whether, or to what extent, NOS activity takes place.

A suitable such assay for identifying agonists of iNOS is a microtiter plate assay in which NOS activity is measured by determining the change in absorbance as NADPH is converted to NADP⁺. This assay comprises:

(a) adding a candidate compound, a known iNOS agonist and a buffer solution to separate microtiter wells;

(b) adding to each well, iNOS enzyme, cofactors therefor, L-arginine and buffer; and

(c) determining the change in absorbance in each well. Typically, the buffer is a HEPES buffer capable of maintaining a pH of about 7, preferably about 7.4. The cofactors comprise oxy hemoglobin, NADPH and BH₄. They may also comprise CaCl₂, MgCl₂, FMN, FAD and/or CaM.

The iNOS may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines. Preferably, iNOS used in the assay is recombinant. More preferably, it is obtained by expression in S 21 cells according to the methodology in Charles et al, Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press, Totowa), vol 100, pgs 51-60.

Step (c) of the assay may be carried out by reading the difference in absorbance between 420 and 405 nm. Typically, this is done by a spectrometer. Comparison of the well containing the candidate compound with the control wells containing a known NOS agonist and no agonist allows %> agonism achieved by the candidate substance to be calculated.

A microtiter assay as set out above is described in detail in Dawson & Knowles, Methods in Molecular Biology (edited by MA. Titheradge, Humana Press, Totowa), vol 100, Chapt. 22, pgs 237-242. Any test substance which is identified as an iNOS agonist using an assay as described above can be used in the present invention. The iNOS agonists used in the present invention typically achieve at least 100%>, for example 200% iNOS agonism, more preferably at least 500%, at least 750%> or at least 1000%) iNOS agonism at a concentration of the agonist of lμg ml^"1, lOμg ml^"1, lOOμg ml^"1, 500μg ml^"1, lmg ml^"1, lOmg ml^"1, lOOmg ml"¹. The percentage agonism represents the percentage increase in activity a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage agonism and concentration of agonist may be used to define an agonist of the invention, with greater agonism at lower concentrations being preferred.

Selective agonists of iNOS can be identified by conducting the above microtiter assay using different purified NOS isoforms.

An agent which reduces the risk of stroke may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The agent which reduces the risk of stroke may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.

The formulation of an agent which reduces the risk of stroke for use in preventing or treating stroke will depend upon factors such as the nature of the exact agent, whether a pharmaceutical or veterinary use is intended, etc. An agent which reduces the risk of stroke may be formulated for simultaneous, separate or sequential use.

Products containing means for determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1, and an agent which reduces the risk of stroke as a combined preparation for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy. Thus, such a product may comprise both means for diagnosis and means for therapy. An agent which reduces the risk of stroke is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. A therapeutically effective amount of an agent which reduces the risk of stroke is administered to a patient. The dose of an agent which reduces the risk of stroke may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. The invention also provides a polynucleotide comprising the sequence set out in SEQ ID NO: 1 or a fragment or variant thereof, wherein the polynucleotide encompasses position 1231 of SEQ ID NO: 1 and wherein the nucleotide at position 1231 of SEQ ID NO: 1 is G. Thus, a polynucleotide of the invention always encompasses position 1231 and the nucleotide at that position is always G. The invention also provides polynucleotides complementary to a polynucleotide of the invention.

The polynucleotide may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

A polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the sequence or the complement of the sequence set out in SEQ ID NO: 1, wherem the nucleotide at position 1231 is G.

A polynucleotide of the invention can hydridize to the sequence or complement of the sequence set out in SEQ ID NO: 1, wherein the nucleotide at position 1231 is G, at a level significantly above background. Background hybridization may occur, for example, because of other DNAs present in a DNA library. The signal level generated by the interaction between a polynucleotide of the invention and the sequence set out in SEQ ID NO: 1, wherein the nucleotide at position 1231 is G, is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the sequence set out in SEQ ID NO: 1, wherein the nucleotide at position 1231 is G. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Selective hybridisation may typically be achieved using conditions of medium to high stringency (for example, 2 x SSC [0.15M sodium chloride and 0.015M sodium citrate] at about 50°C to about 60 °C). Flόwever, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989, supra). For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at about 60 °C to about 65 °C. If lower stringency is required suitable conditions include 2 x SSC at 60 °C.

The sequence of SEQ ID No: 1, wherein the nucleotide at position 1231 is G, may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the sequence set out in SEQ ID NO: 1, wherein the nucleotide at position 1231 is G, will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to that sequence over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length the sequence.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings)

(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the

National Centre for Biotechnology Information (http://w w.ncbi.nim.nih.govΛ. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl Acad. S i. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01 , and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90%> sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95%> sequence identity over 40 nucleotides. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 1, wherein the nucleotide at position 1231 is G. Such fragments will preferably be at least 10, preferably at least 25, or at least 50, for example at least 100, at least 200, or at least 500, or at least 1000 nucleotides in length. They will ^•typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length.

The following Example illustrates the invention:

Example 1. Materials and Methods

(i) Patients and normal controls

255 Caucasian patients with ischaemic stroke or transient ischaemia presenting to a neurological cerebrovascular service were studies and compared with 92 normal Caucasian controls. Controls were recruited from the spouses of the patients where possible and also from community controls randomly selected from health authority lists from the same family practices as the patients. The DNA samples were collected from each of the subjects by Dr. Hugh Markus of Kings College School of Medicine and dentistry. DNA was extracted from blood samples and stored at 4°C until analysis. All of the subjects gave informed consent and collection was approved by the appropriate ethics committee.

(ii) Probe and oligonucleotides

The probes used in this project are found within intron 2 of the iNOS gene. Polymorphism arises from the presence of a single point mutation (SNP). This SNP allows the genotype of individuals to be deduced following PCR and restriction enzyme digestion using the restriction endonuclease Rsal.

The sequences of the PCR primers used in the project are F = 5'-gatttggatt cttcatgtcc ctcc-3' (SEQ ID NO: 2) and R = 5'-tcaccattgt taccctcttc ttat-3' (SEQ ID NO: 3). The PCR product produced is 441 base pairs in length.

(Hi) Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) was carried out by incubating the DNA samples with the appropriate reagents at three different temperatures. The three temperatures correspond to the three main steps in PCR, denaturation, annealing and extension. The cycle used in these experiments was as follows: 96°C for 35 seconds,

56°C for 2 minutes, 72°C for 2 minutes. The cycle was repeated 30 times, after which samples were held at 72°C for 10 minutes. The PCR machine used in these experiments was the Primus 96 (MWG BIOTECH). Each PCR contained lμl DNA sample (approximately lOOng); 2μl buffer [lOmM Tris-HCl (pH 9.0 at 25°C), 50mM KC1, 1.5mM MgCl₂ and 0.1% TritonX- 100], 2μl dNTP (each nucleotide at a concentration of lOOmM in water, pH7.5), 0.5μl of each of the two primers (25 pmol), 0.5μl Taq polymerase (0.5 units) and was made up to a total volume of 20 μl with distilled water.

(iv) Electrophoresis

2% agarose gels containing ethidium bromide were prepared for electrophoresis. The gels were run at 200V for approximately 20 minutes and then visualised under ultraviolet light.

(v) Restriction enzyme digestion

The PCR product was then subjected to restriction enzyme digestion using the restriction enzyme Rsal (New England Biolabs). The recognition site of this enzyme is : 5'....GT1AC....3' 3'....CAtTG....5\ The enzyme generates a blunt end cut between the T and A residues.

Digestion with Rsal allows the characterisation of the genotype of the subjects with respect to the iNOS gene. Samples with a "b" allele contain DNA with an Rsal site (GTAC). Following digestion, this results in the generation of two bands, whereas samples with an "a" allele (GTGC) can not be cut as this site is not recognised by Rsal.

Restriction enzyme digestion reactions were carried out as follows: 20 μl of the PCR product for each subject sample (i.e. the total volume of each PCR reaction was mixed with lOμl of reagent mix (40μl Rsal, 192μl lOxNEBuffer and 400μl distilled water) and incubated at 37°C for 2 hours.

(vi) Electrophoresis and the counting of alleles

The restriction products were then run on a 2%> agarose gel in the manner described above. The gels were visualised under UV light and photographs of the gels were taken. The number and position of the bands on the photographs allowed the genotype of the sample with respect to the iNOS gene to be identified as follows:

- Genotype "aa", one band is visible as the "a" allele does not possess the recognition site necessary for Rsal to cut the DNA;

- Genotype "bb", two bands are present, because the "b" allele has the recognition site required for the restriction enzyme to cut the DNA into two fragments; and

- Genotype "ab", three bands are visible: one due to the "a" allele (which is not cut) and two due to the "b" allele (which is cut).

From these results, the frequency of the various genotypes and also the allele frequency can be calculated in both stroke patients and normal controls. Various statistical techniques were then used to determine whether any significant differences existed between the two sample populations.

(vii) Statistical analysis

The chi-squared test and Fisher's exact test were used to analyse the data and calculate the significance of the genotype differences and the allele frequency differences between the control group and the group of patients with stroke history. The chi-squared test was used to analyse the data for the genotype frequencies and Fisher's exact test was used to analyse the data of the allele frequencies in the two groups.

2. Results

Figures 2a and 2b show photographs taken under UV light of two gels following electrophoresis. Figure 2a shows the PCR product prior to restriction enzyme digestion. A lkb ladder is included as a size marker. The restriction products were of the following lengths: allele "a", 441 base pairs; and allele "b", 177 base pairs and 264 base pairs.

(i) The results of the experiments were as follows:

Table 1 : Number of subjects in the stroke group and control group presenting each of the genotypes.

Table 2: Percentage of subjects in the stroke group and the control group presenting each of the genotypes (see also Figure 3).

Table 3: Frequency of each allele in the control group and the stroke group. The frequency is in numbers not percentages.

Table 4: Percentage of each allele present in the control group and the stroke group (see also Figure 4).

The above tables summarise the results obtained from the photographs taken following electrophoresis of the restriction products. They state the values which are necessary to carry out further analysis. Statistical tests were then carried out to assess the significance of these results.

(ii) Chi-squared test for the significance of differences between the genotypes of the control group and the stroke group:

Null hypothesis: The genotype of the stroke group is the same as the genotype of the control group.

- Table size: 3 rows and 2 columns.

- The chi-squared value was calculated using a computer software program called Graph Pad Instat 2.01.

- Chi-squared value = 17.089

- Degrees of freedom = 2 - P value = 0.0002

At a 5% significance level, a P value of 0.0002 indicates that there is a highly significant association between the genotype and stroke history of a patient. These results suggest that genotype "aa" is more common in stroke patients than in the normal patients whereas genotypes "ab" and "bb" are more common in the normal control patients.

(Hi) Fisher's exact test for the significance of differences in the allele frequencies between the stroke group and the control group:

See previous Table 3:

Null hypothesis: There is no association between alleles present in an individual and the stroke history of an individual.

Calculations were carried out using a software program called GraphPad Instat 2.01. The two-sided P value calculated was O.0001. At 5% significance level, this is considered extremely significant. This suggests that there is significant association between the allele type and stroke history of the patient. The results indicate that the "a" allele appears a lot more frequently in the genotype of stroke patients than normal patients. In addition, the "b" allele is a lot more apparent in normal subjects than in stroke patients.

Claims

1. A method for determining the susceptibility of a subject to stroke, which method comprises determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1, thereby to determine the susceptibility of the subject to stroke.

2. A method according to claim 1, wherein the absence or presence of G at position 1231 is determined, the presence of G indicating susceptibility of the subject to stroke.

3. A method according to claim 1 or 2, wherein the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject is determined for both alleles of the subject.

4. A method according to claim 3, wherein the absence or presence of G at position 1231 is determined for both alleles, the presence of G at one or both alleles indicating susceptibility of the subject to stroke.

5. A test kit suitable for use in a method for determining the susceptibility of a subject to stroke, which test kit comprises means for determining the sequence of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1.

6. A test kit according to claim 5, wherein the means for determining the sequence of the polynucleotide at position 1231 comprises two oligonucleotides which can be used to amplify a polynucleotide comprising all or part of the sequence set out in SEQ ID NO: 1 or an allelic variant thereof, wherein the polynucleotide amplified comprises the nucleotide at position 1231 of the sequence set out in SEQ ID NO: 1 or an allelic variant thereof.

7. A test kit according to claim 5 or 6 , wherein the oligonucleotides have the sequences set out in SEQ ID NO: 2 and SEQ ID NO: 3.

8. A test kit according to claim 7, wherein the test ldt further comprises the restriction enzyme Rs l.

9. A test kit according to any one of claims 5 to 8, wherein the test kit further comprises an agent which reduces the risk of stroke.

10. A test kit according to claim 9, wherein the agent is an anti-hypertensive agent and/or a modulator of iNOS.

11. An agent which reduces the risk of stroke for use in a metliod of treatment of a subject identified as susceptible to stroke by therapy.

12. An agent according to claim 11, wherein the subject is identified as susceptible to stroke by use of a method according to any one of claims 1 to 4.

13. An agent according to claim 11 or 12 which is an anti-hypertensive agent and/or a modulator of iNOS.

14. Use of an agent which reduces the risk of stroke in the manufacture of a medicament for use in a method of treatment of a subject identified as susceptible to stroke.

15. Use according to claim 14, wherein the subject is identified as susceptible to stroke by use of a method according to any one of claims 1 to 4.

16. Use according to claim 14 or 15, wherein the agent is an anti-hypertensive agent and/or a modulator of iNOS.

17. A method for reducing the risk of stroke in a subject, which method comprises:,

(a) identifying whether the subject is susceptible to stroke; and

18. A method according to claim 17, wherein step (a) is carried out by a method according to any one of claims 1 to 4.

19. A method according to claim 17 or 18, wherein the agent in step (b) is an anti-hypertensive agent and/or a modulator of iNOS.

20. Products containing means for determining the identity of the nucleotide at position 1231 of the iNOS (NOS2A) gene intron 2 of the subject, which position is defined by reference to SEQ ID NO: 1, and an agent which reduces the risk of stroke as a combined preparation for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy.

21. Products according to claim 20 wherein the agent is an anti-hypertensive agent and/or a modulator of iNOS.

22. A polynucleotide comprising the sequence set out in SEQ ID NO: 1 or a fragment or derivative thereof, wherein the polynucleotide encompasses position

1231 of SEQ ID NO: 1 and wherein the nucleotide at position 1231 of SEQ ID NO: 1 is G.