WO2001053522A2

WO2001053522A2 - (CA)n POLYMORPHISMS IN AN INTRON OF THE ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE AND THEIR USE IN DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

Info

Publication number: WO2001053522A2
Application number: PCT/EP2001/000593
Authority: WO
Inventors: Karl Stangl; Ivar Roots; Ingolf Cascorbi
Original assignee: Epidauros Biotechnologie Ag
Priority date: 2000-01-20
Filing date: 2001-01-19
Publication date: 2001-07-26
Also published as: WO2001053522A3; AU2001240521A1

Abstract

Provided is an in vitro screening method for determining the presence of atherosclerosis, preferably coronary artery disease or susceptibility to such a disease in a subject comprising determining the CA repeat number in an intron of the endothelial nitric oxide synthase (eNOS) gene in a sample from a subject, the number of the CA repeat being indicative of increased risk of coronary artery disease. Furthermore, the use of oligo- or polynucleotides for the detection of the presence of the CA repeat number in an intron of the eNOS gene and determining the risk of coronary artery disease in the subject is described. In addition, assay kits and apparatus adapted and useful for carrying out the method of the invention are provided.

Description

Title of the invention

(CA)_n Polymorphisms in an intron of the endothelial nitric oxide synthase gene and their use in diagnostic and therapeutic applications

Field of the invention

The present invention relates generally to means and methods of diagnosing and treating the phenotypic spectrum as well as the overlapping clinical characteristics with several forms of inherited abnormal expression and/or function of the human endothelial nitric oxide synthase (eNOS) gene. In particular, the present invention relates to an in vitro screening method for determining the presence of atherosclerosis, preferably coronary artery disease or susceptibility to such a disease in a subject comprising determining the CA repeat number in an intron of the endothelial nitric oxide synthase eNOS) gene in a sample from a subject, the number of the CA repeat being indicative of increased risk of atherosclerosis, preferably coronary artery disease. Furthermore, the present invention relates to the use of an oligo- or polynucleotide for the detection of the presence of the CA repeat number in an intron of the eNOS gene and determining the risk of atherosclerosis, preferably coronary artery disease in the subject. In addition, the present invention relates to assay kits and apparatus adapted and useful for carrying out the method of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

Background of the invention

Nitric oxide (NO) plays a key role in vascular homeostasis mediating shear stress- induced endothelium-dependent vasodilation (Murad, 1986; Ignarro, 1990; Moncada et al., 1991 ; Nathan, 1992; Schmidt, 1994; Lowenstein et al., 1994; Busse and Fleming, 1995; Sase and Michel, 1997). Moreover, NO possesses a broad spectrum of antiatherogenic properties as it inhibits platelet adhesion and aggregation (Radomski et al., 1987), leukocyte adherence to the endothelium (Kelly et al., 1996), growth factor-induced proliferation and migration of vascular smooth muscle cells (Garg and Hassid, 1989; Sarkar et al., 1996), extracellular matrix turn over (Murell et al., 1995) and neointima formation following vessel injury (van der Leyen et al., 1995). In normal vessels, the main source of NO production is the Ca²⁺-calmodulin dependent endothelial isoform (eNOS) of nitric oxide synthase enzymes (Nathan and Xie, 1994). Like the cytochrome P450 gene families the NO synthases belong to heme proteines containing cysteine thiolate-liganded protoporphyrin heme prosthetic groups (Masters et al., 1996). The gene encoding eNOS, comprises 26 exons, spans 21 kb and is located on chromosome 7q35-36 (Marsden et al., 1992; Nadaud et al., 1994; Miyahara et al., 1994). The key role of NO in regulating vascular tone has led to studies investigating whether polymorphisms of the eNOS gene may be associated with arterial hypertension in humans. In a French population, the (CA)_n polymorphism and two biallelic markers at intron 18 failed to show association with essential hypertension (Bonnardeaux et al., 1995), whereas a linkage was reported between a variable number of tandem repeat polymorphism at intron 4 in Japanese (Uwabo et al., 1998). Moreover, as NO inhibits key processes in the pathogenesis of atherosclerosis, and a loss of endothelial NO production with consecutive endothelial dysfunction occurs at an early stage of disease and may promote further progression (Lϋscher et al., 1993; Harrison, 1993), eNOS represents an attractive candidate gene for coronary artery disease. A 27-bp tandem-repeat polymorphism at intron 4 as a smoking-dependent atherogenic risk factor in an Australian population has accordingly been described (Wang et al., 1996). Moreover, a Glu289Asp exchange was found to be over- represented in a French but not in a Northern Irish population enrolled in the ECTIM study (Poirier et al., 1999). This study, which investigated 10 single nucleotide polymorphisms, reported no further association with coronary excess risk (Poirier et al., 1999). Accordingly, there is a need for means and methods for determining putative atherogenic risk factors, such methods being helpful for ameliorating atherosclerosis, preferably coronary artery diseases or susceptibility to such diseases. Thus, the technical problem of the present invention is to comply with the needs described above.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Summary of the Invention

The present invention is based on the finding of novel, so far unknown correlation between (CA)_n polymorphism in an intron, preferably intron 13 of the endothelial nitric oxide synthase (eNOS) gene and increased risk of coronary artery disease of subjects and the population distribution of these alleles. Based upon the knowledge of this novel coronary risk determining factor diagnostic tests and reagents for such tests were designed for the specific detection and genotyping of eNOS alleles in humans, including homozygous as well as heterozygous, frequent as well as rare alleles of an intron, preferably intron 13 of the eNOS gene. The determination of the eNOS gene intron allele status of humans with such tests is useful for the optimization of therapies of and useful in the determination of the individual predisposition to several common vascular diseases.

In a first embodiment, the invention provides an in vitro screening method for determining the presence of atherosclerosis, preferably coronary artery disease or susceptibility to such a disease in a subject comprising determining the CA repeat number in an intron of the endothelial nitric oxide synthase (eNOS) gene in a sample from a subject, the number of the CA repeat being indicative of increased risk of atherosclerosis, preferably coronary artery disease. In yet another embodiment, the invention provides an assay kit for carrying out the method of the invention, said kit comprising means for assaying the CA repeat number in an intron, preferably intron 13 of the eNOS gene.

In a further embodiment, the invention provides an apparatus for the method of the invention, said apparatus comprising means adapted for receiving the data of measurement of a subject's level of CA repeat number in an intron of the eNOS gene and computer means for comparing the data of measurement of the level of the CA repeat number to a set of reference data to determine increased risk of atherosclerosis, preferably corony artery disease.

The methods, uses, kits and apparatus of the invention are useful for the diagnosis and treatment of atherosclerosis, preferably coronary artery diseases and provide the potential for the development of a pharmacodynamic profile of drugs for a given patient.

Description of the invention

The finding and characterization that variations in the intron 13 of the eNOS gene in human individuals is a putative coronary risk factor provide a very potent tool for improving the diagnostic and preventive therapy of atherosclerosis, preferably coronary artery diseases. The diagnosis of the individual allelic eNOS gene status permits a more focused therapy, e.g., by opening the possibility to apply individual dose regiments of drugs before the onset of the disease. Furthermore, diagnostic tests to genotype eNOS gene intron variants may help to correlate genotypes with drug activity or side effects.

Accordingly, the invention relates to an in vitro screening method for determining the presence of atherosclerosis, preferably coronary artery disease or susceptibility to such a disease in a subject comprising determining the CA repeat number in an intron of the endothelial nitric oxide synthase (eNOS) gene in a sample from a subject, the number of the CA repeat being indicative of increased risk of coronary artery disease. Preferably, said intron is intron 13 of the eNOS gene.

In the context of the present invention the term "increased risk" of atherosclerosis, preferably coronary disease as used herein means that carriers of high CA repeat numbers have an approximately twofold higher risk to suffer from atherosclerosis, preferably coronary artery disease compared to carriers of lower CA repeat numbers.

The term "atherosclerosis" comprises a common disease of the vessel wall, best characterized in Libby et al., Molecular biology of atherosclerosis. Int. J. Cardiol.

1997; 62 Suppl 2: S23-9.

As the product of endothelial nitric oxide synthase (eNOS), nitric oxide, possesses vasodilatory and antiantherogenic properties, an altered eNOS function could promote atherosclerosis. Preferably, the disease to be determined in accordance with the method of the present invention invention is atherosclerosis or a related disease such as general atherosclerosis, coronary artery disease, stroke, endothelial dysfunction or peripheral arterial occlusive disease.

In accordance with the present invention, it has surprisingly been found that the (CA)_n polymorphism in intron 13 is associated with excess risk of coronary artery disease. While the (CA)_n polymorphism in intron 13 was known (Nadaud et al.,

1994) its putative function, if any, had not been described. In accordance with the present invention, the association between variations in CA repeat copy number [(CA)_n polymorphism] in intron 13 of the eNOS gene and the risk of coronary artery disease was investigated in 1000 consecutive patients with angiographically confirmed coronary artery disease and 1000 age- and gender-matched controls by a PCR based fragment length calculation. 28 different alleles were identified containing 17 to 44 CA repeats. Presence of one allele containing >38 repeats was associated with an excess risk of coronary artery disease (odds ratio: 1.94; 95% confidence interval: 1.31 -2.86; p=0.001 ). Carriers of alleles containing >38 CA repeats were, in particular, overrepresented in the subgroup without common cardiovascular risk factors, odds ratio 3.39 (95% confidence interval: 1.30-8.86, p=0.009). Logistic regression analysis disclosed, that the (CA)_n polymorphism proved to be an independent risk factor (relative risk: 2.17; 95% confidence interval: 1.44- 3.27, p=0.0002). The findings obtained in accordance with the present invention indicate that high numbers of CA repeats in an intron of the eNOS gene are associated with an excess risk of atherosclerosis, preferably coronary artery disease. Thus, the present invention provides a simple and reliable screening method for determining increased risk of atherosclerosis, preferably coronary artery disease. For example, it is a well known fact that genomic DNA of individuals, which harbor the individual genetic makeup of all genes, including can easily be purified from individual blood samples. These individual DNA samples are then used for the analysis of the sequence composition of the eNOS gene intron alleles that are present in the individual which provided the blood sample. The sequence analysis was carried out by PCR amplification of relevant regions of intron 13 of the eNOS gene, subsequent purification of the PCR products, followed by automated DNA sequencing with established methods (ABI PRISM® dye terminator cycle sequencing).

One parameter that should be considered in the attempt to determine the individual genotype by direct DNA-sequencing of PCR-products from human blood genomic DNA is the fact that each human harbors (usually, with very few abnormal exceptions) two gene copies of each autosomal gene (diploidy). Because of that, great care should be taken in the evaluation of the sequences to be able to identify unambiguously not only homozygous sequence variations but also heterozygous variations.

The details of the different steps of the screening method of the present invention are described in the examples below. The methods of the mutation analysis followed standard protocols and are described in detail in the examples. In general such methods to be used in accordance with the present invention for evaluating the phenotypic spectrum in patients with mutations in the eNOS gene encompass for example haplotype analysis, single-strand conformation polymorphism analysis (SSCA), PCR and direct sequencing. On the basis of thorough clinical characterization of many patients the phenotypes can then be correlated to these mutations as well as to mutations that had been described earlier.

As described in the examples, patients being at risk of having atherosclerosis, preferably a coronary artery disease can be identified by comparing their (CA)_n polymorphism with that of patients known to have such a disease. Thus, in one embodiment of the screening method of the invention, the indication of increased risk of atherosclerosis, preferably coronary artery disease results from comparing said subjects measured level of CA repeat number to a set of reference data.

In a preferred embodiment of the present invention, the screening method described above further comprises assaying a subject for (a) common atherogenic risk factor(s) including arterial hypertension, hypercholesterolemia, diabetes and/or smoking, the results of the assay for the CA repeat number and the assay for atherogenic risk factor(s) being indicative of increased risk of atherosclerosis, preferably coronary artery disease.

Usually the step of comparing a patient's data with reference data comprises incorporating the data of measurement(s) into a probability density function generated from the set of reference data by a linear discriminant analysis procedure. Preferably, logistic regression analysis as described in the examples is used. The reference data reflects the CA repeat number in the intron of eNOS gene for subjects having atherosclerosis, preferably a coronary artery disease and/or the level of corresponding CA repeat number of normal, unaffected subjects. As will be generally understood by those skilled in the art, methods for screening for increased risk of atherosclerosis, preferably coronary artery disease are processes of decision- making by comparison. For any decision-making process, reference values based on patients having the disease or condition of interest and/or patients not having the disease or condition of interest are needed. In the present invention the reference value is preferably a CA repeat number of carriers of alleles containing >38 CA repeats, who were overrepresented in a subgroup without common cardiovascular risk factors. However, it is understood by the person skilled in the art, that depending on the population investigated a new set of reference data can be established by collecting the reference values for a number of samples. It will be apparent to those skilled in the art, that the set of reference data will improve by including increasing numbers of reference values. Furthermore, the step of comparing the subjects measured level of CA repeat number to a set of reference data may comprise a step of comparing the log of the data of measurement(s) to the set of reference data. To determine whether the patient is at increased risk of having or being susceptible to atherosclerosis, preferably a coronary artery disease, a cut-off must be established. It is apparent to those skilled in the art that a cut-off established to determine whether a patient is at increased risk of having or being susceptible to a atherosclerosis, preferably coronary artery disease may be established by the laboratory, the physician or on a case by a case basis by each patient. The cut-off level can be based on several criteria such as described in the examples. The cut-off level could be established using a number of methods, including: percentiles, mean plus or minus standard deviation(s), multiples of medium value, patient's specific risk or other methods known to those skilled in the art. Statistical and mathematical techniques for calculating the reference parameters, cut-off etc. are known to the person skilled in the art and are described in the examples. Furthermore, as mentioned above, in any one of the embodiments of the present invention, changing the risk cut-off level of a positive or using the different a priori risks which may apply to different subgroups in the population, could change the results of the discriminant analysis for each patient.

In a particularly preferred embodiment of the screening method of the present invention, a CA repeat number equal to or greater than 38 is indicative of increased risk of atherosclerosis, preferably coronary artery disease. Preferably, said disease is atherosclerosis or related thereto; see supra.

In accordance with the screening method of the present invention, the method of determining the CA repeat number in an intron of the eNOS gene can be performed by using a polynucleotide or a nucleic acid molecule capable of hybridizing to a coding region of - preferably exon 13 and/or 14 - of the eNOS gene or to a non-coding region, preferably intron 13. The nucleotide sequence of the eNOS gene is described in Miyahara et al., Cloning and structural characterization of the human endothelial nitric-oxide-synthase gene. Eur. J. Biochem. 1994: 223, 719-726 and available from the database, e.g., GenBank Ace. No. D 26607. Thus, a primer or oligonucleotide derived from the nucleotide sequence of the eNOS gene can be used for the detection of the (CA)_n polymorphism in an intron of the eNOS gene.

The polynucleotides and nucleic acids used for detection can, of course, be conveniently labeled by incorporating or attaching, e.g., a radioactive or other marker. Such markers are well known in the art. The labeling of said nucleic acid molecules can be effected by conventional methods. Additionally, the presence or expression of variant eNOS gene can be monitored by using a primer pair that specifically hybridizes to eNOS nucleic acid sequences and by carrying out a PCR reaction according to standard procedures. Specific hybridization of the above mentioned probes or primers preferably occurs at stringent hybridization conditions. The term "stringent hybridization conditions" is well known in the art; see, for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual" second ed., CSH Press, Cold Spring Harbor, 1989; "Nucleic Acid Hybridisation, A Practical Approach", Hames and Higgins eds., IRL Press, Oxford, 1985. Furthermore, the genomic DNA obtained from the subject may be directly sequenced to identify the CA repeat number in intron 13 of the eNOS gene. The present invention further comprises methods wherein such a fingerprint may be generated by RFLPs of DNA obtained from the subject, optionally the DNA may be amplified prior to analysis, the methods of which are well known in the art; see, e. g., Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Further modifications of the above-mentioned embodiment of the invention can be easily devised by the person skilled in the art, without any undue experimentation from this disclosure; see, e.g., the examples. In a preferred embodiment of the present invention, the above described methods comprise PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques or hybridization techniques.

Over the past 20 years, genetic heterogeneity has been increasingly recognized as a significant source of variation in drug response. Many scientific communications (Meyer, Ann. Rev. Pharmacol. Toxicol. 37 (1997), 269-296 and West, J. Clin. Pharmacol. 37 (1997), 635-648) have clearly shown that some drugs work better or may even be highly toxic in some patients than in others and that these variations in patient's responses to drugs can be related to molecular basis. This "pharmacogenomic" concept spots correlations between responses to drugs and genetic profiles of patient's (Marshall, Nature Biotechnology, 15 (1997), 954-957; Marshall, Nature Biotechnology, 15 (1997), 1249-1252). In this context of population variability with regard to drug therapy, pharmacogenomics has been proposed as a tool useful in the identification and selection of patients which can respond to a particular drug without side effects. This identification/selection can be based upon molecular diagnosis of genetic polymorphisms by genotyping DNA from leukocytes in the blood of patient, for example, and characterization of disease (Nebert, Clin. Genet. 56 (1999), 247-258; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103). For the providers of health care, such as health maintenance organizations in the US and government public health services in many European countries, this pharmacogenomics approach can represent a way of both improving health care and reducing overheads because there is a large cost to unnecessary therapies, ineffective drugs and drugs with side effects.

Furthermore, determining the risk of atherosclerosis, preferably coronary artery disease and susceptibility to such a disease can help to prevent the outbreak of the disease by applying drugs that counteract, e.g., other risk factors such as arterial hypertension, hypercholesterolemia, diabetes and smoking.

Thus, a particular object of the present invention concerns drug/pro-drug selection and formulation of pharmaceutical compositions for the treatment of atherosclerosis, preferably coronary artery diseases which are amenable to chemotherapy taking into account the polymorphism of the variant form of an intron of the eNOS gene that cosegregates with the affected phenotype of the patient to be treated and optionally other risk factors such as those mentioned above. This allows the safe and economic application of drugs which for example were hitherto considered not appropriate for therapy of, e.g., atherosclerosis due to either their side effects in some patients and/or their unreliable pharmalogical profile with respect to the same or different phenotype(s) of the disease. The means and methods described herein can be used, for example, to improve dosing recommendations and allows the prescriber to anticipate necessary dose adjustments depending on the considered patient group.

In a further embodiment of the above-described screening methods, a further step is included comprising administering to a subject determined to be at risk of having atherosclerosis, preferably a coronary disease, a medicament to abolish or alleviate said disease. This embodiment allows treatment of a given coronary artery disease before the onset of clinical symptoms due to the phenotype response caused by the eNOS gene and/or other risk factors. In a preferred embodiment of the method of the invention said medicament are chemotherapeutic agents such as ACE - inhibitors, Beta HGM reductase inhibitors ("Statins"), Aspirin, Clopidogrel, Antioxidants.

Furthermore, the present invention relates to the use of an oligo- or polynucleotide for the detection of the presence of the CA repeat number in intron 13 of the eNOS gene and determining the risk of coronary artery disease in the subject. Such oligo- and polynucleotides can be easily derived from the nucleotide sequence of the eNOS gene, preferably from the region of exon 13 - intron 13 - exon 14. Preferably, said oligo- or polynucleotide is about 15 to 50, preferably 20 to 40, more preferably 20 to 30 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 1 or 2 or a complementary sequence. Hence, in a still further embodiment, the present invention relates to a primer or probe consisting of an oligonucleotide as defined above. In this context, the term "consisting of" means that the nucleotide sequence of the oligo- and polynucleotide described above and employed for the primer or probe of the invention does not have any further nucleotide sequences of the eNOS gene immediately adjacent at its 5' and/or 3' end. However, other moieties such as labels, e.g., biotin molecules, histidin flags, antibody fragments, colloidal gold, etc. as well as nucleotide sequences which do not correspond to the eNOS gene may be present in the primer and probes of the present invention. Furthermore, it is also possible to use the above described particular nucleotide sequences and to combine them with other nucleotide sequences derived from the eNOS gene wherein these additional nucleotide sequences are interspersed with moieties other than nucleic acids or wherein the nucleic acid does not correspond to nucleotide sequences of the eNOS gene. Furthermore, it is evident to the person skilled in the art that the oligonucleotide can be modified, for example, by thio-phosphate- backbones and/or base analogs well known in the art (Flanagan, Proc. Natl. Acad. Sci. USA 96 (1999), 3513-8; Witters, Breast Cancer Res. Treat. 53 (1999), 41-50; Hawley, Antisense Nucleic Acid Drug Dev. 9 (1999), 61 -9; Peng Ho, Brain Res. Mol. Brain Res. 62 (1998), 1-11 ; Spiller, Antisense Nucleic Acid Drug Dev. 8 (1998), 281- 93; Zhang, J. Pharmacol. Exp. Ther. 278 (1996), 971-9; Shoji, Antimicrob. Agents Chemother. 40 (1996), 1670-5; Crooke, J. Pharmacol. Exp. Ther. 277 (1996), 923- 37).

Thanks to the present invention atherosclerosis, preferably coronary artery disease status can be determined independent from other, for example environmental risk factors. The dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients at the wrong dose.

Furthermore, the present invention relates to an assay kit for carrying out the method of the invention, said kit comprising means for assaying the CA repeat number in an intron of eNOS gene and optionally suitable means for detection. The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals. The kit of the invention may advantageously be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art. The kit or diagnostic compositions may be used for methods for detecting (CA)_n polymorphism in an intron of the eNOS gene in accordance with any one of the above-described methods of the invention, employing, for example, nucleic acid hybridization and/or amplification techniques such as those described herein before and in the examples.

In a still further embodiment, the present invention relates to an apparatus for the method of the invention, said apparatus comprising means adapted for receiving the data of measurement of a subject's level of CA repeat number in an intron of the eNOS gene and computer means for comparing the data of measurement of the level of the CA repeat number to a set of reference data to determine increased risk of coronary artery disease. These and other embodiments are disclosed or are obvious from and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on Internet, e.g. under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

The methods, uses, kits and apparatus of the invention can be used for the diagnosis of all kinds of atherosclerosis, preferably coronary artery diseases hitherto unknown as being related to or dependent on (CA)_n polymorphism in an intron, preferably intron 13 of the eNOS gene. Furthermore, as discussed in section 3 of the examples, the present invention provides the novel finding that CA repeats provide an important element for mRNA splicing and that this function strongly depends on the CA repeat length. Therefore, the present invention also relates to and enables a generally applicable in vitro screening method for determining the presence of a disease, disorder or susceptibility to a disease or disorder comprising determining the CA repeat number in an intron of a given gene in a sample from a subject, wherein the number of the CA repeat co-segregates with the disease, disorder or susceptibility to the disease or disorder. Thus, embodiments which make use of the in vitro screening method, uses, kits and apparatus except that instead of a eNOS gene a different gene is assayed are also in the scope of the teaching of the present invention. The methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein. Brief description of the figures

Figure 1 : Frequency of eNOS alleles in 974 cases and 979 controls containing different numbers of CA repeats. CAD indicates coronary artery disease.

Figure 2: Odds ratios (OR) and 95% confidence intervals at different cut offs.

The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

Examples

Genomic samples, isolated by standard techniques from Caucasian patients were obtained under consideration of all legal, ethical and medical requirement of the Charite University Medical Center of Berlin.

1. Methods

Study population

The present study was a matched case-control study with a multiple endpoint consisting of 7 genetic traits. The design of the study has been described in detail elsewhere (Laule et al., 1999). 1000 consecutive Caucasian patients from the Berlin area, who had been admitted for coronary angiography to the Charite University Medical Center, at the Humboldt University of Berlin, were enrolled between October of 1995 and January of 1997. An additional 1000 patients also admitted to this hospital served as controls. They were matched by age (±3 years), gender, and time of admittance (within two weeks). The disease spectrum of the control group reflects that of a large university hospital which provides all internal and surgical disciplines. Exclusion criteria were evidence of coronary or peripheral artery disease, or of any kind of vasculitis. All severe disease states potentially affecting atherogenic risk- factor profile and/or coagulation status were excluded. For the purpose of this study, all controls were screened on a basis of patient history, physical examination, ECG, and echocardiogram. This study was approved by the Charite Hospital Ethics Committee in 1994 and all participating subjects provided written consent.

Operational definitions

To determine the severity of coronary artery disease, the number of affected vessels with a stenosis >50% in a major coronary artery or in a major branch was used. The extent of coronary artery disease was expressed as number of affected arteries: i.e., as 1 -, 2- or 3-vessel disease. Diagnosis of myocardial infarction was established by evaluating patients' case notes on the basis of WHO criteria (1979) as well as by assessing angiographic findings. Body mass index was calculated as weight (kg) divided by the height (m) squared (kg/m²). Subjects were defined as smokers if they currently smoked or had smoked within the last 10 years. All others were classified as nonsmokers. Subjects were defined to be at "low risk" for coronary artery disease, if they had no common atherogenic risk factors including arterial hypertension, hypercholesterolemia, diabetes and smoking.

Laboratory measurements and techniques

Blood was collected in the morning after an overnight fast of at least 10 hours. EDTA-containing tubes served for analysis of plasma lipids. Serum total cholesterol and triglycerides were measured by enzymatic methods (Boehringer Mannheim, Germany). HDL-cholesterol was analyzed enzymatically (Boehringer Mannheim, Germany); and LDL-cholesterol, apolipoprotein A1 , and B by immunoturbidimetric assays (Tina-quant, Boehringer Mannheim, Germany). An immunoassay served for analysis of von Willebrand factor (Biomed Labordiagnostik, Schleissheim, Germany). 10 ml venous blood samples, drawn in EDTA as anticoagulant, were obtained from each subject and stored at -20°C. After thawing, erythrocytes were disrupted for 30 min in a hypoosmolaric buffer containing 155 mmol/L NH₄CI, 10 mmol/L KH₂C0₃, 0.1 mmol/L EDTA, at pH 8.0. Leukocytes were isolated by 30 min of centrifuging at 1000 g at 4°C, resuspended in 20 mmol/L Tris/HCI, 2 mmol/L EDTA, 30 mmol/L NaCI, at pH 7.5, and stored at -20°C. DNA was isolated manually by standard 3-step phenol/chloroform extraction after digestion with proteinase K (Boehringer, Mannheim, Germany), or with an 341 A Applied Biosystems^® (Weiterstadt, Germany) DNA extractor. DNA was dissolved overnight at 55°C in 10 mmol/L Tris and 1 mmol/L EDTA buffer (pH 8.0), and was stored at 4°C until further analysis.

The number of CA repeats in intron 13 was determined by a modified procedure of Nadaud (Nadaud et al., 1994). Approximately 50 ng of genomic DNA were amplified using 5 pmol of primers GT NOS (5'-TGAGGAGAGACTCAGAATTGGA, SEQ ID NO: 1 ) and fluorescence- labeled GT NOS BR (5'-FAM-GCTTGTGTGGGGTTTCAGGCT, SEQ ID NO: 2) (TIB Molbiol, Berlin, Germany), 0.2 mmol/L dNTP (Boehringer-Mannheim, Germany), 3 mmol/L MgCI₂, and 1 unit AmpliTaq^® (Perkin Elmer, Weiterstadt, Germany) in a volume of 25 μl containing appropriate buffer. After initial 2 min at 94°, 20 cycles were performed for 20 s at 95°C and 30 s at 60°C, further 10 cycles for 20 s at 95°C and 30 s at 58°C, and finally 5 cycles for 20 s at 95°C, and 30 s at 54°C. Amplification efficiency was checked by 3% MetaPhor^® (FMC Rockland, Maine, USA) gel electrophoresis. The fragment size was analyzed with an Applied Biosystems 373A^® sequencer with a denaturing 6% polyacrylamide gel using GeneScan^® software in presence of an internal DNA size standard GS500 (Perkin Elmer).

For correlation of fragment size with the number of CA repeats, 55 selected samples with apparently homozygous alleles (same number of repeats) were sequenced after amplification using the same primers as above, but without fluorescence label. The products were purified with 30000 NMWL ultrafiltration filter units (Millipore, Eschborn, Germany) and 3 μl were processed with a ABI PRISM^® dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Weiterstadt, Germany). The number of CA repeats was plotted versus the fragment size as obtained by GeneScan^® analysis. Since the number of CA repeats and the fragment size showed a linear relation, the correlation of the fragment size to the number of CA repeats could be calculated by the equation: (CA)_n = Vz (fragment length - 106).

Statistical analysis

The study was a matched case-control study. Samples were controlled ccording to age and gender. Values are presented as median and as 25^th and 75^th percentiles. Genotype frequencies were compared by χ²- or by Fisher's exact test. Differences between cases and controls were investigated by means of the distribution of the CA repeat numbers. All further calculations were made for presence of at least one allele. The relative risk of repeat copy numbers was cumulatively calculated at different cutoff values by logistic regression analysis adjusted for age, body mass index, gender, diabetes, smoking, hypertension, and hypercholesterolemia. Logistic regression was performed to determine the influence of CA repeat copy numbers as an independent atherogenic risk factor in comparison to other established risk factors. Association of CA repeats with the extent of disease (3-vessel versus 1 -vessel disease), early disease manifestation (<40 years), acute coronary syndromes, as well as acute and previous myocardial infarction was analyzed by logistic regression. To evaluate the impact of eNOS genotypes on anthropometric and biochemical parameters, multiple linear regression analysis was employed. In case of small or sparse data sets, data with ties or large but unbalanced data, exact nonparametric inference was applied. All analyses were performed by means of the Windows SPSS 7.5 package and StatXact-3^®, from Cytel.

2. Results

Patient characteristics

Table 1 depicts baseline characteristics of the case patients and the control subjects. Both groups differed significantly with respect to the higher prevalence of atherogenic risk factors including diabetes, hypercholesterolemia, hypertension and smoking in the case group. Moreover, cases had significantly higher levels of total cholesterol and LDL- cholesterol, apolipoprotein B and A1 , and triglycerides. Genotype was not available in 26 cases and in 21 controls.

Cases Controls

(n=1000) (n=1000)

Age (years) 60.6 (55.1 -67.1 ) 60.5 (54.5-66.5) 0.477

Female (%) 24.1 24.1

History (%)

Diabetes 22.8 11.4 <0.00

1

Smoking 44.0 35.2 <0.00

1

Hypertension 55.2 35.9 <0.00

1

Hypercholesterolemia 52.7 30.3 <0.00

1

Age of manifestation < 40 years (%) 6.8

Acute coronary syndrome. > (%) 23.5

Mvocardial infarction (%) 66.9

Acute 9.1

History of 57.8

Severity of coronary artery disease

(%)

1 -vessel 29.7

2-vessel 36.5

3-vessel 33.8

Body mass index 26.3 (24.3-28.6) 26.0 (24.0-28.7) 0.182

Total cholesterol (mg/dL) 224 (197-255) 212 (181 -243) <0.00

1

LDL cholesterol (mgl/dL) 147 (120-174) 139 (112-162) <0.00

1 l

HDL cholesterol (mg/dL) 42 (35-54) 42 (35-54) 0.727

Apolipoprotein B (g/L) 1.2 (1.0-1.4) 1 .1 (0.9-1.3) <0.01

Apolipoprotein A1 (g/L) 1.2 (1.0-1.4) 1.1 (0.9-1.3) <0.00

1 I

Triglycerides (mg/L) 151 (1 15-213) 133 (97-177) <0.00

Von Willebrand factor (%) 244 (181 -320) 246 (173-340) 0.472 Table 1 : Characteristics of cases and controls. Continuous values are expressed as median, 25^th and 75^th percentiles.

(CA)n polymorphism and coronary artery disease

Figure 1 shows the allele frequencies in cases and controls. 28 different alleles were identified containing 17 to 44 CA repeats. The odds ratios and 95% Cl at different cut off points starting from 24 CA repeats to 40 CA repeats are depicted in Figure 2.

Cutoff value Cases Controls Univariate analysis Logistic regression

Number of CA < Cutoff > Cutoff < Cutoff > Cutoff Odds ratio P Relative risk* P repeats n n (%) n n (%) (95% Cl) (95% Cl)

34 440 534 (54.8) 487 492 (50.3) 1.20 (1.01-1.44) 0.043 1.28 (1.06-1.55) 0.010 t t-

35 587 387 (39.7) 640 339 (34.6) 1.25 (1.04-1.50) 0.020 1.34 (1.10-1.63) 0.004

36 731 243 (29.4) 795 184 (18.8) 1.44 (1.16-1.78) 0.001 1.54 (1.22-1.93) 0.0003

37 831 143 (14.6) 876 103 (10.5) 1.46 (1.12-1-92) 0.006 1.66 (1.25-2.22) 0.0005

38 898 76 (7.8) 938 41 (4.2) 1.94 (1.31-2.86) 0.001 2.17 (144-3.27) 0.0002

39 936 38 (3.9) 957 22 (2.2) 1.77 (1.04-3.01) 0.034 2.00 (1.14-3.50) 0.016

40 960 14 (1.4) 974 5 (0.51) 2.84 (1.02-7.92) 0.037 3.61 (1.24-10.5) 0.018

Table 2: Frequencies of subjects, odds ratios and relative risks of the CA repeat copy number for coronary artery disease at different cutoff values. Cl denotes confidence interval. *Adjusted for age, body mass index, gender, diabetes, hypercholesterolemia, hypertension, and smoking, f Values are expressed as a percentage of the 974 available genotypes in the case group. $ Values are expressed as a percentage of the 979 available genotypes in the control group.

As shown in Table 2, presence of one allele including >34 CA repeats was associated with an excess risk of coronary artery disease with the highest significance being reached at a cutoff value >38 (univariate analysis: odds ratio: 1.94, 95% confidence interval: 1.31-2.86, p=0.001); logistic regression: relative risk: 2.17; 95% confidence interval: 1.44-3.27; p=0.0002). We therefore performed further analyses using this cutoff value. Among cases, 7.8% carried one allele containing >38 repeats, as did 4.2% among controls.

Cases Controls Odds ratio p

CA repeats CA repeats

<38 >38 <38 >38 (95% Cl) n n n n

All 898 76 938 41 1.94 (1.31-2.86) 0.001

Male gender 678 61 714 31 2.07 (1.33-3.23) 0.001

Female gender 220 15 224 10 1.53 (0.67-3.47) 0.309

Diabetes 208 11 108 2 2.86 (0.62-13.1) 0.233

Hypertension 502 37 336 15 1.65 (0.89-3.06) 0.107

Hypercholesterolemia 480 35 282 15 1.71 (0.88-3.36) 0.112

Smoking 393 37 333 15 2.09 (1.13-3.88) 0.017

At low risk 72 9 244 9 3.39 (1.30-8.86) 0.009 Table 3: Frequencies of subjects and odds ratios of the (CA)_n polymorphism for the cutoff value >38 in subgroups stratified according to gender and common atherogenic risk factors. Cl denotes confidence interval.

As depicted in Table 3, stratification to gender and to the presence or absence of common atherogenic risk factors including diabetes, hypercholesterolemia, hypertension, and smoking revealed, that carriers of alleles including >38 repeats were, in particular, overrepresented in the subgroup lacking common cardiovascular risk factors with an odds ratio of 3.39 (95% confidence interval: 1.30-8.86, p=0.009). In a further step, logistic regression was performed to evaluate the importance of the (CA)n polymorphism at the cutoff >38 repeats for subgroups of case patients at higher risk.

Relative risk* P

(95% Cl)

Disease severityf 0.77 (0.4-1.5) 0.435

Age of manifestation <40 years 1.32 (0.6-3.0) 0.506

Acute coronary syndromes 0.99 (0.7-1.4) 0.686

Unstable angina 1.04 (0.5-2.0) 0.899

Acute myocardial infarction 0.88 (0.3-2.5) 0.816

History of myocardial infarction 1.50 (0.9-2.6) 0.145

Table 4: Relative risk of the (CA)_n polymorphism for subgroups of coronary artery disease patients at higher risk. Cl denotes confidence interval. *Adjusted for age (not for subgroup: age of manifestation), body mass index, gender, diabetes, hypertension, hypercholesterolemia, and smoking. The calculations were made for the cutoff value >38 CA repeats, f comparing 3-vessel versus 1 - vessel disease.

As shown in Table 4, the (CA)_n polymorphism was not associated with an increased risk for developing 3-vessel disease, early disease manifestation, acute coronary syndromes, or myocardial infarction. The impact of the (CA)_n polymorphism and of common risk factors on the development of coronary artery disease was calculated by logistic regression analysis (Table 5).

Relative risk P

(95% Cl)

Hypercholesterolemia 2.58 (2.12-3.13) 0.0001

Diabetes 2.20 (1 .69-2.86) 0.0001

(CA)_n polymorphism^* 2.17 (1 .44-3.27) 0.0002

Hypertension 2.12 (1 .74-2.59) 0.0001

Smoking 1.68 (1 .37-2.06) 0.0001

Body mass index 0.98 (0.95-1 .01 ) 0.223 Table 5: Risk factors of coronary artery disease as calculated by logistic regression analysis. Cl denotes confidence interval, "calculated for the cutoff value >38 CA repeats.

Beside hypercholesterolemia, diabetes, hypertension, and smoking, the (CA)_n polymorphism proved to be an independent risk factor with a relative risk of 2.17 (95% confidence interval: 1.44-3.27, p=0.0002).

(CA)n polymorphism and clinical parameters

Furthermore, investigated the relationship of the (CA)_n polymorphism to patient characteristics was investigated which included age, systolic and diastolic blood pressure and body mass index, and to biochemical parameters such as triglycerides, total cholesterol, LDL- and HDL-cholesterol, apolipoprotein A1 and B, and von Willebrand factor. In association with the (CA)_n polymorphism, multiple regression analysis revealed no differences among carriers and non-carriers of alleles >38 CA repeats with regard to these parameters. Of particular interest, with medians of 130 mmHg (25^th-75^th percentiles: 120-145 mmHg) versus 135 mmHg (120-145 mmHg), p=0.70, and 80 mmHg (75-90 mmHg) versus 80 mmHg (75-90 mmHg), p=0.75, no significant differences in systolic and diastolic blood pressure were found between the two groups.

Discussion

Endothelial nitric oxide synthase plays a key role in vascular wall homeostasis and there is accumulating evidence that derangements of the eNOS pathway may contribute to the pathogenesis of atherosclerosis (Cooke and Dzau, 1997). Thus, it is intriguing to focus on eNOS gene as a candidate for coronary artery disease and to investigate eNOS gene polymorphisms as potential inherited risk factors. In corroboration of this concept, Wang et al. described a variable number of tandem repeats at intron 4 as a smoking dependent atherogenic risk factor in an Australian population (Wang et al., 1996).

The present invention provides first evidence that the (CA)_n polymorphism in intron 13 of the eNOS gene is associated with an excess risk of coronary artery disease. The results, based on large sample size of 2000 cases and controls, indicate that high CA repeat copy numbers manifested in an increased coronary risk. As the most significant differences were yielded for CA repeat numbers >38, logistic regression was employed for this cutoff value to determine the importance of the (CA)_n polymorphism on development of coronary artery disease.

It was a key finding of the present invention that the (CA)_n polymorphism proved to be an independent risk factor with an impact comparable to that of established parameters including hypercholesterolemia, diabetes, hypertension, and smoking. In all strata in cases listed in Table 3, more carriers of high CA repeats (>38) have been found than in controls. These differences achieved significance only among men, smokers, and subjects at low risk. At present, no explaination can be given for the finding of a smaller odds ratio among women, except in terms of the larger confindence intervals for the smaller number of cases.

The importance of the (CA)_n polymorphism became most apparent in the "low risk" group which lacked potentially confounding common cardiovascular risk factors.

The observed coronary excess risk at high repeat numbers was based on the presence of only one allele, a fact, which is suggestive for a dominant or gain- of-function type mechanism. The (CA)_n polymorphism showed a lack of linkage with premature disease onset and extent of disease, and it, furthermore, did not produce significant alterations in laboratory and biometrical parameters. Consistent with an earlier report (Bonnardeaux et al., 1995) no differences in systolic and diastolic blood pressure associated with the (CA)_n polymorphism were detected. Putative mechanisms

Molecular mechanisms that underlie the association between the (CA)_n polymorphism and cardiovascular excess risk are not yet elucidated. The most difficult issue to address is, whether it is a direct effect of the repeat itself, or the repeat is in linkage disequilibrium with some other causal element. One possibility is, that the (CA)_n polymorphism is perverse, decreases eNOS expression and/or activity and leads to derangements of the eNOS pathway, in turn promoting atherosclerosis. For example, variation in the CA repeat number may affect eNOS gene expression on the mRNA splicing level by acting as an important regulatory element. As recently shown, CTG expansions in a non-coding region of the myotonic dystrophy gene sequester a protein which acts as a splicing regulator on different pre- mRNAs, such as that of troponin T, thereby leading to aberrant splicing causing disease (Philips et al., 1998). Since A/C-rich motifs have been identified as splicing enhancers (Coulter et al., 1997), CA expansions at intron 13 of the eNOS gene could also sequester a factor that plays a role in splicing of the eNOS pre-mRNA or of another crucial pre-mRNA in the vascular wall tissue. Consistent with this hypothesis, it could be demonstrated through in vitro studies that CA repeats function as a novel intronic splicing enhancer element: Minigenes derived from eNOS were constructed that contain exons 13 and 14 as well as intron 13 with various CA repeats. In vitro splicing revealed that no splicing activity could be detected without CA repeats, and that the splicing efficiency could be dramatically stimulated by the presence of CA repeats; significantly, the extent of splicing stimulation clearly correlated with the number of CA repeats. These findings support the hypothesis that the CA repeats provide an important element for eNOS mRNA splicing and that this function strongly depends on the CA repeat length. In another scenario, the (CA)_n polymorphism merely tracks with coronary artery disease without causal relation to it and the observed association, moreover, reflects a linkage disequilibrium with a causal variation in the eNOS gene itself or in a neighboring gene. However, up to now, there are no data on a linkage between the (CA)_n polymorphism and to the two polymorphisms described as potentially functional - i.e., the variable number of tandem repeats at intron 4 (Wang et al., 1996) and Glu298Asp (Poirier et al.,1999) - or to any other reported eNOS polymorphism.

In conclusion, the present invention provides evidence that the (CA)_n polymorphism of the eNOS gene is associated with an excess risk of coronary artery disease and represents an independent risk factor with an impact comparable to that of established cardiovascular risk factors. Among the putative underlying molecular mechanisms, the role of high numbers of CA repeats as regulatory element for splicing of the eNOS gene requires further clarification.

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Claims

1. An in vitro screening method for determining the presence of atherosclerosis, preferably coronary artery disease or susceptibility to such a disease in a subject comprising: determining the CA repeat number in an intron, preferably intron 13 of the endothelial nitric oxide synthase (eNOS) gene in a sample from a subject, the number of the CA repeat being indicative of increased risk of atherosclerosis, preferably coronary artery disease.

2. The method of claim 1 , wherein the indication of increased risk of atherosclerosis, preferably coronary artery disease results from comparing said subjects measured level of CA repeat number to a set of reference data.

3. The method of claim 1 or 2 further comprising: assaying a subject for (a) common atherogenic risk factor(s) including arterial hypertension, hypercholesterolemia, diabetes and/or smoking, the results of the assay for the CA repeat number and the assay for the atherogenic risk factor(s) being indicative of increased risk of atherosclerosis, preferably coronary artery disease.

4. The method of claim 2 or 3, wherein the step of comparing comprises incorporating the data of measurement(s) into a probability density function generated from the set of reference data by a linear discriminant analysis procedure.

5. The method of any one of claims 2 to 4, wherein the step of comparing comprises comparing the log of the data of measurement(s) to the set of reference data.

6. The method of any one of claims 1 to 5, wherein a CA repeat number equal to or greater than 38 is indicative of increased risk of atherosclerosis, preferably coronary artery disease.

7. The method of any one of claims 1 to 6, wherein said disease is atherosclerosis or related thereto.

8. The method of any one of claims 1 to 7 comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques or hybridization techniques.

9. The method of any one of claims 1 to 8, further comprising administering to a subject determined to be at risk of having atherosclerosis, preferably a coronary artery disease, a medicament to abolish or alleviate said disease.

10. Use of an oligo- or polynucleotide for the detection of the presence of the CA repeat number in an intron of the eNOS gene and determining the risk of atherosclerosis, preferably coronary artery disease in the subject in accordance with a method of any one of claims 1 to 9.

11. The use of claim 10, wherein said oligonucleotide is about 15 to 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 1 or 2.

12. An assay kit for carrying out the method of any one of claims 1 to 9, said kit comprising means for assaying the CA repeat number in an intron of the eNOS gene.

13. An apparatus for the method of any one of claims 1 to 9, said apparatus comprising means adapted for receiving the data of measurement of a subject's level of CA repeat number in an intron of eNOS gene and computer means for comparing the data of measurement of the level of the CA repeat number to a set of reference data to determine increased risk of atherosclerosis, preferably coronary artery disease.