METHOD AND KIT FOR DIAGNOSIS OF AN INCREASED RISK OF CARDIOVASCULAR DISEASES

Abstract: The invention relates to a method for indicating groups at risk of cardiovascular diseases. In particular, the invention offers the possibility of determining whether a person, by smoking, has an increased risk of cardiovascular disease on the basis of a genetic predisposition. A polymorphism has now been found which is clearly associated with an increased risk of cardiovascular disease in smokers in contrast to non-smokers. Smokers who possess a specific allele are at appreciably greater risk than smokers who do not possess this allele.

Description: Method and kit for diagnosis of an increased risk of cardiovascular diseases The invention relates to a method for indicating groups at risk of cardiovascular diseases. In particular, the invention offers the possibility of determining whether a person, by smoking, has an increased risk of cardiovascular disease on the basis of a genetic predisposition. A polymorphism has now been found which is clearly associated with an increased risk of cardiovascular disease in smokers in contrast to non- smokers. Smokers who possess a specific allele are at appreciably greater risk than smokers who do not possess this allele. Cardiovascular diseases are some of the major causes of disease and death in the world, in particular in the western world. It is generally accepted that myocardial infarctions are caused by blockages in the coronary arteries. A large number of suggestions for finding genetic causes for various cardiovascular diseases have been made. In "The Journal of Biological Chemistry", Vol. 271, no. 22, pp. 13055-13060, Shu Ye et al. indicate that a 5A/6A polymorphism in the stromelysin promoter can be responsible for the course of coronary arteriosclerosis. In "Coronary Arterie Decisie" (1996; 7:p.51~55), Arterie Padua et al. state that a deletion polymorphism in the gene for the angiotensin-converting enzyme constituted a risk factor for cardiovascular disease in individuals who otherwise had a low risk of such an abnormality. What is relevant here is the presence or absence of an Alu repetitive sequence of 287 base pairs , as described by Nieuwenhuizen et al. in their 1995 European Patent Application on t-PA- polymorphism. It is suspected that a reduced fibrinolytic capacity increases the risk of myocardial infarctions. For instance, it has been found that polymorphism in fibrinogen is possibly associated with an increased risk of myocardial infarction and thrombosis (de Maat M.P.M. et al. Regress. September 23, 1996). It is also reported that smoking has no effect on plasma fibrinogen contents in CAD patients (patients suffering from cardiovascular arteriosclerosis) (de Maat MPM, Pietersma A, Kofflard M, Sluiter W., Kluft C. Association of plasma fibrinogen levels with coronary artery disease, smoking and inflammatory markers. Atherosclerosis 1996.121: 185-191 and Cortellaro M, Boschetti C, Cofrancesco E, Zanussi C, Catalano M, de Baetano G, Gabrielli L, Lombardi B, Specchia G, Tavazzi L, Tremoli E, Delia Volpe A, Polli E, and the PLAT study group. The PLAT Study: a multidisciplinary study of haemostatic function and conventional risk factors in vascular disease patients. Atherosclerosis 1991;90:109-118). The fibrinolytic capacity can be reduced by a raised concentration of plasminogen activator inhibitor (PAI) in the blood. Although raised concentrations of PAI type I sometimes appear to be associated with an increased risk of myocardial infarctions, a consistent relationship has not yet been demonstrated. A review of the state of the art in this field is given, for instance, by Heinrich J, Balleisen L, Schulte H, Assmann G, van de Loo J. Fibrinogen and factor VII in the prediction of coronary risk. Results from the PROCAM Study in healthy men. Atheroscler Thromb 1994; 144;54-59. correction: 1994; 144:1392. An increasing amount of research is being performed into genetic predisposition to cardiovascular diseases. However, none of this research provides any teaching on the effect of smoking in combination with one or more of these genetic factors or polymorphisms . Recently it has been found that polymorphism occurs at site - 675 in the promoter region of the PAI-1 gene, one allelic variant containing four successive guanosine residues (4G) and the other five (5G) . In this context it was found that the 4G allele is associated with raised PAI-1 plasma levels (Dawson et al., J. Biol. Chem. 268 (1993) 10739)- Other polymorphisms of PAI-1 have also been disclosed. (Ernst E, Matrai A, Marshall M. Blood rheology in patients with transient ischemic attacks. Stroke, 1988;19:634-636; Qizilbash N, Jones L, Warlow Ch, Mann J. Fibriogen and lipid concentrations as risk factors for transient ischaemic attacks and minor ischaemic strokes. Br. Med. J. 1991;303 605 _ 609; Koster T, Rosendaal FR, Reitsma PH. van de Velden PA, Briet E, Vandenbroucke JP. Factor VII and fibrinogen levels as risk factors for venous thrombosis. Thromb Haemostas 199*+ ;71:719-722) . These have not been linked with MI, nor has any attention been paid to the relevance thereof with regard to smoking. In an article by Eriksson et al. (Proc . Natl. Acad. Sc i . US 92 (1995) 1851 ) it is demonstrated that the 4G allele occurs more frequently in patients suffering from MI. These findings point to the role of PAI-1 in the origin of myocardial infarctions. However, a relationship between the 4G-5G polymorphism and the risk of myocardial infarctions is contradicted by other studies (Ye et al. Thromb. Haemostas. 7 (1995) 837; Ridker et al., 1996 (personal notification) . The Eriksson article makes no mention of or suggestion with regard to smoking. The aim of the invention is to provide methods and means for reducing the risk of cardiovascular diseases by identifying groups at risk of such diseases and establishing whether an individual belongs to such a risk group. Once it has been established that a person belongs to a risk group, preventive measures can be taken. Examples of relevant cardiovascular diseases are thrombosis , cerebral and myocardial infarctions, angina pectoris, peripheral arteriosclerosis and vascular dementia. Infarctions are of particular importance. It has now been found that homozygotism for the 4G allele of PAI-1 (genotype 4G4G) is associated with a slightly increased risk (+ 60 % ) of cardiovascular disease, in particular myocardial infarction, compared with 5G homozygotes (genotype 5G5G) , but that for smokers this increase is by a factor of approximately 5, whilst heterozygous smokers (genotype 4G5G) appear to have approximately 1 £ times the risk of myocardial infarction. The invention therefore relates to a method with which such risk groups can be identified or individuals belonging to such groups can be detected. Said method is characterised in that persons are selected who come into contact, have come into contact or possibly will come into contact with tobacco smoke in such a way that they can be designated as smokers and the nature of the 4G/5G polymorphism in the promoter region of the gene of the plasminogen activator inhibitor type I (PAI-1) of the selected persons is then determined. The selected persons can be active or passive smokers. Passive smokers are those who, because they are frequently in an environment where there are one or more smokers, inhale a quantity of smoke which can possibly also potentially be hazardous to them. Cancer research carried out on passive smokers and smokers has revealed that passive smoke contains higher concentrations of toxic and carcinogenic substances than directly inhaled smoke. 75 % of the nicotine in a cigarette passes into the atmosphere. On diagnosis of increased risk, this group can possibly force a change in the passive smoking conditions , for example a change in the working conditions or the like on the grounds of medical indication. A smoker or a passive smoker can be defined as someone who has a raised cotinine level in the saliva or urine. The detection of tar in the lungs is also a good gauge of whether someone has to be regarded as a (passive) smoker. These tests are known to a person skilled in the art in the field of medical research on smokers. Such a person skilled in the art will know which persons belong to the category of smokers and passive smokers. In any event regular users of cigarettes, cigars and pipes must be designated as smokers. Regular means that they consume at least one standard pack of tobacco product, such as 20 cigarettes, in 1 week or consume an equivalent harmful quantity of one or more other tobacco products. In principle, the test will be able to be used on anyone who regards him or herself as a smoker, in other words on the basis of subjective criteria. The test persons can also be a man/woman couple who have a child or wish to have a child and who want to establish whether their child (potentially) belongs to the risk group. Depending on the finding it is possible to determine what genotype the child has or will have and it is also possible to determine whether preventive measures are needed. Consequently the child itself does not always have to be tested. The person can also be a pregnant woman. After all, a foetus is dependent on the quality and quantity of the vascular system of the womb. By virtue of this method, targeted preventive treatment to prevent cardiovascular diseases in risk groups can be proposed. In particular, this method can also be used on young persons before they start to smoke or at an early stage, so that actual prevention can take place before any damage has been caused. The term young persons covers people aged between 0 and 18. Preventive measures include stopping or preventing smoking, but also a change in smoking habits, for example cutting down on smoking, changing tobacco product or nicotine source to a less aggressive type and the provision of information which can assist in stopping smoking. This includes, for example, stopping with the aid of chewing gum. Another possibility is slow reduction in nicotine consumption by the temporary use of nicotine patches or other forms of nicotine release not associated with smoking. Preventive measures in the first instance also include the provision of information on polymorphism and the associated consequences for smokers. This information can be given in the form of a leaflet included with the kit or can be provided orally before, during or after the test. The leaflet can also provide information on the preventive measures to be taken, as have already been indicated above. The kit can be provided with information on the risk of smoking, prevention of smoking or a change in smoking habits . Sources from which such information can be obtained can also be listed in the kit. The kit possibly contains information on courses or alternative sources of information for assistance in taking measures to change smoking habits. For example, a web site on the Internet can be a potential source of information. Reference to courses in this field and the names of the bodies providing such courses can, for example, also form part of the kit in the form of a leaflet or something similar. Determination of the nature of the 4G/5G polymorphism in the promoter region of the gene of the plasminogen activator inhibitor type I (PAI-1) can be carried out in a manner known per se analogously to other polymorphism detection methods. Said determination can, for example, be carried out by means of allele-specific PCR amplification (Mansfield et al. Thromb. Haemostas . 74 (1995) 1032) or using another amplification technology such as the generally known NASBA. A person skilled in art can also use other technology for detection of specific sequences. Suitable probes are, for example, probes with a sequence of 12- 20, such as CACGTGGGGAGTCA and ACGTGGGGGAGTCA or the complementary sequences for, respectively, 4G and 5G variants (see also Dawson et al. and Eriksson et al., cited above). Only one or more probes which is or are specific for one of the alleles has to be used, the absence of reaction indicating homozygotism for the other allele. Preferably, a probe is used which indicates whether the 4G allele is present. If the 4G allele is absent, the person is homozygous for 0 and does not have the increased risk of myocardial infarction as a consequence of smoking which can be associated with the 4G allele. It is also preferable to select a probe such that it becomes clear whether homozygotism or heterozygotism occurs. After all, a person homozygous for the 4G allele has a greater risk than does a person who is heterozygous. The heterozygous person, in turn, has an increased risk compared with the 5G/ G homozygous person. The selection of a usable probe or usable probes which can provide information on this can readily be made by a person skilled in the art without any inventive effort, on the basis of the data presented here and the cited literature with the sequences for the PAI-I alleles in combination with general knowledge on allelic polymorphism. For the sake of completeness it is pointed out that the content of the cited literature is incorporated here by reference. In addition to the possibility of preventing cardiovascular diseases associated with smoking, there is now also the possibility of obtaining reassurance if one is 5G/5G homozygous and thus has no or only a slightly increased risk. Furthermore, it is now also possible to develop tobacco products with which the risk of cardiovascular diseases associated with the 4G allele can be avoided. After all, there is now a test which can be used to verify that this objective has been achieved. In addition, this knowledge offers a basis for unravelling the mechanism which is responsible for increased risk. The possibilities also include screening groups or individuals in combination with other risk factors. For instance, within the risk group a subdivision can be made into sub-groups with various levels of risk. Such risk factors can also be genetic in nature, but can also be different. For instance, by way of example it is possible to screen for factors which are associated with diabetes mellitus, hyperhomocysteinaemia, obesity, high blood pressure, insulin resistance, cancer, little exercise and poor diet. Both the "American Medical Association" and Dutch general practitioners have guidelines for such factors and those skilled in the art can therefore be considered to have such information at their disposal. In addition to genetic tests for more than one gene, it is thus possible, by way of example, to test for 4G/5G polymorphism in combination with blood pressure and/or vessel wall thickness and/or high blood lipids. Various test methods are known and commercial kits available for all of the risk factors mentioned above. One or more test methods for determination of diabetes mellitus, hyperhomocysteinaemia, obesity, high blood pressure, insulin resistance, cancer, little exercise, vessel wall thickness and blood lipids can be carried out in combination with a 4G/5G polymorphism determination and in this combination forms part of the invention. In general, a method in which a 4G/5G polymorphism determination is carried out with another test for determination of a risk factor associated with smoking is an embodiment of the invention. A kit with which such a combination method has to be carried out likewise forms part of the invention. A kit in which means are present for performing a 4G/5G polymorphism determination of the PAI-I promoter in site -675 in combination with a leaflet at least containing information on the risk of smoking in combination with the various -675 morphisms with respect to cardiovascular diseases also forms part of the invention, both as such and in combination with other means for determination of at least one other risk factor associated with cardiovascular disease, as indicated above. A kit in which means are present for carrying out a 4G/5G polymorphism determination of the PAI-1 promoter in site -675 in combination with means for determination of a factor which indicates that a person belongs to the category of smokers likewise forms part of the invention. Such a factor can be cotinine in a human sample of, for example, urine or saliva. Said kit can, of course, also contain elements from the other kits which form part of the invention. Such a kit is of interest for passive smokers in particular. Example 132 persons with a history of myocardial infarction and 265 control persons were obtained from the Rotterdam study group of 7983 men and women aged 55 and older. The -675 (4G/5G) polymorphism in the PAI-1 gene was determined in blood cells. The population A study was carried out on test persons who were selected from the Rotterdam study. The Rotterdam study is a population which is based on a study of 7 83 persons. Between March 1990 and July 1993 all persons aged 55 and older who lived in Ommoord, a suburb of Rotterdam in The Netherlands, were asked to take part. 78 % responded to this request. The study was approved by the Medical Ethics Committee of the Erasmus University and written consent was obtained from all participants. The objective and the design of the study have been described elsewhere (Ref. 2). Patients (n=150) were selected from this group on the basis of the presence of an infarction pattern on an ECG using the Modular ECG Analysis System diagnostic classification system (Ref. 3, O > independently of a history of chest pain. Two control test persons per case were arbitrarily selected from a group of the same age, such a group covering a period of 5 years and the myocardial infarction cases originating from a sample of the participants who had no history of cardiovascular disease, that is to say no history of myocardial infarction, angina pectoris or infarction and who had a normal ECG and no peripheral arterial disease (ankle/arm index 0.9), Test persons who were taking anticoagulants were excluded. Clinical trial All test persons were first visited at home. Information on current state of health, medical history, use of drugs and smoking habits was obtained by means of a computerised questionnaire which comprised the Dutch version of the Rose cardiovascular questionnaire (Ref. 5) • The home interview was followed by two visits to the test centre between 8 a.m. and 4 p.m.. The test persons were asked not to fast and not to stop smoking. Various cardiovascular risk indicators were determined during these visits. For instance, height and weight were determined and the body mass index was calculated in kg per m 2 . Blood pressure when seated was determined in the right upper arm using an arbitary zero sphygmomanometer. The average of two measurements per time was taken. The systolic blood pressure was determined in the ankles (rear tibia vein) with the test person lying down, using a standard sleeve for adults just above the malleoli with an 8 MHz Doppler transducer device (Ref. 6) . The ankle/arm index is the ratio of the systolic blood pressure in the ankle to the systolic pressure in the arm. Peripheral arterial disease was defined as a right or left ankle/arm index of less than or equal to 0.9- Laboratory study The taking of blood samples and the storage thereof has been described elsewhere (Ref. 7)- Blood samples were collected using CTAD vacutainers (0.11 M citrate, 15 mM theophylline, 3-7 mM adenosine and 0.198 mM dipyridamole ; Diatube H, Becton and Dickinson, Mylan, Cedex, France (Ref. 8)). The total, serum and HDL cholesterol was determined using an automated enzymatic determination method. The PAI-1 antigen concentration was measured in CTAD plasma using Innotest PAI-1 ( Innogenetics , Zwijndrecht, Belgium) (Ref. 9), Genomic DNA was isolated from blood cells and the -675 (4G/5G) PAI-1 genotype was determined by means of allele-specific PCR amplification (Ref. 10) . Statistical analysis After excluding persons for whom there were no data with regard to either 4G/5G polymorphism or PAI-1 antigen concentration, the population tested was made up of 132 cases and 265 control persons. There was a positive deviation in the distribution of plasma concentration of PAI-1 antigen and, therefore, statistical analysis was carried out on the logarithmic transformations, but the average value and the 95 % confidence intervals (CI) which are presented were back-transformed to the original scale. Quantities of PAI-1 antigen and averages and ratio of potential confounders (the accepted cardiovascular disease risk factors) were calculated for the three genotypes in the control group and differences between the genotypes were investigated using variance analysis. Logistic regression was used to calculate the relative risk (estimated as the odds ratio) of myocardial infarction with increasing PAI-1 antigen in steps of 50 ng/ml. The relative risks applied were calculated by means of logistic regression models in which checks were made for age, sex, current smoking habits, total cholesterol and HDL cholesterol. To investigate the modifying effects of age, sex, smoking, body mass index, serum cholesterol and blood pressure on the basis of the strength of the association between the 4G/5G polymorphism and the risk of myocardial infarction, the test population was subdivided into men and women and into two age categories, with a limit of 75 years old, into current smokers (persons who answered in the affirmative when asked "Do you smoke?") and non-smokers (that is to say the remainder, including former smokers and those who have never smoked) and according to increasing quantities of body mass index (limits 25 kg/m 2 and 27 kg/m 2 ), systolic blood pressure (limits 130 mm Hg and 145 mm Hg) , total cholesterol (limits 5-5 mmol/1 and 7-0 mmol/1) and HDL cholesterol (limits 1.0 mmol/1 and 1.5 mmol/1). The limits for categories of continuous variables with logical steps were arbitrarily chosen for an investigation of the distribution of these variables in our study population. Results Correct selection from the population source was checked by comparing the detected genotype distribution in the control group with the anticipated value according to a Hardy Weinburg equilibrium (Table 1 ) . The frequency of the 4G allele was 53 % , that of the 5G allele was 47 % and the distribution of the genotypes agreed with the Hardy Weinburg equilibrium; as anticipated, 77 test persons (29 % ) who were homozygous for the 4G allele were found, 129 (49 % ) who were heterozygous were found and 59 (22 % ) who were homozygous for the 5G allele were found (Table 1). Characteristics of the entire population studied are presented in Table 2. Total PAI-1 antigen plasma concentrations in men were somewhat lower than in women; age-adjusted averages were 51 ng/ml (CI 47.56) and 56 ng/ml (52.61). Furthermore, there was no clear difference in PAI-1 quantities between current smokers and non- (current) smokers; the age and weight-adjusted values were 58 ng/ml (50.67) and 53 ng/ml (50.57). The 4G allele was associated with higher PAI-1 antigen concentrations. In the control test persons, the highest PAI-1 antigen quantity of 61 ng/ml (53-70) was found for the 4G homozygous persons; the heterozygous persons had intermediate PAI-1 quantities of 51 ng/ml (46.53); and the test persons who were homozygous for the 5G allele had the lowest PAI-1 quantities of 46 ng/ml (39-54). A similar trend was detected in the case test persons; a value of 65 ng per ml (CI 56.75) was detected for persons with 4G4G, a value of 54 ng/ml (47-63) was found for the 4G5G and a value of 48 ng/ml (38.59) was determined for the 5G G persons. The risk of non-fatal myocardial infarction appeared to increase with increasing quantities of PAI-1 antigen; test persons having a PAI-1 of below 50 ng/ml were defined as the reference group with a risk of 1; compared with this reference group, test persons with values of between 0 and 100 ng/ml had a relative risk (odds ratio) of myocardial infarction of 1.11 (CI 0.70, 1-77) and test persons with PAI-1 higher than 100 ng/ml had a relative risk of 1.64 (CI 0.86, 3.11). However, after adjustment for smoking, body mass index, systolic blood pressure, serum ckolesterol, total cholesterol and HDL cholesterol these risks were no longer increased (odds ratios 0.82 (CI 0.48, 1.39) and 1.00 (CI 0.49, 2.05). The relative risk (odds ratio) of myocardial infarction was increased in test persons with 4G4G genotype compared with persons homozygous for the 5G allele (Table 3) • In order to investigate whether the association between the 4G/5G polymorphism and the risk of myocardial infarction could be influenced by other (known) factors, the established accepted risk factors for myocardial infarction were compared in the three genotypes for the control test persons (Table 4) . Smoking was appreciably differently distributed over the genotypes. In those homozygous for the 4G allele the ratio of smokers was 8 % , compared with 21 % for those homozygous for the 5G allele. Total cholesterol and HDL cholesterol were somewhat higher in the genotype with high risk but in total no significant differences in the other cardiovascular risk factors could be found between the genotypes. In addition, the relative risks of non-fatal myocardial infarction for the various genotypes were adjusted for age, sex and smoking, HDL and total cholesterol (Table 3). Additional adjustments for plasma quantities for the PAI-1 antigen did not change the association (Table 3) • The total percentage of smokers was 18 % (34 cases and 39 controls). The relatively low ratio of smokers in the control test persons with the 4G4G genotype suggests that the test persons with the 4G4G genotypes who smoke may have died (Table 4). In contrast, in the cases the ratio of smokers amongst the test persons with the 4G4G genotype was much higher and for the 5G5G, 4G5G and 4G4G persons the percentages of smokers were 16, 25 and 32 respectively. This suggested effect modification of smoking on the association between the polymorphism and myocardial infarction. Stratification for age and sex gave no prominent differences in the estimated risks (data not shown) . Stratification for smoking showed that the increased risk of myocardial infarction associated with the 4G allele was appreciably higher in the case of smokers (Table 5)- Stratification for body mass index, systolic blood pressure and for the total and HDL cholesterol after exclusion of smokers showed that the increased risk of myocardial infarction which was associated with the 4G allele did not differ for the various strata (data not shown). In the entire population studied, smokers had twice the risk of myocardial infarction compared with non- (current) smokers (odds ratio 2.0 (95 % CI 1.2, 3-5))- To determine whether the relative risk of smoking for myocardial infarction differed for the various genotypes, the population was stratified according to the three genotypes. In test persons who are homozygous for the 4G allele the relative risk of myocardial infarction associated with smoking was appreciably increased (odds ratio 4.9 (1.8, 1.3)); for heterozygous test persons who smoked the risk of myocardial infarction increased by 50 % ; an odds ratio of 1.5 (0.7. 3-3). In the case of those homozygous for the 5G allele the risk of myocardial infarction was not significantly increased by smoking, with an odds ratio of 1.1 (0.3, 3-7). Discussion It can be seen from this study that in particular in the case of smokers the 4G allele of the PAI-1 gene was associated with an increased number of non-fatal myocardial infarctions. This association was independent of plasma concentrations of PAI-1 antigen and other known risk factors. The study which was carried out here has limitations. Firstly, the case group comprises only the non-fatal myocardial infarction cases. The disproportionate distribution of smokers between the cases and the control persons, however, suggests that many smokers with 4G allele have developed a non-fatal myocardial infarction or have died. Therefore, it is assumed that comparable results would have been found for fatal cases of myocardial infarction. Secondly, one source of deviation in this study can be a change n the risk profile of the cases after infarction. Although this could be important for the plasma concentrations of PAI-1, our main interest was, however, the influence of the 4G/5G polymorphism on the risk of myocardial infarction. With regard to this, no deviation was expected because genotypes do not change in the case of myocardial infarction. With regard to smoking, some persons who have had a myocardial infarction may have given up smoking thereafter. This then reduces the relative risk of myocardial infarction that is associated with smoking, but it is improbable that the proportion of those who have given up smoking differs for the various genotypes. Therefore, the relative risk of myocardial infarction that is associated with the various genotypes in smokers will provide a valid estimate of the true association between polymorphism and risk. In the group of non-smokers, the relative risk may possibly be subject to overestimation because of the presence of ex-smokers in this category. The association between the 4G5G polymorphism and myocardial infarction has been studied previously, but could not be confirmed in two other studies (Refs 1, 11). Differences between the investigated populations in terms of distribution of risk-modifying factors could perhaps explain the differences in the findings. In any event, our findings give a new aspect with regard to the mechanism of association between 4G/5G polymorphism and the risk of myocardial infarction. The deletion of one guanine at -675 in the PAI-1 promoter could perhaps have a direct effect on increased PAI-1 transcription in persons with the 4G allele. This would be confirmed by the finding that the 4G allele is associated with raised PAI-1 plasma quantities. In addition, being homozygous for the 4G allele is associated with an increased risk of myocardial infarction in smokers. However, this association appears to be independent of plasma quantities of PAI-1. Furthermore, despite the strong association between 4G/5G polymorphism and the risk of myocardial infarction in smokers, smoking appears to exert no effect on plasma PAI-1 quantities. It is possible that the polymorphism in the PAI-1 gene influences the risk of myocardial infarction through a mechanism which is not reflected in the plasma concentrations of the PAI-1 antigen but possibly at a more local cellular level. The 4G G polymorphism itself can have an effect on PAI-1 function. There may possibly be genetic mutation in complete linkage disequilibrium with polymorphous locus within or close to the PAI-1 gene. Smokers with the 4G4G genotype appear to have five times the risk of non-fatal myocardial infarction compared with other smokers. Preference should perhaps be given to providing smokers with the easily detectable 4G allele with information on their noticeably increased risk of developing myocardial infarction. List of references relating to the example 1. Ye S. Green F. Scarabin PY. Nicaud V. Bara L, Dawson SJ, Humphries SE, Evans A, Luc G, Cambou JP, Arveiler D, Henney AM, Cabien F. The 4G5G polymorphism of the plasminogen activator inhibitor-1 gene is associated with differences in plasma PAI-1 activity but not with risk of myocardial infarction. Thromb Haemostas 1995;74:837-41. 2. Hofman A, Grobee DE, de Jong PTVM, van den Ouweland FA. Determinants of disease and disability in the elderly: the Rotterdam Elderly Study, Eur. J. Epidemiol 1991 , 7:403-422. 3. Bemmel JH van, Kors JA, Herpen van G. Methodology of the Modular ECG Analyis System MEANS. Meth Inform Med 1990;29: 3 353- 4. Willems JL., Abreu-Lima C, Arnaud P., van Bemmel JH, Brohet C, Degani R, et al. The diagnostic performance of computer programs in the interpretation of electrocardiograms. N Engl. J. Med. 1991; 325:1767-1773- 5- Rose GA, Blackburn H. Gillum RF. Cardiovascular survey methods. World Heath Organisation, Geneva, Switzerland, 1982. 6. Bots ML, van Swieten JC, Breterler MM, de Jong PTVM, van Gijn J., Hofman A, Grobbee DE. Cerebral white matter lesions and atherosclerosis in the Rotterdam Study. Lancet 1993 s 3^1: 1232- 1237- 7. van der Bom JG, Bots ML, de Bruijn AM, Hofman A, Grobee DE. Measurement of β-thromboglobulin in the elderly. Findings from Rotterdam Study. Fibrinolysis 1994;8 (Suppl 2):157-159. 8. Ranby M, Sundell IB, Nilsson TK. Blood collected in strong acidic citrate anticoagulant used in a study of dietary influence on basalt to activity. Thromb Haemostas. 19δ9;62:917 _ 922. 9. Meijer P, Pollet DE, Wauters J, Kluft C. Specificity of antigen assays of plasminogen activator inhibitor in plasma: Innotest PAI-1 immunoassay evaluated. Clin. Chim. 1994; 40/1:110-5. 10. Mansfield MW, Sticklang MH, Grant PJ. Plasminogen activator inhibitor-1 (PAI-1) promoter polymorphism and coronary artery disease non-insulin dependent diabetes. Thromb Haemostas 1995;74:1032-4. 11. Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Miletich JP. Arterial and venous thrombosis is not associated with the 4G/polymorphism in the promoter of the plasminogen activator inhibitor (PAI-1) gene in a large cohort of US men. (Personal notification) . Table 1 Table 2 Values are ratios or averages with standard deviations and ranges. Table 3 Relative risk of myocardial infarction for the various 4G5G genotypes (4G4G = homozygous for the 4G allele; 4G5G heterozygous ; 5G5G = homozygous for the G allele). Adjusted for age, sex, smoking, serum, HDL and total cholesterol and PAI-1 antigen quantity Reference risk Table 4 Average values and ratios for the cardiovascular disease risk factors according to the various genotypes for the control persons. (With adjustment for age and sex) . (4G4G = homozygous for the 4G allele; 4G5G = heterozygous; 5G5G = homozygous for the G allele). Values are percentages or averages (standard deviation) p * Value of the F trial Table 5 Relative ratio (odds ratio adjusted for age and sex) of myocardial infarction associated with the 4G allele for current smokers and non- ( current) smokers.

Claims: Claims 1. Method for determining the risk of cardiovascular disease in a person, characterised in that a person is selected who smokes, has smoked or wants to smoke tobacco and/or a man/woman couple who have a child or wish to have a child is selected and/or a pregnant women is selected and the 4G/5G polymorphism in the promoter region of the gene of the plasminogen activator inhibitor type I (PAI-1) is determined for the selected person. 2. Method for determining the risk of cardiovascular disease in a person, characterised in that a person who smokes, has smoked or wants to smoke tobacco is selected and the 4G/5G polymorphism in the promoter region of the gene of the plasminogen activator inhibitor type I (PAI-1) is determined for the selected person. 3. Method according to Claim 1 or 2, wherein the persons who want to or can start to smoke are selected from the group of young people aged between 0 and 18. 4. Method according to Claim 1, wherein the person who smokes or has smoked is a passive smoker or an active smoker. 5. Method according to one of the preceding claims, wherein cardiovascular diseases comprise thrombosis, cerebral and myocardial infarctions , angina pectoris , peripheral arteriosclerosis and vascular dementia. 6. Method according to one of the preceding claims, wherein cardiovascular diseases comprise cerebral and myocardial infarctions, in particular myocardial infarctions. 7. Method according to one of the preceding claims, wherein a 4G/5G polymorphism determination is carried out with one or more other tests for determination of a risk factor associated with smoking. 8. Method according to Claim 7, wherein the method used as the other test is a test method for determination of diabetes mellitus, hyperhomocysteinaemia, obesity, high blood pressure, insulin resistance, cancer, little exercise, vessel wall thickness and blood lipids. 9. Method according to Claim 7 and/or 8, wherein the establishment of living conditions which constitute a risk factor associated with smoking, such as stress and diet, is used as test method. 10. Method according to Claim 1 or 2, wherein a person who smokes or has smoked tobacco is selected by detection of cotinine in a biological fluid obtained from the test person using a method known per se for such detection and in accordance with medical standards. 11. Kit for carrying out a method according to one of the preceding claims, which comprises means for differentiating a 4G allele and a 5G allele in the promoter region of PAI-1. 12. Kit according to Claim 11, which is also provided with information on the risk of smoking, prevention of smoking or a change in smoking habits or which indicates where or how such information is obtainable . 13» Kit according to Claim 11 or 12, which also contains a leaflet at least containing information on the risk of smoking in combination with the various -675 morphisms with regard to cardiovascular diseases or a leaflet which indicates an information source from which such information can be obtained. 14. Kit according to one of Claims 11-13, which further contains aids for carrying out a DNA test on a human sample. 15. Kit according to one of Claims 11-14, which further contains means for determining at least one other risk factor associated with smoking. 16. Kit according to Claim 15, which contains, as further means, means for carrying out the determination of diabetes mellitus, hyperhomocysteinaemia, obesity, high blood pressure, insulin resistance, cancer, little exercise, vessel wall thickness or blood lipids. 17. Kit according to one of the preceding kit claims containing means for determining a factor which indicates that a person belongs to the category of smokers or the category of people who have smoked. 18. Kit according to Claim 17, wherein the factor is cotinine that occurs in a human sample, for example urine or saliva. -0-0-0-

Inventors: GROBBEE DIEDERICK EGBERTUS, KLUFT CORNELIS, GROBBEE, DIEDERICK, EGBERTUS, KLUFT, CORNELIS
Applicants: GROBBEE DIEDERICK EGBERTUS, KLUFT CORNELIS, TNO, GROBBEE, DIEDERICK, EGBERTUS, KLUFT, CORNELIS, NEDERLANDSE ORGANISATIE VOOR TOEGEPAST-NATUURWETENSCHAPPELIJK ONDERZOEK TNO
Application year: 1998
Main classification: C12Q1
IPC codes: A61B10, C12N15, C12Q1