WO2005042776A1

WO2005042776A1 - Method of determining predisposition towards atherosclerosis

Info

Publication number: WO2005042776A1
Application number: PCT/GB2004/004471
Authority: WO
Inventors: Christian Alexander Vedeler; Kjell-Morten Myhr; Ottar NYGÅRD
Original assignee: Forinnova As; Owen, Deborah
Priority date: 2003-10-23
Filing date: 2004-10-22
Publication date: 2005-05-12
Also published as: GB0324778D0

Abstract

The invention provides a method of determining predisposition towards atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases which involves determining the genotype of a human or non-human mammalian subject for at least one Fc receptor and identifying whether the determined genotype signifies or corresponds to a positive or negative disposition towards said disease. Methods of prognosis are also provided. A preferred Fc receptor genotype to be determined in accordance with the methods of the invention is the FcϜRIIIA receptor.

Description

METHOD OF DETERMINING PREDISPOSITION TOWARDS ATHEROSCLEROSIS

This invention relates to a method of determining predisposition towards disease, in particular predisposition towards atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis). Atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases such as those discussed above, and diseases associated therewith such as those discussed above are relatively common and in order to optimise treatment of patients, methods of determining whether or not an individual has a disposition towards developing such a disease or is susceptible to developing such a disease are in demand. If for example a test can be carried out which identifies a patient as being disposed towards contracting such a disease then, if desired, appropriate preventative treatments, if known, or other therapeutic interventions for optimal long term benefits may be carried out. In addition, when contracted by a patient, such diseases show a widely different pattern of development over time, which varies from patient to patient. Thus, some patients may have the disease but show minor or infrequent symptoms over many years while others, with apparently the same disease, may suffer relatively rapid deterioration leading even to total incapacity or death. Accordingly, there is also a need for methods by means of which the future severity of (i.e. prognosis of) such diseases may be predicted for the individual patient so that, where the prognosis is good (i.e. disease progress is likely to be benign), unnecessary treatment may be avoided and where it is bad (i.e. disease progress is likely to be non-benign), preventative action (e.g. diagnostic scanning, surgical intervention, etc.) may be taken for at-risk patients and therapeutic or palliative treatment may be given to early (and later) stage disease sufferers. Whilst genotyping of Fc receptors has been shown to be useful to diagnose predisposition to certain diseases of the immune system, for example certain autoimmune diseases, surprisingly and advantageously, it has now been found that determining the genotype of an individual's Fc receptors provides the basis of the determination of predisposition towards atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis). Thus viewed from one aspect the invention provides a method of determining predisposition towards atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), which involves determining the genotype of a human or non-human mammalian subject for at least one Fc receptor and identifying whether the determined genotype signifies or corresponds to a positive or negative disposition towards said disease. By positive disposition or predisposition it is meant that the individual in question has a higher than average likelihood or probability of contracting or being affected by or suffering from said disease, i.e. the individual has a higher than average relative susceptibility to developing said disease. By negative disposition or predisposition, it is meant that the individual in question has a lower than average likelihood or probability of contracting or being affected by or suffering from said disease, i.e. the individual has a lower than average relative susceptibility to developing said disease. If carrying out such a method leads to a patient being identified as having a positive disposition towards a particular disease then careful monitoring of the patient is generally desired in order that an early diagnosis or detection of said disease can occur as and when the patient contracts the disease or starts being affected by or suffering from said disease. Indeed, if appropriate prophylactic, preventative or therapeutic treatment for such diseases are available then these can be carried out once a positive disposition towards disease is determined. In addition, if appropriate tests exist to determine whether or not a patient has contracted or is suffering from a particular disease, then these can also be carried out once a positive disposition towards disease is determined. If, by way of such tests, it is found that the patient has already contracted or is suffering from the disease then appropriate therapeutic treatment can be carried out. Thus, the methods of the invention can be used to reinforce diagnosis based on other tests, symptoms or indicators. Thus, viewed from a further aspect the invention provides a method of prophylaxis or therapy of a human or non-human mammalian subject to combat a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), which method comprises determining the genotype of said subject for at least one Fc receptor, identifying whether the determined genotype signifies or corresponds to a positive or negative disposition towards said disease, and, where said determined genotype corresponds to a positive disposition, carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject. Said diagnostic procedure can be any appropriate procedure for the disease in question and may include for example any appropriate physical examination, a diagnostic imaging procedure or a biological assay which may involve taking a sample from the patient and carrying out a genetic or other biological diagnostic test. As well as determining the presence or absence of a particular disease, such diagnostic procedures also include the detection of other factors associated with said disease or with an increased or decreased risk of said disease. By way of example if a positive disposition for atherosclerosis, or other vessel diseases, or diseases associated therewith such as those discussed above, is determined, early diagnostic imaging of the patient's vasculature may be recommended and, if stenoses are detected, surgical intervention, e.g. percutaneous transluminal angioplasty (PCTA), may reduce the likelihood of vessel stenosis or occlusion and thereby infarction, which again reduce the future health care costs and will improve the patient's future quality of life. Alternatively, or in addition, the determination of a positive disposition according to the present invention, optionally coupled with detection of other risk factors such as high blood cholesterol, high homocysteine, high triglycerides, high blood pressure, and smoking may assist an individual to effect life style changes which will reduce the likelihood of development or further development of atherosclerosis or of other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and such other vessel diseases, including the likelihood of brain or heart infarction or stenosis. Such changes may include cessation of smoking, change of diet, increase in regular exercise, reduction of stress, etc. The invention is particularly concerned with the genotypes for Fey receptors, i.e. for receptors for the Fc portion of immunoglobulin G (IgG). Such receptors occur on many cells, in particular leukocytes (e.g. monocytes, lymphocytes, and granulocytes), macrophages and microglia in brain, and on other cells such as endothelial cells, trophoblasts, keratinocytes and Schwann cells. Three main classes of human leukocyte FcyR have been identified, namely FcyRI (CD64), FcγRII (CD34) and FcγRIII (CD16). These show variability in their distribution on different cell types, in their strength of binding to IgG and their capability to bind to different IgG sub-classes.

Within the FcyR classes, 8 genes and alternative splicing variants lead to a variety of receptor isoforms that have differences in structure and have distinct functional capacities. In addition to this variety, certain FcyR genes have allelic variants which affect their receptor function. Thus for example the FcγRII receptor has two known isoforms, FcyllA and FcyRIIB. FcyRIIA is expressed on cells such as macrophages and neutrophils and has several allelic forms leading to FcyRIIA polymorphism.

One variant contains histidine (131 H) while another contains arginine (131

R). The H/H variant has higher affinity for lgG2 than the R/R variant.

FcyRIIB is present on cells such as B cells and polymorphic variants (e.g. lle232Thr substitution) may regulate the affinity for IgG subclasses and the degree of inhibition of immune response. Similarly, the FcyRIII receptor has two known isoforms, FcyRIIIA and FcyRIIIB. FcyRIIIA is expressed on cells such as monocytes and macrophages and the 158 Val(V) allelic variant has higher affinity for lgG1 and lgG3 than the 158 Phe(F)-type receptor.

FcγRIIIB, which is expressed on cells such as neutrophils, has several allelic forms with individuals homozygous for FcyRIIIB neutrophil antigen (NA)1 being more efficient in binding lgG1 and lgG3 than individuals homozygous for the NA2 allele. The FcR genotype determined according to the invention is preferably an Fey receptor and preferably the FcyRIII receptors. More preferably, the Fey receptor genotype determined is FcyRIIIA. A particularly preferred

FcyRIIIA genotype to be analysed is the FA/ genotype at position 158. Prognostic methods are also provided by the present invention. In contrast to determining susceptibility or disposition towards particular disease states, such prognostic methods allows a prediction to be made of the severity and course of the disease should the individual contract it or already have contracted it. Thus, a further aspect of the invention provides a method of disease prognosis for a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), which method comprises determining the genotype of a human or non-human mammalian subject for at least one Fc receptor, and identifying whether the determined genotype corresponds to a benign or non-benign prognosis for said disease. By benign and non-benign prognoses, it is meant that the prognoses are more or less benign, e.g. good or not so good or bad or worse, etc. If genetic markers for susceptibility or disposition towards the disease in question are known, for example the FcR genetic markers as described elsewhere herein, or any other appropriate genetic markers, then a further aspect of the invention provides a method of disease prognosis for a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), which method comprises determining the presence or absence of a genetic marker for susceptibility to said disease in a human or non-human mammalian subject, for example by analysing the DNA or RNA of said subject, and determining the genotype of said subject for at least one Fc receptor, and identifying whether the determined Fc receptor genotype corresponds to a benign or non-benign prognosis for said disease. Where a non-benign prognosis is determined and a genetic marker for susceptibility is present (in embodiments where this parameter is analysed), then the above described methods of prognosis may optionally also involve carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject. Examples of appropriate diagnostic procedures and therapeutic or surgical interventions, etc., for the various diseases are described above. Thus, viewed from a yet further aspect the invention provides a method of prophylaxis or therapy of a human or non-human mammalian subject to combat a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), which method comprises determining the genotype of said subject for at least one Fc receptor, identifying whether the determined genotype corresponds to a benign or non-benign prognosis for said disease, and, where said determined genotype corresponds to a non-benign prognosis, carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject. In the methods of prognosis as described herein the Fc receptors determined are preferably Fey receptors, and preferably FcγRIII receptors. More preferably, the Foγ receptor genotype identified is FcγRIIIA. A particularly preferred FcγRIIIA genotype to be analysed is the FΛ genotype at position 158. In particular where methods of prognosis or methods of determining predisposition to atherosclerosis, or other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis) are concerned the genotype determined is preferably FcγRIIIA. A particularly preferred FcγRIIIA genotype to be analysed is the FA/ genotype at position 158. Viewed from a yet further aspect, the invention provides a predisposition (susceptibility) assay or a prognostic assay for a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), said assay comprising obtaining a sample (for example a DNA or RNA sample) from a human or non-human mammalian subject (e.g. involving separating or isolating such a sample from a body fluid such as blood); and identifying or determining the genotype of that sample (for example the DNA or RNA sample) for a Fc receptor, for example by amplifying a segment of that DNA containing at least a characteristic part of the gene for that receptor and identifying the allele or alleles of the gene for that receptor present in that DNA. Preferred Fc receptors to be genotyped in such predisposition or prognostic assays are as defined above. Thus, the assays of the present invention are generally carried out on a sample which has been removed from the patient (i.e. are in vitro assays) and are not practised on the human or animal body perse. Optionally such prognostic assays (or any of the prognostic methods described herein) may also involve identifying the presence or absence of a marker for susceptibility to the selected disease. Optionally such susceptibility assays (or methods of determining disposition as described herein) may also involve identifying the presence or absence of an additional or further marker for susceptibility to the selected disease. Preferably said susceptibility markers are genetic markers, for example found in the DNA or RNA of a subject. In the embodiments of the invention where these additional susceptibility markers are identified as being present and a non-benign and/or positive disposition (as appropriate) has been identified, then the above described methods of determining disposition and prognosis may optionally also involve carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject. Examples of appropriate diagnostic procedures and therapeutic or surgical interventions, etc., for the various diseases are described above. Determining whether a particular FcR genotype corresponds to a positive or negative disposition to a particular disease or corresponds to a benign or non-benign genotype for a particular disease can be carried out by any appropriate method, but generally involves comparing the relative frequencies of the different genotypes in relevant populations of affected and non-affected individuals. For example, in the case of the methods to determine disposition towards the selected disease, where positive and negative dispositions need to be determined, this may be done by comparing the relative frequencies of the different genotypes in a population of patients who have contracted the particular disease or are suffering from a particular disease state, to a population not affected by the disease. ln the case of the methods of prognosis, where benign and non- benign genotypes need to be determined, this may be done by comparing the relative frequency of the different genotypes in populations of late-stage, early stage and possibly mid-stage disease patients as well as mild, moderate and severe forms of the disease and thereby identifying which genotype or genotypes have significant occurrence in the sections of the population for which the disease progression has been benign or non- benign. For example, for atherosclerosis or other vessel diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease or atherosclerosis of the carotid artery, genotypes for patients who have several cardiac or cerebral blood vessels (or other relevant vessels) affected by disease (e.g. 1 , 2, 3 or more vessels affected), can be compared with patients who have fewer or no cardiac or cerebral vessels (or other relevant vessels) affected. (The number of vessels affected can be regarded as an indicator of the severity of the disease, i.e. the more vessels which are affected, the more severe the disease). Affected vessels can be determined by any appropriate test, for example an affected vessel might conveniently be defined as a vessel which has a diameter stenosis of at least 50%. Where cardiac vessels are concerned, such analysis can conveniently be carried out using a cardiac angiogram. Alternatively, the genotypes of patients with severe atherosclerosis, e.g. patients who are found to have stenosis of greater than 70% of the relevant blood vessels, e.g. the carotid or vertebral arteries, can be compared with the genotypes of patients with less severe atherosclerosis or no detectable atherosclerosis. Thus, appropriate affected and non-affected individuals for comparison and individuals with different stages of disease can readily be identified by a person skilled in the art, e.g. an appropriate clinician, using standard and known disease criterion/ definitions. For example, for coronary artery disease an appropriate disease definition is given in the methods section of Example 1. From the above discussion it can be seen that the methods to determine disposition or prognosis as described herein may comprise the steps of: determining the genotype of a nucleic acid (e.g. DNA or RNA) encoding at least one Fc receptor, wherein said nucleic acid is obtained from a test mammalian subject; and comparing the thus determined genotype to the genotype of a nucleic acid (e.g. DNA or RNA) encoding a corresponding Fc receptor obtained from a normal mammalian subject or the genotype of a nucleic acid (e.g. DNA or RNA) encoding a corresponding Fc receptor obtained from a diseased mammalian subject, wherein said diseased mammalian subject is a mammalian subject afflicted with a disease or a certain stage of a disease selected from the group consisting of atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), wherein when the determined genotype for the nucleic acid obtained from the test mammalian subject corresponds to the genotype of nucleic acid obtained from said normal mammalian subject, a negative disposition or benign prognosis (as appropriate) is made for the test mammalian subject; and wherein when the determined genotype for the nucleic acid obtained from the test mammalian subject corresponds to the genotype of the nucleic acid obtained from said diseased mammalian subject, a positive disposition or non-benign prognosis (as appropriate) is made for the test mammalian subject. Any differences in the genotypes of the Fc receptors between different categories of healthy and non-healthy patients can be determined (for example by the above methods) and used in the methods of the invention. Generally these differences will involve allelic differences, for example the presence of one or more mutations or polymorphisms or variations in one allele for a particular Fc receptor gene which are not found in other alleles. By comparing such allelic patterns, differences can be found which are associated with disease predisposition or different types of prognosis. Some allelic differences or allelic variants in Fc receptors and in particular Foγ receptors are already known in the art, see for example the above discussion of the H and R alleles of the FcγRIIA, the NA1/NA2 alleles of the FcγRIIIB, and the Val and Phe alleles for the FcγRIIIA, and these (and indeed any other known allelic differences or allelic variants) can be compared in different categories of healthy and non-healthy patients. Although the above described known allelic differences or allelic variants are the preferred Fc genotypes to be determined in the methods described herein, the methods of the invention are not limited to the use of known allelic differences or variants and any allelic differences or variants which are seen with a greater frequency in certain categories of healthy or non-healthy patients and are associated with disease predisposition or different types of prognosis can be used. Preferred allelic differences or variants to be analysed when assessing predisposition to or prognosis of the specific diseases with which the methods of the invention are concerned are those wherein the differences or variations affect receptor function. More preferred allelic differences or variants to be analysed are discussed elsewhere herein. For example preferred allelic differences or variants to be analysed are differences or variants in the FcγRIIIA receptor alleles. The most preferred genotype to be analysed is the F/V genotype at position 158 of FcyRIIIA. To determine the genotype of an individual for an Fc receptor, it is necessary to obtain a sample of the DNA of that individual. For this it is preferable to use FcR allele-specific binders (e.g. PCR primers or other materials (e.g. oligonucleotide or nucleic acid probes or antibodies) capable of selectively binding to DNA or DNA fragments containing the particular FcR allele). Accordingly, viewed from a further aspect, the invention provides the use of an FcR allele-specific binder for the manufacture of a composition for use in a method of determining disposition to disease, a method for prognosis of a disease, or methods of prophylaxis or therapy according to the invention. Viewed from a further aspect the invention provides an FcR allele- specific binder for use in a method of determining disposition to disease, a method for prognosis of a disease, or methods of prophylaxis or therapy according to the invention. Preferred FcR allele-specific binders are those which bind specifically to appropriate allelic variants of the Foγ receptors as described elsewhere herein. Viewed from a still further aspect, the invention provides the use of a material prophylactically or therapeutically effective against a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis) for the manufacture of a medicament for use in the method of prophylaxis or therapy according to the invention. Viewed from a still further aspect the invention provides the use of an

Fc genotype in a method of determining disposition to or prognosis of a disease selected from atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis). Preferred Fc genotypes and diseases are described elsewhere herein. Therapeutic treatment as referred to herein includes treatment to alleviate or reduce the occurrence of disease symptoms (i.e. palliative treatment) as well as curative treatment. Viewed from a yet further aspect the invention provides a diagnostic or prognostic or predisposition kit for carrying out the methods of the invention, i.e. for determining disposition towards or prognosis of atherosclerosis, other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis) in accordance with the methods of the invention, said kit comprising at least one (preferably 2 or more, more preferably 4 or more, e.g. up to 12) FcR allele-specific binders (e.g. PCR primers or other materials, for example antibodies or allele-specific oligonucleotide or nucleic acid probes, capable of selectively binding to DNA or DNA fragments containing the particular FcR allele or the Fc receptors encoded thereby) and instructions for the performance of a method of determination, prognosis, prophylaxis or therapy according to the invention. Preferred FcR allele specific binders are those which are specific for (i.e. capable of selectively binding to) allelic variants of Fc receptors as described elsewhere herein. More preferred kits will comprise FcR binder sequences capable of binding to FcγRIIIA alleles and especially preferably FcR binder sequences capable of selectively binding to allelic variants of FcyRIIIA. Specific preferred FcR allele specific binder sequences are outlined below. The FcR genotype of an individual may be determined from a sample of the individual's DNA. Appropriate samples and methods of isolating DNA therefrom are well known and documented in the art. Typically the genotype may be determined by taking a body fluid (e.g. blood, saliva or urine) or body tissue sample. Preferably the sample taken will be a blood sample. Preferably, the DNA will be separated from other non-aqueous components of the sample, for example by cell lysis, solvent extraction and centrifugation. Th e separated DNA may then be tested directly or may be amplified, e.g. using PCR with FcR allele specific primers which are specific for relevant allelic variants of the FcR allele in question, before determination. For direct testing, an allele-specific binder which is specific for the allelic variant of the FcR allele in question which carries or is conjugatable to a reporter (e.g. a radiolabel, a chromophore or an enzyme) should be used as in conventional direct or indirect binding assays. If DNA amplification is used, the amplified product may be separated on a gel. This is preferably done together with a standard DNA fragment produced by simultaneous amplification using a second primer effective for all subjects so as to avoid occurrence of false negatives for the particular FcR allele. Although it is preferable to use an FcR allele specific binder which is specific for a relevant allelic variant to determine the genotype of an individual for an Fc receptor, other appropriate methods of genotyping can be used. For example, more general PCR primers which are capable of binding to a number of FcR alleles can be used, after which the particular allele present in an individual can be determined by an appropriate sequencing technique. Alternatively, such general primers can be used to amplify the FcR alleles which are present, after which the particular allele present in an individual can be determined by hybridising the amplified alleles with an allele variant specific oligonucleotide probe which can be detected directly or indirectly. Many FcR genes have been identified in the literature and thus selection of appropriate allele-specific binder sequences is not problematic. Thus for FcyRIIA, FcyRIIIA and FcyRIIIB for example the following PCR primers may be used:

FcyRIIA

EC2-131R : 5'CCA GAA TGG AAA ATC CCA GAA ATT CTC TCG3' EC2-131H : 5'CCA GAA TGG AAA ATC CCA GAA ATT CTC TCA3' TM1(antisense): 5'CCA TTG GTG AAG AGC TGC CCA TGC TGG GCA3' Control 1 : 5'GAT TCA GTG ACC CAG ATG GAA GGG3' Control 2 : 5'AGC ACA GAA GTA CAC CGC TGA GTC3'

The control sequences give rise to an amplified fragment of 270 bp and can be used as an internal positive control. The other primers amplify a 980 bp fragment.

FcyRIIIB

NA1 : 5'CAG TGG TTT CAC AAT GTG AA3'

NA2 : 5'CAA TGG TAC AGC GTG CTT 3'

Reverse primer : 5'ATG GAC TTC TAG CTG CAC 3' Control 1 : 5'CAG TGC CTT CCC AAC CAT TCC CTT A 3' Control 2 : 5'ATC CAC TCA CGG ATT TCT GTT GTG TTT C3'

The control sequences give rise to an amplified fragment of 439 bp and can be used as an internal positive control. The other primers amplify a 141 bp fragment (NA1 ) or a 219 bp fragment (NA2). Since there is a substantial difference in length between the NA1 specific and NA2 specific reaction products, if desired, both alleles can be detected in the same reaction.

FcyRIIIA

F-specific primer: 5'- CTG AAG ACA CAT TTT TAC TCC CAA C-3' V-specific primer: 5'- CTG AAG ACA CAT TTT TAC TCC CAA A-3' Reverse primer: 5'- TCC AAA AGC CAC ACT CAA AGA C-3' Control 1 : GAG-ACT-GAA-AAA-CCC-TTG-GAA-TC Control 2: GCT-TTC-TCA-GAC-CTC-CAT-GTA-G

The control sequences give rise to an amplified fragment of 406 bp. The other primers amplify a 73 bp fragment. Allelic variant specific sequences such as these (e.g. the EC2-131 R, EC2-131 H, NA1 , NA2, F-specific and V-specific sequences) or sequences with a high degree of homology therewith may be used as the allele-specific binders or as the binding domain of allele-specific binders in the kits of the invention. Put another way the allele specific binders as described herein may comprise sequences such as the EC2-131 R, EC2-131 H, NA1 , NA2, F- specific and V-specific sequences, or sequences with a high degree of homology therewith. Preferred allele specific binders are those which are specific to allelic variants of FcyRIIIA, e.g. F specific and V specific sequences. For atherosclerosis and other vessel diseases, e.g. other cerebrovascular or cardiovascular diseases, e.g. other cerebrovascular or cardiovascular diseases and in particular diseases with vessels affected by atherosclerosis such as coronary artery disease and atherosclerosis of the carotid artery, and diseases associated with atherosclerosis and other vessel diseases such as brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities (e.g. stenosis), the genotype FcyRIIIA V V is indicative of a negative disposition or benign prognosis. The genotypes V/F and/or F/F are indicative of a positive disposition or a non-benign prognosis (i.e. the increased likelihood of contracting the disease or the likelihood that the progression of the disease or the extent of the disease will be more severe). Particularly preferred diseases for which disposition or prognosis is determined in accordance with the invention are atherosclerosis and/or coronary artery disease. The invention will now be described further with reference to the following Example: EXAMPLE 1

FcvRlllA polymorphism as a risk factor for coronary artery disease and atherosclerosis

Methods

Patient population and control subjects

A total of 882 consecutive adult Norwegian Caucasian patients who had an elective diagnostic coronary angiography at the Department of Heart Disease, Haukeland University Hospital in Bergen, Norway in 2000 were included in the study. For patients who were admitted for recatheterization during that period, only the first angiography was considered.

All patients completed a self-administered questionnaire that provided information about medical history, risk factors, medication, hypertension, diabetes mellitus, previous myocardial infarction, and smoking habits. The information was checked against the medical records, and in all cases with discrepancies or missing information, the patients were telephoned for clarification. History of hypertension indicated subjects currently being treated for hypertension according to clinical criteria, and diabetes mellitus included both type I and type II. The diagnosis of previous myocardial infarction was based on the medical history and records or on the findings of typical sequelae of infarction on ventricular angiography. Smokers were identified as past and/or present smokers and non-smokers. All routine laboratory analyses were performed by standard assays at the Department of Clinical Biochemistry, Haukeland University Hospital.

The study was approved by the Regional Research Ethics committee.

Angiographic evidence of coronary artery disease

Coronary Angiograms were performed by cardiologists who were unaware of the patients' risk-factor profiles, and coronary stenoses were confirmed in orthogonal views. Significant coronary artery disease (CAD) was defined as a diameter stenosis of at least 50% in any of the main coronary arteries (the left main coronary artery or the left anterior descending coronary artery with its major diagonal branches, the right coronary artery, or the circumflex coronary artery with its major marginal branch). Depending on dominance, the descending or posterior descending coronary artery was included as part of the right coronary artery or the circumflex coronary artery. The extent of CAD was scored as 0 (no vessel disease), 1 (single vessel disease), 2 (double vessel disease) or 3 (triple vessel disease) according to the number of main vessels with stenosis. A left main-stem artery stenosis without stenosis of the right coronary artery was classified as double vessel disease.

FcyR genotype analysis

Genomic DNA was extracted from whole blood with QIAamp™ Blood Kit (Qiagen GmbH, Hilden, Germany) as described by the manufacturer.

The FcyRIIA genotypes were determined using amplification refractory mutation system-polymerase chain reaction (BoHo et al., Clin. Exp. Immunol., 1996, 104:264-268). Briefly, the two allele specific primers EC2-131 R (5'-CCA GAA TGG AAA ATC CCA GAA ATT CTC TCG-3') and EC2-131 H (5'-CCA GAA TGG AAA ATC CCA GAA ATT CTCTCA-3') in combination with the antisense/reverse primer TM1 (5'CCA TTG GTG AAG AGC TGC CCA TGC TGG GCA-3') were used to amplify a 980-bp fragment in separate PCR reactions. A 270 bp product amplified from the T 22 gene served as an internal positive control. The PCR conditions were denaturation for 5 min at 94 C, followed by 45 cycles of 94 C for 45 sec, 63 C for 30 sec and 72 C for 90 sec, and a final extension at 72 C for 10 min.

FcyRIIIA was detected using a PCR based method with Ampli Taq Stoffel enzyme as described previously (Leppers-van de Straat et al., J. Immunol. Methods, 2000, 242:127-132). Two PCR reactions were performed using allele specific primers for (V): 5'- CTG AAG ACA CAT TTT TAC TCC CAA C-3' and (F): 5'- CTG AAG ACA CAT TTT TAC TCC CAA A-3' and the reverse primer 5'- TCC AAA AGC CAC ACT CAA AGA C-3'. The same internal positive control was used as in the case of FcyRIIA. PCR conditions were denaturation for 5 min at 95 C, followed by 35 cycles of 94 C for 30 sec, 64 C for 30 sec and 72 C for 30 sec, and a final extension at 72 C for 8 min.

FcyRIIIB was amplified using the NA1- specific primer (5'-CAG TGG TTT CAC AAT GTG AA-3') and NA2-specific primer (5'-CAA TGG TAC AGC GTG CTT -3') with the common reverse primer (5'-ATG GAC TTC TAG CTG CAC-3') modified after Bux et al. (Transfusion. 1995, 35:54-57). The 141 bp product for NA1 and 219 bp product for NA2 were amplified in the same reaction. A 439 bp product from the human growth hormone served as an internal positive control. PCR conditions were denaturation for 3 min at 94 C, followed by 30 cycles of 94 C for 1 min, 57 C for 2 min and 72 C for 1 min, and a final extension at 72 C for 10 min.

All PCR products were analyzed by electrophoresis, using 1.5% agarose gel with ethidium bromide and visualized by ultraviolet light.

Other laboratory analyses

All routine laboratory analyses were performed by standard assays at the Department of Clinical Biochemistry, Haukeland University Hospital.

Statistical analysis

Continuous variables are reported as means and categorical variables as counts (%). Investigation of the dependence between factors related to atherosclerosis and sex was performed by Fishers exact test for categorical and by the Mann Whitney test for categorical and continuous variables, respectively. Age and triglycerides were log-transformed to achieve equality of variance. Logistic regression was used to study multiple risk factors for CAD. In these analyses, patients with single, double, and triple vessel disease were combined. The analysis was performed with SPSS 11.0.

Results

Characteristics of the patients

The mean age of the 260 women and 622 men was 64 years and 61 years, respectively. A total of 273 patients (31 %) had a previous myocardial infarction, 86 (9.8%) had undergone coronary artery bypass grafting, and 92 (10.4%) had been treated with coronary angioplasty. Diabetes mellitus was previously diagnosed in 96 patients (10.9%), 339 (38.4%) were currently treated for hypertension, and 519 (58.8%) were current or former smokers. Established risk factors for CAD such as lipid levels are also included in Table 1. Sedimentation rate was included as a parameter of inflammation since CRP (C-reactive protein) was not determined.

FcyR genotypes related to angiography

Angiography revealed that 183 patients (20.7%) had minimal or no CAD, 170 (19.3%) had single-vessel disease, 205 (23.2%) had two-vessel disease, and 324 (36.7%) had three-vessel disease. The distribution of patients according to genotype and CAD is presented in Table 2, and the results demonstrate no association between the FcyRIIA or FcyRIIIB genotypes and CAD. The genotype distribution for FcyRIIIA was, however, significantly associated with disease. Among the 873 patients successfully genotyped, homozygosity for the V V polymorphism was detected in 73 (8.4%). The proportion of patients carrying two V alleles was highest in those without CAD (12.7%) and lowest among those with triple vessel disease (5.7%). A highly significant trend toward a protective effect of the polymorphism was demonstrated (p=0.002). In analysis restricted to patients with CAD, a similar association was demonstrated (p=0.035). Put another way, there was a correlation between number of patients homozygote for FcyRIIIA V V and vessel disease compared to those who were either heterozygote or homozygote for the F/F (p=0.085). The F/F and F/V groups were also significantly correlated to more severe vessel disease than the V/V group (p=0.002).

The potential bias from an association of the FcyRIIIA genotype with other risk factors was evaluated by logistic regressions. In addition to established risk factors, several other potential predictors of CAD were evaluated. Factors significantly related to CAD in univariate analyses, which failed to reach significance in multivariate analysis, were not included in the final model. The only exception from this rule was smoking habits. The other risk factors that failed in the multivariate model were the concentration of glucose, uric acid, platelet count, and tryglycerides (all univariate pθ.027). Body mass index, fibrinogen, creatinine and leukocyte count showed no significant association with CAD in the univariate analysis.

The results of the univariate and multivariate analysis are summarised in Table 3. In addition to established risk factors, sedimentation rate and homozygosity for FcγRIIIA-158V were significantly associated with CAD and the protective effect of FcyRIIIA V/V was upheld in the multivariate model. Thus, compared to those being heterozygous, or homozygous for the F allele, patients homozygous for the V allele had significantly reduced risk: Odds Ratio (OR), 0.53; (Cl, 0.32 to 0.90). Additional adjustment for classical risk factors and sedimentation rate did not affect the results.

Discussion

We have assessed polymorphisms in three different FcyR genes as potential genetic determinants of CAD among patients referred for diagnostic coronary angiography. Two polymorphisms in the FcyRIIA and FcyRIIIB genes had no influence, whereas a polymorphism in the FcyRIIIA gene was associated with a strong protective effect. Patients homozygous for the FcγRIIIA-V158 allele were half as likely of having significant coronary stenosis, and the V allele was also associated with less extensive disease.

Data presented here describe results derived from a cross-sectional study, and the question of causality may never be answered by this design. Our results must therefore be reproduced in population-based studies before inferences of a direct causal effect may be drawn with greater confidence. However, in a previous study from the same geographical area, the prevalence of homozygosity for the V allele was similar among healthy controls (15.1 %) (Vedeler et al., Neurology. 2000, 55:705-707), as in patients without CAD in the current investigation (12.7%). Thus, the finding of a significantly lower frequency in CAD patients strongly suggests a protective effect.

This is the first study that shows a significant association between FcyR polymorphism and CAD. In particular, we have for the first time showed a significant correlation between the gene for FcyRIIIA and angiographically verified coronary artery disease. The data thus provides evidence for an association between FcyRIIIA allelic variants and atherosclerosis.

Table 1. Baseline characteristics of the study population

Female Male P (n=268) (n=622) Age, y 64.1 ± 11.3 61.2 ± 10.5 <0.001^t FcγRmA (W) 28 (10.8) 45 (7.3) 0.108 CABG 18 (7.0) 68 (11.1) 0.063 PCI 14 (5.4) 78 (12.8) 0.001 Previous myocardial infarction 51 (19.8) 222 (36.4) < 0.001 Diabetes mellitus 25 (9.7) 71 (11.7) 0.478 Hypertension 111 (43.0) 228 (37.4) 0.128 Smoking (past and/or present) 100 (38.8) 419 (68.9) < 0.001 Total cholesterol (mmol/L) 5.63 ± 1.22 5.06 ± 1.06 < 0.001 HDL cholesterol (mmol/L) 1.59 ± 0.84 1.33 ± 0.72 < 0.001 LDL cholesterol (mmol/L) 3.33 ± 1.22 2.95 ± 1.11 < 0.001 Triglycerides (mmol L) 1.67 ± 0.89 1.83 ± 1.01 0.016^f Sesdimentationrate (mm) 19.7 ± 12.9 14.65 ± 14.3 < 0.001 t Variables log transformed to achieve equal variance. For categorical variables, number and percentage and Fishers exact test are reported. For continuous variables, mean and standard deviation within each group is calculated. The Mann Whitney test is used to compare the two genders. CABG: Coronary artery bypass grafting. PCI: Percut. translum. coronary angioplasty

Table 2. Distribution of the FcyRIIA, FcγRmB and FcγRJQTIA genotypes according to the extent of coronary artery disease (CAD).

Extent of CAD Genotype No significant Single vessel Double vessel Triple vessel n(%) p* P- (n=183) (n=170) (n=205) (n=324) trend# FcγRHAt • H/H 38 (20.8) 31 (18.2) 32 (15.6) 64 (19.8) 165 (18.7) HZR 88 (48.1) 85 (50.0) 09 (43.9) 155 (48.0) 418 (47.4) FJR 57 (31.1) 54 (31.8) 83 (40.5) 104 (32.2) 298 (33.8) 0.42 0.54 FcγRπϊA§ V/V 23 (12.7) 19 (11.2) 13 (6.4) 18 (5.7) 73 (8.4) V/F 69 (38.1) 68 (40.0) 92 (45.1) 135 (42.5) 364 (41.7) F F 89 (49.2) 83 (48.8) 99 (48.5) 165 (51.9) 436 (49.9 0.085 0.002 FcγRIQBJ NAl/NAl 22 (12.0) 24 (14.3) 23 (11.3) 36 (11.1) 105 (12.0) NA1 NA2 83 (45.4) 76 (45.2) 98 (48.0) 153 (47.4) 410 (46.7) NA2 NA2 78 (42.6) 68 (40.5) 83 (40.7) 134 (41.5) 363 (41.3) 0.97 0.82

* Pearson chi-square test

# The Jonckheere-Te stra trend test for extent of CAD compares patients being homozygous for the various alleles (H H, NAl NAl, and V V) against the other genotypes as reference. f Genotyping was successfully performed in 881. patients % Genotyping was successfully performed in 878 patients § Genotyping was successfully performed in 873 patients

Table 3. Odds ratio (with 95% confidence intervals) for coronary artery disease in univariate and multivariate analysis

Risk factors Univariate OR Multivariate ORf (n=882) (n=749)

FcγiπA (W vs FF+VF) 0.53 (0.32-0.90) 0.45 (0.23-0.87)

Male gender 4.87 (3.45-6.87) 7.30 (4.60-11.6)

Age (years) 1.04 (1.03-1.06) 1.06 (1.04-1.08)

Diabetes mellitus 2.80(1.38-5.67) 3.93 (L45-10.7)

Smoking 1.98 (1.42-2.76) 1.43 (0.92-2.21)

Hypercholesterolemia* 3.46 (2.41-4.96) 4.83 (3.11-7.5)

Sedimentation rate (mm) 1.02 (1.01-1.03) 2.12 (1.24-3.6) ^♦Total cholesterol >6.5 mmol/L or treatment with hpid lowering drugs f The model includes all variables in the table

Claims

Claims:

1. A method of determining predisposition towards atherosclerosis or other vessel diseases, or diseases associated with atherosclerosis or other vessel diseases, which involves determining the genotype of a human or non-human mammalian subject for at least one Fc receptor and identifying whether the determined genotype signifies or corresponds to a positive or negative disposition towards said disease.

2. A method of disease prognosis for a disease selected from atherosclerosis and other vessel diseases, and diseases associated with atherosclerosis and other vessel diseases, which method comprises determining the genotype of a human or non-human mammalian subject for at least one Fc receptor, and identifying whether the determined genotype corresponds to a benign or non-benign prognosis for said disease.

3. A method of prophylaxis or therapy of a human or non-human mammalian subject to combat a disease selected from atherosclerosis and other vessel diseases, and diseases associated with atherosclerosis and other vessel diseases, which method comprises determining the genotype of said subject for at least one Fc receptor, identifying whether the determined genotype signifies or corresponds to a positive or negative disposition towards said disease or a benign or non-benign prognosis for said disease, and, where said determined genotype corresponds to a positive disposition and/or a non-benign prognosis, carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject.

4. The method of claim 1 wherein said method further comprises identifying the presence or absence of an additional or further marker for susceptibility to the selected disease.

5. The method of claim 2 wherein said method further comprises identifying the presence or absence of a marker for susceptibility to the selected disease.

6. The method of claim 4 or claim 5, wherein when said susceptibility markers are identified as being present and a non-benign and/or positive disposition has been identified, said method also involves carrying out a diagnostic procedure on said subject, carrying out surgical intervention on said subject, and/or administering a prophylactically or therapeutically effective amount of a material prophylactically or therapeutically effective against said disease to said subject.

7. The method of any one of claims 1 to 6 wherein said method comprises the steps of determining the genotype of a nucleic acid encoding at least one Fc receptor, wherein said nucleic acid is obtained from a test mammalian subject; and comparing the thus determined genotype to the genotype of a nucleic acid encoding a corresponding Fc receptor obtained from a normal mammalian subject or the genotype of a nucleic acid encoding a corresponding Fc receptor obtained from a diseased mammalian subject, wherein said diseased mammalian subject is a mammalian subject afflicted with a disease or a certain stage of a disease selected from the group consisting of atherosclerosis and other vessel diseases, and diseases associated with atherosclerosis and other vessel diseases, wherein when the determined genotype for the nucleic acid obtained from the test mammalian subject corresponds to the genotype of the nucleic acid obtained from said normal mammalian subject, a negative disposition or benign prognosis (as appropriate) is made for the test mammalian subject; and wherein when the determined genotype for the nucleic acid obtained from the test mammalian subject corresponds to the genotype of the nucleic acid obtained from said diseased mammalian subject, a positive disposition or non-benign prognosis (as appropriate) is made for the test mammalian subject.

8. A predisposition (susceptibility) assay or a prognostic assay for a disease selected from atherosclerosis and other vessel diseases, and diseases associated with atherosclerosis and other vessel diseases, said assay comprising obtaining a sample from a human or non-human mammalian subject and identifying or determining the genotype of that sample for a Fc receptor and optionally identifying the presence or absence of a marker for susceptibility to the selected disease.

9. The use of an FcR allele-specific binder for the manufacture of a composition for use in a method of determining disposition to disease, a method for prognosis of a disease, or methods of prophylaxis or therapy as claimed in any one of claims 1 to 8.

10. The method, assay or use of any one of claims 1 to 9 wherein said determination involves the detection of an allelic variant of an Fc receptor which corresponds to a positive or negative disposition towards said disease, or a benign or non-benign prognosis for said disease.

11. The method, assay or use of any one of claims 1 to 10, wherein said Fc receptor is an Fey receptor.

12. The method, assay or use of claim 11 , wherein said Fc receptor is an FcyRIII receptor.

13. The method, assay or use of claim 12, wherein said Fc receptor is FcγRIIIA.

14. The method, assay or use of claim 13, wherein the FcγRIIIA genotype to be analysed is the F V genotype.

15. The method, assay or use of claim 14, wherein the genotype FcyRIIIA V/V is indicative of a negative disposition or benign prognosis.

16. The method, assay or use of claim 14, wherein the genotypes FcyRIIIA V/F and/or F/F are indicative of a positive disposition or a non- benign prognosis.

17. The method, assay or use of any one of claims 1 to 16 wherein said other vessel diseases are other cerebrovascular or cardiovascular diseases.

18. The method, assay or use of any one of claims 1 to 17 wherein said disease is coronary artery disease or atherosclerosis of the carotid artery.

19. The method, assay or use of any one of claims 1 to 18 wherein said associated disease is a disease selected from brain (cerebral) infarct (stroke), heart (cardiac) infarct, and aorta and arterial disorders of the extremities.

20. The method, assay or use of claim 19 wherein said disorder of the extremities is stenosis.

21. A diagnostic or prognostic kit for determining disposition towards or prognosis of atherosclerosis and other vessel diseases, and diseases associated with atherosclerosis and other vessel diseases, said kit comprising at least one FcR allele-specific binder and instructions for the performance of a method of determining predisposition, prognosis, prophylaxis or therapy as claimed in any one of claims 1 to 20.

22. The kit of claim 21 wherein said FcR binder is capable of selectively binding to allelic variants of FcγRIIIA.