WO2006002930A2

WO2006002930A2 - FcϜRIIa POLYMORPHISM AND ITS USE IN DIAGNOSIS

Info

Publication number: WO2006002930A2
Application number: PCT/EP2005/007073
Authority: WO
Inventors: Bernhard Stockmeyer; Ahmed Sheriff; Udo Gaipl; Reinhard Voll; Joachim Robert Kalden; Werner G. Daniel; Dorette Raaz; Christoph Garlichs; Martin Herrmann; Peter Kern
Original assignee: Friedrich Alexander Universitaet Erlangen Nuernberg
Current assignee: Friedrich Alexander Universitaet Erlangen Nuernberg
Priority date: 2004-06-30
Filing date: 2005-06-30
Publication date: 2006-01-12
Anticipated expiration: 2006-12-30
Also published as: WO2006002930A3

Abstract

The present invention is directed to a method of determining the risk of a patient with various diseases of developing or suffering from acute coronary syndrome as well as an antibody for use in this method. The present invention is furthermore directed to a diagnosic kit and particles for use in this method as well as the use thereof and the diagnosis and prevention of several diseases.

Description

FcγRIIa polymorphism and its use in diagnosis

The present invention is directed to a method of determining the risk of a patient of developing or suffering from acute coronary syndrome as well as to an antibody for use in this method. The present invention is furthermore directed to a diagnostic kit and particles for use in this method as well as the use thereof in the diagnosis and prevention of several diseases.

Coronary artery disease (CAD) is the leading cause of death in the industrialized world. CAD can progress into destabilization of atherosclerotic plaques, leading clinically to acute coronary syndromes (ACS) consisting of unstable angina pectoris (UAP), non-ST- elevation myocardial infarction or ST-elevation myocardial infarction. Development of ACS is accompanied by high cardiovascular mortality and morbidity. Therefore, an early and specific prediction of coronary risk is essential for the stratification of the treatment.

Atherosclerosis, by nature, is a chronic inflammatory disease (1). Therefore, the pathophysiological role of C-reactive protein (CRP) within the chronic stable phase as well as within ACS has been studied extensively. Chronically elevated serum levels of CRP implicate increased risk for destabilization or death.

Recent studies assume an active role of CRP as an important mediator of tissue damage (2,3). CRP induces complement activation via the classical pathway. This in turn triggers the influx of neutrophils. It decorates the surface of the ligand with opsonizing complement fragments and enhances phagocytosis of the cells that have bound CRP and complement. CRP also acts as an opsonin by interacting with Fc receptors (4). In addition to binding to the membranes of injured viable cells, CRP also binds to the membranes and nuclear constituents of necrotic cells. However, genetically determined factors with regard to the action of CRP in CAD have rarely been studied yet. FcγRIIa, a receptor well established to interact with the Fc portions of IgG has recently been reported also to directly bind CRP (5). However, there are conflicting data on this issue (6,7).

FcγR bridge the humoral and cellular arms of the immune system. Three main classes of human leukocyte FcγR (FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD 16)) are encoded by eight FcγR genes (8). FcγR use the specificity of antibodies and endow the cells with potent cellular effector functions. They are crucial for the clearance of immune complexes. They regulate antibody production and are involved in the regulation of B cell and T cell development (9). The heterogeneity of FcγR is essential for regulation and fine-tuning of the immune response (10). Polymorphisms of FcγRIIa, FcγRIIIa, and FcγRIIIb further increase the functional heterogeneity of these IgG receptors. FcγRIIa is expressed on most myeloid cells, and bears either an arginine (R131) or a histidine (H131) at extracellular amino acid position 131 (11). This substitution dictates receptor binding capacity of complexed IgG2 and IgG3, FcγRIIa- R131 being the receptor with lower binding capacity (12,13).

Detailed analyses showed that FcγRIIa, containing the arginine (FcγRIIa-R131), displays a high avidity for CRP, whereas FcγRIIa-H131 displays a low avidity (14). Recently, the associations of the FcγRIIa genotypes with the manifestation of other chronic inflammatory diseases (15) and with their clinical course (8) have been reported.

Gardemann et al. (20) hypothesized that the FcγRIIa Rl 3 IH polymorphism has an impact on coronary heart disease in a German cohort. This polymorphism was analysed in 510 healthy man and 2391 male participants with coronary angiography. No associations were observed between Fc7RIIa-R131H polymorphism and the risks of coronary artery disease and myocardial infarction following genotyping in the total sample. Only when the study sample was restricted to low-risk groups of all the individuals the FcγRIIa-R13 IH polymorphism was linked to a risk of CAD and MI. However, the conclusion of Gardemann et al. was that the above polymorphism is not an independent risk indicator of CAD and MI and that the impact of this gene variation is restricted to older individuals who are at low risk for this disease.

Thus, it is a problem underlying the present invention to provide a method for determining the risk of an acute coronary syndrome which method in particular can be used in the early and follow-up diagnosis of unstable angina pectoris (UAP) and myocardial infarction (MI). It is a further problem underlying the invention to provide a diagnostic method and a diagnostic kit for use in medicine, in particular for the rapid determination of the FcγRIIa polymorphism and, thus, for the individual risk of the patient of developing or suffering from unstable angina pectoris, myocardial infarction or other related diseases.

These problems are solved by the subject matter of the independent claims. Preferred embodiments are set forth in the dependent claims.

The identification of clinically relevant determinants for the destabilization of CAD promises a high reduction of ACS-associated mortality. In order to achieve this, the inventors genotyped the FcγRIIa alleles of Caucasians with CAD. They observed a significant association of FcγRIIa-R/R131 with UAP. It is thus concluded that testing for FcγRII polymorphism allows further risk stratification in CAD.

Surprisingly and unexpectedly, it turned out that the FcγRIIa polymorphism provides a means for determining the individual risk of a patient of developing or suffering from ACS and in particular UAP and MI. Thus, in contrast to the teachings of the prior art, in particular of Gardemann et al, this polymorphism has a strong and statistically significant relation to ACS without being restricted to a subpopulation such as older individuals or distinct ethnic groups. It is noted that the present invention shows the inventive effects by means of a study population which is comparable to that of Gardemann et al., i. e. patients of Caucasian ethnicity. Therefore, the FcγRIIa polymorphism can be used for a precise and rapid detection of the individual risk to suffer from acute coronary syndrome as well as many related diseases.

According to the invention, the geno- and/or phenotyping of FcγR in patients with atherosclerosis results in the following, clinically significant benefits:

It provides a clinically important information, whether or not a patient will develop atherosclerotic complications (figure 1). Examples for such complications are: development of an acute coronary syndrome or stroke or transient ischemic attack. These disease entities result in an increased mortality. Similar to the benefits of established risk markers, i.e. analysis the ECG (changes of ST-segments), the analysis of heart-specific enzymes (particularly Troponin I), the clinical symptoms, geno- and/or phenotyping of FcγR allows a significantly improved risk stratification for the patient.

Herewith, clinical decisions can be made with higher accuracy thereby improving the prognosis of patients' life and reducing costs for further diagnostic and therapeutical steps:

an improved risk stratification by using geno- and/or phenotyping of FcγR positively influences decisions about the necessity of hospitalisation and the intensity of medical care (for example: do the patients have to be monitored on the expensive intensive care unit or is it justified by improved risk stratification to monitor the patient less frequently on the less expensive regular ward; i.e. improved risk stratification by using geno- and/or phenotyping of FcγR reduces costs for hospitalisation);

an improved risk stratification by using geno- and/or phenotyping of FcγR strongly influences the specific kind and spectrum of medical therapy. Patients with the 'unfavourable' FcγRII-allele would undergo an expensive intensified regimen, which consequently will improve the patients' prognosis. In patients with the 'favourable' FcγRII-allele and therefore better prognosis as compared to patients with the 'unfavourable' FcγRII-allele, expensive therapy is without any additional effect with regard to the patients' prognosis, but could do even harm the patients (i.e. increased incidence of bleeding under a strong anticoagulation within the therapy of acute vascular complications);

improved risk stratification by using geno- and/or phenotyping of FcγR improves primary and secondary prevention in patients with atherosclerotic risk profile.

Patients with the 'unfavourable' FcγRII-allele should aim at a more intensive modulation of their atherosclerotic risk factors as compared to patients with lower cardiovascular risk in order to improve their prognosis. Vice versa, in patients with the 'favourable' FcγRII-allele broad and expensive treatment of atherosclerotic risk factors may not have prognostic relevance (figure 2). The risk stratification by using geno- and/or phenotyping of FcγR can start very early in life (i.e. with birth), thus making primary prevention with consecutive large cost reductions more efficient;

improved risk stratification by using geno- and / or phenotyping of FcγR improves anti¬ inflammatory medical therapy. Since the level of CRP itself in the blood does not sufficiently explain its effect in atherosclerosis, functional aspects of the CRP receptor FcγR adds an important additional information for the anti-inflammatory therapy, which therefore can be tailored more accurately to each patient;

improved risk stratification by using geno- and / or phenotyping of FcγR improves significantly anti-platelet therapy in atherosclerosis, the basis of medical treatment for atherosclerosis (i.e. acetylsalicylacid, clopidogrel). FcγRII is expressed on platelets. Herewith, platelets aggregate and release prothrombotic substances leading to thrombotic complications of atherosclerosis. Evaluation of FcγRII geno- and / or phenotyping allows better dosage of the anti-platelet therapy in patients with atherosclerosis;

evaluation of FcγR geno- and / or phenotype allows a more distinguished interpretation of the prognostic value of increased CRP blood levels in patients with atherosclerosis. It allows a genetic explanation, why CRP levels differ interindividually resulting in distinguished prognosis among patients with equally increased CRP levels.

The present invention introduces a method for the diagnosis (first diagnosis or at follow up) of atherosclerosis- related risks by using a system for determining the FcγRIIa geno- and/or phenotype. This test system is used for the risk stratification of patients in the primary or secondary prevention or for decision making in the therapy of the following diseases:

Coronary artery disease and its clinical manifestations such as stable angina, unstable angina, non-Q-wave infarction (non-ST-segment myocardial infarction), Q-wave infarction (ST-segment myocardial infarction), and ischemic cardiomyopathy;

Coronary artery disease with regard to early complications of myocardial infarction (i.e. re-infarction, left ventricular failure, cardiogenic shock, rhythm disturbances); coronary artery disease with regard to late complications of myocardial infarction (i.e. ventricular septum defect, ventricular rupture); vasospastic angina (Prmzmetal-angina); in patients with coronary artery disease after bypass-surgery; for risk stratification of sudden cardiac death; in patients with atherogenic risk factors such as arterial hypertension, diabetes mellitus, hyperlipoproteinemia, the metabolic syndrome, increase of Lipoprotein (a), hyperfibrinogenemia, and hyperhomocysteinemia; in antiphosholipid-antibody syndromes; in genetically caused tissue-plasminogen activator deficiencies; in patients with vasculitis, which are associated with increased risk for atherosclerotic complications; in patients with chronic renal insufficiency of various origins, which are associated with increased risk for atherosclerotic complications; in transplant vasculopathy, i.e. in patients after organ transplantation (particularly transplantation of the heart and kidney); in clinical studies about the pathophysiology of atherosclerosis, particularly about the pathophysiology of coronary artery disease or stroke and their sub forms; in various clinical subforms of gestosis; in patients with aortic valve stenosis or aortic valve sclerosis.

The use of the present diagnostic methods and kits etc. for first or follow-up diagnosis of the above diseases is falling within the scope of the present invention.

Thus, a diagnostic system for the determination of the Fc7 receptor (FcR) polymorphisms (FcγRIIa -R131 (arginine at position 131), FcγRIIa -H131 (histidine at position 131)), respectively, which comprises a mixture containing at least one substance which is able to detect Fcγ receptor (FcR) polymorphisms (FcγRIIa -Rl 31 (arginine at position 131), FcRIIa -H131 (histidine at position 131)), respectively, especially, for the diagnosis of atherosclerotically caused risks, is disclosed herein.

In this mixture, nucleic acids for the detection of the polymorphism or monoclonal antibodies against the R- and/or the H-type of the FcγRIIa and/or CRP and/or CRP derivatives and/or CRP parts and/or IgG, like eg. human IgG2 and/or murine IgGl can be included. To these molecules, markers like eg. dyes, especially, fluorescent dyes/molecules can be coupled.

The mixtures, substances, molecules described above may be used in a diagnostic system for the analysis of cells, especially, blood cells like antigen presenting cells (monocytes, dendritic cells, macrophages, B cells), granulocytes, eosinophiles. This kind of application can contain a method for nucleotide analysis of FcγRIIa eg. PCR, RT-PCR, Southern Blot, Northern Blot, RFLP analysis, sequencing. An application like this can alternatively contain a method for protein analysis of FcγRIIa eg. SDS gel electrophoresis, Western Blot, protein sequencing. A diagnostic application like this can also contain a method for protein expression analysis of FcγRIIa eg. ELISA, FACS analysis, immune histochemical analysis.

In the diagnostic method of the present invention, monoclonal antibodies against the R- and/or the H-type of the FcγRIIa, and/or CRP and/or CRP derivatives and/or CRP parts and/or IgG, like eg. human IgG2 and/or murine IgGl, which is coupled/coated to particles (eg. beads, latex beads) are used. In the ideal situation, the incubation of the cells which shall be analysed with the monoclonal antibodies against the R- and/or the H-type of the FcγRIIa and/or CRP and/or CRP derivatives and/or CRP parts and/or IgG, like eg. humane IgG2 and/or murine IgGl, will be performed at a temperature of 37°C or below 37°C, especially, at temperatures between 2 - 25⁰C.

Besides, additional particles can be used which show typical expression patterns and expression levels of FcγRIIa phenotypes like RR (with two alleles with arginine at position 131), or RH (one allele arginine and the other histidine), or HH (two alleles histidine). These particles can be cells (eukaryotic and/or prokaryotic), fixed cells, embedded cells, beads of divers materials, liposomes. The material used must contain the appropriate mixture of FcγRIIa and a, for carriers of the characteristics, typical expression level. The particles can then serve as standards for protein expression analyses.

From the substances and mixtures described above, a kit can be compiled which serves for the determination of the polymorphism.

The present invention in particular is directed to the following aspects and embodiments:

In a first aspect, an ex-vivo method of determining the risk of a patient of developing or of suffering from acute coronary syndrome (ACS) is provided comprising the steps of: a) detecting the FcγRIIa genotype or phenotype of said patient at amino acid position 131 (R/H); b) determining and classifying the individual risk of said patient in the following order:

H/H < H/R < R/R.

By this method, a precise risk assessment for an individual patient can be made. For example, a blood sample is taken from the patient soon after he or she arrived at the medical institution or the physician. Subsequently a genotyping of the respective polymorphism is done for example by means of PCR. As soon as the results of this analysis are known (and thus the patient's individual risk regarding ACS), the physician in charge may initiate the required therapeutical steps in order to provide the best possible and most cost-effective treatment of said patient.

In this connection, it is in particular referred to Tables 2, 3 and 5, wherein the indication of the relative risk of developing UAP and MI is clearly highest for the R/R genotype.

According to a preferred embodiment the method can be used for the detection of the individial risk of developping or suffering from unstable angina pectoris (UAP), non- ST-elevation and ST-elevation myocardial infarction (MI).

In the present method, step a), one or more substances are preferably used which are capable of detecting the FcγRIIa polymorphism 131 R/H. Perferably, said one or more substances are selected from nucleic acids, antibodies raised against the R and/or H type of FcTRIIa, CRP or derivatives or fragments thereof, and/or IgG.

In particular the following combinations of antibodies may be used herein:

1. Antibodies against R and antibodies against H;

2. Antibodies against R+H and antibodies against H;

3. Antibodies against R+H and antibodies against R.

This combination allows a quantitative determination of arginine and/or histidine (R/H) in FcγRIIa.

The antibodies used in this invention may be selected from a group, which consists of polyclonal antibodies, monoclonal antibodies, chimeric antibodies and synthetic antibodies. It is noted that monoclonal antibodies are most preferred in this invention.

The antibody according to the invention can be additionally linked to a detectable agent.

The term "antibody", is used herein for intact antibodies as well as antibody fragments, which have a certain ability to selectively bind to an epitop. Such fragments include, without limitations, Fab, F(ab')₂ und Fv antibody fragment. The term "epitop" means any antigen determinant of an antigen, to which the paratop of an antibody can bind. Epitop determinants usually consist of chemically active surface groups of molecules (e.g. amino acid or sugar residues) and usually display a three-dimensional structure as well as specific physical properties.

The antibodies according to the invention can be produced according to any known procedure. For example the pure complete FcγRIIa receptor according to the invention or a part of it can be produced and used as immunogen to immunize an animal and to produce specific antibodies.

The production of polyclonal antibodies is commonly known. Detailed protocols can be found for example in Green et al, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, editor), pages 1 - 5 (Humana Press 1992) und Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992). hi addition, the expert is familiar with several techniques regarding the purification and concentration of polyclonal antibodies, as well as of monoclonal antibodies (Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

The production of monoclonal antibodies is as well commonly known. Examples include the hybridoma method (Kohler and Milstein, 1975, Nature, 256:495-497, Coligan et al., section 2.5.1 - 2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988).), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

In brief, monoclonal antibodies can be attained by injecting a mixture which contains the receptor of the invention into mice. The antibody production in the mice is checked via a serum probe. In the case of a sufficient antibody titer, the mouse is sacrificed and the spleen is removed to isolate B-cells. The B cells are fused with myeloma cells resulting in hybridomas. The hybridomas are cloned and the clones are analyzed. Positive clones which contain a monoclonal antibody against the protein are selected and the antibodies are isolated from the hybridoma cultures. There are many well established techniques to isolate and purify monoclonal antibodies. Such techniques include affinity chromatography with protein A sepharose, size-exclusion chromatography and ion exchange chromatography. Also see for example, Coligan et al., section 2.7.1 - 2.7.12 and section ,,Immunglobulin G (IgG)", in Methods In Molecular Biology, volume 10, pages 79 - 104 (Humana Press 1992).

CRP and IgG are used in their protein form in order to evaluate the binding behavior of FcγRIIa. Human IgG 2 binds to FcγRIIa 13 IH with higher affinity and murine IgG 1 binds to FcTRIIa 13 IR with higher affinity.

C-reactive protein (CRP) is an acute phase protein of human beings. CRP is a member of the class of acute phase reactants as its levels rise dramatically during inflammation in the human body. It was suggested that patients with elevated basal levels of CRP are at an increased risk for stable coronary heart disease (by a factor of 2).

The amount of CRP produced by the body varies from person to person, and this is affected by an individual's genetic charcteristics and lifestyle. Higher CRP levels tend to be found in individuals who smoke, have high blood pressure, are overweight and don't exercise, whereas lean, athletic individuals tend to have lower CRP levels.

FcγRII is the main receptor of CRP on human monocytes and PMN. However, the binding behavior of CRP to different receptor allotypes is differing remarkebly and is thus leading to an obvious functional heterogeneity due to the polymorphism of FC7RII. Therefore, CRP protein or functional fragments or derivatives may act to evaluate the binding behavior of the patients FcTRIIa and thus the individual FcγRIIa allele of a patient. As also mentioned above, the substances my be detectably labelled. Preferably, the label is a dye or a coloured molecule. More preferably, the dye is a fluorescent or chemiluminescent dye. All dyes well known in the art may be used for this purpose.

According to a preferred embodiment, the genotype of the patient is determined by evaluating the FcγRIIa polymorphism 131 by a method of nucleic acid analysis. This method preferably is selected from PCR, RT-PCR, Southern Blotting, Northern Blotting, RFLP-analysis, and/or nucleic acid sequencing. For further details see also McPherson et al. (ed.), PCR, A Practical Approach, Oxford, IRL Press 1995.

As an alternative, the FcγRIIa polymorphism 131 is detected by a method of amino acid analysis, preferably by ELISA, FACS-analysis, and/or immunohistochemical analysis (as mentioned above).

The one or more substances used in the present method may be coupled or coated onto particles, preferably beads or latex beads.

The Fc7RIIa genotype or phenotype of the patient is detected in body cells sampled from the patient. Those cells preferably are blood cells, most preferably antigen presenting cells (APCs), granulocytes and/or eosinophils. The APCs preferably are selected from monocytes, dendritic cells, macrophages and/or B cells.

In the method of othe present invention, the incubation of the patient's body cells with the one or more substances as defined herein is performed at a temperature of 37°C or below 37⁰C. More precisely, the temperature preferably is from about 2 to about 25°C, more preferably from 15 to 22°C, 17 to 22°C, and most preferably 18 - 2O⁰C. Usually, the temperature may be ambient temperature.

According to a second aspect, the present invention provides an antibody which is directed against the 131 H type of FcγRIIa. As mentioned above, that antibody may be selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and a synthetic antibody. The monoclonal antibody is most preferred.

In a third aspect, the invention provides a diagnostic kit comprising one or more of the substances as defined above.

Furthermore, a particle is encompassed, which is comprising expression patterns or levels of expression of the Fc7R.Ha genotypes 131 R/R, 131 H/H and/or 131 R/H. Those particles might be used a standards for the present methods (see also above). This particle preferably is selected from the group consisting of cells (procaryotic and/or eucaryotic), fixed cells, embedded cells, beads and/or liposomes, wherein the beads may preferably comprise latex, sepharose, agarose and/or polystyrene.

In a further aspect, the invention provides the use of the method as defined above, of the antibody as disclosed herein or the above mentioned kit for the diagnosis of acute coronary syndromes (ACS). As already mentioned above, the ACS is selected from the group consisting of unstable angina pectoris (UAP), non-ST-elevation and ST-elevation myocardial infarction (MI).

Furthermore the method, antibody, kit of the invention may be used for the diagnosis/prognosis of early and late complications of ACS, for the diagnosis of an individual's risk of sudden cardiac death and for the early diagnosis (preventive diagnosis) of catastrophic antiphospholipid syndrome (Asherson's Syndrome), systemic lupus erythematosus, rheumatoid arthritis, vasculitis, multiple sclerosis, immune- mediated thrombocytic purpura and myasthenia gravis. Additionally, the use encompasses the various medical applications indicated above, i.e. the use in the diagnosis/prevention of vasospastic angina (Prinzmetal-angina); in patients with coronary artery disease after bypass-surgery; in patients with atherogenic risk factors such as arterial hypertension, diabetes mellitus, hyperlipoproteinemia, the metabolic syndrome, increase of Lipoprotein (a), hyperfibrinogenemia, and hyperhornocysteinemia; in antiphosholipid-antibody syndromes; in genetically caused tissue-plasminogen activator deficiencies; in patients with vasculitis, which are associated with increased risk for atherosclerotic complications; in patients with chronic renal insufficiency of various origins, which are associated with increased risk for atherosclerotic complications; in transplant vasculopathy, i.e. in patients after organ transplantation (particularly transplantation of the heart and kidney); in clinical studies about the pathophysiology of atherosclerosis, particularly about the pathophysiology of coronary artery disease or stroke and their subforms; in various clinical subforms of gestosis; in patients with aortic valve stenosis or aortic valve sclerosis.

The present invention will be further described with reference to the following figures and examples; however, it is to be understood that the present invention is not limited to such figures and examples.

The figures are showing the following:

Figure IA shows the significantly altered distribution of the FcγRIIa genotype (RR, HR or HH) within the patients with CAD and ACS as compared to NHD.

Figure IB illustrates the three different pathways (1, 2, and 3) to develop an AMI are indicated by arrows. A significantly higher part of patients with FcγRIIa-R/R131 genotype develop an AMI going through the stadium of UAP (pathway T).

Examples

Methods

Study Population

We recruited 91 consecutive patients of Caucasian ethnicity with CAD, among which 25 had SAP, 19 UAP (without any elevation of cardiac enzyme markers), and 47 acute myocardial infarction (AMI) for this study. The latter suffered from chest pain with significant elevation of cardiac enzyme markers with or without significant ECG changes [Table 4]. Blood collection

Immediately after admission and at several time points afterwards, blood was drawn for the classification of the CAD. Furthermore, we collected blood into sodium citrate containing tubes (Sarstedt, Germany) and purified the peripheral blood mononuclear cells (PBMC) using the Vacutainer™ system (BD BioSource; Heidelberg, Germany). Caucasian normal healthy blood donors (NHD) served as controls. The study was approved by the institutional ethics committee for human subjects.

Analysis of FcγRIIa alleles

Genomic DNA was isolated from PBMC using the Wizzard™ DNA purification kit (Promega, USA) according to the manufacturer's instruction. Identification of the FcγRIIa polymorphism was performed by PCR as described previously (16). Briefly, for the allele-specific amplification, primers P5G (5'-GAA AAT CCC AGA AAT TTT TCC G) (for the FcγRIIa-R131 allele; SEQ ID NO: 1) and P4A (5'-GAA AAT CCC AGA AAT TTT TCC A) (for the FcγRIIa-H131 allele; SEQ ID NO: 2) were combined with the common antisense primer, P 13 (5'-CTA GCA GCT CAC CAC TCC TC) (SEQ ID NO: 3).

Statistical Analyses

Statistical analyses were performed using the χ² test, or Fisher's exact test when indicated. For the relative risk, the 95% confidence interval (CI) was calculated as well. A value of PO.05 was considered significant.

Results

The detailed analysis of the FcγRIIa genotype revealed a significantly altered distribution (reduced FcγRIIa-H/H) within the patients with CAD and ACS as compared to NHD [Figure 1 A/Table 2 and 5]. Strikingly, there was a highly significant difference between patients with UAP when compared to those within SAP (P<0.01) [Figure IB/Table 2 and 5]. The FcγRIIa-R/R genotype was almost exclusively found in the patients with UAP. However, there was no significant difference of the frequency of FcγRIIa-R131 between patients with AMI and NHD. The allele frequencies and the significances of the detailed analyses are shown in Table 2 and 5. Additional analyses showed a markedly reduced frequency of the FcγRJIa-R/R genotype in SAP and FcγRIIa-H/H genotype in ACS patients. Statistical analyses proofed the sensitivity and specificity for the Non-RR genotype to detect SAP and for the Non-HH genotype to detect ACS in all patients with CAD. The Non-RR genotype has a sensitivity of 96 % with a specificity of 29 % to detect SAP. The Non-HH genotype has a sensitivity of 86 % with a specificity of 24 % to detect ACS.

Detailed analyses of relative risk, confidential interval, significance, sensitivity, and specificity are shown in Table 6. In general, the FcγRIIa-R/R131 genotype had a significantly higher frequency in ACS patients. This was especially prominent comparing patients with UAP and SAP. In addition, the FcγRIIa-H/H131 genotype turned out to be protective for controls to develop ACS. Throughout all our patients, distribution of FcγRIIa genotypes were not significantly related to the serum levels of CRP or the age of the patients in which patients finally developed their acute coronary syndrome [not shown].

In this application, the inventors show that destabilization of CAD is related to the underlying genetic polymorphism of FcγRIIa. The role for CRP in risk stratification of CAD is well established. However, the mechanistic link between CRP levels and disease is still elusive (17). Here , an association of the FcγRIIa, a receptor for CRP (6), with manifestations of CAD is described. Leukocyte IgG receptors (FcγR) serve as a link between humoral and cellular branches of the immune system and mediate potent leukocyte effector functions like pro- and anti-inflammatory reactions (8). If these leukocyte functions could also be mediated by direct CRP binding to FcγRIIa, our findings could indicate a direct pathophysiological role of CRP and of FcγRIIa in the development and progression of CAD.

The genetically determined interindividual variability of FcγR function has been extensively studied in chronic inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, or multiple sclerosis. There is a clear association between FcγR genotypes and chronic inflammatory diseases, which is discussed to be due to clearance deficiency of IgG2-containing immune complexes by the FcγRIIa- R131. Another concept assumes a detrimental inflammatory response through highly efficient FcγR - IgG interaction due to variations in the affinity to the IgG2 subclass.

For atherosclerosis and its complications, the relevance of the FcγRIIa genotypes can be derived from the following: FcγRIIa is expressed on monocytes/macrophages, neutrophils, and platelets which all play a role in atherogenesis and atherosclerosis (18). Binding of FcγRIIa on platelets causes platelet-aggregation and release of platelet granula. Thereby, genetically determined variability in CRP binding to FcγRIIa may translate into various biological effects of CRP on FcγRIIa bearing cells and finally influence clinical outcome decisively.

Whatever the exact underlying pathophysiological role of FcγRIIa polymorphisms in chronic inflammatory diseases is, our data allow clinically highly interesting risk stratiiϊcation in patients with CAD. The development from NHD to AMI patient can basically follow 3 pathways, via SAP ((1) in Figure IB), via UAP (2), or directly (3). The FcγRIIa-R/R genotype highly significantly favors pathway 2 over pathway 1. In synopsis with the patient's history, symptoms, ECG, and heart specific markers, determination of the patient's FcγRIIa genotype may allow improved risk stratification with consecutive therapeutical consequences. Notably, with regard to AMI, there might be a referral bias: the cohort contains only patients, who were hospitalized for their AMI. The genotype of patients, who die before admission to the hospital because of sudden cardiac death remains elusive.

Beyond patients with known CAD, determination of FcγRIIa genotypes may also allow risk stratification in the setting of primary prevention. According to our results, the FcγRIIa-H/H genotype had a significantly lower frequency for ACS as compared to healthy controls. Here, early genotyping together with stratification of classical atherogenic risk factors may improve the predictive value. In a broader context, the protective value of FcγRIIa-H/H against ACS may contribute to the well known reduced incidence of ACS in the Japanese population (19), which has a highly increased frequency of the FcγRIIa-H131 allele (R/R131 : 3.6%, R/H131 : 31.4%, H/H131 : 65%) (15).

In conclusion, the R/R genotype has a high frequency in patients with UAP when compared to those with SAP, NHD, or AMI. In contrast, the FcγRIIa-H/H genotype is protective against ACS. Therefore, genotyping of FcγRIIa may become part of the risk stratification in patients prone for the development of ACS.

Tables:

Table 1

FcγRIIa genotype distribution in 187 healthy controls and 91 patients with coronary artery disease (i.e. patients with stable and unstable angina pectoris as well as patients with acute myocardial infarction)

FcγRIIa genotype Controls Coronary Artery Disease (SAP, UAP, AMI) (n = 187) (« = 91)

FcγRIIa-R/R131 51 (27)* 20 (22) FcγRIIa-R/H131 84 (45)* 56 (62) FcγRIIa-H/H131 52 (28)* 15 (16)

P-value P < 0.05

Percentages in parathesis. *Distribuation of controls according to: Rascu et al, Annals New York Academy of Sciences 1997.

SAP = stable Angina pectoris; UAP = unstable Angina pectoris; AMI = acute Myocardial infarction.

Table 2

FcγRIIa genotype distribution stratified according to the clinical phenotype (i.e. patients with stable coronary artery disease versus patients with acute coronary syndrome [= unstable Angina pectoris or acute myocardial infarction])

Acute coronary syndrome

FcγRIIa Genotype SAP all UAP AMI

FcγRIIa-R/R131 1 (4) 19 (29) 7 (37) 12 (25.5) FcγRIIa-R/H131 18 (72) 38 (58) 10 (53) 28 (59.5) FcγRIIa-H/H131 6 (24) 9 (14) 2 (10) 7 (15)

P-Value P = 0.032 P < 0.01 Ns

Table 3

Relative risk of each FcγRIIa genotype to develop an acute coronary syndrome (i.e. unstable Angina pectoris or acute myocardial infarction).

FcγRIIa genotype All [CI 95%]

FcγRIIa-R/R131 1.65 rø.89 - 3.0τT

FcγRIIa-R/H131 1.06 [0.61 - 1.84]

FcγRIIa-H/H131 0.49 IO.23 - 1.061 Table 4: Patients' characteristics. Data are mean ± SD. SAP = stable angina pectoris; UAP = unstable angina pectoris; AMI = acute myocardial infarction; ns = non¬ significant.

SAP UAP AMI P-Value

(n = 25) (n = 19) (n = 47)

Age, y (admission) 63.9 + 10 67.2 ± 10.2 62.6 ± 13 ns

Age, y (first coronary event) 58.6 ± 13.9 62.2 ± 9.9 61.8 ± 9.7 ns

Male sex, % 80 91.3 79.1 ns

Creatinine Kinase (U/L) 1.45 ± 73.64 125.93 ± 97.78* 2010.42 ± 1766.28 1 * ns (vs SAP)

Troponin I (ng/mL) 0.02 ± 0.01 1.15 ± 2.94* 36.18 + 32.871 ± P < 0.001

(vs SAP/UAP)

Table 5: Distribution of genotypes m patients with coionary artery disease (CAD) and normal healthy controls (NHD), see also Table 2 Percentages in parenthesis ACS = acute coronary syndrome; SAP = stable angina pectoris, UAP = unstable angina pectoris; AMI = acute myocardial infarction.

NHD CAD

--

FcγRIIa total CAD SAP total ACS , UAP Z AMI genotype n 187 91 25 66 19 47

R/R131 51 (27) 20 (22) 1 (4) . 19 (29) 7 (37) 12 (25.5)

H/R131 84 (45) 56 (62) 18 (72) ' 38 (58) - 10 (53) 28 (59.5)

H/H131 52 (28) 15 (16) 6 (24) 9 (14) ' 2 (10) 7 (15)

Table 6: Analyses of relative risk, confidential interval, significance, sensitivity, and specificity in the patients' groups. Ns = non significant, P value calculated by *Fisher' s exact test

References

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6. Saeland E, van Royen A, Hendriksen K et al. Human C-reactive protein does not bind to fc{ {gamma}} RIIa on phagocytic cells. J Clin Invest. 2001;107:641-642.

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11. Warmerdam PA, van De Winkel JG, Gosselin EJ, Capel PJ. Molecular basis for a polymorphism of human Fc gamma receptor II (CD32). J Exp Med. 1990; 172: 19- 25.

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14. Zuniga R, Markowitz GS, Arkachaisri T, Imperatore EA, D'Agati VD, Salmon JE. Identification of IgG subclasses and C-reactive protein in lupus nephritis: the relationship between the composition of immune deposits and FCgamma receptor type IIA alleles. Arthritis Rheum. 2003;48:460-470.

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Claims

P 19204Patent claims:

1. An ex-vivo method of determining the risk of a patient of developing or of suffering from acute coronary syndrome (ACS) comprising the steps of: a) detecting the FcγRIIa genotype or phenotype of said patient at amino acid position 131. b) determining and classifying the individual risk of said patient in the following order:

H/H < H/R < R/R.

2. The method of claim 1, wherein the ACS is selected from the group consisting of unstable angina pectoris (UAP), non-ST-elevation and ST-elevation myocardial infarction (MI).

3. The method of claim 1 or 2, wherein in step a), one or more substances are used capable of detecting the Fc7R.Ha polymorphism 131 H/H, R/H und R/R.

4. The method of claim 3, wherein the one or more substances are selected from nucleic acids, antibodies raised against the R and/or H type of FcτRIIa, CRP or derivatives or fragments thereof, and/or IgG.

5. The method of claim 4, wherein the IgG is selected from human IgG2 and/or murine IgGl.

6. The method of claim 4 or 5, wherein the substances are detectably labelled.

7. The method of claim 6, wherein the label is a dye or a coloured molecule.

8. The method of claim 7, wherein the dye is a fluorescent or chemiluminescent dye.

9. The method of one or more of the preceding claims, wherein the FcγRIIa polymorphism 131 is detected by a method of nucleic acid analysis.

10. The method of claim 9, wherein the method is selected from PCR, RT-PCR, Southern Blotting, Northern Blotting, RFLP-analysis, and/or nucleic acid sequencing.

11. The method of one or more of claims 1-8, wherein the FcγRIIa polymorphism 131 is detected by a method of amino acid analysis.

12. The method of claim 11, wherein the method is selected from ELISA, FACS- analysis, and/or immunohistochemical analysis.

13. The method of claim 12, wherein one or more substances of one or more of claims 4-8 are used, which are coupled or coated onto particles.

14. The method of claim 13, wherein the particles are beads or latex beads.

15. The method of one or more of the preceding claims, wherein the FcγRIIa genotype or phenotype of the patient is detected in body cells sampled from the patient.

16. The method of claim 15, wherein the cells are blood cells.

17. The method of claim 16, wherein the cells are selected from the group consisting of antigen presenting cells (APCs), granulocytes and/or eosinophils.

18. The method of claim 17, wherein the APCs are selected from monocytes, dendritic cells, macrophages and/or B cells.

19. The method of one or more of the preceding claims, wherein the incubation of the patient's body cells with one or more substances of claims 4-8 is performed at a temperature of 37°C or below 37°C.

20. The method of claim 19, wherein the temperature is from about 2 to about 25°C.

21. The method of claim 20, wherein the temperature is from 15 to 22°C.

22. The method of claim 21 the temperature is from 17- 22°C, preferably 18 - 20⁰C.

23. An antibody, which is directed against the 131 H type of Fc7RIIa.

24. The antibody of claim 23, which is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and a synthetic antibody.

25. A diagnostic kit comprising one or more of the substances of claims 4-8 or 23 and 24.

26. A particle, which is comprising expression patterns or levels of expression of the FcγRIIa genotypes 131 R/R, 131 H/H and/or 131 R/H.

27. The particle of claim 26, which is selected from the group consisting of cells (procaryotic and/or eucaryotic), fixed cells, embedded cells, beads and/or liposomes.

28. The bead of claim 27, which comprises latex, sepharose, agarose and/or polystyrene.

29. Use of the method of one or more of claims 1-22, of the antibody of claim 23 and 24, or the kit of claim 25 for the diagnosis of acute coronary syndromes (ACS).

30. The use of claim 29, wherein the ACS is selected from the group consisting of unstable angina pectoris (UAP), non-ST-elevation and ST-elevation myocardial infarction (MI).

31. The use of claim 29 or 30 for the diagnosis/prognosis of early and late complications of ACS.

32. The use of claim 29 for the diagnosis of an individual's risk of sudden cardiac death.

33. The use of claims 29 for the diagnosis in patients suffering from catastrophic antiphospholipid syndrome (Asherson's Syndrome), systemic lupus erythematosus, rheumatoid arthritis, vasculitis, multiple sclerosis, immune- mediated thrombocytic purpura, myasthenia gravis, vasospastic angina (Prinzmetal-angina); coronary artery disease after bypass-surgery; in patients with atherogenic risk factors such as arterial hypertension, diabetes mellitus, hyperlipoproteinemia, the metabolic syndrome, increase of Lipoprotein (a), hyperfibrinogenemia, and hyperhomocysteinemia; antiphosholipid-antibody syndromes; genetically caused tissue-plasminogen activator deficiencies; in patients with vasculitis, which are associated with increased risk for atherosclerotic complications; in patients with chronic renal insufficiency of various origins, which are associated with increased risk for atherosclerotic complications; in transplant vasculopathy, i.e. in patients after organ transplantation; in various clinical subforms of gestosis; in patients with aortic valve stenosis or aortic valve sclerosis.