WO2006027260A1

WO2006027260A1 - Use of a gene modification in the gene of the human cdca3 protein in medical diagnosis and therapy

Info

Publication number: WO2006027260A1
Application number: PCT/EP2005/009716
Authority: WO
Inventors: Dieter Rosskopf
Original assignee: Universität Duisburg-Essen
Priority date: 2004-09-10
Filing date: 2005-09-09
Publication date: 2006-03-16
Also published as: DE102004044374A1

Abstract

The invention relates to the use of a gene modification, particularly a polymorphism, in the gene of the human cell division cycle-associated protein 3 (CDCA3 protein) for diagnosing diseases and/or determining the risk of contracting a disease and/or predicting individual effects and side effects of medicaments.

Description

Use of a gene change in the gene of the human CDC45 protein in medical diagnostics and therapy

The present invention relates to the use of a gene modification or a nucleolide sequence change (base sequence change), in particular a polymorphism, in the gene of the human cell division cycle-associated protein 3 {cell division cycle associated protein 3; CDCA3 protein), in particular the use of a gene modification or a nucleolide sequence change in the promoter region of the CDC A3 gene, for diagnosis and therapy, for the prediction of disease risks, courses of disease and / or for the prediction of the response of medicaments.

In addition, the present invention allows statements on the optimal dosage of drugs and for the prediction of side effects of drugs. The invention also makes it possible to find further gene modifications in the gene of CDCA3 and the mono- and polygenetic haplo-type analysis for the provision of methods for the diagnosis of disease risks, disease progression and the response to drugs.

A still largely unsolved problem in clinical medicine is the prediction of which patients are at high risk for a disease, what their disease will progress to, and what therapeutic options best suit the patient. Many such risks are genetic. Only the reliable prediction of disease risks allows the sensible development and implementation of preventive programs.

In the development and application of medicaments (but also other therapy method), it is therefore an unresolved problem to predict which patients benefit most from which therapy. Thus, it is known that only roughly roughly 50% of the treated patients respond to therapy for most drugs. According to the current state of knowledge, genetic influences play a major role here. In conducting clinical-scientific studies - eg. For example, in drug development and drug approval, large numbers of participants are often required. to provide the corresponding statistical evidence. Reliable selection of appropriate study participants makes drug trials faster, more efficient, and more cost-effective. For this purpose, genetic tests in the context of so-called pharmacogenetics are indispensable. The same applies to the prediction of patients who carry a high risk for adverse drug reactions due to their genetic endowment.

Another current problem in drug development is the definition of target structures for the development of new drugs (so-called drug targets). Genetic information and the understanding of relevant mechanisms which are causally involved in the development of diseases form the basis for the successful identification of such drug targets.

To date, only a few systems exist for disease-related genetic risk prediction and for the field of so-called pharmacodynamic pharmacogenetics (ie the branch of pharmacogenetics that deals less with drug metabolism and transport, but rather with the genetic basis of individual effects on the actual target structures of corresponding drugs ). For example, in the field of stroke prediction, there are only a few genetic risk markers that are predominantly non-specific. Therefore, the search for genetic markers for pharmacogenetic and diagnostic purposes is a field of science in the current focus of biomedical basic research. The main problem currently is that few suitable markers are available for the above purposes.

The object of the present invention is, on the one hand, to make it possible to predict which patients are at high risk of being affected by diseases, in particular cardiovascular diseases such as cardiac arrhythmias or strokes, in particular ischemic stroke attacks, and on the other hand Moreover, the inherent principle of predicting the course and risk of disease in other diseases can be used. In addition to diseases from the cardiovascular area, all diseases in question whose formation, expression and course of a controlled or unregulated cell growth are decisive. dend, in particular diseases from the oncological, immunological and inflammatory spectrum. Based on the risk prediction possible in the context of the present invention and the understanding of the fundamental pathogenetic mechanisms, suitable patients or test persons for corresponding drug studies can be selected in order to make drug development more efficient and safer under a pharmacogenetic approach. In addition, the invention is suitable for the improved allocation of therapeutic resources.

The present invention is thus - according to a first aspect of the present invention - the use of a gene modification, in particular a polymorphism, in the located on chromosome 12 gene of the human cell division cycle-associated protein 3 (CDCA3 protein) for the diagnosis of diseases and / or to determine the risk of contracting a disease, and / or to predict individual drug effects and / or side effects according to claim 1. Further, according to the invention advantageous embodiments are the subject of the dependent use claims.

In other words, the subject matter of the invention is, inter alia, the use of a gene modification, in particular a polymorphism, in the gene of the human cell division cycle-associated protein 3 (CDC A3 protein) located on chromosome 12 for the production of a diagnostic agent for the diagnosis of diseases and / or for determining the risk of contracting a disease and / or for predicting individual drug effects and / or side effects according to claim 17.

Another object of the present invention is - according to a second aspect of the present invention - a method for determining the risk of subjects to contract a disease and / or for the prediction of individual drug effects and / or -nebenwirkun¬ gene according to claim 13. Other Advantageous embodiments of the method according to the invention which are advantageous according to the invention are the subject of the dependent method claims. - A -

Finally, the present invention is - according to a third aspect of the present invention - defined in claim 18 neducleotide nucleotide sequence, in particular in the chromosome 12 lokali¬ sierte gene of the human cell division cycle-associated protein 3 (CDC A3 - protein) and / or its promoter, which is characterized by a gene change, in particular a polymorphism, as defined in detail below.

The present invention is intended in particular to enable the detection of a genetic polymorphism with standard genetic-diagnostic methods for medical-diagnostic and pharmacogenetic purposes as well as for purposes of therapy.

The present invention relates on the one hand to the definition of the genetic modification per se, on the other hand to the description of the associated biological principle and finally to its successful application in investigations on humans.

In the context of the present invention, it was possible to show for the first time that genetic polymorphisms in the growth-regulating genes (apart from oncological applications, such as in oncogenes) have a significant effect on the course and expression of (folk) diseases, such as, for example, , As cardiovascular diseases have. The present invention allows the efficient genetic identification of individuals for diagnostic and pharmacogenetic purposes using routine diagnostic methods.

The present invention relates in particular to a method for diagnosis (ie more precisely the use of the aforementioned gene change for the diagnosis) or determination of the risk, the course and the expression of diseases as well as for the prediction of desired and undesired drug effects by gene analysis of the Cell growth regulie¬ generating genes, in particular by gene analysis of the CDCA3 protein codie¬ gene gene. The corresponding gene is synonymously named TOME-I (Trigger of Mitotlc Entry) or gene C-8 and is well known in the art. For this, the publications of Ayad NG, Rankin S, Muraka- mi M, Jebanathirajah J, Gygi S, Kirschner MW, "Tome-1, a trigger of mitotic entry, is degraded during Gl via the APC", Cell, 2003, Vol. 113, pp. 101 to 1 13; Ansari-Lari MA, Shen Y, Muzny DM, Lee W, Gibbs RA, "Large-scale sequencing in human chromosomes 12pl3: experimental and computational gene structure determination", Genome Res. 7, No. 3, 1997, pages 268 to 280 and Ansari-Lari MA, Muzny DM, Lu J, Lu F, Lilley CE, Spanos S., Malley T, Gibbs RA, "A gene-rich cluster between the CD4 and trisphosphate isomerase genes at human chromosomes 12p 13 ", Genome Res., 6 (4), 1996, pages 314 to 326, the entire disclosure of which is hereby incorporated by reference. The name CDC A3 was chosen by the curators of the human genome project and will be used in the following.

It has long been known that cells undergo a so-called cell cycle as part of their growth regulation, in which the phases G1, S, G2 and M are passed through in normally controlled form (FIGS. 1 to 10). Here, "S" denotes the synthesis phase during which reduplication of the DNA occurs, and "M" the mitotic phase, i. the actual division of the cells. "Gl" and "G2" mark phases of putative inactivity ("gap phases") during which substantial preparations are pending for the upcoming synthesis and partition phases. In addition, nonproliferating cells can remain in a G0 phase. It is also known that the passage from one to the next phase requires passing through so-called checkpoints (checkpoints). The basis for this is the controlled interaction of a complex biochemical control loop of numerous proteins, whereby according to current knowledge, inter alia, cyclins, cyclin-dependent kinases (cdk) and many other factors are involved. Mechanistically, phosphorylation and dephosphorylation processes as well as the formation and degradation of corresponding factors play a role. This means that the amount of protein present as a result of synthesis and degradation is critical to the control of such processes. The regulation of the degradation is often carried out via ubiquitin-dependent proteolysis.

As exemplified in FIG. 3, the active complex of cyclin-dependent kinase 1 (cdkϊ) and cyclin acts as a checkpoint for entry into the nucleus

Mitosis. Normally, cdkl is started by phosphorylation inactivated by the kinase weel (Figure 4). CDC A3 or TOME-I combines in the cell with the protein skpl to form a complex and can induce the partial synthesis of weel (FIG. 7). The lack of weel eliminates inactivation of cdkl and the cell can enter M phase (Figures 5 and 6). The presence of CDCA3 or TOME-I is therefore crucial for the controlled entry into the mitotic phase of cells. Fig. 10 ver¬ shows that the cellular CDCA3 concentration increases during the cell cycle. As the duration of the mitotic phase increases, the activity of another multiprotein complex in the cell, called the anaphase-promoting complex (APC), increases. APC mediates the polyubiquitinylation of proteins, which then undergo proteolytic degradation. The target proteins of the APC also include CDCA3 (Figures 2, 8 and 9). This shows that the end of the mitosis phase is due to the decrease in the cellular content of CDCA3. In the absence of the CDCA3 / sÄp / complex, weel can again contribute to the inhibition of cdkl. Thus, the cellular level of CDCA3 is decisively involved in the regulation of entry and end of the cellular mitosis phase. The functional elimination of CDCA3 leads to mitosis inhibition or to a strong slowing down of the mitosis phase. The original work of Ayad NG, Rankin S, Murakami M, Jebanathirajah J, Gygi S, Kirschner MW, "Tome-1, a Trigger of Mitotic Entry, Is Degraded during Gl via the APC" serves as a reference for these explanations. , Cell, 2003, 13, pages 101 to 13, the entire disclosure of which is hereby incorporated by reference. Reference is also made to the following conceptual comments, the entire disclosure of which is hereby incorporated by reference: Lim HH, Supra U, "Tome-1, weel, and the onset of mitosis: coupled destruction for minimal entry", Mol. Cell., 2003, 11, pp. 845-846 and Kraft C, "Mitotic entry: tipping the balance", Curr. Biol., 2003, 13, R445-446.

Data base analysis (GEO, National Center for Biotechnology Information) shows that CDCA3 - depending on the cell cycle - is found in almost all human and mouse and rat cells.

It can be seen from the above that the amount of CDCA3 in the cell is crucial for the beginning and end of the mitosis phase. The CDCA3 gene is found on the short arm of chromosome 12 and has 6 exons (Fig. 1). Like the transcription of all genes, that of CDCA3 is also controlled by a promoter. Changes in the promoter activity influence the transcription rate of the CDCA3 gene and thus the amount of protein - in equilibrium from synthesis and degradation - from the beginning to the end of the mitosis phase.

The invention thus also relates to a gene or nucleotide sequence change or a polymorphism in the CDCA3 gene, in particular in its promoter region, which influences the transcription rate of the CDC A3 protein. A schematic representation of the CDC A3 gene is shown in Fig. 1 to entneh¬ men.

It has now surprisingly been found that this gene modification in the CDCA3 gene, in particular in the promoter region of the CDCA3 gene, is suitable for the diagnosis of diseases. It is characterized in that the gene or nucleotide sequence of the human CDCA3 gene, in particular of the promoter of the human CDCA3 gene (CDCA3 promoter), of Proban¬ the gekenn in sequence listing 1 (Fig. 10) by arrow marking gekenn ¬ compared position with the reference sequence of human chromosome 12 compares and in the event that at this position a thymidine (T) instead of an adenine (A) is present assigns the subject an increased risk of disease or an altered response to drugs. Sequence Listing 1 (FIG. 10) shows a section of the gene or nucleotide sequence of the short arm of chromosome 12-corresponding to the definition of the orientation of chromosome 12-with a part of the CDCA3 gene or the CDCA3 promoter and the labeled one Site of the invention relevant morphism. A corresponding, alternative, synonymous representation as in Sequence Listing 1 according to Fig. 10 can be found in the attached so-called SEQUENCE LISTING, there in the represented SEQ. ID. No. 1, where this invention relevant polymorphism in position 164 is present.

With reference to the numbering of the nucleotide sequence of chromosome 12 in its definition according to the orientation of the assembly of the human genome {Human Genome Project) in version build 35.1 of 04 June 2004, the subjects are classified as having an increased risk of disease or an altered response to drugs if at position 6.831.154 there is a thymidine (T) instead of an adenine (A) (Figure 2).

Based on the definition of chromosome orientation derived from Kontig NTJ009759 Version 15 of August 20, 2004, the corresponding base substitution or polymorphism is located at position 6.815.154 of the nucleotide sequence (Figure 3 and Figure 4).

As such, the CDC A3 gene is read in the reverse direction with respect to the orientation of chromosome 12, so that the complementary strand is read relative to the orientation of chromosome 12. The CDCA3 gene thus has an opposite orientation to chromosome 12. Consequently, the invention is characterized-in other words-by the fact that the gene or nucleotide sequence of the human CDCA3 gene, in particular the promoter of the human CDCA3 gene (CDCA3 promoter), of probands of the type shown in Sequence Listing 2 (FIG. Fig. 11) indicated by arrow mark position with the reference sequence of the CDCA3 gene according to its orientation and compares in the event that at this position an adenine (A) instead of a thymidine (T), the subject an increased disease risk or a changed Assigning claims to medicinal products. The position indicated by arrow marking in sequence listing 2 according to FIG. 11 corresponds to or is the same as the appended so-called SEQUENCE LISTING, there in the represented SEQ. ID. No. 2 with the position or base position 141.

In other words, the subjects are assigned an increased risk of disease or an altered response to drugs, if in the gene or nucleotide sequence of the CDCA3 gene in orientation of the CDCA3 gene at position -776, based on the start codon (ATG) , an adenine (A) instead of a thymidine (T) (Sequence Listing 3 according to Fig. 12 and Fig. 5). Base position -776, relative to the start codon (ATG), according to sequence protocol 3 (FIG. 12) corresponds or is equivalent to the appended so-called SEQUENCE LISTING, there in the represented SEQ. ID. No. 3 with the position or base position 347. In addition, Sequence Listing 4 (FIG. 13) shows the complete CDCA3 gene in the chromosomal orientation, wherein the promoter region is shown in italics after prediction; the representation of sequence listing 4 according to FIG. 13 corresponds or is equivalent to the enclosed so-called SEQUENCE LISTING, there with the represented SEQ. ID. No. 4, where the previously described polymorphism at position or base position 3155 is localized. Sequence Listing 5 (Figure 14) shows the complementary sequence towards the CDCA3 gene; the representation of sequence listing 5 according to FIG. 14 corresponds or is equivalent to the appended so-called SEQUENCE LISTING, there with the represented SEQ. ID. No. 5, where the polymorphism in question is localized at position or base position 847.

About 50% of Caucasian individuals homozygous carry the wild-type (AA) of this gene change, 40% are heterozygous carriers of gene alteration and 10% homozygous carriers of the gene change in CDCA3 gene. The exact sequence description can be found in the sequence protocols 1 to 3 (FIGS. 10 to 12); these sequence protocols 1 to 3 according to FIGS. 10 to 12 correspond to the alternative, synonymous representations according to the attached so-called SEQUENCE LISTING, there SEQ. ID. Nos. 1 to 3.

Without wishing to be bound by theory, the base exchange affects the consensus sequence of the binding site of transcription factors, in particular as shown in FIG. Thus, the Applicant was able to show that in the case of variant A (adenine instead of thymidine), based on the orientation of the CDCA3 gene, the binding of the transcription factors CdxA and CF2-II are omitted (Figure 6). As a consequence of this influence on the binding sequence for certain transcription factors, the transcription rate of CDCA3 cDNA molecules is significantly influenced.

Figure 7 shows how, depending on the genotype, the amount of transcript of CDCA3 in human lymphoblasts behaves. Human B-lymphoblasts from healthy Caucasian donors served as starting cells. Detection was carried out by quantitative RT-PCR using specific oligonucleotide primers using standard methods known to the person skilled in the art. dardverfahren. These results indicate that depending on the genotype, the amount of CDCA3 transcript varies. The Applicant has now surprisingly been able to show that the genotypic formation of the CDC A3 protein-encoding gene makes it possible to draw conclusions about the risk of developing diseases, in particular ischemic diseases, such as ischemic stroke, and other cardiovascular diseases.

Cellular growth processes play an important role in the genesis, progression, severity and risk of many diseases. In addition, cellular proliferation is a crucial target for many drugs and other therapies that aim to either stimulate or inhibit cell growth. Such diseases and their specific therapies are, inter alia, cardiovascular diseases, immunological diseases, diseases in which inflammatory processes play an important role, diseases in which regeneration processes are involved, and oncological diseases In addition to influencing the risk of disease, the function of a disease-modifying gene or, with regard to therapy and pharmacogenetic implications, the function of a therapy-controlling gene is gene modification.

Examples of such diseases and their therapy are to be mentioned according to the invention: arteriosclerosis, wall thickening and changes of blood vessels, cardiac hypertrophy, cardiac insufficiency, arterial hypertension, stroke, especially ischemic stroke, heart remodeling processes, cardiac arrhythmias, atrial fibrillation, cardiovascular disorders ¬ illnesses;

Metabolic disorders, such as obesity and its consequences, in particular diabetes mellitus, insulin resistance, dyslipidemia, hyperuricemia and gout; - Immune diseases, such. Rheumatoid arthritis, autoimmune diseases, transplant rejection, excessive transfusion reactions as well as the specific and nonspecific defensibility against a variety of pathogens, such as viruses, bacteria, fungi and protozoa;

- Diseases in the formation and expression of inflammatory processes play an important role, including Wundheilungs¬ processes, fracture healing, pathogen-related inflammation, and the healing of various lesions in the human organism, eg. After myocardial infarction or after stroke; - Diseases where remodeling and remodeling processQ play an important role. Osteoporosis, myocardial hypertrophy, changes in the wall of blood vessels, the regeneration of parenchymatous organs (eg, the liver), but also remodeling in the brain, eg. As after lesions, or in the context of physiological see conversion and the formation of new synaptic connections.

A further use according to the invention of the gene modification is the prediction of response rates, dosage guidelines and side effects of medicaments which are used for the abovementioned disorders. On the other hand, the invention is suitable for predicting the effects of medicaments on medicaments whose therapeutic objectives, effects or undesired effects are an influence on cell proliferation and cellular remodeling and remodeling freezes. Examples which may be mentioned include: steroid hormones, their pharmaceutical derivatives and blockers and modulators of the signaling pathways influenced by them;

- cytostatics; - Medicines that influence the remodeling of cardiovascular systems sen, such. ACE inhibitors, ATI blockers, β-blockers, Cα antagonists, antiarrhythmics, diuretics;

- Immunopharmaceuticals;

- Medicines for the treatment of inflammatory processes, such. B. COX inhibitors. In the method according to the invention for determining the gene change, body material is taken from a subject and DNA is prepared therefrom as carrier of the genetic information. The structure of the CDC A3 promoter is determined from the isolated DNA and compared with the corresponding reference sequence at the corresponding site. Various methods known to those skilled in the art, which predominantly require PCR amplification, but can also do without PCR amplification, are suitable for determining the change in the sequence. Suitable detection methods are, for example: sequencing of the DNA strand; Methods based on a restriction length comparison; Processes in which DNA fragments are separated by mass spectroscopic methods; Pyrose-quencing method; specific methods for detecting DNA differences, such as Taqman PCR; selective hybridization methods and all other known and yet to be developed methods for the detection of sequence differences.

Alternatively, methods are also contemplated that directly the amount of CDCA3 protein in cells and tissues of humans z. For example, they can be measured with immunological methods in order to conclude the present genotype or to conclude that there is a present disease risk or to infer the individual drug response.

Alternatively, methods which directly determine the amount of CDC A3 transcripts in cells and tissues of humans by suitable molecular biological methods (eg quantitative RT-PCR, Northern blot, RNAse protection assay, various hybridization methods) are also suitable derive the genotype or to conclude on the present disease risk or to conclude the individual drug response.

Alternatively, methods which clone the promoter of CDCA3 and conclude by measuring the promoter activity on the genotype or close to a present disease risk or close to the individual drug response are also contemplated. In the context of the present invention, it is equally possible, for example with the aid of a preferably automated screening method, in particular a high-throughput screening method (HTS), to identify specific substances or pharmacologically active compounds which can be used for the targeted prophylaxis or therapy of disorders associated with Fehlregulati¬ on the CDC A3 protein nen, which in the context of this preferably automated screening system, a high throughput of samples is possible. Furthermore, the substances which can be used for targeted prophylaxis or therapy of disorders associated with a defective regulation of the CDCA3 protein or their corresponding precursors can be provided from a corresponding substance library (substance bank), which is in particular a protein protein. or gene library ("protein or gene bank"). Furthermore, within the scope of the present invention, a gene therapy approach for the targeted prophylaxis or therapy of disorders associated with a dysregulation of the CDC A3 protein is also possible.

Further embodiments, modifications, variations and advantages of the present invention will be readily apparent to those skilled in the art upon reading the description without departing from the scope of the present invention.

The present invention will be illustrated by the following embodiments, which by no means limit the present invention.

EXAMPLES

Example 1: Stroke is one of the most common human cardiovascular diseases in the Western world. Important causes are the arterial hypertension and arteriosclerotic remodeling processes with in part inflammatory processes of vessel walls. Another cause of stroke is cardioembolic processes, i. H. Blood clots from the heart are "hurled" and can close blood vessels in the head area. Cardioembolic strokes are therefore distinguished from noncardioembolic, i. predominantly arteriosclerotic strokes. In one study, 494 stroke patients were genotyped at the CDC A3 promoter locus and compared to the genotypes of 100 healthy blood donors. With regard to the healthy blood donors, carriers of the CDCA3 promoter T allele had a 1.3 times higher risk of developing a noncardioembolic stroke. Homozygous carriers of the T allele suffered strokes about 8 years earlier than heterozygous carriers of the T allele and homozygous carriers of the wild-type A allele. After evaluation of the data using a Cox Proprotional Hazard Model, the age-related risk of the homozygous T-allele carriers was twice as high as that of the heterozygous carriers or the homozygous carriers of the A-allele. In Fig. 8 the corresponding results are graphically represented (there representation of the relative risk as reciprocal, that is, homozygous carriers of the A allele, have only half the risk as homozygous carriers of the T allele). It is clear from these results that the determination of the CDCA3 promoter polymorphism is suitable for the risk prediction of contracting a non-cardioembolic stroke. Based on such a prediction, targeted drug and non-drug preventive studies can be planned.

A related study is shown in Figures 11 and 12, in which 474 patients with ischemic stroke have been studied. The {pdd ratios} shown in Figure 12 show the relative risk for the respective factors (age: age, diab: diabetes, TOME-I: corresponds to CDC A3 protein, hypjip: elevated blood lipid levels, hyperion: hypertension, sex: sex ). There the study was carried out sexually or age-specifically, there are statistically insignificant values for the corresponding factors (age, sex).

Example 2:

In a second study, the frequency of CDCA3 promoter polymorphism alleles in patients with atrial fibrillation was analyzed in comparison to healthy volunteers. Atrial fibrillation is the most common cardiac arrhythmia in humans. In addition to general heart disease, genetic factors play an important role. In addition to electrophysiological remodeling, structural remodeling in the atria also plays an important role in the genesis of the disease. Common therapies for atrial fibrillation and, in particular, therapies to stabilize normal heart rhythms are performed according to the principles of trial and error. A pharmacogenetic prediction or classification of atrial fibrillation according to etiological and genetic aspects is of great importance.

From FIG. 9 it can be seen that homozygous and heterozygous carriers of the T allele suffer from atrial fibrillation less frequently than homozygous carriers of the A allele, that is to say they have a relative protection against the occurrence of the disease.

Example 3:

The T / A polymorphism of the CDCA3 gene, particularly the CDCA3 promoter, was demonstrated by restriction fragment length analysis. Here, the corresponding region with the primers 5'-GATGGTTTTCCTGAGGCGAGCGATAG-S 'and

5'-GGTCGGAAAGTCGAGGGACAGTTGTCTTGGTATTCG-S amplified using the PCR technique. The result is a 140 bp PCR fragment. This is digested with the restriction enzyme ex. In the case of the wild-type genotype AA (based on the chromosomal nomenclature), the fragment remains undigested (140 bp). In the case of carriers of the homozygous variant (TT), two fragments of 104 and 36 bp are formed. In the heterozygous carriers, a mixed image of fragments 140, 104 and 36 is formed. The method was validated by comparison with sequenced samples.

Claims

claims:

1. Use of a gene modification, in particular polymorphism, in the gene of the human cell division cycle-associated protein 3 (CDC A3 protein) located on chromosome 12 for the diagnosis of

Illnesses and / or to determine the risk of developing a disease, and / or for the prediction of individual drug effects and / or side effects.

2. Use according to claim 1, characterized in that the genetic modification, in particular the polymorphism, is located in the promoter region of the gene coding for the human cell division cycle-associated protein 3 (CDCA3 protein) gene.

3. Use according to claim 1 or 2, characterized in that the gene modification, in particular the polymorphism, an exchange of the base adenine by the base thymidine, based on the definition of the orientation of chromosome 12, comprises (Sequence Listing 1, Fig. 10 and / or SEQUENCE LISTING SEQ ID NO: 1).

4. Use according to one of the preceding claims, characterized ge indicates that the gene modification, in particular the polymorphism, is located at the ge in Sequence Listing 1 (Fig. 10) marked by arrow marking position and / or that the gene modification, in particular the polymorphism, at base position 6,831,154 of

Chromosome 12, based on the assembly of the human genome in the build version 35.1 of June 4, 2004, is localized and / or that the gene change, in particular the polymorphism, at Basenpositi¬ on 6.815.154 of chromosome 12, based on version Kontig NT_009759 from August 20, 2004, and / or that the gene modification, in particular the polymorphism, in position 164 of SEQ. ID. No. 1 is localized according to SEQUENCE LISTING. 5. Use according to one of the preceding claims, characterized ge indicates that the gene change, in particular the polymorphism, is located at the ge in Sequence Listing 2 (Fig. 11) marked by arrow marking position and / or that the gene modification, in particular the polymorphism is located at base position -776 relative to the start codon of the CDCA3 gene (Sequence Listing 3 according to Fig. 12 and Fig.

5) and / or that the gene modification, in particular the polymorphism, at base position 141 of SEQ. ID. No. 2 is localized according to SEQUENCE LISTING and / or that the gene alteration, in particular the polymorphism, at base position 347 of

SEQ. ID. No. 3 is located according to SEQUENCE LISTING.

6. Use according to one of the preceding claims, characterized ge indicates that the disease is associated with the misregulation and / or mis-expression of the CDC A3 protein.

7. Use according to one of the preceding claims, characterized ge indicates that the disease is a cardiovascular disease.

8. Use according to claim 6, characterized in that the kardio¬ vascular disease is an ischemic disease, in particular an ischemic stroke, and / or a heart disease, in particular an atrial fibrillation and / or a cardiac arrhythmia.

9. Use according to one of the preceding claims, characterized ge indicates that subjects who have the gene modification, as defined in the preceding claims, in particular the polymorphism, in their genome, have an increased risk of disease, in particular as defined in claims 6 to 8, to fall ill.

10. Use according to one of the preceding claims, characterized ge indicates that the amount of CDCA3 protein and / or its Vor¬ runners in cells and / or tissues of the human body is quantified in order thereby to the diseases and / or the risk of illness, and / or to conclude individual drug effects and / or side effects.

1 1. Use according to one of the preceding claims, character- ized in that the gene activity, in particular the promoter activity, of the CDCA3 gene is quantified, thereby to the diseases and / or the risk of developing a disease, and / or to the individual drug effects and / or side effects.

12. Use according to one of the preceding claims, characterized gekenn¬ characterized in that the amount of CDCA3 protein and / or its precursor in cells and / or tissues of the human body and / or the gene activity, in particular the promoter activity, of the CDC A3 gene quantified in order to be able to infer the genotype of the gene change, in particular of the polymorphism, as defined in the preceding claims.

13. Method for determining the risk of a subject suffering from a disease, in particular as defined in claims 6 to 8, and / or for predicting individual drug effects and / or side effects in a subject, characterized gekennzeich¬ net, that at least the position of the CDC A3 gene of the subject indicated in sequence listing 1 and / or 2 (Fig. 10 and / or Fig. 1 1) is analyzed by arrow marking and / or that at least the

Position 164 of SEQ. ID. No. 1 and / or position 141 of SEQ. ID. No. 2 is analyzed in accordance with SEQUENCE LISTING of the CDC A3 gene of the subject, in particular wherein, in the event that a thymidine, based on the definition of the orientation of chromosome 12, is present at this position instead of an adenine, the subject has an increased level Associated with the disease risk and / or the subject ver¬ altered drug effects and / or side effects are assigned.

14. The method according to claim 13, characterized in that the Proban¬ the body material, in particular cell and / or tissue material, ent taken.

15. The method according to claim 13 or 14, characterized in that the DNA to be examined from the body material, in particular ZeIl- and / or tissue material, isolated and then sequenced.

16. Method for determining a gene change, in particular a polymorphism, in the gene of the human cell division cycle-associated protein 3 (CDCA3 protein) localized on chromosome 12, characterized in that at least those in sequence listing 1 (FIG. and / or in Sequence Listing 2 (FIG. 11) an arrowheaded position of the CDC A3 gene for the presence of a gene change, in particular a polymorphism, in particular as defined in the claims, and / or that at At least position 164 of SEQ. ID. No. 1 and / or position 141 of SEQ. ID. No. 2 according to SEQUENCE LISTING of the CDCA3 gene for the presence of a gene alteration, in particular a polymorphism, in particular as defined in the preceding claims.

17. Use of a gene modification, in particular polymorphism, in the gene of the human cell division cycle-associated protein 3 (CDC A3 protein) located on chromosome 12 for the production of a diagnostic agent for the diagnosis of diseases and / or for detection the risk of contracting a disease, and / or predicting individual drug effects and / or side effects, in particular as defined in any one of claims 1 to 12.

18. Nucleotide sequence, in particular a gene localized in chromosome 12 on the gene of the human cell division cycle-associated protein 3 (CDCA3 protein) and / or its promoter, characterized by a gene modification, in particular a polymorphism, as defined in the claims ,