EP1751308A2 - Use of a genetic modification in the human gnaq gene for predicting risk of disease, the course of a disease, and reaction to treatments - Google Patents

Abstract

In order to determine the risk of disease, the course of a disease, the action of medicaments, the side-effects of medicaments and drug targets, a base substitution is identified in the non-translated region 5' of the gene for the G alpha q sub-unit of human G proteins, preferably the presence of two or three of the polymorphisms GC(-909/-908)TT, G(-382)A or G(-387)A being detected.

Description

Use of a gene modification in the human GNAQ gene to predict disease risks, disease courses and to predict the response to disease therapies

TECHNICAL FIELD TO WHICH THE INVENTION RELATES

The invention relates to methods for determining the presence of various polymorphisms in the human Gαq gene (GNAQ) for predicting disease risks, disease courses and selecting individually suitable therapy methods.

All cells of the human body have membrane receptors on their surface that control all cell functions. Such receptors include the so-called heptahelical receptors for hormones, neurotransmitters and che okine. There are also a variety of receptors for growth factors and receptors with intrinsic tyrosine kinase activity, for example receptors for insulin, insulin-like growth factor, epidermal growth factor, platelet-derived growth factor and many more. Furthermore, there are many receptors that are responsible for regulating blood formation, such as the receptor for erythropoietin. Cell receptors, motility, gene expression, apoptosis and chemotaxis are controlled via such receptors. The receptors mentioned transmit their signals into the cell interior via the activation of so-called heterotrimeric G proteins. These G proteins consist of a large family of different isoforms and are each composed of different α, β and γ subunits. At present 5 β subunits, 13 γ subunits and more than 20 α subunits are known which are encoded by different genes (Farfel et al., 1999). The combination of these different α, β and γ subunits creates a large number of different heterotrimeric G proteins.

The isoform combination determines which heterotrimer can be activated by a specific receptor. The ßγ subunits are to be considered functionally as a monomer. At rest, the α subunit bound GDP (Figure 1). After activating a coupling receptor, the α subunit releases GDP in exchange for GTP and the β subunits dissociate from the α subunits. Both the free a and the ßγ subunits can control the activity of a variety of different effectors. These include, for example, ion channels, adenylyl cyclase, PI3 kinase, different MAP kinases etc. The α subunits have an intrinsic GTPase activity which hydrolyzes the GTP bound after activation to GDP. Subsequently, the released β-subunits reassociate with the α-subunit, thereby ending the activation cycle. The heterotrimer is thus available for a renewed activation cycle (Bourne, 1997). A scheme of the G protein cycle is shown in Figure 1. The activation of such G proteins is the crucial step for cell activation. Due to the paramount importance of G proteins, it is immediately obvious that mutations or genetic polymorphisms in genes coding for G proteins must have a lasting influence on the activatability of cells if these mutations influence the function or expression of G protein subunits. This also has a decisive influence on the risk of illness or the course of illness. In addition, the response to the therapy of diseases, be it through pharmaceuticals or other measures such as radiation, diets, operations, invasive interventions, etc. depends on the activability of G proteins.

Meaning of the Gαq subunit

The Gαq subunit is expressed in all human body cells. Their stimulation leads, among other things, to the activation of phospholipase C and thus to an increase in the intracellular Ca ⁺ concentration (Figure 1). So z. B. Ca ²⁺ -dependent processes can be activated. Gαq can also regulate the activity of ion channels, for example potassium or calcium channels. Almost all known receptors couple to Gαq, for example the receptors for acetylcholine, adenosine, adrenaline, angiotensin, bradykinin, endothelin, histamine, noradrenaline, P2-purinergic receptors, opioids, dopamine, epidermal growth factor, FSH, VIP, thyroliberin, glucagon , Vasopressin, histamine and many more. After stimulation Gαq-coupled receptors, apoptosis is induced in many cell types, so ^'is that a relationship to tumor diseases and their progression and response to therapy, but also with inflammatory and immunological diseases and their progression and treatment response is obtained. In addition, a variety of metabolic pathways are regulated by Gαq. Changes in expression of Gαq (overexpression or lack of expression) lead to a number of disease states or phenotypes in animal experiments or at the cellular level:

1. Overexpression of Gαq in the heart leads to hypertrophy, heart failure and apotosis;

2. Constitutively active Gαq induces apoptosis via the protein kinase C pathway;

3. Knockout of Gαq inhibits platelet aggregation, leads to ataxia and disrupts motor coordination

4. Knockout of Gαq leads to obesity and Gαq is involved in the signal transduction of insulin;

5. Constitutively active Gαq subunits are oncogenic (De et al., 1992) and are involved in the regulation of glucose metabolism.

These few examples already show that function-changing mutations in Gαq or under- or overexpression of the protein can also lead to different diseases and / or functional disorders in humans.

SUMMARY OF THE INVENTION

The object on which the invention is based is to find polymorphisms and to clarify their physiological or pathophysiological significance. Therefore a. to provide function-changing genomic polymorphisms and haplotypes in the GNAQ gene which either lead to an amino acid exchange, or b. that affect the splicing behavior, or c. which lead to a change in the protein expression or to a change in the expression of splice variants, or d. which are suitable for finding and / or validating further polymorphisms or haplotypes in the GNAQ gene. e. To provide nucleotide exchanges and haplotypes that are suitable to generally predict disease risks and courses f. To provide nucleotide exchanges and haplotypes that are suitable for generally predicting drug response and side effects. G. To provide nucleotide exchanges and haplotypes that are suitable to generally predict the effects of other forms of therapy (radiation; heat, heat, cold, movement, etc.)

Because of the fundamental importance of Gαq for signal transduction, such polymorphisms or haplotypes are suitable for generally predicting disease risks or disease courses in all diseases or for predicting therapy response / therapy failure or undesirable side effects for all pharmaceuticals or non-pharmacological therapies.

This object is achieved by a method for the determination of disease risk, progression of a disease, the effects of drugs, drug side effects and drug targets which ^'is connected to a base substitution in the gene GNAQ, the subunit Gαq encoding the human G proteins, in which is in the 5 'untranslated region of the gene for the Gαq subunit human G proteins identified a base exchange (polymorphism).

Another object of the invention is a genetic test containing a probe for identifying one or more of the polymorphisms in the 5 'untranslated region of the GNAQ gene.

DESCRIPTION OF THE FIGURES

Figure 1 - The Gαq signaling pathway. The diagram shows how the Gαq pathway after receptor stimulation is linked to a variety of signal transduction components, including ion channels, transcription factors and the synthesis of eicosanodes. PLC, phospholipase C; IP3, inositol triphosphate; PKC, protein kinase C; PLA2, phospholipase A2; AA, arachidonic acid; MLCK, myosin light chain kinase; CaM, calmodulin; p42 / p44; p42 and p44 MAP kinase.

Figure 2 - Intron / exon structure of human GNAQ and position of GC (-909 / -908) TT polymorphism (not to scale).

Figure 3 - Putative binding sites for transcription factors in the promoter of the GNAQ gene; The numbers on the right represent the reference to. ATG, the numbers on the left refer to the transcription start point.

Figure 4 - Results of the Electrophoretic Mobility Shift Assay (EMSA) with constructs containing the GC or the TT genotype in the GNAQ promoter. After adding nuclear extract, an increased binding can be seen of core protein and the "GC construct", which has a further binding site for the transcription factor SP-1. The binding is specifically inhibited by an anti-SP-1 antibody or in the presence of a displacing SP-1 oligonucleotide.

Figure 5 - Constructs for measuring promoter activity using secreted alkaline phosphatase (SEAP). The constructs used for the reporter assay are described in the left part of the figure. The right part of the figure shows the SEAP activity measured 24 hours after transfection of HEK cells and smooth muscle cells of the rat aorta (A-10).

Figure 6 - Genotype-dependent activity of the GNAQ promoter. The promoter construct -798 / + 89, each with the GC or the TT genotype, was transfected in HEK cells. The secretion of alkaline phosphatase was increased after stimulation with serum or angiotensin. One recognizes an increased activation of the promoter with the GC genotype.

Figure 7 - Expression of GNAQ mRNA in tissue depending on the GC (-909 / -908) TT polymorphism. The quotient Gαq / β-actin mRNA is shown.

Figure 8 - Protein / DNA ratio in the human heart in atrial fibrillation (VF) and sinus rhythm (SR) and dependence of the protein / DNA ratio on GC (-909 / - 908) TT polymorphism.

Figure 9 - GNAQ GC (-909 / -908) TT polymorphism and Ca ²⁺ increases in skin fibroblasts after stimulation with bradykinin. You can see higher increases in the cytoplasmic table-free Ca ²⁺ concentration in cells from subjects with at least one GC allele.

Figure 10 - GNAQ GC (-909 / -908) TT polymorphism and circulatory parameters in healthy people. The stroke volume of the heart (left) and total peripheral resistance (right) are shown depending on the genotype.

Figure 11 - GNAQ GC (-909 / -908) TT polymorphism and disease progression in patients with chronic heart failure. The time from the initial diagnosis to the heart transplant is shown as a measure of the disease progression. One recognizes the more favorable course of the disease with the GC / GC genotype.

Figure 12 - Expression of GNAQ mRNA in adipose tissue depending on the GC (-909 / -908) TT polymorphism. The quotient Gαq / β-actin mRNA is shown.

Figure 13 - Inhibitory effect of insulin on isoprenaline-induced lipolysis depending on the GC (-909 / -908) TT polymorphism. The glycerol release is shown as a marker for lipolysis.

Figure 14 - BMI, HOMA-IR and insulin concentration in PCOS patients depending on the GC (-909 / -908) TT polymorphism.

Figure 15 - GNAQ GC (-909 / -908) TT polymorphism and serum cholesterol in healthy individuals.

Figure 16 - GNAQ GC (-909 / -908) TT polymorphism and disease progression in patients with CLL. The time from the initial diagnosis to the start of therapy is shown as a measure of disease progression. One recognizes the more favorable course of the disease with the TT / TT genotype.

Figure 17A - GNAQ GC (-909 / -908) TT polymorphism and time to metastasis in patients with bladder cancer.

Figure 17B - GNAQ GC (-909 / -908) TT polymorphism and time to tumor progression in patients with bladder cancer.

Figure 17C - GNAQ GC (-909 / -908) TT polymorphism and survival in patients with bladder cancer.

Figure 18 - GNAQ GC (-909 / -908) TT polymorphism vasoconstriction after injection of noradrenaline, angiotensin, or endothelin into the skin. The change in skin circulation following the injection is shown.

DETAILED DESCRIPTION OF THE INVENTION

In the method according to the invention, one or more polymorphisms are identified in the 5 'untranslated region of the GNAQ gene. Polymorphisms which are preferably identified by the method according to the invention include a GC (-909 / -908) TT, a G (-382) A or a G (-387) A polymorphism or two or three of these polymorphisms.

According to a preferred embodiment, the genetic test according to the invention contains one or more probes for identifying one or more of the polymorphisms GC (-909 / -908) TT, G (-382) A or G (-387) A in the 5 'untranslated region of the GNAQ gene.

The human Gαq gene (GNAQ) is located on chromosome 9q21 (GenBank Accession number NM_002072, Fig. 2). An essential element of the invention is the finding of the gene polymorphisms GC (-909 / -908) TT, G (-382) A and G (-387) A located in front of exon 1 in the promoter of the gene, which are obtained by systematic sequencing of DNA Samples of people have been found. For this purpose, gene sequences which are located before exon 1 of GNAQ were amplified by means of a PCR reaction and sequenced according to the Sanger method. The PCR reactions can be carried out according to the so-called slowdown method and the corresponding PCR and / or sequencing primer according to a method described in Bachmann et al. , Pharmacogenetics 13 (12) 759-766, December 2003 algorithm described and established. Reference is made to the disclosure of this publication for the purposes of the invention.

The polymorphisms found according to the invention show a substitution of guanine by thymine (G-909T) at position -909 and at the same time a substitution of cytosine by thymine at position -908 (C-908T). The exchanges G (-909) T and C (- 908) T always occur simultaneously, so that the genotypes TT / TT, GC / GC and TT / GC result. In the G (-382) A polymorphism at position -382 there is a substitution of guanine by adenine, in the G (-387) A polymorphism there is a substitution of guanine by adenine at position -387. The corresponding partial sequences are accordingly for the GC (-909 / -908) TT polymorphism: GGTGCGGGAG CAGTAGGCGT CCGCAGAGCC CGCGGGGGCC GGCCCAGCCC -

901 and

GGTGCGGGAG CAGTAGGCGT CCGCAGAGCC CGCGGGGGCC GTTCCAGCCC -

901

The partial sequences for the G (-382) A and G (-387) A polymorphisms are:

GTAGGGGAGC CTCGCAGGCG GCGGCGGCGG -361 and

GTAAGGGAÄC CTCGCAGGCG GCGGCGGCGG -361

These SNPs are numbered in such a way that the nucleotide A of the start codon ATG is assigned the number +1. Since the number 0 does not exist in accordance with the convention, the number -1 is assigned to the nucleotide located in front of the A of the start codon ATG.

The detection of these SNPs in the sense of their use according to the invention can be demonstrated using any method known to the person skilled in the art, for example direct sequencing, PCR with subsequent restriction analysis, reverse hybridization, dot blot or slot blot method, mass spectrometry, Taqman®- or Light-Cycler® technology, Pyrosequencing®. Invader® technology, Luminex method, etc. Furthermore, these gene polymorphisms can be detected simultaneously after multiplex PCR and hybridization on a DNA chip. Distribution of the GC (-909 / -908) TT, G (-382) A and G (-387) A polymorphisms among different ethnic groups and use of these genotypes to find other relevant polymorphisms and haplotypes.

For this purpose, different DNA samples from Caucasians, Black Africans and Chinese were genotyped. The result is shown in the following table

This genotype distribution is highly significantly different in the Chi test with a Chi 86.1 and a P <0.0001. The GC genotype is most common in black Africans.

From this distribution it can be concluded that the GC (-909 / - 908) represents the "original state" in terms of development history (based on Caucasians). Such differences in genotype distribution among different ethnic groups usually indicate that associated phenotypes are important for evolution and it brought the carriers a certain advantage. It is known to the person skilled in the art that ethnically different genotype distributions are an indication that certain genotype and haplotypes with certain diseases or physiological chemical and pathophysiological responses or response to therapy, e.g. associated with pharmaceuticals.

This genotype distribution is highly significantly different in the chi ² test between Caucasians and Chinese with a Chi 18.30 and a P <0.001. The G (-382) allele frequency (% G) is highest in Chinese, followed by Black Africans and Caucasians.

This genotype distribution is significantly different in the chi ² test between Caucasians and Chinese with a chi 7.7 and a P <0.01. The G (-387) allele frequency (% G) is highest in Chinese, followed by Black Africans and Caucasians. Another analysis shows a coupling imbalance between the three polymorphisms in Caucasians. Coupling imbalance is the occurrence of allele combinations (haplotypes) that statistically clearly occur more frequently or less frequently than would be expected in relation to their frequency.

The following ^'table shows for Caucasians, the distribution of G (-387) A stratified by genotype G (-382) A genotypes.

There is no statistically significant connection between these polymorphisms. There is no coupling imbalance between the G (-382) A and the G (-387) SNP. The following table shows the distribution of G (-382) A genotypes stratified for GC (- 909 / -908) TT genotypes.

Coupling analyzes show a significant coupling imbalance between the GC (-909 / -908) TT and the G (- 382) A SNP (Chi-Square = 18.3; p = 0.01). The following table shows the distribution of G (-387) A genotypes stratified for GC (-909 / -908) TT genotypes.

Coupling analyzes show a significant coupling imbalance between the GC (-909 / -908) TT and the G (- 387) A SNP (Chi-Square = 14.1; p = 0.01). In summary, there is a significant coupling imbalance between the GC (-909 / -908) TT with both the G (-382) A and the G (-387) A SNP. In contrast, the G (-382) A and the G (-387) A SNP are inherited independently of one another.

Another object of the invention is that these newly found polymorphisms can be used to detect and validate further relevant genomic gene changes in GNAQ or neighboring genes, which are, for example, in coupling imbalance with genotypes in the GNAQ gene. These can also be genes that are also located on chromosome 9, but at a great distance from the GNAQ gene. To do this, proceed as follows: 1. For certain phenotypes (cellular properties, disease states, disease courses, drug responses, etc.), an association with the polymorphisms GC (-909 / -908) TT or the G (- 387) A or the G (-382) A is first established ,

2. For newly detected gene changes in GNAQ or neighboring genes, it is examined whether existing associations are strengthened or weakened using the genotypes or haplotypes described above.

Functional importance of the GC (-909 / -908) T polymorphism

It was examined which functional changes gene changes in the gene GNAQ can be assigned. A correlation to alternative splicing, tissue-specific expression or an overexpression of the Gαq protein depending on genotypes of the GC (-909 / -908) TT polymorphism are conceivable here, for example. For this purpose, a computer program was first used to investigate whether the nucleotide changes found could influence the binding of transcription factors. Transcription factors bind to specific consensus sequences and can increase or decrease the promoter activity, so that an increased or decreased transcription of the gene results and thus the expression level of the encoded protein is increased or decreased. As shown in Figure 3, the GC is (-909 / -908) polymorphism in TT ^'of a consensus sequence of the binding site for the transcription factor SP-1, its binding ability may be affected by the polymorphism. This impairment relates to the genotype (-909 / -908) TT. The appearance of this genotype leads to the loss of an SP-1 binding site to its consensus sequence: GGGGCGGGGC. A so-called EMSA (electrophoretic mobility shift assay) is carried out to experimentally investigate this effect. In this experiment, short nucleic acid sections that contain the polymorphism are incubated with cell nucleus extracts. Transcription factor proteins that are in these extracts now bind to the nucleic acid sections with different intensities. The binding to the DNA is finally made visible in the X-ray film. A strong bond results in an intense bond. Figure 4 shows the result of this experiment with specific constructs containing either the TT or the GC genotype. The stronger intensity of the GC construct band shows a stronger binding of a transcription factor to this region. The disappearance of the band by adding an SP-1 antibody and the displacement of the binding by a commercial SP-1 oligonucleotide shows that the binding transcription factor is SP-1.

For the functional detection of a changed promoter activity depending on certain genotypes, different fragments of the promoter with the GC or with the TT genotype were cloned into the vector pSEAP in order to quantify the promoter activity by means of a so-called "reporter assay" after expression of the vector in HEK cells (Figure 5) For this purpose, the constructs are cloned in front of a gene which codes for secreted alkaline phosphatase (SEAP) .If the construct has a promoter activity, the SEAP gene is increasingly transcribed and the increased secretion of alkaline phosphatase into the cell culture medium can be measured. As Figure 5 shows, construct -798 / + 89 exhibits the highest opened (the numbers refer to the transcription start point, which is -214 in the GNAQ gene). Since the polymorphism is in this region (-694 / -695 relative to the transcription start point), which shows the highest reporter activity and also a transcription factor binding site is influenced by the nucleic acid exchange, it has now been investigated whether stimulation of HEK cells unites Influences the reporter activity of these constructs. For this purpose, the construct -789 / + 89 with the polymorphisms GC (- 909 / -908) and TT (-909 / -908) was transfected into HEK cells and the cells were stimulated with serum and angiotensin II (Figure 6) , In constructs with the GC genotype, stimulation with serum or 10 nM angiotensin II leads to a significantly (p <0.05) 2 to 4-fold increased promoter activity compared to the TT genotype. The GC polymorphism in the promoter of the GNAQ gene thus means that the promoter activity is increased and, accordingly, the Gαq protein is increasingly expressed. In order to check whether this regulation also takes place in vivo, the expression of Gαq was examined at the mRNA level by means of real-time PCR in cardiac tissue.

For this purpose, mRNA was obtained from human surgical tissue during cardiac surgery and was transcribed into cDNA using reverse transcriptase. The process is familiar to the person skilled in the art. The expression level was subsequently determined using real-time PCR (Taqman method) and compared with the expression level of the housekeeping gene β-actin. The results are shown in Figure 7. The GCGC genotype leads to an increase in Gαq transcription of at least 25% compared to the T allele. To further investigate whether GC (-909 / -908) TT polymorphism can be involved in the development of cardiac hypertrophy, the protein content was determined in comparison to the DNA content of heart samples from patients with chronic atrial fibrillation and patients with sinus rhythm. Fig. 8 shows that the relative cellular protein content in samples from patients with atrial fibrillation is increased compared to samples from patients in the sinus rhythm. Since the occurrence of cardiac hypertrophy in patients with chronic atrial fibrillation is higher than in patients with sinus rhythm, the protein / DNA index represents a marker for cardiac hypertrophy. Fig. 8 shows the protein / DNA index, divided according to the GC (-909 / - 908) TT polymorphism. This figure shows that both samples from patients with chronic atrial fibrillation and samples from patients with sinus rhythm have the highest protein / DNA index in GC / GC genotypes.

It was thus proven that there are gene changes in the GNAQ gene which, on the one hand, change the expression of

Gαq in the heart tissue and on the other hand can lead to a change in the amount of protein in the heart. This can be the GC (-909 / -908) TT polymorphism or polymorphisms that are in coupling imbalance with them (eg the G (-382) A or the G (-387) A polymorphism). Part of the invention described here is therefore also to quantify the expression of Gαq at the mRNA level or protein level, to associate it with known polymorphisms of the GNAQ and to discover and validate new, even more suitable polymorphisms. The results shown here of a genotype-dependent expression of Gαq and a changed total amount of protein in human hearts is extremely important. In transgenic Animals overexpressing Gαq leads to increased apoptosis of cardiac myocytes as the cause of heart failure (Adams and Brown, 2001; Mende et al., 1998). The same effects can therefore also be expected in people who overexpress the Gαq protein due to a gene change in GNAQ. Here we expect an increased cardiovascular risk. This includes an increased risk of obesity, hypertension, stroke, coronary artery disease, myocardial infarction, pre-eclampsia etc. We also expect a changed response to substances whose receptors activate Gαq or substances which indirectly induce agonists whose effects are mediated via Gαq. The changed tendency to apoptosis can influence a variety of disease courses (e.g. tumor and immune diseases) and determine the response to pharmaceuticals.

Furthermore, the overexpression of Gαq when carrying the GC allele leads to a consecutively increased signal transduction after stimulation of cells with agonists whose signal transduction comprises the Gαq protein. The detection was carried out in human skin fibroblasts loaded with the Ca ²⁺ indicator Fura-2 after stimulation with the hormone bredykinin, in whose effect Gq is known to be involved (Fig. 9). It can clearly be seen that the extent of the increase in the intracellular Ca ²⁺ concentration increases with an increasing number of GC alleles. In principle, the detection of gene changes in the GNAQ gene is suitable for predicting the strength of the activation of Gq and thus the efficiency of signal transduction via Gq-coupled receptors. Use of a gene modification in GNAQ to predict disease risks and disease courses

Because of the key function of the Gαq subunit for cell activation, it is an essential part of the invention that disease changes and disease courses can be generally predicted using gene changes in GNAQ. Human heterotrimeric G proteins are composed of the subunits α, β and γ. A number of isoforms are in turn known which are encoded by different genes. For example, there are 13 different γ-isoforms (γl - γl3), at least 5 different ß-isoforms (ßl- ß5) and a variety of different α-isoforms (αs (short and long), αo, αil-3, αq, αll- 16, αolf etc.) Since G proteins are known to play a central role in controlling the function of all human cells, regardless of which cell receptors are activated, it can be expected that the course of diverse and very different diseases in a genetically determined, increased activability of G proteins is influenced. Especially with the diverse functions of G-proteins, function-changing mutations become particularly important and predictive. This is in contrast to mutations in other genes which are responsible for other proteins, e.g. Encode hormones or hormone receptors.

This means that gene changes in proteins that are expressed in all human body cells and concentrate incoming hormone signals at a central point and thereby regulate cell functions, determine all physiological and pathophysiological processes. be influenced or at least modulated. In addition, responses to pharmaceuticals are influenced in a special way. This affects both undesirable and undesirable drug effects.

It has been repeatedly postulated in the scientific literature that changes in the function of G proteins have a lasting influence on diverse diseases or on the course of various diseases. Such gene changes can be structure-changing mutations in G-protein subunits which, for example, alter the activatability by a receptor, affect the enzymatic GTPase activity or influence the dimerization of β-subunits. In addition, such changes could change the composition of heterrotrimeric G proteins. Furthermore, the expression level of such G-protein subunits can be changed or splice variants with changed function can occur (Farfel et al., 1999; Iiri and Bourne, 1998; Iiri et al., 1998; Spiegel, 1999; Spiegel, 1997; Spiegel, 1996).

The following can be seen from the examples given above:

1. Genetic changes in genes that code for ubiquitously expressed proteins influence a variety of diseases or cause a variety of disease risks.

2. G proteins control almost all signal transduction processes in the human body.

3. While it is clear from the literature cited that it can generally be assumed that mutations and polymorphisms in genes which code for G proteins cause such diseases a connection with genomic mutations in the GNAQ gene for the Gαq subunit of heterotrimeric G proteins with disease risks was neither described nor assumed in the literature.

The following can be mentioned as diseases which are associated with a gene change in GNAQ and which are determined, for example, by a changed expression level of Gαq protein:

1. Cardiovascular diseases. These include, in particular, hypertension, stroke, coronary heart disease and myocardial infarction, heart failure, cardiac arrhythmia, preeclampsia or gestosis. 2. Enocrinological and metabolic diseases. These include, in particular, obesity, metabolic syndrome, type 2 diabetes mellitus, gout, osteoporosis, thyroid diseases such as hyper- and hypothyroidism and M.Basedow, hyper- and hypoparathyroidism, Cushing's disease, hyper- and hypoaldosteronism and many others more; 3. Psychiatric disorders such as depression, schizophrenia, alcoholism and anxiety disorders, phobias, neuroses; 4. Neurological diseases such as Parkinson's disease, multiple sclerosis, epilepsy; 5. Dermatological diseases such as psoriasis, neurodermatitis 6. Tumor diseases. Use of gene changes in the GNAQ gene to predict the risk of cardiovascular diseases

In transgenic animals that overexpress Gαq in the heart, an increased tendency to heart hypertrophy, heart failure and apoptosis is observed. It can therefore be assumed that an increased expression of Gαq in humans, as is the case with the GC genotype of the GC (-909 / -908) TT polymorphism, leads to an increased cardiovascular risk. As shown in Figure 10, young men who have the GC genotype have an increased heart stroke volume with reduced peripheral resistance. As is well known, this is an expression of a hyperdynamic circulatory situation that is associated with an increased risk of hypertension and cardiac hypertrophy. In the elderly we find a clear dependence of the left ventricular mass index on the GNAQ GC (-909 / -908) TT polymorphism (p <0.05):

Thus there is an increased risk of left heart hypertrophy for GC genotypes with an increased risk of atrial flickering, other rhythm disturbances and sudden cardiac death. Use of gene changes in the GNAQ gene to predict the risk of heart failure in chronic heart failure (heart failure) The role of Gq in the development of cardiac hypertrophy has been well proven by tests on transgenic mice. However, the course of this disease is influenced by several factors: ejection volume of the heart, neurohumoral activability of the heart, physiological conditioning and genetic variability (Molkentin and Dorn II, 2001; Givertz et al., 2004). These and other factors determine the course of the disease, which can lead to death or a heart transplant due to chronic heart failure. It can therefore be assumed that a changed activatability of the Gq gene via neurohumoral activation, eg via angiotensin II, as is the case with the GC (-909 / -908) TT polymorphism, has an influence on the course of the disease Heart failure can result. In patients with chronic heart failure due to cardiomyopathy, the stronger neurohumoral activability is shown in terms of a better ejection performance of the heart in homozygous GC carriers:

These differences in the ejection output of the heart go hand in hand with differences in the course of the disease: the median time to a heart transplant for heart failure due to cardiomyopathy is in homo- zygote GC subjects 7 years, heterozygotes 5 years and homozygous TT subjects only 1.5 years (Figure 11).

The role of GNAQ in the action of insulin and endothelin in adipose tissue

Normally, glucose uptake in adipose tissue is regulated by tyrosine phosphorylation of IRS-1 followed by binding to the p85 subunit of PI-3 kinase. However, some studies also showed IRS-1 independent glucose transport into the cell. This is mediated by either insulin or endothelin. The endothelin-mediated glucose uptake is Gq dependent, but PI-3K independent. (Kanzaki et al., 2000; Wu-Wong et al., 1999; Rachdaoui and Nagy, 2003). TNFalpha is considered to be an important mediator for insulin resistance (Hotamisligil, 2000), with adipose tissue being one of the main sites of action for TNFalpha (Ruan et al., 2002). In fat cells it could be shown that chronic treatment with TNF alpha led to a reduced endothelin-1 dependent glucose uptake. It also resulted in decreased Gq / 11 expression, and this decreased expression resulted in decreased cell glucose uptake (Rachdaoui and Nagy, 2003). It can therefore be assumed that a genotype-dependent change in Gq expression in adipose tissue leads to different insulin responses and to the pathogenesis of insulin resistance, as can contribute to diseases such as type II diabetes mellitus or polycystic ovarian syndrome (PCOS). Use of gene changes in the GNAQ gene to predict insulin effects in adipose tissue

Based on the molecular analysis of the GC (-909 / - 908) TT polymorphism (greater activability of the promoter in GC alleles and increased mRNA expression in GCGC genotypes), it was assumed that the effects observed could also be transferred to Gq-mediated effects in adipose tissue are. To confirm this hypothesis, the genotype-dependent mRNA expression in human adipose tissue was first examined. For this purpose, mRNA was obtained from human subcutaneous adipose tissue using breast reduction plastics and was transcribed into cDNA using reverse transcriptase. The process is familiar to the person skilled in the art. The expression level was subsequently determined using real-time PCR (Taqman method) and compared with the expression level of the housekeeping gene β-actin. As shown in Figure 12, GC homozygotes show around 40% increased Gq mRNA expression in human adipose tissue compared to heterozygote or homozygote TT carriers.

The inhibitory effect of insulin on lipolysis was investigated in cultivated human fat cells. For this purpose, lipolysis in human fat cells was induced ex vivo with isoprenaline and the inhibitory influence of insulin was examined by adding it. Lipolysis was quantified based on the release of glycerol (Hauner et al., 2002). This shows the strongest inhibitory insulin effect in fat cells of individuals with the GCGC genotype (Figure 13). This is true with the observation of an increased gene expression of Gq in this this genotype. In addition, these results are an indication that gene changes in the GNAQ gene are suitable for predicting drug effects, as shown here by way of example for insulin.

Insulin resistance using the example of PCOS patients

Polycystic Ovarian Syndrome (PCOS) is a common endocrine disorder characterized by chronic anovulation and hyperandrogenism. About 5% of all premenopausal women are affected. Most women with PCOS have insulin resistance, which increases the risk of a metabolic syndrome (obesity, type 2 diabetes, fat metabolism disorders, and hypertension). The body mass index (BMI) and insulin resistance, measured according to the HOMA-IR method (homeostasis model assessment for insulin resistance), were examined using Gq genotypes of the GC (-909 / -908) TT polymorphism. Figure 14 clearly shows that TT / TT homozygous patients have a significantly increased BMI, HOMA-IR and insulin concentration in the serum.

This shows that gene changes in the GNAQ gene can be used to predict insulin resistance. ^'

Risk of metabolic disorders

One of the most common metabolic disorders is hypercholesterolemia, which is known to be associated with an increased risk of coronary artery disease, myocardial infarction, stroke and Alzheimer's disease. Even in healthy young people, it can be found that the GC (~ 909 / ~ 908) TT polymorphism is associated with a changed cholesterol concentration in the serum (Figure 15). Basic properties of malignant tumors

In malignant tumors, also known as cancer, there are characteristic changes in basic functions that promote the growth of such cells in an unfavorable manner. Cancer cells are characterized by a loss of contact inhibition and uncontrolled cell growth. Such changes are triggered by a large number of noxae, known as carcinogens, which damage the genome. Such chemicals include many chemicals, tobacco smoke, but also UV light. In addition, genetic factors play an outstanding role in the development of cancer. In addition to their uninhibited growth, cancer cells are also characterized by the tendency to colonize daughter tumors (metastases) in other organs. The metastases spread regularly via the bloodstream or lymphatic vessels. Cancer diseases are incurable in most cases and lead to death. Therapeutically, attempts are made to surgically remove the initial tumor and metastases. In addition, tumors can be irradiated. So-called cytostatic agents, antibodies against certain proteins or surface markers or immunomodulating substances (cytokines, interferons) are used to try to kill the rapidly dividing cancer cells or to convert them to programmed cell death (apoptosis). The therapeutic measures currently available in most cases only lead to prolonged survival, not to definitive healing.

Cancer prognostic factors

It is of great medical relevance to define prognostic factors for the course of cancer that Provide information about the response to certain forms of therapy or which are predictive of the occurrence of metastases, tumor progression and survival. Up to now, prognostic factors that are generally known to the person skilled in the art have been used in medicine. These include, for example, the size of the tumor, its depth of penetration into the surrounding tissue, growth beyond organs, penetration into blood or lymphatic vessels or lymph nodes, and the degree of differentiation of the tumor cells. There are also some relatively non-specific serological markers. The procedure for classifying the tumors is generally referred to as "staging" and "grading". In general, the presence of distant metastases and a low degree of differentiation are prognostically very unfavorable parameters. Nevertheless, it is the general experience in medicine that patients with the same tumor stage have drastically different disease courses. While rapid progression of the disease and the appearance of metastases and recurrences are observed in some patients, the disease comes to a standstill in other patients for unclear reasons. Metastases can occur locally, regionally and far from the mother tumor. To do this, it is necessary for a large number of tumor cells to reach the adjacent lymph or blood path by simply flushing them into liquid or by clapping into neighboring tissue. Recurrence tendency is the recurrence of a tumor after incomplete or partial removal of the tumor. This is not a new malignant transformation, but the regrowth of a tumor tissue that has not been completely removed. Recurrence is also possible through metastases, which can remain latent for many years. Progression is the recurrence of a tumor with higher grading (more dedifferentiation) or the emergence of metastases.

Obviously, there are a large number of individual, undetected biological variables that largely determine the course of a tumor disease, regardless of staging and grading. Such factors include genetic host factors.

In addition, it is desirable to develop genetic markers that are predictive of the occurrence of tumors. Such markers fulfill the function of promptly introducing the affected individuals to further screening measures (serology, X-ray, ultrasound, NMR, etc.). This enables early-stage cancer to be identified and treated therapeutically, whereby the chances of recovery and survival in early-stage tumors are much better than in advanced-stage tumors.

Types of tumors

In general, all cells of the human body can malignant and lead to cancer. The statements made here and below describe general mechanisms of tumor progression, metastasis and therapeutic response. In this respect, the mechanisms and claims described here apply to all tumors in humans, for example to the following tumors:

Urogenital tract tumors: These include bladder carcinoma, renal cell carcinoma, prostate carcinoma and seminoma. Tumors of the female genital organs: breast carcinoma, carcass carcinoma, ovarian carcinoma, cervical carcinoma.

Tumors of the gastrointestinal tract: oral carcinoma, esophageal carcinoma, gastric carcinoma, liver carcinoma, bile duct carcinoma, pancreatic carcinoma, colon carcinoma, rectal carcinoma.

Tumors of the respiratory tract: larynx carcinoma, bronchial carcinoma.

Skin tumors: malignant melanoma, basalioma, T-cell lymphoma

Tumor diseases of the hematopoietic system: Hodgkin and non-Hodgkin lymphomas, acute and chronic leukaemias etc. Tumor diseases of the brain or nerve tissue: glioblastoma, neuroblastoma, medulloblastoma, meningeal sarcoma, astrocytoma.

Soft tissue tumors, such as sarcomas and head and neck tumors.

G proteins and malignant transformation of cells

Experimental in vitro studies show that mutations in G protein subunits can cause a malignant transformation of cells. The expression of a constitutively active Gαq subunit with a lack of GTPase activity leads to the malignant transformation of fibroblasts (Kalinec et al., 1992).

G protein mutations and human cancers

In humans, some rare adenomas have shown somatic mutations in the Gas and Gai2 subunits. These are called Gip2 (Gαi2- Subunit) or called Gsp (gas subunit). In contrast, these are not genetic host factors that modulate the course of the disease, but causal factors (overview in: (Farfel et al., 1999).

Use of gene changes in the GNAQ gene to predict the course of tumor diseases

An essential part of the present invention is the provision of diagnostically relevant gene changes in the GNAQ gene as a prognostic factor for all tumor diseases in humans. Naturally, not all tumor diseases can be described here. The principle is therefore explained using selected examples that demonstrate its general applicability:

The following examples serve to further explain the invention.

Example 1: Chronic Lymphocytic Leukemia (CLL)

Chronic lymphoblastic leukemia is a chronic form of leukemia. A large number of degenerate lymphocytes are characteristic of the disease. A total of 30% of all leukemic diseases are chronic lymphatic leukemias. The median age of onset is 65 years. Only ten percent of the patients are younger than 50 years. Men are affected two to three times more often than women. Risk factors for the development of a CLL are not known. However, the disease rarely occurs in Japan and China. Japanese people who immigrated to the USA very rarely develop CLL. This fact suggests that genetic factors are a Role-play. Therapy depends on the stage of the disease. A CLL can be benign for up to 20 years, which means that the patient shows no symptoms apart from enlarged lymph nodes and possible fatigue and loss of appetite. Treatment only begins when the number of lymphocytes begins to rise above a certain level, the proportion of red blood cells and the number of platelets decreases or other complications occur. Early treatment does not affect the course and outcome of the disease. The main therapeutic measure is chemotherapy. In certain cases, the patient also has to have radiation or surgery. Patients can live with the diagnosis of CLL for up to 20 years without showing serious symptoms. However, the further the disease has progressed, the greater are the health disorders due to changes in the organ system. Depending on the Binet stage of the disease, the doctor can estimate the prognosis. The stage of CLL is characterized, among other things, by how many lymphocytes there are in the blood and bone marrow, how large the spleen and liver are and whether or not there is anemia. CLL leads to changes in the immune system, making people suffering from this disease more at risk of developing other types of cancer. At the same Binet stage, however, patients show very different disease courses. It is part of the invention to show that gene changes in the GNAQ gene are suitable for predicting the course of the CLL. For this purpose, patients with CLL were genotyped with regard to the described gene changes in GNAQ and the gene status was compared with the disease progression. Under progress Here we define the time interval between the initial diagnosis of CLL and the need for therapy.

Figure 16 shows a significant difference in therapy freedom depending on the GNAQ GC (-909 / - 908) TT polymorphism, whereby the CC genotype is associated with a two-fold accelerated disease progression (hazard ratio 2.07; p = 0.02).

Example 2: Bladder cancer

Bladder cancer is a malignant tumor of the urinary bladder. Bladder cancer most often occurs between the ages of 60 and 70. Men are affected three times as often as women. Bladder cancer is the third most common form of cancer in men after lung and prostate cancer. Bladder cancer can be caused by external factors. Risk factors include smoking, constant exposure of the organism to chemicals such as dyes, painkiller abuse. For many patients, the studies show that it is a superficial tumor. This can be surgically removed using the cystoscope. More than 70% of patients treated for superficial bladder cancer show a tumor recurrence in the course. Recurring tumors with non-muscle-invasive disease occur in more than half of them. These can be treated or controlled curatively by transurethral resection. It is therefore important to recognize these lesions early and to follow up the patient regularly and closely. The first priority is cystoscopy with urine cytology. Elimination urograms are used at regular intervals to check for possible tumor manifestations in the kidney pelvis and ureters. So far there are hardly any valid markers for the further course of the disease are predictive. Therefore, the classic factors such as depth of penetration, degree of differentiation, metastasis, lymph node involvement etc. are currently used for the prognosis. Genetic markers for tumor progression, tendency to relapse, probability of survival and response to therapy would significantly improve the care of patients with bladder cancer.

According to the invention, gene changes in GNAQ are used to predict the further course of the disease. Figure 17A shows the time until the occurrence of metastases depending on the GNAQ GC (-909 / -908) TT polymorphism. The risk of metastasis in patients with a C allele is increased by about two times. The median time to metastasis is 108 months for the GC / TT and GC / GC genotypes, but 64 months for the TT / TT genotype. A similar relationship is found when examining the time to tumor progression (Figure 17B). By this we mean the occurrence of metastases or the recurrence of the tumor with higher staging or grading. The curve shape is significantly different for the genotypes (p = 0.045, log rank test, with the GC genotype carriers being assigned the more favorable shape. Finally, the relationship between GNAQ GC (-909 / -908) TT polymorphism and survival is shown (Figure 17C.) Again, it can be seen that patients with the GNAQ TT // TT genotype die earlier than patients with the GC allele, while the median survival time is 102 months for patients with the GC allele, however only 66 months for the TT / TT genotype. Use of gene changes in the GNAQ gene to predict disease risks and courses

Since the diverse functions of Gαq are well known, gene changes in the GNAQ gene can increase the risk of many different diseases or influence the course of diseases. It is generally not possible to examine all human diseases and their course. However, we have shown this as an example for three different diseases: left heart hypertrophy, CLL, and bladder cancer. These data clearly demonstrate the usability of the gene changes in the GNAQ gene for the purpose described here. These diseases have no connection a priori.

Pharmacogenetics - Diagnostics of the effectiveness of pharmaceuticals, the potency and efficiency of pharmaceuticals and the occurrence of undesirable effects

Basics and goals of pharmacogenetics

The effectiveness of drugs and / or the occurrence of undesirable side effects is defined in addition to the specific substance properties of the chemically defined products by a number of parameters. Two important parameters, the achievable plasma concentration and the plasma half-life largely determine the effectiveness or ineffectiveness of pharmaceuticals or the occurrence of undesirable effects. The plasma half-life is determined, among other things, by determining the rate at which certain pharmaceuticals are metabolized in the liver or other body organs to form effective or ineffective metabolites and at which they leave the body can be excreted, whereby the excretion can take place via the kidneys, via the breathing air, over the sweat, over the sperm fluid, over the stool or over other body secretions. In addition, the effectiveness when given orally is limited by the so-called "first pass effect", since after absorption of pharmaceuticals via the intestine, a certain proportion in the liver is metabolized to ineffective metabolites.

Mutations or polymorphisms in genes of metabolizing enzymes can change their activity by changing their amino acid composition, which increases or decreases the affinity for the metabolizing substrate and thus the metabolism can be accelerated or slowed down. Similarly, mutations or polymorphisms in transport proteins can change the amino acid composition in such a way that the transport and thus the excretion from the body is accelerated or slowed down.

To select the most suitable substance for a patient, the optimal dosage, the optimal dosage form and to avoid undesirable, sometimes Knowledge of genetic polymorphisms or mutations that lead to changes in gene products is of paramount importance in terms of harmful or fatal side effects.

The effect of hormones in the human body and the importance of polymorphisms in hormone receptors

A large number of hormones and peptide hormones in the human body, but also receptor antagonists, exert their effect on so-called receptors of the body cells. These are proteins of different compositions. After activation of these receptors, these signals must be directed into the cell, which is mediated by the activation of heterotrimeric G proteins. Such G proteins are composed of different α, β and γ subunits. These receptors can be divided into specific groups, depending on the activability by defined hormones. Those skilled in the art are aware that mutations or polymorphisms in certain receptors can determine the effectiveness of certain agonists or antagonists at these receptors. For example, a common GlylδArg polymorphism in the gene coding for the β2-adrenoceptor influences the strength of the response to the β2-sympathomimetic salbutamol (Martinez et al., 1997). Polymorphisms in the D2 receptor gene determine the frequency of occurrence of dyskinesia in the treatment of Parkinson's disease (Parkinson's disease) with Levadopa (Oliveri et al., 1999). Polymorphisms in the μ-opiate receptor gene determine the analgesic effectiveness of opiates (Uhl et al., 1999).

The gene changes mentioned in specific receptors can only be used to diagnose the effects of pharmaceuticals if they are specific agonists or antagonists at the receptors under consideration. On the other hand, it is desirable to individually diagnose the general responsiveness to all pharmaceuticals and to individually predict the risk of undesirable effects under therapy with pharmaceuticals. The diagnosis of the activatability of G proteins allows a general diagnosis of the effectiveness of pharmaceuticals, their optimal dosage and the occurrence of side effects.

We generally understand pharmaceuticals as substances that are supplied to the human body from the outside in order to produce certain conditions. Such substances can be hormones, low or high molecular substances, peptides or proteins, antibodies and others. Most of the pharmaceuticals used to treat diseases, physical malfunctions or mental disorders are hormones, agonists on hormone receptors, antagonists on hormone receptors or other substances which directly or indirectly influence the expression of receptors or the concentration of hormones. A number of pharmaceuticals exert this influence in that physiological counterregulations take place during therapy with such substances, which increase the concentrations of hormones that activate G protein-coupled receptors. Therapy with diuretics (diuretics), in particular loop diuretics and thiazide diuretics, may be mentioned here as a generally known example. The loss of table salt that occurs during therapy and the lowering of blood pressure lead to the activation of the renin-angiotensin-aldosterone system. The increased hormone angiotensin II stimulates an increased absorption of sodium in the kidney, stimulates salt absorption, increases blood pressure through a direct vasoconstrictive effect on smooth vascular muscle cells and induces proliferation processes. It is well known that these mechanisms, which are caused by angiotensin II, occur after the hormone has been coupled to receptors, which Mediate effect via activation of heterotrimeric G proteins. The efficiency of these effects can be predicted if the strength of the activability of G proteins can be diagnosed. Other drugs exert their effect in that they inhibit the reuptake of transmitters released from neurons, for example noradrenaline, adrenaline, serotonin or dopamine. The pharmacon sibutramine, which inhibits the reuptake of serotonin and norepinephrine in the central nervous system, can be mentioned as an example, thereby reducing the feeling of hunger and increasing thermogenesis. Accordingly, sibutramine can be used to treat obesity. Since noradrenaline and serotonin activate G protein-coupled receptors, the diagnosis of the activability of G proteins is particularly suitable for predicting the effectiveness of sibutramine and the occurrence of typical side effects associated with sibutramine (eg increase in heart rate and blood pressure) ,

According to the invention, a method is now provided which is generally suitable for the diagnosis of the activatability of G proteins. For this purpose, one or more polymorphisms in the GNAQ gene are examined, which codes for the human Gαq subunit of heterotrimeric G proteins. Polymorphisms which diagnose the occurrence or non-occurrence of an alternative splicing process of the gene or a modified one are particularly suitable for this

Predict expression of Gαq. Overexpression predictably leads to an increased activation of heterotrimeric G proteins and an increased activation of all cells of the human body. A determination of the presence of polymorphisms in GNAQ thus allows the diagnosis of the effectiveness and undesired Effects of drugs, especially agonists and antagonists of all receptors, the effects of which are mediated by heterotrimeric G proteins. In addition, such polymorphisms in GNAQ can be used to diagnose the effects of drugs that either indirectly or as a result of body counter-regulation mechanisms increase or decrease the concentrations of endogenous hormones whose receptors activate heterotrimeric G proteins. Thus, the invention allows a diagnosis of effects and undesirable effects of all pharmaceuticals and is not limited to pharmaceuticals that influence specific receptors in an agonistic or antagonistic manner. In addition, the diagnosis of the allelic or haplotype status in GNAQ can be used to determine the individually optimal and tolerable dosage of drugs.

The detection of the GC (-909 / -908) TT polymorphism is used either alone or in all conceivable combinations for the diagnosis of an increased or reduced activatability of G proteins. In addition, all further gene changes in GNAQ can be used for diagnosis, which are in a coupling imbalance to these polymorphisms and / or which additionally promote or inhibit the alternative splicing process or expression.

These gene mutations can use any, known to the expert procedure be detected, such as direct sequencing, restriction analysis, reverse hybridization, dot blot or slot-blot method, spectrometry mass, Taqman ^® - or Light Cycler ^® - technology, Pyrosequencing etc Furthermore, these gene polymorphisms can be carried out simultaneously after multiplex PCR and hybrid detection on a DNA chip. In addition, other methods can be used for the diagnosis of an increased activatability of G proteins, which enable the direct detection of the expression level of Gαq or splice variants of Gαq.

The method mentioned is particularly suitable for diagnosing the action of agonists or antagonists on receptors, the effects of which are known to be mediated by G proteins. The following examples are given here, although the list of examples could be extended:

1.Adrenergic receptors, especially α and β adrenoceptors and their isoforms and subgroups, i.e. αl and α2 adrenoceptors as well as ßl, ß2, ß3 and ß4 adrenoceptors

2. Muscarinic receptors and their isoforms, e.g. ml, m2, m3, m4 and m5 muscarinic receptors and their subtypes. Typical antagonists on muscarinic receptors are, for example, atropine, scopolamine, ipratroprium, pirencezine and N-butylscopolamine. Typical agonists are carbachol, bethanechol, pilocarpine etc.

3. dopamine receptors, e.g. D1, D2, D3, D4, and D5 receptors and their isoforms and splice variants

4. Serotonin receptors, e.g. 5-HT1- 5-HT2, 5-HT3, 5-HT4, 5HT-5, 5HT-6 and 5-HT7 receptors and their subtypes, isoforms and splice variants. Typical agonists are sumatriptan and cisapride, antagonists are for example ondansetron, methysergide, buspirone and U-rapidil.

5. Endothelin receptors and their subtypes, isoforms and splice variants 6. Bradykinin receptors, for example Bl and B2 receptors and their subtypes, isoforms and splice variants

7. Angiotensin receptors, e.g. AT II type and type 2 receptor, typical antagonists on the AT II receptor are losartan and other sartans.

8. Receptors for endorphins and opiates, e.g. the μ-opiate receptor

9. Chemokine receptors CCR1-12 and CXCR1-8 for e.g. Interleukin-1/2/3/4/5/6/7/8/9/10/11/12, RANTES, MlP-lα, MlP-lß, stromal cell-derived factor, MCP1-5, TARC, Lymphotactin, Fractalkine, Eotaxin 1-2, NAP-2, LIX etc.

10. Adenosine receptors and their subtypes, isoforms and splice variants

11. Receptors for thrombin (protease-activated receptors)

12. Receptors for lysophosphatidic acid, phosphatidic acid, receptors for sphingosine phosphates and their derivatives

13. Receptors for prostaglandins and thromboxanes, e.g. B. for PGE1, PGE2, PGF, PGD2, PGI2, PGF2α, Thromboxan A2, etc.

14. Receptors for neuropetides, e.g. NPY1-5

15. histamine receptors, e.g. Hl-H3 receptors

16. Receptors for platelet activating factor (PAF receptor)

17. Receptors for leukotrienes

18. Receptors for insulin, glucagon, insulin-like growth factor (IGF-1 and IGF-2), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)

19. Receptors for growth hormone (GH), somatostatin (SSTR1-5), thyreotropic hormone (TSH), oxytocin, prolactin, gonadotropins

20. Receptors for cytokines, eg interferons 21. Receptors for purines

22. Orphan receptors, the effects of which are mediated by G proteins.

23. Receptors for leptin

24. CpG oligonucleotides

The effects of pharmaceuticals which influence the reuptake, breakdown or re-synthesis of neurotransmitters or which have changes in the expression or responsiveness of the abovementioned receptors (e.g. sibutramine, fluoxetine) during therapy can also be predicted. The effects of all pharmaceuticals can also be diagnosed, which directly or indirectly change the concentrations of agonists which activate the above-mentioned receptors as a result of a physiological counter-reaction. The influence of radiation therapy in cancer patients can also be predicted.

In particular, the effects and undesirable effects of the following drugs from the following indication areas can be diagnosed:

1. Antihypertensives, e.g. β-blockers (propanolol, bisproolol, etc.), diuretics (hydrochlorothiazide and other thiazide diuretics; furosemide, piretanide and other loop diuretics, chlorothalidone), αl adrenoreceptor blockers (e.g. doxazosin), angiotensin, prazosin Zeptor blocker (for example losartan), ACE inhibitors (enalapril, captopril, ramipril, etc.), Ca ² + channel blockers (eg Ni fedipin, verapamil, amlodipine, felodipine), clonidine, reserpine, renin inhibitors

2. Pharmaceuticals for the treatment of heart failure, for example β-blockers (for example propanolol, metoprolol), ACE inhibitors (eg captopril, enalapril, ramipril, etc.), angiotensin receptor blockers (eg losartan), digitalis glycosides, catecholamines, diuretics. 3. Pharmaceuticals for the treatment of low blood pressure or heart failure, eg α- and β-smpathomimetics (Effortil, adrenaline, noradrenaline, dobutamine, β-adrenoceptor blockers, ACE inhibitors, angiotensin II receptor blockers.)

4. Pharmaceuticals for the treatment of migraines, e.g. Sumatriptan, rizatriptan, zolmitriptan and other agonists at serotonin receptors, ß-blockers (propanolol, timolol), ergotamine and dihydroergotamine

5. Morphine-type analgesics (morphine, codeine, etc.)

6. Pharmaceuticals for the treatment of coronary heart disease such as adenosine, β-blockers (eg propanolol, acebutolol), nitrates and Ca ²⁺ channel blockers

7. Pharmaceuticals for the treatment of psychiatric disorders (schizophrenia, manic-depressive disorders, psychoses, depression) and addictive disorders such as alcoholism (e.g. fluoxetine, paoxetine, imipramine, desipramine, doxepin, Mianserin, trazodone, lofepramine), anxiety syndromes (diazepam, etc.), which, for example affect the dopaminergic, serotonergic or adrenergic system. But also pharmaceuticals that mediate their effect via receptors for GABA, glycine or glutamate or their derivatives.

8.Drugs for the treatment of Alzheimer's disease (e.g. tacrine) and for the treatment of Parkinson's disease (e.g. bromocriptine, L-DOPA, carbidopa, biperiden, seleginil, etc.) which transmitter concentrations at e.g. muscarinic or dopaminergic substances

9. Pharmaceuticals for the treatment of bronchial asthma, which, for example, either directly bronchodilate or have an anti-inflammatory effect, for example salbutamol, terbutalin, albuterol, theophylline, montelukast, zafirlukast, cromoglicinic acid, ipratropium bromide. Such pharmaceuticals also include antibodies directed against certain proteins and receptors.

10. Pharmaceuticals for the treatment of gastric or intestinal motility disorders and pharmaceuticals for the treatment of irritable bowel syndrome (irritable bowel syndrome) e.g. N-butylscopolamine, pirenzepin, metoclopramide)

11. Pharmaceuticals for the treatment of obesity, which either directly activate lipolytically active receptors, e.g. β3-adrenergic agonists, or are centrally active, e.g. Sibutramine, or similar substances that change the feeling of satiety or affect thermogenesis. This includes pharmaceuticals that affect gastric emptying.

12. Pharmaceuticals for the treatment of chronic inflammatory processes or disorders of the immune system, e.g. Cytokines (interferons) in the treatment of viral hepatitis or interleukin-2 for HIV infection. Such diseases also include Crohn's disease, ulcerative colitis, asthma, psoriasis, neurodermatitis, hay fever. This also includes antibodies against cytokines or against cytokine receptors, e.g. against TNFα

13. Pharmaceuticals for the treatment of gestosis and preeclampsia / eclampsia and HELLP syndrome

14. Pharmaceuticals for the treatment of fertility disorders or for the elimination of menstrual disorders or for contraception

15. Pharmaceuticals for the treatment of cardiac arrhythmia

16. Antidiabetic drugs (acarbose, insulin, troglitazone, metformin, etc.)

17. Hypnotics, antiemetics and antiepileptics 18. Pharmaceuticals for the treatment of disorders of sexual life, e.g. erectile dysfunction, female sexual dysfunction, libido deficiency, orgasm disorders (phosphodiesterase inhibitors such as sildenafil, prostaglandin El, agonists on dopamine receptors, e.g. apomorphine, yohimbine, phentolamine)

19. Pharmaceuticals for the treatment of cancer and chemotherapeutic agents, e.g. 5-fluorouracil, antibodies against proteins and receptors (e.g. against HER-2), substances that block tyrosine kinases, etc.

20. Pharmaceuticals for the treatment of allergic and tumor diseases, in which the effect is achieved by the administration of CpG nucleotides.

21. Pharmaceuticals for the treatment of obesity, metabolic syndrome or diabetes, e.g. B. sibutramine, orlistat, leptin, topiramate, glinide, glitazone, biguanide etc.

22. Pharmaceuticals for the treatment of HIV infection, including antibodies and receptor blockers. Prediction of the development of lipodystrophy under therapy with proteinase inhibitors.

Of course, it is not possible within the scope of the invention described here to provide evidence that all pharmaceutical effects are determined by the GNAQ gene status. Naturally, one cannot investigate the genotype-dependent effects of pharmaceuticals that will only be developed and used in the future. In contrast, examples of pharmaceuticals with different mechanisms of action are to be shown here by way of example, so that these findings can be generalized. As an example of the general applicability of the GNAQ GC (-909 / -908) TT polymorphism to predict drug effects, the vasoconstrictors angiotensin II, noradrenaline or endothelin were injected into the skin in humans and the subsequent change in blood flow was recorded ( Figure 18). After administration of all three hormones, which couple to different receptors, it can be seen that the reduction in blood flow to the skin is most pronounced in the GC / GC genotype, which can be explained by the increased expression of Gαq.

Further evidence of the general use of gene changes in the GNAQ gene to predict pharmacological effects also results from the observed genotype-dependent disease courses in bladder carcinoma. These patients were all treated with different pharmaceuticals. The different disease courses made visible by using gene changes in the GNAQ gene demonstrate a different response to these forms of therapy.

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Claims

PATE NT REQUESTS

1. A method for the determination of disease risk, ^• course of a disease, the effects of drugs, drug side effects and which is drug targets, connected to a base substitution in the gene GNAQ encoding the Gαq subunit of human G-proteins characterized in that A base exchange (polymorphism) was identified in the 5 'untranslated region of the gene for the Gαq subunit of human G proteins.

2. The method according to claim 1, characterized in that one identifies a GC (-909 / -908) TT polymorphism.

3. The method according to claim 1, characterized in that one identifies a G (-382) A polymorphism.

4. The method according to claim 1, characterized in that one identifies a G (-387) A polymorphism.

5. The method according to claim 1, characterized in that the presence of two or three of the polymorphisms GC (-909 / -908) TT, G (-382) A or G (-387) A identi ized.

6. The method according to any one of claims 1 to 5, characterized in that to identify one or more polymorphism / men in the GNAQ gene, the same is carried out.

7. The method according to any one of claims 1 to 5, characterized in that the polymorphism in the gene GNAQ is examined or detected by reverse hybridization.

8. The method according to any one of claims 1 to 5, characterized in that the polymorphism in the GNAQ gene is examined or detected by cleavage with restriction enzymes.

9. The method according to claim 6, characterized in that prior to sequencing, a gene section of the GNAQ gene which contains one or more of positions -909, -908, -382 or -387 is amplified.

10. Genetic test, containing a probe for identifying one or more polymorphisms in the 5 'untranslated region of the GNAQ gene.

11. Genetic test according to claim 10, characterized by a probe for identifying one or more of the polymorphisms GC (-909 / -908) TT, G (-382) A or G (-387) A.