DE10030945A1 - Use of a gene modification in the gene for the beta3 subunit of the human G protein - Google Patents

Abstract

The invention relates to the use of a mutation in the gene for the beta 3 subunit of human G proteins. Adenine is substituted by thymine at site 1, in position 657, in exon 9 and/or guanine is substituted by adenine at site 1, in position 814, in exon 10 and/or adenine is substituted by guanine at site 1, in position (-350), in the promoter area and/or adenine is substituted by cytosine at site 1, in position 3882, in intron 9 and/or guanine is substituted by adenine at site 1, in position 5177, in intron 9 and/or guanine is substituted by adenine at site 1, in position 5249, in intron 9 and/or there is an insert consisting the nucleotides cytosine-adenine-cytosine-adenine at site 1, in position 6496, in intron 10, for predicting physiological and pathophysiological conditions in the human body.

Description

The invention relates to new sequence variants of the human gene of the G protein subunit Gβ3 and its Use to predict physiological and pathophysiological processes in the human body, especially for the diagnosis of a wide range of Diseases, especially to determine the Disposition to high blood pressure and obesity Disposition to a variety of diseases associated with malfunctioning of the immune system, which Diposition to psychiatric illnesses as well as to Therapeutic development and targeted use of well-known and pharmaceuticals still to be developed on the basis principles of pharmacogenetics.

The human Gβ3 subunit is an important one Component in signal transmission systems, in all Human cells occur. The sequence variants in GNB3 are with the accumulation of the G protein β3 Gβ3s and Gβ3s2 splice variants associated. These are responsible for an increased signal transmission in Cells expressing Gβ3s and Gβ33s2.

Since Gβ3 occurs in all cells examined so far, carries out the increased signal transmission mediated Gβ3s and Gβ3s2 to increase physiological and pathophysiological responses to a variety of non-hormonal and hormonal stimuli.  

This change in function takes a wide range Spectrum of central nervous and peripheral functions noticeable, so in the cardiovascular system, in metabolic Functions, central nervous functions and the Neurosecretion. So Gβ3, Gβ3s and Gβ3s2 are interesting Attack points for pharmaceuticals and therapeutic agents with one broad focus of indication.

Various findings indicate that Gβ3, Gβ3s and Gβ 3s2 a role in pathogenesis / pathophysiology a number of common diseases, such as B. the Hypertension, heart attack, coronary artery disease and other cardiovascular diseases, stroke, metabolic diseases such as obesity and the Diabetes play a role. They also play one Role in other malfunctions such. B. the erectile dysfunction. The variants are special involved in changes in immune regulation and suitable therefore to predict the diposition to or of Course of a variety of immunological diseases, where an increased or reduced function of Immune cells is significant. To such diseases belong u. a. HIV infection, asthma, psoriasis, so-called atopic diseases such as atopic dermatitis, Hepatitis B and hepatitis C, graft rejection, Crohn's disease, ulcerative colitis, u. v. m. Play next to it these variants play a role in preeclampsia / gestosis, spontaneous abortion, the success of an in vitro Fertilization, etc.

The aim of the invention is to create variants, polymorphisms, Mutations and resulting haplotypes in DNA Determine sequence of the human Gβ3 gene (GNB3) and their correlation with disease dispositions  determine. Based on this correlation, a Procedures for diagnosing these diseases, to predict severity, course and Survival time, a system for predicting the individual responsiveness to different Pharmaceutical classes and a system for the development of a effective class of Gβ3 or Gβ3s and Gβ3s2 Therapeutics, as well as the development of test systems for Research into pathophysiological relationships and Development obg. Therapeutics are being developed. In summary, one for each GNB3 genotype individually predicted optimal therapeutic agent or be developed.

The structure of the GNB3 gene is shown in Fig. 1. It consists of 11 exons, with the start codon ATG located in exon 3, while the stop codon is located in exon 11. The cDNA encoding the Gβ3 protein has been previously described (MA Levine, PM Smallwood, PT Moen, Jr., LJ Helman, and TG Ahn. Molecular cloning of beta 3 subunit, a third form of the G protein beta subunit polypeptides, Proc. Natl. Acad. Sci. USA 87 (6): 2329-2333, 1990). In deviation from this published cDNA sequence, the applicant previously described the mutations C825T ( Fig. 1) and C1429T ( Fig. 1) (cf. DE 19 61 9 362 A1, DE 196 37 518 A1 and the subsequently published PCT / EP99 / 06534). The numbering of the above-mentioned polymorphisms relates to the cDNA sequence, with the start codon ATG being assigned position +1. In addition, it was previously described that alternative splicing of exon 9 creates a new splicing variant with 6WD repeats (Gβ3s) (W. Siffert, D. Rosskopf, G. Siffert, S. Busch, A. Moritz, R. Erbel, AM Sharma, E. Ritz, HE Wichmann, KH Jakobs, and B. Horsthemke. Association of a human G-protein beta3 subunit variant with hypertension. Nat. Genet. 18 (1): 45-48, 1998) and that alternative splicing of Exon 10 leads to the creation of a further splice variant with 6WD repeats (cf. the post-published PCT / EP99 / 06534). It was found that the 825T allele and the 1429T allele are predictive for the generation of these splice variants. Since the expression of these splice variants in cells generally leads to an increased activatability of G proteins, the strength of the intracellular signal transduction can be predicted by genotyping the C825T or the C1429T polymorphism.

In addition, the genomic sequence of GNB3 was published (M.A. Ansari-Lari, D.M. Muzny, J. Lu, F. Lu, C.E. Lilley, S. Spanos, T. Malley, and R.A. Gibbs. A gene rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13. Genomes Research 6: 314-326, 1996)

It has now been found that the sequence of the human Gβ3 gene (GNB3) in deviation from that of Ansari-Lari et al. described sequence in addition to Mutations in the cDNA (C825T, A657T, G814A, C1429T) and a mutation in the promoter (A-350G) further variants available. It has also been found that this genetic variants with the disposition for various diseases, u. a. High blood pressure, obesity, Overweight, correlate.

The invention then relates to the sequence of human Gβ3 gene located at positions 657, 814, 825 and 1429 of the cDNA or at positions (-350), 3382,  5177, 5249 and 6496 of the genomic sequence in whole or is partially mutated.

Fig. 1 of the drawing shows the gene structure of GNB3 and new polymorphisms.

The exon-intron structure of GNB3 is shown. The Gen consists of 11 exons, previously described Polymorphisms are found in exon 10 (C825T) and exon 11 (C1429T). The numbering corresponds to the cDNA Sequence, with the start codon ATG in exon 3 the position +1 is assigned. The start of the is also shown Transcription in exon 1. Related to this Starting point of transcription is a mutation in Promoter (A-350G) and the mutations shown in Intron 9, A3382C, G5177A, and G5249A. Under use this numbering (transcription starting point = 1) the C825T exchange at position 5500. Still takes place there is a CACA insertion at position 6496.

The areas of exon 9 and exon are also shown 10, if the mutations are at position 825 and 1429 of the cDNA and 3382, 5177, 5249 and / or 6496 of the Alternatively, the DNA sequence can be spliced. In angular The numbering of brackets is shown Polymorphisms if as position 1 of the Transcription start point is set in exon 1.

The mutations are characterized in detail below. The numbering refers to the transcription start point of GNB3 ( Fig. 1). This starting point corresponds to nucleotide 52221 that of Ansari-Lari et al. published sequence of a section of human chromosome 12 (MA Ansari-Lari, DM Muzny, J. Lu, F. Lu, CE Lilley, S. Spanos, T. Malley, and. RA Gibbs. A gene-rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13. Genome Research 6: 314-326, 1996). The polymorphism C825T corresponds, for example, to the genomic sequence C5500T.

Base exchange A3382C: the sequence within intron 9

is mutated into the following base sequence:

Base exchange G5177A: the sequence within intron 9

is mutated into the following base sequence:

Base exchange G5249A: the sequence within intron 9

is mutated into the following base sequence:

The CACA insert: the sequence within intron 10

is mutated into the base sequence

(see also Fig. 1).

The following sequences (haplotypes) are particularly important
Sequence with mutations 825C-1429C-3382A-5249G and without the CACA insert in intron 10
Sequence with mutations 825T-1492T-3382C-5249G and with the CACA insert in intron 10

It was also found that all examined Individuals with an 825T allele and / or a 1429T Allele simultaneously a 3382C allele, a 5249A allele and wear a CACA insert. So there is a 100% coupling imbalance between the Positions 825 and 1429 of the cDNA and positions 3382  and 5249 of the DNA sequences within intron 9 and the CACA insert at position 6496 of the DNA sequences within from Intron 10. The Genotyping at positions 3382 (intron 9) and / or 5249 (intron 9) and / or 6496 (intron 10) as well like genotyping at cDNA positions 825 and 1429 the prediction of an alternative splicing process, which is used for alternative splicing of exons 9 and 10 leads.

The invention further relates to a method for Determination of disease dispositions, all Sequences and variants of GNB3 from the single mutation up to all possible variants (including each any absolute number of variants with can be included) can be genotyped and the corresponding statements about disease disposition allow.

The method is characterized in that the DNA of a subject isolated and at least on one of the exchanged postions genotyped and subsequently is compared with the reference DNA sequence. Prefers are embodiments in which at least the position 3382, position 5177, position 5249 or the Position 6496, at least the two positions 825 and 3382, 825 and 5177, 825 and 5249 or 825 and 6496, at least the three positions 825, 3382 and 5249, 825, 3382 and 6496, 825, 5249 and 6496, at least the four Positions 825, 3382, 5249 and 1429, and 825, 3382, 5249, respectively and 6496 or at least the five positions 825, 3382, 5249, 1429 and 6496 can be genotyped.

Genotyping is done by sequencing or through other methods used for the detection of  Point mutations are suitable. This includes PCR-based Genotyping procedures such as B. allele-specific PCR, PCR reactions and restriction fragment length analysis, PCR reactions using the Taqman system or Molecular beacons, genotyping procedure under Use of oligonucleotides (examples would be here dot blotting and hybridization, single nucleotide primer Extension analyzes, oligonucleotide ligation assays), Method using restriction enzymes and Single nucleotide polymorphism (SNP) analysis using Matrix-assisted laser desorption / ionization mass Spectrometry (MALDI), and in principle any future available method for variant detection including gene chip technology in all of theirs technological designs.

Based on this, the method according to the invention is for Determination of a wide range of different Dispositions suitable ex vivo.

In one embodiment, the Provision to clarify a disposition for Hypertension, predicting the individual Blood pressure as such, and other cardiovascular Diseases, including myocardial infarction, Heart failure and stroke as well as from Irregular heartbeat. In a broader sense, too Development of end-stage renal failure (Need for dialysis) or risk of mortality under dialysis including.

In a further embodiment variant, the Disposition for metabolic diseases like Obesity, obesity and type II diabetes mellitus,  including a prediction of the weight range as such or a disposition for weight changes, finally a prediction of the relationship of the Body mass as such, as z. B. in body mass Express index (BMI), determined.

In addition, the method is used to predict the Weight gain after incisive personal Events such as B. after births in women or in convalescence after serious illnesses. Farther the procedure is used to estimate the possible Birth weight and weight development after Birth. The method also serves to predict Premature birth, weight gain of premature babies, the risk of preeclampsia / gestosis, with or without HELLP syndrome, from abortion, the success of an in vitro Fertilization.

The diagnosis is in a further embodiment variant for disposition to changed immune reactions intended. This includes predicting the Response to vaccination, the course of infection and Autoimmune diseases and transplant rejection Organs. As infectious and immune diseases are here to name, for example, the HIV infection and the Progression to AIDS, Kaposisarcoma, hepatitis C and B, allergic asthma and other atopic diseases, such as Crohn's disease, ulcerative colitis, atopic dermatitis and psoriasis. Furthermore, are to be stated here Rheumatic diseases, e.g. B. the rheumatoid arthritis.

In a further embodiment variant, the Diagnostics regarding the predisposition to emergence  malignant tumors and their course, especially the Tendency to metastasis. As such are tumor diseases in particular to list breast cancer Colon cancer, gastric and pancreatic cancer, maligue Melanoma of the skin, leukemia, T-cell lymphoma, Kaposis sarcoma, lung and larynx carcinomas.

The method also allows the determination the course and severity of diseases, as well as the prediction of survival with and after serious medical conditions, e.g. B. after Myocardial infarction, heart failure, stroke and / or Heart failure.

Furthermore, the applicability of the first teaching according to the invention for pharmacogenetics received, d. H. the diagnosis 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 unwanted side effects will besides the specific ones Material properties of the chemically defined products defined by a number of parameters. Two important ones Parameters, the achievable plasma concentration and the Plasma half-lives largely determine that Effectiveness or ineffectiveness of parameters or that Occurrence of undesirable effects. The Plasma half-life is determined, among other things, by at what speed certain drugs in the Liver or other body organs to be effective or metabolized inactive metabolites and with which  Speed they are excreted from the body the excretion via the kidneys, via the Breathing air, over the sweat, over the sperm fluid, via the stool or other body secretions can. In addition, the effectiveness with oral administration is determined by limits the so-called "first pass effect" because after Absorption of pharmaceuticals through the intestine a certain Inactive metabolites in the liver is metabolized.

Mutations or polymorphisms in metabolizing genes Enzymes can limit their activity in this way change that their amino acid composition changes becomes, causing affinity for the metabolizing substrate is increased or decreased and thus the metabolism can be accelerated or slowed down. More like that Way, mutations or polymorphisms in Transport proteins the amino acid composition in the Change the way that the transport and thus the Elimination from the body accelerates or slows down becomes.

To select the most suitable for a patient Substance, the optimal dosage, the optimal Dosage form and to avoid unwanted, e.g. T. harmful or fatal side effects knowledge of genetic polymorphisms or of Mutation that leads to a change in gene products from of outstanding importance.  

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

A variety of hormones and peptide hormones of the human body exert their effect on so-called Receptors of the body cells. These are proteins different composition. After activation These receptors have to send these signals into the cell interior be guided what about the activation of heterotrimeric G proteins is mediated. Such G- Proteins are made up of different α-, β- and γ- Subunits are composed. The receptors can, depending on the activatability, be defined by Hormones, divided into certain groups. The specialist it is known that mutations or polymorphisms in certain receptors the effectiveness of certain Agonists or antagonists at these receptors can determine. So one affects more often Gly16Arg polymorphism in the gene responsible for the β2- Adrenoceptor encodes the level of responsiveness the β2 sympathomimetic salbutamol (Martinez FD, et al. Association between genetic polymorphisms of the beta2- adrenoceptor and response to albuterol in children with and without a history of wheezing. J Clin Invest. 1997 Dec 15; 100 (12): 3184-8). 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 (olives RL, et al .; Dopamine D2 receptor gene polymorphism and the risk of levodopa induced dyskinesias in PD. Neurology. 1999 Oct. 2253 (7): 1425-30): Polymorphisms in the µ-opiate receptor gene determine the analgesic effectiveness of opiates (Uhl GR, et al. The mu opiate receptor as a candidate gene for pain: polymorphisms, variations in expression,  nociception, and opiate responses. Proc Natl Acad Sci USA. 1999 Jul 6; 96 (14): 7752-5).

The gene changes mentioned in specific receptors can only be used to diagnose the effects of Drugs are used as these drugs are specific Agonists or antagonists on the considered Are receptors. However, one is desirable individual diagnosis of general responsiveness towards pharmaceuticals and the individual prediction of 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 and side effects.

Most used to treat physical diseases Malfunctions or mental disorders Pharmaceuticals are hormones, agonists on hormone receptors, Antagonists to hormone receptors or other substances, which the expression of receptors or the Concentration of hormones directly or indirectly influence. A number of pharmaceuticals exert this influence characterized in that under therapy with such substances physiological counter-regulations take place, which the Levels of hormones that increase G protein activate coupled receptors. As a well known An example is therapy with diuretics Substances (diuretics), especially loop diuretics and called thiazide diuretics. The one with therapy occurring loss of saline and lowering blood pressure lead to the activation of the renin-angiotensin Aldosterone system. The increased hormone  Angiotensin II stimulates increased absorption of Sodium in the kidney, stimulates salt intake, increases blood pressure through a direct vasoconstrictor Effect on smooth vascular muscle cells and induced Proliferation processes. It's common knowledge that these mechanisms caused by angiotensin II Coupling of the hormone to receptors takes place, which your Effect via activation of heterotrimeric G proteins convey. The efficiency of these effects are then predictable if the level of activatability of G- Proteins can be diagnosed. Other pharmaceuticals exert their effect by resuming of transmitters released from neurons, e.g. B. Inhibit norepinephrine, adrenaline, serotonin or dopamine. The pharmacon sibutramine can be mentioned here as an example which is the resumption in the central nervous system inhibits of serotonin and noradrenaline, thereby the Feeling of hunger reduced and thermogenesis increased. Accordingly, sibutramine can be used to treat obesity be used. Since norepinephrine and sibutramine G- Activating protein-coupled receptors is that Diagnostics of the activatability of G proteins for Predicting the effectiveness of sibutramine and the Occurrence of typical, sibutramine-associated Side effects (e.g. increase in heart rate and Blood pressure) is particularly suitable.

Furthermore, the first teaching of the invention enables Predict which specific drugs are defined in Illnesses work.

The invention is based on the fact that a method was developed that generally for the diagnosis of Activability of G proteins is suitable. For this become one or more polymorphisms in the GNB3 gene  investigated that for the human Gβ3 subunit encoded heterotrimeric G proteins. Particularly suitable are polymorphisms that diagnose the Occurrence or non-occurrence of an alternative Predict splicing of the gene, creating a new one Splice variant of the gene and protein with a maximum of 6WD Repeat domains arise or suppress their creation becomes. When this splice variant occurs predictable to an increased activatability of heterotrimeric G proteins and for enhanced All cells of the human body can be activated. A determination of the presence of Polymorphisms in GNB3 the diagnosis of effectiveness and adverse effects of drugs, in particular Agonists and antagonists of all receptors, their Effects mediated via heterotrimeric G proteins become. Such polymorphisms can also be used in GNB3 used the effects of pharmaceuticals too diagnose, either indirectly or as a result of Counter-regulation mechanisms of the body Increase concentrations of endogenous hormones or lower their receptors heterotrimeric G proteins activate. The invention thus allows diagnostics effects and undesirable effects of all pharmaceuticals and is not limited to pharmaceuticals in agonistically or antagonistically specific Affect receptors.

For the diagnosis of an increased or reduced Activatability of G proteins serves in particular Detection of the polymorphisms C825T, C1429T, A657T and G814A alone or in any combination as Haplotype analysis (numbering according to the cDNA, where position +1 is assigned to the start codon ATG).  

In addition, all other gene changes in GNB3 that are in a coupling imbalance to C825T or C1429T and / or which promote or inhibit the alternative splicing process can be used for diagnosis. This applies in particular to genetic polymorphisms in intron 9 of GNB3, e.g. B. A3382C, G5177A, G5249A and the CACA insert in intron 10 ( Fig. 1).

These gene changes can be with any, the Methods familiar to a person skilled in the art are detected, e.g. B. direct sequencing, restriction analysis, reverse Hybridization, dot blot or slot blot method, Mass spectrometry, etc. Furthermore, these Gene polymorphisms simultaneously after multiplex PCR and Hybridization to a DNA chip can be detected. Besides can be used to diagnose increased activatability other methods of G proteins are also used, which provides direct evidence of the formation and expression of Isoforms of the Gβ3 subunit serve which Show splice variants.

Carrying out the analysis for pharmacogenetic Investigation includes the prediction of the Responding to therapy e.g. B. with antihypertensive drugs (including ACE inhibitors, AT1 receptor blockers, β-blockers, α- Receptor blockers, Ca antagonists, vasodilators), Medicines to treat heart failure, Medicines to treat irregular heartbeat, Asthma, depression, schizophrenia, Alzheimer's disease, the use of vaccines, the treatment of erectile Dysfunction.

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 applicability of this method has already been demonstrated for the α2-adrenergic receptor, the f.MLP receptor and the interleukin-8 receptor (see PCT / EP99 / 06534) and published (Baumgart D, et al. G protein beta3 subunit 825T allele and enhanced coronary vasoconstriction on alpha (2) -adrenoceptor activation. Circ Res. 1999 Nov 12; 85 (10): 965-9; Virchow S. et al., The G protein beta3 subunit splice variant Gbeta3-s causes enhanced chemotaxis of human neutrophils in response to interleukin-8. Naunyn Schmiedebergs Arch Pharmacol. 1999 Jul; 360 (1): 27-32; Virchow S. et al. Enhanced fMLP-stimulated chemotaxis in human neutrophils from individuals carrying the G protein beta3 subunit 825 T-alleles. FEBS Lett. 1998 Oct 2; 436 (2): 155-8) and can be applied to other receptors. The following examples are given:

• - Adrenergic receptors, especially α- and β- Adrenoceptors and their isoforms and subgroups, d. H. α1 and α2 adrenoceptors as well as β1, β2, β3 and β4 Adrenoceptors
• - Muscarinic receptors and their isoforms, e.g. B. m1, m2, m3 and m4 muscarinic receptors and their subtypes. Typical antagonists to muscarinic receptors are for example atropine, scopolamine, ipratroprium, Pirenzepin and N-butylscopolamine. Typical agonists are carbachol, bethanechol, pilocarpine etc.
• - Dopamine receptors, e.g. B. D1-, D2-, D3-, D4-, and D5- Receptors and their isoforms and splice variants  
• - Serotonin receptors, e.g. B. 5-HT1- 5-HT2-, 5-HT3- and 5- HT4 receptor and its subtypes. Typical agonists are sumatriptan and cisapride, which are antagonists for example ondansetron, methysergide, buspirone and Urapidil.
• - endothelin receptors
• - Bradykinin receptors, e.g. B. B1 and B2 receptors
• - angiotensin receptors, e.g. B. AT II Type1 and Type2 Receptor, typical antagonists on the AT II receptor Losartan and other sartans.
• - Receptors for endorphins and opiates, e.g. B. the µ- Opiate receptor
• - Chemokine receptors CCR1-12 and CXCR1-8 for e.g. B. Interleukin-1/2/3/4/5/6/7/8/9/10/11/12, RANTES, MIP-1α, MIP-1β, stromal cell-derived factor, MCP1-5, TARC, Lymphotactin, Fractalkine, Eotaxin 1-2, NAP-2, LIX etc.
• - adenosine receptors
• - Thrombin receptors
• - receptors for lysophosphatidic acid, phosphatidic acid, Receptors for sphingosine phosphates and their derivatives
• - Receptors for prostaglandins and thromboxanes, e.g. B. for PGE1, PGE2, PGF, PGD2, PGI2, PGF2α, Thromboxan A2, Etc.  
• - Receptors for neuropeptides, e.g. B. NPY1-5
• - histamine receptors, e.g. B. H1-H3 receptors
• - receptors for platelet activating factor (PAF- Receptor)
• - Receptors for leukotrienes
• - Receptors for insulin, glucagon, insulin-like growth factor (IGF-1 and IGF-2), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)
• - growth hormone (GH) receptors, somatostatin (SSTR1-5), thyreotropic hormone (TSH), oxytocin, Prolactin, gonadotropins
• - receptors for interferons
• - Purine receptors
• - Orphan receptors, their effects through G proteins mediated.

The effects of can also be diagnosed Pharmaceuticals, which are the resumption, the dismantling or the Influence new synthesis of neurotransmitters or at those under therapy changes in expression or responsiveness of the above receptors occur (e.g. sibutramine, fluoxetine). Diagnosed the effects of all pharmaceuticals, which in a direct or indirect way also in a row a physiological backlash the concentrations  of agonists that have the above receptors activate, change.

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

• - antihypertensive drugs, e.g. B. β-blockers (propanolol, bisoprolol, etc.), diuretics (hydrochlorothiazide and other thiazide diuretics; furosemide, piretanide and other loop diuretics, chlorthalidone, spironolactone), α1-adrenoceptor blockers (e.g. doxazosin, prazerosin), angiotensin receptor e.g. Losartan), ACE inhibitors (enalapril, captopril, ramipril etc.), Ca ²⁺ channel blockers (e.g. nifedipine, verapamil, amlodipine, felodipine), clonidine, reserpine
• - Pharmaceuticals for the treatment of heart failure, e.g. B. β- Blockers (e.g. propanolol, metoprolol), ACE inhibitors (e.g. Captopril, enalapril, ramipril, etc.), angiotensin Receptor blockers (e.g. losartan).
• - Medicines used to treat low blood pressure or heart failure, e.g. B. α- and β-smpathomimetics (Etilefrin, adrenaline, noradrenaline, dobutamine)
• - Medicines to treat migraines, e.g. B. Sumatriptan, Rizatriptan, Zolmitriptan and other agonists Serotonin receptors, β-blockers (propanolol, timolol), Ergotamine and dihydroergotamine
• - morphine-type analgesics (morphine, codeine, etc.)  
• - Pharmaceuticals for the treatment of coronary artery disease such as Adenosine, β-blockers (e.g. propanolol, acebutolol), Nitrate esters and other NO donors (e.g. molsidomine), Antiplatelet agents
• - Pharmaceuticals for the treatment of psychiatric disorders like alcoholism, schizophrenia, manic-depressive Diseases, psychoses, depression (e.g. fluoxetine, Paoxetine, imipramine, desipramine, doxepin, Mianserin, Trazodone, lofepramine), anxiety syndromes (diazepam, etc.), which z. B. the dopaminergic, serotonergic or adrenergic Affect system
• - Pharmaceuticals 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, selegiline, etc.) which transmitter concentrations at z. B. muscarinic or dopaminergic substances, influence.
• - Pharmaceuticals for the treatment of bronchial asthma, which e.g. B. either directly bronchodilating or act anti-inflammatory, e.g. B. salbutamol, Terbutaline, albuterol, theophylline, montelukast, Zafirlukast, cromoglicic acid, ipratropium bromide
• - Pharmaceuticals for the treatment of motility disorders of the Stomach or intestines (e.g. N-butylscopolamine, pirenzepin, Metoclopramide)
• - Pharmaceuticals to treat obesity, which either directly activate lipolytically active receptors, e.g. B.  β3-adrenergic agonists, or are centrally active, e.g. B. Sibutramine, or similar substances
• - Pharmaceuticals for the treatment of chronic inflammatory processes or disorders of the immune system, e.g. B. Interferons in the treatment of viral hepatitis or interleukin 2 for HIV infection
• - Pharmaceuticals for the treatment of gestosis and Preeclampsia / eclampsia and HELLP syndrome
• - Pharmaceuticals for the treatment of fertility disorders or to eliminate cycle disorders in women
• - Pharmaceuticals for the treatment of irregular heartbeat
• - antidiabetic drugs (acarbose, insulin, troglitazone, Metformin, etc.)
• - hypnotics, antiemetics and antiepileptics
• - Pharmaceuticals to treat erectile dysfunction (Sildenafil, prostaglandin E1, apomorphine)

According to a second and third teaching, the invention relates to the use of a gene modification in the human G-protein subunit Gβ3 (gene: GNB3) for the discovery of new disease genes and for the discovery and development of new targets for drugs (drug targets) people in whom one of the named Polymorphisms are present in GNB3, e.g. B. A (-350) G, C825T, A3382C, G5177A, G5249A, C1429T or the CACA insert in intron 10 ( FIG. 1) or further gene changes in GNB3 or combinations of the alleles mentioned (haplotypes) are characterized in that in their body cells undergo a predictable increase or decrease in signal transduction via heterotrimeric G proteins (S. Virchow, N. Ansorge, D. Rosskopf, H. Rübben, and W. Siffert. The G protein beta3 subunit splice variant Gbeta3-s causes enhanced chemotaxis of human neutrophils in response to interleukin-8. Naunyn Schmiedebergs Arch. Pharmacol. 360 (1): 27-32, 1999. S. Virchow, N. Ansorge, H. Rübben, G. Siffert, and W. Siffert. Enhanced fMLP-stimulated chemotaxis in human neutrophils from individuals carrying the G protein beta3 subunit 825 T-alleles. FEBS Lett. 436 (2): 155-158, 1998. W. Siffert, D. Rosskopf, G. Siffert, S. Busch, A. Moritz, R. Erbel, AM Sharma, E. Ritz, HE Wichmann, KH Jakobs, and B. Horsthemke. Association of a human G-protein beta3 subunit variant with hypertension. Nat. Genet. 18 (1): 45-48, 1998).

The gene status can be determined by means of suitable, the expert demonstrate known methods. The prediposition for certain diseases in which an increased or decreased G protein activation is significant through different mechanisms.

• a) People who experience one or more of the gene changes mentioned increasingly form splice variants of the Gβ3 protein, e.g. B. the previously described variants Gβ3s-1 and Gβ3s-2, which were described in earlier patent applications. Such splice variants with at most 6WD repeats occur as functional proteins in human cells and can be expressed in a variety of expression systems using suitable vectors, e.g. B. in HEK-TS cells. G-protein βγ subunits can interact biochemically with a variety of cellular effector systems, e.g. B. with ion channels, phospholipase C isoforms, the PI3 kinase, the adenylyl cyclase, etc. (N. Gautam, GB Downes, K. Yan, and O. Kisselev. The G protein betagamma complex. Cell. Signal. 10 (7: 447-455, 1998) The splicing variants mentioned interact more intensely with such proteins, as is shown in FIG. 2 on the basis of the activation of the MAP kinase.
  First, HEK cells are transfected with a hemagglutin-labeled MAP kinase construct (HA-erk), the hemagglutin being used for the specific precipitation of the construct by specific antibodies. In the following it is examined whether the Gβ3s-1 splice variant has an increased activation of the MAP kinase compared to the wild type (Gβ3). For this purpose, the cells of the expression system are additionally transfected with vectors which code for Gβ3-s1 or Gβ3. The activation of the MAP kinase is quantified after immunoprecipitation using standard biochemical methods known to those skilled in the art. After immunoprecipitation, a significantly increased activation of the MAP kinase upon co-transfection with Gβ3-s1 can be seen, which is almost twice as strong as the activation after co-transfection with Gβ3 wild type. The underlying methods of transfection and the detection of the activation of such proteins by immunoprecipitation are carried out here using standard methods which are known to the person skilled in the art. The result shows that such splice variants of Gβ proteins with only 6WD repeats are able to activate certain interaction partners to an increased extent. This has the consequence that z. B. Chemicals are identified and pharmaceuticals are developed which specifically influence the interaction of Gβγ-dimers, which contain Gβ3s-1 or Gβ3s-2, with such effector proteins or other biochemically defined interaction partners (enzymes, tonal channels, etc.). Such pharmaceuticals can therefore inhibit or reduce the increased interaction of these splice variants with effector proteins and can therefore be used to treat a large number of disease states. The method mentioned can be extended to any other interaction partner of Gβγ subunits. In addition, different cellular transfection systems (bacteria, yeast or mammalian cells) and a large number of different vectors can be used, which are familiar to the person skilled in the art. In addition, the yeast two hybrid system known to the person skilled in the art or similar systems can be used to identify new interaction partners.
  In the example shown, suitable drug targets are identified by examining the interaction of Gβ3s and Gβ3s2 with known biochemical interaction partners and / or reaction pathways.
• b) In addition, the signal transduction via heterotrimeric G proteins not only causes a direct interaction of α- and βγ-subunits with specific components of the intracellular signal transduction, which lead to a rapid influencing of effector systems of a cell that does not require the transcription of genes, e.g. B. Opening or closing ion channels (P. Kofuji, N. Davidson, and HA Lester. Evidence that neuronal G-protein-gated inwardly rectifiying K ⁺ channels are activated by Gβγ subunits and function as heteromultimers. Proc. Natl. Acad. Sci. USA 92: 6542-6546, 1995). Rather, it is also known that G protein-mediated signal transduction can activate or inhibit the transcription of a large number of genes. For example, the expression of defined, constitutively active G protein α subunits leads to an increased activation of the prolactin promoter (J. Tian, J. Chen, and C. Bancroft. Expression of constitutively active G _S subunits in GH3 pituitary cells stimulates prolactin promoter activity. J. Biol. Chem. 269: 33-36, 1994). G-protein-βγ subunits also control the translocation of other effectors into the cell nucleus, which can then control transcription there (Metjian A, Roll RL, Ma AD, Abrams CS: Agonists cause nuclear translocation of phosphatidylinositol 3-kinase gamma. A Gbetagamma -dependent pathway that requires the p110gamma amino terminus. J Biol Chem 1999 Sep 24; 274 (39): 27943-7). An increased activatability of G proteins, which is present in carriers of the GNB3 825T allele and other polymorphisms and haplotypes of GNB3 and can be detected by genetic analysis, thus leads to an increased inhibition or suppression of the transcription of certain genes. This makes it possible to record differential gene activation (quantitative or qualitative) by genotyping and to identify and define new drug targets after identifying the increased, decreased or differentially expressed genes and after identifying the encoded gene products (e.g. proteins). The procedure required for this is described using two examples.

example 1

The applicant has previously described (cf. PCT / EP99 / 06534) that gene changes in the GNB3 gene are associated with an accelerated progression to AIDS disease, with an increased increase in human immunodeficiency virus also being observed in these individuals. This effect is caused by an increased activatability of heterotrimeric G proteins. The following method, which is shown schematically in FIG. 3, can now be used, for example, to identify new genes or increased or decreased transcribed genes.

In Fig. 3 of the drawing a strategy for finding new drug targets is shown based on the genotyping on GNB3 locus. The procedure that can be used to develop new drugs against HIV is shown as an example. For this purpose, cells from 825T allele carriers and C825 allele carriers are inoculated with HI virus and the virus multiplication is quantified. Reduced virus replication is found in 825T allele carriers. The total mRNA is extracted from the infected cells of 825T and C825 allele carriers and rewritten into cDNA. This is followed by a comparative quantitative and qualitative analysis of gene expression. Since genes 3, 4, and 5 are expressed equally, their gene products are eliminated as drug targets. In contrast, the differently expressed genes 1 and 2 are potential drug targets. Their gene products are identified and these can then be used for drug screening.

First, to produce an expression system, T-lymphocytes, for example CD ⁴⁺ -positive T-lymphocytes, or macrophages from homozygous C825 or 825T allele carriers are isolated and brought into culture. This is followed by an infection with HI virus (T or M tropic viruses) or suitable laboratory strains. After this infection, there is an increase in such viruses, which can be quantified by methods known to those skilled in the art, e.g. B. via quantitative detection of the virus genome by RT-PCR, Northern blot analysis or detection of p24 antigen. This shows that a reduced reproduction of HI virus in cells of homozygous 825T allele carriers can be observed in vitro. From this it can be concluded that if there is an increased signal transduction, the HIV virus replication is reduced in vitro, which is caused by a differential activation and transcription of genes in cells of 825T allele carriers in contrast to C825 allele carriers. Techniques for identifying the relevant genes that are increased, decreased or differentially activated are known to the person skilled in the art. For this purpose, e.g. B. quantitative RT-PCR methods, Northern blot analyzes, hybridization on DNA chips after reverse transcription, differential display, etc. can be used. In this way, genes and gene products can be identified which influence the propagation of the HI virus if a defined genotype or haplotype of the GNB3 gene is present. After identification of the gene products, chemicals, and therefore pharmaceuticals, can be developed which influence the transcription of the corresponding genes, their expression or the function of their gene products in the desired manner. It is therefore the subject of the invention to develop new drug targets by determining the gene status at the GNB3 locus.

Example 2

It is known that 825T allele carriers have an increased risk for the development of overweight and obesity (W. Siffert et al .: Worldwide ethnic distribution of the G protein beta3 subunit 825T alleles and its association with obesity in Caucasian, Chinese, and Black African individuals. J. Am. Soc. Nephrol. 10th (9): 1921-1930, 1999). It happens to those affected to a strong increase in body fat (R. A. Hegele, C. Anderson, T.K. Young, and P.W. Connelly. G protein beta3 subunit gene splice variant and body fat distribution in nunavut inuit. Genome Res. 9 (10): 972-977, 1999). The fat cells of 825T allele carriers show thereby an increased tendency to accumulate fat and a decreased lipolysis, and their preadipocytes show an increased tendency to proliferation and terminal differentiation. To identify the for Genes responsible for these processes become preadipocytes or adipocytes after genotyping accordingly Subjects using standard procedures to form a Expression system introduced into the cell culture. For example, the terminal differentiation can be too Adipocytes or an inhibition of lipolysis by adding of insulin into the culture medium. From the Preadipocytes or adipocytes of C825- or 825T- Allele carriers can be quantitatively different or different differential expression of genes for example reverse transcription of the mRNA to cDNA using quantitative PCR method, Northern blot method, differential display, hybridization on DNA chips or more similar, more suitable and known to the person skilled in the art Techniques are examined. The identification for example with 825T allele carriers increased or differentially expressed genes lead to identification  the gene products for the definition of such "drug targets", their inhibition by suitable pharmaceuticals of the increased Counteracts adipogenesis or reduced lipolysis. These can then be used therapeutically for medicinal purposes Treatment of obesity can be used.

Accordingly, the use of the invention Genotyping at the GNB3 locus and use and detection a new one of the described variants and haplotypes Procedures to ensure the creation of all of the responsible for the diseases mentioned Identify proteins, genes and their gene products and to develop suitable pharmaceuticals for their therapy.

The procedures described under a) and b) Determination of the activability of interaction partners or genes that are affected in their transcription can be derived from the gene for the β3 subunit of human G proteins transferred to other genes.

The invention further relates to the following teachings:

• - A gene that represents the intron sequence of intron 9 shown and Intron 10 with all possible permutations contains, for the purpose of introducing the gene into human or non-human cells or tissues to induce of alternative splicing and expression of Splice variants with the aim of increasing Induce signal transduction.
• - The production of antisense constructs or Medicines against the intron structure shown the goal of preventing alternative splicing.

Claims (16)

1. Use of a gene change in the gene for the β3- Subunit of the human G protein, with 9 in the exon Item 657 in Appendix 1 a substitution of adenine is present by thymine and / or in exon 10 Item 814 in Appendix 1 a substitution of guanine present by adenine and / or being in the promoter region at position (-350) in Appendix 1 a substitution of Adenine is present by guanine and / or in the intron 9 at position 3382 in Appendix 1 a substitution of adenine is present by cytosine and / or being in the intron 9 Item 5177 in Appendix 1 a substitution of guanine due to adenine and / or being in the intron 9 Item 5249 in Appendix 1 a substitution of guanine is present by adenine and / or in the intron 10 Item 6496 in Appendix 1 an insert consisting of the Nucleotides cytosine-adenine-cytosine-adenine is present for Prediction of physiological and pathophysiological Processes in the human body. 2. Use according to claim 1, characterized in that the risk of an illness is determined. 3. Use according to claim 1 or 2, characterized in that a course of the disease is predicted. 4. Use according to one of claims 1 to 3, characterized in that  that is the responsiveness of a subject to pharmaceuticals is predicted. 5. Use according to one of claims 1 to 4, characterized in that the side effects that occur in a subject the administration of a drug can be predicted. 6. Use according to one of claims 1 to 5, characterized in that a substitution in exon 10 at position 825 in Appendix 1 of cytosine by thymine and / or that in exon 11 at position 1429 in Appendix 1 a substitution of Cytosine by thymine is present. 7. Use of at least two expression systems with at least different proportions more different Expression of one or more genes, the Activation of an interaction partner by the differently encoded genes encoding proteins depending on the expression of the genes through the comparison the activation of the interaction partner in the Expression systems is determined. 8. Use according to claim 7, characterized in that which can be activated as a function of the genes identified interaction partner as drug target be used. 9. Use according to claim 7 or 8, characterized in that both expression systems with at least one Interaction partner of the different  pronounced genes encoded proteins or be injected. 10. Use of at least two expression systems, especially of cells with at least different ones Proportion of different characteristics of one or more Genes, with transcription dependent on other genes of the expression of the differently expressed genes by comparing the transcription in the Expression systems is determined. 11. Use according to claim 10, characterized in that the one transcription depends on the different pronounced genes as drug target be used. 12. Use according to one of claims 7 to 11, characterized in that as a differently expressed gene, the gene for the β3- Subunit of the G protein is used. 13. Use according to one of claims 7 to 12, characterized in that the expression systems are human Expression systems. 14. Use according to one of claims 7 to 13, characterized in that the expression systems chemically, e.g. B. hormonally, or be physically stimulated. 15. Use according to one of claims 7 to 14, characterized in that  the expression systems by pathogens, e.g. B. Viruses, get infected. 16. Use according to one of claims 7 to 15, characterized in that the different forms of the genes Transfection or injection is made.