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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1841—Transforming growth factor [TGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • G01N2333/91205—Phosphotransferases in general
  • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

• the present invention relates to the use of a single-stranded or double-stranded nucleic acid containing a fragment of the hsgk for the diagnosis of hypertension, the said fragment being at least 10 nucleotides / base pairs long and the said fragment further comprising a polymorphism which results from the presence or Absence of an insertion of nucleotide G at position 732/733 in intron 2 of the hsgkl gene results.
• the present invention further relates to the use of the direct correlation between the overexpression or the functional molecular modification of human homologs of the sgk family and the length of the Q / T time for the diagnosis of Long Q / T syndrome, and the use of the nucleic acid a human homologue of the sgk gene family or one of its fragments for the diagnosis of Long-Q / T syndrome.
• polymorphisms of single nucleotides single nucleotides
• single nucleotide polymorphisms SNP
• the invention relates to the use of a functional activator or a transcription factor which increases the expression of the genes of the sgk family for the manufacture of a medicament for the therapy and / or prophylaxis of Long-Q / T syndrome.
• the human version of the sgk was cloned from liver cells (Waldegger et al., 1997). It was shown that the expression of the hsgkl is influenced by the regulation of the cell volume. Such a dependence on the cell volume has so far not been demonstrated for the expression of the rat sgk. Furthermore, it was found that the rat kinase stimulates the epithelial Na + channel (ENaC) (Chen et al, 1999; Naray-Pejes-Toth et al, 1999). The ENaC in turn plays a crucial role in renal Na excretion. An increased activity of the ENaC leads to an increased renal retention of sodium ions, and in this way to the development of hypertension, as shown by WO02 / 074987 A2.
• hsgk2 and hsgk3 Two other members of the human sgk gene family were cloned, the hsgk2 and hsgk3 (Kobayashi et al., 1999), both of which - like the hsgkl - are activated by Ins ⁇ lin and IGF1 via the PI3 kinase pathway. Electrophysiological experiments showed that coexpression of the hsgk2 and hsgk3 also resulted in a significant increase in the activity of the ENaC.
• hsgkl is a considerable diagnostic tool in many diseases in which cell volume changes play a decisive pathophysiological role, such as, for example, hypernatremia, hyponatremia, diabetes mellitus, renal insufficiency, hypercatabolism, hepatic encephalopathy and microbial or viral infections Has potential.
• hsgkl activates the endothelial Na + channel, which increases renal Na + absorption. Since this increased renal Na + absorption is associated with hypertension, it was assumed here that an increased expression of the hsgkl should lead to hypertension, a reduced expression of the hsgkl should ultimately lead to hypotension.
• DE 100 421 37 also describes a similar relationship between the overexpression or overactivity of the human homologues hsgk2 and hsgk3 with the overactivation of the ENaC, the resulting increased renal Na absorption and the hypertension that develops from this. Furthermore, the diagnostic potential of the kinases hsgk2 and hsgk3 regarding arterial hypertension has already been discussed.
• WO02 / 074987 A2 discloses the relationship between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgkl gene with a genetic predisposition to hypertension.
• SNP single nucleotide polymorphism
• the object of the present invention is therefore to provide a further polymorphism in the hsgkl gene, the occurrence of which in one version or the other may correlate even better with the phenotypic occurrence of hypertension in the patient than the two known polymorphisms in exon 8 and intron 6.
• the invention relates to the use of an isolated single- or double-stranded nucleic acid which contains a fragment of the nucleic acid sequence according to SEQ ID No. 1 or according to SEQ ID No. 2 comprises, for the diagnosis of hypertension, wherein said fragment is at least 10 nucleotides / base pairs, preferably at least 15 nucleotides / base pairs, in particular at least 20 nucleotides / base pairs long, and wherein said fragment has the polymorphism in intron 2 of the hsgkl gene either with or without the insertion of nucleotide G at position 732/733.
• SEQ ID No. 1 describes the genomic DNA sequence of the hsgkl without the insertion of the nucleotide G (or GTP) at position 732/733 in intron 2 of the hsgkl gene, which describes the so-called “wild type (WT)” sequence and SEQ ID No. 2 the genomic DNA sequence of the hsgkl with the insertion of the nucleotide G (or GTP) at position 732/733 in intron 2 of the hsgkl gene, the so-called “insertion G (InsG)” sequence.
• the present invention relates to a kit for diagnosing hypertension, comprising at least one isolated single- or double-stranded nucleic acid which contains a fragment of the sequence according to SEQ ID No. 1 or 2 includes.
• the said fragment from SEQ ID No. 1 or 2 is at least 10 nucleotides / base pairs, preferably at least 15 nucleotides / base pairs, in particular at least 20 nucleotides / base pairs.
• said fragment from SEQ ID No. 1 or 2 comprise the polymorphism in intron 2 of the hsgkl gene either with or without the insertion of nucleotide G at position 732/733.
• the kit for the diagnosis of hypertension in addition to or instead of the single or double-stranded nucleic acid mentioned above — may also contain at least one antibody which is directed against such a region of the hsgk protein whose presence in the hsgkl protein is dependent on the presence of the insertion of nucleotide G at position 732/733 in intron 2 of the corresponding coding hsgk gene.
• an antibody directed against precisely this spliced-out protein region could be used to detect the polymorphism version of the individual , With such an antibody, a predisposition to develop hypertension could therefore be diagnosed.
• the invention in a third aspect, relates to a method for diagnosing hypertension, which comprises the following method steps: a) taking a body sample from an individual, b) optionally isolating and / or amplifying genomic DNA, cDNA or mRNA from the body sample according to a) , c) Quantification of alleles which have an insertion of nucleotide G at position 732/733 in intron 2 of the hsgkl gene.
• a body sample is taken from a test individual, who is preferably a mammal, in particular a human.
• a test individual who is preferably a mammal, in particular a human.
• blood samples or also saliva samples are preferably used as body samples of the patient, which comprise cellular material and can be obtained by the patient with relatively little effort.
• body samples that also include cells, such as tissue or cell samples, etc., can also be used.
• step b either genomic DNA or cDNA or also mRNA is prepared from the body sample from a) using standard methods (Sambrook-J and Russell-DW (2001) Cold Spring Harbor, NY, CSHL Press) and / or optionally amplified. All suitable methods known to the person skilled in the art can be used here.
• This DNA isolation step or DNA amplification step can optionally also be dispensed with, in particular if detection methods are used in step c) which themselves contain a PCR amplification step.
• step c) the number of alleles is finally quantified which have an insertion of the nucleotide G at position 732/733 in intron 2 of the hsgkl gene.
• Individuals who have two WT alleles are likely to be predisposed to training of hypertension.
• the quantification / identification of the alleles with regard to the polymorphism at position 732/733 in intron 2 of the hsgkl gene can be carried out using various methods which are known to the person skilled in the art. Some preferred methods are explained in more detail below. However, the quantification of the number of alleles that have an insertion of nucleotide G at position 732/733 in intron 2 of the hsgkl gene is not limited to the preferred methods below.
• the genotype (or the number of alleles) can be identified with respect to the polymorphism at position 732/733 by direct sequencing of the DNA, preferably the genomic DNA, from the body sample at said position 732/733 in intron 2 of the hsgkl gene ,
• short oligonucleotides with sequences from the vicinity of position 732/733 of the hsgkl gene must be made available as sequencing primers according to known sequencing methods.
• Another, likewise preferred method for identifying the genotype (or for quantifying the number of alleles) with regard to the polymorphism at position 732/733 are all known methods which are based on the hybridization of the genomic DNA from the body sample with specific hybridization probes.
• An example of such a hybridization method is e.g. the southern blot. If, for example, the presence of the G insert at position 732/733 in intron 2 of the hsgkl gene destroys or also forms an interface for a restriction endonuclease, nucleic acid fragments with lengths that differ from the corresponding fragment lengths in the WT alleles differ. A specific genotype could thus be detected with regard to the polymorphism in question at position 732/733.
• the genotype for the polymorphism in question at position 732/733 could also be determined with the help of a specific hybridization probe Splice variant missing exon can be detected.
• Another example of a hybridization method is the hybridization of the genomic DNA from the body sample with a labeled, single-stranded oligonucleotide, preferably 15-25 nucleotides in length, which either has a G insertion at position 732/733 or does not have it. Under very specific hybridization conditions that can be tested experimentally for each individual oligonucleotide by known methods, a completely hybridizing oligonucleotide can then be distinguished from an oligonucleotide with a single base mismatch.
• an oligonucleotide could be provided which contains the sequence of a fragment from SEQ ID No. 2 and at its 3 'end has the G at polymorphism position 732/733. If this oligonucleotide was hybridized with a sample fragment of the WT allele (without G insertion), this could not be extended and ultimately amplified in a subsequent PCR reaction due to the mismatch at the 3 'end.
• a ligation assay is ultimately based on the same principle as the PCR oligonucleotide elongation assay: only those double-stranded nucleic acid fragments can be ligated to another double-stranded nucleic acid fragment that have an exact base pairing at the end.
• the occurrence of a specific ligation product can therefore be made dependent on the presence or absence of the G insertion at position 732/733 in intron 2 of the hsgkl gene.
• the Q, R and S waves that can be detected with the ECG measuring device represent measured values for assessing the spread of excitation.
• the so-called Q / T time is defined as the time that elapses with the help of an EKG measuring device from the start of the spread of the T - Wave (the occurrence of the Q deflection) is to be detected until the end of the excitation propagation, which is characterized by the end of the T wave.
• the Q / T time thus represents the time that passes between the beginning of a new state of excitation of the heart and the return to the resting state. A significantly longer Q / T time therefore leads to cardiac arrhythmias and ultimately to the Long Q / T syndrome mentioned above.
• the invention therefore furthermore relates to the use of the direct correlation between the overexpression or the functional molecular modification of human homologs of the sgk family, in particular the hsgkl gene, and the length of the Q / T time for diagnosing the Long-QT syndrome.
• a human homologue of the sgk family which comprises a functional molecular modification in the above sense, means a homologue of the sgk family which is mutated in such a way that the properties, in particular the catalytic properties or the substrate specificity of the corresponding protein can also be changed.
• Equipment of the human homologues of the sgk family implies that individual mutations in the genes hsgkl, hsgk2 or hsgk3 could occur in individual patients, which alter the level of expression or the functional properties of the kinases hsgkl, Modify hsgk2 or hsgk3, and thus lead to a genetically caused extension of the Q / T time and ultimately to a predisposition to the development of Long Q / T syndrome.
• Such mutations could occur, for example, in the regulatory gene regions or also in intron sequences of the sgk gene locus.
• individual differences in the genetic makeup of the sgk locus could also affect the coding gene area.
• Mutations in the coding region could then possibly lead to a functional change in the corresponding kinase, for example to modified catalytic properties of the kinase, which ultimately also influence the Q / T time. Accordingly, both types of mutation described above could cause an extension of the Q / T time and thus ultimately the predisposition to the development of the Long Q / T syndrome.
• SNP single nucleotide polymorphisms
• SNPs in the intron region or in regulatory sequences of the hsgk genes can, in their mutated version, possibly lead to a change in the expression level of the corresponding kinase.
• SNPs in the intron region could also lead to a functional modification of the kinase if they influence the alternative splicing of the immature mRNA.
• Another object of the invention relates to the use of a single- or double-stranded nucleic acid which comprises the sequence of a human homologue of the sgk family or one of its fragments, in particular the hsgkl gene itself or one of its fragments, for the diagnosis of a predisposition to the formation of the long -Q / T syndrome.
• the single- or double-stranded nucleic acid is preferably at least 10 nucleotides / base pairs in length.
• certain antibodies which are directed against substrates of the human homologues of the sgk family, in particular against substrates of the hsgkl, are also suitable for diagnosing a predisposition to the formation of Long-Q / T syndrome and hypertension.
• This diagnostic Antibodies are preferably directed against such an epitope of the human homologues of the sgk family, in particular the hsgkl, which contains the phosphorylation site of the substrate either in phosphorylated form or in non-phosphorylated form.
• the ubiquitin protein ligase Nedd4-2 (Acc No. BAA23711) is used as the substrate of the human homologue of the sgk family.
• This ubiquitin protein ligase is a protein which is specifically phosphorylated by the human homologues of the sgk family [Debonneville et al., Phosphorylation of Nedd4-2 by Sgkl regulates epithelial Na (+) channel cell surface expression. EMBO J., 2001; 20: 7052-7059; Snyder et al., Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na (+) channel. J. Biol.
• Phosphorylation sites for the hsgkl have the consensus sequence (R X R X X S / T), where R is arginine, S is serine, T is threonine and X is any amino acid.
• Nedd4-2 Acc No. BAA23711
• the abovementioned antibodies for diagnosing a predisposition to the formation of the Long-Q / T syndrome are therefore preferably directed against the substrate Nedd4-2 and particularly preferably against a protein region of Nedd4-2 with the sequence of the potential phosphorylation site for the hsgkl, the consensus Sequence (RXRXXS / T).
• these antibodies are directed against those Nedd4-2 protein regions which comprise at least one of the two potential phosphorylation sites serine at amino acid position 382 and / or serine at amino acid position 468.
• kits for the diagnosis of Long-QT syndrome or other diseases which manifest themselves in an extension of the Q / T time preferably contains antibodies, or in particular, directed against the human homologs of the sgk protein family
• kits can also contain antibodies that are directed against the human homologs of the sgk protein family and nucleic acids that hybridize with the human homologues of the sgk gene family under stringent conditions.
• the kit according to the invention for diagnosing Long-Q / T syndrome can also be antibodies which are directed against the hsgkl protein, or
• hybridization under stringent conditions is understood to mean hybridization under such hybridization conditions with regard to the hybridization temperature and formamide content of the hybridization solution as is described in the relevant specialist literature (Sambrook-J and Russell-DW (2001) Cold Spring Harbor, NY, CSHL Press) has been described.
• the diagnostic kit can contain single or double-stranded nucleic acids as hybridization probes that have a sequence according to SEQ ID No. Have 1 or 2 which are at least 10 nucleotides / base pairs long and which comprise the polymorphism at position 732/733 in intron 2 of the hsgkl gene either with or without the insertion of the nucleotide G.
• those antibodies are provided which are specifically directed against regions of the hsgkl protein whose presence in the hsgkl protein depends on the presence of the G insertion at position 732/733 in intron 2 of the hsgkl gene.
• regions which are alternatively spliced out in the immature mRNA due to the presence or absence of this G insertion and therefore are not present in the mature mRNA and in the protein resulting therefrom are suitable as immunogenic epitopes against which diagnostic antibodies are directed can.
• nucleic acid regions of the hsgkl gene are also particularly suitable as diagnostic hybridization probes which can hybridize with such a gene region spliced out at position 732/733 depending on the G insertion.
• the kit for diagnosing Long-Q / T syndrome can preferably also contain such nucleic acid fragments as specific hybridization probes that the known SNPs in the hsgkl gene, in particular the SNP in exon 8 (C2617T, D240D), the SNP in intron 6 (T2071C ) and / or the SNP in intron 2 at position 732/733 (insertion of G).
• SNPs in the hsgkl gene in particular the SNP in exon 8 (C2617T, D240D), the SNP in intron 6 (T2071C ) and / or the SNP in intron 2 at position 732/733 (insertion of G).
• a “functional activator” is to be understood as a substance that activates the physiological function of the corresponding kinase of the sgk family.
• a “positive transcription regulator” is to be understood as a substance that activates the expression of the corresponding kinase of the sgk family.
• Another object of the invention thus relates to the use of a functional activator or a positive transcription regulator of a human homologue of the sgk family, in particular the hsgkl, for lowering the Q / T time and in particular for the therapy and / or prophylaxis of the Long-QT syndrome .
• Known functional activators and / or positive transcription regulators of the human human homologues of the sgk family, in particular the hsgkl are glucocorticoids, mineral corticoids, aldosterone, gonadotropins, and a number of cytokines, in particular TGF-ß.
• the invention therefore further relates to the use of substances selected from the group consisting of glucocorticoids, mineral corticoids, aldosterone, gonadotropins and cytokines, in particular TGF- ⁇ , for the manufacture of a medicament for the therapy and / or prophylaxis of Long-QT syndrome.
• the invention further relates to a medicament containing a substance selected from the group of substances mentioned above for the therapy and / or prophylaxis of the Long-Q / T syndrome.
• test subjects were all members of the German-Caucasian breed and came from different parts of Germany. Blood was taken from the twins and their parents for verification of the bipolar structure and for further molecular genetic analyzes. Each participating subject was previously examined by a doctor. No chronic medical illness was known to any of the test subjects. After 5 min, the blood pressure of the test person in the sitting position was measured by a trained doctor using a standardized mercury sphygmomanometer (2 measurements with a time interval of 1 min). The mean of the two measurements was used as the blood pressure value.
• dizygot twins for correlation studies is that they match in age and that the external influences on their phenotypes can be considered minimal (Martin et al., Nat Genet 1997, 17: 387-392).
• PCR polymerase chain reaction
• hsgkl gene For the molecular genetic analysis of the target gene, here the hsgkl gene, three further microsatellite marker regions (d6s472, d6sl038, d6s270) in the immediate vicinity of the hsgkl locus were amplified by PCR and then compared with the corresponding samples from the other twin and the parents , In this way it was possible to decide whether the twins had inherited identical or different alleles from their parents with regard to the allele examined.
• the correlation analysis was carried out using the so-called "structural equation modeling" (SEM) model (Eaves et al., Behav Genet 1996, 26: 519-525; Neale, 1997: Mx: Statistical modeling.
• SEM structural equation modeling
• This model is based on variance-covariance matrices of the test pairs by the probability of them either none, one or two identical alleles are characterized.
• the variance regarding the phenotype was divided into a variance based on the genetic background of all genes (A), a variance based on the genetic background of the target gene (Q), here the hsgkl gene, and the variance due to external influences (E ) is based.
• VAR A 2 + Q 2 + E 2
• the differences between models that take the genetic variance with respect to the target gene hsgkl into account or not take it into account were calculated as ⁇ 2 statistics.
• the allele ratios for each pair and each locus were calculated using the so-called "multipoint" model (MAPMAKER / SIBS; Kruglyak et al., Am J Hum Genet 1995, 57: 439-454) based on the parental genotypes.
• the low values for the error probabilities p determined which do not or only slightly exceed the accepted error probability of p ⁇ 0.01, prove the direct correlation between the genetic variance with regard to the hsgkl locus and the phenotypically determined variance the measured blood pressure.
• the SNPs in the hsgkl gene published in databases were first examined to determine whether they were real SNPs - and not pure sequencing errors - and whether the SNPs were sufficient are polymorphic to provide the basis for diagnostic evidence of a predisposition to hypertension.
• Blood samples were taken from a sample of the 75 pairs of twins. After amplification of the genomic DNA of the hsgkl gene from the blood samples by means of PCR the exons and introns (but not the promoter region) of the hsgkl gene were sequenced directly and completely using suitable sequencing primers. In a sequence comparison of the hsgkl genes, which originated from different subjects, a further polymorphism in intron 2 was noticed, consisting of the insertion of an additional nucleotide G in position 732/733.
• test subjects showed this the rarer WT / WT genotype significantly shorter Q / T times than heterozygous WT / InsG subjects and these in turn significantly shorter Q / T times than subjects with the more common InsG / InsG genotype (see Table 3). Longer Q / T times increase the risk of developing cardiac arrhythmias, in particular the Long Q / T syndrome.

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