EP1044268A1 - Nouvelles variantes de sequences du gene recepteur beta2-adrenergique humain et leur utilisation - Google Patents

Nouvelles variantes de sequences du gene recepteur beta2-adrenergique humain et leur utilisation

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Publication number
EP1044268A1
EP1044268A1 EP98966818A EP98966818A EP1044268A1 EP 1044268 A1 EP1044268 A1 EP 1044268A1 EP 98966818 A EP98966818 A EP 98966818A EP 98966818 A EP98966818 A EP 98966818A EP 1044268 A1 EP1044268 A1 EP 1044268A1
Authority
EP
European Patent Office
Prior art keywords
beta2
diseases
sequence
disposition
variants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98966818A
Other languages
German (de)
English (en)
Inventor
Margret Hoehe
Bernd Timmermann
Karla KÖPKE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Publication of EP1044268A1 publication Critical patent/EP1044268A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the invention relates to new sequence variants of the human beta2-adrenergic receptor gene and their use for the diagnosis of a spectrum of diseases, in particular for the determination of hypertension dispositions and for the development of therapeutic agents on the basis of pharmacogenetic principles.
  • the human beta2-adrenergic receptor is an important component of the sympathetic nervous system and, as such, regulates a spectrum of central and peripheral functions, e.g. Cardiovascular functions, metabolic functions, central nervous functions and neurosecretion. It is the target of pharmaceuticals / therapeutic agents with a wide range of indications, which are among the most commonly prescribed drugs.
  • This receptor could play a role in the pathogenesis / pathophysiology of a number of common diseases, e.g. hypertension and other cardiovascular diseases, various neuropsychiatric diseases such as Depression, and metabolic diseases such as Obesity (Insel PA (Ed) (1987) Adrenergic receptors in man, Marcel Dekker, New York, Basel).
  • the aim of the invention is to determine variants, polymorphisms, mutations and resulting haplotypes in the DNA sequence of the human beta2-adrenergic receptor gene and to determine their correlations with disease dispositions. Based on these correlations, a method for diagnosing these disease predispositions, for predicting severity, course and survival time, a system for predicting individual responsiveness to beta2 active therapeutic agents, for developing individually specific beta2 receptor agonists and antagonists, and a system for developing a new one Class of beta2 effective therapeutic agents, as well as the development of test systems for researching pathophysiological relationships and development of the above-mentioned therapeutic agents.
  • an individually optimal therapeutic agent can be predicted or developed for each beta2 genotype.
  • the object is achieved according to the claims, the subclaims are preferred variants. 2
  • the invention then relates to the sequence of the human beta2-adrenergic receptor gene, which is at positions 159, 245, 565, 934, 1120, 1221, 1541, 1568, 1633, 1666, 1839, 2078, 2110, 2640, and 2826 in whole or is partially mutated.
  • sequences are particularly important:
  • the invention furthermore relates to a method for determining disease dispositions, it being possible for all sequences and variants of the beta2-adrenergic receptor gene from the individual mutation to all possible combinations of all variants (including any absolute number of variants which can be included) to be genotyped and enable the corresponding statements about disease dispositions.
  • the method is characterized in that the DNA of a test subject is isolated and genotyped at least in one of the exchanged positions and subsequently compared with the reference DNA sequence.
  • Embodiments are preferred in which at least position 1633, at least the three positions 1541, 1633 and 1666 or the four last-mentioned positions (1541, 1568, 1633 and 1666) or at the seven positions 245, 565 934, 11541, 1568, 1633 and genotyped in 1666. 3
  • the method can also be varied by genotyping at least 3 of the 4 positions 1541, 1568, 1633 and 1666 and subsequently comparing them with the reference DNA sequence. Genotyping of positions 1541, 1633 and 1666 is preferred here.
  • Genotyping is performed by sequencing or by other methods that are suitable for the detection of point mutations. This includes PCR-based genotyping methods such as B. allele-specific PCR, other genotyping methods using ohgonucleotides (examples would be 'dot blotting' or 'oligonucleotide ligation assays' (OLA)), methods using restriction enzymes, and' single nucleotide polymorphism '(SNP) analysis using' matrix assisted laser desorption / ionization mass spectrometry (MALDI), as well as in principle any future method for variant detection including chip technology in all its technological versions.
  • PCR-based genotyping methods such as B. allele-specific PCR, other genotyping methods using ohgonucleotides (examples would be 'dot blotting' or 'oligonucleotide ligation assays' (OLA)), methods using restriction enzymes, and' single nucleotide polymorphism '
  • the method according to the invention is suitable for determining a broad spectrum of the most diverse disease dispositions.
  • a disposition for high blood pressure e.g. to determine a disposition for high blood pressure (or the prediction of the range of individual blood pressure values as such), and other cardiovascular diseases, including myocardial infarction and stroke, in the broadest sense the development of end-stage renal failure (requiring dialysis).
  • Another preferred embodiment variant allows e.g. the determination of a disposition for neuropsychiatric diseases such as depression and anxiety disorders, attention deficit disorder (with hyperactivity), eating disorders, e.g. for anorexia nervosa and bulimia, or disorders caused by post-traumatic stress; or for diseases of the autonomic nervous system, e.g. Bradbury-Eggleston, Sky-Drager and Riley-Day syndrome as well as selective noradrenergic and baroreceptor dispositions, or migraines.
  • neuropsychiatric diseases such as depression and anxiety disorders, attention deficit disorder (with hyperactivity), eating disorders, e.g. for anorexia nervosa and bulimia, or disorders caused by post-traumatic stress
  • diseases of the autonomic nervous system e.g. Bradbury-Eggleston, Sky-Drager and Riley-Day syndrome as well as selective noradrenergic and baroreceptor dispositions, or migraines.
  • Another area of application is the determination of a disposition for metabolic diseases such as obesity (as well as familial 'morbid obesity'), including a prediction of the weight range as such or a disposition for weight change, and finally a prediction of the ratio of the body measurements as such, as they are e.g. Express 'body mass index' (BMI). 4
  • the method also allows the course and severity of diseases to be determined, and the prediction of survival after serious medical diseases, e.g. after myocardial infarction, heart failure and / or stroke.
  • a further preferred embodiment variant enables the determination of an individually different reactivity of the autonomic nervous system, in particular on endogenous and exogenous stress (as expressed, for example, in particular by an individually different disposition to changes in blood pressure and / or heart rate (deflections) on endogenous and exogenous stress comes), or by individually different blood pressure changes to endogenously or exogenously induced changes in the salt concentration in the blood (individually different salt sensitivity or resistance), and in the broadest sense also by individually different salt and water regulation or reabsorption in the kidney (related volume regulation ) is expressed.
  • Another important object of the invention is the use of the claimed sequence variants a) for predicting the individually different responsiveness to previously known therapeutic agents (beta2 receptor ligands) and the individually different responsiveness to the endogenous ligands adrenaline and noradrenaline; b) preferably for the development of individually specific beta2 receptor agonists and antagonists; c) in particular also for the development of a new class of therapeutic agents which are directed to the beta2 receptor gene at the 5 'regulatory region, promoter region, in particular e.g. attack the leader peptide and act by regulating transcription, translation and by influencing their efficiency, primarily by regulating expression.
  • another object of the invention is the prediction of individual habituation to drug administration (tachyphylaxis), as well as a different disposition for drug side effects. Overall, it is possible to predict individually optimal therapeutic agents, which are based on different mechanisms of action.
  • kits or methods can advantageously be used to predict individual disease disposition or individual responsiveness to various beta2 therapeutics.
  • Cultures (cells) that express the various combinations of individual ß2 variants mentioned can thus serve as test models for the development of individually specific therapeutic agents (ß2 agonists and antagonists, as well as beta2 expression-regulating DNA therapeutic agents). This corresponds to test models in vitro, but also in vivo test models are included (transgenic animals that carry these individual receptor variants).
  • the three mutations described have a significant effect on phenotypic parameters such as heart rate, noradrenaline concentrations, blood pressure changes on experimentally induced physical and mental stress, 'coping styles' and personality dimensions, as well as weight and weight change.
  • phenotypic parameters such as heart rate, noradrenaline concentrations, blood pressure changes on experimentally induced physical and mental stress, 'coping styles' and personality dimensions, as well as weight and weight change.
  • an association of the leader peptide mutation with hypertension was also shown.
  • beta2-agonist-induced vasodilation and beta2 receptor mutations, preferably at position 16 of the amino acid sequence and a relationship between beta2-receptor expression in fibroblast cultures of genotyped individuals and beta2 receptor mutations, preferably at position 16 of the amino acid sequence, could be established.
  • Combination 1 1541 T (Cys Allel), 1568 T, 1633 A (Arg Allel), 1666 C (Gin Allel)
  • Combination 2 1541 C (Arg Allel), 1568 C, 1633 G (Gly Allel), 1666 G (Glu Allele)
  • combination 3 1541 T (Cys Allele), 1568 T, 1633 G (Gly Allele), 1666 C (Gin Allele)
  • Combination 1 was observed significantly more frequently in individuals who were inherited with hypertension and is therefore a genetic risk factor.
  • Detection of specific beta2 'haplotypes' consisting of seven variants Taking all variants into account, 'haplotypes' consisting of seven variants (including the three mutations mentioned) could be extracted; The calculations were based on the goal of identifying 'haplotypes' from the entirety of the genome which were sufficient to distinguish the patient group from the control group. A specific 'haplotype', combination 1, can be observed more frequently in cases of genetic hypertension, and this can be extended to other phenotypes.
  • Combination 1 245 G, 565 G, 934 A, 1541 T (Cys allele), 1568 T, 1633 A (Arg allele),
  • Combination 2 245 A, 565 A, 934 G, 1541 C (Arg allele), 1568 C, 1633 G (Gly allele),
  • Combination 3 245 G, 565 G, 934 G, 1541 T (Cys Allel), 1568 T, 1633 G (Gly Allel),
  • the multiplex PCR sequencing method was used for the collection of the entire polymorphic spectrum of the beta2-receptor gene.
  • the entire promoter region known to date and the coding region were divided into eight fragments and amplified by means of PCR (see FIG. 1). These PCR fragments were pooled and sequenced simultaneously.
  • the fragments of the terrination reactions were separated on a sequence gel and transferred to a nylon membrane by direct transfer electrophoresis (DTE).
  • DTE direct transfer electrophoresis
  • the individual sequence ladders were decoded by successive hybridization with specific oligonucleotides.
  • fragment II was amplified with hooves of the two primers ADRBR-F2: 5 ' -GCATACCCCCGCTCCAGATAAA-3 ' and ADRBR-R2: 5 ' -GCACGCACATACAGGCACAAATAC-3 ' .
  • fragment III it was the two primers ADRBR-F3: 5 ' -GGCCGCGTTTCTGTGTTGG-3 ' and ADRBR-R3: 5 ' -AGTGCGTTCTGCCCGTTATGTG-3 ' .
  • fragment VIII the two primers ADRBR-F8: 5 ' -GGTACTGTGCCTAGCGATAAC-3 ' and ADRBR-R8: 5 ' - TAAAATACCCCGTGTGAGCAAATAAGAG-3 ' .
  • the reaction conditions for these four fragments were as follows: 10 x PCR buffer (100 mM Tris-HCl, 15 mM MgCl 2 x 6 H 2 O, 500 mM KC1, pH 8.3), dNTP 2 mM, 30 ⁇ M Primer F, 30 ⁇ M primer R, 50 ng genomic DNA and 5 U of a Taq DNA polymerase. Three fragments were amplified with the following temperature profile: 94 ° C for 4 min; 35 cycles: 94 ° C 30 sec, 60 ° C 30 sec, 72 ° C 1 min and finally 72 ° C 10 min.
  • Fragment IV was generated using the two primers ADRBR-F4: 5 - GGGGAGGGAAAGGGGAGGAG-3 ' and ADRBR-R4: 5 ' -
  • TACCCTCAAGTTAAATAGTCTGTT-3 used.
  • the conditions for these two PCR reactions were as follows: 10 x PCR buffer (160 mM (NH 4 ) 2 SO 4 , 0.1% Tween-20, 500 mM KOH, pH), dNTP 2 mM, 30 ⁇ M Primer F , 30 ⁇ M Primer R, 50 ng genomic DNA and 4 U of a mixture of the Taq DNA polymerase and a thermostable inorganic pyrophosphatase from Thermits thermophilus.
  • Fragment V was amplified using the two primers ADRBR-F5: 5 - ATGCGCCGGACCACGAC-3 ' and ADRBR-R5: 5 - GTAGAAGGACACGATGGA-3, fragment VI using the two primers ADRBR-F6: 5 - GCTACTTTGCCATTACTTCACC-3 ' and ADRBR-R6: 5 ' -
  • AAATCTGGGCTCCGGCAGTAGATAAG-3 ' AAATCTGGGCTCCGGCAGTAGATAAG-3 ' .
  • These two fragments were amplified using Perkin Elmer's 'AmpliTaq Gold Kit'.
  • the temperature profile for these two fragments was as follows: 94 C 10 min; 35 cycles: 94 C 30 sec, 56 ° C [fragment V] or 58 ° C [fragment VI] 30 sec, 72 ° C 1 min and finally 72 ° C 10 min.
  • the sequencing was done using the 'Thermo Sequenase cycle sequencing kit' from Amersham.
  • the PCR primers described above were used as sequencing primers.
  • the sequencing was carried out in four multiplex pools.
  • Pool 1 contained the sequencing primers ADRBR-Fl, ADRBR-F3, ADRBR-F5 and ADRBR-F7;
  • Pool 2 the sequencing primers ADRBR-Rl, ADRBR-R3, ADRBR-R5 and ADRBR-R7.
  • the PCR fragments I, III, V and VII were used in both sequencing pools.
  • Pool 3 on the other hand, contained the sequencing primers ADRBR-F2, F4, F6 and F8;
  • Pool 4 the sequencing primers ADRBR-R2, R4, R6 and R8.
  • the fragments ⁇ , IV, VI and VIII were inserted into these two pools.
  • Parola AL and KobUa BK The peptide product of a 5 ' leader cistron in the beta 2 adrenergic receptor mRNA inhibits receptor synthesis. J Biol Chem. 269 (6): 4497-505 (1994).

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne de nouvelles variantes de séquences du gène récepteur bêta2-adrénergique humain et leur utilisation pour diagnostiquer un ensemble de maladies, notamment pour constater des prédispositions à l'hypertension artérielle, pour diagnostiquer une réactivité, différente selon les individus, à des agents thérapeutiques et pour développer des agents thérapeutiques sur la base de principes pharmacogénétiques.
EP98966818A 1997-12-30 1998-12-30 Nouvelles variantes de sequences du gene recepteur beta2-adrenergique humain et leur utilisation Withdrawn EP1044268A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19758401 1997-12-30
DE19758401 1997-12-30
PCT/DE1998/003818 WO1999037761A1 (fr) 1997-12-30 1998-12-30 Nouvelles variantes de sequences du gene recepteur beta2-adrenergique humain et leur utilisation

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EP1044268A1 true EP1044268A1 (fr) 2000-10-18

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EP (1) EP1044268A1 (fr)
AU (1) AU2510799A (fr)
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Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197505B1 (en) 1997-04-04 2001-03-06 Pyrosequencing Ab Methods for assessing cardiovascular status and compositions for use thereof
EP1121462A2 (fr) * 1998-10-14 2001-08-08 Eurona Medical AB Genes d'evaluation d'etat cardio-vasculaire et compositions d'utilisations associees
US6861217B1 (en) * 1998-11-25 2005-03-01 Genaissance Pharmaceuticals, Inc. Variation in drug response related to polymorphisms in the β2-adrenergic receptor
AU2001243526A1 (en) * 2000-03-10 2001-09-24 University Of Cincinnati Adrenergic receptor overexpression in airway tissues for the treatment of airwayobstructive diseases
EP1280814A4 (fr) * 2000-04-13 2003-05-14 Genaissance Pharmaceuticals Association d'haplotypes du recepteur adrenergique beta2 reagissant aux medicaments
US6586183B2 (en) 2000-04-13 2003-07-01 Genaissance Pharmaceuticals, Inc. Association of β2-adrenergic receptor haplotypes with drug response
EP1417338A4 (fr) * 2001-07-16 2005-06-29 Price Foundation Ltd Genes et polymorphismes nucleotidiques simples associes a des troubles du comportement alimentaire
CA2579574A1 (fr) * 2004-07-23 2007-01-04 The University Of North Carolina At Chapel Hill Procedes et materiaux pour determiner la sensibilite a la douleur et prevoir et traiter des troubles apparentes
SE0402198D0 (sv) * 2004-09-13 2004-09-13 Astrazeneca Ab Method
CN102154272B (zh) 2006-11-30 2014-03-12 爱科来株式会社 肥胖基因扩增用引物对、含有其的肥胖基因扩增用试剂及其用途

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Title
See references of WO9937761A1 *

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DE19860930A1 (de) 1999-09-16
AU2510799A (en) 1999-08-09

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