EP1100961A1 - Polymorphismes genetiques dans le gene du recepteur humain des neurokinines 2 et leur utilisation dans le diagnostic et traitement de maladies - Google Patents

Polymorphismes genetiques dans le gene du recepteur humain des neurokinines 2 et leur utilisation dans le diagnostic et traitement de maladies

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Publication number
EP1100961A1
EP1100961A1 EP99934891A EP99934891A EP1100961A1 EP 1100961 A1 EP1100961 A1 EP 1100961A1 EP 99934891 A EP99934891 A EP 99934891A EP 99934891 A EP99934891 A EP 99934891A EP 1100961 A1 EP1100961 A1 EP 1100961A1
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Prior art keywords
seq
nucleic acid
acid sequence
nk2r
polymo
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EP99934891A
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German (de)
English (en)
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John Craig Smith
Rakesh Anand
John Edward Norris Morten
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AstraZeneca AB
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AstraZeneca AB
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Priority claimed from GBGB9816194.6A external-priority patent/GB9816194D0/en
Priority claimed from GBGB9816836.2A external-priority patent/GB9816836D0/en
Priority claimed from GBGB9905462.9A external-priority patent/GB9905462D0/en
Priority claimed from GBGB9910647.8A external-priority patent/GB9910647D0/en
Application filed by AstraZeneca AB filed Critical AstraZeneca AB
Publication of EP1100961A1 publication Critical patent/EP1100961A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • 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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to novel sequence and polymorphisms in the human neurokinin 2 receptor (NK2R) gene, including its promoter region.
  • the invention also relates to methods 5 and materials for analysing allelic variation in the NK2R gene and its promoter region, and to the use of NK2R polymorphism in the diagnosis and treatment of NK2R ligand-mediated diseases such as asthma, depression, anxiety, emesis and urinary incontinence.
  • the reader is referred to the following publications for background information: The neurokinin A (substance K) receptor, Gerard et al, J.Biol.Chem 265, 20455-20462 (1990);
  • the NK2R gene is organised into five exons interrupted by 4 introns. Gerard et al,
  • exon 2 and exon 4 and flanking sequence have been published by EMBL as follows: Exon 2, Accession Number M75102, 632 bp; Exon 4, Accession Number
  • SEQ ID No. 1 (1414 bp) identifies the revised and new sequence of, and flanking, exon 3 of the human NK2R gene.
  • the novel sequence itself, or parts thereof, can be used, inter alia, as a hybridisation probe to identify clones harbouring the NK2R gene, for use in genetic linkage studies or for design and use as amplification primers suitable, for example, to amplify the complete NK2R gene with or without its promoter element using an amplification reaction such as the PCR.
  • the full extent of known sequence upstream of the initiating ATG codon is disclosed in Graham et al, (Biochem Biophys Res Commun. 177:8-16,1991; EMBL Accession Number M75101).
  • the inventors have extended this known sequence upstream by approximately 870 bases, and have identified in excess of 20 differences with the documented upstream sequence disclosed in Graham et al.
  • the novel promoter sequence itself, or parts thereof can be used, inter alia, as a hybridisation probe to identify clones harbouring the NK2R gene and/or promoter, for use in genetic linkage studies or for design and use as amplification primers suitable, for example, to amplify the complete NK2R gene and promoter element using an amplification reaction such as the PCR.
  • the promoter element is also useful for heterologous expression studies as well as for homologous NK2 receptor expression.
  • the sequence can be used as a target to identify compounds that modify (i.e. inhibit or enhance) expression from the promoter.
  • SNPs in the NK2R gene have been described. There is an SNP in exon 1 which leads to a variation at amino acid 23 in the NK2R of either isoleucine or threonine (Graham et al, Biochem Biophys Res Commun 177:8-16, 1991). There is an SNP in exon 5 which results in a variation in the polypeptide sequence at amino acid 375 of either an arginine or a histidine (Graham et al, Biochem Biophys Res Commun 177:8-16, 1991).
  • the present invention is based on the discovery of novel promoter sequence of the human NK2 receptor (SEQ ID No. 6, positions 1-874 of SEQ ID No 5) as well as revised promoter sequence (SEQ ID No. 7) and ten novel single nucleotide polymorphisms (SNPs) within the promoter region. Relative to SEQ ID No. 5, the promoter SNPs are located at nucleotide position: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304.
  • the presence of one or more of these polymorphisms in the promoter region may have an impact on NK2R gene expression and thus on the amount of NK2 receptor within cells.
  • the efficacy of an agonist or antagonist therapeutic will likely depend on the amount of receptor available for interaction.
  • the present invention is also based on the discovery of three novel SNPs in the NK2R gene, and novel sequence (as well as revised sequence) at and around exon 3 of the human NK2 receptor gene.
  • One SNP has been found in the intron sequence flanking exon 2 at position 423, as defined by the position in EMBL accession number M75102
  • another SNP has been found in the intron sequence flanking exon 3 at position 553, according to SEQ ID No. 1 herein (corresponding to position 54, in EMBL accession number M75103); and, the other SNP has been found in the intron sequence flanking exon 4 at position 72, as defined by the position in EMBL accession number M75104.
  • each of the polymorphisms (emboldened; only the unpublished allele illustrated) and sequence immediately flanking each polymorphism site is as follows: a) (exon 2 flank polymorphism) GGATTGTGGAGGACAACAGTGTGTG (SEQ ID No. 9) b) (exon 3 flank polymorphism) GGGAGGCGCC'AGCGTGGGCCCGCAG (SEQ ID No. 10) c) (exon 4 flank polymorphism) CCCAGGTGGGTGTAAGGGGTCCTCT (SEQ ID No. 11) d) (position 153 promoter polymorphism) AATGAAGAACATGGCCCCCCCGACT (SEQ ID No.
  • the positions of the promoter polymorphisms are relative to SEQ ID No. 5.
  • a method for the diagnosis of a single nucleotide polymorphism in NK2R in a human comprises determining the sequence of the nucleic acid of the human at one or more of positions 423 (as defined by the position in EMBL accession number M75102), 553 (according to SEQ ID No.
  • a method for the diagnosis of a single nucleotide polymorphism in NK2R in a human comprises determining the sequence of the nucleic acid of the human at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (all relative to SEQ ID No. 5), and determining the status of the human by reference to polymorphism in the NK2R promoter.
  • a method for the diagnosis of one or more single nucleotide polymorphism(s) in NK2R gene in a human comprises determining the sequence of the nucleic acid of the human at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 , 1304 (each according to their position in SEQ ID No. 5 herein), 423 (according to the position in EMBL accession number M75102), 553 (according to the position in SEQ ID No. 1 herein) and 72 (according to the position in EMBL accession number M75104), and determining the status of the human by reference to polymorphism in the NK2R gene.
  • the term human includes both a human having or suspected of having a NK2R ligand-mediated disease and an asymptomatic human who may be tested for predisposition or susceptibility to such disease. At each position the human may be homozygous for an allele or the human may be a heterozygote.
  • the NK2R gene includes exon coding sequence, intron sequences intervening the exon sequences and, 3' and 5' untranslated region (3' UTR and 5' UTR) sequences, including the promoter element of the NK2R gene.
  • the method for diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 423 (according to the position in EMBL accession number M75102) is the presence of G and/or A.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 553 (according to the position in SEQ ID No. 1) is the presence of G and/or C.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 72 (according to the position in EMBL accession number M75104) is the presence of C and or T.
  • the method for diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 153 (according to the position in SEQ ID No. 5) is insertion of G.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 159 (according to the position in SEQ ID No. 5) is the presence of C and/or G.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 384 (according to the position in SEQ ID No. 5) is the presence of G and/or A.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 719 (according to the position in SEQ ID No. 5) is the presence of G and/or C.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 921 (according to the position in SEQ ID No. 5) is the presence of T and/or C.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 956 (according to the position in SEQ ID No. 5) is the presence of C and/or T.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 1002 (according to the position in SEQ ID No. 5) is the presence of T and/or C.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 1010 (according to the position in SEQ ID No. 5) is the presence of C and/or T.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 1158 (according to the position in SEQ ID No. 5) is the presence of G and/or A.
  • the method of diagnosis described herein is one in which the single nucleotide polymo ⁇ hism at position 1304 (according to the position in SEQ ID No. 5) is the presence of C and/or T.
  • the inventors have noted linkage between the polymo ⁇ hism at position 153 (-/+ G) and that at position 384 (G/A), such that if a G residue is inserted at position 153 (according to the position in SEQ ID No. 5), an A residue will be present at position 384 (according to the position in SEQ ID No. 5).
  • the method for diagnosis is preferably one in which the sequence is determined by a method selected from amplification refractory mutation system (ARMSTM-allele specific amplification), allele specific hybridisation (ASH), ohgonucleotide ligation assay (OLA) and restriction fragment length polymo ⁇ hism (RFLP).
  • a method for the diagnosis of NK2R ligand-mediated disease comprises: i) obtaining sample nucleic acid from an individual; ii) detecting the presence or absence of a variant nucleotide at one or more of positions 423 (as defined by the position in EMBL accession number M75102), 553 (according to SEQ ID No. 1 herein), and 72 (as defined by the position in EMBL accession number M75104), in the NK2R gene; and, iii) determining the status of the individual by reference to polymo ⁇ hism in the NK2R gene.
  • a method for the diagnosis of NK2R ligand-mediated disease comprises: i) obtaining sample nucleic acid from an individual, ii) detecting the presence or absence of a variant nucleotide at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 according to SEQ ID No. 5, in the NK2R promoter; and, determining the status of the individual by reference to polymo ⁇ hism in the NK2R promoter.
  • a method for the diagnosis of NK2R ligand-mediated disease comprises: i) obtaining sample nucleic acid from an individual, ii) detecting the presence or absence of a variant nucleotide at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158, 1304 (each according to the position in SEQ ID No. 5 herein), 423 (according to the position in EMBL accession number M75102), 553 (according to the position in SEQ ID No. 1 herein), and 72 (according to the position in EMBL accession number M75104), in the NK2R; and, determining the status of the individual by reference to polymo ⁇ hism in the NK2R gene.
  • EMBL accession M75101 contains exon 1, 258bp of intron 1 sequence and upstream sequence (including some promoter sequence)
  • EMBL accession number M75102 contains exon 2 and flanking sequences
  • EMBL accession number M75103 contains exon 3 and flanking sequences
  • EMBL accession number M75104 contains exon 4 and flanking sequences.
  • the variants discovered in the present invention are at position 423 in sequence M75102, position 553 (according to the position in SEQ ID No. 1 herein), position 72 in sequence M75104, and positions 153, 159, 384, 719, 921, 956, 1002, 1010, 5 1158, 1304 (each according to the position in SEQ ID No. 5 herein).
  • Allelic variation at position 423 in sequence M75102 consists of a single base substitution from G (the published base), for example to A.
  • Allelic variation at position 553 (according to SEQ ID No. 1) consists of a single base substitution from G (the published base), for example to C.
  • Allelic variation at position 72 in M75104 consists of a single base 0 substitution from C (the published base), for example to T.
  • Allelic variation at position 153 in SEQ ID No. 5 consists of a single base addition of, for example a G between nucleotides 153 (T) and 154 (G) of the sequence depicted in SEQ ID No. 5.
  • Allelic variation at position 159 (according to SEQ ID No.
  • NK2R promoter polymo ⁇ hisms and their locations in SEQ ID No.
  • the inventors have identified the following SNPs: C159G, G384A, G719C, T921C, C956T, T1002C, CIOIOT, G1158A and C1304T.
  • the 'normal' residue at a particular position is identified as such herein on the basis of it being the most common residue at that position amongst the limited number of individuals tested. It is to be appreciated that this is merely an arbitrary designation.
  • the invention resides in the 5 identification of the existence of different alleles at particular loci. The status of the individual may be determined by reference to allelic variation at one or more of the above loci.
  • a haplotype is a set of alleles found at linked polymo ⁇ hic sites (such as within a gene) on a single (paternal or maternal) chromosome. If recombination within the gene is random, 0 there may be as many as 2 n haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs.
  • One approach to identifying mutations or polymo ⁇ hisms which are correlated with clinical response is to carry out an association study using all the haplotypes that can be identified in the population of interest.
  • the frequency of each haplotype is limited by the frequency of its rarest allele, so that SNPs with low frequency alleles are particularly useful as markers of low frequency haplotypes.
  • low frequency SNPs may be particularly useful in identifying these mutations (for examples see: Linkage disequilibrium at the cystathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma levels of homocysteine.
  • CBS cystathionine beta synthase
  • vWF von willebrand factor
  • test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before use in the analysis of sequence variation.
  • allelic variation requires a mutation discrimination technique, optionally an amplification reaction and a signal generation system.
  • Table 1 lists a number of mutation detection techniques, some based on the PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in Table 2. Further amplification techniques are listed in Table 3. Many current methods for the detection of allelic variation are reviewed by Nollau et al., Clin. Chem.
  • Fluorescence Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)
  • Preferred mutation detection techniques include ARMSTM-allele specific amplification, ALEXTM, COPS, Taqman, Molecular Beacons, RFLP, OLA, restriction site based PCR and FRET techniques. Particularly preferred methods include ARMSTM-allele specific amplification, OLA and RFLP based methods.
  • the allele specific amplification technique known in the art as ARMSTM is an especially preferred method.
  • ARMSTM- allele specific amplification (described in European patent No. EP-B- 332435, US patent No. 5,595,890 and Newton et al. (Nucleic Acids Research, Vol. 17, p.2503; 1989)), relies on the complementarity of the 3' terminal nucleotide of the primer and its template.
  • the 3' terminal nucleotide of the primer being either complementary or non- complementary to the specific mutation, allele or polymo ⁇ hism to be detected.
  • primer extension from the primer whose 3' terminal nucleotide complements the base mutation, allele or polymo ⁇ hism. Those primers which have a 3' terminal mismatch with the template sequence severely inhibit or prevent enzymatic primer extension.
  • Polymerase chain reaction or unidirectional primer extension reactions therefore result in product amplification when the 3' terminal nucleotide of the primer complements that of the template, but not, or at least not efficiently, when the 3' terminal nucleotide does not complement that of the template.
  • the mammalian tachykinins also referred to as neurokinins, are a family of peptides consisting of substance P, neurokinin A and neurokinin B. They are found widely distributed throughout the central and peripheral nervous system, where they function as neurotransmitters and neuromodulators. They have been implicated in a number of biological processes including pain transmission, smooth muscle contraction, vasodilation and activation of the immune system. Therapeutic intervention in the action of the neurokinins is believed to be beneficial in a number of disease conditions, for a review see: Longmore et al (1997) Can. J. Physiol. Pharmacol. 75, 612-621. A number of pharmaceutical companies are researching neurokinin antagonists. NK1 antagonists have been explored by Glaxo, Pfizer, Merck, Parke-Davis, Lilly,
  • NK2 antagonists have been explored by a number of pharmaceutical companies including Glaxo Wellcome, Merck, Parke-Davis, Pfizer, Sanofi and Yamanouchi, primarily for CNS indications. For examples of NK2 antagonists see Swain (1996) Exp. Opin. Ther. Patents 6(4), 367-378.
  • dual NK1/ NK2 receptor antagonist will have some clinical utility, particularly for asthma. As compared with conventional therapies, it is expected that a dual NK1/NK2 receptor antagonist will better control airways hyperresponsiveness and neurogenic inflammation (extravasation and hypersecretion), both of which are characteristic manifestations of asthma. This multifaceted approach improves upon other therapies that are designed to treat only a single clinical manifestation of this disease. Other therapeutic opportunities for NK1/NK2 receptor antagonist exist in pain, migraine, anxiety, depression, urinary incontinence, and inflammatory bowel disease.
  • NK1/NK2 receptor antagonists Two of the compounds are peptides: FK-224 (Fujisawa) and SI 6474 (Servier). The other two, MDL- 105,212 (Marion Merrell Dow) and a recent compound from Merck, are structurally related to the selective NK2 antagonist, SR48968 (Sanofi). Neurokinin receptor antagonists have been reviewed by C J Swain (1996) in Exp. Opin. Ther. Patents, 6, 367; and by Elliot & Seward (1997) in Exp. Opin. Ther. Patents, 7, 43.
  • the diagnostic methods of the invention are used to assess the efficacy of therapeutic compounds in the treatment of asthma, depression, anxiety, urinary incontinence, emesis and other NK2R ligand-mediated diseases.
  • the polymo ⁇ hisms identified in the present invention occur in the flanking intron sequence of exon 2, the flanking intron sequence of exon 3, the flanking intron sequence of exon 4 and the promoter region of the NK2R gene.
  • the changes are not expected to alter the amino acid sequence of the NK2R, but may affect the transcription and/or message stability of the sequences and thus affect the level of the receptor in cells.
  • Assays for example reporter-based assays, may be devised to detect whether one or more of the above polymo ⁇ hisms affect transcription levels and/or message stability.
  • NK2R polymo ⁇ hism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity of the NK2R.
  • the polymo ⁇ hisms may also affect the response to agents acting on other biochemical pathways regulated by a NK2R ligand. The diagnostic methods of the invention may therefore be useful both to predict the clinical response to such agents and to determine therapeutic dose.
  • the diagnostic methods of the invention are used to assess the predisposition and/or susceptibility of an individual to diseases mediated by an NK2R ligand.
  • NK2R gene polymo ⁇ hism may be particularly relevant in the development of asthma, depression, anxiety, urinary incontinence, emesis and other diseases modulated by an NK2R ligand.
  • the present invention may be used to recognise individuals who are particularly at risk
  • the diagnostic methods of the invention are used in the development of new drug therapies which selectively target one or more allelic variants of the NK2R gene. Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design
  • Drugs may be designed to regulate the biological activity of variants implicated in the disease process whilst minimising effects on other variants.
  • the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes.
  • nucleic acid comprising any one of the following polymo ⁇ hisms:
  • nucleic acid comprising any one of the following polymo ⁇ hisms: the nucleic acid sequence with G insertion at position 153; the nucleic acid with G at position 159; the nucleic acid sequence with A at position 384; the nucleic acid sequence with C at position 719; the nucleic acid sequence with C at position 921; the nucleic acid sequence with T at position 956; the nucleic acid sequence with C at position 1002; the nucleic acid sequence with T at position 1010; the nucleic acid sequence with A at position 1158 and, the nucleic acid sequence with T at position 1304, (all according to the position in SEQ ID No. 5) or a complementary strand thereof or a fragment thereof of at least 20 bases comprising at least one of
  • nucleic acid comprising any one of the following polymo ⁇ hism containing sequences: the nucleic acid disclosed in EMBL Accession Number M75102 with A at position 423; the nucleic acid sequence disclosed in SEQ ID No. 1 with C at position 553; the nucleic acid sequence disclosed in EMBL Accession Number M75104 with T at position 72; the nucleic acid sequence disclosed in SEQ ID No. 5 with G insertion at position 153; the nucleic acid disclosed in SEQ ID No. 5 with G at position 159; the nucleic acid sequence disclosed in SEQ ID No. 5 with A at position 384; the nucleic acid sequence disclosed in SEQ ID No.
  • Fragments are at least 17 bases more preferably at least 20 bases, more preferably at least 30 bases.
  • the invention further provides nucleotide primers which detect the NK2R gene polymo ⁇ hisms of the invention.
  • Such primers can be of any length, for example between 8 and 100 nucleotides in length, but will preferably be between 12 and 50 nucleotides in length, more preferable between 17 and 30 nucleotides in length.
  • an allele specific primer capable of detecting an NK2R gene polymo ⁇ hism at one or more of positions 423 (as defined by the position in EMBL accession number Ml 7502), 553 (according to SEQ ID No. 1 herein) and 72 (as defined by the position in EMBL accession number M75104) in theNK2R gene.
  • an allele specific primer capable of detecting an NK2R promoter polymo ⁇ hism at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to their position in SEQ ID No. 5) in the NK2R promoter.
  • an allele specific primer capable of detecting an NK2R gene polymo ⁇ hism at one or more of positions: 423 (as defined by the position in EMBL accession number Ml 7502), 553 (according to SEQ ID No.
  • a suitable amplification primer used to detect the presence or absence of the insertion of G at position 153 might be one which is designed to allow primer extension from new position 154, i.e. wherein the 3'- terminal two bases are G-G, the first G representing the inserted G at position 153 (the polymo ⁇ hism) and the second G representing the normal G at position 154 which has shunted to position 155 due to the insertion of the G at position 153 in the polymo ⁇ hic DNA.
  • Such a primer will allow amplification from nucleic acid containing the polymo ⁇ hism at 153, but with the normal allele, amplification will be inhibited because the 3 '-terminal base (G) of the primer will not hybridise to the complementary base and thus will not allow primer extension when used with a polymerase enzyme that lacks 3' to 5' proof-reading activity (such as Klenow or Taq DNA polymerase).
  • An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as PCR, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e.g. as used for ARMSTM allele specific amplification assays.
  • the allele specific primer is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.
  • An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof are also contemplated wherein about 6-8 of the nucleotides at the 3' terminus correspond with the allele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the primer. Often the nucleotide at the -2 and/or -3 position (relative to the 3' terminus) is mismatched in order to optimise differential primer binding and preferential extension from the correct allele discriminatory primer only Primers may be manufactured using any convenient method of synthesis.
  • an allele-specific oligonucleotide probe capable of detecting a NK1R gene polymo ⁇ hism of the invention.
  • an allele-specific oligonucleotide probe capable of detecting an NK2R gene polymo ⁇ hism at one or more of positions: 423 (according to the position in EMBL accession number M17502), 553 (according to the position in SEQ ID No. 1), 72 (according to the position in EMBL accession number M75104) and, 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to their positions in SEQ ID No. 5) in the NK2R gene.
  • the allele-specific oligonucleotide probe is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.
  • Such probes will be apparent to the molecular biologist of ordinary skill.
  • Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length.
  • such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene.
  • Suitable oligonucleotide probes might be those consisting of or comprising the sequences depicted in SEQ ID Nos. 9 - 21, or sequences complementary thereto.
  • the emboldened nucleotide (the polymo ⁇ hism site) as illustrated in these SEQ ID's (see above) could be altered to ensure specific hybridisation to, and thus detection of, the variant allele.
  • the probes of the invention may carry one or more labels to facilitate detection, such as in Molecular Beacons.
  • a diagnostic kit comprising one or more allele-specific primers of the invention and/or one or more allele-specific oligonucleotide probe of the invention.
  • kits may comprise appropriate packaging and instructions for use in the methods of the invention. Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase. Such kits may also comprise companion primers and/or control primers or probes.
  • a companion primer is one that is part of the pair of primers used to perform PCR. Such primer usually complements the template strand precisely.
  • the NK2R gene has been mapped to chromosome lOq 11-21 (JEN Morten et al,
  • the single nucleotide polymo ⁇ hisms of this invention may be used as genetic markers for this region in linkage studies. This particularly applies to the polymo ⁇ hisms at position 553 (according to the position SEQ ID No. 1), position 72 (according to the position in EMBL accession number M75104), position 159 (according to the position in SEQ ID No. 5) and position 1010 (according to the position in SEQ ID No. 5) because of their relatively high frequency. Those polymo ⁇ hisms that occur relatively infrequently are useful as markers of low frequency haplotypes.
  • a method of treating a human in need of treatment with an NK2R ligand antagonist drug comprises: i) diagnosis of a single nucleotide polymo ⁇ hism in NK2R gene in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No.
  • a method of treating a human in need of treatment with an NK2R ligand antagonist drug comprises: i) diagnosis of a single nucleotide polymo ⁇ hism in NK2R promoter in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to their position in SEQ ID No. 5) in the NK2R promoter, and determining the status of the human by reference to polymo ⁇ hism in the NK2R promoter; and, ii) administering an effective amount of an NK2R ligand antagonist drug.
  • a method of treating a human in need of treatment with an NK2R ligand antagonist drug comprises: i) diagnosis of a single nucleotide polymo ⁇ hism in NK2R promoter in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions: 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No. 1 herein), 72 (as defined by the position in EMBL accession number, M75104) and, 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to their position in SEQ ID No. 5) in the NK2R promoter, and determining the status of the human by reference to polymo ⁇ hism in the NK2R promoter; and, ii) administering an effective amount of an NK2R ligand antagonist drug.
  • NK2R ligand antagonist drug in preparation of a medicament for treating a NK2 -mediated disease in a human diagnosed as having a single nucleotide polymo ⁇ hism at one or more of positions 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No. 1 herein) and 72 (as defined by the position in EMBL accession number, M75104).
  • an NK2R ligand antagonist drug in preparation of a medicament for treating a NK2 -mediated disease in a human diagnosed as having a single nucleotide polymo ⁇ hism at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to SEQ ID No. 5) in the NK2R promoter.
  • an NK2R ligand antagonist drug in preparation of a medicament for treating a NK2 -mediated disease in a human diagnosed as having a single nucleotide polymo ⁇ hism at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158, 1304 (all according to SEQ ID No. 5), 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No. 1 herein) and 72 (as defined by the position in EMBL accession number, M75104) in the NK2R gene.
  • a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymo ⁇ hism at one or more of positions 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No. 1 herein) and 72 (as defined by the position in EMBL accession number, M75104).
  • a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymo ⁇ hism at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to SEQ ID No. 5), in the NK2R promoter.
  • a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymo ⁇ hism at one or more of positions: 153, 159, 384, 719, 921, 956, 1002, 1010, 1158, 1304 (all positions according to SEQ ID No. 5), 423 (as defined by the position in EMBL accession number, M75102), 553 (as defined by the position in SEQ ID No. 1) and 72 (as defined by the position in EMBL accession number, M75104), in the NK2R gene.
  • Suitable NK2R ligand antagonist drugs are drugs whose main activity is towards NK2R for example those described in Swain (1996) Exp Opin Ther Patents 6(4):367-378 and drugs which interact with the NK2R in addition to other receptors for example mixed NK1/NK2 receptor antagonists, examples of which are described in Elliott & Seward (1997) Exp Opin Ther Patents 7(l):43-54.
  • SEQ ID No. 1 (1414 bp) herein, represents the revised and extended nucleotide sequence of and around exon 3 of the human NK2 receptor gene.
  • SEQ ID No. 2 (corresponding to positions 1-500 of SEQ ID No. 1) represents novel sequence (500 bp) upstream of exon 3 of the human NK2 receptor gene.
  • SEQ ID No. 3 (corresponding to positions 905-1414 of SEQ ID No. 1) represents novel sequence (510 bp) downstream of exon 3 of the human NK2 receptor gene.
  • SEQ ID No. 4 (corresponding to positions 501-909 of SEQ ID No.
  • SEQ ID No. 1 represents the revised sequence of exon 3 of the human NK2 receptor gene inco ⁇ orating the 46 differences from the sequence disclosed in EMBL M75103, most of which occur at the 3' end of the sequence in EMBL (see alignment in Figure 2).
  • SEQ ID No. 5 (2023 bp) identifies the revised human NK2R promoter sequence, exon 1 of the human NK2R gene and the first 72bp of intron 1. Accordingly, reference to the NK2R promoter region and the position of the promoter polymo ⁇ hisms are identified herein with reference to SEQ ID No. 5.
  • SEQ ID No. 6 represents novel promoter sequence.
  • SEQ ID No. 8 is an amalgamation of SEQ ID Nos 6. and 7 (Positions 1-1559 of SEQ ID No5). It represents the complete promoter element upstream of the initiating ATG codon of exon 1 of the NK2R gene. This promoter element can be utilised in heterologous or autologous expression studies according to standard methods, and using standard vehicles i.e. plasmids, host cells etc., known in the art (See for example "Molecular Cloning - A Laboratory Manual” Second Edition, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, 1989).
  • an isolated nucleic acid sequence comprising the sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID No. 2 or 3; (ii) a nucleotide sequence having at least 90% sequence identity to (i); (iii) the nucleotide sequence of SEQ ID No. 4; (iv) the nucleotide sequence of SEQ ID No. 1 ; (v) an isolated fragment of (i), (ii), (iii) or (iv); and (vi) a nucleotide sequence fully complementary to (i), (ii), (iii), (iv) or (v).
  • nucleic acid sequence comprising the sequence selected from the group consisting of:
  • nucleic acid sequence comprising the sequence selected from the group consisting of:
  • group (ii) relates to variants of the polynucleotide depicted in group (i).
  • the variant of the polynucleotide may be a naturally occurring allelic variant, from the same species or a different species, or a non-naturally occurring allelic variant.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a deletion, addition or substitution of one or more nucleotides.
  • Sequence identity can be assessed by best-fit computer alignment analysis using suitable software such as Blast, Blast2, FastA, Fasta3 and PILEUP.
  • Preferred software for use in assessing the percent identity, i.e how two polynucleotide sequences line up is PILEUP.
  • Identity refers to direct matches.
  • two polynucleotide sequences with 90% identity have 90% of the nucleotides being identical and in a like position when aligned optimally allowing for up to 10, preferably up to 5 gaps.
  • the present invention particularly relates to polynucleotides which hybridise to one or other of the polynucleotide sequences depicted in SEQ ID No. 2, 3 or 6, their complementary sequences, or fragment thereof, under stringent conditions.
  • stringent conditions are those conditions which enable sequences that possess at least 80%, preferably at least 90%, more preferably at least 95% and more preferably at least 98% sequence identity to hybridise together.
  • nucleic acids which can hybridise to one or other of the nucleic acids of SEQ ID No. 2, 3 or 6, or their complementary antisense strand thereof include nucleic acids which have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% sequence identity and most preferably 100%, over at least a portion (at least 20, preferably 30 or more consecutive nucleotides) of the polynucleotide sequence depicted in SEQ ID No. 2, 3 or 6.
  • nucleic acid fragments thereof useful for example as oligonucleotide primers to amplify the NK2R promoter region and/or the NK2R promoter and gene sequences using any of the well known amplification systems such as the polymerase chain reaction (PCR), or fragments that can be used as diagnostic probes to identify corresponding nucleic acid sequences are also part of this invention.
  • the invention thus includes polynucleotides of shorter length than the novel NK2R sequences depicted in SEQ ID Nos. 2, 3 or 6 that are capable of specifically hybridising to the sequences depicted herein.
  • Such polynucleotides may be at least 17 nucleotides in length, preferably at least 20, more preferably at least 30 nucleotides in length and may be of any size up to and including or indeed, comprising the complete sequences depicted in SEQ ID Nos. 2, 3 or 6.
  • An example of a suitable hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe nucleic acid is greater than 300 bases or base pairs, say 500 bp, is: 6 x SSC (saline sodium citrate), 0.5% SDS (sodium dodecyl sulphate), lOO ⁇ g/ml denatured, sonicated salmon sperm DNA.
  • 6 x SSC saline sodium citrate
  • SDS sodium dodecyl sulphate
  • lOO ⁇ g/ml denatured, sonicated salmon sperm DNA 6 x SSC (saline sodium citrate), 0.5% SDS (sodium dodecyl sulphate), lOO ⁇ g/ml denatured, sonicated salmon sperm DNA.
  • An example of a suitable hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 12 and 50 bases is: 3M trimethylammonium chloride (TMAC1), 0.01M sodium phosphate (pH 6.8), lmM EDTA (pH 7.6), 0.5% SDS,100 ⁇ g/ml denatured, sonicated salmon sperm DNA and 0.1% dried skimmed milk.
  • TMAC1 trimethylammonium chloride
  • the hybridisation can be performed at 68°C for at least 1 hour and the filters then washed at 68°C in 1 x SSC, or for higher stringency, 0.1 x SSC/0.1% SDS.
  • Hybridisation techniques are well advanced in the art.
  • a fragment can be any part of the full length sequence and may be single or double stranded or may comprise both single and double stranded regions.
  • a fragment is a restriction enzyme fragment.
  • SEQ ID No. 7 positions 875-1559 of SEQ ID No 5 corresponds to the corrected non-coding sequence upstream of the initiating ATG codon of exon 1 of the NK2R gene (the promoter element).
  • SEQ ID No. 8 is an amalgamation of SEQ ID Nos 6. and 7 (Positions 1-1559 of SEQ ID No5). It represents a larger promoter region comprising novel and corrected promoter sequence upstream of the initiating ATG codon of exon 1 of the NK2R gene.
  • a modulator is any substance, particularly chemical compounds, that has an affect on NK2R expression.
  • the NK2R promoter sequence may be used in biochemical assays to identify agents which modulate the expression of NK2 receptor.
  • the design and implementation of such assays will be evident to the biochemist of ordinary skill.
  • the promoter sequence may conveniently be used to express a reporter gene in a convenient prokaryotic or eukaryotic host cell.
  • Test compounds can then be introduced into the test system and measurements made to determine their effect on expression from the NK2R promoter. Active compounds can then be further assessed for their activity on NK2 receptor expression in vivo.
  • test compound or library of test compounds may be used in conjunction with the test assay.
  • Particular test compounds include low molecular weight chemical compounds (preferably with a molecular weight less than 1500 daltons) suitable as pharmaceutical or veterinary agents for human or animal use.
  • NK2R promoter sequences of SEQ ID Nos 7 and 8 can also be used in homologous (NK2R) or heterologous expression studies.
  • the person skilled in the art of protein and peptide expression would be able to utilise the NK2R promoter sequences of SEQ ID Nos 7 or 8, or fragments thereof, for expression studies without inventive input.
  • NK2R promoter element of SEQ ID No. 7 or 8, or fragments thereof in expression studies.
  • an expression vector including a nucleic acid sequence comprising SEQ ID No.7 or SEQ ID No. 8, or fragments thereof.
  • the nucleic acid sequences of the invention particularly those relating to and identifying the single nucleotide polymo ⁇ hisms identified herein represent a valuable information source with which to identify further sequences of similar identity and characterise individuals in terms of, for example, their identity, haplotype and other sub- 5 groupings, such as susceptibility to treatment with particular drugs.
  • nucleic acid sequences of the invention are particularly useful as components in databases useful for sequence identity, genome mapping, pharmacogenetics and other search analyses.
  • sequence information relating to the nucleic acid sequences and polymo ⁇ hisms of the invention may be reduced to, converted into or stored in a tangible medium, such as a computer disk, preferably in a computer readable form.
  • the invention provides a computer readable medium having stored thereon one or more nucleic acid sequences of the invention.
  • a computer readable medium comprising and having stored thereon a member selected from the group consisting
  • nucleic acid comprising the sequence of a nucleic acid of the invention, a nucleic acid consisting of a nucleic acid of the invention, a nucleic acid which comprises part of a nucleic acid of the invention, which part includes at least one of the polymo ⁇ hisms of the invention, a set of nucleic acid sequences wherein the set includes at least one nucleic acid sequence of the invention, a data set comprising or consisting of a nucleic acid sequence of the invention
  • the computer readable medium can be any composition of matter used to store information or data, including, for example, floppy disks, tapes, chips, compact disks, digital disks, video disks, punch cards and hard drives.
  • a nucleic acid comprising any of the sequences of SEQ ID No. 1 to SEQ ID No. 21; a set of nucleic acids wherein at least one of said sequences comprises a sequence selected from SEQ ID Nos. 1 to 21; a data set representing a nucleic acid sequence comprising any of the sequences of SEQ ID No. 1 to SEQ ID No. 21; a nucleic acid selected from the group consisting of any of SEQ ID Nos. 1 to 21; a set of nucleic acids wherein at least one of said sequences consists of the sequence of SEQ ID Nos. 1 to 21; a nucleic acid comprising any part (i.e. a fragment of at least 20 bases) of a sequence of SEQ ID No. 1 to 21, which part includes at least one of the polymo ⁇ hisms identified herein.
  • a computer based method for performing sequence identification, said method comprising the steps of providing a nucleic acid sequence comprising a polymo ⁇ hism of the invention in a computer readable medium; and comparing said polymo ⁇ hism containing nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to identify identity (homology), i.e. screen for the presence of a polymo ⁇ hism.
  • identity identity
  • Such a method is particularly useful in pharmacogenetic studies and in genome mapping studies.
  • a method for performing sequence identification comprising the steps of providing a nucleic acid sequence comprising a sequence selected from the group consisting of: SEQ ID No. 1 to SEQ ID No. 21 in a computer readable medium; and comparing said nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to identify identity.
  • a method for performing sequence identification comprising the steps of providing one or more of the following polymo ⁇ hism containing nucleic acid sequences: the nucleic acid disclosed in EMBL Accession Number M75102 with A at position 423; the nucleic acid sequence disclosed in SEQ ID No. 1 with C at position 553; the nucleic acid sequence disclosed in EMBL Accession Number M75104 with T at position 72; the nucleic acid sequence disclosed in SEQ ID No. 5 with G insertion at position 153; the nucleic acid disclosed in SEQ ID No. 5 with G at position 159; the nucleic acid sequence disclosed in SEQ ID No. 5 with A at position 384; the nucleic acid sequence disclosed in SEQ ID No.
  • nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 719 the nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 921; the nucleic acid sequence disclosed in SEQ ID No. 5 with T at position 956; the nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 1002; the nucleic acid sequence disclosed in SEQ ID No. 5 with T at position 1010; the nucleic acid sequence disclosed in SEQ ID No. 5 with A at position 1158 and, the nucleic acid sequence disclosed in SEQ ID No.
  • nucleic acid sequence 5 with T at position 1304, or a complementary strand thereof or a fragment thereof of at least 20 bases comprising at least one of the polymo ⁇ hisms, and comparing said nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to determine identity.
  • a method for performing sequence identification comprising the steps of providing one or more of the following polymo ⁇ hism containing nucleic acid sequences: the nucleic acid disclosed in EMBL Accession Number M75102 with A at position 423; the nucleic acid sequence disclosed in SEQ ID No. 1 with C at position 553; the nucleic acid sequence disclosed in EMBL Accession Number M75104 with T at position 72; the nucleic acid sequence disclosed in SEQ ID No. 5 with G insertion at position 153; the nucleic acid disclosed in SEQ ID No. 5 with G at position 159; the nucleic acid sequence disclosed in SEQ ID No. 5 with A at position 384; the nucleic acid sequence disclosed in SEQ ID No.
  • nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 719 the nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 921; the nucleic acid sequence disclosed in SEQ ID No. 5 with T at position 956; the nucleic acid sequence disclosed in SEQ ID No. 5 with C at position 1002; the nucleic acid sequence disclosed in SEQ ID No. 5 with T at position 1010; the nucleic acid sequence disclosed in SEQ ID No. 5 with A at position 1158 and, the nucleic acid sequence disclosed in SEQ ID No.
  • AMPLITAQTM available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.
  • Figure 1 represents SEQ ID No. 1 (1414 bp) illustrating: novel sequence flanking exon 3 (SEQ ID No. 2, positions 1-500; and, SEQ ID No. 3, positions 905-1414); SEQ ID No. 4 (positions 501-909, emboldened) representing corrected Accession Number M75103 sequence inco ⁇ orating the 46 differences; exon 3 sequence (underlined) which starts at position 667 (GTA) and ends at position 829 (AAG); and, the polymo ⁇ hism at position 553 [square bracketed].
  • Figure 2 represents an alignment of the published sequence in EMBL accession No.
  • the sequence of the regions flanking exon 3 of the NK2 Receptor gene and promoter region was determined by dye terminator sequencing in both directions on BAC clone 110P24 (Research Genetics). Sequencing was performed using standard protocols (FM Ausebel and
  • Promoter primers Reverse 1113-1088, 594-570 (positions are defined according to their location in SEQ ID No. 5).
  • DNA was prepared from frozen blood samples collected in EDTA following protocol I (Molecular Cloning: A Laboratory Manual, p392, Sambrook, Fritsch and Maniatis, 2 nd Edition, Cold Spring Harbor Press, 1989) with the following modifications.
  • the thawed blood was diluted in an equal volume of standard saline citrate instead of phosphate buffered saline to remove lysed red blood cells.
  • Samples were extracted with phenol, then phenol/chloroform and then chloroform rather than with three phenol extractions.
  • the DNA was dissolved in deionised water.
  • Templates were prepared by PCR using the oligonucleotide primers and annealing temperatures set out below.
  • the extension temperature was 72° and denaturation temperature 94°.
  • 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR.
  • these primers were modified to include the Ml 3 forward and reverse primer sequences (ABI protocol P/N 402114, Applied Biosystems) at the 5' end 0 of the forward and reverse oligonucleotides respectively.
  • Dye-primer sequencing using Ml 3 forward and reverse primers was as described in the ABI protocol P/N 402114 for the ABI PrismTM dye primer cycle sequencing core kit with "AmpliTaq FS”TM DNA polymerase, modified in that the annealing temperature was 45° and 5 DMSO was added to the cycle sequencing mix to a final concentration of 5 %.
  • Novel promoter polymorphisms positions according to SEQ ID No. 5
  • the nine substitution polymo ⁇ hisms are identified in Table 4 which indicates the position of the polymo ⁇ hism (relative to SEQ ID No. 5), the types of residue (different alleles) at each position, the frequency of each allele and the number of individuals sampled.
  • the polymo ⁇ hism flanking exon 3 disrupts a BssKI (New England Biolabs) or BstNI (New England Biolabs) recognition site (CCAGG). Either enzyme could therefore be used in a diagnostic test.
  • the PCR product (293 bp) containing the wild type (most frequent) sequence (CCAGC), generated using the primers indicated above is not cleaved by BssKI (New England Biolabs), a single band of 293 bp will therefore be observed following incubation of the PCR product containing the wild type sequence with BssKI. Digestion of a heterozygote product however, will generate products of 293 bp, 273 bp and 20 bp.
  • a homozygous variant product (CCAGG) will be cleaved to products of 273 bp and 20 bp. Comparable fragment sizes for BstNI would be 293 bp, 271 bp and 22bp.
  • Promoter position 719 (according to SEQ ID No. 5) A PCR product was generated using the primers and conditions described:
  • a PCR product was generated using the primers and conditions described:
  • Promoter position 956 (according to SEQ ID No. 5)
  • a PCR product was generated using the primers and conditions described:
  • Polymo ⁇ hism at position 956 creates an Ava II site (GGTCC).
  • a PCR product (563 bp) containing the wild type sequence was not digested with Ava II (New England Biolabs). Digestion of a heterozygote product generates products of 563 bp, 379 bp and 184 bp. Digestion of a homozygote variant generates products of 379 bp and 184 bp.
  • a PCR product was generated using the primers and conditions described:
  • Promoter position 1010 (according to SEQ ID No. 5)
  • a PCR product was generated using the primers and conditions described:
  • a PCR product was generated using the primers and conditions described:
  • a PCR product (563 bp) containing the wild type sequence was digested with Taq I (New England Biolabs) generating products of 384 bp,159 bp and 20 bp. Digestion of a heterozygote product generates bands of 543 bp,
  • Promoter position 1304 (according to SEQ ID No. 5)
  • a PCR product was generated using the primers and conditions described: 30
  • Promoter position 159 (according to SEQ ID No. 5)
  • a Nae I site (GCCGGC) can be created at the polymo ⁇ hic site (CCCC/GCGAC), the 10 engineered base is emboldened.
  • a PCR product (172 bp) containing the wild type sequence is resistant to digestion with Nae I (New England Biolabs), digestion of heterozygote product gives bands of 172 bp, 148 bp and 24 bp, digestion of the homozygous variant gives bands of
  • Promoter position 384 (according to SEQ ID No. 5)
  • a Sma I site( CCCGGG) can be created at the polymo ⁇ hic site (CCC G/A GG), the engineered base is emboldened.
  • Digestion of the PCR product generated from a wild type 20 template with Sma I (New Engalnd Biolabs) generates bands of 384 bp and 27 bp, digestion of heterozygote product gives bands of 411 bp, 384 bp and 27 bp.
  • the homozygote variant product is resistant to digestion with Sma I.
  • Polymo ⁇ hic variants that do not create or modify restriction enzyme recognition sites could be detected using alternative technologies, such as ARMSTM- allele specific amplification, and the like.

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Abstract

L'invention concerne une nouvelle séquence ainsi que des polymorphismes du gène du récepteur humain des neurokinines 2 (NK2R), notamment la région promoteur de ce gène. L'invention concerne également des procédés et matériaux d'analyse de la variation allélique, dans le gène de NK2R et la région promoteur de celui-ci, ainsi que l'utilisation du polymorphisme de NK2R dans le diagnostic et traitement de maladies induites par le ligand de NK2R, telles que l'asthme, la dépression, l'angoisse, les vomissements et l'incontinence urinaire.
EP99934891A 1998-07-25 1999-07-20 Polymorphismes genetiques dans le gene du recepteur humain des neurokinines 2 et leur utilisation dans le diagnostic et traitement de maladies Withdrawn EP1100961A1 (fr)

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