WO2000006767A1

WO2000006767A1 - Genetic polymorphisms in the human neurokinin 2 receptor gene and their use in diagnosis and treatment of diseases

Info

Publication number: WO2000006767A1
Application number: PCT/GB1999/002336
Authority: WO
Inventors: John Craig Smith; Rakesh Anand; John Edward Norris Morten
Original assignee: Astrazeneca Ab
Priority date: 1998-07-25
Filing date: 1999-07-20
Publication date: 2000-02-10
Also published as: AU5052599A; JP2002521061A; EP1100961A1

Abstract

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 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.

Description

GENETIC POLYMORPHISMS IN THE HUMAN NEUROKININ 2 RECEPTOR GENE AND THEIR USE IN DIAGNOSIS AND TREATMENT OF DISEASES

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);

10 Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends, Graham et al, Biochem Biophys Res Commun 177, 8-16 (1991); Cloning and expression of the substance K receptor and analysis of its role in mitogenesis. Kris et al, Cell Growth and Differentiation 2, 15-22 (1991); A single mutation in the neurokinin 2 receptor prevents agonist-induced desensitization Nemeth and Chollet, J. Biol Chem 270, 27601-

15 27605 (1995); Structure and function of G protein-coupled receptors, CD Strader et al, Annual Reviews Biochemistry 63,101-132 (1994); and The evolution and structure of aminergic G protein-coupled receptors, D Donnelly et al, Receptors and Channels 2, 61-78 (1994).

The NK2R gene is organised into five exons interrupted by 4 introns. Gerard et al,

20 265, 20455-20462 (1990). International patent application WO 92/16220 (Children's Medical Center) teaches cloning of cDNA and genomic DNA encoding the human NK-2 receptor and discloses exon and intron/exon junction sequence.

The sequences for 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

25 M75104, 466 bp. All positions herein relate to the positions indicated therein unless stated otherwise or apparent from the context. The sequence of exon 3 and flanking sequence has been published in EMBL (390 bp) under accession No. M75103. However, the inventors have identified a number of errors in this sequence and have identified novel flanking sequence. Accordingly, reference to exon 3, its flanking sequences and the position of the

30 polymorphism is identified herein with reference to SEQ ID No. 1.

The sequence for exon 1 and some upstream non-coding region (putative promoter) has been published in EMBL (size 1327) under accession No. M75101. However, the inventors have identified a number of errors in this sequence and have extended this sequence by 874 bp. Accordingly, the positions of the promoter polymorphisms identified herein is made with reference to SEQ ID No. 5.

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. In addition, the sequence can be used as a target to identify compounds that modify (i.e. inhibit or enhance) expression from the promoter. This is conveniently done by measuring expression levels of a reporter gene (for example beta- galactosidase) under the control of the NK2R promoter in transfected host cells in the presence or absence of test compounds. The use of knowledge of polymorphisms to help identify patients most suited to therapy with particular pharmaceutical agents is often termed "pharmacogenetics". Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al. (1997), Clinical Chemistry, 43:254; Marshall (1997), Nature Biotechnology, 15:1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al, (1998), Nature Biotechnology, 16:33.

Some 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).

Clinical trials have shown that patient response to drugs is heterogeneous. Thus there is a need for improved approaches to pharmaceutical agent design and therapy. 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.

For the avoidance of doubt the location of 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. 12) e) (position 159 promoter polymorphism) AGAACATGCCCCGCCGACTCCCGCT (SEQ ID No.13) f) (position 384 promoter polymorphism) GCTGTGGTTCCCGAAAGAGCCCTGA (SEQ ID No. 14) g) (position 719 promoter polymorphism) C AAACC AGGCTCC AACTCCTTCTGT (SEQ ID No. 15) h) (position 921 promoter polymorphism) AGCCCCCCCTCGCGAGC AGGC AGC A

(SEQ ID No. 16) i) (position 956 promoter polymorphism) GGCCCCCACTGGTCCTGCCCCAGAG

(SEQ ID No. 17) j) (position 1002 promoter polymorphism) AGGGCTGGCAGCCGGCAGAGCCCTG

(SEQ ID No. 18) k) (position 1010 promoter polymorphism) C AGCTGGC AGAGTCCTGAGC ACCC A

(SEQ ID No. 19)

1) (position 1158 promoter polymorphism) AAACCTGGGTTCAAGTCCTAACTTG (SEQ ID No. 20) m) (position 1304 promoter polymorphism) TCTGTGTTTCCATATGATATTCGAG

(SEQ ID No. 21)

¹ the published sequence disclosed in EMBL accession number M75103 has an additional cytosine at this location (position 50 in M75103) which is not present in this corrected sequence.

The positions of the promoter polymorphisms are relative to SEQ ID No. 5. According to one aspect of the present invention there is provided a method for the diagnosis of a single nucleotide polymorphism in NK2R in a human, which method 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.

1 herein) and 72 (as defined by the position in EMBL accession number M75104), and determining the status of the human by reference to polymorphism in the NK2R gene. According to another aspect of the present invention there is provided a method for the diagnosis of a single nucleotide polymorphism in NK2R in a human, which method 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.

According to another aspect of the present invention there is provided a method for the diagnosis of one or more single nucleotide polymorphism(s) in NK2R gene in a human, which method 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.

As defined herein, 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.

In one embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at position 423 (according to the position in EMBL accession number M75102) is the presence of G and/or A.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 553 (according to the position in SEQ ID No. 1) is the presence of G and/or C.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 72 (according to the position in EMBL accession number M75104) is the presence of C and or T. In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at position 153 (according to the position in SEQ ID No. 5) is insertion of G. In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 159 (according to the position in SEQ ID No. 5) is the presence of C and/or G.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 384 (according to the position in SEQ ID No. 5) is the presence of G and/or A.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 719 (according to the position in SEQ ID No. 5) is the presence of G and/or C. In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 921 (according to the position in SEQ ID No. 5) is the presence of T and/or C.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 956 (according to the position in SEQ ID No. 5) is the presence of C and/or T.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 1002 (according to the position in SEQ ID No. 5) is the presence of T and/or C.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 1010 (according to the position in SEQ ID No. 5) is the presence of C and/or T.

In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism at position 1158 (according to the position in SEQ ID No. 5) is the presence of G and/or A. In another embodiment of the invention preferably the method of diagnosis described herein is one in which the single nucleotide polymoφhism 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 polymoφhism 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 (ARMS™-allele specific amplification), allele specific hybridisation (ASH), ohgonucleotide ligation assay (OLA) and restriction fragment length polymoφhism (RFLP). In another aspect of the invention we provide a method for the diagnosis of NK2R ligand-mediated disease, which method 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 polymoφhism in the NK2R gene.

In another aspect of the invention we provide a method for the diagnosis of NK2R ligand-mediated disease, which method 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 polymoφhism in the NK2R promoter. In another aspect of the invention we provide a method for the diagnosis of NK2R ligand-mediated disease, which method 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 polymoφhism in the NK2R gene.

The published sequence of the NK2R gene is presently divided into several records on the EMBL database. 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 and 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. 5) consists of a single base substitution from C (the published base) for example to G. The following nomenclature is adopted herein to indicate the normal 5 residue, the possible variant residue and the sequence position (according to SEQ ID No. 5): N#V, wherein the normal residue and the possible variant are identified before and after the residue number respectively such that for example, G384A indicates that the normal residue at position 384 is G and the variant is A (although it could also be C or T). Thus, with respect to the NK2R promoter polymoφhisms and their locations in SEQ ID No. 5, in addition to the G 0 residue insertion at position 153, 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 polymoφhic 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 2ⁿ haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs. One approach to identifying mutations or polymoφhisms 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. As particular mutations or polymoφhisms associated with certain clinical features, such as adverse or abnormal events, are likely to be of low frequency within the population, 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. Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse IF, London J, Stansbie D, Humphries SE, and Gudnason V; and Variation at the von willebrand factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of three novel single nucleotide polymoφhisms in the vWF gene promoter. Blood (1999) 93:4277-83, Keightley AM, Lam YM, Brady JN, Cameron CL, Lillicrap D). The test sample of nucleic acid is conveniently a sample of blood, bronchoalveolar lavage fluid, sputum, urine or other body fluid or tissue obtained from an individual. It will be appreciated that the 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.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of one or more of the polymoφhisms identified herein. In general, the detection of 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. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997. Abbreviations:

Table 1 - Mutation Detection Techniques

General: DNA sequencing, Sequencing by hybridisation

Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage * Note: not useful for detection of promoter polymoφhisms.

Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Ohgonucleotide arrays (DNA Chips)

Solution phase hybridisation: Taqman™ - US-5210015 & US-5487972 (Hoffmann-La Roche), Molecular Beacons - Tyagi et al (1996), Nature Biotechnology, 14, 303; WO

95/13399 (Public Health Inst., New York), ASH

Extension Based: ARMS™-allele specific amplification (as described in European patent

No. EP-B-332435 and US patent No. 5,595,890), ALEX™ - European Patent No. EP 332435

Bl (Zeneca Limited), COPS - Gibbs et al (1989), Nucleic Acids Research, 17, 2347. Incorporation Based: Mini-sequencing, APEX

Restriction Enzyme Based: RFLP, Restriction site generating PCR

Ligation Based: OLA- Nickerson et al. (1990) P.N.A.S. 87:8923-8927.

Other: Invader assay

Table 2 - Signal Generation or Detection Systems

Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998 (Zeneca Limited)

Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation protection assay, Mass spectrometry, SERRS - WO 97/05280 (University of Strathclyde).

Table 3 - Further Amplification Methods

SSR, NASBA, LCR, SDA, b-DNA

Preferred mutation detection techniques include ARMS™-allele specific amplification, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, OLA, restriction site based PCR and FRET techniques. Particularly preferred methods include ARMS™-allele specific amplification, OLA and RFLP based methods. The allele specific amplification technique known in the art as ARMS™ is an especially preferred method.

ARMS™- 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 polymoφhism to be detected. There is a selective advantage for primer extension from the primer whose 3' terminal nucleotide complements the base mutation, allele or polymoφhism. 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,

RPR, and Sanofi, primarily for CNS indications. In a recent clinical trial it was reported that a single dose of Pfizer CP- 122,721 inhibits emesis associated with chemotherapy and was well tolerated with no adverse effects (Kris et al, JNCI, 89, 817, 1997). Most recently Merck has announced positive Phase II studies with a NK1 antagonist in depression/anxiety. 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.

It is believed that 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.

Four companies have published on mixed 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.

In a further aspect, 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 polymoφhisms 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 polymoφhisms affect transcription levels and/or message stability.

Individuals who carry particular allelic variants of the NK2R gene, especially those within the promoter element, may therefore exhibit differences in receptor levels under different physiological conditions and will display altered abilities to react to different diseases. In addition, differences in receptor level arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy. NK2R polymoφhism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity of the NK2R. However, the polymoφhisms 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. 5 In a further aspect, 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 polymoφhism 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

10 from developing these conditions.

In a further aspect, 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

15 of new drugs. Drugs may be designed to regulate the biological activity of variants implicated in the disease process whilst minimising effects on other variants.

In a further diagnostic aspect of the invention the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes. The person of ordinary skill will be able to design and

20 implement diagnostic procedures based on the detection of restriction fragment length polymoφhism due to the loss or gain of one or more of the sites (see for example, Examples 3 and 4 herein).

According to another aspect of the present invention there is provided a nucleic acid comprising any one of the following polymoφhisms:

25 the nucleic acid disclosed in EMBL Accession Number M75102 with A at position 423 in the flanking sequence of exon 2 according to the nucleotide positioning therein; the nucleic acid sequence disclosed in EMBL Accession Number M75103 with C at position 54 in the flanking sequence of exon 3 according to the nucleotide positioning therein; and, the nucleic acid sequence disclosed in EMBL Accession Number M75104 with T at position

30 72 in the flanking sequence of exon 4 according to the nucleotide positioning therein; or a complementary strand thereof or a fragment thereof of at least 20 bases comprising at least one of the polymoφhisms. According to another aspect of the present invention there is provided a nucleic acid comprising any one of the following polymoφhisms: 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 the polymoφhisms

According to another aspect of the present invention there is provided a nucleic acid comprising any one of the following polymoφhism 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. 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. 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 polymoφhisms.

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 polymoφhisms 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. According to another aspect of the present there is provided an allele specific primer capable of detecting an NK2R gene polymoφhism 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.

According to another aspect of the present there is provided an allele specific primer capable of detecting an NK2R promoter polymoφhism 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. According to another aspect of the present there is provided an allele specific primer capable of detecting an NK2R gene polymoφhism 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), 72 (as defined by the position in EMBL accession number M75104 and, positions 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to the position in SEQ ID No. 5), in the NK2R gene.

Because the polymoφhism at position 153 (according to SEQ ID No. 5) is addition of a G nucleotide, identical to the normal nucleotide at position 154, 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 polymoφhism) 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 polymoφhic DNA. Such a primer will allow amplification from nucleic acid containing the polymoφhism 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 ARMS™ 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. Examples of such methods may be found in standard textbooks, for example "Protocols for Oligonucleotides and Analogues; Synthesis and Properties," Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1^st Edition. If required the primer(s) may be labelled to facilitate detection. According to another aspect of the present invention there is provided an allele- specific oligonucleotide probe capable of detecting a NK1R gene polymoφhism of the invention.

According to another aspect of the present invention there is provided an allele- specific oligonucleotide probe capable of detecting an NK2R gene polymoφhism 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.

The design of 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. In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene. However, if required one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected. 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 polymoφhism 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.

According to another aspect of the present invention there is provided 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.

The diagnostic 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,

Human Genetics 88, 200-203 (1991)).

In another aspect of the invention, the single nucleotide polymoφhisms of this invention may be used as genetic markers for this region in linkage studies. This particularly applies to the polymoφhisms 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 polymoφhisms that occur relatively infrequently are useful as markers of low frequency haplotypes.

According to another aspect of the present invention there is provided a method of treating a human in need of treatment with an NK2R ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymoφhism 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. 1 herein) and 72 (as defined by the position in EMBL accession number, M75104), and determining the status of the human by reference to polymoφhism in the NK2R gene; and ii) administering an effective amount of an NK2R ligand antagonist drug.

According to another aspect of the present invention there is provided a method of treating a human in need of treatment with an NK2R ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymoφhism 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 polymoφhism in the NK2R promoter; and, ii) administering an effective amount of an NK2R ligand antagonist drug.

According to another aspect of the present invention there is provided a method of treating a human in need of treatment with an NK2R ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymoφhism 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 polymoφhism in the NK2R promoter; and, ii) administering an effective amount of an NK2R ligand antagonist drug.

Preferably determination of the status of the human is clinically useful. Examples of clinical usefulness include deciding which NK2R ligand antagonist drug or drugs to administer and/or in deciding on the effective amount of the drug or drugs. According to another aspect of the present invention there is provided use of 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 polymoφhism 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).

According to another aspect of the present invention there is provided use of 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 polymoφhism 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.

According to another aspect of the present invention there is provided use of 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 polymoφhism 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.

According to another aspect of the present invention there is provided a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymoφhism 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).

According to another aspect of the present invention there is provided a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymoφhism 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.

According to another aspect of the present invention there is provided a pharmaceutical pack comprising an NK2R ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymoφhism 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. 1) represents the revised sequence of exon 3 of the human NK2 receptor gene incoφorating 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 polymoφhisms are identified herein with reference to SEQ ID No. 5. SEQ ID No. 6 (corresponding to positions 1-874 of SEQ ID No. 5) represents novel promoter sequence. SEQ ID No. 7 (positions 875-1559 of SEQ ID No 1) corresponds to the corrected non-coding sequence upstream of the initiating ATG codon of exon 1 of the NK2R gene (the inventors have identified 21 deviations from the sequence disclosed in Graham et al. (supra)). 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).

According to another aspect of the invention there is provided 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).

According to another aspect of the invention there is provided an isolated nucleic acid sequence comprising the sequence selected from the group consisting of:

(i) the nucleotide sequence of SEQ ID No. 6; (ii) a nucleotide sequence having at least 90% identity to (i);

(iii) the nucleotide sequence of SEQ ID No. 7;

(iv) the nucleotide sequence of SEQ ID No. 8;

(v) a fragment of (i), (ii), (iii) or (iv); and

(vi) a nucleotide sequence fully complementary to (i), (ii), (iii), (iv) or (v). According to another aspect of the invention there is provided an isolated nucleic acid sequence comprising the sequence selected from the group consisting of:

(i) the nucleotide sequence of SEQ ID No. 2, 3 or 6;

(ii) a nucleotide sequence having at least 90% sequence identity to (i);

(iii) the nucleotide sequence of SEQ ID No. 1, 4, 5, 7 or 8; (iv) a fragment of (i), (ii) or (iii);and

(v) a nucleotide sequence fully complementary to (i), (ii), (iii) or (iv).

In the above three paragraphs, 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. As known in the art 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. In the context of the present invention, 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. As used herein, 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. Thus, 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.

As well as the full novel sequences depicted in SEQ ID Nos. 2, 3 and 6, smaller 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), lOOμg/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,100μg/ml denatured, sonicated salmon sperm DNA and 0.1% dried skimmed milk. 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. The person skilled in the art will be able to adapt the hybridisation conditions to ensure hybridisation of sequences with 80%, 90%) or more identity. 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. In a preferred embodiment, a fragment is a restriction enzyme fragment. As mentioned above, 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.

In a further aspect of the invention we provide the use of a nucleotide sequence comprising SEQ ID No.7 or SEQ ID No. 8 in assays to identify modulators of NK2R expression. A modulator is any substance, particularly chemical compounds, that has an affect on NK2R expression.

As described earlier, 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. For example, 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.

Any convenient 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.

The 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.

According to a further aspect of the invention there is provided the use of the NK2R promoter element of SEQ ID No. 7 or 8, or fragments thereof, in expression studies.

According to a further aspect of the invention there is provided 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 polymoφhisms 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. These approaches are most easily facilitated by storing the sequence information in a computer readable medium and then using the information in standard macromolecular structure programs or to search sequence databases using state of the art searching tools such as GCG (Genetics Computer Group), BlastX BlastP, BlastN, FASTA (refer to Altschul et al. J. Mol. Biol. 215:403-410,

10 1990). Thus, the nucleic acid sequences of the invention are particularly useful as components in databases useful for sequence identity, genome mapping, pharmacogenetics and other search analyses. Generally, the sequence information relating to the nucleic acid sequences and polymoφhisms 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. For

15 example, chromatographic scan data or peak data, photographic scan or peak data, mass spectrographic data, sequence gel (or other) data.

The invention provides a computer readable medium having stored thereon one or more nucleic acid sequences of the invention. For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting

20 of: a 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 polymoφhisms 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

25 or a part thereof comprising at least one of the polymoφhisms identified herein. 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.

In a particular embodiment of the invention there is provided a computer readable

30 medium having stored thereon a member selected from the group consisting of: 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 polymoφhisms identified herein.

A computer based method is also provided for performing sequence identification, said method comprising the steps of providing a nucleic acid sequence comprising a polymoφhism of the invention in a computer readable medium; and comparing said polymoφhism 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 polymoφhism. Such a method is particularly useful in pharmacogenetic studies and in genome mapping studies.

In a particular embodiment of the invention there is provided a method for performing sequence identification, said method 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.

In another embodiment of the invention there is provided a method for performing sequence identification, said method comprising the steps of providing one or more of the following polymoφhism 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. 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. 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 polymoφhisms, and comparing said nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to determine identity.

In another embodiment of the invention there is provided a method for performing sequence identification, said method comprising the steps of providing one or more of the following polymoφhism 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. 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. 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 polymoφhisms, in a computer readable medium; and comparing said nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to determine identity.

The invention will now be illustrated but not limited by reference to the following Examples and Figures. All temperatures are in degrees Celsius.

In the Examples below, unless otherwise stated, the following methodology and materials have been applied.

AMPLITAQ™, available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.

General molecular biology procedures can be followed from any of the methods described in "Molecular Cloning - A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989). Electropherograms were obtained in a standard manner: data was collected by ABI377 data collection software and the wave form generated by ABI Prism sequencing analysis (2.1.2).

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 incoφorating the 46 differences; exon 3 sequence (underlined) which starts at position 667 (GTA) and ends at position 829 (AAG); and, the polymoφhism at position 553 [square bracketed]. Figure 2 represents an alignment of the published sequence in EMBL accession No.

75103 with that of the newly identified sequence from BAC clone 110P24 (SEQ ID No. 4). Using the Martinez/Needleman-Wunsch alignment algorithm, the inventors have identified a number of revisions (underlined) including a couple of large insertions in the G rich region at the 3' end of M75103. There is an insertion of 16 bases after position 352 of M57103 and an insertion of 4 bases after position 378 of M75103. The novel polymoφhism is marked by an asterisk.

EXAMPLES

Example 1 Identification of Polymorphisms

1. Methods

Sequencing

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

LM Albright. DNA Sequencing in Current Protocols in Molecular Biology, Vol 1 (1995) edited by FM Ausebel). BAC sequencing primers Forward 715-738; Reverse 681-704

(positions are defined according to their location in SEQ ID No. 1).

Promoter primers Reverse 1113-1088, 594-570 (positions are defined according to their location in SEQ ID No. 5). DNA Preparation

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.

Template Preparation

Templates were prepared by PCR using the oligonucleotide primers and annealing temperatures set out below. The extension temperature was 72° and denaturation temperature 94°. Generally 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR.

¹ Numbering for exon 2 is as defined by the position in EMBL accession number, M75102

² Numbering for exon 3 is as defined by the position in SEQ ID No. 1

³ Numbering for exon 4 is as defined by the position in EMBL accession number, M75104 Promoter region

Fragment Forward Primer Reverse Primer Annealing Temp Time

13-479 13-32 460-479 58 60 sec

300-882 300-319 863-882 58 60 sec

5 772-1335 772-791 1316-1644 60 60 sec

1172-1644 1172-1191 1625-1644 58 60 sec

Numbering according to the position in SEQ ID No. 5.

For dye-primer sequencing 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

Dye-primer sequencing using Ml 3 forward and reverse primers was as described in the ABI protocol P/N 402114 for the ABI Prism™ dye primer cycle sequencing core kit with "AmpliTaq FS"™ 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 %.

The extension reactions for each base were pooled, ethanol/sodium acetate precipitated, washed and resuspended in formamide loading buffer.

4.25 % Acrylamide gels were run on an automated sequencer (ABI 377, Applied Biosystems). 0

2. Results

Novel exon 2, 3 and 4 flanking sequence polymoφhisms

(1) Exon 2 Flanking intron sequence (position relative to EMBL Accession Number 5 M75102)

Nucleotide 423 G/A Allele frequency G 97%

A 3% (2) Exon 3 Flanking intron sequence (position relative to SEQ ID No. 1)

Nucleotide 553 C/G Allele frequency C 53% G 47%

(3) Exon 4 Flanking intron sequence (position relative to EMBL Accession Number M75104)

Nucleotide 72 C/T Allele frequency C 74% T 26%

Allele frequencies were determined from 37 individuals.

Novel promoter polymorphisms (positions according to SEQ ID No. 5)

(4) Position 153 (+/-) insertion of G 34 individuals tested.

CATGC - 83.8%

CAT(G)GC + 16.2%

(5) - (13)

The nine substitution polymoφhisms are identified in Table 4 which indicates the position of the polymoφhism (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.

Table 4

3. Diagnostic Assays

Position 553 (according to SEQ ID No. 1

The polymoφhism 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. For example, 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:

Fragment Forward Primer Reverse Primer Annealing Temp Time

300-882 300-319 863-882 58 60 sec

Polymoφhism at position 719 (G/C) disrupts a Taq I site (TCGA). A PCR product

(582 bp) containing the wild type sequence was digested with Taq I (New England Biolabs) generating bands of 418 bp and 164 bp., digestion of a heterozygote product will generate bands of 582 bp, 418 bp and 164 bp whereas a homozygous variant product will be resistant to digestion with Taq I. Promoter position 921 (according to SEQ ID No. 51

A PCR product was generated using the primers and conditions described:

Fragment Forward Primer Reverse Primer Annealing Temp Time 772-1335 772-791 1316-1335 60 60 sec

Polymoφhism at position 921 (T/C) creates a Nru I site (TCGCGA). A PCR product (563 bp) containing the wild type sequence was not digested by Nru I (New England Biolabs). Digestion of a heterozygote product generates products of 563 bp, 414 bp and 149 bp whereas digestion of a homozygous variant product generates products of 414 bp and 149 bp.

Promoter position 956 (according to SEQ ID No. 5)

A PCR product was generated using the primers and conditions described:

Fragment Forward Primer Reverse Primer Annealing Temp Time

772-1335 772-791 1316-1335 60 60 sec

Polymoφhism at position 956 (C/T) 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.

Promoter position 1002 (according to SEQ ID No.

A PCR product was generated using the primers and conditions described:

Fragment Forward Primer Reverse Primer Annealing Temp Time

772-1335 772-791 1316-1335 60 60 sec

Polymoφhism at position 1002 (T/C) disrupts a Pvu II site (CAGCTG). A PCR product (563 bp) containing the wild type sequence was digested by Pvu II (New England Biolabs) generating products of 333 bp and 230 bp. Digestion of a heterozygote product generates products of 563 bp, 333 bp and 230 bp. A homozygous variant product is resistant to digestion with Pvu II.

Promoter position 1010 (according to SEQ ID No. 5)

5 A PCR product was generated using the primers and conditions described:

Fragment Forward Primer Reverse Primer Annealing Temp Time

772-1335 772-791 1316-1335 60 60 sec

10 Polymoφhism at position 1010 (C/T) disrupts a Ban II site (GAGCCC). A PCR product (563 bp) containing the wild type sequence was digested by Ban II (New England Biolabs) generating products of 325 bp and 238 bp. Digestion of a heterozygote products generates products of 563 bp, 325 bp and 238 bp. A homozygous variant product is resistant to digestion with Ban II.

15 Promoter position 1158 (according to SEQ ID No. 5)

A PCR product was generated using the primers and conditions described:

Fragment Forward Primer Reverse Primer Annealing Temp Time

772-1335 772-791 1316-1335 60 60 sec

20

Polymoφhism at position 1158 (G/A) disrupts a Taq I site (TCGA). A non- polymoφhic Taq I site is located at positions 1312-1315. 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,

25 384 bp, 159 bp and 20 bp. Digestion of the homozygous variant product generates bands of 543 bp and 20 bp

Promoter position 1304 (according to SEQ ID No. 5)

A PCR product was generated using the primers and conditions described: 30

Fragment Forward Primer Reverse Primer Annealing Temp Time

1172-1644 1172-1191 1625-1644 58 60 sec Polymoφhism at position 1304 (C/T) creates a Nde I site ( CAT ATG). A PCR product (472 bp) containing the wild type sequence was resistant to digestion with Nde I (New England Biolabs). Digestion of a heterozygote product generates bands of 472 bp, 340 bp and 132 bp. Digestion of a homozygous variant product generates bands of 340 bp and 5 132 bp.

4. Engineered Restriction Sites

Promoter position 159 (according to SEQ ID No. 5)

A Nae I site (GCCGGC) can be created at the polymoφhic 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

148 and 24 bp.

Fragment Forward Primer Engineered Primer Annealing Temp Time 15 13-185 13-32 160-185 58 60 sec

Promoter position 384 (according to SEQ ID No. 5)

A Sma I site( CCCGGG) can be created at the polymoφhic 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.

Fragment Forward Primer Engineered Primer Annealing Temp Time

25 13-411 13-32 385-411 58 60 sec

No diagnostic test for the polymoφhism at position 153 (G insertion) was performed. Confirmation was obtained by direct sequencing.

Polymoφhic variants that do not create or modify restriction enzyme recognition sites could be detected using alternative technologies, such as ARMS™- allele specific amplification, and the like.

Claims

CLAIMSWhat is claimed is:

1. A method for diagnosis of one or more single nucleotide polymo╧åhism(s) in NK2R gene in a human, which method 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 polymo╧åhism in the NK2R gene.

2. A method according to claim 1 in which the single nucleotide polymo╧åhism at position 423 (according to the position in EMBL accession number M75102) is the presence of G and/or A, the single nucleotide polymo╧åhism at position 553 (according to the position in SEQ ID No. 1) is the presence of G and/or C, the single nucleotide polymo╧åhism at position 72 (according to the position in EMBL accession number M75104) is the presence of C and/or T, the single nucleotide polymo╧åhism at position 153 (according to the position in SEQ ID No. 5) is insertion of G, the single nucleotide polymo╧åhism at position 159 (according to the position in SEQ ID No. 5) is the presence of C and/or G, the single nucleotide polymo╧åhism at position 384 (according to the position in SEQ ID No. 5) is the presence of G and/or A , the single nucleotide polymo╧åhism at position 719 (according to the position in SEQ ID No. 5) is the presence of G and/or C, the single nucleotide polymo╧åhism at position 921 (according to the position in SEQ ID No. 5) is the presence of T and/or C, the single nucleotide polymo╧åhism at position 956 (according to the position in SEQ ID No. 5) is the presence of C and/or T, the single nucleotide polymo╧åhism at position 1002 (according to the position in SEQ ID No. 5) is the presence of T and/or C, the single nucleotide polymo╧åhism at position 1010 (according to the position in SEQ ID No. 5) is the presence of C and/or T, the single nucleotide polymo╧åhism at position 1158 (according to the position in SEQ ID No. 5) is the presence of G and/or A, the single nucleotide polymo╧åhism at position 1304 (according to the position in SEQ ID No. 5) is the presence of C and/or T

3. A method as claimed in claim 1 or 2, wherein the region containing the potential polymo╧åhism is amplified by polymerase chain reaction prior to determining the sequence.

4. A method as claimed in any of claims 1 - 3, wherein the presence or absence of the polymo╧åhism is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes.

5. A method according to claim 1 or claim 2, in which the sequence is determined by a method selected from ARMS-allele specific amplification, allele specific hybridisation, oligonucleotide ligation assay and restriction fragment length polymo╧åhism (RFLP).

6. A method as claimed in any of the preceding claims for use in assessing the predisposition and/or susceptibility of an individual to diseases mediated by NK2R ligands.

7. A nucleic acid comprising any one of the following polymo╧åhism 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. 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. 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╧åhisms.

8. A diagnostic nucleic acid primer capable of detecting a polymo╧åhism in the NK2R gene 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), 72 (as defined by the position in EMBL accession number M75104 and, positions 153, 159, 384, 719, 921, 956, 1002, 1010, 1158 and 1304 (according to the position in SEQ ID No. 5), in the NK2R gene.

9. A diagnostic primer as claimed in claim 8 which is an allele specific primer adapted for use in ARMS.

10. An allele-specific oligonucleotide probe capable of detecting a polymo╧åhism in the NK2R gene at one or more of positions: 423 (according to the position in EMBL accession number Ml 7502), 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.

11. A diagnostic kit comprising one or more diagnostic primer(s) as defined in claim 8 or 9 and/or one or more allele-specific oligonucleotide probes(s) as defined in claim 10.

12. A method of treating a human in need of treatment with an NK2R ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymo╧åhism 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╧åhism in the NK2R promoter; and, ii) administering an effective amount of an NK2R ligand antagonist drug.

13. Use of 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╧åhism 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.

14. 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╧åhism 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

5 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.

15. An isolated nucleic acid sequence comprising the sequence selected from the group consisting of:

10 (i) the nucleotide sequence of SEQ ID No. 2, 3 or 6;

(ii) a nucleotide sequence having at least 90% sequence identity to (i);

(iii) the nucleotide sequence of SEQ ID No. 1, 4, 5, 7 or 8;

(iv) a fragment of (i), (ii) or (iii);and

(v) a nucleotide sequence fully complementary to (i), (ii), (iii) or (iv). 15

16. Use of a nucleotide sequence comprising SEQ ID No.7 or SEQ ID No. 8 in assays to identify modulators of NK2R expression.

17. Use of the NK2R promoter element of SEQ ID No. 7 or 8, or fragments thereof, in 20 expression studies.

18. An expression vector including a nucleic acid sequence comprising SEQ ID No.7 or SEQ ID No. 8, or fragments thereof.

19. A computer readable medium having stored thereon a member selected from the group consisting of: a nucleic acid comprising any of the sequences of SEQ ID No. 1 to SEQ ID No.

25 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 of a sequence of SEQ ID No. 1 to 21 which part includes at least one of the polymo╧åhisms identified in claim 1.

20. A method for performing sequence identification, said method comprising the steps of 5 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.

10 21. A method for performing sequence identification, said method comprising the steps of providing one or more of the following polymo╧åhism 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

15 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. 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

20 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. 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╧åhisms, and comparing said nucleic acid

25 sequence to at least one other nucleic acid or polypeptide sequence to determine identity.

22. A method for performing sequence identification, said method comprising the steps of providing one or more of the following polymo╧åhism 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. 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. 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╧åhisms, in a computer readable medium; and comparing said nucleic acid sequence to at least one other nucleic acid or polypeptide sequence to determine identity.