GPR50 is a human G protein coupled receptor (GPCR) first identified (Reppert et al., 1996, F. E. B. S. Lett., 386 219-224) as being related to the Melatonin family of GPCR's. The GPR50 gene was identified using a polymerase chain reaction (PCR) approach to identify novel members of the Melatonin receptor family from human genomic DNA. PCR primers were designed against certain highly conserved regions in the third and sixth transmembrane domains of known Melatonin receptors and used to clone a fragment of the gene. This fragment was used as a probe to isolate cDNA clones from a pituitary cDNA library. One of the resultant cDNA's identified was termed H9 and subsequently GPR50. This contains an open reading frame encoding a protein of 613 amino acids with 7 hydrophobic (transmembrane) domains. The cDNA sequence encoding GPR50 has been submitted to the EMBL database under accession number U52219. In this study the authors showed that when the cDNA encoding GPR50 was transfected into heterologous mammalian cells, these cells did not bind radio labelled Melatonin. Hence, GPR50 is referred to as an"orphan GPCR"as the identity of the native ligand is unknown.

Subsequent studies showed that the GPR50 gene is localised to human chromosome Xq28 (Gubitz & Reppert, 1999, Genomics, 55, 248-251). Also the expression of GPR50 in human, sheep and rodent tissues, is localised to hypothalamic structures in the brain, the pituitary, the retina and peripheral tissues (Reppert at al., ibid ; Drew et al. , 1998, J. of Neuroendocrinology, 1998, 10, 651-661; Drew et al. , 2001, J. of Neuroendocrinology, 13, 453-458).

Drugs which change the level of a GPR50 mediated response or change the biological activity of GPR50 are expected to be useful in the treatment of all conditions in which the GPR50 plays a pathophysiological role. Such drugs are particularly useful in the treatment of

conditions associated with the regulation of metabolism such as obesity, non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia and atherosclerosis.

DNA polymorphisms are variations in DNA sequence between one individual and another. DNA polymorphisms may lead to variations in amino acid sequence and consequently to altered protein structure and functional activity. Polymorphisms may also affect mRNA synthesis, maturation, transportation and stability. Polymorphisms which do not result in amino acid changes (silent polymorphisms) or which do not alter any known consensus sequences may nevertheless have a biological effect, for example by altering mRNA folding or stability.

Knowledge of polymorphisms may be used to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed"pharmacogenetics").

Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymorphisms may be 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 97/40438, Spectra Biomedical; and Schafer et al.

(1998), Nature Biotechnology, 16,33.

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

Point mutations in polypeptides will be referred to as follows: natural amino acid (using 1 or 3 letter nomenclature), position, new amino acid. For (a hypothetical) example "D25K"or"Asp25Lys"means that at position 25 an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one polypeptide will be shown between square brackets with individual mutations separated by commas.

Nucleotide or amino acid insertion will be referred to as follows: position/position.

For a hypothetical example,"220/221"means that there is a nucleotide (s) or amino acid (s) insertion between positions 220 and 221 as defined by the identified SEQ ID NO.

Insertion of amino acid (s) will also be referred to as follows: position, amino acid (s), position. For a hypothetical example,"220 (GG) 221"or"220 (GlyGly) 221"means that two glycine residues are inserted between the amino acids at positions 220 and 221 as defined by the identified SEQ ID NO.

A cDNA encoding GPR50 is set out as SEQ ID NO : 2, with the first nucleotide of the GPR50 coding region accorded position 70.

The genomic DNA sequence of the human GPR50 gene has been determined and submitted to the EMBL database under accession number AF003625. The genomic sequence comprises one intron (2,863 bp) interrupting the cDNA sequence between nucleotide positions 256-257 as defined in SEQ ID NO : 2. Part of the genomic DNA sequence of the human GPR50 gene is set out as SEQ ID NO: 1, and shows the last 100 nucleotides of intron 1, with the 2, 764th nucleotide of intron 1 accorded position 1. Note that the cDNA sequence from nucleotide position 257 onwards, and as defined in SEQ ID NO: 2, is also set out in SEQ ID NO: 1, but with nucleotide position 257 accorded position 101 in SEQ ID NO: 1.

All positions herein of polymorphisms in intron 1 and in the human GPR50 gene transcribed into mRNA (and thence cDNA) are defined with reference to SEQ ID NO : 1 unless stated otherwise or apparent from the context.

All positions herein of polymorphisms in the GPR50 polypeptide are defined with reference to SEQ ID NO : 3 unless stated otherwise or apparent from the context.

The present invention is based on the discovery of four polymorphisms in the human GPR50 gene transcribed into mRNA (and thence cDNA) and one polymorphism in the intronic sequence of the human GPR50 gene.

According to one aspect of the present invention there is provided a method for the detection of a polymorphism in GPR50 in a human, which method comprises determining the sequence of the human at any one of the following positions: positions 86,468, 1418,1495 and 1717 of SEQ ID NO : 1; positions 502,528 and 602 of SEQ ID NO: 3.

The term"human"includes both a human having or suspected of having a GPR50 mediated response and an asymptomatic human who may be tested for predisposition or susceptibility to such a response. As the GPR50 gene is in the X chromosome, at each position the human female may be homozygous for an allele or the female human may be a heterozygote. The male human can only be homozygous for the alleles.

The term"detection of a polymorphism"refers to determination of the genetic status of an individual at a polymorphic position (in which the individual may be homozygous or heterozygous at each position).

The term"GPR50 mediated response"means any disease in which changing the level of a GPR50 mediated response or changing the biological activity of GPR50 would be of therapeutic benefit.

The term"polymorphism"includes single nucleotide substitution, nucleotide insertion and nucleotide deletion, which in the case of insertion and deletion includes insertion or deletion of one or more nucleotides at a position of a gene and variable numbers of a repeated DNA sequence.

In one embodiment of the invention preferably the polymorphism is further as: polymorphism at position 86 is presence of C and/or T; polymorphism at position 468 is presence of C and/or T; polymorphism at position 1418 is presence of T and/or insertion of CCACTGGCCACA ; polymorphism at position 1495 is presence of G and/or A; polymorphism at position 1717 is presence of G and/or A; polymorphism at position 502 is presence of Ile and/or insertion of Thr-Thr-Gly-His ; polymorphism at position 528 is presence of Ala and/or Thr; and polymorphism at position 602 is presence of Val and/or Ile.

The polymorphism at position 1418 of SEQ ID NO: 1 is the result of an insertion- deletion event defined as insertion of CCACTGGCCACA between positions 1417 and 1418 (1417/1418). This results in an overall extra 12 bases and it will be appreciated by the skilled person that this will have an effect on the numbering of positions downstream of this. For example, position 1418 of SEQ ID NO: 1 (the deletion variant of GPR50) becomes position 1430 of SEQ ID NO : 42 (the insertion variant of GPR50) after the insertion. It will also be appreciated by the skilled person that it may not be necessary to sequence the entire insertion at this position to distinguish between the two alleles. For example, position 1418 is either a T or a C when comparing the sequence of the two alleles.

The polymorphism at position 502 of SEQ ID NO: 3 is the result of an insertion- deletion event defined as insertion of Thr-Thr-Gly-His between positions 501 and 502 (501/502). This results in an overall extra 4 amino acids and it will be appreciated by the skilled person that this will have an effect on the numbering of positions downstream of this.

For example, position 502 of SEQ ID NO: 3 becomes position 506 of SEQ ID NO : 43 after the insertion (the addition of the insertion of four amino acids Thr-Thr-Gly-His at position 501/502 changes the amino acid sequence of GPR50 from that of SEQ ID NO : 3 to that set out as SEQ ID NO : 43). It will also be appreciated by the skilled person that it may not be necessary to sequence the entire insertion at this position to distinguish between the two alleles. For example, position 502 is either an Ile or a Thr when comparing the sequence of the two alleles.

Preferred methods for detection of nucleic acid polymorphism are amplification refractory mutation system and restriction length polymorphism.

More preferably the polymorphism at position 86 is used to investigate blood triglyceride levels or a disease related thereto.

More preferably the polymorphism at position 1418 and/or the polymorphism at position 502 is used to investigate blood triglyceride levels or a disease related thereto.

More preferably the polymorphism at position 1495 and/or the polymorphism at position 528 is used to investigate blood triglyceride levels or a disease related thereto.

More preferably the polymorphism at position 1717 and/or the polymorphism at position 602 is used to investigate blood triglyceride levels or a disease related thereto.

In another aspect of the invention we provide a method for the diagnosis of GPR50- mediated disease, which method comprises: i) obtaining sample nucleic acid from an individual, ii) detecting the presence or absence of a variant nucleotide at any one of the following positions: positions 86,468, 1418,1495 and 1717 in the GPR50 gene as defined by the positions in SEQ ID NO : 1 ; iii) determining the status of the individual by reference to polymorphism in the GPR50 gene.

Allelic variation at each position in the GPR50 gene, including preferred variation is described herein.

The status of the individual may be determined by reference to allelic variation at any combination of one, two, three, four or all five positions optionally in combination with any other polymorphism in the gene that is (or becomes) known.

The test sample of nucleic acid is conveniently present in a sample of blood, sputum, skin, or other body fluid or tissue obtained from an individual. It will be appreciated that the test sample may equally comprise 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 analysis of allelic variation.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures that may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. A number of mutation detection

techniques, some based on PCR, may be used in combination with a number of signal generation systems, a selection of these are highlighted below. 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", 2nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations : ALEXTM Amplification refractory mutation system linear extension APEX Arrayed primer extension ARMSTM Amplification refractory mutation system b-DNABranched DNA CMC Chemical mismatch cleavage COPS Competitive oligonucleotide priming system DGGE Denaturing gradient gel electrophoresis dHPLC Denaturing High Performance Liquid Chromatography FRET Fluorescence resonance energy transfer GPCR G protein coupled receptor LCR Ligase chain reaction MASDA Multiple allele specific diagnostic assay NASBA Nucleic acid sequence based amplification OLA Oligonucleotide ligation assay PCR Polymerase chain reaction PTT Protein truncation test RFLP Restriction fragment length polymorphism SDA Strand displacement amplification SSCP Single-strand conformation polymorphism analysis SSR Self sustained replication TGGE Temperature gradient gel electrophoresis UTR Untranslated region WAVE See-www. transgenomic. com Mutation Detection Tech) 1iques

General: DNA sequencing, Sequencing by hybridisation Scanning: PTT*, SSCP, DGGE, TGGE, dHPLC (using WAVE), Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage * Note: not useful for detection of promoter polymorphisms.

Hybridisation Based: Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips).

Solution phase hybridisation: TaqmanTM-US-5210015 & US-5487972 (Hoffinann-La Roche), Molecular Beacons-Tyagi et al (1996), Nature Biotechnology, 14,303 ; WO 95/13399 (Public Health Inst., New York).

Extension Based: ARMS, ALEXTM-European Patent No. EP 332435 B1 (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 Other: Invader assay Signal Generation or Detection Systems Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation-United Kingdom Patent No. 2228998 (Zeneca Limited) Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Ultra Violet absorption (WAVE) Colorimetric, Hybridisation protection assay, Mass spectrometry.

Further Al1lplif cation Metllods SSR, NASBA, LCR, SDA, b-DNA Protein variation detection methods Immunoassay Immunohistology Peptide sequencing Immunoassay techniques are known in the art e. g. A Practical Guide to ELISA by D M Kemeny, Pergamon Press 1991; Principles and Practice of Immunoassay, 2nd edition, C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the United Kingdom. Histological techniques are described in Theory and Practice of Histological Techniques by J D Bancroft & A Stevens, 4th Edition,

Churchill Livingstone, 1996. Protein sequencing is described in Laboratory Techniques in Biochemistry and Molecular Biology, Volume 9, Sequencing of Proteins and Peptides, G Allen, 2nd revised edition, Elsevier, 1989.

Preferred mutation detection techniques include DNA Sequencing, dHPLC & WAVE, ARMS, ALEXTM, COPS, Taqman, Molecular Beacons, RFLP, restriction site based PCR and FRET techniques, polyacrylamide gel electrophoresis and capillary electrophoresis.

Particularly preferred methods include ARMSTM and RFLP based methods. ARMSTM is an especially preferred method.

In a further aspect, the diagnostic methods of the invention are used to assess the efficacy of therapeutic compounds in the treatment of GPR50-mediated diseases particularly disease states associated with the regulation of metabolism such as obesity, non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia and atherosclerosis.

Assays, for example reporter-based assays, may be devised to detect whether one or more of the above polymorphisms affect transcription levels and/or message stability.

Individuals who carry particular allelic variants of the GPR50 gene may exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and may display altered abilities to react to different diseases. In addition, differences in protein regulation and/or the protein's properties arising as a result of allelic variation may have a direct effect on the response of an individual to drug therapy. The diagnostic methods of the invention may be useful both to predict the clinical response to such agents and to determine therapeutic dose.

In a further aspect, the diagnostic methods of the invention are used to assess the predisposition of an individual to diseases mediated by GPR50. This may be particularly relevant in the regulation of metabolism and in diseases that are mediated by GPR50 such as obesity, non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia and atherosclerosis. The present invention may be used to recognise individuals who are particularly at risk from developing these conditions.

In particular, an association has been observed between the presence of the nucleotide/s at each of the positions 86, 1418 and 1495 of GPR50 of SEQ ID NO : 1 and an increased circulating triglyceride level of the individual. (See, example 3). This association was present in both men and women. The results indicate that the presence of each of these polymorphisms in the GPR50 gene of an individual may be used to detect the presence of or likely future development of raised circulating triglyceride levels. Therefore in a preferred

aspect of the invention we provide the use of the polymorphisms at positions 86,1418 and 1495 in the GPR50 gene of SEQ ID NO : 1, to detect the presence of or possible future development of elevated circulating triglycerides.

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 GPR50 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 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.

According to another aspect of the invention there is provided a human GPR50 gene or its complementary strand comprising a allelic variant polymorphism at one or more of positions defined herein or a fragment thereof of at least 20 bases comprising at least one novel polymorphism.

Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bases.

According to another aspect of the present invention there is provided a polynucleotide comprising at least 20 contiguous bases of the human GPR50 gene and comprising an allelic variant selected from any of the following: Region Variant Position of SEQ ID NO : 1 Intron 1 T 86 Exon 2 T 468 Exon 2 CCACTGGCCACA 1418 Exon 2 A 1495 Exon 2 1717 According to another aspect of the present invention there is provided a human GPR50 gene or its complementary strand comprising a polymorphism, preferably corresponding with one or more the positions defined herein or a fragment thereof of at least 20 bases comprising at least one polymorphism.

Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bases.

The scope of the invention does not extend to any nucleic acid as it is found in nature.

A nucleic acid of the invention is preferably in isolated form, for example through being at least partially purified from any substance with which it occurs naturally.

Novel sequence disclosed herein, may be used in another embodiment of the invention to regulate expression of the gene in cells by the use of antisense constructs. To enable methods of down-regulating expression of the gene of the present invention in mammalian cells, an example antisense expression construct can be readily constructed for instance using the pREP10 vector (Invitrogen Corporation). Transcripts are expected to inhibit translation of the gene in cells transfected with this type of construct. Antisense transcripts are effective for inhibiting translation of the native gene transcript, and capable of inducing the effects (e. g. , regulation of tissue physiology) herein described.

Oligonucleotides, which are complementary to and hybridisable with any portion of novel gene mRNA disclosed herein, are contemplated for therapeutic use. U. S. Patent No.

5,639, 595, "Identification of Novel Drugs and Reagents", issued Jun. 17,1997, wherein methods of identifying oligonucleotide sequences that display in vivo activity are thoroughly described, is herein incorporated by reference.

Expression vectors containing random oligonucleotide sequences derived from previously known, polynucleotides are transformed into cells. The cells are then assayed for a phenotype resulting from the desired activity of the oligonucleotide. Once cells with the desired phenotype have been identified, the sequence of the oligonucleotide having the desired activity can be identified. Identification may be accomplished by recovering the vector or by polymerase chain reaction (PCR) amplification and sequencing the region containing the inserted nucleic acid material. Antisense molecules can be synthesised for antisense therapy.

These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O- alkylRNA, or other oligonucleotide mimetics. U. S. Patent No. 5,652, 355, "Hybrid Oligonucleotide Phosphorothioates", issued July 29,1997, and U. S. Patent No. 5,652, 356, "Inverted Chimeric and Hybrid Oligonucleotides", issued July 29,1997, which describe the synthesis and effect of physiologically-stable antisense molecules, are incorporated by reference. Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.

The invention further provides nucleotide primers which can detect the polymorphisms of the invention.

According to another aspect of the present invention there is provided an allele specific primer capable of detecting a GPR50 gene polymorphism, at one or more of the positions as defined herein.

An allele specific primer is used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position e. g. as used for ARMSTM 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.

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"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 GPR50 gene polymorphism at one or more of the positions defined herein.

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. The probes of the invention may carry one or more labels to facilitate detection.

According to another aspect of the present invention there is provided an allele specific primer or an allele specific oligonucleotide probe capable of detecting a GPR50 gene polymorphism at one of the positions defined herein.

According to another aspect of the present invention there is provided a diagnostic kit comprising an allele specific oligonucleotide probe of the invention and/or an allele-specific primer 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), nucleotides, and polymerase (s) such as thennostable polymerases, for example taq polymerase.

In another aspect of the invention, a polymorphism of this invention may be used as a genetic marker in a linkage study. This particularly applies to polymorphisms of relatively high frequency.

Low frequency polymorphisms may be particularly useful for haplotyping as described below. A haplotype is a set of alleles found at linked polymorphic sites (such as within a gene) on a single (paternal or maternal) chromosome. If recombination within the gene is random, there may be as many as 2"haplotypes, where 2 is the number of alleles at each polymorphic position and n is the number of polymorphic positions. One approach to identifying mutations or polymorphisms, 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 polymorphisms with low frequency alleles are particularly useful as markers of low frequency haplotypes. As particular mutations or polymorphisms associated with certain clinical features, such as adverse or abnormal events, are likely to be of low frequency within the population, low frequency polymorphisms may be particularly useful in identifying these mutations (for examples see: De Stefano V et al., Ann Hum Genet (1998) 62: 481-90; and Keightley AM et al., Blood (1999) 93: 4277-83.

According to another aspect of the present invention there is provided a method of treating a human in need of treatment with a GPR50 drug in which the method comprises: i) detection of a polymorphism in GPR50 in a human, which detection comprises determining the sequence of the nucleic acid at one or more of positions 86,468, 1418,1495 and 1717 of SEQ ID NO : 1; positions 502,528 and 602 of SEQ ID NO : 3, and determining the status of the human by reference to polymorphism in GPR50; and ii) administering an effective amount of the drug.

The term"GPR50 drug"means any drug which changes the level of a GPR50 mediated response or changes the biological activity of GPR50. For example the drug may be an agonist or an antagonist of a natural ligand for GPR50.

Preferably determination of the status of the human is clinically useful. Examples of clinical usefulness include deciding which drug or drugs to administer and/or establishing the effective amount of the drug or drugs.

Drugs which change the activity of GPR50 may be of value in a number of disease conditions, including disease states associated with the regulation of metabolism such as obesity, non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia and atherosclerosis.

According to another aspect of the present invention there is provided use of a GPR50 drug in the preparation of a medicament for treating a GPR50-mediated disease in a human determined as having a polymorphism defined herein.

According to another aspect of the present invention there is provided a pharmaceutical pack comprising a GPR50 drug and instructions for administration of the drug to humans tested for a polymorphism therein, preferably at one or more of the positions defined herein.

According to another aspect of the present invention there is provided a computer readable medium comprising at least one novel polynucleotide sequence of the invention stored on the medium. The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis or any other bioinformatic analysis. The reader is referred to Bioinformatics, A practical guide to the analysis of genes and proteins, Edited by A D Baxevanis & B F F Ouellette, John Wiley & Sons, 1998. Any computer readable medium may be used, for example, compact disk, tape, floppy disk, hard drive or computer chips.

The polynucleotide sequences of the invention, or parts thereof, particularly those relating to and identifying the polymorphisms identified herein represent a valuable information source, for example, to characterise individuals in terms of haplotype and other sub-groupings, such as investigation of 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 bioinfonnatics programs or to search sequence databases, using state of the art searching tools such as "GCC". Thus, the polynucleotide sequences of the invention are particularly useful as

components in databases useful for sequence identity and other search analyses. As used herein, storage of the sequence information in a computer readable medium and use in sequence databases in relation to'polynucleotide or polynucleotide sequence of the invention' covers any detectable chemical or physical characteristic of a polynucleotide of the invention that may be reduced to, converted into or stored in a tangible medium, such as a computer disk, preferably in a computer readable form. For 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 polynucleotide sequences of the invention. For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of : a polynucleotide comprising the sequence of a polynucleotide of the invention, a polynucleotide consisting of a polynucleotide of the invention, a polynucleotide which comprises part of a polynucleotide of the invention, which part includes at least one of the polymorphisms of the invention, a set of polynucleotide sequences wherein the set includes at least one polynucleotide sequence of the invention, a data set comprising or consisting of a polynucleotide sequence of the invention or a part thereof comprising at least one of the polymorphisms identified herein.

A computer based method is also provided for performing sequence identification, said method comprising the steps of providing a polynucleotide sequence comprising a polymorphism of the invention in a computer readable medium; and comparing said polymorphism containing polynucleotide sequence to at least one other polynucleotide or polypeptide sequence to identify identity (homology), i. e. screen for the presence of a polymorphism.

Three of the polymorphisms of the present invention result in variation in the amino acid sequence of the translated protein. Polymorphism at position 1417/1418 as defined in SEQ ID NO: 1 results in a polypeptide having inserted therein four additional amino acids Thr-Thr-Gly-His. The amino acid sequence of this insertion variant of GPR50 is set out as SEQ ID NO: 43 with the four additional amino acids accorded positions 502-505.

Polymorphism at position 1495 as defined in SEQ ID NO: 1 results in an amino acid change from alanine to threonine at corresponding position 528 of the translated protein (Ala528Thr) as defined in SEQ ID NO : 3. Polymorphism at position 1717 as defined in SEQ ID NO : 1

results in an amino acid change from valine to isoleucine at corresponding position 602 of the translated protein (Val602Ile) as defined in SEQ ID NO : 3.

Thus according to another aspect of the present invention there is provided an allelic variant of human GPR50 polypeptide having a threonine at position 528 of SEQ ID NO : 3 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at position 528 of SEQ ID NO : 3.

According to another aspect of the present invention there is provided an allelic variant of human GPR50 polypeptide comprising a threonine-threoniile-glycine- histidine at positions 502-505 of SEQ ID NO : 43 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at positions 502-505 of SEQ ID NO : 43.

According to another aspect of the present invention there is provided an allelic variant of human GPR50 polypeptide comprising an isoleucine at position 602 of SEQ ID NO: 3 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at position 602 of SEQ ID NO : 3.

Fragments of GPR50 polypeptide are at least 10 amino acids, more preferably at least 15 amino acids, more preferably at least 20 amino acids. The polypeptides of the invention do not encompass naturally occurring polypeptides as they occur in nature, for example, the polypeptide is at least partially purified from at least one component with which it occurs naturally. Preferably the polypeptide is at least 30% pure, more preferably at least 60% pure, more preferably at least 90% pure, more preferably at least 95% pure, and more preferably at least 99% pure.

According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human GPR50 polypeptide having a threonine at position 528 of SEQ ID NO : 3 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at position 528 of SEQ ID NO: 3.

According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human GPR50 polypeptide comprising a threonine-threonine- glycine-histidine at positions 502-505 of SEQ ID NO : 43 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at positions 502- 505 of SEQ ID NO : 43.

According to another aspect of the present invention there is provided an antibody specific for an allelic variant of human GPR50 polypeptide comprising an isoleucine at

position 602 of SEQ ID NO: 3 or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises the allelic variant at position 602 of SEQ ID NO : 3.

Antibodies can be prepared using any suitable method. For example, purified polypeptide may be utilised to prepare specific antibodies. The term"antibodies"includes polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F (ab') 2, Fab and single chain Fv. Antibodies are defined to be specifically binding if they bind the antigen with a Ka of greater than or equal to about 107 M-1. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. N. Y. Acad. Sci., 51: 660 (1949).

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunisations, small samples of serum are collected and tested for reactivity to antigen.

Examples of various assays useful for such determination include those described in: Antibodies : A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988 ; as well as procedures such as counter current immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U. S. Patent Nos. 4,376, 110 and 4,486, 530.

Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U. S. Patent Nos. RE 32, 011, 4, 902, 614,4, 543,439 and 4,411, 993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds. ), (1980).

The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al. ,"Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3 : 1-9 (1990) which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example"A Practical Guide to

ELISA"by D. M. Kemeny, Pergamon Press, Oxford, England.

According to another aspect of the invention there is provided a diagnostic kit comprising an antibody of the invention.

The invention will now be illustrated but not limited by reference to the following Examples and Figure. General molecular biology procedures can be followed from the methods described in"Molecular Cloning-A Laboratory Manual"Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or"Current Protocols in Molecular Biology", Volumes 1-3, Edited by F M Asubel, R Brent & R E Kingston, published by John Wiley, 1998.

EXAMPLES Example 1 Identification of GPR50 polymorphisms in cohorts of obese and "normal" individuals Sequencing of DNA from 94 extremely obese individuals and 29 normal individuals identified the following five polymorphisms in the GPR50 DNA sequence. Four of these polymorphisms were single nucleotide substitutions described in the table below and one polymorphism was a 12 base pair insertion/deletion. Because of the small sample size, allele frequencies are referred to as common (>5%) or rare (-1%). Position Region Reference Variant Resultant Protein sequence Frequency of SEQ allele allele codon SEQ ID NO : 3 variant. ID change allele NO : 1 86 Intron C T N/a N/a Common 1 468 Exon 2 C T AAC-AAT Asnl 85Asn Rare 1495 Exon 2 G A GCT-ACT Ala528Thr Common 1717 Exon 2 G A GTT-ATT Val602Ile Common 1418 Exon 2 Deletion Insertion CCACTGGC 501ThrThrGlyHis Common CACA 502

Example 2 GPRSO polyniofphisms in a"nornial"population The frequencies of the four"common"polymorphisms at positions 86,418, 1495 and 1717 of SEQ ID NO : 1, were determined in 1162"normal"individuals. Position of Reference Second Resultant Resultant Second SEQ ID allele allele codon Amino Allele NO : 1 change Acid Frequezcy Change 86 C T N/a N/a 10% 1418 deletion insertion CCACTGGC Thr-Thr-58% CACA Gly-His 1495 G A GCT-ACT Ala-Thr 57% 1717 G A GTT-ATT Val-Ile 40% The C to T polymorphism occurring at position 86 of SEQ ID NO : 1 was present in 10% of the alleles. This level of occurrence was similar to that observed in the obese individual cohort in example 1.

The polymorphism occurring at position 1418 of SEQ ID NO : 1 was present in 58% of the alleles. This level of occurrence was similar to that observed in the cohorts used in example 1.

The G to A polymorphism occurring at position 1495 of SEQ ID NO : 1 was present in 57% of the alleles. This level of occurrence was similar to that observed in the obese individual cohort in example 1.

The G to A polymorphism occurring at position 1717 of SEQ ID NO : 1 was present in 40% of the alleles. This level of occurrence was similar to that observed in the cohorts used in example 1.

Example 3 Association of GPRSOpolymo7phisms, in a"normal"population, with circalatingplasma triglyceride concentrations.

An association was observed between polymorphisms at positions 86,1418 and 1495 of SEQ ID NO: 1 and an increased circulating triglyceride level in the"normal"population.

The results indicate that the presence of these polymorphisms in the GPR50 gene of an individual may be used to diagnose the presence of or likely future development of cardiovascular disease.

The association of circulating plasma triglyceride levels in individuals with 2 GPR50 alleles containing a C at position 86, compared with either individuals with one C and one T, and individuals where both alleles contain a T at position 86, is shown below. Genotype C/C C/T and T/T P value Log of fasting serum 0. 19 0. 32 p=0. 0008 Triglycerides levels Standard Deviation 0. 02 0. 03

The association of circulating plasma triglyceride levels in individuals with 2 GPR50 alleles containing a G at position 1495, compared with either individuals with one G and one A, and individuals where both alleles contain an A at position 1495, is shown below. Genotype G/G G/A and A/A P value Log of fasting serum 0. 27 0. 19 p=0. 012 Triglycerides levels) Standard Deviation 0. 03 0. 02

The association of circulating plasma triglyceride levels in individuals with 2 GPR50 alleles with the deletion at position 1418, compared with either individuals with an insertion and a deletion, and individuals where both alleles contain the insertion at position 1418, is shown below. Genotype Mean Fasting Lower 95% CI Upper 95% CI P value serum Triglycerides level (nmol/1) ins/ins 1.21 1.17 1. 26 ins/del 1. 19 1. 12 1. 26 del/del 1.31 1.24 1.37 0.011

Example 4 Experimental procedures used to identify polymorphism in GPR50 of obese and "normal" individuals 94 obese individuals were screened for mutations within GPR50 Exons 1 and 2.

Mutation within Exon 1 were screened for by sequencing PCR products obtained from amplification of these 94 individuals with the primers as defined by SEQ ID NO : 4 and SEQ ID NO : 5 using an ABI 377 sequencer according to the manufacturers instructions (Perkin Elmer, Cheshire, UK).

Alternative procedures for sequencing DNA are well known in the art and are thoroughly described in Maniatis et al. , ibid. The primers used for PCR and sequencing were: GPR50 exon 2 was screened for mutations by amplification of the exon in 7 overlapping fragments using primers as defined by SEQ ID Nos: 6-19 inclusive. Each fragment was than analysed by dHPLC using a Transgenomic Wave machine (www. transgenomic. com). Twenty nine"normal"individuals were screened for polymorphisms within exons 1 and 2 of GPR50 by sequencing of PCR products from each individual separately. Exon 1 was amplified using primers with SEQ ID NO: 24 and 25 and Exon 2 was amplified as overlapping PCR products using primer pairs with SEQ ID NO: 26 to 35 inclusive.

Forward primers included an M13F sequence tag and reverse primers included an M13R sequence tag. PCR products were sequenced by dye-terminator sequencing using M13F and M13R primers for forward and reverse sequence respectively (Protocol E79000M, DYEnamic ET Primer DNA Sequencing for MegaBace, Amersham Biosciences). Sequencing reactions were run on a MegaBace capillary sequencer. Sequence traces were analysed for polymorphisms after assembly and quality calling with phred/phrap/consed package (University of Washington).

Example 5 Experimentalprocedures used to type polymorphisnzs in GPR50 in the"nornzal" population Following identification, the polymorphism at position 86 of SEQ ID NO: 1 was typed using the following method. Genotyping of this polymorphism was carried out by PCR amplification and restriction digestion of the amplified products using standard molecular

biological protocols. Restriction sites were introduced into the DNA by designing PCR primer pairs with one or two deliberately introduced mismatches.

The primers used for this PCR are as defined in SEQ ID NO: 20 and SEQ ID NO : 21.

The enzyme Xho I was used for digestion of product. The PCR and restriction enzyme digestion were conducted in 10F1 volumes and performed using standard protocols. For the polymorphism at position 86 of SEQ ID NO : 1 only amplified DNA containing the reference allele (C) would be digested whereas the second allele (T) would give an undigestable PCR product. Digested samples were run on a 3% (w/v) agarose gel for 2 hours, which allowed the digested PCR product to be distinguished from undigested PCR product, and scored blind on 2 occasions.

Following identification, the polymorphism at position 1495 of SEQ ID NO : 1 was typed using the following method. Genotyping of this polymorphism was carried out by PCR amplification and restriction digestion of the amplified products using standard molecular biological protocols. Restriction sites were introduced into the DNA by designing PCR primer pairs with one or two deliberately introduced mismatches. The primers used for this PCR are as defined in SEQ ID NO: 22 and SEQ ID NO : 23. The enzyme Sac II was used for digestion of product.

The PCR and restriction enzyme digestion were conducted in 10, ul volumes and performed using standard protocols. For the polymorphism at position 1495 of SEQ ID NO : 1 only amplified DNA containing the reference allele (G) would be digested whereas the second allele (A) would give an undigestable PCR product. Digested samples were run on a 3% (w/v) agarose gel for 2 hours, which allowed the digested PCR product to be distinguished from undigested PCR product, and scored blind on 2 occasions.

The insertion/deletion at position 1418 of SEQ ID NO : 1 was genotyped using a fluorescent PCR method using primers with SEQ ID NO : 36 and SEQ ID NO: 37 to detect products of 113 or 125 bp in the absence or presence of the 12bp insertion sequence, respectively: The forward primer was labelled with FAM and the products resolved using an ABI 3700 capillary sequencer.

The polymorphism at position 1717 of SEQ ID NO : 1 was genotyped using a TaqMan assay established using the Applied Biosystems Assays-by-Design service. PCR products were generated using primers with SEQ ID NO: 38 and 39.

The A allele was detected with FAM labelled probe of SEQ ID NO : 40. The G allele was detected with VIC labelled probe of SEQ ID NO : 41.

The assay was run as an end point fluorescence assay measured on an ABI 7900.

PCR Products Primers used for the amplification of the human GPR50 gene for sequencing and genotyping. PCR forward oligo PCR reverse oligo SEQ ID NO : 4 SEQ ID NO : 5 SEQ ID NO : 6 SEQ ID NO:7 SEQ ID NO:8 SEQ ID N0 : 9 SEQ ID NO : 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO : 13 SEQ ID NO: 14 SEQ ID NO : 15 SEQ ID NO : 16 SEQ ID NO : 17 SEQ ID NO:18 SEQ ID NO : 19 SEQ ID NO : 20 SEQ ID NO : 21 SEQ ID NO : 22 SEQ ID NO : 23 SEQ ID NO:24 SEQ ID NO : 25 SEQ ID NO : 26 SEQ ID NO : 27 SEQ ID NO : 28 SEQ ID NO : 29 SEQ ID NO : 30 SEQ ID NO : 31 SEQ ID NO : 32 SEQ ID NO : 33 SEQ ID NO : 34 SEQ ID NO : 35 SEQ ID NO : 36 SEQ ID NO : 37 SEQ ID NO : 38 SEQ ID NO : 39