WO2000017393A1

WO2000017393A1 - Polymorphisms in the human beta1 integrin subunit gene, suitable for diagnosis and treatment of integrin ligand mediated diseases

Info

Publication number: WO2000017393A1
Application number: PCT/GB1999/003066
Authority: WO
Inventors: John Edward Norris Morten
Original assignee: Zeneca Ltd; AstraZeneca AB
Current assignee: Syngenta Ltd; AstraZeneca AB
Priority date: 1998-09-19
Filing date: 1999-09-15
Publication date: 2000-03-30
Anticipated expiration: 2001-03-19
Also published as: EP1114183A1; JP2002526090A; AU5876399A

Abstract

This invention relates to polymorphisms in the human β1 integrin subunit gene, in particular at positions 590, 751, 1206 and 1282 in the promoter as defined by EMBL ACCESSION NO. X68969, positions 562, 886, 1189, 1279 and 1942, in the coding region as defined by EMBL ACCESSION NO. X07979, and position 2502 in the 3'-untranslated region as defined by EMBL ACCESSION NO. X07979. The invention also relates to methods and materials for analysing allelic variation in the β1 integrin subunit gene, and to the use of β1 integrin subunit polymorphism in the diagnosis and treatment of integrin ligand mediated diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis and allergic asthma.

Description

POLYMORPHISMS IN THE HUMAN BETAl INTEGRIN SUBUNIT GENE, SUITABLE FOR DIAGNOSIS AND TREATMENT OF INTEGRIN LIGAND MEDIATED DISEASES

This invention relates to polymorphisms in the human βi integrin subunit gene. The invention also relates to methods and materials for analysing allelic variation in the βi integrin subunit gene, and to the use of βi integrin subunit polymoφhism in the diagnosis and treatment of integrin ligand mediated diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis and allergic asthma.

The integrins are a family of heterodimeric cell surface receptors that are composed of noncovalently associated glycoprotein subunits (α and β) and are involved in the adhesion of cells to other cells or to extracellular matrix. The interactions between integrins and their protein ligands are fundamental for maintaining cell function, for example by tethering cells at a particular location, facilitating cell migration, or providing survival signals to cells from their environment. Ligands recognised by integrins include extracellular matrix proteins, such as collagen and fibronectin; plasma proteins, such as fibrinogen; and cell surface molecules, such as transmembrane proteins of the immunoglobulin superfamily and cell-bound complement. There are at least 15 different human integrin α subunits and at least 8 different β subunits and each β subunit can form a heterodimer with one or more subunits. The specificity of the interaction between integrin and ligand is governed by the α and β subunit composition.

The integrin family can be subdivided into classes based on the β subunit. The largest ofthese is the βi integrin class, also known as the Very Late Antigens (VLA). The βi integrin subunit, also known as CD29, comprises 778 arnino acids and is formed from a 798 amino acid precursor by the cleavage of a 20 amino acid N-terminal signal peptide. The extracellular domain comprises amino acid residues 1-708, the transmembrane domain residues 709-731 and there is a short intracellular domain comprising residues 732-778. The C-terminal portion of the extracelluar domain contains four cysteine-rich domains, residues 446-615, which form internal disulphide bonds. Close to the N-terminus is a domain (residues 130-295) which is related to the von Willebrand factor A-domain and which contains sequences thought to interact with divalent cations and ligands.

The βi subunit forms a heterodimer with at least 8 different subunits (α.ι, α₂, α₃, α₄, α₅, α₆, α.ιo and α_v). Most members of the βi integrin family are expressed widely in the body and are involved in cell-extracellular matrix interactions with a range of specificities. For example α₅βι and α_vβι bind fibronectin and α₆βι binds laminin, while otiβi and α₂βι bind laminin and collagen and α₃βι binds laminin, collagen and fibronectin. It is thought that these βi integrins have a role in cell survival, differentiation and motility and could be important in development, wound repair, tissue homeostasis and cancer metastsis. Another βj integrin family member, α₄βι, also known as VLA-4 or CD49d/CD29, is unusual in its expression pattern and its ligand specificity. It is expressed on numerous hematopoietic cells, including hematopoietic precursors, peripheral and cytotoxic T lymphocytes, B lymphocytes, monocytes, thymocytes and eosinophils, and established cell lines, o βi has two main ligands, Vascular Cell Adhesion Molecule-1 (VCAM-1), also known as CD 106, an immunoglobulin superfamily member expressed on the surface of activated vascular endothelial cells and a variety of other cells including dendritic cells, macrophages and fibroblasts, and an isoform of fibronectin containing the alternatively spliced type III connecting segment (CS-1 fibronectin).

The activation and extravasation of blood leukocytes plays a major role in the development and progression of inflammatory diseases. Cell adhesion to the vascular endothelium is required before cells migrate from the blood into inflamed tissue and is mediated by specific interactions between cell adhesion molecules on the surface of vascular endothelial cells and circulating leukocytes. o βi is believed to have an important role in the recruitment of lymphocytes, monocytes and eosinophils during inflammation. The affinity of leukocyte integrins for their ligands is normally low but activation of leukocytes increases integrin affinity. At sites of inflammation, leukocyte integrins are thought to be activated by chemokines which act via receptors on the leukocyte surface. Integrin affinity is thought to be regulated by conformational changes in the integrin subunits induced by intracellular signalling pathways acting on the integrin cytoplasmic tails. Expression of α₄βι ligands is upregulated at sites of inflammation. VCAM-1 expression is upregulated on endothelial cells in vitro by inflammatory cytokines and in human inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, allergic asthma and atherosclerosis. CS-1 fibronectin expression is upregulated in rheumatoid arthritis.

Monoclonal antibodies directed against α₄βι have been shown to be effective in a number of animal models of human inflammatory diseases including multiple sclerosis, rheumatoid arthritis, allergic asthma, contact dermatitis, transplant rejection, insulin-dependent diabetes, inflammatory bowel disease, and glomerulonephritis. α₄βj /ligand binding has also been implicated in T-cell proliferation, B-cell localisation to germinal centres, haematopoietic progenitor cell localisation in the bone marrow, angiogenesis, placental development, muscle development and tumour cell metastasis.

Integrins recognise short peptide motifs in their ligands. The minimal α βι binding epitope in CS-1 is the tripeptide leucine-aspartic acid-valine (LDV) while VCAM-1 contains the similar sequence isoleucine-aspartic acid-serine (IDS). Small molecule inhibitors of ligand binding to ouβi have been designed based on these short peptide motifs. βι antagonists, monoclonal antibodies directed against α₄βι or its ligands and inhibitors of α₄βι ligand expression may have utility in the treatment of autoimmune, allergic and vascular inflammatory diseases, the prevention of tumour metastasis and in mobilisation of haematopoietic progenitor cells from bone marrow prior to tumour chemotherapy.

A DNA sequence comprising the βi integrin subunit gene promoter has been cloned and published as a EMBL Accession number: X68969 (1891 bp); (this is also listed in EMBL under accession number S 56437 but for convenience the former sequence alone will generally be cited) . A cDNA encoding the βi integrin subunit has been cloned and published as a EMBL Accession number: X07979 (3614 bp). All positions herein relate to the position in the relevant EMBL Accession number unless stated otherwise or apparent from the context. When considering positional relationships in the presence of a single base deletion the introduction of an appropriate gap is required.

One approach is to use knowledge of polymorphisms 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 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. Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous. Thus there is a need for improved approaches to pharmaceutical agent design and therapy.

Variations in polypeptide sequence will be referred to as follows: original 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.

The present invention is based on the discovery of four single nucleotide polymoφhisms (SNPs) in the promoter region of the human β, integrin subunit gene, five single nucleotide polymorphisms (SNPs) in the coding region of the human βi integrin subunit gene and one single nucleotide polymoφhism (SNP) in the 3 ^' untranslated region of the human βi integrin subunit gene.

According to one aspect of the present invention there is provided a method for the diagnosis of a single nucleotide polymorphism in a βi integrin subunit gene in a human, which method comprises determining the sequence of the nucleic acid of the human at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942, in the coding region of the β, integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or position 2502 in the 3 '-untranslated region of the β, integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and determining the status of the human by reference to polymorphism in the βi integrin subunit gene.

According to another aspect of the present invention there is provided a method for the diagnosis of a single nucleotide polymorphism in a βi integrin subunit gene in a human, which method comprises determining the sequence of the nucleic acid of the human at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 and determining the status of the human by reference to polymorphism in the βi integrin subunit gene.

The term human includes both a human having or suspected of having a βi integrin subunit 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.

In one embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at promoter position 590 is presence or absence of G. In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at promoter position 751 is presence of A and/or G.

In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at promoter position 1206 is presence of G and/or C .

In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at promoter position 1282 is presence of C and/or A.

In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at coding region position 562 is presence of T and/or C.

In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at coding region position 886 is presence of T and/or C. In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at coding region position 1189 is presence of A and/or C.

In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at coding region position 1279 is presence of A and/or C . In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymoφhism at coding region position 1942 is 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 polymorphism in the 3 '-untranslated region at position 2502 is presence of T and/or G.

The method for diagnosis is preferably one in which the sequence is determined by a method selected from amplification refractory mutation system and restriction fragment length polymorphism. In another aspect of the invention we provide a method for the diagnosis of βi integrin subunit 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 590,

751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969 and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 and/or at position 2502 , in the 3 '-untranslated region of the β, integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 iii) determining the status of the individual by reference to polymorphism in the βi integrin subunit gene.

Allelic variation at promoter position 590 consists of a single base (G) deletion. Allelic variation at promoter position 751 consists of a single base substitution from A (the published base), preferably to G. Allelic variation at promoter position 1206 consists of a single base substitution from G (the published base), preferably to C. Allelic variation at promoter position 1282 consists of a single base substitution from C (the published base), preferably to

A.

Allelic variation at coding region position 562 consists of a single base substitution from T (the published base), preferably to C. Allelic variation at coding region position 886 consists of a single base substitution from T (the published base), preferably to C. Allelic variation at coding region position 1189 consists of a single base substitution from A (the published base), preferably to C. Allelic variation at coding region position 1279 consists of a single base substitution from A (the published base), preferably to C. Allelic variation at coding region position 1942 consists of a single base substitution from C (the published base), preferably to T. Allelic variation at 3' untranslated region position 2502 consists of a single base substitution from T (the published base), preferably to G.

The status of the individual may be determined by reference to allelic variation at any one, two, three, four, five, six, seven, eight, nine, ten or all eleven positions optionally in combination with any other polymorphism that is or becomes known. The test sample of nucleic acid is conveniently a sample of blood, bronchoalveolar lavage fluid, sputum, 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 analysis of allelic 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 variant nucleotides at one or more polymoφhic 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. 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 polymorphisms. Hybridisation Based Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide 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)

Extension Based: ARMS™, 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

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

Table 3 - Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

Preferred mutation detection techniques include ARMS™, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.

Particularly preferred methods include ARMS™ and RFLP based methods. ARMS™ 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 βi integrin subunit ligand mediated diseases such as autoimmune, allergic and vascular inflammatory diseases. Some polymorphisms identified in the present invention occur in the promoter region of the βi integrin subunit gene. The changes are not expected to alter the amino acid sequence of βi integrin subunit , but several of the polymoφhisms affect transcription sites within the promoter region and thus may affect the transcription of the βi integrin subunit gene.

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 βi integrin subunit gene may therefore exhibit differences in their ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react to different diseases. In addition, differences in protein regulation 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 and/or susceptibility of an individual to diseases mediated by βi integrin subunit ligands. This may be particularly relevant in the development of autoimmune, allergic and vascular inflammatory diseases and other diseases which are modulated by βi integrin subunit interactions. The present invention may be used to recognise individuals who are particularly at risk 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 βi integrin subunit 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. In the accompanying Example 2 we provide details of convenient engineered restriction enzyme sites that are lost or gained as a result of a polymorphism of the invention.

According to another aspect of the present invention there is provided a nucleic acid comprising any one of the following polymoφhisms: -lithe nucleic acid of EMBL ACCESSION No. X68969 with absence of G at position 590 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with G at position 751 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with C at position 1206 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with A at position 1282 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 562 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 886 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 1189 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 1279 sequence as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with T at position 1942 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with G at position 2502 as defined by the position in EMBL ACCESSION No. X07979; or a complementary strand thereof or an antisense sequence to at least one of the above polymorphisms in the coding region or 3 '-untranslated region 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 nucleic acid of the invention does not encompass naturally occuring nucleic acid as it occurs in nature, for example, the nucleic acid is at least partially purified from at least one component with which it occurs naturally. Preferably the nucleic acid 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. 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 pREPIO vector (Invitrogen Coφoration). Transcripts are expected to inhibit translation of the gene in cells transfected with this type 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 hybridizable with any portion of novel gene rnRNA 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.nucleotide molecules can be synthesized 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. According to another aspect of the invention there is provided use of a nucleic acid sequence comprising at least one of the polymorphisms in the promoter disclosed herein to identify compounds that modify expression of the human βi integrin subunit gene. Modification of expression includes inhibition or enhancement of expression. This is conveniently done by measuring expression levels of a reporter gene (for example beta- galactosidase) under the control of the promoter in transfected host cells in the presence or absence of test compounds. Suitable test compounds include polynucleotides capable of binding to the promoter through triplex strand formation. Accordingly suitable compounds can be identified for therapeutic use which alter native gene expression either up or down as appropriate for the relevant disease to be treated. The reader is directed to the following references on nucleic acid triplex formation and uses: Progress in developments of Triplex- Based strategies: Giovannangeli C; Helene C: Antisense and Nucleic Acid Drug Development / 7/4 (413-421) /1997; Recent developments in triple-helix regulation of gene expression: Neidle S: Anti-Cancer Drug Design / 12/5 (433-442) /1997; Triplex DNA: Fundamentals, advances, and potential applications for gene therapy: Chan PP; Glazer PM: Journal of Molecular Medicine / 75/4 (267-282) /1997; Oligonucleotide directed triple helix formation: Sun J-S; Garestier T; Helene C: Current Opinion in Structural Biology / 6/3 (327-333) /1996; C Mayfield, M Squibb, D Miller (1994) Inhibition of nuclear protein binding to the human Ki- ras promoter by triplex-forming oligonucleotides Biochemistry 33,3358-3363; WM Olivas, LJ Maher (1996) Binding of DNA oligonucleotides to sequences in the promoter of the human bcl-2 gene Nucleic Acids Research 24, 1758-1764; C Mayfield, S Ebinghaus, J Gees, D Jones, B Rodu, M Squibb, D Miller (1994) Triplex formation by the human HA-ras promoter inhibits Sp 1 binding and in vitro transcription J Biol Chem 269, 18232- 18238 ; and JE Gee, GR

Revankar, TS Rao, ME Hogan (1995) Triplex formation at the rat neu gene utilizing imidazole and 2'-deoxy-6-thioguanosine base substitutions Biochemistry 34,2042-2048.

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, 1988. 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 single nucleotide polymoφhisms 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^hen using the information in standard bioinformatics 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 a 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 polymoφhisms 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 polymoφhism 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.

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 βi integrin subunit gene polymorphism at one or more of positions 590, 751, 1206 and 1282 in the promoter of the β, integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

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 ARMS™ 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^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 βi integrin subunit gene polymorphism at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

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 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) and polymerase(s) such as thermostable polymerases, for example taq polymerase. In another aspect of the invention, the single nucleotide polymoφhisms of this invention may be used as genetic markers in linkage studies. This particularly applies to the polymorphism at promoter position 590 because of its informative frequency (see below). The βi integrin subunit gene has been mapped to chromosome lOpl 1.2 (Goodfellow et al, Ann. Hum. Genet. 53: 15-22, 1989). Low frequency polymoφhisms may be particularly useful for haplotyping as described below. 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, 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 polymorphisms 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 JF, 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 IN, Cameron CL, Lillicrap D). According to another aspect of the present invention there is provided a method of treating a human in need of treatment with a β, integrin subunit ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymorphism in β, integrin subunit gene in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and determining the status of the human by reference to polymoφhism in the βi integrin subunit gene; and ii) administering an effective amount of a βi integrin subunit ligand antagonist . Preferably determination of the status of the human is clinically useful. Examples of clinical usefulness include deciding which antagonist drug or drugs to administer and/or in deciding on the effective amount of the drug or drugs. βi integrin subunit ligand antagonist drugs have been disclosed in the following publications: international patent application WO 97/49731, Zeneca Limited; international patent application WO 97/02289, Zeneca Limited; international patent application WO 96/20216, Zeneca Limited; US patent 5510332, Texas Biotechnology; international patent application WO 96/01644, Athena Neurosciences; international patent application WO 96/01644, Athena Neurosciences and; international patent application WO 96/00581, Zeneca Limited. A βj integrin subunit ligand antagonist drug may act directly at βi integrin subunit heterodimer and/or at a ligand, such as VCAM or CS-1 fibronectin which binds to β, integrin subunit containing heterodimer, such as VLA-4. VLA-4 antagonists as anti-inflammatory agents have been reviewed by Lin KC & Castro AC in Curr. Opin. Chem. Biol. (1998), 2: 453- 457.

According to another aspect of the present invention there is provided use of a βi integrin subunit ligand antagonist drug in preparation of a medicament for treating a βi integrin subunit ligand mediated disease in a human diagnosed as having a single nucleotide polymorphism at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

According to another aspect of the present invention there is provided a pharmaceutical pack comprising a βi integrin subunit ligand antagonist drug and instructions for administration of the drug to humans diagnostically tested for a single nucleotide polymorphism at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 and/or. at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

The invention will now be illustrated but not limited by reference to the following

Examples. 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). Example 1

Identification of Polymorphisms

1. Methods

Genomic 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. C-DNA Preparation

RNA was prepared from lymphoblastoid cell lines from Caucasian donors using standard laboratory protocols (Chomczynski and Sacchi, Anal. Biochem. 162, 156-159, 1987) and used to generate first strand cDNA (Gubler and Hoffman, Gene 25, 263-269, 1983). 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°, each step was 1 minute. For analysis of the promoter region generally 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR. For analysis of the coding region generally 100 pg cDNA was used in each reaction and subjected to 40 cycles of PCR.

Promoter Region, X68969

Coding Region, X07979

For dye-primer sequencing the forward primers were modified to include Ml 3 forward sequence (ABI protocol P/N 402114, Applied Biosystems) at the 5' end of the oligonucleotides. Dye Primer Sequencing

Dye-primer sequencing using Ml 3 forward primer 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 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).

2. Results Novel Polymorphisms

Integrin B eta- 1 Promoter

EMBL Accession NosX68969; S56437

ID HSBEIN

Ref: Cervella etal, J. Biol. Chem. 268: 5148-5155, 1993

'TF = transcription factor

Frequency is the allele frequency of the variant allele in European control subjects.

Integrin Beta- 1 cDNA

EMBL Accession No X07979

LD HSFNRB

Ref: Argaves et al, J. Cell Biol. 105: 1183-1190,1987

Chromosome 1 Op 11.2 Ref Goodfellow et α/, Ann. Hum. Genet. 53: 15-22, 1989

Frequency is the allele frequency of the variant allele in control subjects. "eng"' = engineered RFLP Example 2

Engineered restriction site primers for detection of polymorphisms

Standard methodology can be used to detect the polymorphism at positions 886, 1189, 1279 and 1942 (as defined by the position in EMBL ACCESSION NO. X07979) based 5 on the materials set out below using a cDNA template.

Primer Sequence 5 '-3'

887-910 Fsp I ATTCCTCCAGCCAATCAGTGATGC (SEQ ID NO:l)

10 1190-1213 Mfe I CAACTGAATTACATTGCTACAATT (SEQ ID NO: 2)

1255-1603 Mlu I GGAAAACGGCAAATTGTCAGAACG (SEQ ID NO: 3)

1918-1941 Nru I GATCTGCAATGGCCGGGGCATTCG (SEQ ID NO: 4)

C at position 886 creates a Fsp I site in diagnostic fragment 471-910 described above. 15 C at position 1189 creates a Mfe I site in diagnostic fragment 961-1213 described above. C at position 1279 creates a Mlu I site in diagnostic fragment 1255-1603 described above. C at position 1942 creates a Nru I site in diagnostic fragment 1918-2101 described above. Sequence Listing Free Text <223> Description of Artificial Sequence:PCR primer

Claims

1 A method for the diagnosis of a single nucleotide polymorphism in a βi integrin subunit gene in a human, which method comprises determining the sequence of the nucleic acid of the human at: one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969; and/or at one or more of positions 562, 886, 1189, 1279 and 1942, in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979; and/or position 2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the position in EMBL ACCESSION NO. X07979; and determining the status of the human by reference to polymorphism in the βi integrin subunit gene.

2 A method for diagnosis according to claim 1 in which the single nucleotide polymorphisms are further defined as: the single nucleotide polymoφhism at promoter position 590 is presence or absence of G; the single nucleotide polymoφhism at promoter position 751 is presence of A and/or G; the single nucleotide polymoφhism at promoter position 1206 is presence of G and/or C; the single nucleotide polymorphism at promoter position 1282 is presence of C and/or A; the single nucleotide polymoφhism at coding region position 562 is presence of T and/or C; the single nucleotide polymoφhism at coding region position 886 is presence of T and/or C; the single nucleotide polymorphism at coding region position 1189 is presence of A and/or C; the single nucleotide polymorphism at coding region position 1279 is presence of A and/or C; the single nucleotide polymoφhism at coding region position 1942 is presence of C and/or T; and the single nucleotide polymoφhism in the 3 '-untranslated region at position 2502 is presence of T and/or G.

3 A method for diagnosis according to any preceding claim in which the sequence is determined by a method selected from amplification refractory mutation system and restriction fragment length polymorphism.

4 A nucleic acid comprising any one of the following polymorphisms: the nucleic acid of EMBL ACCESSION No. X68969 with absence of G at position 590 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with G at position 751 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with C at position 1206 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X68969 with A at position 1282 as defined by the position in EMBL ACCESSION No. X68969; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 562 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 886 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 1189 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with C at position 1279 sequence as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with T at position 1942 as defined by the position in EMBL ACCESSION No. X07979; the nucleic acid of EMBL ACCESSION No. X07979 with G at position 2502 as defined by the position in EMBL ACCESSION No. X07979; or a complementary strand thereof or an antisense sequence to at least one of the above polymorphisms in the coding region or 3 '-untranslated region or a fragment thereof of at least

20 bases comprising at least one polymoφhism.

5 A computer readable medium comprising at least one nucleic acid sequence as defined in claim 4 stored on the medium.

6 An allele specific primer capable of detecting a βi integrin subunit gene polymoφhism at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979. 7 An allele-specific oligonucleotide probe capable of detecting a βi integrin subunit gene polymoφhism at one or more of: positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979: and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

8 Use of a nucleic acid sequence comprising at least one of the polymoφhisms at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969 to identify compounds that modify expression of the human βi integrin subunit gene.

9 Use of a βi integrin subunit ligand antagonist drug in preparation of a medicament for treating a βi integrin subunit ligand mediated disease in a human diagnosed as having a single nucleotide polymoφhism at: one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979 and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979.

10 A method of treating a human in need of treatment with a βi integrin subunit ligand antagonist drug in which the method comprises: i) diagnosis of a single nucleotide polymoφhism in β, integrin subunit gene in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions 590, 751, 1206 and 1282 in the promoter of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X68969, and/or at one or more of positions 562, 886, 1189, 1279 and 1942 , in the coding region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and/or at position.2502 in the 3 '-untranslated region of the βi integrin subunit gene as defined by the positions in EMBL ACCESSION NO. X07979, and determining the status of the human by reference to polymoφhism in the βi integrin subunit gene; and ii) administering an effective amount of a βi integrin subunit ligand antagonist .