EP1114182A1 - Polymorphismes du gene humain vcam-1, appropries au diagnostic et traitement des maladies mediees par un ligand vcam-1 - Google Patents

Polymorphismes du gene humain vcam-1, appropries au diagnostic et traitement des maladies mediees par un ligand vcam-1

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EP1114182A1
EP1114182A1 EP99946341A EP99946341A EP1114182A1 EP 1114182 A1 EP1114182 A1 EP 1114182A1 EP 99946341 A EP99946341 A EP 99946341A EP 99946341 A EP99946341 A EP 99946341A EP 1114182 A1 EP1114182 A1 EP 1114182A1
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Prior art keywords
vcam
embl accession
gene
nucleic acid
positions
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EP99946341A
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German (de)
English (en)
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John Edward Norris Morten
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AstraZeneca AB
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AstraZeneca AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to polymorphisms in the human Vascular Cell Adhesion Molecule-1 (VCAM-1) gene.
  • the invention also relates to methods and materials for
  • VCAM-1 ligand mediated diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis and allergic asthma.
  • VCAM-1 also known as CD 106, is a 90-110 kDa glycoprotein member of the immunoglobulin superfamily expressed mainly on the surface of activated vascular endothelial
  • VCAM-1 is also found as a soluble form in serum, probably as a result of proteolytic cleavage of endothelial cell surface VCAM-1.
  • Cell adhesion molecules have been reviewed in Mousa et al. (1997), DDT, 2, 187-199.
  • VCAM-1 is a ligand for the ⁇ integrins, ⁇ 4 ⁇ , also known as Very Late Antigen-4
  • VLA-4 or CD49d/CD29
  • ⁇ ⁇ 7 integrins are members of a family of heterodimeric cell surface receptors that are composed of non-covalently associated glycoprotein subunits ( ⁇ and ⁇ ) and are involved in the adhesion of cells to other cells or to extracellular matrix. Integrin ⁇ 4 ⁇ is expressed on numerous haematopoietic cells, including
  • Integrin ⁇ ⁇ 7 is expressed on lymphocytes that preferentially home to gastrointestinal mucosa and gut-associated lymphoid tissue.
  • the ⁇ integrins recognise a short amino acid sequence, glutamine-isoleucine-aspartic acid-serine- proline (QIDSP), exposed on the C-D loop of immunoglobulin domains 1 and 4.
  • QIDSP glutamine-isoleucine-aspartic acid-serine- proline
  • 25 binding site may also be located in the adjacent immunoglobulin domain.
  • VCAM-1 on unactivated vascular endothelial cells is low or absent but is upregulated in human inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, allergic asthma and atherosclerosis. Soluble VCAM-1 is elevated in serum and cerebrospinal fluid of multiple sclerosis patients and in serum during inflammatory bowel disease and after
  • endothelial cells can be induced to express VCAM-1 by inflammatory cytokines such as tumour necrosis factor and interleukin-1 or by oxidative stress.
  • VCAM-1 gene expression in vascular endothelial cells during inflammation is regulated by transcriptional activation, thought to involve the dimeric transcription factor, nuclear factor- ⁇ B (N - ⁇ B).
  • vascular endothelium 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 inflammed tissue and is mediated by specific interactions between cell adhesion molecules on the surface of vascular endothelial cells and circulating leukocytes.
  • ⁇ integrin/VCAM-l binding is believed to have an important role in the recruitment of lymphocytes, monocytes and eosinophils during inflammation.
  • Monoclonal antibodies directed against the ⁇ 4 integrin subunit 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.
  • ⁇ 4 ⁇ /VCAM-1 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.
  • Small molecule inhibitors of VCAM-1 binding to ⁇ integrins have been designed based on the QIDSP motif in VCAM-1 and similar motifs in other ⁇ 4 integrin ligands fibronectin and mucosal addressin cell adhesion molecule- 1 (MAdCAM-1).
  • Small molecule and monoclonal antibody inhibitors of VCAM-1 binding to ⁇ integrins and inhibitors of VCAM-1 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.
  • VCAM-1 VCAM-1 gene has been cloned and published as a EMBL Accession number: M92431 (2396 bp) and all positions herein relate to the position therein unless stated otherwise or apparent from the context.
  • 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) r 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.
  • the present invention is based on the discovery of six single nucleotide polymorphisms (SNPs) in the VCAM-1 gene.
  • a method for the diagnosis of a single nucleotide polymorphism in VCAM-1 in a human comprises determining the sequence of the nucleic acid of the human at one or more of positions 278, 647, 707, 748, 829 and 1467 in the VCAM-1 gene as defined by the positions in EMBL ACCESSION NO. M92431, and determining the status of the human by reference to polymorphism in the VCAM-1 gene.
  • the term human includes both a human having or suspected of having a VCAM-1 ligand mediated disease and an asymptomatic human who may be tested for predisposition or susceptibility to such disease. At each position the human may be homozygous for an allele or the human may be a heterozygote.
  • the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 278 is presence of T and/or C.
  • the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 647 is presence of A and/or G.
  • the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 707 is presence of T and/or C.
  • the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 748 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 position 829 is presence of G 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 position 1467 is presence of T and/or C.
  • 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.
  • a method for the diagnosis of VCAM-1 ligand-mediated disease comprises: i) obtaining sample nucleic acid from an individual, ii) detecting the presence or absence of a variant nucleotide at one or more of positions 278, 647, 707, 748, 829 and 1467 (as defined by the position in EMBL accession number M92431), in the VCAM-1 gene and iii) determining the status of the individual by reference to polymorphism in the VCAM-1 gene.
  • Allelic variation at position 278 consists of a single base substitution from T (the published base), preferably to C.
  • Allelic variation at position 647 consists of a single base substitution from A (the published base), preferably to G.
  • Allelic variation at position 707 consists of a single base substitution from T (the published base), preferably to C.
  • Allelic variation at position 748 consists of a single base substitution from T (the published base), preferably to C.
  • Allelic variation at position 829 consists of a single base substitution from G (the published base), preferably to A.
  • Allelic variation at position 1467 consists of a single base substitution from T (the published base), preferably to C.
  • the status of the individual may be determined by reference to allelic variation at any one, two, three, four, five or all six positions.
  • 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.
  • 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.
  • Hybridisation Based Solid phase hybridisation Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips)
  • Fluorescence Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom
  • Patent No. 2228998 (Zeneca Limited) Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric,
  • Preferred mutation detection techniques include ARMSTM, ALEXTM, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.
  • Particularly preferred methods include ARMSTM and RFLP based methods.
  • ARMSTM is an especially preferred method.
  • the diagnostic methods of the invention are used to assess the efficacy of therapeutic compounds in the treatment of VCAM-1 ligand mediated diseases such as autoimmune, allergic and vascular inflammatory diseases.
  • VCAM-1 ligand mediated diseases such as autoimmune, allergic and vascular inflammatory diseases.
  • the polymo ⁇ hisms identified in the present invention occur in the promoter region of the VCAM-1 gene. The changes are not expected to alter the amino acid sequence of VCAM-1, but several of the polymorphisms affect transcription sites within the promoter region and thus may affect the transcription of the
  • VCAM-1 gene For example the changing of the nucleotide at position 748 (as defined by the position in EMBL ACCESSION NO. M92431) from T to C results in the gain of a Ela-F rev site and the loss of a TATA box.
  • 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.
  • allelic variants of the VCAM-1 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.
  • 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.
  • the diagnostic methods of the invention are used to assess the predisposition and/or susceptibility of an individual to diseases mediated by VCAM-1 ligands. This may be particularly relevant in the development of autoimmune, allergic and vascular inflammatory diseases and other diseases which are modulated by VCAM-1 interactions.
  • the present invention may be used to recognise individuals who are particularly at risk from developing these conditions.
  • 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 VCAM-1 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.
  • the presence or absence of variant nucleotides is detected by reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes.
  • Example 1 we provide details of convenient engineered restriction enzyme sites that are lost or gained as a result of a polymorphism of the invention.
  • nucleic acid comprising any one of the following polymorphisms: the nucleic acid of EMBL ACCESSION No. M92431 with C at position 278 in the promoter sequence as defined by the position in EMBL ACCESSION No. M92431; the nucleic acid of EMBL ACCESSION No. M92431 with G at position 647 in the promoter sequence as defined by the position in EMBL ACCESSION No. M92431; the nucleic acid of EMBL ACCESSION No. M92431 with C at position 707 in the promoter sequence as defined by the position in EMBL ACCESSION No.
  • 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.
  • 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.
  • nucleic acid sequence comprising at least one of the polymorphisms in the promoter disclosed herein to identify compounds that modify expression of the VCAM-1 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.
  • a reporter gene for example beta-galactosidase
  • 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.
  • 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.
  • polynucleotide sequences of the invention or parts thereof, particularly those relating to and identifying the single nucleotide 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 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.
  • 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.
  • a tangible medium such as a computer disk
  • 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.
  • 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
  • a computer based method for performing sequence identification, said method comprising the steps of providing a polynucleotide sequence comprising a polymo ⁇ hism 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
  • the invention further provides nucleotide primers which can detect the polymorphisms of the invention.
  • an allele specific primer capable of detecting a VCAM-1 gene polymorphism at one or more of positions 278,
  • VCAM-1 gene 20 647, 707, 748, 829 and 1467 in the VCAM-1 gene as defined by the positions in EMBL ACCESSION NO. M92431.
  • 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
  • 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,
  • primer 30 2 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.
  • an allele-specific oligonucleotide probe capable of detecting a VCAM-1 gene polymorphism at one or more of positions 278, 647, 707, 748, 829 and 1467 in the VCAM-1 gene as defined by the positions in EMBL ACCESSION NO. M92431.
  • the allele-specific oligonucleotide probe is preferably 17- 50 nucleotides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.
  • probes will be apparent to the molecular biologist of ordinary skill.
  • Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8-15 bases in length.
  • such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene.
  • 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.
  • 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.
  • the single nucleotide polymorphisms of this invention may be used as genetic markers in linkage studies. This particularly applies to the polymorphism at 278 (as defined by the position in EMBL ACCESSION NO. M92431) because of its relatively high frequency (see below).
  • the VCAM-1 gene has been mapped to chromosome lp31-32 (Cybulsky et al Proc. Natl. Acad. Sci. USA 88, 7859-7863, 1991).
  • 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 n haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs.
  • One approach to identifying mutations or 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.
  • 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.
  • SNPs with low frequency alleles are particularly useful as markers of low frequency haplotypes.
  • mutations or polymo ⁇ hisms 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.
  • CBS cystathionine beta synthase
  • a method of treating a human in need of treatment with a VCAM-1 ligand antagonist drug comprises: i) diagnosis of a single nucleotide polymorphism in VCAM-1 gene in the human, which diagnosis comprises determining the sequence of the nucleic acid at one or more of positions 278, 647, 707, 748, 829 and 1467 (as defined by the position in EMBL accession number M92431), and determining the status of the human by reference to polymorphism in the VCAM-1 gene; and ii) administering an effective amount of a VCAM-1 ligand antagonist .
  • 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.
  • VCAM-1 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.
  • VCAM-1 ligand antagonist drug may act directly at VCAM-1 and/or at a ligand, such as VLA-
  • VCAM- 1 ligand antagonist drug in preparation of a medicament for treating a VCAM-1 ligand mediated disease in a human diagnosed as having a single nucleotide polymorphism at one or more of positions 278, 647, 707, 748, 829 and 1467 (as defined by the position in EMBL accession number M92431).
  • a pharmaceutical pack comprising VCAM-1 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 278, 647, 707, 748, 829 and 1467 (as defined by the position in EMBL accession number M92431).
  • AMPLITAQTM 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).
  • DNA Preparation DNA was prepared from frozen blood samples collected in EDTA following protocol I
  • 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. All reactions contained 1 mM MgCl 2 . Generally 50 ng of genomic DNA was used in each reaction and subjected to 35 cycles of PCR.
  • the forward primers were modified to include Ml 3 forward sequence (ABI protocol P/N 402114, Applied Biosystems) at the 5' end of the oligonucleotide.
  • DNA polymerase Amplitaq GoldTM , Perkin Elmer Cetus was used to generate products 595-1151 and 1264-1804.
  • Dye-primer sequencing using Ml 3 forward and reverse primers was as described in the ABI protocol P/N 402114 for the ABI PrismTM dye primer cycle sequencing core kit with "AmpliTaq FS”TM DNA polymerase, modified in that the annealing temperature was 45° and 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.
  • Standard methodology can be used to detect the polymorphism at positions 647 (as defined by the position in EMBL ACCESSION NO. M92431) based on the materials set out below.
  • Reverse primer sequence CCCAGAGGTCCTTTACAGCGAT (SEQ ID NO:l).
  • the product generated by these primers will include a Pvu I site, only if the diagnostic fragment contained a G allele at position 647.

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Abstract

L'invention concerne des polymorphismes du gène humain de la Molécule d'Adhésion Cellulaire Vasculaire-1 (VCAM-1), notamment à au moins une position 278, 647, 707, 748, 829 et 1467 dans le gène VCAM-1 tel que défini par les positions dans L'ENTREE EMBL NO.M92431. L'invention concerne également des techniques et des matériaux d'analyse de variation allélique dans le gène VCAM-1, et l'utilisation du polymorphisme VCAM-1 dans le diagnostic et le traitement des maladies médiées par le ligand VCAM-1 telles que la sclérose en plaques, l'arthrite rhumatoïde, l'athérosclérose et l'asthme allergique.
EP99946341A 1998-09-19 1999-09-15 Polymorphismes du gene humain vcam-1, appropries au diagnostic et traitement des maladies mediees par un ligand vcam-1 Withdrawn EP1114182A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9820338.3A GB9820338D0 (en) 1998-09-19 1998-09-19 Chemical compounds
GB9820338 1998-09-19
PCT/GB1999/003057 WO2000017392A1 (fr) 1998-09-19 1999-09-15 Polymorphismes du gene humain vcam-1, appropries au diagnostic et traitement des maladies mediees par un ligand vcam-1

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EP1114182A1 true EP1114182A1 (fr) 2001-07-11

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EP99946341A Withdrawn EP1114182A1 (fr) 1998-09-19 1999-09-15 Polymorphismes du gene humain vcam-1, appropries au diagnostic et traitement des maladies mediees par un ligand vcam-1

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EP (1) EP1114182A1 (fr)
JP (1) JP2002526089A (fr)
AU (1) AU5875599A (fr)
GB (1) GB9820338D0 (fr)
WO (1) WO2000017392A1 (fr)

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US8133859B2 (en) 2006-09-27 2012-03-13 The United States Of America As Represented By The Department Of Health And Human Services SCGB3A2 as a growth factor and anti-apoptotic agent
US10947311B2 (en) 2015-11-20 2021-03-16 The Board Of Trustees Of The Leland Stanford Junior University VCAM-1 mediated methods and compositions for treating aging-associated impairments

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DK162890D0 (da) * 1990-07-06 1990-07-06 Novo Nordisk As Polypeptid
JP2000508912A (ja) * 1996-04-19 2000-07-18 スペクトラ バイオメディカル，インコーポレイテッド 多型形態と複数の表現型との相関付け
GB9613112D0 (en) * 1996-06-21 1996-08-28 Zeneca Ltd Chemical compounds

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AU5875599A (en) 2000-04-10
WO2000017392A1 (fr) 2000-03-30
JP2002526089A (ja) 2002-08-20
GB9820338D0 (en) 1998-11-11

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