WO2007055636A1 - Polymorphismes à nucléotide unique dans le gène ckrx associés à une maladie respiratoire - Google Patents

Polymorphismes à nucléotide unique dans le gène ckrx associés à une maladie respiratoire Download PDF

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WO2007055636A1
WO2007055636A1 PCT/SE2006/001260 SE2006001260W WO2007055636A1 WO 2007055636 A1 WO2007055636 A1 WO 2007055636A1 SE 2006001260 W SE2006001260 W SE 2006001260W WO 2007055636 A1 WO2007055636 A1 WO 2007055636A1
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ckrx
seq
individual
polypeptide
fragment
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PCT/SE2006/001260
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Simon Smith
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • the present invention relates to single nucleotide polymorphisms in the CKlRX gene and their association with respiratory diseases such as asthma.
  • the present invention also relates to the identification of corresponding haplotypes and their association with respiratory diseases such as asthma.
  • the present invention also identifies a role for CKRX in human disorders where previously its function was unknown.
  • the present invention also provides methods and assays for identifying compounds which interact with CKRX and which may be used for treating respiratory diseases.
  • Respiratory diseases include Acute Lung Injury, Acute Respiratory Distress Syndrome (ARDS), occupational lung disease, lung cancer, tuberculosis, fibrosis, pneumoconiosis, pneumonia, emphysema, Chronic Obstructive Pulmonary Disease (COPD) and asthma.
  • ARDS Acute Respiratory Distress Syndrome
  • COPD Chronic Obstructive Pulmonary Disease
  • Asthma is generally defined as an inflammatory disorder of the airways with clinical symptoms arising from intermittent airflow obstruction. It is characterised clinically by paroxysms of wheezing, dyspnea and cough. It is a chronic disabling disorder that appears to be increasing in prevalence and severity. It is estimated that 15% of children and 5% of adults in the population of developed countries suffer from asthma.
  • beta agonists reduce the symptoms, i.e., transiently improve pulmonary functions, but do not affect the underlying inflammation so that lung tissue remains in jeopardy.
  • beta agonists results in desensitization which reduces their efficacy and safety.
  • the agents that can diminish the underlying inflammation, the anti-inflammatory steroids have their own known list of disadvantages that range from immunosuppression to bone loss.
  • Glycophorin A 5 cyclosporin, and a nonapeptide fragment of IL-2 all inhibit interleukin-2 dependent T lymphocyte proliferation and therefore, IL-9 production, however, they are known to have many other effects.
  • cyclosporin is used as a immunosuppressant after organ transplantation. While these agents may represent alternatives to steroids in the treatment of asthmatics, they inhibit interleukin-2 dependent T lymphocyte proliferation and potentially critical immune functions associated with homeostasis.
  • CKRX is a member of a gene family called G-protein coupled receptors or GPCRs. CKRX is also known as CCRL2. GPCRs encode polypeptides composed of an N- terminal extracellular domain, seven transmembrane hydrophobic domains, three extracellular and three intracellular loops, and an intracellular C-terminus. They are important in mediating the effects of chemokines, small to medium-sized proteins and glycoproteins that are vital regulators of physiogic processes such as immunity and inflammation. For many GPCRs, the ligand (chemokine) with which they interact is known but for CKRX 3 among others, the ligand(s) is (are) not known and the GPCR is known as an orphan GPCR in recognition of this fact.
  • the nucleotide sequence of CKRX was first entered into the publically accessible sequence databases in 1997 by Ansari-Lari et al (sequence # AF014958). The nucleotide sequence is set out in SEQ ID NO: 1.
  • the nucleotide sequence in SEQ ID NO:1 is the genomic sequence of CKRX and includes exon 1 (spanning residues 900 to 1244) and exon 2 (spanning residues 1810 to 3264).
  • the amino acid sequence of the encoded CKRX protein has been deduced from the nucleotide sequence of the gene and is present hi the protein sequence database TREMBL (sequence # 000421). The amino acid sequence is set out in SEQ ID NO:2.
  • International patent publication number WO2004/083232 discusses a method of determining whether an individual is predisposed to inflammatory bowel disease, which method comprises identifying whether the individual has specified polymorphisms in the CCRL2 polynucleotide or protein, which polymorphism is associated with inflammatory bowel disease.
  • International patent publication number WO97/41225 discusses polynucleotides which encode chemokine receptors called MMLR-CCR (also known as CCR5) and MPHG-CCR (also known as CCRL2 or CKRX).
  • US patent publication number 2002/0076760 discusses Human G-Protein Coupled Receptor (HNFDS78, also known as CCRL2 or CKRX) polypeptides and DNA encoding them.
  • HNFDS78 Human G-Protein Coupled Receptor
  • CCRL2 also known as CKRX
  • Oostendorp et al J Histochem Cytochem, 2004 Mar, 52(3): 401-10 discuss the localization and enhanced mRNA expression of the orphan chemokine receptor L- CCR in the lung in a murine model of ovalbumin-induced airway inflammation.
  • L- CCR may be considered as a mouse orthologue to human CCRL2 or CKRX (approximately 53% identity).
  • the present invention identifies polymorphisms that are associated with asthma and, therefore, provides direct evidence for a role for CKRX in respiratory diseases such as asthma. This allows the development of methods associated with the diagnosis and therapy of respiratory diseases such as asthma.
  • SNPs Single nucleotide polymorphisms
  • SNPs Single nucleotide polymorphisms
  • the present invention identifies polymorphisms that are associated with asthma and, therefore, provides direct evidence for a role for CKRX in respiratory disease such as asthma.
  • a method for detecting the presence of a respiratory disease in an individual which method comprises the steps of
  • step (b) comprises detecting for the presence of one or more of the following nucleotides: A at position 2323 of SEQ ID NO:1; T at position 3135 of SEQ ID NO: 1; A at position 3753 of SEQ ID NO:1; T at position 3898 of SEQ ID NO: 1 ; and G at position 3931 of SEQ ID NO: 1.
  • a method for detecting the presence of a respiratory disease in an individual comprises the steps of a) providing a nucleic acid sample that has been removed from the individual; and b) determining the nucleotide of the individual at one or more of the following positions: positions 2323, 3135, 3753, 3898, and 3931 of SEQ ID NO:1; and c) detecting the presence of a respiratory disease if one or more of the following nucleotides are detected: A at position 2323 of SEQ ID NO:1; T at position
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 2323, 3135, 3753 and 3898 of SEQ ID N0-.1.
  • the method comprises determining the nucleotide of the individual at position 2323 of SEQ ID NO : 1.
  • the method comprises determining the nucleotide of the individual at position 3135 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 3753 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 3898 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 2323 and position 3931 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 3135 and position 3931 of SEQ ID NO : 1.
  • the method comprises determining the nucleotide of the individual at position 3753 and position 3931 of SEQ ID NO: 1. In one embodiment, the method comprises determining the nucleotide of the individual at position 3898 and position 3931 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at each of the following positions: positions 2323, 3135, 3753, 3898 and 3931 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 3135, 3753 and 3898 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 3135 and 3898 of SEQ ID NO: 1.
  • the respiratory disease is asthma.
  • the genomic sequence of CKRX is set out in SEQ ID NO: 1.
  • This sequence is also referred to as the "locus" for CKRX gene.
  • locus for CKRX gene.
  • Identification of SNP locations (i.e. polymorphic sites) in similar sequences are contemplated. The person skilled in the art can readily line up a similar sequence and locate the same SNP locations.
  • the position of the SNP refers to the position in SEQ ID NO: 1 where the first nucleotide in the sequence listed is position 1.
  • the presence of one or more of these polymorphisms in an individual may also therefore indicate the susceptibility of an individual to a disease condition.
  • An individual who has a genetic predisposition to a disease is one whose gene sequences identify that that individual is susceptible to a particular disease, i.e. has a higher risk of incurring the disease during their lifetime than the population as a whole.
  • the present invention provides a method for determining a genetic predisposition of an individual to the presence of a respiratory disease, which method comprises: a) providing a nucleic acid sample that has been removed from the individual; and b) determining the nucleotide of the individual at one or more of the following positions: positions 2323, 3135, 3753, 3898, and 3931 of SEQ ID NO:1.
  • step (b) comprises detecting for the presence of one or more of the following: A at position 2323 of SEQ ID NO:1; T at position 3135 of SEQ ID NO:1; A at position 3753 of SEQ ID NO:1; T at position 3898 of SEQ ID NO: 1 ; and G at position 3931 of SEQ ID NO: 1.
  • a method for determining a genetic predisposition of an individual to the presence of a respiratory disease comprises the steps of a) providing a nucleic acid sample that has been removed from the individual; and b) determining the nucleotide of the individual at one or more of the following positions: positions 2323, 3135, 3753, 3898, and 3931 of SEQ ID NO:1; and c) determining a genetic predisposition of the individual to the presence of a respiratory disease if one or more of the following nucleotides are detected: A at position 2323 of SEQ ID NO:1; T at position 3135 of SEQ ID NO:1; A at position 3753 of SEQ ID NO:1; T at position 3898 of SEQ ID NO:1; and G at position 3931 of SEQ ID NO:1
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 2323, 3135, 3753 and 3898 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at position 2323 of SEQ ID NO: 1. In one embodiment, the method comprises determining the nucleotide of the individual at position 3135 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at position 3753 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at position 3898 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 2323 and position 3931 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 3135 and position 3931 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at position 3753 and position 3931 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at position 3898 and position 3931 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at each of the following positions: positions 2323, 3135, 3753, 3898 and 3931 of SEQ ID NO: 1.
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 3135, 3753 and 3898 of SEQ ID NO:1.
  • the method comprises determining the nucleotide of the individual at one or more of the following positions: positions 3135 and 3898 of SEQ ID NO: 1.
  • the respiratory disease is asthma.
  • a SNP in a coding sequence may alter the sequence of the polypeptide, giving rise to a defective or variant isoform which may be associated with a disease condition.
  • the present invention identifies that one of the polymorphisms identified as being associated with asthma results in a variant of CKRX protein in which Valine at amino acid position 168 of SEQ ID NO:2 is substituted by Methionine (V168M).
  • V168M Methionine
  • V168M refers to the amino acid change wherein Valine at amino acid position corresponding to position 168 of SEQ ID NO:2 is substituted by Methionine.
  • CKRX V168M refers to a CKRX protein in which Valine at amino acid position corresponding to position 168 of SEQ ID NO:2 is substituted by Methionine.
  • CKRX V168M also encompasses other CKRX forms, for example those with one or more of the further amino acid changes identified in Table 1 but which also have Methionine at the position corresponding to position 168.
  • a method for determining a genetic predisposition of an individual to a respiratory disease comprises taking a biological sample from an individual and contacting the sample with an antibody that specifically recognises a CKRX form having an amino acid change V168M (ie a CKRX form in which Valine at amino acid position corresponding to position 168 of SEQ ID NO:2 is substituted by Methionine).
  • Respiratory disease is meant a disease of the respiratory system. Respiratory diseases include Acute Lung Injury, Acute Respiratory Distress Syndrome (ARDS), occupational lung disease, lung cancer, tuberculosis, fibrosis, pneumoconiosis, pneumonia, emphysema, Chronic Obstructive Pulmonary Disease (COPD) and asthma.
  • the respiratory disease is asthma.
  • a phased 5' to 3' sequence of nucleotides found at two or more polymorphic sites in a locus on a single chromosome from a single individual is referred to as a "haplotype".
  • the present invention identifies a number of haplotypes for the CKRX locus each of which comprise different combinations of the 8 polymorphic sites within the gene. The haplotypes identified are set out in Table 3 in the Examples section herein.
  • an isolated nucleic acid molecule comprising any one of the following 4 CKRX haplotypes:
  • each of haplotypes 2-5 above comprise SEQ ID NO:1 with the exception that the nucleotides specified in the above table (and Table 3) for each haplotype are present at the corresponding position within SEQ ID NO:1.
  • an isolated nucleic acid molecule selected from the group consisting of haplotypes 2 and 3, i.e.
  • a method for haplotyping the CKRX gene in an individual comprising the steps of: a) providing a nucleic acid sample that has been removed from the individual; b) determining the nucleotides present at one or more of the positions 2229, 2321, 2323, 2548, 3135, 3753, 3898 and 3931 of the individual's copy of CKJRX gene, wherein the position numbers are determined by comparison to SEQ ID NO:!; c) assigning the individual a particular haplotype by comparison of the nucleotides present at said positions to those shown in Table 3.
  • Either determining that the nucleotide present at position 2229 is C or determining that the nucleotide present at position 2321 is A indicates the individual is haplotype 1. Determining that the nucleotide present at position 3931 is A indicates the individual is haplotype 2. Determining that the nucleotide present at position 2323 is A or determining that the nucleotide present at position 3135 is C or determining that the nucleotide present at position 3753 is A or determining that the nucleotide present at position 3898 is T indicates the individual is haplotype 3. Determining that the nucleotide present at position 2548 is G indicates that the individual is haplotype 4.
  • determining that the nucleotide present at position 2229 is A and determining that the nucleotide present at position 2323 is G and determining that the nucleotide present at position 2548 is A and determining that the nucleotide present at position 3931 is G indicates the individual is haplotype 5.
  • a method for haplotyping the CKRX gene in an individual comprising the steps of: a) providing a nucleic acid sample that has been removed from the individual; b) determining the nucleotides present at positions 2229, 2321, 2323, 2548, 3135, 3753, 3898 and 3931 of the individual's copy of CKRX gene, wherein the position numbers are determined by comparison to SEQ ID NO:1; c) assigning the individual a particular haplotype by comparison of the nucleotides present at said positions to those shown in Table 3.
  • An individual's genotype is the unphased 5' to 3' sequence of nucleotide pairs found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual. Accordingly, the present invention also includes methods of genotyping an individual comprising isolating from the individual a nucleic acid mixture comprising two copies of the CKRX gene present in the individual and determining the identity of the nucleotide pair at the polymorphic sites identified herein in the two copies to assign a CKRX genotype to the individual.
  • compositions useful in performing the genotyping or haplotyping methods include oligonucleotide primers and probes designed to specifically hybridise to a target region containing the polymorphic sites described herein. Suitable oligonucleotides are described herein.
  • haplotypes As identified herein, the presence of certain of these haplotypes in an individual correlates with, those individuals having a respiratory disease such as asthma.
  • individuals of haplotype 3 may have a genetic predisposition to the presence of a respiratory disease such as asthma.
  • individuals of haplotype 2 may have a genetic predisposition to the absence of a respiratory disease such as asthma.
  • a method for detecting the presence or absence of a respiratory disease in an individual comprising identifying the haplotype using a method in accordance with the invention wherein determining haplotype 3 (referred to above as “Hap 3”) is predictive of the presence of the disease in an individual, and determining haplotype 2 (referred to above as “Hap 2”) is predictive of the absence of the disease in an individual.
  • Hap 3 determining haplotype 3
  • Hap 2 determining haplotype 2
  • the method is for detecting the presence or absence of asthma in an individual. "Detecting the presence of asthma” can also be described as diagnosing an asthmatic condition.
  • oligonucleotide probes to a nucleic acid sample derived from an individual.
  • the probe may comprise a nucleotide sequence which binds specifically to a particular allele of one of the polymorphisms whilst not binding specifically to other alleles of the polymorphisms.
  • an oligonucleotide which hybridises specifically to an allele of a nucleotide sequence which comprises one or more positions of single nucleotide polymorphism selected from the group consisting of positions 2229, 2321, 2323, 2548, 3135, 3753, 3898 and 3931 of SEQ ID NO:1.
  • the oligonucleotides are less than 100 nucleotides in length.
  • Preferred oligonucleotides are between 15 to 30 nucleotides in length and, most preferably, between 20 and 25 nucleotides in length.
  • an oligonucleotide which hybridises specifically to an allele of a nucleotide sequence which comprises one or more positions of single nucleotide polymorphism selected from the group consisting of positions 2323, 3135, 3753, 3898 and 3931 of SEQ ID NO:1.
  • Such an oligonucleotide may correspond in sequence to a region of the CKRX gene, or its complement, which contains one or more of the single nucleotide polymorphisms described herein. Under suitably stringent conditions, specific hybridisation of such a probe is predictive of the presence of the sequence alteration in the test nucleic acid.
  • an oligonucleotide which hybridises specifically to an allele defined by reference to one or more single nucleotide polymorphisms in SEQ ID NO: 1 is capable of predicting for the presence of those one or more single nucleotide polymorphisms in a test nucleic acid.
  • the probe oligonucleotide may be labelled to facilitate detection of the probe once bound to the test sample nucleic acid.
  • a probe oligonucleotide may also be used as a primer for use in the amplification of the region of sample nucleic acid comprising a polymorphism.
  • the region of interest may be amplified using a pair of oligonucleotide primers wherein the first of such a pair comprises a nucleotide sequence which hybridises to a complementary sequence located 5' to the polymorphic site of interest and the second of such a pair comprises a nucleotide sequence which hybridises to a complementary sequence located 3' to the polymorphic site of interest.
  • the amplification products of such probes can subsequently be sequenced to determine the identity of the nucleotide at the polymorphic site of interest.
  • Determining the presence of the polymorphisms identified herein as well as the specific haplotypes provides a method for identifying a genetic basis for an inter- individual variation to treatment with a range of disease therapeutics that are used or being developed.
  • the provision of such information for pharmacogenetics i.e. the study of genetic variations in drug response will be useful for identifying the specific therapy regime that will be most effective in an individual.
  • a diagnostic kit for diagnosing disease, in particular asthma, in an individual comprising one or more oligonucleotides as described herein.
  • the kit comprises two or more oligonuleotides, wherein at least two of said oligonucleotides are capable of detecting a different single nucleotide polymorphism from another of said at least two oligonucleotides, and wherein the single nucleotide polymorphisms are selected from the group consisting of positions 2323, 3135, 3753, 3898 and 3931 of SEQ ID NO:1.
  • one oligonucleotide may be completely complimentary to a portion of SEQ ID NO: 1 containing one of the above defined SNPs and another oligonucleotide may be completely complimentary to another portion of SEQ ID NO: 1 containing another of the above defined SNPs.
  • the present invention identifies for the first time an association of the gene, CKRX, with the respiratory disease asthma. Before this association was recognised, it was known that CKRX encoded a polypeptide that may function as a G protein coupled receptor. However, CKRX is an "orphan" receptor meaning that to date, no ligand had been identified and therefore a role for CKRX has not previously been known. The present invention therefore identifies a functional role for CKRX.
  • a method for treating a respiratory disease in an individual which method comprises modifying CKRX expression or functional activity.
  • the disease is asthma.
  • the present invention also provides an assay for identifying a compound as a potential treatment of a respiratory disease, which assay comprises determining that the compound interacts with CKRX.
  • CKlRX is meant protein encoded by the gene whose sequence is set out in SEQ ID NO: 1 as well as any of the isogenes having SNPs as identified herein.
  • the present invention identifies that those individuals having a polymorphism in the CKRX gene defined by the presence of A at position 2323 of SEQ ID NO:1 are susceptible to disease. This suggests that the polymorphism, which results in a modified form of CKRX that has an amino acid change V168M, provides an altered form of the protein. It can therefore be deduced that the modified form has an aberrant function which results in the symptoms of disease, for example asthma.
  • the method for treating a respiratory disease comprises treating the individual so as to modify the function or expression of the CKRX polypeptide having the V168M amino acid change (ie the CKRX polypeptide in which Valine at amino acid position corresponding to position 168 of SEQ ID NO:2 is substituted by Methionine) .
  • suitable individuals for such treatment can be identified by an immunoassay to detect the presence of the modified form.
  • a method of detecting the presence in a sample of a CKRX polypeptide having an amino acid change V168M comprising:
  • RNA precursor that encodes a ribonucleic acid (RNA) precursor
  • the precursor comprises: (i) a first stem portion comprising a sequence that is identical to 15 to 40 consecutive nucleotides of an RNA molecule transcribed from 15 to 40 consecutive nucleotides of the DNA sequence identified in SEQ ID NO:1 and wherein the 15 to 40 consecutive nucleotides of the DNA sequence identified in SEQ ID NO:1 comprises one or more of the following polymorphisms: A at position 2323; T at position 3135; A at position 3753; T at position 3898; G at position 3931;
  • a second stem portion comprising a sequence that is identical to 15 to 40 consecutive nucleotides of an RNA molecule transcribed from 15 to 40 consecutive nucleotides of the DNA sequence identified in SEQ ID NO:1 and wherein the 15 to 40 consecutive nucleotides of the DNA sequence identified in SEQ ID NO:1 comprising one or more of the following polymorphisms: A at position 2323; T at position 3135; A at position 3753; T at position 3898; G at position 3931; and wherein the first and second stem portions can hybridize with each other to form a duplex stem; and (iii) a loop portion that connects the two stem portions.
  • CKRX function Other methods for modifying CKRX function include introducing a compound which interacts with the gene product. Such compounds can be identified in screening assays.
  • the present inventors have identified an association between CKRX and a respiratory disease, in particular asthma. Accordingly, the CKRX protein has been identified as a suitable target with which to screen for diseases, and in particular respiratory diseases such as asthma. Accordingly, in another aspect of the invention there is provided an assay for identifying a compound as a potential compound that modulates the function the CKRX polypeptide, and which can be used for the treatment of diseases such as respiratory diseases.
  • the present invention provides a method of identifying an agent that modulates the function of the CKRX polypeptide, which method comprises:
  • the method comprises: (a) providing a sample containing CKRX polypeptide or a homologue thereof or a fragment of either, and a candidate agent; and
  • fragment refers to a subsequence of the full length sequence that comprises at least 25, preferably at least 50, more preferably at least 100 consecutive amino acids of the sequence depicted in SEQ ID NO: 2, preferably the fragment is a polypeptide that is the CKRX protein with either or both C-terminal and N-terminal truncations. It is understood that the polypeptide for use in the invention may be both a fragment and a homologue of the CKRX protein.
  • the screening methods of the invention are carried out using a polypeptide comprising an amino acid sequence as depicted in SEQ ID NO: 2, or a sequence possessing, in increasing order of preference, at least 80%, 85%, 90%, 95%, 97%, 98% and 99% amino acid sequence identity thereto.
  • Such variants are herein referred to as "homologues”.
  • sequence identity between two sequences can be determined by pair-wise computer alignment analysis, using programs such as, BestFit, Gap or FrameAlign.
  • the preferred alignment tool is BestFit.
  • suitable software such as Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, Fasta3 and PILEUP 3 and a scoring matrix such as Blosum 62.
  • Such software packages endeavor to closely approximate the "gold-standard" alignment algorithm of Smith- Waterman.
  • the preferred software/search engine program for use in assessing similarity i.e. how two primary polypeptide sequences line up, is Smith- Waterman. Identity refers to direct matches, similarity allows for conservative substitutions.
  • the present invention provides a method of identifying an agent that modulates the function of the CKRX V168M polypeptide, which method comprises: (a) providing a sample containing the CKRX V168M polypeptide or a homologue thereof or a fragment of either, and a candidate agent; and (b) detecting the binding of the CKRX V168M polypeptide, homologue or fragment, to the candidate agent in the sample.
  • the method comprises:
  • a homologue or a fragment of the CKRX V168M polypeptide refers to a homologue or a fragment of CKRX as defined above but wherein the homologue or a fragment contains Methionine at a position which, when the homologue or fragment is aligned with SEQ ID NO:2, corresponds to position 168 of SEQ ID NO:2.
  • a method of treatment of a patient suffering from a respiratory disease such as asthma comprising administration to said patient of an effective amount of a compound identified according to a screening method of the invention.
  • a model animal system for studying a respiratory disease such as asthma by providing an animal having a mutation or knock out for CKRX.
  • Methods for generating suitable transgenic animals are well known to those skilled in the art.
  • a non-human host mammal model for asthma comprising CKRX gene disrupted.
  • the non-human host mammal has a knock out for CKRX.
  • the gene has nucleotide A at a position corresponding to position 2323 of SEQ ID NO: 1.
  • the present invention provides a recombinant mammalian cell comprising a nucleic acid of SEQ ID NO: 1 but wherein the nucleic acid has nucleotide A at position 2323.
  • Allele refers to a particular form of a genetic locus, distinguished from other forms by its particular nucleotide or amino acid sequence.
  • Antibodies can be whole antibodies, or antigen-binding fragments thereof.
  • the invention includes fragments such as Fv and Fab, as well as Fab' and F(ab') 2 , and antibody variants such as scFv, single domain antibodies, Dab antibodies and other antigen-binding antibody-based molecules.
  • “Expression” refers to the transcription of a genes DNA template to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product (i.e., a peptide, polypeptide, or protein).
  • the term “activates gene expression” refers to inducing or increasing the transcription of a gene in response to a treatment where such induction or increase is compared to the amount of gene expression in the absence of said treatment.
  • the terms “decreases gene expression” or “down-regulates gene expression” refers to inhibiting or blocking the transcription of a gene in response to a treatment and where such decrease or down- regulation is compared to the amount of gene expresssion in the absence of said treatment.
  • “Functional activity" of a protein in the context of the present invention describes the function the protein performs in its native environment. Altering the functional activity of a protein includes within its scope increasing, decreasing or otherwise altering the native activity of the protein itself. In addition, it also includes within its scope increasing or decreasing the level of expression and/or altering the intracellular distribution of the nucleic acid encoding the protein, and/or altering the intracellular distribution of the protein itself.
  • Gene is a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
  • Gene is an unphased 5' to 3' sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.
  • Haplotype pair refers to the two haplotypes found for a locus in a single individual.
  • Haplbtype is a phased 5 1 to 3' sequence of nucleotides found at two or more polymorphic sites in a locus on a single chromosome from a single individual.
  • isolated nucleic acid refers to material removed from its original environment (for example, the natural environment in which it occurs in nature), and thus is altered by the hand of man from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • the term "isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the nucleic acids of the present invention.
  • Locus refers to a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • Nucleic acid refers to single stranded or double stranded DNA and RNA molecules including natural nucleic acids found in nature and/or modified, artificial nucleic acids having modified backbones or bases, as are known in the art.
  • Phhased as applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is known.
  • Polymorphic site is a position within a locus at which at least two alternative sequences are found in a population.
  • Polymorphism refers to the sequence variation observed in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Single Nucleotide Polymorphism refers, typically, to the specific pair of nucleotides observed at a single polymorphic site. In rare cases, three or four nucleotides may be found.
  • “Stringent hybridisation conditions” refers to an overnight incubation at 42 0 C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulphate, and 20 pg/nil denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 0 C.
  • Unphased as applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is not known.
  • Variant or “derivative” in relation to CKRX gene or polypeptide includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic or amino acids from or to the nucleotide or polypeptide sequence of CKRX.
  • RFLP restriction fragment length polymorphism analysis
  • SSCP single strand conformation polymorphism analysis
  • ASO allele specific oligonucleotide hybridisation
  • OLA oligonucleotide ligation assay
  • the read out from these assays can from any of a number of types: radioactive, fluorescent, chemilurninescent, enzymatic, analysis of size, charge or mass etc.
  • DNA amplification methods are known, most of which rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self- sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • an enzymatic chain reaction such as a polymerase chain reaction, a ligase chain reaction, or a self- sustained sequence replication
  • PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247-252).
  • Self- sustained sequence replication (3SR) is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874).
  • Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. In the Q ⁇ Replicase technique, RNA replicase for the bacteriophage Q ⁇ , which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197.
  • rolling circle amplification (Lizardi et al., (1998) Nat Genet 19:225) is an amplification technology available commercially (RCATTM) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
  • RCATTM rolling circle amplification
  • SDA strand displacement amplification
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • primer pair 1 5'-CAATT ACACG CTGGC ACCAG AG-3' and 5'- CCCAC GAAGT ACAGT CCAAT GA-3', amplify a portion of exon 2; primer pair 2, 5'-TCATTG GACTG TACTT CGTGG G-3' and 5'-ACAGG AGAGG GTTGA TGCAG C-3 ⁇ amplify a portion of exon 2; primer pair 3, 5'-CTCAG GCACC GTGCA AGGCT-3' and 5'-GAACT TCGAC AGAAC AAGTTA CC-3', amplify a portion of exon 2 and 3' flanking sequence; and primer pair 4, 5'-CATAC CTCAG GCCTC ACCAG C-3' and 5'-GCCGT GAACG TGTGC CTGAT G-3', amplify 3' flanking sequence only.
  • oligonucleotide primers are less than 100 nucleotides in length.
  • Preferred oligonucleotides are between 15 to 30 nucleotides in length and, most preferably, between 20 and 25 nucleotides in length.
  • Such oligonucleotides must be capable of specifically hybridising to a target region of a CKRX polynucleotide.
  • specific hybridisation means that the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridising conditions, while failing to form such a structure when incubated with a non-target region or non-CKRX polynucleotide under the same hybridising conditions.
  • CKRX oligonucleotides of the present invention may also be arrayed onto a solid surface so as to provide an ordered array for rapid screening of samples for polymorphisms.
  • Array techniques are known in the art and described, for example, in WO 98/20020 and WO 98/20019.
  • Suitable samples for SNP and/or haplotype analysis are taken from genomic samples from an individual of interest. Accordingly, methods according to some aspects of the invention may include obtaining a genomic sample.
  • a test sample of genomic nucleic acid may be obtained, for example, by extracting nucleic acid from cells or biological tissues or fluids, saliva, tears, urine, sweat, buccal cell samples, hair or skin.
  • SNPs and haplotypes are associated with an individual's susceptibility to respiratory disease, particularly asthma. Accordingly, a determination of SNPs or haplotypes of individuals can be used as a diagnostic test for identifying asthma in a patient or for identifying a predisposition.
  • SNP and haplotype information can also be useful in determining interindividual variation to the effects of treatment with a particular drug.
  • information may be useful in determining or predicting an individual's response to a particular disease therapeutic agent, in particular, drugs for the treatment of a respiratory disease such as asthma.
  • the ability to predict a patient's response to a particular therapeutic agent is useful for physicians in making a decision as to how to treat an asthma patient, for example.
  • An asthma patient whose haplotype pair indicates that the patient will respond well to a particular therapeutic agent is a better candidate for that treatment than a patient who is likely to exhibit a low or intermediate response.
  • CKRX The functional activity of CKRX may be modified by suitable molecules/agents which bind either directly or indirectly to CKRX protein, or to the nucleic acid encoding it.
  • Agents may be naturally occurring molecules such as peptides and proteins, for example antibodies, or they may be synthetic molecules.
  • Methods of modulating the level of expression of CKRX include, for example, using antisense techniques. Antisense constructs are described in detail in US 6,100,090 (Monia et al), and Neckers et al., 1992, CritRev Oncog 3(1-2):175-231, the teachings of which document are specifically incorporated by reference. Other methods of modulating gene expression are known to those skilled in the art and include dominant negative approaches as well as introducing peptides or small molecules which inhibit gene expression or functional activity.
  • CKRX changes in events immediately down-stream of CKRX activity, such as the modulation of intracellular messengers or expression of genes whose transcription is regulated by CKRX expression, can be used as an indication that a molecule in question affects the functional activity of CKRX.
  • CKRX protein encoded by the gene whose sequence is set out in SEQ ID NO: 1 as well as any of the isogenes having SNPs as identified herein. Assays
  • the present invention also provides a method of screening compounds to identify antagonists to CKRX or modified forms of CKRX including those modified forms identified herein such as the variants having valine or methionine at amino acid residue 168.
  • Candidate compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, peptide and gene libraries, and natural product mixtures.
  • Chemical libraries include combinatorial chemistry libraries and, in particular, a combinatorial chemical library comprising compounds that interact with GPCRs.
  • Such antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the CKRX receptor; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • Compounds having inhibitory, activating, or modulating activity can be identified using in vitro and in vivo assays for CKRX activity and/or expression, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
  • the screening method may simply measure the binding of a candidate compound to the CKRX, or to cells or membranes bearing the CKRX receptor, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the CKRX receptor, using detection systems appropriate to the cells bearing the receptor. For example, a cell or membrane preparation expressing a CKRX receptor may be contacted with a compound of interest. The ability of the compound to generate a response, eg. a rapid release of intracellular (cytosolic) calcium following interaction with the CKRX receptor is then measured.
  • a parallel sample which does not receive the test compound is also monitored as a control.
  • the treated and untreated cells or membranes are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, chemotaxis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of en2ymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds.
  • suitable phenotypic criteria including but not limited to microscopic analysis, chemotaxis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of en2ymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds.
  • Such methods are known in the art (eg Neote K, et al., Cell, 72:415-25 (1993)).
  • a compound which binds but does not elicit a response identifies that compound as an antagonist.
  • An antagonist compound is also one which binds and produces an opposite response.
  • a compound which binds and elicits a response is identified as an agonist.
  • Inhibitors of activation are generally assayed in the presence of a known agonist for example chemokines and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constitutively active polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activity of the CKRX receptor. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing CKRX receptor, to form a mixture, and determining whether its ability to bind CKRX protein is reduced.
  • Fusion proteins such as those made from Fc portion and CKRX receptor, may also be used for high-throughput screening assays to identify antagonists for CKRX receptor function (see D. Bennett et al., JM?/ Recognition, 8:52-58 (1995); and K. johanson et al., J Biol Chem, 270(16):9459-9471 (1995)). Expressing CKRX in cells
  • CKRX in its normal or any of its polymorphic forms may be expressed in cells by introducing expression vectors encoding the CKRX polypeptide. Recombinant methods for expressing proteins in this way are well known to those skilled in the art.
  • Vectors for expressing proteins are known for expression in prokaryotic cells, in yeast cells, typically S. cerevisiae and in mammalian cells and each include the specifc genetic elements for expression in the particular cell type.
  • CKRX protein can be expressed and purified from systems such as these for use in methods for detecting molecules which interact with CKRX.
  • Recombinant organisms i.e., genetically modified animals, expressing a CKRX or a variant CKRX gene comprising a polymorphism are prepared using standard procedures known in the art.
  • a construct comprising CKRX or the variant human gene having polymorphism 3 is introduced into a non-human animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Genetically-modified animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector comprising the transgene, see e.g., U.S. Pat. No. 5,610,053. Another method involves directly injecting a transgene into the embryo. A third method involves the use of embryonic stem cells.
  • the genetic modification process results in replacement of the animal's CKRX gene with the human CKRX gene.
  • animals into which the human CKRX isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see “The Introduction of Foreign Genes into Mice” and the cited references therein, In: Recombinant DNA, Eds. J. D. Watson, M. Gilrnan, J. Witkowski, and M. Zoller; W. H. Freeman and Company, New York, pages 254- 272).
  • Recombinant nonhuman animals stably expressing a human CKRX isogene and producing human CKRX protein can be used as biological models for studying diseases related to abnormal CKRX expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • these non-human animals can be used as models for diseases including asthma.
  • the invention also relates to pharmaceutical compositions for treating disorders affected, such as respiratory diseases, including asthma, by expression or function of a CKRX isogene described herein.
  • a suitable pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these CKRX isogenes; an antisense oligonucleotide directed against one of the CKRX isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which activates or inhibits expression of a CKRX isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • therapeutically effective amount is meant that one or more of the symptoms relating to disorders related to the expression or function of a CKRX isogene is reduced and/or eliminated.
  • the composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water. Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • a positive association occurs when a particular sequence variant or allele occurs more frequently among asthmatics than in people without asthma, or, in a collection of asthma families, when a particular allele is transmitted from parents to affected children more often than expected by chance.
  • a positive association implies that a particular allele behaves as a risk factor itself for asthma, increasing disease risk by modulating gene function, or is co-inherited with a second polymorphism which is in fact the risk-modifying variant. Either way, establishment of a statistically significant genetic association provides a direct link between a gene and asthma, suggesting that the gene or other genes working in the same pathway, are worthy targets for therapy.
  • the human CKRX gene lies on chromosome band 3pl4 and comprises two exons. Exon 1 is small and the entire coding sequence is present in exon 2.
  • SNPs single nucleotide polymorphisms
  • the nucleotide sequence of CKRX is set out in SEQ ID NO:1
  • the SNPs were identified by searching the various publicly accessible SNP databases for likely polymorphic sites. These databases included the TSC (the SNP consortium,), the NCBI (the National Center of Biotechnology Information), and the EBI (European Bioinformatics Institute). While the SNP databases are a useful guide for identifying polymorphisms, most of the SNPs described therein have not been formally validated, and only about 50% are eventually confirmed as polymorphic in the Caucasian population. For CKRX, the search for SNPs focussed around the coding sequence and up to 1000 bp of 3' flanking sequence. Within this region, eight database SNPs were identified and PCR primers were designed to amplify gene segments spanning these SNPs for DNA sequencing.
  • PCR primers including their nucleotide sequences, are detailed above. Amplification reactions were set up using genomic DNA as template from 14 unrelated DNA samples, and the resulting DNA products subjected to DNA sequencing. Each of the eight database SNPs were confirmed as sites of nucleotide variation, and these SNPs were selected for genotyping.
  • SNPs identified in CKRX are shown in Table 1. Importantly, three of the SNPs were predicted to change the amino acid sequence of the CKRX protein and, as such, may alter CKRX function, making them good candidates for genetic association studies.
  • Table 1 Genetic association of polymorphisms in CKRX to asthma SNP in Table 1 refers to the precise position of the polymorphism within SEQ ID NO:1.
  • Each of the CKRX polymorphisms was genotyped in a collection of 530 Scandinavian asthma families. The families were ascertained for two or more siblings with a doctor's diagnosis of asthma. At least of one the siblings must have been receiving regular anti-inflammatory treatment for at least a year prior to recruitment or, in the case of seasonal asthma, received anti-inflamatory treatment throughout the course of two consecutive seasons, or had a doctor's diagnosis of asthma plus a provocation test demonstrating a PD20 value of ⁇ 10 ⁇ mol methacholine.
  • a PD20 value is the amount of methacholine (in ⁇ mol) at which the forced expiratory volume in 1 second (FEV 1 ) is reduced by 20% and is used as a measure of asthma severity. Although responsiveness to methacholine is just one measure of asthma, severity of asthma is judged to be greater the smaller the PD20 value.
  • Biologic parents of asthmatic siblings were also recruited and genotyped for the above-noted CKRX polymorphisms.
  • Genotyping of the CKRX polymorphisms was performed for six of the SNPs using a Taqman allelic discrimination assay.
  • the presence of one or both alleles at a polymorphic site was accomplished using oligonucleotide probes that can discriminate between two sequences that differ by only 1 nucleotide.
  • the probes are labeled with different fluorochromes so that information on both alleles can be captured simultaneously and the process can be automated.
  • SNPshot primer extension approach
  • TDT transmission disequilibrium test
  • the number of transmissions for each of the CKRX SNPs is shown in Table 1. Five of the SNPs showed a distortion in the expected number of transmissions that reached statistical significance. The associated SNPs occurred at positions 2323, 3135, 3753, 3898 and 3931, the former two SNPs falling within exon 2 of CKPvX. The rarer alleles of the SNPs at 2323, 3135, 3753 and 3898 were over-transmitted to offspring with asthma, while the rarer form of the SNP at 3931 was under-transmitted. Importantly, the SNP at position 2323 was one of three amino acid changing SNPs identified, the other two SNPs not being associated with asthma. This genetic association provided the first direct evidence linking CKRX with respiratory disease, specifically asthma, in man.
  • bronchial hyper- responsiveness BHR
  • atopy a PD20 value of ⁇ 10 ⁇ mol methacholine was used to define a BHR phenotype.
  • Atopy is measured by reaction to various antigens in a skin prick test, and a single positive skin prick test was used to define atopy. The association of polymorphisms in CKRX to BHR and atopy was tested in the same way as described for asthma above (Table 2).
  • haplotype 3 The rarer form of the SNP at 3931, which was also associated with asthma, was located on a different haplotype (haplotype 2). Inspection of the haplotypes for transmission to offspring with asthma revealed that haplotype 3 was over-transmitted while haplotype 2 was under-transmitted.
  • Lys Ser Ser Tyr Asn Leu Asp Lys Ser VaI His lie Thr Lys Leu He 275 280 285 Ala Thr Thr His Cys cys lie Asn Pro Leu Leu Tyr Ala Phe Leu Asp 290 295 300

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Abstract

La présente invention concerne des polymorphismes à nucléotide unique du gène CKRX et leur association à des maladies respiratoires telles que l'asthme. La présente invention concerne aussi l'identification d'haplotypes correspondants et leur association à des maladies respiratoires telles que l'asthme. Donc, la présente invention identifie aussi une fonction pour le CKRX dans des troubles humains pour lesquels sa fonction était inconnue auparavant. À cet égard, la présente invention fournit aussi des méthodes et des tests pour identifier des composés qui interagissent avec le CKRX et qui peuvent être utilisés pour traiter des maladies respiratoires.
PCT/SE2006/001260 2005-11-08 2006-11-06 Polymorphismes à nucléotide unique dans le gène ckrx associés à une maladie respiratoire WO2007055636A1 (fr)

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WO1997041225A2 (fr) * 1996-04-26 1997-11-06 Incyte Pharmaceuticals, Inc. Recepteurs mixtes mammaliens de lymphocytes et de chimoquine
US20020076760A1 (en) * 1996-07-22 2002-06-20 Derk Bergsma G-protein coupled receptor HNFDS78 polypeptides
WO2004040000A2 (fr) * 2002-09-09 2004-05-13 Nura, Inc Recepteurs couples a la proteine g et leurs utilisations
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