TARGET

FIELD OF THE INVENTION

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. Thus, the present invention also identifies a role for CKRX in human disorders where previously its function was unknown. In this regard,, the present invention also provides methods and assays for identifying compounds which interact with CKRX and which may be used for treating respiratory diseases.

BACKGROUND OF THE INVENTION

The essential function of the lungs requires a fragile structure with enormous exposure to the environment, including pollutants, microbes, allergens, and carcinogens. Host factors, resulting from interactions of lifestyle choices and genetic composition, influence the response to this exposure. Damage or infection to the lungs can give rise to a wide range of diseases of the respiratory system (or respiratory diseases). A number of these diseases are of great public health importance. 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.

Among the most common respiratory diseases is asthma. 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.

The mechanism of susceptibility to respiratory diseases such as asthma remains unknown. Interestingly, while most individuals experience similar environmental exposures, only certain individuals develop an allergic response characterised by the symptoms of asthma.

Current treatments suffer their own set of disadvantages. The main therapeutic agents, 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. In addition, constant use of 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.

Furthermore, because of the problems associated with conventional therapies, alternative treatment strategies have been evaluated. 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. For example, 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.

There is therefore a need for the identification of genes, or other genes working the same pathway, that are involved in respiratory diseases such as asthma and represent targets for therapy.

While a number of studies document a heritable component to atopy and asthma, to date, family studies have been difficult to interpret since these disorders are significantly influenced by age and gender, as well as many environmental factors

such as allergens, viral infections, and pollutants. Moreover, because there is no known biochemical defect associated with susceptibility to these disorders, the mutant genes and their abnormal gene products can only be recognized by the anomalous phenotypes they produce.

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. International patent publication number WO2004/040000 discusses GPCR polypeptides and polynucleotides, including 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).

SUMMARY OF THE INVENTION

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.

Single nucleotide polymorphisms (SNPs) are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in normal individuals in a population. It is recognised that SNPs may be responsible for variations between individuals, including variations which predispose an individual to a disease or cause it. Approximately half of all coding sequence SNPs result in synonymous (i.e. silent) codon changes. Even though these SNPs may have no effect on protein function, they are potentially useful for tracking other variations nearby as adjacent stretches of DNA tend to be inherited together ('linkage disequilibrium').

The single nucleotide polymorphisms identified in the present invention (by reference to their position in SEQ ID NO:1) are listed below:

Polymorphism Position Change from the nucleotide in SEQ ID NO:1 to

1 2229 A

2 2321 T

3 2323 A

4 2548 G

5 3135 T

6 3753 A

7 3898 T

8 3931 A

As mentioned above, 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.

Accordingly, in a first aspect of the invention, there is provided a method for detecting the presence of a respiratory disease in an individual, which method 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.

In one embodiment, 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.

Accordingly, there is provided a method for detecting the presence of a respiratory disease in an individual, which method 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

3135 of SEQ ID NO:1; A at position 3753 of SEQ ID NO:1; T at position 3898 of SEQ ID NO:l; and G at position 3931 of SEQ ID NO:l.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3753 of SEQ ID NO: 1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3898 of SEQ ID NO: 1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 2323 and position 3931 of SEQ ID NO: 1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3135 and position 3931 of SEQ ID NO : 1.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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.

In a further embodiment, 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. However, it will be understood that the invention is not intended to be limited to the exact sequence as set out in that listing but includes variants and derivatives thereof. 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, as set out in the Table above, 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.

Accordingly, in a second aspect, 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.

In one embodiment of this second aspect, 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.

Accordingly, there is provided a method for determining a genetic predisposition of an individual to the presence of a respiratory disease, which method 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

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3753 of SEQ ID NO:1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3898 of SEQ ID NO: 1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 2323 and position 3931 of SEQ ID NO: 1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3135 and position 3931 of SEQ ID NO:1.

In one embodiment, the method comprises determining the nucleotide of the individual at position 3753 and position 3931 of SEQ ID NO: 1.

hi one embodiment, the method comprises determining the nucleotide of the individual at position 3898 and position 3931 of SEQ ID NO:1.

In one embodiment, 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.

Li one embodiment, 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.

Li one embodiment, 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.

In a further embodiment, 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). Antibodies to such a variant protein can be generated using standard methods.

It should be noted that hereinafter 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. It should also be noted that hereinafter 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.

Accordingly, in a further aspect of the invention, there is provided a method for determining a genetic predisposition of an individual to a respiratory disease, which method 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).

By "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. In a particularly preferred embodiment of the methods of the present invention, 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.

Accordingly, in a further aspect, there is provided an isolated nucleic acid molecule comprising any one of the following 4 CKRX haplotypes:

As will be clear to the skilled addressee, 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.

In a preferred embodiment, there is provided an isolated nucleic acid molecule selected from the group consisting of haplotypes 2 and 3, i.e.

In a further aspect, there is provided 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.

From Table 3, it can be seen that each of the following nucleotides at the specified positions in CKJRX indicate which haplotype is present:

Position Allele Haplotvpe

2229 C 1

2321 A 1

2323 A 3 2548 G 4

3135 T 3

3753 A 3

3898 T 3

3931 A 2

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.

From Table 3 it can also be seen that 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.

In one aspect, there is provided 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.

As identified herein, the presence of certain of these haplotypes in an individual correlates with, those individuals having a respiratory disease such as asthma. In particular, 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.

Accordingly, in another aspect of the invention, there is provided 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.

In a preferred embodiment, 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.

Methods for determining the sequences of nucleic acid sequences and determining the identity of nucleotides at particular positions within a sequence will be recognised to those skilled in the art and suitable methods are described herein. A number of these methods employ binding an oligonucleotide probe 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.

Accordingly, in another aspect of the invention there is provided 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.

Suitably, 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.

In a preferred embodiment, there is provided 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.

More than one probe may be used on the same test sample. Accordingly, within the context of the invention, 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.

Suitably 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. In this aspect, 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.

In another aspect of the invention there is provided a diagnostic kit for diagnosing disease, in particular asthma, in an individual comprising one or more oligonucleotides as described herein.

In one embodiment of this aspect, 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.

For example, 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.

Accordingly, in another aspect of the invention there is provided a method for treating a respiratory disease in an individual, which method comprises modifying CKRX expression or functional activity. In one embodiment, 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.

By 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.

In particular, 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.

Accordingly, in one embodiment, 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) .

Furthermore, suitable individuals for such treatment can be identified by an immunoassay to detect the presence of the modified form.

Thus, in another aspect of the invention, there is provided a method of detecting the presence in a sample of a CKRX polypeptide having an amino acid change V168M, the method comprising:

(a) providing an antibody capable of binding to CKRX Vl 68M;

(b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and

(c) detecting an antibody-antigen complex comprising said antibody; wherein detection of an antibody-antigen complex indicates the presence in the sample of CKRX Vl 68M.

Suitable methods of treatment include those methods in which the gene expression of CKRX is modified.

Accordingly, in another aspect, there is provided a method of modifying gene expression of CKRX in an individual, the method comprising: introducing into the individual a nucleic acid sequence that encodes a ribonucleic acid (RNA) precursor, wherein 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;

(ii) 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.

Other methods for modifying CKRX function include introducing a compound which interacts with the gene product. Such compounds can be identified in screening assays.

As mentioned above, 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.

Thus, the present invention provides a method of identifying an agent that modulates the function of the CKRX polypeptide, which method comprises:

(a) providing a sample containing CKRX polypeptide or a homologue thereof or a fragment of either, and a candidate agent; and

(b) detecting the binding of the CKRX polypeptide, homologue or fragment, to the candidate agent in the sample.

In an embodiment of this aspect, the method comprises: (a) providing a sample containing CKRX polypeptide or a homologue thereof or a fragment of either, and a candidate agent; and

(b) measuring the binding of the CKRX polypeptide, homologue or fragment to the candidate agent in the sample; and

(c) comparing the binding of CKRX polypeptide, homologue or fragment to the candidate agent in the sample with the binding of the CKRX polypeptide, homologue or fragment to a control agent, wherein the control agent is known to not bind to the CKRX polypeptide; wherein an increase in the binding of the CKRX polypeptide, homologue or fragment to the candidate agent in the sample relative to the binding of the CKRX polypeptide, homologue or fragment to the control agent indicates that the candidate agent modulates the function of CKRX polypeptide.

The term "fragment" as used herein 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.

In a preferred embodiment, 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".

The 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. In practice, when searching for similar/identical sequences to the query search, from within a sequence database, it is generally necessary to perform an initial identification of similar sequences using 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. Thus, 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.

In a further aspect, 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.

In an embodiment of this aspect, the 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) measuring the binding of said CKRX V168M polypeptide, homologue or fragment to the candidate agent in the sample; and

(c) comparing the binding of said CKRX V168M polypeptide, homologue or fragment to the candidate agent in the sample with the binding of said CKRX polypeptide, homologue or fragment to a control agent, wherein the control agent is known to not bind to the CKRX V168M polypeptide; wherein an increase in the binding of said CKRX V168M polypeptide, homologue or fragment, to the candidate agent in the sample relative to the binding of the CKRX V168M polypeptide, homologue or fragment, to the control agent indicates that the candidate agent modulates the function said CKRX Vl 68M polypeptide.

It should be noted that 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.

According to a further aspect of the invention, there is provided 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.

According to a further aspect of the invention, there is provided 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.

Thus in a further aspect of the invention there is provided a non-human host mammal model for asthma comprising CKRX gene disrupted.

In one embodiment, the non-human host mammal has a knock out for CKRX.

In another embodiment, the gene has nucleotide A at a position corresponding to position 2323 of SEQ ID NO: 1.

In a further aspect, 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.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods. See, generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and

Ausubel et al, Short Protocols in Molecular Biology (1999) 4 ^th Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N. Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, NJ.). These documents are incorporated herein by reference.

Definitions

"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. For example, 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. Similarly, 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.

"Genotype" 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, as referred to herein, 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. For example, 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. Preferably, 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", as used herein, 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.

"Phased" 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 (SNP)" 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.

Methods for measuring SNPs and haplotvpes

A wide variety of assays for identifying and characterising SNPs in a sample are currently used. These include restriction fragment length polymorphism analysis (RFLP), single strand conformation polymorphism analysis (SSCP) (Orita et al. P.N.A.S. USA, 1989, 86: 2766-2770) allele specific oligonucleotide hybridisation (ASO) (Saiki et al. P.N.A.S. USA, 1989, 86:6230-6234) oligonucleotide ligation assay (OLA) (Landegren et al. 1988, Science 241; 1077-1080), primer extension or mini- sequencing type assays, Syvanen et al. 1999; Hum. Mutat 13:1-10), TaqMan® (Livak et al. 1995; Nat. Genet. 9: 341-342), molecular beacons (Tyagi et al. 1998; Nat.

Biotechnol. 16:49-53), nuclease (Goldrick 2001; Hum. Mutat. 18;190-204) and structure-specific nuclease invader technology (Fors et al. 2000; Pharmacogenomics; 1:219-229)

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.

A variety of technology platforms have been developed to increase throughput. A number of such platforms are reviewed, for example by Weiner and Hudson in BioTechniques 32; S4-S13 (June 2002).

Most assays and platforms for SNP and haplotype analysis start off with genomic DNA and require some form of amplification step.

Many 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.

Many target and signal amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al., Science 242:229- 237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).

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 β Replicase technique, RNA replicase for the bacteriophage Q β, which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197.

Alternative amplification technology can be exploited in the present invention. For example, rolling circle amplification (Lizardi et al., (1998) Nat Genet 19:225) is an amplification technology available commercially (RCAT™) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. A further technique, strand displacement amplification (SDA; Walker et al., (1992) PNAS (USA) 80:392) begins with a specifically defined sequence unique to a specific target.

Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

The following primers (SEQ ID NOs: 3-10) have been used to amplify segments of the CKRX gene: 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.

Suitably, 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. As used herein, 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.

Use of SNP or haplotype information

As described herein particular 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. In particular, with reference to the SNPs identified herein., 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.

In order to identity a correlation between a SNP or haplotype as identified herein and a clinical response to a particular therapeutic agent, it is necessary to obtain data on clinical responses exhibited by a population of individuals who received the treatment such as a population in a clinical trial. SNP or haplotype determinations will be made for each individual in the trial and a correlation identified using statistical methods.

Modifying the functional activity of CElRX

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.

In addition, 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.

By CKRX 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.

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)).

Techniques such as analytical centrifugation, affinity binding studies involving chromatography or electrophoresis can be used to detect molecules which interact directly with CKRX. Other techniques that allow the identification of protein-protein interactions include immunoprecipitation and yeast two hybrid studies.

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. Alternatively, the screening method may involve competition with a labeled competitor. Further, 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. 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.

Model systems for determining CKRX activity

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. Preferably, 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.

Preferably the genetic modification process results in replacement of the animal's CKRX gene with the human CKRX gene. Examples of 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. In particular, these non-human animals can be used as models for diseases including asthma.

Pharmaceutical compositions

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.

Preferably, the composition contains the active ingredient in a therapeutically effective amount. By 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.).

For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, 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.

The present invention will now be described with reference to the following non- limiting examples.

EXAMPLES

Direct evidence linking CKRX with asthma has come from genetic association studies which seek to question whether polymorphisms in the nucleotide sequence of a gene increase risk for disease. Polymorphisms are variations in the nucleotide sequence of a gene, which may have a subtle effect on gene function, and are common in the population.

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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.

A search for polymorphisms revealed multiple single nucleotide polymorphisms (SNPs) in exon 2 and in the sequence flanking the 3 ' end of the gene. SNPs are the most frequent form of nucleotide sequence variation identified in man and each identifies a unique position at which either of two nucleotides may occur.

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. Details of the 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.

The 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. Base change indicates the sequence variation at each polymorphic site; Numbers of transmissions indicates the number of times the rarer allele of each polymorphism was transmitted or not transmitted to affected offspring; and P = indicates the confidence in the reported associations.

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 μmol methacholine. A PD20 value is the amount of methacholine (in μmol) 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. In this approach, 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. For two of the SNPs, at positions 2321 and 2323, this approach was not possible since each SNP would likely interfere with the assay of the other, so a primer extension approach, called SNPshot, was employed. In both techniques, genotypes on >90% of the DNA samples was collected with high accuracy.

To assess genetic association of SNPs with asthma, the transmission disequilibrium test (TDT) was performed. In the TDT, the number of times each allele at a

polymorphic site is transmitted from parents to an affected offspring is counted. No association is characterised by approximately equal transmission of either allele for a given SNP. Although the number of transmissions is rarely identical for two alleles of a given SNP, the difference would not be expected to reach statistical significance. In contrast, a genetic association is characterised by a bias in the transmission of one allele over the alternate allele which reaches statistical significance. The strength of the association is indicated by the significance level of the statistical test used.

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.

To examine the association of CKRX SNPs further, we more closely defined the asthma phenotype based upon its two major clinical attributes: bronchial hyper- responsiveness (BHR) and atopy. BHR is measured using the methacholine challenge test and a PD20 value of < 10 μmol 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). Each of the polymorphisms that were previously positive with asthma were positive for BHR and atopy, and the strength of the association increased for all but one of the SNPs (3931). The fact that the association increased with more precisely defined disease phenotypes strengthened the evidence that CKRX is involved in the disease pathway(s) leading to asthma.

Table 2. Genetic association of CKlRX polymorphisms to bronchial hyper- responsiveness and atopy.

Examination of the CKRX SNPs for co-inheritance on common haplotypes revealed that 5 haplotypes represented greater than 99% of the variation in the gene (Table 3). Notably, the rarer alleles of the asthma-associated SNPs at 2323, 3135, 3753, and 3898 all lay on the same haplotype (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.

Table 3. CKRX haplotypes.

All publications mentioned in the above specification, and references cited in said publications, are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

The CKRXnucleotide sequence (SEQ ID NO: 1) is shownbelow.

gatgtcattt aatcaatata taaattctaa gcaaataggt ctgatcccca aattaggtta 60 gtcacagctg ctgagtcgtt gacccaagag aagctcatct agattttttc attattttca 120 agttcctctt ctcggttcgt ccttcttcca gaccatgccc tccccgtccc actctcttcc 180 cacagcctcc cctccccaca gcctcctccc accattccaa atctgggctg ttctctcaat 240 ttccttctct ctggactcaa acctacccta gcccccagcc tcagtttggg gttaaacttg 300 tcctcctcac attctctccc acccaacttg atgtcgcctc tgtgtcatca ccacgggatt 360 ttcctcctct gggttctcct tttccgagtg gggtcagctc ccccatgagt cacagcacca 420 atcacttctg gctgcttgca aacccctttg ctttcctcag tgttgacacc cagggcagcc 480 ctatgctcac tgccgctgag accccacctc tgcccctggc cttttcccag ctgacatcac 540 cctgtggctt ccattttcct aaaattctct tttgaggcct cagtcttaac caaagcacac 600 agtgcccctc aaaaatgaca ataaaaaccc aaacacaccg tgactgtcat ggcaggttcc 660 tggtccccgt attgaatcag cgggtgggtt tctgcgaaca ctggtgagag gccgcattag 720 agggtcagga ccctcaggtc tggactcgtg gtcaccacat accttcctcc ctgctgacag 780 tagctggtac ctgttaccta ctcagagtgt cacatgccac aagccagagc gtcttggcag 840 ttctcagcac cttgacatca cttccttgct accactcaga gcggcagtga cacagttccc 900 ttatctcaga aggccagaag acggctgtca aaggtcacag ggaaatcaaa ggcggggtac 960 agggccagag ggaggaggaa acaacttccc ggttgctttc agacgcttca gagatcctct 1020 ggaggcctgg gggagctttt gagtacttta tttcagttgg tccctgagct cggtgagtgg 1080 ggcgggtaga gccaccaggg gaatcaacag tggtttctcg tgcccctcag ggtcaggagc 1140 agtctgatca aaaggagggc atccactgtc cggggccatt cccacagctc ccggatgctg 1200 ggtctggagg ctgcgccctt cccctgcagg agctcagccc agtggtaagt catctgtgtg 1260 tcatctatgt atttaacccc ttatggccat gttgatgctg agcatggttt cacttttgca 1320 aacatttatt tatacccttc gagagaaaaa cgtctcagct gtcacaggaa gctgcttcgg 1380

ggggtgagca aactttttaa aatgcagaaa ttatgatcta cacccgtttc ttaaaagtaa 1440 gccatcgtac ttggttctct ttaattatat attttcttac atattgtgtt catgtaggca 1500 agtcctgttt ctgctaaaag aaggtaagtt ctaccaaggc ggtgtcatgc cagctttatt 1560 tcccgtggca cctggcacac tgctaagcac ttacatgctt aacaactaga ttgggaatgg 1620 tgctgctctg gggaagtggg cacacgttaa agaaatgttt atttcagtct tctgaaatag 1680 ggaattactc tggctaaaat gtagctccag aaagggaaag tggggctgta tgaatccagg 1740 tccagtttgt tgtttcctcc aggataaggc agctgtcgga ggggaaaatc atctcccatt 1800 tctccacagg gcagtctgaa gatggccaat tacacgctgg caccagagga tgaatatgat 1860 gtcctcatag aaggtgaact ggagagcgat gaggcagagc aatgtgacaa gtatgacgcc 1920 caggcactct cagcccagct ggtgccatca ctctgctctg ctgtgtttgt gatcggtgtc 1980 ctggacaatc tcctggttgt gcttatcctg gtaaaatata aaggactcaa acgcgtggaa 2040 aatatctatc ttctaaactt ggcagtttct aacttgtgtt tcttgcttac cctgcccttc 2100 tgggctcatg ctgggggcga tcccatgtgt aaaattctca ttggactgta cttcgtgggc 2160 ctgtacagtg agacattttt caattgcctt ctgactgtgc aaaggtacct agtgtttttg 2220 cacaagggca actttttctc agccaggagg agggtgccct gtggcatcat tacaagtgtc 2280 ctggcatggg taacagccat tctggccact ttgcctgaat acgtggttta taaacctcag 2340 atggaagacc agaaatacaa gtgtgcattt agcagaactc ccttcctgcc agctgatgag 2400 acattctgga agcattttct gactttaaaa atgaacattt cggttcttgt cctcccccta 2460 tttattttta catttctcta tgtgcaaatg agaaaaacac taaggttcag ggagcagagg 2520 tatagccttt tcaagcttgt ttttgccata atggtagtct tccttctgat gtgggcgccc 2580 tacaatattg catttttcct gtccactttc aaagaacact tctccctgag tgactgcaag 2640 agcagctaca atctggacaa aagtgttcac atcactaaac tcatcgccac cacccactgc 2700 tgcatcaacc ctctcctgta tgcgtttctt gatgggacat ttagcaaata cctctgccgc 2760 tgtttccatc tgcgtagtaa caccccactt caacccaggg ggcagtctgc acaaggcaca 2820 tcgagggaag aacctgacca ttccaccgaa gtgtaaacta gcatccacca aatgcaagaa 2880

gaataaacat ggattttcat ctttctgcat tatttcatgt aaattttcta cacatttgta 2940 tacaaaatcg gatacaggaa gaaaagggag aggtgagcta acatttgcta agcactgaat 3000 ttgtctcagg caccgtgcaa ggctctttac aaacgtgagc tccttcgcct cctaccactt 3060 gtccatagtg tggataggac tagtctcatt tctctgagaa gaaaactaag gcgcggaaat 3120 ttgtctaaga tcacataact aggaagtggc agaactgatt ctccagccct ggtagcattt 3180 gctcagagcc tacgcttggt ccagaacatc aaactccaaa ccctggggac aaacgacatg 3240 aaataaatgt attttaaaac atctatttaa tgtattttaa aataatttgt aagttgattt 3300 taaaaccaat ttaactacat tccaaattat agacagccca tttatatggg agtaactttt 3360 caggctcatt gcctcgccgg tgatgagaag aactagctag ctggaagctg tgggaaaaag 3420 aggtaaggta acttgttctg tcgaagttct ctaaattctc ttgcttactt gccacacccc 3480 taggccccca gcttccccta acccaaggtt tctggtattt tctcgtactt tatcaagact 3540 atggaatctt aggagactta acaaaagcaa atgagaaatt atgtttagaa atgtctaaca 3600 aaatgaattc tttgtccttt taagtataac acatacctca ggcctcacca gcacataact 3660 acaaaaggtt gtcccacttc ctttctgtgg ctgagttagt agaacacagg ctcccacctg 3720 ccacatcagc agaaggtcac ctcaacatgt gagctacctc cccggagacc ccccagatcc 3780 gtaaggatga tgcatccttg atcctaaaaa cattttcctg ttcctggtgt tcagaattgg 3840 actccacact cactggtctc tttataatct tgcttctggc cctttgaggc ctcaaagcta 3900 ccagggcctt gctgccaggg gacaatcacc gctcccggct gagtcctgca gacatagggc 3960 ctgggctggc tgcctcctcc tgctggccca gcatcttgct tgcactaaag agagctggag 4020 gcttggcact gatgcttgct aaaaacctac ccaggccgcc cactgctgcc gcactgcagg 4080 gcaccagccc acactcctcc cctcctggca tcaggcacac gttcacggca ctaaacctta 4140 caggaaagca gtggaaccct gtctttcctt cacaggaggc actttccttc ctaggcaaaa 4200 acgatgatac ccatgggcac agcatctcac atagggacac atagagatgt ctgactcatt 4260 aaa 4263

The CKRX amino acid sequence (SEQ ID NO: 2) is shown below.

Met Al a Asn Tyr Thr Leu Al a Pro Gl u Asp Gl u Tyr Asp VaI Leu li e 1 5 10 15

Gl u Gl y Gl u Leu Gl u Ser Asp Gl u Al a Gl u Gi n cys Asp Lys Tyr Asp 20 25 30

Al a Gi n Ala Leu Ser Al a Gi n Leu VaI Pro Ser Leu Cys Ser Ala VaI 35 40 45

Phe VaI li e Gl y VaI Leu Asp Asn Leu Leu VaI VaI Leu li e Leu VaI

50 55 60

Lys Tyr Lys Gly Leu Lys Arg VaI Glu Asn lie Tyr Leu Leu Asn Leu 65 70 75 80

Ala VaI Ser Asn Leu Cys Phe Leu Leu Thr Leu Pro Phe Trp Ala His 85 90 95

Ala Gly Gly Asp Pro Met cys Lys lie Leu lie Gly Leu Tyr Phe VaI 100 105 110 Gly Leu Tyr Ser Glu Thr Phe Phe Asn cys Leu Leu Thr VaI Gin Arg 115 120 125

Tyr Leu VaI Phe Leu His Lys Gly Asn Phe Phe ser Ala Arg Arg Arg

130 135 140

VaI Pro Cys Gly lie lie Thr Ser VaI Leu Ala Trp VaI Thr Ala lie 145 150 155 160

Leu Ala Thr Leu Pro Glu Tyr VaI VaI Tyr Lys Pro Gin Met Glu Asp 165 170 175

Gin Lys Tyr Lys Cys Ala Phe Ser Arg Thr Pro Phe Leu Pro Ala Asp 180 185 190 Glu Thr Phe Trp Lys His Phe Leu Thr Leu Lys Met Asn lie Ser VaI 195 200 205

Leu VaI Leu Pro Leu Phe lie Phe Thr Phe Leu Tyr VaI Gin Met Arg

210 215 220

Lys Thr Leu Arg Phe Arg Glu Gin Arg Tyr Ser Leu Phe Lys Leu VaI 225 230 235 240

Phe Ala lie Met VaT VaI Phe Leu Leu Met Trp Ala Pro Tyr Asn lie 245 250 255

Ala Phe Phe Leu Ser Thr Phe Lys Gl u His Phe Ser Leu Ser Asp cys 260 265 270

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

Gly Thr Phe Ser Lys Tyr Leu Cys Arg cys Phe His Leu Arg Ser Asn 305 310 315 320

Thr Pro Leu Gin Pro Arg Gly Gin Ser Ala Gin Gly Thr Ser Arg Gl u 325 330 335

Glu Pro Asp His Ser Thr Glu VaI 340