WO2010141999A1

WO2010141999A1 - Agents and methods for diagnosing and treating ankylosing spondylitis

Info

Publication number: WO2010141999A1
Application number: PCT/AU2010/000729
Authority: WO
Inventors: Matthew Arthur Brown
Original assignee: The University Of Queensland
Priority date: 2009-06-12
Filing date: 2010-06-11
Publication date: 2010-12-16

Abstract

This invention discloses methods and agents for diagnosing the presence or risk of development of ankylosing spondylitis (AS). More particularly, the present invention discloses the use of NR4A2, TNFAIP3, RORA and CD69 as diagnostic markers of AS. The invention has practical use in early diagnosis of AS, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected subjects.

Description

TITLE OF THE INVENTION

AGENTS AND METHODS FOR DIAGNOSING AND TREATING ANKYLOSING

SPONDYLITIS

FIELD OF THE INVENTION

[0001] This invention relates generally to methods and agents for diagnosing the presence or risk of development of ankylosing spondylitis (AS). More particularly, the present invention relates to the use of Nuclear Receptor subfamily 4, group A, member 2 (NR4A2), Tumour Necrosis Factor, Alpha-Induced Protein 3 (TNFAIP3), RAR-related orphan receptor A (RORA) and CD69 as diagnostic markers of AS. The invention has practical use in early diagnosis of AS, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected subjects.

BACKGROUND OF THE INVENTION

[0002] AS affects 1-9 per 1000 Caucasian individuals, making it one of the most common causes of inflammatory arthritis (Van der Linden, S. et al., 1983, Br J Rheumatol, 22: 18-19 and; Braun, J. et al, \99S, Arthritis Rheum, 41: 58-67). The condition principally affects the axial skeleton including the spine and sacroiliac joints, causing pain, stiffness, and eventually bony ankylosis. Peripheral joints and tendon insertions (entheses) are commonly affected, and approximately one-third of patients develop acute anterior uveitis.

[0003] Genetic factors play a major role in the pathogenesis of AS (Brown, M. A. et al., 1997, Arthritis Rheum, 40: 1823-1828) and there is a striking tendency towards familial clustering and a connection with human leukocyte antigen (HLA)-B27 (Reville, J. D., 2006, Current Opinion in Rheumatology 18: 332-341). The major susceptibility gene, HLA-B27, is present in >95% of Caucasians with AS, yet only 1-5% of HLA-B 27 carriers develop AS, and HLA-B27 carriage alone does not explain the pattern of disease recurrence in families, (Brown, M. A. et al., 2000, Ann Rheum Dis, 59: 883-886).

[0004] Current nucleic acid based diagnostic methods for determining the risk of developing AS or diagnosing subjects with AS rely on detecting the presence of the HLA-B27 gene. However, as discussed above, this screening method is extremely unreliable since a large proportion of subjects who carry the HLA-B 27 gene never develop AS. [0005] Accordingly, there is a recognized need for more effective markers for detecting the presence or diagnosing the risk of AS. It would be highly advantageous to have a reliable screening method to enable better treatment and management decisions to be made in subjects with AS or a predisposition to developing AS. [0006] The present invention is predicated in part on the discovery that

NR4A2, TNFAIP3, RORA and CD69 are useful in diagnosing the presence or risk of development of AS. In particular, the present inventors have determined that NR4A2, TNFAIP3, RORA and CD69 are down regulated in subjects with AS, as compared to their expression in subjects lacking AS. The inventors have reduced these discoveries to practice in novel diagnostic assays that detect aberrant expression of NR4A2,

TNFA1P3, RORA and CD69 for diagnosing the presence or risk of development of AS.

SUMMARY OF THE INVENTION

[0007] The present invention represents a significant advance over current technologies by providing better surrogate markers of AS, as well as facile methods of diagnosing or detecting the presence or risk of developing AS. The present invention discloses methods of diagnosing the presence or risk of development of AS in a subject through detecting gene expression. Advantageous embodiments involve monitoring the expression of certain genes in peripheral leukocytes of the immune system, which may be reflected in changing patterns of RNA levels or protein production that correlate with the presence or risk of development of AS. As such, these methods are suitable for widespread screening of symptomatic and asymptomatic subjects.

[0008] Accordingly, in one aspect, the present invention provides methods for diagnosing the presence or risk of development of AS in a subject. These methods generally comprise detecting in the subject aberrant expression of an AS marker gene selected from NR4A2, TNFAIP3, RORA and CD69, which indicates the presence or risk of development of AS. Generally, the subject is one that is suspected of having AS or at risk of having AS.

[0009] In some embodiments, the methods comprise detecting aberrant expression of an AS marker polynucleotide. Non-limiting examples of AS marker polynucleotides can be selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 17-23 or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence similarity with at least a portion of the sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 wherein the portion comprises at least 15 contiguous amino acid residues of that sequence; and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under at least medium or high stringency conditions. In illustrative examples of this type, the methods comprise detecting aberrant expression of an AS marker polynucleotide(s) selected from the group consisting of; (a) a polynucleotide comprising a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 13 and 17-23 or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence similarity with at least a portion of the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 wherein the portion comprises at least 15 contiguous amino acid residues of that sequence; and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under at least medium or high stringency conditions.

[0010] In specific examples of this type, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, which indicates the presence or risk of development of AS.

[0011] In still other specific examples, the methods comprise detecting aberrant expression of a TNFAIP3 polynucleotide, which indicates the presence or risk of development of AS. [0012] In still other specific examples, the methods comprise detecting aberrant expression of a CD69 polynucleotide, which indicates the presence or risk of development of AS. [0013] In still other specific examples, the methods comprise detecting aberrant expression of a RORA polynucleotide, which indicates the presence or risk of development of AS.

[0014] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a TNFAIP3 polynucleotide, which indicates the presence or risk of development of AS.

[0015] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS. [0016] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

[0017] In still other specific examples, the methods comprise detecting aberrant expression of a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.

[0018] In still other specific examples, the methods comprise detecting aberrant expression of a TNFAlP 3 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

[0019] In still other specific examples, the methods comprise detecting aberrant expression of a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

[0020] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS. [0021] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

[0022] In still other specific examples, the methods comprise detecting aberrant expression of a TNFAIP 3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS. [0023] In still other specific examples, the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS. [0024] In some embodiments, such aberrant expression is detected by: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level or functional activity of at least one AS marker expression product; and (3) comparing the measured level or functional activity of the or each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more subjects lacking AS, wherein a lower level or functional activity of the or each expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of AS in the subject. [0025] In some embodiments, the methods further comprise diagnosing the presence of AS in the subject when the measured level or functional activity of the, or each expression product is lower than the measured level or functional activity of the corresponding expression product. In these embodiments, the lower expression typically represents an at least about 9/10, 4/5, 7/10, 3/5, 1/2, 2/5, 3/10, 1/5, 1/10, 1/20, 1/50, 10^"2, 10^"3, 10^"4, 10^"5, 10^"6, 10^'7, 10-⁸, 10^"9, 10^"10, 10^'", 10^'12, 10^"13, 10^"14, 10^"15, 10-¹⁶, 10^"17, 10^"18, 10^"19 or 10^"20 of the level or functional activity of an individual corresponding expression product, which is referred to as "aberrant expression."

[0026] In some embodiments, the methods further comprise diagnosing the absence of AS when the measured level or functional activity of the or each expression product is the same as or similar to the measured level or functional activity of the corresponding expression product. In these embodiments, the measured level or functional activity of an individual expression product varies from the measured level or functional activity of an individual corresponding expression product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1 %, which is hereafter referred to as "normal expression."

[0027] Suitably, the biological sample comprises tissues, cells or cell lines, bodily fluids, blood or peripheral blood mononuclear cells (PBMCs). Suitably, the expression product is selected from a RNA molecule or a polypeptide. In some embodiments, the expression product is the same as the corresponding expression product. In other embodiments, the expression product is a variant (e.g., an allelic variant) of the corresponding expression product.

[0028] In certain embodiments, the expression product or corresponding expression product is a target RNA (e.g., mRNA) or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least medium or high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of a polynucleotide. In these embodiments, the measured level or abundance of the target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample. Suitably, the nucleic acid probe is immobilized on a solid or semi-solid support. In illustrative examples of this type, the nucleic acid probe forms part of a spatial array of nucleic acid probes. In some embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization (e.g., using a nucleic acid array). In other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g., using a polymerase chain reaction (PCR)). In still other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay.

[0029] In other embodiments, the expression product or corresponding expression product is an AS marker polypeptide whose level is measured using at least one antigen-binding molecule that is immuno-interactive with the AS marker polypeptide. In these embodiments, the measured level of the AS marker polypeptide is normalized to the level of a reference AS marker polypeptide that is present in the same sample. Suitably, the antigen-binding molecule is immobilized on a solid or semi-solid support. In illustrative examples of this type, the antigen-binding molecule forms part of a spatial array of antigen-binding molecules. In some embodiments, the level of antigen-binding molecule that is bound to the target polypeptide is measured by immunoassay (e.g., using an ELISA or RIA).

[0030] In some embodiments, the diagnostic methods of the present invention further comprise detecting expression of at least one other AS marker, illustrative examples of which include HLA-B27. [0031] In a related aspect, the present invention provides methods for treating or inhibiting the development or progression of AS in a subject. These methods generally comprise detecting aberrant expression of at least one AS marker expression product in the subject, and administering to the subject at least one therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of AS in the subject. Representative examples of such therapies include nonsteroidal antiinflammatory drugs (NSAIDS) examples of which include Sulfasalazine (Azulfidine), medication, exercise, physical therapy and surgery.

[0032] In still another aspect, the present invention provides probes for interrogating nucleic acid for the presence of an AS marker polynucleotide as described for example above for use in the diagnostic methods of the present invention. These probes generally comprise a nucleotide sequence that hybridizes under at least medium or high stringency conditions to a NR4A2, TNFAIP3, RORA or CD69 polynucleotide. In some embodiments, the probes consist essentially of a nucleic acid sequence which corresponds or is complementary to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, wherein the portion is at least 15 nucleotides in length. In other embodiments, the probes comprise a nucleotide sequence which is capable of hybridizing to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 under at least medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length. In still other embodiments, the probes comprise a nucleotide sequence that is capable of hybridizing to at least a portion of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 17-23 under at least medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.

[0033] In a related aspect, the invention provides a solid or semi-solid support for use with the diagnostic methods of the present invention, wherein the solid or semisolid support comprises at least one nucleic acid probe as broadly described above immobilized thereon. In some embodiments, the solid or semi-solid support comprises a spatial array of nucleic acid probes immobilized thereon.

[0034] Still a further aspect of the present invention provides an antigen- binding molecule that is immuno-interactive with an AS marker polypeptide as described for example above for use in the diagnostic methods of the present invention. [0035] In a related aspect, the invention provides a solid or semi-solid support comprising at least one antigen-binding molecule as broadly described above immobilized thereon. In some embodiments, the solid or semi-solid support comprises a spatial array of antigen-binding molecules immobilized thereon. [0036] Still another aspect of the invention provides the use of one or more

AS marker polynucleotides as described for example above, or the use of one or more probes as broadly described above, or the use of one or more AS marker polypeptides as described for example above, or the use of one or more antigen-binding molecules as broadly described above, in the manufacture of a kit for diagnosing the presence of AS in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a graphical representation of unsupervised hierarchical clustering of 34 samples based on expression levels detected in the microarray experiment described in the Examples using 17,308 probes flagged as present. Samples are clustered on the horizontal axis with the vertical axis representing the degree of correlation between samples. The lengths of the branches are indicative of the similarities between samples or genes.

[0038] Figure 2 is a graphical representation of unsupervised hierarchical clustering of 34 samples based on expression levels detected in the microarray experiment using the 485 probes differentially expressed between AS and control samples. AS samples are in red and control samples are blue. Samples are clustered on the horizontal axis and genes clustered on the vertical axis. The lengths of the branches in the dendrograms represent the degrees of correlation between samples or genesets. For expression levels, yellow represents overexpressed genes and red underexpressed genes.

[0039] Figure 3 is a graphical representation showing quantitative RT-PCR analysis of NR4A2, TNFAIP3, RORA and CD69 expression in subjects without AS (control) and patients with AS. Graph (A) the samples used for the array analysis were (n=17 AS and 17 controls) and in graph (B) a second population of patients and controls with wider disease severity and non-age and sex-matched were used (n=35 AS and 18 controls). Boxed numbers represent fold-changes in expression (AS/control. * = p<0.05, ** = pO.Ol, AS vs. Control). [0040] Figure 4 is a graphical representation illustrating a Receiver Operator Curve(ROC) showing predictive power of the identified gene sets. Graph (A) illustrates a ROC curve generated from array expression values for the 452 genes significantly different between AS and control samples. Graph (B) illustrates ROC curves generated from the combined quantitative RT-PCR data for NR4A2, TNFAIP3 and CD69 from the discovery and confirmation sample sets. Area under curve (AUC) represents overall ability of the test to discriminate between those individuals with the disease and those without the disease. Inset in (B) are the AUC values from the ROC curves for the individual candidate genes.

TABLE A

BRIEF DESCRIPTION OF THE SEQUENCES

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. [0042] The articles "a" and "an" are used herein to refer to one or to more than one {i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0043] The term "aberrant expression," as used herein to describe the expression of NR4A2, TNFAIP3, RORA or CD69 refers to the down regulation of at least one NR4A2, TNFAIP3, RORA or CD69 gene relative to a 'normal' level of expression of at least one NR4A2, TNFAIP 3, RORA or CD69 gene or allelic variant thereof in healthy or normal cells or in cells obtained from a subject lacking AS, and/or to a level of at least one NR4A2, TNFAIP 3, RORA or CD69 gene product (e.g., transcript or polypeptide) in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from at least one subject lacking AS. In some embodiments, a gene of the present invention is aberrantly- or under-expressed if its level of expression is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of expression of the corresponding gene in healthy or normal cells or in cells obtained from a subject without AS, or in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking AS.

[0044] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. [0045] The term "amplicon" refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an "amplicon" may include the sequence of probes or primers used in amplification. [0046] The term "ankylosing spondylitis (AS)" as used herein includes subjects suffering from undifferentiated spondylarthritis (USpA). A subject is generally considered as suffering from USpA if he/she fulfils the criteria for spondylitis as defined in the European Spondyloarthropathy Study Group (ESSG) classification, the Amor criteria or the ASAS Criteria. ESSG classification includes inflammatory spinal pain or synovitis (asymmetrical, predominantly in lower limbs) and any one or more of the following: positive family history, psoriasis, inflammatory bowel disease, alternate buttock pain and enthesopathy. Amor criteria includes clinical symptoms or past history of any one or more of: lumbar or dorsal pain at night or lumbar or dorsal morning stiffness; asymmetrical oligoarthritis; buttock pain; sausage-like finger or toe; heel pain; iritis; non-gonococcal urethritis or cervicitis accompanying, or within 1 month before, the onset of arthritis; acute diarrhea accompanying, or within 1 month before, the onset of arthritis; presence of history of psoriasis and/or balanitis and/or of inflammatory bowel disease (ulcerative colitis, Crohn's disease). Radiological findings; Sacroiliitis (grade >2 if bilateral, grade >3 if unilateral). Amor criteria may further include genetic background; presence of HLA-B27 and/or family history of ankylosing spondylitis, reactive arthritis, uveitis, psoriasis or chronic inflammatory bowel disease. Amor criteria may further include response to therapy; definite improvement of musculoskeletal complaints with non-steroidal anti-inflammatory drugs (NSAIDs) in less than 48 h or relapse of the pain in less than 48 h if NSAIDs discontinued. The ASAS criteria comprises axial spondyloarthritis as either sacroiliitis on magnetic resonance imaging (MRI) or on x-ray, plus one or more clinical feature of spondyloarthritis, or carriage of HLA-B27 plus two or more clinical features of spondyloarthritis. Clinical features of spondyloarthritis may include presence of inflammatory back pain, arthritis, enthesitis of the heel, uveitis, dactylitis, psoriasis, Crohn's disease or ulcerative colitis, marked reduction in back pain with 24-48 hours of a full dose of a non-steroidal anti-inflammatory drug, a family history of spondyloarthritis, carriage of HLA-B27, or an elevated c-reactive protein level. AS is classified by the modified New York Criteria (1984) which includes any one or more of: low back pain and stiffness for more than 3 months, which improves with exercise, but is not relieved by rest; limitation of motion of the lumbar spine in both the sagittal and frontal planes; limitation of chest expansion relative to normal values correlated for age and sex. AS may further include radiological criterion Sacroiliitis grade 2 bilaterally or grade 3—4 unilaterally. AS is generally present if the radiological criterion is associated with at least one clinical criterion.

[0047] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. [0048] As used herein, the term "immuno-interactive" and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample. Specific binding to an antigen under such conditions may require an antigen-binding molecule that is selected for its specificity for a particular antigen. For example, antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample. A variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno-interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0049] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal. The biological sample may include a biological fluid such as peripheral blood and the like. In certain embodiments, the biological sample comprises cells from a tissue biopsy. [0050] "Cells" and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0051] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0052] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. [0053] By "corresponds to" or "corresponding to" is meant a polynucleotide having a nucleotide sequence that is substantially identical (e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity) to all or a portion of a reference polynucleotide sequence or of a complement thereof.

[0054] By "effective amount", in the context of treating or preventing a condition is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. [0055] The terms "expression" or "gene expression" refer to either production of RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention. [0056] As used herein, the term "functional activity" generally refers to the ability of a molecule (e.g., a transcript or polypeptide) to perform its designated function including a biological, enzymatic, or therapeutic function. In certain embodiments, the functional activity of a molecule corresponds to its specific activity as determined by any suitable assay known in the art. [0057] The term "gene" as used herein refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g., tRNA, rRNA, regulatory RNAs such as ribozymes, post-transcription gene silencing- (PTGS) associated RNAs etc) as well as associated non-coding regions and optionally regulatory regions. In certain embodiments, the term "gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The gene sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extra-chromosomal maintenance or for integration into the host.

[0058] By "housekeeping gene" is meant a gene that is expressed in virtually all cells since it is fundamental to the any cell's functions (e.g., essential proteins and RNA molecules). [0059] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base- pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances as known to those of skill in the art.

[0060] The phrase "hybridizing specifically to" and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0061] Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.

[0062] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an

"isolated polynucleotide", as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. [0063] As used herein, a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally-occurring nucleic acid molecule can encode a protein that occurs in nature.

[0064] By "obtained from" is meant that a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source. For instance, the extract may be isolated directly from a biological tissue or fluid of the subject.

[0065] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof, including nucleotides with modified or substituted sugar groups and the like) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a variant nucleic acid sequence. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.

[0066] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages {e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages {e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties {e.g., polypeptides), intercalators {e.g., acridine, psoralen, etc.), chelators, alkylators and modified linkages {e.g., α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. RNA forms of the genetic molecules of the present invention are generally mRNA or iRNA including siRNAs. The genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules. The terms "nucleotide sequence," "polynucleotide" and "nucleic acid" used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. [0067] The terms "polynucleotide variant" and "variant" refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.

[0068] "Polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.

[0069] The term "polypeptide variant" refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter.

[0070] By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides. Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary," it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotides may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotides or a stretch of non- complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.

[0071] "Probe" refers to a molecule that binds to a specific sequence or sub- sequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope. As used herein, the term "probe" encompasses primers which can be used for example in template- dependent nucleic acid extension, ligation or amplification reactions.

[0072] By "promoter" is meant a region of DNA, which controls at least in part the initiation and level of transcription. Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including a TATA box and CCAAT box sequences, as well as additional regulatory elements (i. e. , activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a transcribable sequence (e.g., a coding sequence or a sequence encoding a functional RNA), the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters according to the invention may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected. The term "promoter" also includes within its scope inducible, repressible and constitutive promoters as well as minimal promoters. Minimal promoters typically refer to minimal expression control elements that are capable of initiating transcription of a selected DNA sequence to which they are operably linked. In some examples, a minimal promoter is not capable of initiating transcription in the absence of additional regulatory elements (e.g., enhancers or other cis-acting regulatory elements) above basal levels. A minimal promoter frequently consists of a TATA box or TATA-like box. Numerous minimal promoter sequences are known in the literature. For example, minimal promoters may be selected from a wide variety of known sequences, including promoter regions from fos, CMV, SV40 and IL-2, among many others. Illustrative examples are provided which use a minimal CMV promoter or a minimal IL2 gene promoter (-72 to +45 with respect to the start site; Siebenlist, 1986).

[0073] The term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.

[0074] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.

[0075] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. [0076] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A infra. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0077] Terms used to describe sequence relationships between two or more polynucleotides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a

"comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0,

Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i. e. , resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15. [0078] "Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences.

[0079] "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.

[0080] The terms "subject" or "individual" or "patient", used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian male subject, for whom therapy or prophylaxis is desired. A preferred subject is a human in need of diagnosis of the presence or absence of AS. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0081] The term "template" as used herein refers to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template" strand. The template may be either RNA and/or DNA, and the complementary strand may also be RNA and/or DNA. In certain embodiments, the complementary strand may comprise all or part of the complementary sequence to the "template," and/or may include mutations so that it is not an exact, complementary strand to the "template". Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part of the invention. [0082] The term "transcribable polynucleotides" or "transcribed nucleic acid sequence" excludes the non-transcribed regulatory sequence that drives transcription. Depending on the aspect of the invention, the transcribable sequence may be derived in whole or in part from any source known to the art, including an animal, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesised DNA. A transcribable sequence may contain one or more modifications in either the coding or the untranslated regions, which could affect the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, insertions, deletions and substitutions of one or more nucleotides. The transcribable sequence may contain an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions. The transcribable sequence may also encode a fusion protein. In other embodiments, the transcribable sequence comprises non-coding regions only. [0083] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i. e. , causing regression of the disease. [0084] By "vector" is meant a nucleic acid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a closed circular plasmid, an extra- chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a marker such as an antibiotic resistance gene that can be used for identification of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0085] The term "normal" is used to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant. [0086] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "NR4A2" shall mean the "NR4A2" gene shall mean the NR4A2 gene, whereas "NR4A2" shall indicate the protein product or products generated from transcription and translation. 2. Markers of AS and uses therefor

[0087] The present invention concerns the detection or diagnosis of AS. Markers of AS, in the form of RNA molecules and polypeptides of specified sequences, of subjects with or susceptible to AS, are disclosed. These markers are indicators of AS and, when aberrantly expressed as compared to their expression in normal subjects or in subjects lacking AS, are diagnostic for the presence or risk of development of AS in tested subjects. Such markers provide considerable advantages over the prior art in this field. In certain advantageous embodiments where cells of the immune system (e.g., leukocytes and leukocyte-containing populations such as peripheral blood mononuclear cells) are used for the analysis, it is possible to conveniently make the diagnosis. [0088] In accordance with the present invention, it has been discovered that aberrant expression of NR4A2, TNFAIP 3, RORA and CD69 as compared to the expression of those genes in normal subjects or in subjects lacking AS, is diagnostic for the presence or risk of AS in tested subjects. [0089] It will be apparent, therefore, that the NR4A2, TNFAIP3, RORA and CD69 expression products will find utility in a variety of applications in detection, diagnosis and treatment of AS. Examples of such applications within the scope of the present disclosure include amplification OΪNR4A2, TNFAIP 3, RORA and/or CD69 transcripts using specific primers, detection of NR4A2, TNFAIP 3, RORA and/or CD69 transcripts by hybridization with oligonucleotide probes and detection of NR4A2, TNFAIP3, RORA and/or CD69 polypeptides.

2.1 NR4A2. TNFAIP 3, RORA and CD69 nucleic acid molecules

[0090] In accordance with the present invention, NR4A2, TNFAIP3, RORA and CD69 nucleic acid sequences find utility inter alia as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of biological samples. In certain embodiments, these probes and primers represent oligonucleotides, which are of sufficient length to provide specific hybridization to a RNA or DNA sample extracted from the biological sample. The sequences typically will be about 10-20 nucleotides, but may be longer. Longer sequences, e.g., of at least about 30, 40, 50, 100, 500, 1000, 5000, 10000, 15000 and even up to full-length, are desirable for certain embodiments.

[0091] Nucleic acid molecules having contiguous stretches of at least about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 or 1000 or 5000 or 10000 or 15000 nucleotides of a sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 13 and 17-23 are contemplated. Molecules that are complementary to the above mentioned sequences and that bind to these sequences under at least medium or high stringency conditions are also contemplated. These probes are useful in a variety of hybridization embodiments, such as Southern and northern blotting. In some cases, it is contemplated that probes may be used that hybridize to multiple target sequences (e.g., allelic variants and/or single nucleotide polymorphisms) without compromising their ability to effectively diagnose the presence or risk of development of AS. In general, it is contemplated that the hybridization probes described herein are useful both as reagents in solution hybridization, as in PCR, for detection or quantification of NR4A2, TNFAIP 3, RORA or CD69 expression, as well as in embodiments employing a solid phase.

[0092] Various probes and primers may be designed around the disclosed nucleotide sequences. For example, in certain embodiments, the sequences used to design probes and primers may include repetitive stretches of adenine nucleotides (poly- A tails) normally attached at the ends of the RNA for the identified marker genes. In other embodiments, probes and primers may be specifically designed to not include these or other segments from the identified marker genes, as one of ordinary skilled in the art may deem certain segments more suitable for use in the detection methods disclosed. In any event, the choice of primer or probe sequences for a selected application is within the realm of the ordinary skilled practitioner.

[0093] Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is desirable. Probes, while perhaps capable of priming, are designed to bind to a target DNA or RNA and need not be used in an amplification process. In certain embodiments, the probes or primers are labeled with radioactive species ³²P, ¹⁴C, ³⁵S, ³H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase).

[0094] The invention also contemplates detection or quantification of naturally-occurring NR4A2, TNFAIP3, RORA and/or CD69 nucleic acid sequences, inclusive of NR4A2, TNFAIP3, RORA or CD69 allelic variants (same locus), homologues (different locus), and orthologues (different organism). NR4A2, TNFAIP3, RORA and CD69 nucleic acid sequences may therefore contain variations such as nucleotide substitutions, deletions, inversions and insertions, relative to the sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23. Variation can occur in either or both the coding (e.g., SEQ ID NO: 17-23) and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the same amino acid sequence. Generally, NR4A2, TNFAIP3, RORA and CD69 polynucleotides will have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and usually at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to reference NR4A2, TNFAIP3, RORA or CD69 nucleotide sequences, as set forth for example in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to their complements, as determined by sequence alignment programs described elsewhere herein using default parameters.

[0095] NR4A2, TNFAIP3, RORA or CD69 polynucleotides will generally hybridize to reference NR4A2, TNFAIP3, RORA or CD69 polynucleotides, as set forth for example in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to a complement thereof, under low stringency, medium stringency, high stringency, or very high stringency conditions. As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al, (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.

[0096] Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45° C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50° C (the temperature of the washes can be increased to 55° C for low stringency conditions). Another embodiment of low stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄ H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5 x Denhardt's reagent [50 x Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 5 x SSPE, 0.1% SDS at 42° C when a probe of about 500 nucleotides in length is employed. [0097] Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60° C. Another embodiment of medium stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄ H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 x Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 1.0 x SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed.

[0098] High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C. Another embodiment of high stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄ H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 x Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1 x SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed.

[0099] In certain embodiments, a NR4A2, TNFAIP3, RORA or CD69 polynucleotide hybridizes to a disclosed nucleotide sequence, as for example set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to a complement thereof, under very high stringency conditions. One embodiment of very high stringency conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C.

[0100] Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et ah, supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to 1.104.

[0101] While stringent washes are typically carried out at temperatures from about 42° C to 68° C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20° C to 25° C below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm are well known in the art (see Ausubel et al., supra at page 2.10.8). In general, the Tm of a perfectly matched duplex of DNA may be predicted as an approximation by the formula:

Tm = 81.5 + 16.6 (loglO M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length)

[0102] wherein: M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The Tm of a duplex DNA decreases by approximately 1° C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at Tm - 15° C for high stringency, or Tm - 30° C for moderate stringency.

[0103] In an illustrative example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42° C in a hybridization buffer (50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe. The membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1% SDS for 15 min at 45° C, followed by 2 x SSC, 0.1% SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68° C. 2.2 NR4A2, TNFAIP3, RORA and CD69 polypeptides

[0104] The present invention contemplates detection or quantification of naturally-occurring NR4A2, TNFAIP3, RORA or CD69 polypeptides, inclusive of NR4A2, TNFAIP3, RORA or CD69 allelic variants, homologues, and orthologues. NR4A2, TNFAIP3, RORA or CD69 polypeptide sequences may therefore contain variations such as amino acid substitutions, deletions and insertions, relative to the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14. NR4A2, TNFAIP3, RORA or CD69 polypeptides will generally have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and usually at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity with a reference NR4A2, TNFAIP3, RORA or CD69 polypeptide, as set forth for example in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, as determined by sequence alignment programs described elsewhere herein using default parameters. A NR4A2, TNFAIP3, RORA or CD69 polypeptide sequence may differ from a reference NR4A2, TNFAIP3, RORA or CD69 polypeptide sequence generally by as much as 60, 50, 40, 30 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

2.3 Anti-NR4A2, TNFAIP3. RORA and CD69 antigen-binding molecules

[0105] The present invention also contemplates the use of antigen-binding molecules that are specifically immuno-interactive with a NR4A2, TNFAIP3, RORA or CD69 polypeptide for diagnosing the presence of AS. In some embodiments, the antigen-binding molecule is a whole polyclonal antibody. Such antibodies may be prepared, for example, by injecting a NR4A2, TNFAIP3, RORA or CD69 polypeptide or portion thereof into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons, Inc, 1991), and Ausubel et al, (1994-1998, supra), in particular Section III of Chapter 11. [0106] In lieu of polyclonal antisera obtained in a production species, monoclonal antibodies may be produced using the standard method as described, for example, by Kohler and Milstein (1975, Nature 256, 495-497), or by more recent modifications thereof as described, for example, in Coligan et al, (1991, supra) by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of NR4A2, TNFAIP3, RORA or CD69 polypeptides.

[0107] The invention also contemplates as antigen-binding molecules Fv, Fab, Fab¹ and F(ab')₂ immunoglobulin fragments. Alternatively, the antigen-binding molecule may comprise a synthetic stabilized Fv fragment. Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a V_# domain with the C terminus or N-terminus, respectively, of a Vj, domain. ScFv lack all constant parts of whole antibodies and are not able to activate complement. ScFvs may be prepared, for example, in accordance with methods outlined in Kreber et al (Kreber et al. 1997, J. Immunol. Methods; 201(1): 35-55). Alternatively, they may be prepared by methods described in U.S. Patent No 5,091,513, European Patent No 239,400 or the articles by Winter and Milstein (1991, Nature 349:293) and Plϋckthun et al (1996, In Antibody engineering: A practical approach. 203-252). In another embodiment, the synthetic stabilized Fv fragment comprises a disulfide stabilized Fv (dsFv) in which cysteine residues are introduced into the W_H and V_/, domains such that in the fully folded Fv molecule the two residues will form a disulfide bond between them. Suitable methods of producing dsFv are described for example in (Glockscuther et al. Biochem. 29: 1363- 1367; Reiter et α/. 1994, J. Biol. Chem. 269: 18327-18331 ; Reiter et α/. 1994, Biochem. 33: 5451-5459; Reiter et al. 1994. Cancer Res. 54: 2714-2718; Webber et al. 1995, MoI. Immunol. 32: 249-258).

[0108] Phage display and combinatorial methods for generating anti-NR4A2, TNFAIP3, RORA or CD69 antigen-binding molecules are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al.

[0109] International Publication No. WO 91/17271 ; Winter et al. International Publication WO 92/20791 ; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281 ; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al (1992) JMoI Biol 226:889-896; Clackson et al (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al (1991) Bio/Technology 9:1373-1377; Hoogenboom et al (1991) Nuc Acid Res 19:4133-4137; and Barbas et al (1991) PNAS 88:7978-7982). [0110] The antigen-binding molecule can be coupled to a compound, e.g., a label such as a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred. An anti-NR4A2, TNFAIP3, RORA or CD69 antigen-binding molecule (e.g., monoclonal antibody) can be used to detect AS marker polypeptides (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. In certain advantageous applications, such antigen-binding molecules can be used to monitor NR4A2, TNFAIP3, RORA or CD69 polypeptide levels in biological samples (including tissues, cells and fluids) for diagnosing the presence or absence of AS. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹1, ³⁵S or ³H. The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

[0111] A large number of enzymes useful as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338. Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution. 3. Methods of detecting NR4A2, TNFAIP3, RORA and CD69 aberrant expression

[0112] The present invention is predicated in part on the determination that NR4A2, TNFAIP3, RORA and CD69 transcripts are down regulated in AS. Accordingly, in certain embodiments, the invention features a method for diagnosing or detecting the presence or absence of AS or the risk of developing AS in a subject, by determining the expression status of NR4A2, TNFAIP 3, RORA and/or CD69 transcripts in a biological sample obtained from the subject and diagnosing the presence or absence of AS in the subject based on the expression status. It is desirable, therefore, to qualitatively or quantitatively determine the levels OΪNR4A2, TNFAIP 3, RORA and/or CD69 transcripts or the level or functional activity of NR4A2, TNFAIP3, RORA and/or CD69 polypeptides in the subject in order to provide the diagnosis. In some embodiments, the presence of AS in the patient is diagnosed when a NR4A2, TNFAIP3, RORA and/or CD69 gene product is expressed at a detectably lower level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking AS. [0113] The corresponding expression product is generally selected from the same gene product, an alternate gene product including splice variants or expression products produced from alternate promoters of the gene, a gene product expressed from a variant gene (e.g., an homologous or orthologous gene) including an allelic variant, or protein products thereof. In some embodiments, the method comprises measuring the level or functional activity of at least one NR4A2, TNFAIP 3, RORA or CD69 expression product.

[0114] In specific embodiments, expression is measured directly (e.g., at the RNA or protein level). In some embodiments, expression is detected in tissue samples. In other embodiments, expression is detected in bodily fluids (e.g., including but not limited to blood and PMBCs). In specific embodiments, the biological sample comprises cells.

[0115] NR4A2, TNFAIP 3, RORA and/or CD69 expression may be detected along with other AS markers, especially, in a multiplex or panel format. Such markers are selected for their predictive value alone or in combination with NR4A2, TNFAIP3, RORA and/or CD69. Thus, in some embodiments, the diagnostic methods of the present invention further comprise detecting expression of at least one other AS marker gene, illustrative examples of which include HLA-B27. 3.1 Nucleic acid-based diagnostics

[0116] Nucleic acids used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al., 1989, supra; and Ausubel et al., 1994, supra). The nucleic acid is typically fractionated (e.g., poly A⁺ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the NR4A2, TNFAIPS, RORA and CD69 sequences present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al. {supra), and in Innis et al, ("PCR Protocols," Academic Press, Inc., San Diego Calif., 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If a cognate NR4A2, TNFAIP 3, RORA or CD69 sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.

[0117] In certain advantageous embodiments, the template-dependent amplification involves the quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology 10: 413-417). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA.

[0118] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No. 320 308. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

[0119] Qβ Replicase, described in PCT Application No. PCT/US87/00880, may also be used. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

[0120] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'α-thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al. , (1992, Proc. Natl. Acad. ScI U.S. A 89: 392-396).

[0121] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of nonspecific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. [0122] Still another amplification method described in GB Application No. 2

202 328, and in PCT Application No. PCT/US89/01025, may be used. In the former application, "modified" primers are used in a PCR-like, template- and enzyme- dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.

[0123] Other nucleic acid amplification procedures include transcription- based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

[0124] Vincent and Kong disclose a method termed helicase-dependent isothermal DNA amplification (HDA) (Vincent and Kong, EMBO Reports, 5(8):795- 800, 2004). This method uses DNA helicase to separate DNA strands and hence does not require thermal cycling. The entire reaction can be carried out at one temperature and this method should have broad application to point-of-care DNA diagnostics.

[0125] Davey et al. , EPO No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

[0126] Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M. A., In: "PCR Protocols: A Guide to Methods and Applications", Academic Press, N. Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sci. U.S.A., 86: 5673-567).

[0127] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used for amplifying target nucleic acid sequences. Wu et al, (1989, Genomics 4: 560).

[0128] Depending on the format, the NR4A2, TNFAIP3, RORA or CD69 nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, JMacromol. Sci. Pure, Appl. Chem., A31(l): 1355-1376).

[0129] In some embodiments, amplification products or "amplicons" are visualized in order to confirm amplification of the NR4A2, TNFAIP 3, RORA or CD69 sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified NR4A2, TNFAIP3, RORA or CD69 sequence. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule. The techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al, 1989, supra and Ausubel et al. 1994, supra). For example, chromophore or radiolabel probes or primers identify the target during or following amplification. [0130] In certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provides different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.

[0131] Following detection/quantification, one may compare the results seen in a given subject with a control reaction (e.g., a statistically significant reference group of normal subjects or of subjects lacking AS; or a statistically significant reference group of subjects with AS). In this way, it is possible to correlate the amount of NR4A2, TNFAIP 3, RORA or CD69 nucleic acid detected with the presence of AS.

[0132] Also contemplated are genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al. (Biotechniques 30(2): 318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry.

[0133] Also contemplated are biochip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14: 441-447) and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A. 91: 5022-5026); Fodor et al. (1991, Science 251: 767-773). Briefly, nucleic acid probes to AS marker polynucleotides are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed NR4A2, TNFA1P3, RORA or CD69 nucleic acids, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate. [0134] As will be appreciated by those of ordinary skill in the art, nucleic acids can be attached to or immobilized on a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non-covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.

[0135] In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.

[0136] The biochip comprises a suitable solid or semi-solid substrate or solid support. By "substrate" or "solid support" is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by practitioners in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluoresce.

[0137] Generally the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.

[0138] In certain embodiments, oligonucleotides probes are synthesized on the substrate, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques can be used. In an illustrative example, the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChip™ technology.

[0139] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more NR4A2, TNFAIP3, RORA or CD69 polynucleotides under conditions favoring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al, 1994, supra, and Sambrook, et al., et al, 1989, supra.

[0140] Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. 1989, supra).

[0141] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.

[0142] Usually the target NR4A2, TNFAIP3, RORA or CD69 polynucleotides are detectably labeled so that their hybridization to individual probes can be determined. The target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu³⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptavidin-reporter molecules.

[0143] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,1 13, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime™ (Pharmacia), Fluoredite™ (Millipore) and FAM (Applied Biosystems International)

[0144] Radioactive reporter molecules include, for example, P, which can be detected by an X-ray or phosphoimager techniques.

[0145] The hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (Homes and Higgins, eds.) (IRL press, Washington D. C, 1985). In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.

[0146] In certain embodiments, high discrimination hybridization conditions are used. For example, reference may be made to Wallace et al (1979, Nucl. Acids Res. 6: 3543) who describe conditions that differentiate the hybridization of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et al (1985, Proc. Natl. Acid. ScL USA 82: 1585) who describe conditions for hybridization of 1 1 to 20 base long oligonucleotides using 3M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al. (supra) describe hybridization conditions that allow stringent hybridization of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as 'locked nucleic acids (Christensen et al, 2001 Biochem J 354: 481-4). [0147] Generally, a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization-optimizing agent, such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant. Examples of isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulfoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts. Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.

[0148] A hybridization mixture containing the target AS marker polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization between the target sequences in the target polynucleotides and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C. For probes longer than 14 nucleotides, 20° C to 50° C is desirable. For shorter probes, lower temperatures are preferred. A sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes. For example, the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days.

[0149] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridized to a target polynucleotide. [0150] The hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a colored colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated color spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for different NR4A2, TNFAIP 3, RORA or CD69 gene products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labeled microbeads, the reaction may be detected using flow cytometry. 3.2 Protein-based diagnostics

[0151] Consistent with the present invention, the aberrant expression of a NR4A2, TNFAIP3, RORA and CD69 protein is indicative of the presence or risk of development of AS. NR4A2, TNFAIP3, RORA or CD69 protein levels in biological samples can be assayed using any suitable method known in the art. For example, antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample. Specific recognition may be provided, for example, by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody. Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., coloured, product. In another suitable method, the primary antibody itself can be detectably labeled. As a result, immunohistological labeling of a tissue section is provided. In some embodiments, a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis. Such an extract (e.g., a detergent extract) can be subjected to western-blot or dot/slot assay of the level of the protein of interest, using routine immunoblotting methods (Jalkanen et ai, 1985, J Cell. Biol. 101: 976-985; Jalkanen et α/., 1987, J. Cell. Biol. 105: 3087-3096). [0152] Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a protein-specific monoclonal antibody, can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify a NR4A2, TNFAIP3, RORA or CD69 protein. The amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et al, 1988, Breast Cancer Research and Treatment 11: 19-30). In other embodiments, two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe. [0153] Additionally, recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins. For example, low-density protein arrays on filter membranes, such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment.

[0154] Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array. The protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array. The protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor. Alternatively, the protein- capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein. Alternatively, the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target. The selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art. Alternatively, the protein- capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo et al., 1998 Anal. Chem. 70:3419- 3425; Cohen et al., 1998, Proc. Natl. Acad. ScL USA 95:14272-14277; Fukuda, et al, 1997 Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic). For example, aptamers are selected from libraries of oligonucleotides by the Selex™ process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding. Alternatively, the in vitro selected protein-capture agent may be a polypeptide (e.g., an antigen) (see, e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. ScL USA, 94:12297-12302). [0155] An alternative to an array of capture molecules is one made through

'molecular imprinting' technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence- specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g., available from ProteinPrint™ and Aspira Biosystems).

[0156] Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et al, 2003 J. Chromatogr. B 787:19-27; Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub.

2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO 03/062444; PCT publication WO 03/077851 ; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210). The antigen-binding molecules of such arrays may recognise at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras- like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases. [0157] Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g., polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Affϊtech and Biosite). Alternatively, 'combibodies' comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations of diabody -producing bacterial clones (e.g., available from Domantis). Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab')₂ immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents. [0158] Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0159] While microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).

[0160] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protein-capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtiter plate or in separate test tubes. [0161] In operation, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186) is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second protein in the sample is already known or known to be invariant. [0162] For analyzing differential expression of proteins between two cells or cell populations, a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding. In an analogous manner, a protein sample of a second cell or population of cells to a second array, is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non-specifically bound components of the sample from the arrays. In a final step, the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array. To determine the differential protein expression pattern of the two cells or populations of cells, the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array.

[0163] In an illustrative example, fluorescence labeling can be used for detecting protein bound to the array. The same instrumentation as used for reading DNA microarrays is applicable to protein-capture arrays. For differential display, capture arrays (e.g. antibody arrays) can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are labeled with different fiuorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from PerkinElmer

Lifesciences). Planar waveguide technology (e.g., available from Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot). Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody). Several alternative readouts have been developed, including adaptations of surface plasmon resonance (e.g., available from HTS Biosystems and Intrinsic Bioprobes), rolling circle DNA amplification (e.g., available from Molecular Staging), mass spectrometry (e.g., available from Sense Proteomic, Ciphergen, Intrinsic and Bioprobes), resonance light scattering (e.g., available from Genicon Sciences) and atomic force microscopy (e.g., available from BioForce Laboratories). A microfluidics system for automated sample incubation with arrays on glass slides and washing has been co-developed by NextGen and Perkin Elmer Life Sciences.

[0164] In certain embodiments, the techniques used for detection of AS marker expression products will include internal or external standards to permit quantitative or semi-quantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols. In specific examples, absolute values for the level or functional activity of individual expression products are determined.

3.3 In vivo Imaging

[0165] In some embodiments, in vivo imaging techniques are used to visualize the expression of NR4A2, TNFAIP3, RORA or CD69 and optionally one or more other AS markers in a patient (e.g., a human or non-human mammal). For example, in some embodiments, AS marker mRNA or protein is labeled using a labeled antigen-binding molecule (e.g., mAb) specific for the AS marker. A specifically bound and labeled antigen-binding molecule can be detected in an individual using an in vivo imaging method, including, but not limited to, radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection.

[0166] The in vivo imaging methods of the present invention are useful in the diagnosis of AS. In vivo imaging is used to visualize the presence and/or amount/level of a NR4A2, TNFAIP3, RORA or CD69 expression product (e.g., NR4A2, TNFAIP3, RORA or CD69 protein). Such techniques allow for diagnosis without the use of an unpleasant biopsy or blood collection and are also useful for providing diagnosis to AS patients. For example, NR4A2, TNFAIP3, RORA and CD69 levels that are indicative of AS can be detected. The in vivo imaging methods of the present invention can further be used to detect AS in other parts of the body.

[0167] In illustrative examples, reagents (e.g., antibodies) specific for NR4A2, TNFAIP3, RORA or CD69 and optionally one or more other AS markers are fluorescently labeled. The labeled reagents are introduced into a subject (e.g., orally or parenterally). Fluorescently labeled antibodies are detected using any suitable method (e.g., using the apparatus described in U.S. Pat. No. 6,198,107, herein incorporated by reference).

[0168] In other embodiments, antibodies are radioactively labeled. The use of antibodies for in vivo diagnosis is well known in the art. Sumerdon et al. (1990, Nucl. Med. Biol, 17:247-254) have described an optimized antibody-chelator for the radioimmunoscintigraphy imaging of tumors using Indium- 11 1 as the label. Griffin et al. (1991, J Clin One 9:631-640) have described the use of this agent in detecting tumors in patients suspected of having recurrent colorectal cancer. The use of similar agents with paramagnetic ions as labels for magnetic resonance imaging is known in the art (Lauffer, 1991, Magnetic Resonance in Medicine 22:339-342). The label used will depend on the imaging modality chosen. Radioactive labels such as Indium- 111, Technetium-99m, or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine- 19 can also be used for positron emission tomography (PET). For MRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.

[0169] Radioactive metals with half-lives ranging from 1 hour to 3.5 days are available for conjugation to antibodies, such as scandium-47 (3.5 days) gallium-67 (2.8 days), gallium-68 (68 minutes), technetiium-99m (6 hours), and indium- 1 1 1 (3.2 days), of which gallium-67, technetium-99m, and indium- 1 1 1 are desirable for gamma camera imaging, gallium-68 is desirable for positron emission tomography. An illustrative method of labeling antibodies with such radiometals is by means of a bifunctional chelating agent, such as diethylenetriaminepentaacetic acid (DTPA), as described, for example, by Khaw et al. (1980, Science 209:295) for In-111 and Tc-99m, and by Scheinberg et al. (1982, Science 215: 1511). Other chelating agents may also be used, but the 1 -(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPA are advantageous because their use permits conjugation without affecting the antibody's immunoreactivity substantially. Another method for coupling DPTA to proteins is by use of the cyclic anhydride of DTPA, as described by Hnatowich et al. (1982, Int. J. Appl. Radiat. Isot. 33:327) for labeling of albumin with In-111, but which can be adapted for labeling of antibodies. A suitable method of labeling antibodies with Tc-99m which does not use chelation with DPTA is the pretinning method of Crockford et al (U.S. Pat. No. 4,323,546). An exemplary method of labeling immunoglobulins with Tc-99m is that described by Wong et al. (1981, J. Nucl. Med., 23:229) for labeling antibodies.

[0170] In still further embodiments, in vivo biophotonic imaging (Xenogen, Almeda, Calif.) is utilized for in vivo imaging. This real-time in vivo imaging utilizes luciferase. The luciferase gene is incorporated into cells, microorganisms, and animals (e.g., as a fusion protein with an AS marker of the present invention). When active, it leads to a reaction that emits light. A CCD camera and software is used to capture the image and analyze it.

3.4 Kits [0171] All the essential materials and reagents required for detecting and/or quantifying NR4A2, TNFAIP3, RORA or CD69 gene expression products, and optionally other AS marker gene products may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microliter plates dilution buffers and the like. For example, a nucleic acid-based detection kit may include: (i) a NR4A2, TNFAIP3, RORA and/or CD69 polynucleotide, and optionally one or more other AS marker polynucleotides, which may be used as a positive control; (ii) a primer or probe that specifically hybridizes to a NR4A2, TNFAIP3, RORA or CD69 polynucleotide, and optionally primers or probes that specifically hybridize to one or more other AS marker polynucleotides. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, Sequenase™ DNA ligase etc depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. Alternatively, a protein-based detection kit may include: (i) a NR4A2, TNFAIP3, RORA and/or CD69 polypeptide, and optionally one or more other AS marker polypeptides, which may be used as a positive control; (ii) an antigen-binding molecule that is immuno-interactive with a NR4A2, TNFAIP3, RORA or CD69 polypeptide and optionally antigen-binding molecules that are immuno-interactive with one or more other AS marker polypeptides. The kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a NR4A2, TNFAIP 3, RORA and/or CD69 AS marker polynucleotide and optionally the expression of one or more other AS marker polynucleotides.

4. Methods of Managing AS

[0172] The present invention also extends to the management of AS, or prevention of further progression of AS, or assessment of the efficacy of therapies in subjects following positive diagnosis for the presence of AS in the subjects. Generally, the management of AS often includes a treatment regime involving medication, exercise, physical therapy and if necessary surgery. Examples of effective medications include but are not restricted to nonsteroidal anti-inflammatory drugs (NSAIDS) such as Sulfasalazine (Azulfidine), Methotrexate (Rheumatrex or Trexall) and Corticosteroids (cortisone); TNF blockers such as etanerce (Enbrel), infliximab (Remicade) and adalimumab (Humira);

[0173] It will be understood, however, that the present invention encompasses the use of any agent or process that is useful for treating or preventing AS and is not limited to the aforementioned illustrative management strategies and compounds.

[0174] Typically, AS-ameliorating agents will be administered in pharmaceutical (or veterinary) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of AS and the prevention of the disease from developing further. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment or prevention of AS, the physician or veterinarian may evaluate severity of any symptom associated with the presence of AS including symptoms related to AS such as for example characterized by acute, painful episodes followed by temporary periods of remission. In any event, those of skill in the art may readily determine suitable dosages of the AS-ameliorating agents and suitable treatment regimens without undue experimentation. [0175] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

[0176] Exemplary subjects for treatment with the methods of the present invention are vertebrates, especially mammals. In certain embodiments, the subject is selected from the group consisting of humans, sheep, cattle, horses, bovine, pigs, dogs and cats. In specific embodiments, the subject is a human.

[0177] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1 ABERRANT EXPRESSION OF NR4A2, TNFΛIP3, RORA and CD69 IN AS

Sample Collection [0178] Approximately 18 patients classified as having AS according to the modified New York criteria, Van Der Linden, S. et αi, (1984, Arthritis Rheum, 27: 361- 368) were used for the study. Cases were selected for relatively active disease, with Ankylosing Spondylitis Disease Index (BASDAI) scores of >4.0, and/or elevated C- reactive protein CRP) levels > 10 mg/L and/or erythrocyte sedimentation rates (ESR) > 25 mm/hr. Other characteristics of the study subjects are shown in Table 1 below which provides a summary of subjects age, sex, disease activity measures, treatment and associated conditions (Ps = psoriasis; IBD = inflammatory bowel disease; PA = peripheral arthritis). Controls were age and gender matched (ND = not done). Eighteen gender- and age-matched healthy controls (±5 yr) were also included. All subjects gave written, informed consent, and the study was approved by the University of Queensland Human Ethics Committee.

[0179] For the validation experiment using quantitative reverse transcription- polymerase chain reaction (qRT-PCR), a larger cohort comprised of another 35 AS patients and 18 healthy controls were studied. All the AS patients met the modified New York criteria for AS diagnosis but had a range of disease severity. Patients and controls were not matched by age or gender (16 females and 37 males; age = 38±10yrs; BASDAI = 3±2.2; CRP = 6.7±8.5; ESR = 12.6±21.0). After informed consent, peripheral blood samples from all participants were collected into heparinised tubes (Greiner, Kremsmϋnster, Austria). PBMCs were immediately isolated from whole blood by Ficoll-Paque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) using a standard protocol (Boyum, A., 1968, Scandinavian Journal of Clinical and Laboratory Investigation, Supplement, 97: 77-89).

RNA extraction and cDNA synthesis

[0180] Total RNA was extracted from 10⁷ PBMCs using the RNeasy Mini Kit (Qiagen, MD) using the manufacturers protocol. The RNA concentration was quantified using a NanoDrop ND- 1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, MA), and the integrity of all total RNA samples was assessed by Agilent 2100 BioAnalyser (Agilent, CA) using RNA Nano Chips. Samples with a RIN above 7.5 were used.

Table 1

[0181] 500ng of total RNA was used as starting material for cRNA amplification using the Illumina TotalPrep cRNA Amplification Kit (Ambion, Applied Biosystems, CA) according to the recommended protocol. A 14 hour in vitro transcription was performed. The concentration of the resulting biotinylated cRNA was quantified by NanoDrop Spectrophotometer. Normal size distribution of the cRNA species was verified with RNA Nano Chips on an Agilent 2100 BioAnalyser.

Microarrav Experiment

[0182] A total of 750ng cRNA for each sample was hybridized to the

Illumina HumanHT-12 V3 Expression BeadChips (Illumina, CA) according to the manufacturer's protocol. The three chips with the 36 samples were processed in parallel for hybridization using the Direct Hyb Assay and read on an Illumina BeadArray Reader.

[0183] Of the 48000 probes on the array, 17308 were found to be expressed in at least one sample and were thus included for analysis. Unsupervised hierarchical clustering using this probeset showed good delineation between AS patients and healthy controls; only 2 patients and 3 controls were misclassified (see Figure 1). Supervised clustering showed samples did not cluster according to gender, age, or their treatment regime, suggesting that affection status was in fact the major determinant of expression patterns.

[0184] To identify the genes determining the clustering, the inventors carried out an unpaired T-test corrected for multiple comparisons. 485 probes representing 452 genes were identified as being significantly different between patients and controls with a p-value <0.0005 (corrected for false discovery rate). The magnitude of differential expression observed was generally low, with only 8 genes showing a significant fold- change >2 (with the maximum fold-change being 2.7) and 42 genes >1.5-fold. Interestingly, all the genes showing >2-fold change and 31 of the 42 genes showing >1.5-fold change were down regulated. To confirm the validity of this probe set, it was then used to re-cluster the samples, resulting in an improved delineation between the AS and control samples, with only 2 controls being misclassified (see Figure 2).

[0185] Candidate genes for further analysis were selected based upon their p- value, fold-change and biological relevance (assisted by Pathways analysis). Four genes were selected for confirmation studies; NR4A2 (AS/Cont = 0.54, p<0.00002), TNFAIP3 (AS/Cont = 0.57, p=0.0003), RORA (AS/Cont = 0.63, p=1.2xlθ^"6) and CD69 (AS/Cont = 0.53, p<0.0005) for further validation by quantitative RT-PCR.

Quantitative RT-PCR measurements

[0186] cDNA was generated from lμg of total RNA using the Bioline cDNA synthesis Kit (Bioline, London, UK) according to manufacturer's instructions. Both candidate and housekeeping gene expression levels were measured in triplicate by quantitative RT-PCR in Micro Amp 384- well optical plates on the ABI TaqMan 7900 Real-time Quantitative PCR platform (both Applied Biosystems, CA). Candidate genes were assayed using the pre-designed TaqMan assays which utilise MGB probes with

FAM dye. For normalisation, expression levels of the housekeeping gene, RPL32 (Kriegova, E et al., 2008, BMC MoI Biol. 9: 69) were measured by SYBR green based quantitative RT-PCR using specific forward and reverse primers (see below). All assays were carried out using SensiMix dT RT-PCR reagent (Quantace, Sydney, Australia) under the following conditions; 50°C for 2 min, 95⁰C for 10 min, and 40 cycles of 95°C for 15 s and 6O⁰C for 60 s. The relative amounts of mRNA for genes of interest were determined using the relative standard curve method, quantitative RT-PCR results were analysed with Mann- Whitney test, /"-values <0.05 were considered significant.

House keeping gene primers

(a) RPL32F - 5'-CCCCTTGTGAAGCCCAAGA-S '

(b) RPL32R - 5'- GACTGGTGCCGGATGAACTT-3'

[0187] The four selected genes were initially validated by quantitative RT- PCR in the RNA samples used for the microarrays, termed the discovery set. As expected the quantitative RT-PCR data showed significant differences between the Control and AS patients and confirmed the direction of the fold changes (Figure 3A).

[0188] For NR4A2 (0.54-fold to 0.17-fold) and TNFAIP3 (0.57-fold to 0.22- fold) these fold-changes also increased significantly. Expression levels of the candidate genes were then measured in a confirmation sample set consisting of a further 35 patient samples which were not selected for disease activity and 18 control samples. All three genes were significantly down-regulated in the AS samples in this dataset as well (Figure 3B). RORA showed a small decrease in the dataset (0.87-fold, p=0.08), however, the analysis was conducted on a small sample size and it is believed that using a larger dataset would provide a more significant result.

[0189] To assess the power of the differentially expressed gene sets to predict AS the inventors generated ROC curves. As would be expected, using the expression levels from the array data for the 452 genes differentially expressed between AS and control samples, the ROC curve generated was an almost perfect classifier with an area under the curve (AUC) of 97% (Figure 4a). The individual predictive power for the three validated candidate genes was then tested within the discovery array set and also showed good predictive power with AUCs -74% (Figure 4b-inset). When these three genes were combined the AUC was 81%, a strong classifier (Figure 4b). The predictive power of the three candidate genes NR4A2, TNFAIPS and CD69 was then applied to the confirmation dataset. With this less selective and more diverse group, the three genes generated individual AUCs of 72-78% (Figure 4b-inset) and a combined ALJC of 77% (Figure 4b).

Data processing and Statistical analyses

[0190] Array data were processed using the Illumina BeadStudio software then the processed data were assessed for quality control and normalised in Lumi (Du, P et al., 2008, Bioinformatics, 24: 1547-1548). Analysis of gene expression patterns was performed in BRB-Array Tools (Simon, R et al., 2007, Cancer Inform, 3: 1 1-17). For quality control scanned images of the arrays were visually inspected for artefacts in Illumina BeadStudio followed by the graphical analysis of density plots in Lumi. One control sample was excluded due to problems during array hybridisation, and one AS sample was a biological outlier probably due to ethnicity (this patient was of Indonesian origin with the other patients and controls being Caucasian), thus statistical analyses were performed on 17 control and 17 AS samples.

[0191] The microarray data were transformed by variance stabilization transformation (VST) (Lin et al., 2008, Nucleic Acids Res; 36: el 1) then normalized by robust spline normalization (RSN) (Workman et al., 2002, Genome Biol, 3: 1-16). Post- normalization quality control was carried out using density plots to ensure all samples had similar distribution and variance. To reduce background noise in the data analysis all probes that were not expressed in any of the samples, i.e. whose intensity was below background in all samples, were excluded from the analysis. This did not exclude probes for genes whose expression was either "switched-on" or "switched-off '.

[0192] Differentially expressed genes were identified by conducting an unpaired Mest with randomized variance model in BRB Array Tools. Multivariate permutation correction included in this package was used to control for false positives with 1000 permutations, and choosing a threshold of maximum of 10 false discoveries and confidence level of 80%.

[0193] Samples were clustered using centred correlation (Pearson correlation) and complete linkage. The predictive power of the differentially expressed genes from the array data was calculated by a Receiver-Operator Characteristic (ROC) curve based on a Bayesian Compound Covariate Predictor in BRB-Array Tools. ROC curves from the qRT-PCR data the individual candidate genes were calculated in SPSSvI 7.0 (SPSS, Chicago, IL). ROC curves were also generated to calculate the predictive power for the combined qRT-PCR data from the 3 candidate genes NR4A2, TNFAIPS and CD69. This was undertaken by first calculating the optimal gene expression level delineating between AS and controls for each gene (Youden Index, J ) (Perkins, N. J and Schisterman E. F., 2006, Am J Epidemiol, 163: 670-675) An integer score was then assigned to each sample representing the number of genes with expression below the Youden index (all 3 genes were down-regulated in AS samples); 1= no genes below J, 2 = 1 gene below J, 3 = 2 genes below J and 4 = all 3 genes below J. A ROC curve was then calculated based upon this data using Jrocfit (Eng J. ROC analysis: web-based calculator for ROC curves, 2006, available from http://www.jrocfit.org) using the Ordinal Rating Scale which has been developed for categorised data. [0194] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0195] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0196] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing the presence or risk of development of AS in a subject, the method comprising detecting in the subject aberrant expression of at least one AS marker gene selected from the group consisting of NR4A2, TNFAIP 3, RORA or CD69, which indicates the presence or risk of development of AS, wherein the AS marker gene is characterized in that it is aberrantly expressed in cells of the immune system in subjects having or at risk of developing AS.

2. A method according to claim 1, comprising detecting aberrant expression of an AS marker polynucleotide selected from the group consisting of a NR4A2 polynucleotide, a TNFAIP 3 polynucleotide, a RORA polynucleotide and a CD69 polynucleotide.

3. A method according to claim 2, wherein the AS marker polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence that shares at least 80% sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 80% sequence identity or similarity with the sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under high stringency conditions.

4. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide, which indicates the presence or risk of development of AS.

5. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a TNFAIP3 polynucleotide, which indicates the presence or risk of development of AS.

6. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a CD69 polynucleotide, which indicates the presence or risk of development of AS.

7. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a RORA polynucleotide, which indicates the presence or risk of development of AS.

8. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide and a TNFAIP 3 polynucleotide, which indicates the presence or risk of development of AS.

9. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.

10. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

1 1. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.

12. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a TNFAIP 3 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

13. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.

14. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP 3 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

15. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a TNFAIP 3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

16. A method according to any one of claims 1 to 3, comprising detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.

17. A method according to any one of claims 1 to 16, wherein aberrant expression is detected by: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level or functional activity of an expression product of at least one AS marker gene; and (3) comparing the measured level or functional activity of the or each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more normal subjects or from one or more subjects lacking AS, wherein a lower level or functional activity of the or each expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of AS in the subject.

18. A method according to claim 17, further comprising diagnosing the presence of AS in the subject when the measured level or functional activity of the expression product is lower than the measured level or functional activity of the corresponding expression product.

19. A method according to claim 17, wherein the method further comprise diagnosing the absence of AS when the measured level or functional activity of the expression product is the same as or similar to the measured level or functional activity of the corresponding expression product.

20. A method according to claim 17, wherein the biological sample comprises tissue, cells or cell lines, bodily fluids or blood.

21. A method according to claim 17, wherein the expression product or corresponding expression product is selected from a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of a NR4A2 polynucleotide, a TNFAIP3 polynucleotide, a RORA polynucleotide or CD69 polynucleotide.

22. A method according to claim 17, wherein the expression product or corresponding expression product is a NR4A2, TNFAIP3, RORA or CD69 polypeptide whose level is measured using at least one antigen-binding molecule that is immuno- interactive with the NR4A2, TNFAIP3, RORA or CD69 polypeptide.

23. A method according to any preceding claim, further comprising detecting aberrant expression of at least one other AS marker.

24. A method according to claim 23, wherein the at least one other AS marker is HLA-B27.

25. A method for treating or inhibiting the development or progression of AS in a subject, the method comprising detecting aberrant expression of at least one AS marker gene selected from NR4A2, TNFAIP3, RORA and CD69 in the subject, and administering to the subject at least one therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of AS in the subject.

26. A method according to claim 25, wherein the therapy is selected from medication, exercise, physical therapy and surgery.