WO2010071405A1

WO2010071405A1 - Markers for detecting predisposition for risk, incidence and progression of osteoarthritis

Info

Publication number: WO2010071405A1
Application number: PCT/NL2008/050817
Authority: WO
Inventors: Hanneke Kerkhof; Andreas Gerardus Uitterlinden; Joyce Berdina Josepha Van Meurs
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2008-12-18
Filing date: 2008-12-18
Publication date: 2010-06-24

Abstract

The present invention relates to a method for the detection of osteoarthritis (OA) or for detection of the risk for developing OA or progression of OA comprising detecting the presence of one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs3815148, rs873598, rs10248619, rs10465850, rs12402320 and SNPs in linkage disequilibrium therewith and/or detecting the differential expression of a gene product from one or more of the genes selected from the group consisting of JAK1, KCNN3, GRB10, COG5, AK3L1, DNAJC6, PRKAR2B, HBP1, GPR22, C10orf46, PRLHR, and DUS4L. Further part of the invention are the use of these genes and/or gene products as target for drug development and pharmaceutical compounds for the prevention or therapy of osteoarthritis.

Description

Title: Markers for detecting predisposition for risk, incidence and progression of osteoarthritis

FIELD OF THE INVENTION

The present invention is in the field of disease diagnostics, including classification and prognosis of disease, in particular osteoarthritis. The invention also relates to methods for screening candidate therapeutic compounds for use in the treatment and prevention of osteoarthritis, and to methods of treatment and prevention of osteoarthritis.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA), the most common form of arthritis, is a complex, chronic disease and a major contributor to functional impairment and reduced independence, particularly affecting the elderly (Peat, G. et al., 2001, Ann. Rheum. Dis. 60:91-97). In the Netherlands, estimated costs for OA are €300 million - 1 billion per year, which is similar to diabetes or asthma. The etiology of OA is not completely understood and at this moment there are no curative and only scarce symptomatic treatments options available for people suffering from OA. The goal of our study is to identify diagnostic and prognostic genetic markers for risk and progression of OA and to identify new therapeutic targets.

Genetics of OA. OA has an estimated heritability of 40% for the knee, 60% for the hip and 65% for the hand (Spector, T. D. and MacGregor A.J., 2004, Osteoarthritis Cartilage 12, Suppl. A: S39-44). This means that a substantial portion of variation in risk for OA can be attributed to genetic variation, i.e. DNA sequence variation in or around genes involved in the etiology of OA. The vast majority of the genes involved are unknown and their identification could explain much about the pathogenesis of OA. Identification of such genes may provide insight into potential pathways not yet recognized to be involved in the onset of OA. There is a need in the art for identification of genetic loci and genes and gene products involved in the pathogenesis of OA Although it has been recognised for a long time that genetic factors play an important role in complex diseases such as OA, findings of studies aiming to identify the genetic origin have been disappointing. A large number of candidate genes have been studied, but up to now candidate gene studies have been difficult to reproduce. Candidate gene association studies are hypothesis based- studies, and since our knowledge of the pathogenesis of OA is limited, candidate gene studies alone will be inadequate to completely explain the genetic basis of this disease. In theory, the most successful approach to identify new genes for OA is a search across the complete genome without an upfront hypothesis. Published genome wide genetic linkage studies have implicated several loci for OA, but also these results have been difficult to replicate. This is probably due to the lack of power of this approach. More recently, a novel and revolutionary approach was developed to search for the most important risk genes involved in a complex trait: a genome-wide association (GWA) study. In a GWA study a dense set of Single Nucleotide Polymorphisms (SNPs) across the genome is genotyped to survey the most common genetic variation for a role in disease. The density of the SNPs in the most advanced commercially available arrays has now increased to 550.000 haplotype tagging SNPs, which cover 95% of the human genome. GWA has already been very successful in identifying genetic risk factors for complex diseases (Richards et al., 2008, Lancet. 3;371(9623):1505-12; Melzer et al., 2008, PIoS Genet. 9;4(5):el000072; Weedon et al., 2008, Nat Genet. 40(5):575-83. Epub 2008 Apr 6). This approach has therefore very high potential to identify the most important common genetic determinants for OA.

The present invention was done using the GWA-approach, through which variation in the DNA were found to be related to the risk for OA. It is an object of the present invention to provide for variants of nucleic acid sequences of (genes harbouring) such SNPs as well as for methods of prognosis, diagnosis and therapy in which these variants are applied.

SUMMARY OF THE INVENTION

By using Genome Wide Association Studies, the present inventors have discovered single nucleotide polymorphisms (SNPs) in the genome that strongly correlate with the susceptibility for osteoarthritis. These SNPs are located in or near genes, which have not previously been associated with osteoarthritis.

In a first aspect, the present invention provides a method of predicting the risk of (progression of) osteoarthritis comprising detecting at least one SNP in or near a gene selected from the group consisting of JAKl, AK3L1, DNAJC6, KCNN3, GRBlO, COG5, PRKAR2B, HBPl, GPR22, C10orf46, PRLHR, and DUS4L.

In a preferred embodiment of a method of the invention, said SNP is selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith In another aspect, the present invention provides an isolated nucleic acid molecule comprising a polymorphic nucleotide position and selectively hybridizing under high stringency conditions to a nucleotide sequence encoding a gene selected from the group consisting of JAKl, AK3L1, DNAJC6, KCNN3, GRBlO, COG5, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L, or to the complement thereof, wherein the polymorphic position is a SNP selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith.

In another aspect, the present invention provides an oligonucleotide that specifically hybridizes to the isolated nucleic acid molecule of the present invention, and wherein the oligonucleotide hybridizes to a portion of the isolated nucleic acid molecule comprising the polymorphic nucleotide position.

In a preferred embodiment of an oligonucleotide of the present invention, said oligonucleotide specifically hybridizes under high stringency conditions to the isolated nucleic acid molecule of the present invention, wherein the oligonucleotide hybridizes to the polymorphic position and wherein the oligonucleotide is between about 18 nucleotides and about 50 nucleotides in length.

In a preferred embodiment of an oligonucleotide of the present invention, a central nucleotide of the oligonucleotide specifically hybridizes with the polymorphic position of the portion of the nucleic acid molecule. In another aspect, the present invention provides a method of genetic screening comprising detecting in a nucleic acid sample the presence of a polymorphic gene wherein at least one oligonucleotide as defined above is allowed to hybridize under stringent conditions to the nucleic acid in said sample.

In a preferred embodiment, said method of genetic screening further comprises the step of amplifying a region of the gene or a portion thereof that contains the polymorphism. In a preferred embodiment of a method of genetic screening of the invention, the polymorphism is identified by a method selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis

(TGGE), Q-PCR, RT-PCR, restriction enzyme analysis and DNA array hybridization.

In another aspect, the present invention provides a kit for identifying an SNP in a polymorphic gene, comprising an oligonucleotide according to any one of claims 7-9 and packaging and instructions for characterizing the genotype of an individual with reference to a polymorphic nucleotide position in a gene selected from the group consisting of JAKl, AK3L1, DNAJC6, KCNN3, GRBlO, COG5, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "single nucleotide polymorphism", abbreviated "SNP", as used herein refers to a DNA sequence variation that involves a substitution, insertion or deletion, generally an alteration, of a single nucleotide position that occurs with a frequency >1% in the population. The term "polymorphism" as used herein refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. It should occur with a frequency >1% in the population.

The term "genotype" in the context of this invention refers to the particular allelic form of a gene, which can be defined by the particular nucleotide (s) present in a nucleic acid sequence at a particular site (s). As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single-or double- stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids).

The term "isolated", as in "isolated nucleic acid molecule", refers to material, such as a nucleic acid, which is substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. An isolated DNA molecule is a fragment of DNA that has been separated and that is no longer integrated in the genomic DNA of the organism from which it is derived. "Nucleotides" are referred to by their commonly accepted single- letter codes following IUPAC nomenclature: A (Adenine), C (Cytosine), T (Thymine), G (Guanine), U (Uracil), W (A or T), R (A or G), K (G or T), Y (C or T), S (C or G), M (A or C), B (C, G or T), H (A, C, or T), D (A, G, or T), V (A, C, or G), N (A, C, G, or T).

A "central nucleotide" refers to a nucleotide positioned essentially in the middle of a target region for hybridization or positioned essentially in the middle of a probe or primer for hybridization.

A "coding" or "encoding" sequence is the part of a gene that codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA.

The terms "hybridise" or "anneal" refer to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary nucleotides. The terms "stringency" or "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower 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 complementary target sequence hybridises to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30⁰C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60⁰C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40⁰C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O.lx SSC at 60⁰C. Hybridization procedures are well known in the art. The terms "under stringent hybridization conditions" and "high stringency conditions" are equivalent.

The term "oligonucleotide" refers to a short sequence of nucleotide monomers (usually 6 to 100 nucleotides) joined by phosphorous linkages (e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), or non- phosphorous linkages (e.g., peptide, sulfamate and others). An oligonucleotide may contain modified nucleotides having modified bases (e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2'-O-methyl ribosyl, 2'-O-methoxyethyl ribosyl, 2'-fluoro ribosyl, 2'-amino ribosyl, and the like). Oligonucleotides may be naturally- occurring or synthetic molecules of double- and single-stranded DNA and double- and single- stranded RNA with circular, branched or linear shapes and optionally including domains capable of forming stable secondary structures (e.g., stem-and-loop and loop-stem-loop structures).

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxy ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. A "pair of bi- directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification, and may be directed to the coding strand of the DNA or the complementary strand.

The term "probe" refers to a single- stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.

The probes and primers herein are selected to be "substantially" complementary (i.e. at least 65%, more preferably at least 80% perfectly complementary) to their target regions present on the different strands of each specific sequence to be amplified. It is possible to use primer sequences containing e.g. inositol residues or ambiguous bases or even primers that contain one or more mismatches when compared to the target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target DNA oligonucleotide sequences, are considered suitable for use in a method of the present invention. Sequence mismatches are also not critical when using low stringency hybridization conditions.

A "complement" or "complementary sequence" is a sequence of nucleotides, which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-paring rules. For example, the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'.

The term "gene", as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein. "Sample" is used in its broadest sense as containing nucleic acids. A sample may comprise a bodily fluid such as blood; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.

By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. The product of amplification is termed an amplicon. Methods of the invention can in principle be performed by using any nucleic acid amplification system. Amplification systems include the Polymerase Chain Reaction (PCR;U.S. 4,683,195, 4,683,202, and 4,800,159) the Ligase Chain Reaction (LCR; EP 0 320 308), Self- Sustained Sequence Replication (3SR), Strand Displacement Amplification (SDA; U.S. 5,270, 184, and 5,455,166), Transcriptional Amplification System (TAS), Q-Beta Replicase, Rolling Circle Amplification (RCA; U.S. 5,871,921), Nucleic Acid Sequence Based Amplification (NASBA), Cleavase Fragment Length Polymorphism (U.S. 5,719,028), Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acid (ICAN), Ramification-extension Amplification Method (RAM; U.S. 5,719,028 and 5,942,391) or other suitable methods for amplification of DNA.

In order to amplify DNA with a small number of mismatches to one or more of the amplification primers, an amplification reaction may be performed under conditions of reduced stringency (e.g. a PCR amplification using an annealing temperature of 38°C, or the presence of 3.5 mM MgC12). The person skilled in the art will be able to select conditions of suitable stringency. The detection of the amplification products can in principle be accomplished by any suitable method known in the art.

"DNA Array" refers to an ordered arrangement of at least two cDNAs on a substrate. At least one of the cDNAs represents a control or standard, and the other, a cDNA of diagnostic or therapeutic interest. The arrangement of two to about 40,000 cDNAs on the substrate assures that the size and signal intensity of each labeled hybridization complex, formed between each cDNA and at least one nucleic acid, is individually distinguishable.

"cDNA" refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single- stranded, represents coding and non-coding 3' or 5' sequence, and lacks introns.

"Portion" refers to any part of a nucleic acid sequence encoding a gene as defined herein used for any purpose; but especially to a fragment of said gene comprising the polymorphic nucleotide position.

In one aspect, the present invention provides a method of predicting the risk of osteoarthritis comprising detecting at least one SNP in or surrounding a specific gene. Methods for detecting a nucleotide change can utilize one or more oligonucleotide probes or primers, including, for example, an amplification primer pair, that selectively hybridize to a target polynucleotide, which contains one or more SNP positions. Oligonucleotide probes useful in practicing a method of the invention can include, for example, an oligonucleotide that is complementary to and spans a portion of the target polynucleotide, including the position of the SNP, wherein the presence of a specific nucleotide at the position (i.e., the SNP) is detected by the presence or absence of selective hybridization of the probe. Such a method can further include contacting the target polynucleotide and hybridized oligonucleotide with an endonuclease, and detecting the presence or absence of a cleavage product of the probe, depending on whether the nucleotide occurrence at the SNP site is complementary to the corresponding nucleotide of the probe. A pair of probes that specifically hybridize upstream and adjacent and downstream and adjacent to the site of the SNP, wherein one of the probes includes a nucleotide complementary to a nucleotide occurrence of the SNP, also can be used in an oligonucleotide ligation assay, wherein the presence or absence of a ligation product is indicative of the nucleotide occurrence at the SNP site. An oligonucleotide also can be useful as a primer, for example, for a primer extension reaction, wherein the product (or absence of a product) of the extension reaction is indicative of the nucleotide occurrence. In addition, a primer pair useful for amplifying a portion of the target polynucleotide including the SNP site can be useful, wherein the amplification product is examined to determine the nucleotide occurrence at the SNP site.

Where the particular nucleotide occurrence of a SNP, or nucleotide occurrences of a haplotype, is such that the nucleotide occurrence results in an amino acid change in an encoded polypeptide, the nucleotide occurrence can be identified indirectly by detecting the particular amino acid in the polypeptide. The method for determining the amino acid will depend, for example, on the structure of the polypeptide or on the position of the amino acid in the polypeptide. Where the polypeptide contains only a single occurrence of an amino acid encoded by the particular SNP, the polypeptide can be examined for the presence or absence of the amino acid. For example, where the amino acid is at or near the amino terminus or the carboxy terminus of the polypeptide, simple sequencing of the terminal amino acids can be performed. Alternatively, the polypeptide can be treated with one or more enzymes and a peptide fragment containing the amino acid position of interest can be examined, for example, by sequencing the peptide, or by detecting a particular migration of the peptide following electrophoresis. Where the particular amino acid comprises an epitope of the polypeptide, the specific binding, or absence thereof, of an antibody specific for the epitope can be detected. Other methods for detecting a particular amino acid in a polypeptide or peptide fragment thereof are well known and can be selected based, for example, on convenience or availability of equipment such as a mass spectrometer, capillary electrophoresis system, magnetic resonance imaging equipment, and the like.

Identification of the nucleotide occurrence can be performed using any method suitable for examining the particular sample. For example, wherein the sample contains nucleic acid molecules, the identification can be performed by contacting polynucleotides in (or derived from) the sample with a specific binding pair member that selectively hybridizes to a region of the polynucleotide that includes the SNP or SNPs, under conditions wherein the binding pair member specifically binds at or near the SNP(s). The binding pair member can be any molecule that specifically binds or associates with the target polynucleotide, including, for example, an antibody or an oligonucleotide.

The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The detection fragments may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.

Alternatively, the DNA fragments may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels that may be associated with nucleotide bases include e.g. fluorescein, cyanine dye or BrdUrd. When using a probe-based detection system, a suitable detection procedure for use in the present invention may for example comprise an enzyme immunoassay (EIA) format (Jacobs et al., 1997, J. Clin. Microbiol. 35, 791-795). For performing a detection by manner of the EIA procedure, either the forward or the reverse primer used in the amplification reaction may comprise a capturing group, such as a biotin group for immobilization of target DNA PCR amplicons on e.g. a streptavidin coated microtiter plate wells for subsequent EIA detection of target DNA -amplicons (see below). The skilled person will understand that other groups for immobilization of target DNA PCR amplicons in an EIA format may be employed.

Probes useful for the detection of the target DNA as disclosed herein preferably bind only to at least a part of the DNA sequence region as amplified by the DNA amplification procedure. Those of skill in the art can prepare suitable probes for detection based on the nucleotide sequence of the target DNA without undue experimentation as set out herein. Also the complementary sequences of the target DNA may suitably be used as detection probes in a method of the invention, provided that such a complementary strand is amplified in the amplification reaction employed.

Suitable detection procedures for use herein may for example comprise immobilization of the amplicons and probing the DNA sequences thereof by e.g. southern blotting. Other formats may comprise an EIA format as described above. To facilitate the detection of binding, the specific amplicon detection probes may comprise a label moiety such as a fluorophore, a chromophore, an enzyme or a radio-label, so as to facilitate monitoring of binding of the probes to the reaction product of the amplification reaction. Such labels are well-known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), β- galactosidase, horseradish peroxidase, streptavidin, biotin, digoxigenin, 35S or 1251. Other examples will be apparent to those skilled in the art. Detection may also be performed by a so called reverse line blot (RLB) assay, such as for instance described by Van den Brule et al. (2002, J. Clin. Microbiol. 40, 779-787). For this purpose RLB probes are preferably synthesized with a 5'amino group for subsequent immobilization on e.g. carboxyl-coated nylon membranes. The advantage of an RLB format is the ease of the system and its speed, thus allowing for high throughput sample processing. Any suitable method for screening the nucleic acids for the presence or absence of polymorphisms is considered to be part of the instant invention. Such methods include, but are not limited to: DNA sequencing, restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis; heteroduplex analysis, single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), real time PCR analysis (e.g. Taqman®), temperature gradient gel electrophoresis (TGGE), primer extension, allele-specific hybridization, and INVADER® genetic analysis assays, cleavase fragment length polymorphism (CFLP) analysis, sequence-characterized amplified region (SCAR) analysis, cleaved amplified polymorphic sequence (CAPS) analysis The use of nucleic acid probes for the detection of specific DNA sequences is well known in the art. Mostly these procedures comprise the hybridization of the target DNA with the probe followed by post- hybridization washings. Specificity is typically the function of post- hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Tm = 81.5 ⁰C + 16.6 (log M) + 0.41 (% GC)- 0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1°C for each 1 % of mismatching; thus, the hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with > 90% identity are sought, the Tm can be decreased 10⁰C. Generally, stringent conditions are selected to be about 5 C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1,2,3, or 4 ⁰C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6,7,8,9, or 10 ⁰C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11,12,13,14,15, or 20⁰C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 ⁰C (aqueous solution) or 32 ⁰C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hvbridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier. New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., supra.

The development of primers and probes useful for the detection of polymorphic positions in a nucleic acid is within the realm of ordinary skill (see for instance Sambrook, J., Russell D. W., Sambrook, J. (2001) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

By using standard DNA technology it is possible to produce probes and primers that directly or indirectly hybridize to the DNA samples to be tested or cDNA produced from RNA by reverse transcription, and which can be used in assays for the detection of the SNPs. Nucleic acid amplification techniques allow the amplification of fragments of nucleic acids, which may be present in very low amounts.

In order to develop nucleic acid-based detection methods, the SNP- specific sequences must be determined for which primers or probes may then be developed. To detect the SNPs by nucleic acid amplification and/or probe hybridization, the nucleic acid may be isolated from any raw sample material, optionally reverse transcribed into cDNA and directly cloned and/or sequenced. DNA and RNA isolation kits are commercially available from for instance QIAGEN GmbH, Hilden, Germany, or Roche Diagnostics, a division of F. Hoffmann-La Roche Ltd, Basel, Switzerland.

A sample useful for practicing a method of the invention can be any biological sample of a subject that contains nucleic acid molecules, including portions of the gene sequences to be examined, or corresponding encoded polypeptides, depending on the particular method. As such, the sample can be a cell, tissue or organ sample, or can be a sample of a biological fluid such as semen, saliva, blood, and the like. A nucleic acid sample useful for practicing a method of the invention will depend, in part, on whether the SNPs of the haplotype to be identified are in coding regions or in non-coding regions. Thus, where at least one of the SNPs to be identified is in a noncoding region or a SNP in LD with the discovered SNPs, the nucleic acid sample generally is a deoxyribonucleic acid (DNA) sample, particularly genomic DNA or an amplification product thereof. However, where heteronuclear ribonucleic acid (RNA), which includes unspliced mRNA precursor RNA molecules, is available, a cDNA or amplification product thereof can be used. Furthermore, while the methods of the invention generally are exemplified with respect to a nucleic acid sample, it will be recognized that particular SNPs can be in coding regions of a gene and can result in polypeptides containing different amino acids at the positions corresponding to the SNPs due to non- degenerate codon changes. As such, in another aspect, the methods of the invention can be practiced using a sample containing polypeptides of the subject. It is also possible that an SNP does not reside in a coding region, but still gives rises to changes in the encoded protein. This can for example be accomplished if the SNP lies in an intron and gives rise to a change in splicing of the mRNA, thereby introduction of an extra amino acid sequence or deletion of a part of the original amino acid sequence.

Using either the cloned nucleic acid as a hybridization probe, using sequence information derived from the clone, or by designing degenerative primers based on the sequence of the SNP and its flanking sequences, nucleic acid hybridization probes and/or nucleic acid amplification primers may be designed an used in a detection assay for detecting the characteristics of the one or more SNPs in a sample as defined herein. The DNA, or alternatively, the cDNA may be PCR amplified by using for instance Pfu and Taq DNA polymerases and amplification primers specific for the SNP DNA sequences. Also complete commercially available systems may be used for PCR (e.g. available form various suppliers such as Roche Diagnostics). A suitable method may for instance include mixing into a suitable aqueous buffering system (e.g. a commercially available PCR buffer) a suitable amount of total DNA as a template (e.g. 1 to 5 μg), a suitable amount (e.g. 10 pmol) of a pair of bidirectional amplification primers, a suitable amount of dNTPs and the DNA polymerase, denaturing the nucleic acids by boiling for 1 min, and performing a cycling reaction of around 10-50 alternating cycles of stringent primer hybridization, strand elongation and denaturing, at suitable temperatures to obtain DNA copies of the DNA template as amplification product. The amount of copies produced upon a certain number of cycles correlates directly to the amount of target DNA in the DNA template.

The skilled person is well aware of the available quantitative PCR methods presently available from commercial suppliers to quantify the amount of target DNA in the template. The term "hybridization signal" as used herein inter alia refers to the amount of amplification product produced upon a certain number of cycles and thus to the amount of target DNA available as template in the reaction.

In another aspect, the invention provides oligonucleotide probes for the detection of the SNP. The detection probes herein are selected to be "substantially" complementary to a single stranded nucleic acid molecule, or to one of the strands of the double stranded nucleic acids generated by an amplification reaction of the invention. Preferably the probes are substantially complementary to the, optionally immobilized (e.g. biotin labelled) antisense strands of the amplicons generated from the target RNA or DNA.

It is allowable for detection probes of the present invention to contain one or more mismatches to their target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target oligonucleotide sequences are considered suitable for use in a method of the present invention.

As is shown in the experimental part, several of the SNPs listed are representative for OA in a general sense, while some reach only statistic significance in OA at one specific site. Thus, the SNPs of the invention comprise the following: rs3815148 minor allele = C: increased risk OA (All OA phenotypes); rs 873598 minor allele = T: decreased risk OA (All OA phenotypes); rslO248619 minor allele = T: increased risk OA (all OA phenotypes); rs 10465850 minor allele = T: increased risk OA (especially for hand

OA); and rs 12402320 minor allele = T increased risk OA (All OA phenotypes);

An additional SNP is rs6088813 (minor allele = C, decreased risk OA (especially for the progression of knee OA).

The genes belonging to these SNPs are, respectively:

C0G5 (rs3815148) MIM: 606821 , GenBank accession no: AF058718.1, GRBlO (rslO248619) MIM: 601523, GenBank accession no: NM_005311.3, JAKl (rsl0465850) MIM: 147795, GenBank accession no: NM_002227.2, AK3L1 (rsl0465850) MIM: 103030, GenBank accession no: NM_013410.2, DNAJC6 (rsl0465850) MIM: 608375, GenBank accession no: AB007942.1, KCNN3(rsl2402320) MIM: 602983 , GenBank accession no: NM_002249.4 PRKAR2B (rs3815148) MfM: 176912, GenBank accession no: NM_002736.2, HBPl (rs3815148) GertefO; 26959, GenBank accession no: NM_012257.3, GPR22 (rs3815148) MIM: 601910, GenBank accession no: NM_005295.2, Cl0orf46 (rs873598) GeneiD: 143384 (no MIM available), GenBank accession no: NM_153810.4,

PRLHR(rs873598) MIM: 600895, GenBank accession no: NM_004248.2, and DUS4L (rs3815148) GeπelD: 11062(no MIM available), GenBank accession no: NM 181581.1 The SNPs rs3815148, rslO248619, and rl2402320 are located intronic in their respective genes, while rsl0465850 lies 347 kb upstream of the JAKl gene and rs873598 lies 68 kb upstream of the PRLHR gene.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins and proteins comprising any other modification, as well as non- glycoproteins and proteins that are otherwise unmodified.

"Protein profile", as used herein, refers to the collection of proteins, protein fragments, or peptides present in a sample. The protein profile may comprise the identities (e.g., specific names or amino acid sequence identities of known proteins, or molecular weights or other descriptive information about proteins that have not been further characterized) of the proteins in a collection, without reference to quantity present. In other embodiments, a protein profile includes quantitative information for the proteins represented in a sample.

"Quantitation", as used herein in reference to proteins in a profile refers to the determination of the amount of a particular protein or peptide present in a sample. Quantitation can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).

"Genetic Marker", "Marker" and "Biomarker" are used interchangeably to refer to an SNP of the invention or a polypeptide that is differentially present in a samples taken from two different subjects, e.g., from a test subject or patient having (a risk of developing) OA, compared to a comparable sample taken from a control subject (e.g., a subject not having (a risk of developing) OA; a normal or healthy subject). The phrase "differentially present" refers to differences in the quantity or frequency (incidence of occurrence) of a marker present in a sample taken from a test subject as compared to a control subject. For example, a marker can be a polypeptide that is present at an elevated level or at a decreased level in samples (of plaque) from risk subjects compared to samples from control subjects. Alternatively, a marker can be a polypeptide that is detected at a higher frequency or at a lower frequency in samples (of plaque) from risk subjects compared to samples from control subjects. A polypeptide is "differentially present" between two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample. For example, a polypeptide is differentially present between two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other.

As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, synthetic antibodies, human antibodies, humanized antibodies, chimeric antibodies, single- chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a polypeptide antigen encoded by a gene comprised in the genomic regions or affected by genetic transformations in the genomic regions listed in Table 1. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG₂, IgGe, IgG/t, IgAi and IgA₂) or subclass of immunoglobulin molecule.

"Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. The phrase "specifically (or selectively) binds" to an antibody or specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. The terms "affecting the expression" and "modulating the expression" of a protein or gene, as used herein, should be understood as regulating, controlling, blocking, inhibiting, stimulating, enhancing, activating, mimicking, bypassing, correcting, removing, and/or substituting said expression, in more general terms, intervening in said expression, for instance by affecting the expression of a gene encoding that protein. The terms "subject" or "patient" are used interchangeably herein and include, but are not limited to, an organism; a mammal, including, e.g., a human, non-human primate, mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; and a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non- mammalian invertebrate.

In its most simple form, a SNP of the invention can be used as a marker for detecting the presence of OA or the predisposition of developing OA or progression of OA. The SNP markers of the present invention may take the form of a single SNP, or the form of a combination of SNPs of the invention and further known SNPs that are predictive for OA. One of the latter is the SNP rs6088813 that has been described by Chapman, K. et al., Human MoI. Genet. 17:1497-1504, 2008 and

Miyamoto, Y. et al., Nature Genetics 39:529-33, 2007. Further, it is stipulated that the proteins encoded by the genes JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L (in or near which the SNPs of the present invention are located) are differentially expressed in subjects having OA or which are in risk of developing OA. The markers provided by the present invention may take the form of single protein biomarkers that are differentially present in risk subjects, or may take the form of unique expression levels or concentrations of combinations of proteins that are indicative, so-called differential protein (expression) profiles. In general terms, the biomarker comprises at least one protein.

Prognostic and diagnostic methods

In a method of the present invention for predicting the risk of a subject developing a medical condition (in the present case osteoarthritis), the marker may be detected in (a sample of) a subject, preferably by in vivo or non-invasive methods or by ex vivo methods, for instance carried out on a sample removed from the test subject. "Detecting" refers to identifying the presence, absence or amount of the marker to be detected. Detection may comprise the demonstration of the presence, in absolute terms or in relative terms (e.g., relative intensity of signals), or of the absence of the marker in (a sample of) the subject. Very suitable, in case of a proteinaceous biomarker, the amount of the biomarker relative to another protein stably present in the subject, such as a household enzyme, may be determined in order to detect the biomarker in a subject.

Non-invasive methods for detecting or measuring proteins in the body of a subject (in vivo) are well known to the artisan. Such methods may include MRI, ultrasound spectroscopy, Raman spectroscopy and/or infra red spectroscopy and generally involve the use of specific labels for detection of the proteins.

Similar methods may be employed when analysing samples for the same purpose. Suitable samples include samples from blood, circulating cells, serum, plasma, urine, saliva, etc.. However, in addition, ex vivo methods may be applied on samples that are obtained by invasive methods, and include the use of mass spectrometry, nucleic acid and protein chip array analysis, antibody array analysis, and/or immunoassay analysis for detection and/or quantification of the expression of gene products, where gene products are defined as any product expressed by the genes JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L including the proteins encoded by such genes and their splice variants and messenger RNA produced by transcription of said genes.

It will be appreciated that the step of measuring the amount of at least one gene product does not need to result in an exact determination of the concentration of mRNA or protein in said sample. It is sufficient that an expression of the amount is obtained relative to the amount present (or not present) in a control sample. Any (semi) quantitative method is suitable, as long as the measured amount can be compared with control or reference values. The present invention also provides a kit of parts for performing the methods as described above. Such kits of parts are based on the detection of the biomarker by in vivo or ex vivo methods as described above. A kit of parts of the present invention comprises a marker, or a detectable binding partner thereof, for instance a probe in the case of detection of a nucleic acid or an antibody in the case of detection of a protein, that binds specifically to the biomarker.

A kit of parts may further comprise components for validating the detection protocol, such as reference or control samples, information on the reference value for the biomarker, peptides capable of binding to the antibody and which can for instance be used in competitive ELISA assays; detectable markers, often containing a labelling moiety, for detecting binding between said biomarker and said antibody. Labelling moieties may include fluorescent, chemiluminescent, magnetic, and radioactive or other moieties suitable for detection by dedicated equipment

The measured concentration may then be compared to reference values available in a database. Such a database may have the form of a listing of SNPs or proteins, wherein to each protein is annotated a reference or threshold value below or above which the risk on the presence or risk for development of OA in a patient is increased. In order to determine the threshold value for each protein, a comprehensive study may be performed between samples from risk-patients (patients that have developed OA) and non-risk patients (that have not developed OA), e.g. such as described herein, and wherein the threshold value is the uppermost or lowest value among the non-risk patients, above which, respectively, below which the statistical chance on the occurrence of OA is significantly increased. The preparation of antibody microarray on e.g. glass slides is known to the skilled person. Antibodies may be spotted on for instance amino-reactive glass slides or other functionalized surfaces. Generally, methods are available to the skilled person to print as many as 20000 spots on a single 2.5 x 7.5 cm glass slide with individual spots being spotted about 300 μm apart. In order to allow the performance of multiple binding experiments on a single slide, a number of grids consisting of a defined group of antibodies can be spotted on one slide. The antibodies may be spotted by any available spotting technique, for instance by contact printing. Tools and technologies developed for the production of DNA microarrays, such as spotter, incubation chambers, differential fluorescent labelling techniques and imaging equipment for quantitative measurement of binding studies, are readily available to the artisan.

In order to prepare antibody microarrays, it will be appreciated that the availability of the purified proteins is not required. By using methods such as peptide immunisation, information on the protein or peptide sequences (e.g. deduced from the above-mentioned genes) is sufficient to design peptides, which upon synthesis and injection into rabbits can be used to raise antibodies. Such antibodies may be used to measure the amount of the native protein in biological samples. After affinity purification, these antibodies may then be used for the preparation of antibody arrays as described above. Procedures for the preparation of antibody arrays based on protein or peptide sequences are commercially available, for instance from Eurogentec, Seraing, Belgium.

As stated, the antibody microarrays may be used for differential protein expression studies (protein profiling). In order to measure the differential expression of proteins in a biological sample under an experimental condition and compare the expression with control samples or reference values, several methods may be used for labelling of the proteins. Very suitable, the proteins from the biological samples are labelled with one or more fluorescent probes (e.g. Cy3 and Cy5) using standard protein labelling protocols. Once the proteins of a biological sample (test and control) have been labelled (preferably differentially labelled using different colour probes for test and control), they can be brought in contact with the antibody microarray. The binding of the antigens to the antibody array may for instance be performed upon incubation of the microarray slide with a small volume (± 50 μl) of labelled biological material, under cover slips. The detection of protein bound to the antibody microarray may be based on the generation of fluorescence. Proteins that bind to the microarray may then be detected using a fluorescent scanner and individual spots of the chip can then be analysed to determine the differential expression between the test and control sample.

In alternative procedures, the antibody microarrays may be used as capturing chips for the quantification of multiple proteins in a biological sample using ELISA methods on the chip. The various proteins identified as biomarkers for assessing OA or the risk for OA as described herein may be measured more quantitatively by such procedures. To determine the concentration of a protein in a biological sample, ELISA techniques are very suitable. Such techniques involve the production of a calibration curve of the fluorescence intensity vs. protein concentration, or the use of a competitive ELISA format, wherein known amounts of unlabelled protein or antigen are provided in the test. When using methods such as peptide immunisation for the preparation of an antibody microarray as described above, the peptides used for immunisation may be used in competitive ELISA experiments on the microarray. Alternatively, multiple sandwich ELISA can be developed using as second antibody, for instance an antibody raised by peptide immunisation against a second epitope of the target protein (a second synthetic peptide). In yet another aspect, the present invention provides the use of a biomarker as defined herein above for predicting the risk of developing OA in a subject. Such use involves either the mere detection of the biomarker in (a sample of) the patient, or the determination whether the amount of biomarker detected is above or below the reference value. Therapeutic methods

In yet another aspect, the present invention provides a method of treating a subject having OA or having an increased risk of developing OA, said method comprising using a proteinaceous marker as defined herein above as a therapeutic target or as a therapeutic agent. Preferably, said use of said protein as a therapeutic target comprises decreasing the amount of at least one protein that is over-expressed in subjects having OA or having an increased risk of developing OA, or increasing the amount of at least one protein that is under-expressed in subjects having OA or having an increased risk of developing OA.

Preferably, said use of said protein as a therapeutic agent comprises increasing the amount of at least one protein that is under- expressed in subjects having OA or having an increased risk of developing OA, and involves administrating said protein to said subject. The present invention also relates to the use of the proteins of the present invention as therapeutic targets. Pharmacogenetics and pharmacogenomics aim at determining the genetic determinants linked to diseases. Most of the diseases are multigenic diseases, and the identification of the genes involved therein should allow for the discovery of new targets and the development of new drugs.

Many physiological diseases are targeted by this novel pharmaceutical approach. One can name the autoimmune and inflammatory diseases, for example Addison's disease, Alopecia Areata, Ankylosing Spondylitis, Behcet's Disease, Chronic Fatigue Syndrome, Crohn's disease, Ulcerative Colitis, Inflammatory Bowel disease, Diabetes and Multiple Sclerosis.

Also osteoarthritis is viewed as a multigenic disease. The SNPs of the present invention have been identified as genetic markers for presence of or predisposition of (progression of) the disease. The identification of these SNPs and their associated genes and gene products provides better information of the patient and allows for the prevention of the development of the disease itself and an improved health, resulting in less disability.

Knowledge of the identity of genes involved in the development and etiology of OA therefore greatly facilitates the development of prophylactic, therapeutic and diagnostic methods for this disease. Diagnosis of the genes responsible for the risk phenotype in a certain subject allows for the design of therapies comprising the use of specific drugs, for instance, drugs directed against the proteins encoded by these genes. It is an aspect of the present invention to use the genetic markers of the present invention and/or the genes associated therewith and/or the proteins encoded by those for the development of inhibitors directed against the genes and/or their expression products (RNA or protein), in particular in the case of over-expression of the biomarker in the subject at risk.

In one embodiment of this aspect, the inhibitors are antibodies and/or antibody derivatives directed against the expression products of said genes. Therapeutic antibodies are for instance useful against gene expression products located on the cellular membrane and can be comprised in a pharmaceutical composition. Also, antibodies may be targeted to intracellular, e.g. cytoplasmic, gene products such as RNA's, polypeptides or enzymes, in order to modulate the activity of these products. Preferably, such antibodies are in the form of intrabodies, produced inside a target cell. In addition, antibodies may be used for deliverance of at least one toxic compound linked thereto to a target cell.

In a preferred embodiment of the present invention, the inhibitor is a small molecule capable of modulating the activity or interfering with the function of the protein expression product of the genes of the invention. In addition, small molecules can also be used for deliverance of at least one linked toxic compound to the target cell.

On a different level of inhibition, nucleic acids can be used to block the production of proteins by destroying the mRNA transcribed from said gene(s). This can be achieved by antisense drugs, ribozymes or by RNA interference (RNAi). By acting at an early stage in the disease process, these drugs are able to prevent the development of the disease. The present invention relates to antisense drugs, such as antisense RNA and antisense oligodeoxynucleotides, ribozymes and RNAi molecules, directed against the genes harbouring or encoding the biomarkers. The expression level of a gene can either be decreased or increased in a risk phenotype. Naturally, inhibitors are used when the expression levels are elevated. However, the present invention also provides for "enhancers", to boost the expression level of a gene harbouring or encoding the biomarkers associated with a risk of suffering developing OA and of which the expression levels are reduced in a risk situation. "Enhancers" may be any chemical or biological compound known or found to increase the expression level of genes, to improve the function of an expression product of a gene or to improve or restore the expression of a gene.

Very suitable therapies to overcome reduced expression levels of a gene or to restore the expression of a gene as disclosed herein include the replacement by gene therapy of the gene or its regulatory sequences that drive the expression of said gene. The invention therefore relates further to gene therapy, in which a dysfunctional gene of a subject encoding the biomarkers or a dysfunctional regulatory sequence of a gene of a subject encoding a biomarker is replaced by a functional counterpart, e.g. by stable integration of for instance a lentiviral vector comprising a functional gene or regulatory sequence into the genome of a subject's host cell which is a progenitor cell of the target cell-line of the subject and grafting of said transfected host cell into said subject. Especially the invention relates to gene therapy of the region in which the SNP of the invention is located by a sequence that is not associated with the risk for OA.

The invention also relates to forms of gene therapy, in which the genes encoding the biomarker are i.a. used for the design of dominant- negative forms of these genes which inhibit the function of their wild- type counterparts following their directed expression from a suitable vector in a target cell.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of patients having OA or having an increased risk of developing OA comprising one or more of the inhibitors, "enhancers", replacement compounds, vectors or host cells according to the present invention as a pharmaceutical reagent or active ingredient. The composition can further comprise at least one pharmaceutical acceptable additive like for example a carrier, an emulsifier, or a conservative.

In addition, it is the object of the present invention to provide a method for treatment of subjects suffering from OA or from an increased risk of developing OA/or progression of OA which method comprises the administration of the pharmaceutical composition according to the invention to patients in need thereof in a therapeutically effective amount.

Small molecule inhibitors

Small molecule inhibitors are usually chemical entities that can be obtained by screening of already existing libraries of compounds or by designing compounds based on the structure of the protein encoded by a gene associated with, harboring or encoding a biomarker of the present invention. Briefly, the structure of at least a fragment of the protein is determined by either Nuclear Magnetic Resonance or X-ray crystallography. Based on this structure, a virtual screening of compounds is performed. The selected compounds are synthesized using medicinal and/or combinatorial chemistry and thereafter analyzed for their inhibitory effect on the protein in vitro and in vivo. This step can be repeated until a compound is selected with the desired inhibitory effect. After optimization of the compound, its toxicity profile and efficacy as therapeutic is tested in vivo using appropriate animal model systems. Differentially expressed genes that do not encode membrane- bound proteins are selected as targets for the development of small molecule inhibitors. To identify putative binding sites or pockets for small molecules on the surface of the target proteins, the three-dimensional structure of those targets are determined by standard crystallization techniques (de Vos et al. 1988, Science 239:888-93; Williams et al. 2001, Nat Struct Biol 8:838-42). Additional mutational analysis may be performed to confirm the functional importance of the identified binding sites. Subsequently, Cerius2 (Molecular Simulations Inc., San Diego, CA, USA) and Ludi/ACD (Accelrys Inc., San Diego, CA, USA) software is used for virtual screening of small molecule libraries (Bohm. 1992 J Comp Aided Molec Design 6:61-78). The compounds identified as potential binders by these programs are synthesized by combinatorial chemistry and screened for binding affinity to the targets as well as for their inhibitory capacities of the target protein's function by standard in vitro and in vivo assays. In addition to the rational development of novel small molecules, existing small molecule compound libraries are screened using these assays to generate lead compounds. Lead compounds identified are subsequently co-crystallized with the target to obtain information on how the binding of the small molecule can be improved (Zeslawska et al. 2000, J MoI Biol 301:465-75). Based on these findings, novel compounds are designed, synthesized, tested, and co-crystallized. This optimization process is repeated for several rounds leading to the development of a high-affinity compound of the invention that successfully inhibits the function of its target protein. Finally, the toxicity of the compound is tested using standard assays (commercially available service via MDS Pharma Services, Montreal, Quebec, Canada) after which it is screened in an animal model system. Ribozymes

Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules possessing endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target message must contain a specific nucleotide sequence. They are engineered to cleave any RNA species site- specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression. Importantly, ribozymes can be used to inhibit expression of a gene of unknown function for the purpose of determining its function in an in vitro or in vivo context, by detecting the phenotypic effect.

One commonly used ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal. Design of the hammerhead ribozyme is disclosed in Usman et al., Current Opin. Struct. Biol. (1996) 6:527-533. Usman also discusses the therapeutic uses of ribozymes. Ribozymes can also be prepared and used as described in Long et al., FASEB J. (1993) 7:25; Symons, Ann. Rev. Biochem. (1992) 61:641; Perrotta et al., Biochem. (1992) 31:16-17; Ojwang et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:10802-10806; and U.S. Pat. No. 5,254,678. Ribozyme cleavage of HIV-I RNA is described in U.S. Pat. No. 5,144,019; methods of cleaving RNA using ribozymes is described in U.S. Pat. No. 5,116,742; and methods for increasing the specificity of ribozymes are described in U.S. Pat. No. 5,225,337 and Koizumi et al., Nucleic Acid Res. (1989) 17:7059- 7071. Preparation and use of ribozyme fragments in a hammerhead structure are also described by Koizumi et al., Nucleic Acids Res. (1989) 17:7059-7071. Preparation and use of ribozyme fragments in a hairpin structure are described by Chowrira and Burke, Nucleic Acids Res. (1992) 20:2835. Ribozymes can also be made by rolling transcription as described in Daubendiek and Kool, Nat. Biotechnol. (1997) 15(3):273-277.

The hybridizing region of the ribozyme may be modified or may be prepared as a branched structure as described in Horn and Urdea,

Nucleic Acids Res. (1989) 17:6959-67. The basic structure of the ribozymes may also be chemically altered in ways familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units. In a therapeutic context, liposome mediated delivery of ribozymes improves cellular uptake, as described in Birikh et al., Eur. J. Biochem. (1997) 245:1-16. Therapeutic and functional genomic applications of ribozymes proceed beginning with knowledge of a portion of the coding sequence of the gene to be inhibited. Thus, for many genes, a nucleic acid sequence provides adequate sequence for constructing an effective ribozyme. A target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5' and 3' nucleotide sequences that flank the cleavage site. Retroviral vectors are engineered to express monomeric and multimeric hammerhead ribozymes targeting the mRNA of the target coding sequence. These monomeric and multimeric ribozymes are tested in vitro for an ability to cleave the target mRNA. A cell line is stably transduced with the retroviral vectors expressing the ribozymes, and the transduction is confirmed by Northern blot analysis and reverse- transcription polymerase chain reaction (RT-PCR). The cells are screened for inactivation of the target mRNA by such indicators as reduction of expression of disease markers or reduction of the gene product of the target mRNA.

Antisense

Antisense polynucleotides are designed to specifically bind to

RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected sequence can interfere with expression of the corresponding gene.

Antisense polynucleotides are typically generated within the cell by expression from antisense constructs that contain the antisense strand as the transcribed strand. Antisense polynucleotides will bind and/or interfere with the translation of the corresponding mRNA. As such, antisense may be used therapeutically to inhibit the expression of the genes associated with, harbouring or encoding the biomarkers of the invention.

Antisense RNA or antisense oligodeoxynucleotides (antisense ODNs) can both be used and may also be prepared in vitro synthetically or by means of recombinant DNA techniques. Both methods are well within the reach of the person skilled in the art. ODNs are smaller than complete antisense RNAs and have therefore the advantage that they can more easily enter the target cell. In order to avoid their digestion by DNAse, ODNs and antisense RNAs may be chemically modified. For targeting to spefically desired target cells, the molecules may be linked to ligands of receptors found on the target cells or to antibodies directed against molecules on the surface of the target cells.

RNAi RNAi refers to the introduction of homologous double stranded

RNA to specifically target the transcription product of a gene, resulting in a null or hypomorphic phenotype. RNA interference requires an initiation step and an effector step. In the first step, input double-stranded (ds) RNA is processed into nucleotide 'guide sequences'. These may be single- or double- stranded. The guide RNAs are incorporated into a nuclease complex, called the RNA-induced silencing complex (RISC), which acts in the second effector step to destroy mRNAs that are recognized by the guide RNAs through base-pairing interactions. RNAi molecules are thus double stranded RNAs (dsRNAs) that are very potent in silencing the expression of the target gene. The invention provides dsRNAs complementary to the genes associated with, harboring or encoding the biomarkers of the present invention.

The ability of dsRNA to suppress the expression of a gene corresponding to its own sequence is also called post-transcriptional gene silencing or PTGS. The only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded mRNA. If the cell finds molecules of double-stranded RNA, dsRNA, it uses an enzyme to cut them into fragments containing in general 21-base pairs (about 2 turns of a double helix). The two strands of each fragment then separate enough to expose the antisense strand so that it can bind to the complementary sense sequence on a molecule of mRNA. This triggers cutting the mRNA in that region thus destroying its ability to be translated into a polypeptide. Introducing dsRNA corresponding to a particular gene will knock out the cell's endogenous expression of that gene. This can be done in particular tissues at a chosen time. A possible disadvantage of simply introducing dsRNA fragments into a cell is that gene expression is only temporarily reduced. However, a more permanent solution is provided by introducing into the cells a DNA vector that can continuously synthesize a dsRNA corresponding to the gene to be suppressed.

RNAi molecules are prepared by methods well known to the person skilled in the art. In general an isolated nucleic acid sequence comprising a nucleotide sequence which is substantially homologous to the sequence of at least one of the genes associated with, harbouring or encoding the biomarkers of the invention and which is capable of forming one or more transcripts able to form a partially of fully double stranded (ds) RNA with (part of) the transcription product of said genes will function as an RNAi molecule. The double stranded region may be in the order of between 10-250, preferably 10-100, more preferably 20-50 nucleotides in length.

RNAi molecules are preferably expressed from recombinant vectors in transduced host cells.

Dominant Negative Mutations

Dominant negative mutations are readily generated for corresponding proteins that are active as multimers. A mutant polypeptide will interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. Thus, a mutation is in a substrate- binding domain, a catalytic domain, or a cellular localization domain. Preferably, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect. In addition, fusion of different polypeptides of various lengths to the terminus of a protein can yield dominant negative mutants. General strategies are available for making dominant negative mutants. See Herskowitz, Nature (1987) 329:219-222. Such a technique can be used for creating a loss of function mutation, which is useful for determining the function of a protein.

Use of Polypeptides to raise Antibodies

The present invention provides in one aspect for antibodies suitable for therapeutic and/or diagnostic use.

Therapeutic antibodies include antibodies that can bind specifically to the expression products of the genes encoding the biomarkers of the invention. By binding directly to the gene products, the antibodies may influence the function of their targets by, for example, in the case of proteins, steric hindrance, or by blocking at least one of the functional domains of those proteins. As such, these antibodies may be used as inhibitors of the function of the gene product. Such antibodies may for instance be generated against functionally relevant domains of the proteins and subsequently screened for their ability to interfere with the target's function using standard techniques and assays (see for instance Schwartzberg, 2001, Crit Rev Oncol Hematol 40:17-24; Herbst et al. Cancer 94:1593-611, 2002).

Alternatively, anti-RNA antibodies may for instance be useful in silencing messengers of the tumor-related genes of the present invention. In another alternative, antibodies may also be used to influence the function of their targets indirectly, for instance by binding to members of signaling pathways in order to influence the function of the targeted proteins or nucleic acids. In yet another alternative, therapeutic antibodies may carry one or more toxic compounds that exert their effect on the target or target cell by virtue of the binding of the carrying antibody thereto. For diagnostic purposes, antibodies similar to those above, preferably those that are capable of binding to the expression products of the genes of the present invention may be used, and that are provided with detectable labels such as fluorescent, luminescent, or radio-isotope labels in order to allow the detection of the gene product. Said antibodies can be targeted to intracellular proteins or even nucleic acids, but preferably such diagnostic antibodies are targeted to proteinaceous targets present on the outer envelop of the cell, such as membrane bound target proteins. The antibodies used in the present invention may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably, the antibodies of the invention are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries (including, but not limited to, synthetic libraries of immunoglobulin sequences homologous to human immunoglobulin sequences) or from mice that express antibodies from human genes. For some uses, including in vivo therapeutic or diagnostic use of antibodies in humans and in vitro detection assays, it may be preferred to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences or synthetic sequences homologous to human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893 and WO98/16654, each of which is incorporated herein by reference in its entirety.

The antibodies to be used with the methods of the invention include derivatives that are modified, i. e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment. Additionally, the derivative may contain one or more non-classical amino acids.

In certain embodiments of the invention, the antibodies to be used with the invention have extended half-lives in a mammal, preferably a human, when compared to unmodified antibodies. Antibodies or antigen- binding fragments thereof having increased in vivo half- lives can be generated by techniques known to those of skill in the art (see, e.g., PCT Publication No. WO 97/34631). In certain embodiments, antibodies to be used with the methods of the invention are single-chain antibodies. The design and construction of a single-chain antibody is described in Marasco et al, 1993, Proc Natl Acad Sci 90:7889-7893, which is incorporated herein by reference in its entirety. In certain embodiments, the antibodies to be used with the invention bind to an intracellular epitope, i.e., are intrabodies. An intrabody comprises at least a portion of an antibody that is capable of immunospecifically binding an antigen and preferably does not contain sequences coding for its secretion. Such antibodies will bind its antigen intracellularly. In one embodiment, the intrabody comprises a single-chain Fv ("sFv"). For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer - Verlag, New York, pp. 269-315 (1994). In a further embodiment, the intrabody preferably does not encode an operable secretory sequence and thus remains within the cell (see generally Marasco, WA, 1998,

"Intrabodies: Basic Research and Clinical Gene Therapy Applications" Springer: New York).

Generation of intrabodies is well known to the skilled artisan and is described for example in U.S. Patent Nos. 6,004,940; 6,072,036; 5,965,371, which are incorporated by reference in their entireties herein.

In one embodiment, intrabodies are expressed in the cytoplasm. In other embodiments, the intrabodies are localized to various intracellular locations. In such embodiments, specific localization sequences can be attached to the intranucleotide polypeptide to direct the intrabody to a specific location.

The antibodies to be used with the methods of the invention or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in WO97/13844; and U.S. Patent Nos. 5,580,717, 5,821,047, 5,571,698, 5,780,225, and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869 and Better et al., 1988, Science 240:1041-1043.

It is also possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of the technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication No. WO 98/24893, which is incorporated by reference herein in its entirety. In addition, companies such as Medarex, Inc. (Princeton, NJ), Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Recombinant expression used to produce the antibodies, derivatives or analogs thereof (e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody and the expression of said vector in a suitable host cell or even in vivo. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods, which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. A variety of host-expression vector systems may be utilized to express the antibody molecules as defined herein

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544). Once an antibody molecule to be used with the methods of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. As stated above, according to a further aspect, the invention provides an antibody as defined above for use in therapy.

For therapeutic treatment, antibodies may be produced in vitro and applied to the subject in need thereof. The antibodies may be administered to a subject by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route and in a dosage which is effective for the intended treatment. Therapeutically effective dosages of the antibodies required for decreasing the rate of progress of the disease or for eliminating the disease condition can easily be determined by the skilled person. Alternatively, antibodies may be produced by the subject itself by using in vivo antibody production methodologies as described above. Suitably, the vector used for such in vivo production is a viral vector, preferably a viral vector with a target cell selectivity for specific target cell referred to herein.

Therefore, according to a still further aspect, the invention provides the use of an antibody as defined above in the manufacture of a medicament for use in the treatment of a subject to achieve the said therapeutic effect. The treatment comprises the administration of the medicament in a dose sufficient to achieve the desired therapeutic effect. The treatment may comprise the repeated administration of the antibody. According to a still further aspect, the invention provides a method of treatment of a human comprising the administration of an antibody as defined above in a dose sufficient to achieve the desired therapeutic effect, wherein the therapeutic effect is the alleviation or prevention of OA and/or the risk of developing OA.

The diagnostic and therapeutic antibodies are preferably used in their respective application for the targeting of kinases or phosphatases, which are often coupled to receptor molecules on the cell's surface. As such, antibodies capable of binding to these receptor molecules can exert their activity-modulating effect on the kinases or phosphatases by binding to the respective receptors. Also transporter proteins may be targeted with advantage for the same reason that the antibodies will be able to exert their activity-modulating effect when present extracellularly. The above targets, together with signalling molecules, represent preferred targets for the antibody uses of the invention as more effective therapy and easier diagnosis is possibly thereby. The diagnostic antibodies can suitably be used for the qualitative and quantitative detection of gene products, preferably proteins in assays for the determination of altered levels of proteins or structural changes therein. Protein levels may for instance be determined in cells, in cell extracts, in supernatants, body fluids by for instance flow- cytometric evaluation of immunostained target cells. Alternatively, quantitative protein assays such as ELISA or RIA, Western blotting, and imaging technology (e.g., using confocal laser scanning microscopy) may be used in concert with the antibodies as described herein for the diagnosis of osteoarthritis.

Pharmaceutical Compositions and Therapeutic Uses Pharmaceutical compositions can comprise polypeptides, antibodies, polynucleotides (antisense, RNAi, ribozyme), or small molecules of the claimed invention, collectively called inhibitor compounds herein. The pharmaceutical compositions will comprise a therapeutically effective amount of either a biomarker protein, an antibody, a polynucleotides or small molecules as described herein.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as pain or loss of cartilage tissue. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician. Specifically, the compositions of the present invention can be used to treat, ameliorate, or prevent the occurrence of an osteoarthritic event in a subject and/or accompanying biological or physical manifestations.

For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the polynucleotide, polypeptide or antibody compositions in the individual to which it is administered. A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the pharmaceutical compositions of the invention can be (1) administered directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) delivered in vitro for expression of recombinant proteins. Direct delivery of the compositions could be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a joint. Other modes of administration include topical, oral, catheterized and pulmonary administration, suppositories, and transdermal applications, needles, and particle guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly lymph cells, macrophages, or dendritic cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Various methods are used to administer the therapeutic composition directly to a specific site in the body. For example, a diseased joint is located and the therapeutic composition injected several times in several different locations within said joint. Alternatively, arteries which serve a joint are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the joint tissues. X-ray imaging may be used to assist in certain of the above delivery methods. Receptor- mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues is also used. Receptor- mediated DNA delivery techniques are described in, for example, Findeis et al., Trends in Biotechnol. (1993) 11:202-205; Chiou et al., (1994) Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.); Wu et al., J. Biol. Chem. (1994) 269:542-46. Preferably, receptor-mediated targeted delivery of therapeutic compositions containing antibodies of the invention is used to deliver the antibodies to specific tissue.

Pharmaceutical compositions containing antisense, ribozyme or RNAi polynucleotides are administered in a range of about 100 ng to about 200 mg of polynucleotides for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of polynucleotides can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a joint, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Ser. No. 08/869,309, which is expressly incorporated herein.

A further aspect of the present invention is a kit-of-parts for use in a method for detecting a polymorphic gene as defined herein in a sample, said kit comprising at least one pair of bidirectional oligonucleotide primers as described herein above, or at least one oligonucleotide probe described herein above; and one of the following: an instruction for performing a method of the present invention, specific packaging material for collecting or storing nucleic acid samples, and a reagent for performing a nucleic acid amplification or hybridization reaction.

EXAMPLES

Genome-Wide Association Study (GWAS) Recently, a new and powerful technique has become available to search the whole human genome for OA risk alleles: a Genome- Wide Association Study (GWAS). This GWAS consists of screening the genome of hundreds to thousands of subjects in a case-control study or population-based cohort study, with >500.000 Single Nucleotide Polymorphisms (SNPs) and subsequently perform an association analysis between the phenotype of interest, in this case OA, and all the genetic markers. Because of the multiplicity of testing with so many markers, certain thresholds have been considered to declare an association "genome-wide significant" (gws). For example, when analyzing 500.000 markers the threshold for a p-value to be considered gws is 5xlO ⁸.

Usually, a typical GWAS consists of a discovery sample with GWA data and a replication sample with either GWA data or that can be genotyped for the particular genetic markers identified in the discovery sample. In this GWAS we used the women of the baseline cohort of the Rotterdam Study (Hofman, A. et al., Eur. J. Epidemiol. 22:819-29, 2007), which were genotyped with the Illumina HumanHap550 Genotyping BeadChip. We genotyped 550.000 SNPs in 644-1659 women, depending on the OA outcome (Table 1), of the Rotterdam Study using Illumina HumanHap 550v3 (Richards, J.B. et al., Lancet 371:1505-12, 2008). After Quality control 535.354 SNPs remained for analyses. Using PLINKvI.01 software (http://pngu.mgh.harvard.edu/purcell/plink/ and Purcell S. et al., Am. J. Hum. Genet. 81:559-75, 2007), we analyzed the association of these SNPs with hip-, knee- and hand-OA and CTX-II levels. In Table 1, the number of women used in each analysis and the genomic inflation factors for each analysis are depicted.

A genomic inflation factor >=1.05 indicates that there might be population stratification present in the study (e.g., there is an admixture of ethnic groups such as Caucasians mixed with Africans). However, for all our analysis the genomic inflation factor was <1.02, indicating no evidence of population stratification Table 1. Number of women in OA GWA + genomic inflation factors

OA Phenotype Nr. Women in GWA Genomic Inflation Factor

In stage 2, SNPs were selected if the following criteria applied:

1. Hand- AND knee- AND hip- OA AND CTX-II (a marker of cartilage breakdown): p-value <= 0.05.

2. One radiographic OA phenotype <= 1.0E-4 AND one other radiographic OA phenotype <= 0.05.

Following this approach, we identified 18 SNPs in 12 loci to be associated with 2 or more OA phenotypes. In Table 2 these 18 SNPs are listed with the p-values for all OA phenotypes and the effect sizes. These 12 loci are not all located within a gene (see Table 3), but even if a SNP is located near a gene, it does not necessarily mean that that particular gene is the causal gene. For example, the SNP could be in Linkage Disequilibrium (LD) with another SNP in a gene 100 kilobases (kb) away. Therefore, all genes or nearby genes mentioned in Table 2 could be involved in osteoarthritis, but also other genes in the surrounding 200kb region of each of the 18 SNPs could be involved. The Growth Differentiation Factor 5 (GDF-5) gene has been reported to be involved in several developmental processes, including chondrogenesis, skeletogenesis and development of joints (Francis-West, P.H. et al., Development 126:1305-15, 1999), adding plausibility for this gene to be functionally involved in osteoarthritis. However, this gene is already found to be involved in osteoarthritis in a few studies now (Miyamoto Y. et al., Nature Genet. 4:529-33, 2007; Chapman, K. et al., Hum. MoI. Genet. 17:1497-1504, 2008) and therefore we do not claim this gene to be newly discovered by our research group.

The other genes mentioned in Table 2 are potentially novel genes involved in OA and have never been reported before by other research groups. Table 2. GWA results on OA: 18 tophits in 12 loci for women of the Rotterdam Study.

Rs-number locus chrom bp-position HWE MAF annotation p-knee p-hip p-hand OR(range) p-ctxll beta CTXII

?føvM i(κ ', Gene Location rs10465850 1 1 65471791 0.86 0.46 JAK1 -347kb 0.009 0.018 0.014 1 .2-1.3 0.018 0.03 rs12402320 2 1 152968030 0.98 0.30 KCN N3 intronic 0.031 0.042 0.014 1.2-1 .2 0.013 0.03 rs4656364 3 1 160545479 0.68 0.10 NOS1 AP intronic 0.040 9x10⁶ >0.05 1.3-1 .9 >0.05 0.01 rs3963342 4 4 43995005 1.00 0.33 KCTD8 intronic 0.040 0.024 0.032 1.2-1 .3 0.035 0.02 rs10248619 5 7 50718584 0.29 0.21 GRB10 intronic 0.002 0.032 0.036 1.3-1 .3 0.01 1 0.04 rs7791286 50824286 0.14 0.17 GRB10 intronic 0.024 0.018 0.010 1.3-1 .3 0.031 0.03 rs3815148 106725656 0.54 0.23 COG5 intronic 7x10 ⁵ >0.05 0.048 1.2-1 .4 >0.05 -1 E-04 rs1548524 6 7 106731799 0.84 0.24 COG5 intronic 9x10 ⁵ >0.05 0.039 1.2-1 .4 >0.05 1 E-04 rs997311 106740269 0.94 0.24 COG5 intronic 8x10 ⁵ >0.05 0.032 1.2-1 .4 >0.05 1 E-04 rs959396 7 9 37001289 0.97 0.38 PAX5 intronic 3x10 ⁶ 0.038 >0.05 0.7-0.8 >0.05 -0.02 rs12352822 8 9 1 19587103 0.32 0.15 TLR4 -69kb 2x10 ⁵ 0.001 >0.05 0.6-0.6 >0.05 -0.02 rs873598 9 10 120413174 0.93 0.45 GPR10 -68kb 0.002 0.004 0.007 0.8-0.8 0.024 -0.02 rs880844 107486497 0.70 0.24 ISCU intronic 0.020 3x10⁵ >0.05 1.2-1 .6 >0.05 0.02

10 12 rs741542 107488184 0.70 0.24 ISCU 3downstream 0.021 3x10⁵ >0.05 1.2-1 .6 >0.05 0.02 rs11651351 11 17 71478457 0.12 0.07 ACOX1 intronic 0.020 0.017 0.027 0.6-0.6 0.012 -0.05

rs4911494 33435328 0.67 0.40 GDF5 coding/intron 0.025 >0.05 2x10⁵ 0.7-0.8 >0.05 -0.009 rs6088813 12 20 33438595 0.67 0.40 GDF5 intronic 0.024 >0.05 1 x10⁵ 0.7-0.8 >0.05 -0.009 rs6087705 33464664 0.65 0.40 GDF5 5upstream 0.025 >0.05 1 x10⁵ 0.7-0.8 >0.05 -0.009 chrom = chromosome; OR(range) = Odds Ratio range for hip OA, knee OA and hand OA; HWE = Hardy- Weinberg equilibrium; MAF = Minor Allele Frequency.

The 18 top-hits identified in the GWA-analysis for OA were tested for other bone parameters and height to discover potential pleiotropic effects of these DNA sequence variations. Those phenotypes are: height, vertebral fracture, non-vertebral fracture, osteoporotic fracture, femoral neck bone mineral density (FN-BMD) and lumbar spine bone mineral density (LS- BMD), see Table 3. The rsl0465850 SNP is borderline significantly associated with vertebral fracture and LS-BMD. The 12402320 SNP is borderline significantly associated with non-vertebral fracture, osteoporotic fracture and LS-BMD. The rslO248619 and rs7791286 SNPs are (borderline) significantly associated with vertebral fracture, non- vertebral fracture and osteoporotic fracture. The rs3815148, rsl548524 and rs997311 SNPs are significantly associated with height and borderline significant with FN-BMD. The rs959396 SNP is associated with FN-BMD and LS-BMD.

Table 3. Association of 18 OA-tophits with bone parameters/height in the Rotterdam Study.

Rs-number p-height (beta) p-vert fx (OR) p-non vert fx (OR) p-OP fx (OR) p-FN BMD (beta) p-LS BMD (beta)

HowA iaci rs10465850 0.57(0.01) 0.12(0.85) 0.70(0.98) 0.74(0.98) 0.87(0.0006) 0.05(0.009)

5 rs12402320 0.93(0.002) 0.90(0.99) 0.16(1.09) 0.08(1.12) 0.33(0.004) 0.09(0.009) rs4656364 0.05(0.08) 0.79(1.05) 0.33(0.90) 0.22(0.88) 0.69(0.002) 0.67(0.003) rs3963342 0.96(0.001) 0.59(1.06) 0.41 (0.95) 0.35(0.94) 0.19(0.005) 0.28(0.003) rs10248619* 0.69 (-0.01) 0.06(1.27) 0.07(1.14) 0.13(1.12) 0.91 (-0.0005) 0.40 (-0.0005) rs7791286* 0.62 (-0.02) 0.05 (1.29) 0.13(1.12) 0.05 (1.17) 0.46 (-0.003) 0.74 (-0.002)

10 rs3815148 0.03 (-0.06) 0.37 (1.12) 0.89(0.99) 0.84 (0.99) 0.06 (-0.008) 0.69 (-0.002) rs1548524 0.04 (-0.06) 0.40 (1.11) 0.86(0.99) 0.82 (0.98) 0.05 (-0.008) 0.73 (-0.001) rs997311 0.05 (-0.06) 0.38 (1.11) 0.96(1.00) 0.89 (0.99) 0.05 (-0.008) 0.73 (-0.001) rs959396 0.93(0.002) 0.79 (0.97) 0.28(1.07) 0.62 (1.03) 0.06 (-0.007) 0.12 (-0.008)

-|κ rs12352822 0.23 (-0.04) 0.97 (1.01) 0.60(1.04) 0.58 (1.05) 0.40 (-0.004) 0.94 (-0.0004) rs873598 0.15(0.04) 0.75 (1.04) 0.80(1.02) 0.42 (0.95) 0.89 (-0.0005) 0.67 (-0.002) rs880844 0.34(0.03) 0.72 (0.96) 0.88(1.01) 0.51 (0.95) 0.31 (0.004) 0.42(0.005) rs741542 0.34(0.03) 0.72 (0.96) 0.89(1.01) 0.51 (0.95) 0.31 (0.004) 0.41 (0.005) rs11651351 0.38 (-0.04) 0.71 (1.08) 0.66(0.95) 0.62 (0.94) 0.35(0.007) 0.92(0.001)

20 Known Lea rs4911494 6x10^"5(0.10) 0.75(1.04) 0.005(1.18) 0.03(1.14) 0.22(0.005) 0.49 (-0.003) rs6088813 7x10^"5(0.10) 0.74(1.04) 0.005(1.18) 0.03(1.14) 0.23(0.004) 0.48 (-0.003) rs6087705 7x10^"5(0.10) 0.74(1.04) 0.005(1.18) 0.03(1.14) 0.23(0.004) 0.499-0.003)

25 OR = odds ratio; p-vert fx = p-value for vertebral fracture; p-non vert fx = p-value non vertebral fracture; p-OP fx = p-value osteoporotic fracture; p-FN BMD = p-value femoral neck bone mineral density; p-LS BMD = p-value lumbar spine bone mineral density.

The top-hits following this search strategy have been replicated within men and women of at least 8 studies of the TREAT-OA consortium (www.TreatOA.eu), which is a recently formed European Consortium to explore the genetics of OA. Of these studies, one study, deCODE has GWA data, the other studies do have DNA available and genetic markers have been genotyped within these studies using the Sequenom system (www.mysequenom.com). From these studies the following results were obtained:

Total population:

Hand OA: OR 1.13 p = 3x10-5 Knee OA: OR 1.19 p = 1x10-7 Hip OA: OR 1.06 p = 0.07 Li^IiI^il^

Total population:

Hand OA: OR 0.95 p = 0.07 Knee OA: OR 0.94 p = 0.05 Hip OA: OR 0.95 p = 0.06

Total population:

Hand OA: OR 1.25 p = 0.003 Knee OA: OR: 1.19 p = 0.03 Hip OA: OR 1.19 p = 0.04

Total population Female population Hand OA: OR 1.04 p = 0.09 OR 1.06 p = 0.04

Total population Hand OA: OR: 1.17 p = 0.001 Hip OA: OR 1.07 p = 0.11 Knee OA: OR 1.05 p = 0.21

Claims

1. A method for the detection of osteoarthritis (OA) or for detection of the risk for developing OA or progression of OA comprising detecting the presence of one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith.

2. A method according to claim 1, wherein rs3815148 is specific for

OA of the hand and the knee and the hip, femoral neck bone mineral density and the height of the subject.

3. A method according to claim 1, wherein rs873598 is specific for OA of the knee, hip and hand.

4. A method according to claim 1, wherein rsl0465850 is specific for OA of the hand and for lumbar spine bone mineral density.

5. A method according to claim 1, wherein rsl2402320 is specific for OA of the hip, hand and knee and for osteoporotic fracture and lumbar spine bone mineral density.

6. A method according to claim 1, wherein rslO248619 is specific for OA of hip, hand and knee and for vertebral and non- vertebral fractures.

7. A method for detecting a nucleotide occurrence for a single nucleotide polymorphism (SNP) indicative of the presence of

OA or the risk for developing OA or progression of OA, comprising: (i) incubating a sample comprising a polynucleotide with a specific binding pair member, wherein the specific binding pair member specifically binds at or near a polynucleotide suspected of being polymorphic, wherein the polynucleotide comprises one of the nucleotide occurrences corresponding to at least one of the polymorphisms selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith; and ii) detecting selective binding of the specific binding pair member, wherein selective binding is indicative of the presence of the nucleotide occurrence, thereby detecting the nucleotide occurrence for the polymorphism.

8. A primer pair for amplifying a polynucleotide comprising a single nucleotide polymorphism (SNP) in the polynucleotide, wherein a forward primer selectively binds the polynucleotide upstream of the SNP position on one strand and a reverse primer selectively binds the polynucleotide upstream of the SNP position on a complementary strand, wherein the SNP is selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith.

9. A primer for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the primer selectively binds the polynucleotide upstream of the SNP position on one strand, wherein the SNP is selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith.

10. A probe for determining a nucleotide occurrence of a single nucleotide polymorphism (SNP) in a polynucleotide, wherein the probe selectively binds to a polynucleotide comprising a particular nucleotide occurrence of an SNP, wherein the SNP is selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith.

11. A kit for identifying at least one single marker allele or haplotype allele of a single nucleotide polymorphism (SNP) as mentioned in claim 1, said kit comprising a primer pair according to claim 8, and optional reagents for amplifying a polynucleotide using said primer pair.

12. A kit for identifying at least one single marker or haplotype allele of one or more single nucleotide polymorphisms (SNPs), said kit comprising an oligonucleotide probe according to claim 10, a primer according to claim 9, or a primer pair according to claim 8, or a combinations thereof, and optional reagents for amplifying a polynucleotide using said primer pair.

13. A method for the detection of osteoarthritis (OA) or for detection of the risk for developing OA or progression of OA comprising detecting the differential expression of a gene product from one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5, , AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L.

14. A method according to claim 13, wherein the differential expression of an mRNA or a protein encoded by said genes is detected.

15. A method according to claim 13 or 14, wherein the detection is performed by using mass spectrometry, protein chip array analysis, antibody array analysis, immunoassay analysis,

MRI, NMR, ultrasound spectroscopy, Raman spectroscopy and/or infra red spectroscopy.

16. An antibody binding to a gene product from a gene selected from the group consisting of JAKl, KCNN3, GRBlO, COG5,

AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L.

17. Use of an SNP according to claim 1, an oligonucleotide probe according to claim 10, a primer according to claim 9, or a primer pair according to claim 8, a gene product according to claim 13, an antibody according to claim 16, or a combination thereof, for the diagnosis of OA or for predicting the risk of developing OA or for progression of OA.

18. A method for screening for therapeutic compounds for the treatment or prevention of OA comprising assaying the effects of compounds on the expression of one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22,

Cl0orf46, PRLHR, and DUS4L.

19. A method for treating or preventing OA comprising modification of the expression of one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5,

AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L.

20. The method of claim 19, wherein said method comprises decreasing the amount of at least one protein that is over- expressed in subjects having OA or having an increased risk of developing OA or progression of OA, or increasing the amount of at least one protein that is under-expressed in subjects having OA or having an increased risk of developing OA or progression of OA.

21. Pharmaceutical composition for the treatment of OA or an increased risk of developing OA or progression of OA , comprising at least one inhibitor compound selected from: an antibody according to claim 16 or derivative thereof, said derivative preferably being selected from the group consisting of scFv fragments, Fab fragments, chimeric antibodies, bifunctional antibodies, intrabodies, and other antibody- derived molecules; a gene product from one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5,

AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L; a small molecule interfering with the biological activity of said gene product; - an antisense molecule, in particular an antisense RNA or antisense oligodeoxynucleotide, which is able to bind to one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L; - an RNAi molecule, which is able to bind to one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L; and a ribozyme, which is able to inhibit a gene product from one or more of the genes selected from the group consisting of JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6,

PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L and a suitable excipient, carrier or diluent.

22. Method of treating a subject, comprising administering to said subject the pharmaceutical composition of claim 22 in an amount effective to decrease or prevent OA.