WO2010071405A1 - Marqueurs pour détecter une prédisposition au risque, une apparition et une progression d'ostéoarthrite - Google Patents

Marqueurs pour détecter une prédisposition au risque, une apparition et une progression d'ostéoarthrite Download PDF

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WO2010071405A1
WO2010071405A1 PCT/NL2008/050817 NL2008050817W WO2010071405A1 WO 2010071405 A1 WO2010071405 A1 WO 2010071405A1 NL 2008050817 W NL2008050817 W NL 2008050817W WO 2010071405 A1 WO2010071405 A1 WO 2010071405A1
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snp
group
polynucleotide
antibodies
snps
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PCT/NL2008/050817
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Hanneke Kerkhof
Andreas Gerardus Uitterlinden
Joyce Berdina Josepha Van Meurs
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Erasmus University Medical Center Rotterdam
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • 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.
  • Osteoarthritis 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.
  • 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.
  • GWA genome-wide association
  • SNPs Single Nucleotide Polymorphisms
  • 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.
  • 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.
  • SNPs single nucleotide polymorphisms
  • 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.
  • said SNP is selected from the group consisting of rs3815148, rs873598, rslO248619, rsl0465850, rsl2402320 and SNPs in linkage disequilibrium therewith
  • 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
  • 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.
  • 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.
  • a central nucleotide of the oligonucleotide specifically hybridizes with the polymorphic position of the portion of the nucleic acid molecule.
  • 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.
  • said method of genetic screening further comprises the step of amplifying a region of the gene or a portion thereof that contains the polymorphism.
  • 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
  • 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.
  • single nucleotide polymorphism 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.
  • polymorphism 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.
  • 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).
  • isolated 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 30 0 C for short probes or primers (e.g.
  • 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 40 0 C.
  • 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 60 0 C. Hybridization procedures are well known in the art. The terms “under stringent hybridization conditions” and “high stringency conditions” are equivalent.
  • 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).
  • phosphorous linkages e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate
  • 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).
  • primer 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.
  • the primer is an oligodeoxy ribonucleotide.
  • 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.
  • 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.
  • the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'.
  • gene 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.
  • 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.
  • TAS Transcriptional Amplification System
  • RCA Rolling Circle Amplification
  • NASBA Nucleic Acid Sequence Based Amplification
  • ICAN Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acid
  • RAM Ramification-extension Amplification Method
  • 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).
  • 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.
  • 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.
  • 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.
  • nucleotide occurrence of a SNP is such that the nucleotide occurrence results in an amino acid change in an encoded polypeptide
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Identification of the nucleotide occurrence can be performed using any method suitable for examining the particular sample.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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), ⁇ - 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.
  • 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.
  • RFLP restriction fragment length polymorphism
  • AFLP amplified fragment length polymorphism
  • SSCP single strand conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • Tm 81.5 0 C + 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 10 0 C. 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.
  • primers and probes useful for the detection of polymorphic positions in a nucleic acid are 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.).
  • nucleic acid amplification techniques allow the amplification of fragments of nucleic acids, which may be present in very low amounts.
  • the SNP-specific sequences must be determined for which primers or probes may then be developed.
  • 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.
  • 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.
  • the nucleic acid sample generally is a deoxyribonucleic acid (DNA) sample, particularly genomic DNA or an amplification product thereof.
  • DNA deoxyribonucleic acid
  • RNA heteronuclear ribonucleic acid
  • a cDNA or amplification product thereof can be used.
  • 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.
  • 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.
  • 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 ⁇ g), 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.
  • hybridization signal 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.
  • 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.
  • 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.
  • detection probes of the present invention it is allowable for detection probes of the present invention to contain one or more mismatches to their target sequence.
  • 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.
  • the genes belonging to these SNPs are, respectively:
  • PRLHR(rs873598) MIM 600895, GenBank accession no: NM_004248.2, and DUS4L (rs3815148) Ge ⁇ elD: 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.
  • polypeptide polypeptide
  • peptide protein
  • polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
  • polypeptide 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 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.
  • a protein profile includes quantitative information for the proteins represented in a sample.
  • Quantitation 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., ⁇ g/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.
  • 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.
  • 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.
  • 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.
  • antibody refers 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.
  • scFv single- chain Fvs
  • Fab fragments F(ab') fragments
  • disulfide-linked Fvs sdFv
  • anti-Id anti-idiotypic antibodies
  • 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, IgG 2 , IgGe, IgG/t, IgAi and IgA 2 ) 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.
  • 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).
  • 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 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.
  • affecting the expression and “modulating the expression” of a protein or gene 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.
  • 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.
  • a non-mammalian vertebrate such as a bird (e.g., a chicken or duck) or a fish, and a non- mammalian invertebrate.
  • 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
  • the proteins encoded by the genes JAKl, KCNN3, GRBlO, COG5, AK3L1, DNAJC6, PRKAR2B, HBPl, GPR22, Cl0orf46, PRLHR, and DUS4L 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.
  • the biomarker comprises at least one protein.
  • 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 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.
  • Suitable samples include samples from blood, circulating cells, serum, plasma, urine, saliva, etc..
  • 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.
  • kits of parts for performing the methods as described above 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • antibody microarrays 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.
  • the antibody microarrays may be used for differential protein expression studies (protein profiling).
  • 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.
  • the proteins from the biological samples are labelled with one or more fluorescent probes (e.g. Cy3 and Cy5) using standard protein labelling protocols.
  • fluorescent probes e.g. Cy3 and Cy5
  • 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 ⁇ l) 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.
  • 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.
  • 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.
  • the peptides used for immunisation may be used in competitive ELISA experiments on the microarray.
  • 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).
  • 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.
  • 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.
  • a proteinaceous marker as defined herein above as a therapeutic target or as a therapeutic agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cytoplasmic gene products
  • RNA's gene products
  • polypeptides or enzymes in order to modulate the activity of these products.
  • antibodies are in the form of intrabodies, produced inside a target cell.
  • antibodies may be used for deliverance of at least one toxic compound linked thereto to a target cell.
  • 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.
  • small molecules can also be used for deliverance of at least one linked toxic compound to the target cell.
  • 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).
  • RNAi RNA interference
  • 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.
  • 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.
  • 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.
  • 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.
  • Trans-cleaving catalytic RNAs 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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 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 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 interference requires an initiation step and an effector 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.
  • RISC RNA-induced silencing complex
  • 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.
  • 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.
  • 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.
  • 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 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.
  • a mutation is in a substrate- binding domain, a catalytic domain, or a cellular localization domain.
  • the mutant polypeptide will be overproduced. Point mutations are made that have such an effect.
  • 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.
  • 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).
  • anti-RNA antibodies may for instance be useful in silencing messengers of the tumor-related genes of the present invention.
  • 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.
  • 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.
  • 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).
  • the antibodies of the invention are human or humanized monoclonal antibodies.
  • 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.
  • 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).
  • 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.
  • 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.
  • the intrabody comprises a single-chain Fv ("sFv").
  • sFv single-chain Fv
  • the intrabody preferably does not encode an operable secretory sequence and thus remains within the cell (see generally Marasco, WA, 1998,
  • 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.
  • 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.
  • 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.
  • IgG, IgA, IgM and IgE antibodies are also possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
  • Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93).
  • PCT publication No. WO 98/24893 which is incorporated by reference herein in its entirety.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a number of viral-based expression systems may be utilized.
  • 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.
  • 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).
  • an antibody molecule to be used with the methods of the invention 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.
  • 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.
  • the invention provides an antibody as defined above for use in therapy.
  • 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.
  • antibodies may be produced by the subject itself by using in vivo antibody production methodologies as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions and Therapeutic Uses 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.
  • therapeutically effective amount 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a diseased joint is located and the therapeutic composition injected several times in several different locations within said joint.
  • 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.
  • receptor-mediated targeted delivery of therapeutic compositions containing antibodies of the invention is used to deliver the antibodies to specific tissue.
  • 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 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g 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.
  • 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.
  • GWAS Genome-Wide Association Study
  • SNPs Single Nucleotide Polymorphisms
  • 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.
  • 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.
  • stage 2 SNPs were selected if the following criteria applied:
  • GDF-5 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.
  • 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 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.

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Abstract

La présente invention porte sur un procédé de détection de l'ostéo-arthrite (OA) ou de détection du risque de développer une OA ou de la progression d'une OA. Ce procédé comprend la détection de la présence d'un ou plusieurs polymorphismes de nucléotide simple (SNP) choisis dans le groupe constitué par rs3815148, rs873598, rs10248619, rs10465850, rs12402320 et des SNP en déséquilibre de liaison avec ceux-ci et/ou la détection de l'expression différentielle d'un produit génique à partir d'un ou plusieurs des gènes choisis dans le groupe constitué par JAK1, KCNN3, GRB10, COG5, AK3L1, DNAJC6, PRKAR2B, HBP1, GPR22, C10orf46, PRLHR et DUS4L. Une autre partie de l'invention porte sur l'utilisation de ces gènes et/ou produits géniques en tant que cible pour un développement de médicament et sur des composés pharmaceutiques pour la prévention ou la thérapie de l'ostéo-arthrite.
PCT/NL2008/050817 2008-12-18 2008-12-18 Marqueurs pour détecter une prédisposition au risque, une apparition et une progression d'ostéoarthrite WO2010071405A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014139723A1 (fr) * 2013-03-14 2014-09-18 Progenika Biopharma S.A. Marqueurs génétiques pour l'arthrose
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JP7137524B2 (ja) 2019-04-24 2022-09-14 ジェネシスヘルスケア株式会社 変形性膝関節症のリスクを判定する方法
CN116735888A (zh) * 2022-11-18 2023-09-12 昆明医科大学第一附属医院 一种特异性多克隆抗体检测cog5的间接elisa方法
CN116735888B (zh) * 2022-11-18 2024-01-12 昆明医科大学第一附属医院 一种特异性多克隆抗体检测cog5的间接elisa方法
RU2816310C1 (ru) * 2022-12-06 2024-03-28 федеральное государственное бюджетное образовательное учреждение высшего образования "Уфимский университет науки и технологий" Способ прогнозирования развития остеопоретических переломов поясничного отдела позвоночника

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