Methods for evaluating osteoarthritis risk

Abstract

Methods are provided for evaluating osteoarthritis (OA), for example for diagnosing OA, to confirm a diagnosis of OA, to assess or prognose progression of OA, determining the severity of a subject who has OA, and determining a subject's risk of developing OA in the future, as are arrays and kits that can be used to practice the methods. In particular examples, the method includes determining an amount of activity (such as an amount of protein present or an amount of expression) of OA risk-related molecules, such as soluble vascular adhesion protein 1 (sVAP-1) or interleukin-15 (IL-15). Also provided are methods of identifying one or more compounds that alter the activity of an OA-related molecule, thereby identifying potential anti-osteoarthritis drugs.

1 . A method of evaluating osteoarthritis (OA) risk in a human subject, comprising:
 detecting expression of OA risk-related nucleic acid molecules in a serum sample of the subject, wherein OA risk-related nucleic acid molecules comprise interleukin-15 (IL-15), matrix metalloproteinase-7 (MMP-7), plasminogen activating inhibitor-1 (PAI-1), and soluble vascular adhesion protein 1 (sVAP-1), and   comparing expression of the OA risk-related nucleic acid molecules in the subject sample to expression of the OA risk-related nucleic acid molecules in a normal control serum sample,   wherein detecting an upregulation of at least IL-15, MMP-7 and sVAP-1 and downregulation of at least PAI-1 in the subject sample as compared to the normal sample indicates that the subject has an increased OA risk.   
     
     
         2 . The method of  claim 1 , wherein the OA risk-related nucleic acid molecules further comprise at least four, at least 10, at least 13, at least 16, or at least 20 nucleic acid molecules listed in any of Tables 8 and 10-13. 
     
     
         3 . The method of  claim 1 , wherein the OA risk-related nucleic acid molecules further comprise D-dimer 5 (DD5), DD6, eotaxin 2 (Eot2), intercellular adhesion molecule-1 (ICAM-1), MMP-2, and P-selectin, wherein the presence of downregulation of PAI-1, DD5, DD6, Eot2, ICAM-1, MMP-2, and P-selectin and the presence of upregulation of IL-15, MMP-7 and sVAP-1 indicates that the subject has an increased risk of developing OA in the future. 
     
     
         4 . The method of  claim 1 , wherein the OA risk-related nucleic acid molecules further comprise interleukin 1 alpha (IL-1a), IL-2, macrophage inhibitory protein (MIP)-1a, B-lymphocyte chemokine (BLC), 6-chemokine (Ckine), fibroblast growth factor (FGF)-7, granulocyte macrophage colony stimulating factor (GM-CSF), insulin-like growth factor binding protein (IGFBP)-2, neurotrophin-4 (NT4), ICAM-3, vascular endothelial (VE)-cadherin, and tissue inhibitors of metalloproteinases 1 (TIMP-1), wherein the presence of downregulation of PAI-1, and the presence of upregulation of IL-15, MMP-7, sVAP-1, IL-1α, IL-2, MIP-1α, BLC, 6-Ckine, FGF-7, GM-CSF, IGFBP-2, NT4, ICAM-3, VE-cadherin, and TIMP-1, indicates that the subject has OA. 
     
     
         5 . The method of  claim 1 , wherein the OA risk-related nucleic acid molecules further comprise macrophage inflammatory protein 1 β (MIP-1β), macrophage inflammatory protein 1 α (MIP-1α), urokinase-type plasminogen activator receptor (UPAR), and vascular cell adhesion molecule-1 (VCAM-1), wherein the presence of upregulation of IL-15, sVAP-1, MMP-7, UPAR, VCAM-1, MIP-1α, and MIP-1 and the downregulation of PAI-1 indicates that the subject has an increased OA risk. 
     
     
         6 . The method of  claim 5 , wherein the OA risk-related nucleic acid molecules further comprise brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), hemofiltrate CC chemokine 1 (HCC1), leptin, and prolactin, wherein the presence of upregulation of IL-15, sVAP-1, MIP-1α,MIP-1β, HCC1, leptin, MMP-7, UPAR, hVCAM-1, and BDNF, and the downregulation of PAI-1, EGF and prolactin indicates that the subject is at risk for developing OA in the future. 
     
     
         7 . The method of  claim 5 , wherein the OA risk-related nucleic acid molecules further comprise IL-2, Eot2, IGFBP-4, ICAM-3, monokine induced by interferon γ (MIG), myeloid progenitor inhibitory factor 1 (MPIF-1), thymus and activation regulated chemokine (TARC), 6-Ckine, and TGFβ receptor III (TGF-β RIII), wherein the presence of upregulation of IL-15, sVAP-1, TGF-β RIII, IL-2, IGFBP-4, ICAM-3, MIG, MMP-7, MPIF-1, UPAR, VCAM-1,6-Ckine, MIP-1α, and MIP-1β, and the downregulation of PAI-1, Eot2 and TARC, indicates that the subject has OA. 
     
     
         8 . The method of  claim 1 , wherein the presence of upregulation in any combination of at least 10 OA risk-related nucleic acid molecules listed in Tables 10-11 as upregulated, indicates that the subject has OA. 
     
     
         9 . The method of  claim 1 , wherein the presence of downregulation in any combination of at least 6 OA risk-related nucleic acid molecules listed in Tables 12-13 as down-regulated, indicates that the subject has an increased risk of developing OA in the future. 
     
     
         10 . The method of  claim 1 , further comprising administering to the subject a treatment to avoid or reduce OA disease if the expression indicates that the subject has increased OA risk. 
     
     
         11 . The method of  claim 10 , wherein the treatment comprises an anti-inflammatory agent. 
     
     
         12 . The method of  claim 1 , wherein the expression of the OA risk-related nucleic acid molecules in the normal control serum sample is represented by a reference value comprising a level of expression for each of the OA risk-related nucleic acid molecules in an absence of OA risk or a range of expression for each of the OA risk-related nucleic acid molecules in subjects of the same gender and in the same age range as the subject. 
     
     
         13 . The method of  claim 1 , wherein the method is a method of determining the progression of OA, wherein detecting expression of the OA risk-related nucleic acid molecules comprises:
 comparing a first and a second expression level of the OA-risk related nucleic acid molecules in the subject determined at a first and a second time point, wherein a statistically significant difference with a p value ≦0.05 between the first and the second expression level of the OA-risk related nucleic acid molecules reflects the progression of OA in the subject.   
     
     
         14 . The method of  claim 1 , wherein detecting expression comprises quantitating an amount of the OA risk-related nucleic acid molecules. 
     
     
         15 . The method of  claim 14 , wherein detecting expression of OA risk-related nucleic acid molecules comprises:
 measuring a quantity of OA risk-related nucleic acid molecules from the subject sample, wherein an increase in the quantity of the OA risk-related nucleic acid molecules in the subject sample relative to a quantity of the OA risk-related nucleic acid molecules in the normal sample is increased expression in those OA risk-related nucleic acid molecules and a decrease in the quantity of the OA risk-related nucleic acid molecules in the subject sample relative to a quantity of the OA risk-related nucleic acid molecules in the normal sample is decreased expression in those OA risk-related nucleic acid nucleic acid molecules.   
     
     
         16 . The method of  claim 15 , wherein a statistically significant difference with a p value ≦0.05 between the quantity in the sample derived from the subject and the reference value indicates that there is differential activity in the OA-risk related nucleic acid molecule. 
     
     
         17 . The method of  claim 1 , wherein the nucleic acid molecules are isolated from the subject sample, thereby generating isolated nucleic acid molecules, and wherein the isolated nucleic acid molecules are hybridized with oligonucleotides that detect the OA risk-related nucleic acid molecules. 
     
     
         18 . The method of  claim 17 , wherein the oligonucleotides are present on an array substrate. 
     
     
         19 . The method of  claim 17 , wherein the oligonucleotides are complementary to any combination of at least four molecules listed in Tables 8 and 10-13. 
     
     
         20 . The method of  claim 1 , wherein evaluating OA risk comprises determining whether the subject has OA, determining whether the subject has an increased OA disease risk, determining the progression of OA in the subject, or determining the severity of OA in the subject. 
     
     
         21 . The method of  claim 1 , wherein detecting expression of OA risk-related nucleic acid molecules comprises quantitatively or qualitatively analyzing nucleic acid molecules obtained from the subject. 
     
     
         22 . A method of evaluating osteoarthritis (OA), wherein the method is a method of classifying age-related OA risk related molecules in a subject, comprising:
 detecting differential activity of at least four OA risk-related molecules in a sample of the subject, wherein the at least four OA risk-related molecules comprise brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), 6Ckine, intercellular adhesion molecule-3 (ICAM-3), TGFβ receptor III (TGF-β RIII), urokinase-type plasminogen activator receptor (UPAR), vascular cell adhesion molecule-1 (VCAM-1), interleukin 2 (IL-2), monokine induced by interferon γ (MIG), matrix metalloproteinase 7 (MMP7), and myeloid progenitor inhibitory factor 1 (MPIF-1), wherein detecting differential activity comprises determining whether there is an upregulation of at least BDNF, 6Ckine, ICAM-3, TGF-β RIII, UPAR, VCAM-1, IL-2, MIG, MMP7, and MPIF-1, and determining whether there is a downregulation of at least EGF, and wherein the presence of differential activity of the at least four OA risk-related molecules indicates that the subject has an increased OA risk that correlates to age.   
     
     
         23 . A method of evaluating OA risk in a human subject, comprising:
 incubating nucleic acid molecules obtained from serum of the subject with an array comprising oligonucleotides complementary to all 36 OA risk-related nucleic acid molecules listed in Tables 10-13 for a time sufficient to allow specific hybridization between the nucleic acid molecules and oligonucleotides, thereby forming nucleic acid molecule: oligonucleotide complexes; and analyzing the nucleic acid molecule: oligonucleotide complexes to determine an amount of each of the nucleic acid molecules present in the serum; and   comparing the amount of each of the nucleic acid molecules in the serum to a reference value, wherein the reference value is an amount of each of the nucleic acid molecules in the absence of OA risk, and wherein the presence of an at least four-fold increase of at least IL-15, MMP-7 and sVAP-1 and an at least four-fold decrease of at least PAI-1 in the serum as compared to the reference value indicates that the subject has increased OA risk.   
     
     
         24 . An array for determining risk of OA, wherein the array consists of antibodies that recognize at least four of the OA-risk related proteins listed in Tables 8 and 10-13 or oligonucleotides that recognize at least four of the OA-risk related nucleic acid molecules listed in Tables 8 and 10-13. 
     
     
         25 . The array of  claim 24 , wherein the at least four OA-risk related proteins or nucleic acid molecules consist of the OA-risk related molecules listed in Table 8, 10, 11, 12 or 13. 
     
     
         26 . A kit for evaluating OA risk in a subject, comprising:
 the array of  claim 24 ; and   a buffer solution, in separate packaging.   
     
     
         27 . A method of identifying an agent that alters an activity of an OA risk-related molecule listed in Tables 8 and 10-13, comprising:
 culturing a cell under conditions sufficient to mimic or induce OA;   contacting the cell with one or more test agents under conditions sufficient for the one or more test agents to alter the activity of an OA risk-related molecule; and   detecting differential activity of the OA risk-related molecule, wherein the presence of differential activity of the OA risk-related molecule indicates that the test agent alters the activity of an OA-related molecule listed in Tables 8 and 10-13.   
     
     
         28 . The method of  claim 27 , wherein the OA risk-related molecule listed in Tables 8 and 10-13 comprises IL-15 or sVAP-1. 
     
     
         29 . The method of  claim 27 , wherein the OA risk-related molecule listed in Tables 8 and 10-13 comprises a nucleic acid sequence, and wherein detecting differential activity of the OA risk-related molecule comprises detecting differential RNA expression, or wherein the OA risk-related molecule listed in Tables 8 and 10-13 comprises a protein sequence, and wherein detecting differential expression of the OA risk-related molecule comprises detecting an amount of protein present. 
     
     
         30 . The method of  claim 27 , wherein the cell is present in a mammal, and wherein culturing the cell under conditions sufficient to mimic OA comprises inducing OA in the mammal or providing a mammal having OA, and wherein contacting the cell with one or more test agents comprises administering the one or more test agents to the mammal.