EP1756317A2 - Procedes d'identification de risque d'osteoarthrite et traitements associes - Google Patents

Procedes d'identification de risque d'osteoarthrite et traitements associes

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Publication number
EP1756317A2
EP1756317A2 EP05768810A EP05768810A EP1756317A2 EP 1756317 A2 EP1756317 A2 EP 1756317A2 EP 05768810 A EP05768810 A EP 05768810A EP 05768810 A EP05768810 A EP 05768810A EP 1756317 A2 EP1756317 A2 EP 1756317A2
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EP
European Patent Office
Prior art keywords
nucleotide sequence
seq
osteoarthritis
polymorphic
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05768810A
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German (de)
English (en)
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EP1756317A4 (fr
Inventor
Steven Mah
Andreas Braun
Stefan M. Kammerer
Matthew Roberts Nelson
Rikard Henry Reneland
Maria L. Langdown
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Sequenom Inc
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Sequenom Inc
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Publication of EP1756317A2 publication Critical patent/EP1756317A2/fr
Publication of EP1756317A4 publication Critical patent/EP1756317A4/fr
Withdrawn legal-status Critical Current

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone

Definitions

  • the invention relates to genetic methods for identifying risk of osteoarthritis and treatments that specifically target such diseases.
  • Osteoarthritis is a chronic disease usually affecting weight-bearing synovial joints. There are approximately 20 million Americans affected by OA and it is the leading cause of disability in the United States. In addition to extensive human suffering, OA also accounts for nearly all knee replacements and more than half of all hip replacements in the United States. Despite its prevalence, OA is poorly understood and there are few treatments available besides anti-inflammatory drugs and joint replacement. [0003] Osteoarthritis (OA) is a disease caused by degeneration of articular cartilage and subsequent joint deformation. In addition to risk factors like body weight, joint injury and age, there is a strong hereditary component to OA, reflected by high heritability estimates from twin studies. So far, few- of the genes responsible for this genetic component have been identified.
  • a subject at risk of osteoarthritis and/or a risk of osteoarthritis in a subject which comprise detecting the presence or absence of one or more polymorphic variations associated with osteoartliritis in or around the loci described herein in a human nucleic acid sample.
  • two or more polymorphic variations are detected in two or more regions of which one is the PADI2, APOB, IL1RL2, IL1RL1, WASPIP, ADAMTS2, BVES, TM7SF3, LOXL1, CASPR4 or APOL3 region or other region in Table B.
  • nucleic acids that include one or more polymorphic variations associated with occurrence of osteoarthritis, as well as polypeptides encoded by these nucleic acids.
  • methods for identifying candidate therapeutic molecules for treating osteoarthritis, as well as methods for treating osteoarthritis in a subject by identifying a subject at risk of osteoarthritis and treating the subject with a suitable prophylactic, treatment or therapeutic molecule are also featured.
  • compositions comprising a cell from a subject having osteoarthritis or at risk of osteoarthritis and/or a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 o ⁇ APOL3 nucleic acid or other nucleic acid referenced in Table B, with a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B.
  • the RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid is designed from a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B that includes one or more polymorphic variations associated with osteoarthritis, and in some instances, specifically interacts with such a nucleotide sequence.
  • nucleic acids bound to a solid surface in which one or more nucleic acid molecules of the array have a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing.
  • compositions comprising a cell from a subject having osteoartliritis or at risk of osteoarthritis and/or a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 polypeptide or other polypeptide referenced in Table B, with an antibody that specifically binds to the polypeptide.
  • the antibody specifically binds to an epitope in the polypeptide that includes a non-synonymous amino acid modification associated with osteoarthritis (e.g., results in an amino acid substitution in the encoded polypeptide associated with osteoarthritis).
  • the antibody selectively binds to an epitope in the PADI2, APOB, IL1RL2, ILIRLI, WASPIP, AOAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 polypeptide, or other polypeptide referenced in Table B, having an amino acid associated with osteoarthritis.
  • an antibody that binds an epitope having an amino acid encoded by rsl367117, rsl041973 and/or rs398829, such as a isoleucine or threonine encoded by rsl 367117 (e.g., a threonine at position 98 in an APOB polypeptide), a glutamic acid or alanine encoded by rsl041973 (e.g., an alanine at position 78 in a ILIRLI polypeptide), a valine or isoleucine encoded by rs398829 (e.g., a valine at position 245 in a ADAMTS2 polypeptide), at the corresponding position in the polypeptide.
  • a isoleucine or threonine encoded by rsl 367117 e.g., a threonine at position 98 in an APOB polypeptide
  • Figures 1A-1J show proximal SNPs in a 100-kb window in PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 ⁇ ndAPOL3 regions of genomic DNA, respectively, that were compared between pools of cases and controls.
  • the x-axis corresponds to their chromosomal position and the y-axis to the test P-values (shown on the -log 10 scale).
  • the continuous bold line presents the results of a goodness-of-fit test for an excess of significance (compared to 0.05) in a 10 kb sliding window assessed at 1 kb increments.
  • Osteoarthritis or degenerative joint disease, is one of the oldest and most common types of arthritis. It is characterized by the breakdown of the joint's cartilage. Cartilage is the part of the joint that cushions the ends of bones, and its breakdown causes bones to rub against each other, causing pain and loss of movement.
  • Type II collagen is the main component of cartilage, comprising 15-25% of the wet weight, approximately half the dry weight, and representing 90-95% of the total collagen content in the tissue. It forms fibrils that endow cartilage with tensile strength (Mayne, R. Arthritis Rhuem. 32:241-246 (1989)). [0011] Most commonly affecting middle-aged and older people, OA can range from very mild to very severe. It affects hands and weight-bearing joints such as knees, hips, feet and the back. Knee OA can be as disabling as any cardiovascular disease except stroke. [0012] Osteoarthritis affects an estimated 20.7 million Americans, mostly after age 45, with women more commonly affected than men.
  • Inclusion or exclusion of samples for an osteoarthritis pool may be based upon the following criteria: ethnicity (e.g., samples derived from an individual characterized as Caucasian); parental ethnicity (e.g., samples derived from an individual of British paternal and maternal descent); relevant phenotype information for the individual (e.g., case samples derived from individuals diagnosed with specific knee, hand or hip osteoarthritis (OA); case samples recruited from an OA knee replacement clinic). Control samples may be selected based on relevant phenotype information for the individual (e.g., derived from individuals free of OA at several sites (knee, hand, hip etc)); and no family history of OA and/or rheumatoid arthritis.
  • ethnicity e.g., samples derived from an individual characterized as Caucasian
  • parental ethnicity e.g., samples derived from an individual of British paternal and maternal descent
  • relevant phenotype information for the individual e.g., case samples derived from individuals diagnosed with specific
  • Additional phenotype information collected for both cases and controls may include age of the individual, gender, family history of OA, diagnosis with osteoarthritis (joint location of OA (e.g., knee, hips, hands and spine), date of primary diagnosis, age of individual as of primary diagnosis), knee history (current symptoms, any major knee injury, menisectomy, knee replacement surgery, age of surgery), HRT history, osteoporosis diagnosis.
  • OA e.g., knee, hips, hands and spine
  • date of primary diagnosis e.g., knee, hips, hands and spine
  • knee history current symptoms, any major knee injury, menisectomy, knee replacement surgery, age of surgery
  • HRT history osteoporosis diagnosis.
  • polymorphic site refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals.
  • a polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example.
  • a polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region.
  • a polymorphic site is often one nucleotide in length, which is referred to herein as a "single nucleotide polymorphism” or a "SNP.”
  • each nucleotide sequence is referred to as a "polymorphic variant” or "nucleic acid variant.”
  • the polymorphic variant represented in a minority of samples from a population is sometimes referred to as a "minor allele” and the polymorphic variant that is more prevalently represented is sometimes referred to as a "major allele.”
  • genotyped refers to a process for determining a genotype of one or more individuals, where a "genotype” is a representation of one or more polymorphic variants in a population.
  • a genotype or polymorphic variant may be expressed in terms of a "haplotype,” which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation.
  • Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position.
  • the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.
  • phenotype refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like.
  • a phenotype is occurrence of osteoarthritis.
  • a polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10%) or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population.
  • a polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid.
  • a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5' untranslated region (UTR), a 3' UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide.
  • DNA e.g., genomic DNA (gDNA) and complementary DNA (cDNA)
  • RNA e.g., mRNA, tRNA, and rRNA
  • Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.
  • polymorphic variants at positions rs910223, rs 1367117, rs 1024791, rs 1041973, rsl465621, rs398829, rsl018810, rsl484086, rs242392, rs8818, rsl395486, rs512294 and/or rsl32659 in the human genome were associated with an increased risk of osteoarthritis, and in specific embodiments, the corresponding allele in the right-most column in Table B for each position is associated with an increased risk of osteoarthritis.
  • polymorphic variants at positions rsl367117, rsl041973 and rs398829 were associated with an increased risk of osteoarthritis, and in specific embodiments, a threonine encoded by rs 1367117, an alanine encoded by rs 1041973, and a valine encoded by rs398829 were associated with an increased risk of osteoartliritis.
  • Polymorphic variants in and around the APOB locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 2 selected from the group consisting of 238, 294, 295, 347, 1425, 4891, 5087, 7041, 7121, 7219, 7443, 7485, 10939, 11367, 11571, 11839, 12551, 12646, 13469, 14913, 15205, 15246, 15695, 17473, 17610, 17828, 18130, 18281, 18623, 18890, 21561, 23100, 23872, 24581, 24582, 24983, 27540, 30846, 31415, 31453, 31899, 37000, 38681, 39287, 42951, 45648, 46222, 46687, 47020, 47593, 48513, 49723, 49986, 53018, 53296, 53547, 53899, 53916, 53933, 54305, 55327, 55895, 56143, 56640, 58486, 59576, 530
  • Polymorphic variants at the following positions in SEQ ID NO: 2 in particular were associated with an increased risk of osteoarthritis: 7219, 7485, 11839, 31899, 37000, 48513, 49986, 56640, 74407, 77398, 93060 and 97627.
  • polymorphic variants in SEQ ID NO: 2 were associated with risk of osteoarthritis: an adenine at position 7219, a guanine at position 7485, an adenine at position 11839, a thymine at position 31899, an adenine at position 37000, a cytosine at position 48513, a guanine at position 49986, a guanine at position 56640, a cytosine at position 74407, a guanine at position 77398, an adenine at position 93060 and an adenine at position 97627.
  • a threonine at amino acid position 98 in an APOB polypeptide was associated with increased risk of osteoarthritis (i.e., an isoleucine to threonine non-synonymous variation).
  • Polymorphic variants in and around the IL1RL2 locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 3 selected from the group consisting of 225, 509, 860, 874, 939, 1483, 1798, 2189, 2215, 2282, 2340, 2963, 3369, 3481, 3564, 3653, 4860, 4941, 4975, 5321, 5346, 5541, 5633, 6007, 6317, 6378, 6382, 6426, 6479, 6641, 6703, 6705, 7963, 8525, 8526, 8598, 8624, 8883, 8980, 13578, 16135, 16141, 16642, 16931, 17004, 17009, 17010, 18713, 18853, 20783, 21335, 22180, 22268, 22285, 25378, 25906, 26015, 26475, 26798 :
  • Polymorphic variants at the following positions in SEQ ID NO: 3 in particular were associated with an increased risk of osteoarthritis: 2215, 3369, 16642, 20783, 52155, 55052, 55941, 74333, 74741, 85366, 85469, 87687, 89660 and 95718, where specific embodiments are directed to position 52155.
  • polymorphic variants in SEQ ID NO: 3 were associated with risk of osteoarthritis: an adenine at position 2215, a deletion at position 3369, a deletion at position 16642, a cytosine at position 20783, a cytosine at position 52155, a cytosine at position 55052, a cytosine at position 55941, a thymine at position 74333, an adenine at position 74741, a deletion at position 85366, a thymine at position 85469, a thymine at position 87687, an adenine at position 89660 and a cytosine at position 95718.
  • Polymorphic variants in and around the ILIRLI locus were tested for association with osteoarthritis. These include polymorphic variants at positions in SEQ ID NO: 4 selected from the group consisting of 207, 6019, 6414, 7341, 10984, 12351, 13335, 16584, 16737, 23897, 24057, 25145, 25300, 26262, 26312, 26589, 27302, 27358, 27451, 27552, 30731, 32085, 32139, 33184, 42382, 42569, 44823, 45217, 45548, 45601, 45722, 45967 ,47367, 47642, 48126, 49218, 49274, 49433, 49610, 51282, 51466, 53757, 53960, 54031, 54574, 55679, 56100, 56182, 59817, 60533, 60656, 72209, 72778, 74293, 77335, 78029, 78374, 7
  • Polymorphic variants at the following positions in SEQ ID NO: 4 in particular were associated with an increased risk of osteoarthritis: 6414, 51282, 54574, 78374, 92029 and 96793, where specific embodiments are directed to position 54574.
  • the following polymorphic variants in SEQ ID NO: 4 were associated with risk of osteoarthritis: an adenine at position 6414, an adenine at positoin 51282, a cytosine at position 54574, a thymine at position 92029 and an adenine at position 96793.
  • Polymorphic variants in and around the WASPIP locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 5 selected from the group consisting of 209, 5908, 7460, 7733, 7855, 7904, 8869, 9480, 13820, 15152, 17713, 17804, 18220, 19083, 19123, 19605, 20247, 20592, 21907, 23273, 23299, 23623, 23669, 23844, 24190, 24486, 24896, 25118, 30551, 30844, 30900, 30942, 31699, 32081, 35078, 36196, 36541, 38356, 45578, 49634, 49774, 51119, 51181, 51652, 54467, 55762, 55999, 57865, 66613, 68377, 69754, 72859, 76512, 76717, 77722, 80998, 82033, 89658, 89960, 94155 and 95679.
  • Polymorphic variants at the following positions in SEQ ID NO: 5 in particular were associated with an increased risk of osteoarthritis: 19083, 30900, 38356, 76512 and 94155, where specific embodiments are directed to positions 30900, 76512 and/or 94155.
  • the following polymorphic variants in SEQ ID NO: 5 were associated with risk of osteoarthritis: a thymine at position 19083, a guanine at position 30900, an adenine at position 38356, an adenine at position 76512 and an adenine at position 94155.
  • Polymorphic variants in and around the ADAMTS2 locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 6 selected from the group consisting of 210, 3608, 3609, 4318, 5593, 5629, 5639, 5640, 8943, 17968, 19887, 21034, 21085, , 21596, 23379, 23432, 24007, 26121, 26273, 26755, 27411, 27710, 27842, 28379, 29603, 31232, 31504, 32583, 32794, 32840, 33044, 33150, 33218, 33513, 33959, 34486, 36289, 36570, 38247, 38477, 38518, 38529, 38667, 39781, 39856, 39927, 40506, 41869, 42452, 44788, 46059, 46846, 47712, 48796, 49441, 49602, 49723, 50050, 50171, 50477, 50818, 50833, 50881, 50882, 51386, 51534, 523
  • Polymorphic variants at the following positions in SEQ ID NO: 6 in particular were associated with an increased risk of osteoarthritis: 5640, 33150, 38247, 38529, 46846, 49723, 50050, 63427, 73889, 189104 and rs428901, where specific embodiments are directed to positions 46846, 73889, 189104 and/or rs428901.
  • polymorphic variants in SEQ ID NO: 6 were associated with risk of osteoarthritis: a cytosine at position 5640, a cytosine at position 33150, an adenine at position 38247, a thymine at position 38529, an adenine at position 46846, a cytosine at position 49723, a cytosine at position 50050, a cytosine a position 63427, a guanine at position 73889, a thymine at position 189104, and an adenine at position rs428901. [0030] Polymorphic variants in and around the BVES locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 7 selected from the group consisting of 241, 801, 899, 2091, 2290, 2440, 4959, 7914, 7969, 7972, 10831, 12399, 13841, 14461, 14680, 16808, 18231, 18394, 18505, 18684, 19257, 20263, 20656, 21499, 21563, 21612, 21834, 22406, 22408, 22685, 23303, 23306, 25139, 25211, 25364, 25381, 25414, 25835, 26214, 27224, 27526, 27934, 28550, 29015, 29879, 29979, 30030, 30585, 31753, 31934, 33227, 33228, 35172, 36901, 36921, 36932, 37061, 37570, 38745, 38970, 39725, 40070, 40460, 41470, 41562, 41956, 42047, 42
  • Polymorphic variants at the following positions in SEQ ID NO: 7 in particular were associated with an increased risk of osteoarthritis: 25414, 25835, 38970, 41470, 44115, 47496, 49038, 50204, 50840, 50964, 50971, 53906, 54149, 58415, 70796, 72325, 75258, 77822, 80002, 85288, 85581, 86904, 90828, 94616, 94712, 95869 and 97856.
  • polymorphic variants in SEQ ED NO: 7 were associated with risk of osteoarthritis: an adenine at position 25414, a cytosine at position 25835, an adenine at position 38970, an adenine at position 41470, an adenine at position 44115, a guanine at position 47496, a cytosine at position 49038, an adenine at position 50204, a thymine at position 50840, a cytosine at position 50964, a cytosine at position 50971, an adenine at position 53906, a guanine at position 54149, a.
  • guanine at position 58415 a thymine at position 70796, a guanine at position 72325, a cytosine at position 75258, an adenine at position 77822, an adenine at position 80002, an adenine at position 85288, an adenine at position 85581, a guanine at position 86904, a guanine at position 90828, an adenine thymine adenine adenine sequence at position 94616, a cytosine at position 94712, a guanine at position 95869 and a cytosine at position 97856.
  • Polymorphic variants in and around the TM7SF3 locus were tested for association with osteoarthritis. These include polymorphic variants at positions in SEQ ID NO: 8 selected from the group consisting of 230, 231, 5330, 6334, 11372, 11456, 11501, 13393, 16666, 17596, 19710, 19800, 20297, 20967, 32514, 33159, 37600, 41259, 41329, 50060, 53292, 53393, 56417, 56435, 58847, 59595, 59661, 60355, 60407, 62357, 68230, 68516, 69055, 72603, 73928, 85897 and 91554.
  • Polymorphic variants at the following positions in SEQ ID NO: 8 in particular were associated with an increased risk of osteoarthritis: 56435, 59595, 53292, 33159 and 41329, with specific embodiments directed to positions 56435 and/or 59595.
  • the following polymorphic variants in SEQ ID NO: 8 were associated with risk of osteoarthritis: a thymine thymine repeat at position 56435, a thymine at position 59595, a cytosine at position 53292, a guanine at position 33159 and a thymine at position 41329.
  • Polymorphic variants in and around the LOXLl locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 10 selected from the group consisting of 213, 249, 1824, 2057, 23O6, 2869, 3976, 4288, 4290, 4434, 5298, 5467, 8486, 8487, 8831, 9036, 9058, 9131, 9732, 9862, 10191, 10270, 16167, 17620, 17751, 17764, 17787, 19401 , 21021, 21902, 22173, 22416, 22653, 24945, 25011, 28563, 48574, 48710, 48880, 50194, 56343, 56455, 56729, 56759, 56895, 57036, 57702, 62515, 62629, 63501, 63547, 64876, 65073, 67149, 67549, 71660, 71906 and 71911.
  • Polymorphic variants in and around the CASPR4 locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 11 selected from the group consisting of 205, 866, 4212, 5934, 11486, 16969, 22509, 22796, 28097, 28626, 28853, 28873, 30155, 30827, 31956, 32404, 32944, 35205, 35227, 35781, 41052, 45051, 46039, 47276, 47678, 47716, 51014, 54408, 54596, 56853, 61851, 62016, 62461, 68257, 69793, 73976, 73999, 74053, 75315, 75729, 76466, 77216, 77217, 79239, 80825, 81060, S1097, 81426, 84787, 84896, 85165, 86502, 86753, 86941, 88787 and 95598.
  • Polymorphic variants at tire following positions in SEQ ID NO: 11 in particular were associated with an increased risk of osteoarthritis: 47716 and 69793.
  • the following polymorphic variants in SEQ ID NO: 11 were associated with risk of osteoarthritis: an adenine at position 47716 and a thymine at position 69793.
  • Polymorphic variants in and around the APOL3 locus were tested for association with osteoarthritis.
  • polymorphic variants at positions in SEQ ID NO: 13 selected from the group consisting of 201, 425, 1095, 2201, 7879, 8395, 8461, 9503, 10304, 10695, 16300, 16444, 17591, 17988, 19116, 19358, 20300, 20669, 20891, 21451, 21978, 22785, 24248, 24770, 24844, 25066, 25096, 25309, 25344, 25529, 25537, 25554, 27963, 28134, 28356, 29648, 29986, 30217, 30267, 30315, 30585, 30724, 30897, 30931, 31080, 31246, 31373, 31463, 31467, 32188, 32288, 32520, 32594, 32657, 32677, 32764, 32784, 32830, 32872, 33121, 33348, 33952, 34184, 34361, 35026, 35192, 35600, 36033
  • Polymorphic variants at the following positions in SEQ ID NO: 13 in particular were associated with an increased risk of osteoarthritis: 20300, 46257, 87958, 89236, 30267, 32657, 36289, 38869, 45051, 54112, 60307, 63499, 20891, 52699, 71768, with specific embodiments directed to position 46257.
  • polymorphic variants in SEQ ID NO: 13 were associated with risk of osteoarthritis: an adenine at position 20300, a thymine at position 46257, an adenine at position 89236, a guanine at position 30267, an adenine at position 32657, a cytosine at position 36289, a guanine at position 38869, a thymine at position 45051, a guanine at position 54112, an adenine at position 60307, a thymine at position 63499, a guanine at position 20891, a guanine at position 52699, and a cytosine at position 71768.
  • polymorphic variants in a region spanning positions 21233000 to 21243000 (approximately 10,000 nucleotides in length) in a APOB locus a region spanning chromosome positions 102456500 to 102471500 (approximately 15,000 nucleotides in length) in a IL1RL2 locus, a region spanning chromosome positions 102570000 to 102583000 (approximately 13,000 nucleotides in length) in a ILIRLI locus, a region spanning chromosome positions 175647734 to 175655734 (approximately 8,000 nucleotides in length) in a WASPIP locus, a region spanning chromosome positions 178746000 to 178751000 (approximately 5,000 nucleotides in length) in a ADAMTS2 locus, a region spanning chromosome positions 105595000 to 105615000 (approximately
  • a method for identifying polymorphic variants proximal to an incident, founder polymorphic variant associated with osteoarthritis comprises identifying a polymorphic variant proximal to the incident polymorphic variant associated with osteoarthritis, where the incident polymorphic variant is in a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B.
  • the nucleotide sequence often comprises a polynucleotide sequence selected from the group consisting of (a) a polynucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (b) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; and (c) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B or a polynucleotide sequence 90% or more identical to the polynucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B.
  • the presence or absence of an association of the proximal polymorphic variant with osteoarthritis then is determined using a known association method, such as a method described in the Examples hereafter.
  • the incident polymorphic variant is a polymorphic variant associated with osteoarthritis described herein.
  • the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database.
  • the polymorphic variant identified is not publicly disclosed and is discovered using a known method, including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic samples.
  • a known association method such as a method described in the Examples hereafter.
  • the incident polymorphic variant is a polymorphic variant associated with osteoarthritis described herein.
  • the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database.
  • the polymorphic variant identified is not publicly disclosed and is discovered using a
  • the proximal polymorphic variant often is identified in a region surrounding the incident polymorphic variant.
  • this surrounding region is about 50 kb flanking the first polymorphic variant (e.g. about 50 kb 5' of the first polymorphic variant and about 50 kb 3' of the first polymorphic variant), and the region sometimes is composed of shorter flanking sequences, sxich as flanking sequences of about 40 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 7 kb, about 5 kb, or about 2 kb 5' and 3' of the incident polymorphic variant.
  • the region is composed of longer flanking sequences, such as flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 9O kb, about 95 kb, or about 100 kb 5' and 3' of the incident polymorphic variant.
  • flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 9O kb, about 95 kb, or about 100 kb 5' and 3' of the incident polymorphic variant.
  • polymorphic variants associated with osteoarthritis are identified iteratively.
  • a first proximal polymorphic variant is associated with osteoarthritis using the methods described above and then another polymorphic variant proximal to the first proximal polymorphic variant is identified (e.g., publicly disclosed or discovered) and the presence or absence of an association of one or more other polymorphic variants proximal to the first proximal polymorphic variant with osteoarthritis is determined.
  • the methods described herein are useful for identifying or discovering additional polymorphic variants that may be used to further characterize a gene, region or loci associated with a condition, a disease (e.g., osteoarthritis), or a disorder.
  • allelotyping or genotyping data from the additional polymorphic variants may be used to identify a functional mutation or a region of linkage disequilibrium.
  • polymorphic variants identified or discovered within a region comprising the first polymorphic variant associated with osteoarthritis are genotyped using the genetic methods and sample selection techniques described herein, and it can be determined whether those polymorphic variants are in linkage disequilibrium with the first polymorphic variant. The size of the region in linkage disequilibrium with the first polymorphic variant also can be assessed using these genotyping methods.
  • methods for determining whether a polymorphic variant is in linkage disequilibrium with a first polymorphic variant associated with osteoarthritis and such information can be used in prognosis/diagnosis methods described herein.
  • Isolated Nucleic Acids Featured herein are isolated PAJ I2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid variants depicted in SEQ ID NO: 1-13, SEQ ID NO: 14-36 or referenced in Table B, and substantially identical nucleic acids thereof.
  • a nucleic acid variant may be represented on one or both strands in a double-stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous).
  • ADAMTS2 exists in two forms, a "long” form comprising a molecule approximately 130 kDa in length (e.g., SEQ ID NO: 21 for cDNA sequence and SEQ ID NO: 44 for amino acid sequence), and a "short” form comprising a molecule approximately 70 kDa in length (e.g., SEQ ID NO: 22 for cDNA sequence and SEQ ID NO: 45 for amino acid sequence).
  • a "long” form comprising a molecule approximately 130 kDa in length
  • SEQ ID NO: 44 for amino acid sequence
  • short comprising a molecule approximately 70 kDa in length
  • nucleic acid includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, siRNA and tRNA) and analogs of DNA or RNA, for ex-ample, by use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded and it is often double-stranded.
  • isolated or purified nucleic acid refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid.
  • isolated includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated.
  • An "isolated” nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 -b, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid fragments As used herein, the term “gene” refers to a nucleotide sequence that encodes a polypeptide. [0043] Also included herein are nucleic acid fragments. These fragments often have a nucleotide sequence identical to a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B, a nucleotide sequence substantially identical to a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B, or a nucleotide sequence that is complementary to the foregoing.
  • the nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B, and may encode a domain or part of a domain of a polypeptide. Sometimes, the fragment will comprises one or more of the polymorphic variations described herein as being associated w th osteoarthritis.
  • the nucleic acid fragment is often 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 10000, 15000, or 20000 base pairs in length.
  • a nucleic acid fragment that is complementary to a. nucleotide sequence identical or substantially identical to a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B and hybridizes to such a nucleotide sequence under stringent conditions is often referred to as a "probe.”
  • Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymo ⁇ hic site as described hereafter.
  • An example of a nucleic acid fragment is an oligonucleotide.
  • oligonucleotide refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides.
  • the backbone and nucleotides within an oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism.
  • Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.
  • Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABITM3900 High Throughput DNA Synthesizer and the EXPEDITETM 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, CA). Analogs and derivatives are exemplified in U.S. Pat. Nos.
  • Oligonucleotides may also be linked to a second moiety.
  • the second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage Ml 3 universal tail sequence), and others.
  • the second moiety may be a non- nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide.
  • Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like.
  • the second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.
  • Nucleic acid coding sequences may be used for diagnostic purposes for detection and control of polypeptide expression. Also, included herein are oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide. Antisense techniques and RNA interference techniques are known in the art and are described herein. [0048] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • hammerhead motif ribozyme molecules may be engineered that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequences or other nucleotide sequences referenced in Table B.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicte d structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including osteoarthritis, resulting from aberrant expression of a target gene described herein.
  • the nucleic acid sequence may be used in hybridization as. says of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or ' Northern blot analysis, in situ hybridization assays).
  • the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide. As addressed, infra, production of functionally impaired polypeptide is the cause of various disease states, such as osteoarthritis.
  • In situ hybridizations using polypeptide as a probe may be employed to predict problems related to osteoarthritis. Further, as indicated, infra, administration of human active polypeptide, recom-binantly produced as described herein, may be used to treat disease states related to functionally impaired polypeptide. Alternatively, gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.
  • nucleic acid vectors are provided herein, which contain a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a sxxbstantially identical sequence thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a p lasmid, cosmid, or viral vector.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.
  • a vector can include a PADI2, APOB, IL1RL2, ILIRLI , WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B in a form suitable for expression of an encoded target polypeptide or target nucleic acid in a host cell.
  • a “target polypeptide” is a polypeptide encoded by aPA2 I2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a substantially identical nucleotide sequence thereof.
  • the recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • the term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like.
  • Expression vectors can be introduced into host cells to produce target polypeptides, including fusion polypeptides.
  • Recombinant expression vectors can be designed for expression of target polypeptides in prokaryotic or eukaryotic cells.
  • target polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • T7 promoter regulatory sequences and T7 polymerase for example, T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S- transferase
  • MST glutathione S- transferase
  • maltose E binding polypeptide or polypeptide A, respectively, to the target recombinant polypeptide.
  • Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for target polypeptides.
  • fusion polypeptide expressed in a refroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks). [0057] Expressing the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide is often used to maximize recombinant polypeptide expression (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Presrs, San Diego, California 185: 119-128 (1990)).
  • Another strategy is to alter the nucleotide sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, Nucleic Acids Res. 20: 2111-2118 (1992)). Such alteration of nucleotide sequences can be carried out by standard DNA synthesis techniques.
  • the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements include an albumin promoter (liver-specific; Pinkert et al, Genes De ⁇ v. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol 43: 235-275 (1 -988)), promoters of T cell receptors (Winoto & Baltimore, EMBOJ.
  • promoters of immunoglobulins (Banerji et al, Cell 33: 129-140 (1983); Queen & Baltimore, Cell 33s 741-748 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477 (1989)), pancreas-specific promoters (Edlund et al, Science 230: 912- 916 (1985)), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166).
  • immunoglobulins (Banerji et al, Cell 33: 129-140 (1983); Queen & Baltimore, Cell 33s 741-748 (1983)
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne & Ruddle,
  • a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, Z OXL1, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B also may be cloned into an expression vector in an antisense orientation.
  • Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus.
  • host cells that include a PADI2, APOB, IL1RL2, IIL1RL1, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B within a recombinant expression vector or a fragment of such a nucleotide sequence which facilitate homologous recombination into a specific site of the host cell genome.
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a target polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vectors can be introduced into host cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • a host cell provided herein can be used to produce (i.e., express) a target polypeptide or a substantially identical polypeptide thereof. Accordingly, further provided are methods for producing a target polypeptide using host cells described herein.
  • the method includes culturing host cells into which a recombinant expression vector encoding a target polypeptide has been introduced in a suitable medium such that a target polypeptide is produced.
  • the metiiod further includes isolating a target polypeptide from the medium or the host cell.
  • cells or purified preparations of cells which include a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 transgene, or other transgene in Table B, or which otherwise misexpress target polypeptide.
  • Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells.
  • the cell or cells include a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 transgene or other transgene referenced in Table B (e.g., a heterologous form of & PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 gene or other gene referenced in Table B, such as a human gene expressed in non-human cells).
  • the transgene can be misexpressed, e.g., overexpressed or underexpressed.
  • the cell or cells include a gene which misexpress an endogenous target polypeptide (e.g., expression of a gene is disrupted, also known as a knockouf .
  • Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed alleles or for use in drug screening.
  • human cells e.g., a hematopoietic stem cells
  • cells or a purified preparation thereof e.g.
  • an endogenous PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B is under the control of a regulatory sequence that does not normally control the expression of the endogenous gene.
  • the expression characteristics of an endogenous gene within a cell e.g., a cell line or microorganism
  • an endogenous corresponding gene e.g., a gene which is "transcriptionally silent,” not normally expressed, or expressed only at very low levels
  • a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell.
  • Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, published on May 16, 1991.
  • Non-human transgenic animals that express a heterologous target polypeptide e.g., expressed from a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B, or substantially identical sequence thereof
  • a heterologous target polypeptide e.g., expressed from a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B, or substantially identical sequence thereof
  • Such animals are useful for studying the function and/or activity of a target polypeptide and for identifying and/or evaluating modulators of the activity of PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acids, other nucleic acids referenced in Table B, and encoded polypeptides.
  • a "transgenic animal” is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird (e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophil ⁇ mel ⁇ nog ⁇ ster), in which one or more of the cells of the animal includes a transgene.
  • a mammal e.g., a non-human primate such as chimpanzee, baboon, or macaque
  • an ungulate such as an equine, bovine, or caprine
  • a rodent such as a rat, a mouse, or an Israeli
  • a transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal.
  • a transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout).
  • a transgenic animal can be one in which an endogenous nucleic acid homologous to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene.
  • tissue-specific regulatory sequences can be operably linked to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B to direct expression of an encoded polypeptide to particular cells.
  • a transgenic founder animal can be identified based upon the presence of a PADU, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B in its genome and/or expression of encoded mRNA in tissues or cells of the animals.
  • a transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B can further be bred to other transgenic animals carrying other transgenes.
  • Target polypeptides can be expressed in transgenic animals or plants by introducing, for example, a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B into the genome of an animal that encodes the target polypeptide.
  • the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.
  • Target Polypeptides which are encoded by a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or a nucleotide sequence referenced in Table B (e.g., SEQ ID NO: 14-36 or a sequence referenced in Table B), or a substantially identical nucleotide sequence thereof.
  • PADI2 PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or a nucleotide sequence referenced in Table B (e.g., SEQ ID NO: 14-36 or a sequence referenced in Table B), or a substantially identical nucleotide sequence thereof.
  • polypeptide examples include proteins and peptides.
  • An "isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language "substantially free” means preparation of a target polypeptide having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-target polypeptide (also referred to herein as a "contaminating protein"), or of chemical precursors or non-target chemicals.
  • a target polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation.
  • target polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight.
  • the APOL3 polypeptide or polypeptide fragment has APOL3 biological activity, for example, apolipoprotein activity.
  • target polypeptide fragments may be a domain or part of a domain of a target polypeptide.
  • the polypeptide fragment may have increased, decreased or unexpected biological activity.
  • the polypeptide fragment is often 50 or fewer, 100 or fewer, or 200 or fewer amino acids in length, and is sometimes 300, 400, 500, 600, 700, or 900 or fewer amino acids in length.
  • the polypeptide fragment sometimes is amino acids 90-396 of SEQ ID NO: 53; amino acids 19-325 of SEQ ID NO: 54; or amino acids 1-196 of SEQ ID NO: 55. Shown in Table A below are examples of polypeptide fragments, where approximate amino acid positions are shown in parenthesis (e.g., a Pellino domain starts at about amino acid 3 and ends at about amino acid 412). Amino acid sequences can be accessed using information in Table B and in SEQ ID NO: 37-55. TABLE A
  • Interleukin 1 receptor-like 1 isoform 1 (SEQ ID NO: 40) is a member of the interleukin 1 receptor family with no known ligand (orphan receptor). ILIRLI exists in soluble (SEQ ID NO: 41-42) and transmembrane forms, suggesting that it may have ligand or ligand scavenging activity.
  • ILIRLI protein agents may be administered to treat or prevent the occurrence of OA.
  • ILIRLI protein agents include ILIRLI polypeptides or fragments thereof that have ILIRLI ligand activity (e.g., recombinant polypeptides of SEQ ID NO: 41-42).
  • ILIRLI protein agents include ILIRLI polypeptides or fragments thereof that have ILIRLI ligand scavenging activity (e.g., recombinant polypeptide of SEQ ID NO: 40).
  • Isolated ILIRLI polypeptides featured herein include the full-length polypeptide, the mature polypeptide (i.e., the polypeptide without the signal sequence MGFWILAILTILMYSTAA) or a polypeptide fragment containing a domain or part of a ILIRLI domain. The polypeptide fragment may have increased, decreased or unexpected biological activity.
  • ADAMTS2 polypeptides having an ADAMTS2 activity e.g., a zinc binding activity, a metalloprotease activity, a procollagen II processing or synthesis activity, or a collagen II synthesis activity in vitro or in vivo.
  • the polypeptides are ADAMTS2 proteins including at least one propeptide domain, at least one metalloproteinase domain, at least one disintegrin-like domain, at least one, two, three, and often four thrombospondin domains, and sometimes having a ADAMTS2 activity, e.g., aADAMTS2 activity as described herein.
  • ADAMTS2 polypeptides and fragments thereof often have biological activity, such as excising the N-propeptide of type II procollagens. Methods for monitoring and quantifying this biological activity are known (e.g., Colige et al., J. Biol. Chem. 270: 16724-16730 (1995)).
  • Human ADAMTS2 protein (SEQ ID NO: 44-45) includes a signal sequence of about 29 amino acids (from amino acid 1 to about amino acid 29 of SEQ ID NO: 44-45).
  • the ADAMTS2 protein without the signal sequence can be approximately 1182 amino acid residues in length (from about amino acid 30 to amino acid 1211 of SEQ ID NO: 44) or approximately 485 amino acid residues in length (from about amino acid 30 to amino acid 514 of SEQ ID NO: 45).
  • Human ADAMTS2 protein includes a "pro" region homologous to the reprolysin family propeptide, which is typically post- translationally cleaved upon conversion of the inactive (or pro-domain containing) protein to the catalytically active metalloprotease.
  • the prodomain region of human ADAMTS2 protein corresponds to about amino acids 30 to 251, 30 to 252, 30 to 253, 30 to 254, 30 to 255, 30 to 256, 30 to 257, 30 to 258 or 30 to 259 of SEQ ID NO: 44-45, where it is understood that the active form of ADAMTS2 does not contain the propeptide domain.
  • catalytically active mature protein can be approximately 960, 959, 958, 957, 956, 955, 954, 953 or 952 amino acids in length (from about amino acid 252, 253, 254, 255, 256, 257, 258, 259 or 260 to amino acid 1211 of SEQ ID NO: 44) or approximately 261, 260, 259, 258, 257,
  • Human ADAMTS2 contains the following regions or other structural features: a signal sequence at about amino acids 1-29 of SEQ ID NO: 44-45; a reprolysin family propeptide domain located at about amino acid residues 30 to 251, 30 to 252, 30 to 253, 30 to 254, 30 to 255, 30 to 256, 30 to 257, 30 to 258 or 30 to 259 of SEQ ID NO: 44-45; a zinc-metalloprotease catalytic domain at about amino acids 251 to 479, 252 to 479, 253 to 479, 254 to 479, 255 to 479, 256 to 479, 257 to 479, 258 to 479 or 259 to 479 of SEQ ID NO: 44-45; a disintegrin domain at about amino acids 480 to 560 of SEQ ID NO: 44; a cysteine-rich domain at about amino acids 618 to 722 of SEQ ID NO:
  • ADAMTS2 polypeptide fragments having activity are selected from amino acids 252-1211, 253-1211, 254-1211, 255-1211, 256-1211, 257-1211, 258-1211, 259-1211 or 260-1211 of SEQ ID NO: 4, where it is understood that the active form of ADAMTS2 does not contain the propeptide domain.
  • Substantially identical target polypeptides may depart from the amino acid sequences of target polypeptides in different manners.
  • conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of target polypeptides.
  • a "conservative amino acid substitution” is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a "non- essential" amino acid is one that can be altered without abolishing or substantially altering the biological function of a target polypeptide, whereas altering an "essential” amino acid abolishes or substantially alters the biological function of a target polypeptide.
  • Amino acids that are conserved among target polypeptides are typically essential amino acids.
  • the polypeptide includes one or more non-synonymous polymorphic variants associated with osteoarthritis, as described above (e.g., a threonine encoded by rsl367117 in an APOB polypeptide).
  • target polypeptides may exist as chimeric or fusion polypeptides.
  • a target “chimeric polypeptide” or target “fusion polypeptide” includes a target polypeptide linked to a non-target polypeptide.
  • a “non-target polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the target polypeptide, which includes, for example, a polypeptide that is different from the target polypeptide and derived from the same or a different organism.
  • the target polypeptide in the fusion polypeptide can correspond to an entire or nearly entire target polypeptide or a fragment thereof.
  • the non-target polypeptide can be fused to the N-terminus or C-terminus of the target polypeptide.
  • Fusion polypeptides can include a moiety having high affinity for a ligand.
  • the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the target polypeptide is fused at the N- or C-terminus to a string of histidine residues.
  • Such fusion polypeptides can facilitate purification of recombinant target polypeptide.
  • Fusion moiety e.g., a GST polypeptide
  • a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B, or a substantially identical nucleotide sequence thereof can be cloned into an expression vector such that the fusion moiety is linked in-frame to the target polypeptide.
  • the fusion polypeptide can be a target polypeptide containing a heterologous signal sequence at its N-terminus.
  • expression, secretion, cellular internalization, and cellular localization of a target polypeptide can be increased through use of a heterologous signal sequence.
  • Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).
  • a serum polypeptide e.g., an IgG constant region or human serum albumin.
  • Target polypeptides can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these target polypeptides can be used to affect the bioavailability of a substrate of the target polypeptide and may effectively increase target polypeptide biological activity in a cell.
  • Target fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a target polypeptide; (ii) mis-regulation of the gene encoding the target polypeptide; and (iii) aberrant post- translational modification of a target polypeptide.
  • target polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify target polypeptide ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of a target polypeptide with a substrate.
  • polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12 -18;310(5973):105-11).
  • a relative short fragment can be synthesized by use of a peptide synthesizer.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g- Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
  • amino acid can be D (dextrorotary) or L (levorotary).
  • Polypeptides and polypeptide fragments sometimes are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like.
  • Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.
  • a detectable label such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.
  • chemically modified derivatives of polypeptides that can provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see e.g., U.S. Pat. No: 4,179,337.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • polymers should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art (e.g., EP 0 401 384 (coupling PEG to G-CSF) and Malik et al. (1992) Exp Hematol. September;20(8): 1028-35 (pegylation of GM-CSF using tresyl chloride)).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • the attachment sometimes is at an amino group, such as attachment at the N-terminus or lysine group.
  • Proteins can be chemically modified at the N-terminus.
  • polyethylene glycol as an illustration of such a composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N- terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • Nucleotide sequences and polypeptide sequences that are substantially identical to a PADI2, APOB, IL1R 2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B and the target polypeptide sequences encoded by those nucleotide sequences, respectively, are included herein.
  • the term "substantially identical" as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively.
  • nucleotide sequences or polypeptide sequences that are 55% or more, 60%> or more, 65% or more, 70% or more, 75%> or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3%> or 4% variability) identical to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence, or other nucleotide sequence referenced in Table B, or the encoded target polypeptide amino acid sequences.
  • One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.
  • Calculations of sequence identity are often performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison pu ⁇ oses).
  • the length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50%> or more, often 60%) or more, and more often 70% or more, 80% or more, 90% or more, or 100% of the length of the reference sequence.
  • nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively are then compared among the two sequences. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotides or amino acids are deemed to be identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences. [0088] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. Mol. Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the http address www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http address www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a set of parameters often used is a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions.
  • stringent conditions refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. , 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used.
  • An example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • stringency conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • An example of a substantially identical nucleotide sequence to a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B.
  • nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes more than 75% or more, 80%) or more, or 85% or more identical to, and often more than 90% or more and 95%o or more identical to a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B.
  • Nucleotide sequences in SEQ ID NO: 1-13 or referenced in Table B and amino acid sequences of encoded polypeptides can be used as "query sequences" to perform a search against public databases to identify other family members or related sequences, for example.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al, J. Mol. Biol. 215: 403-10 (1990).
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(17): 3389-3402 (1997).
  • default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used (see the http address www.ncbi.nlm.nih.gov).
  • SNPs in a sequence substantially identical to a sequence in SEQ ID NO: 1-13 or referenced in Table B can be identified at nucleotide positions that match (i.e., align) with nucleotides at SNP positions in each nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B.
  • insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.
  • Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring.
  • Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
  • Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50% or more, about 55% or more, often about 70-75% or more or about 80-85%) or more, and sometimes about 90-95% or more identical to the amino acid sequences of target polypeptides or a fragment thereof. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B or a fragment of this sequence.
  • nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B can further be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B.
  • substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a target polypeptide in a particular expression system.
  • the nucleic acid can be one in which one or more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae), human (e.g., 293 cells), insect, or rodent (e.g., hamster) cells.
  • Methods for prognosing and diagnosing osteoarthritis are included herein. These methods include detecting the presence or absence of one or more polymorphic variations in a nucleotide sequence associated with osteoarthritis, such as variants in or around the loci set forth herein, or a substantially identical sequence thereof, in a sample from a subject, where the presence of a polymorphic variant described herein is indicative of a risk of osteoarthritis. Determining a risk of osteoarthritis sometimes refers to determining whether an individual is at an increased risk of osteoarthritis (e.g., intermediate risk or higher risk).
  • nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1- 13 or referenced in Table B; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more
  • polymorphic variants at the positions described herein are detected for determining a risk of osteoarthritis, and polymorphic variants at positions in linkage disequilibrium with these positions are detected for determining a risk of osteoarthritis.
  • the terms "SEQ ID NO: 1-13" and other nucleotide sequences "referenced in Table B” refers to individual sequences in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any individual sequence referenced in Table B, or any individual nucleic acid sequence provided in the sequence listing, including SEQ ID NO: 14-36 each sequence being separately applicable to embodiments described herein.
  • Risk of osteoarthritis sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. Risk often is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating risk based upon patient data are well known (see, e.g., Agresti, Categorical Data Analysis, 2nd Ed. 2002. Wiley). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations.
  • determining the presence of a combination of two or more polymorphic variants associated with osteoarthritis in one or more genetic loci (e.g., one or more genes) of the sample is determined to identify, quantify and/or estimate, risk of osteoarthritis.
  • the risk often is the probability of having or developing osteoarthritis.
  • the risk sometimes is expressed as a relative risk with respect to a population average risk of osteoarthritis, and sometimes is expressed as a relative risk with respect to the lowest risk group.
  • Such relative risk assessments often are based upon penetrance values determined by statistical methods, and are particularly useful to clinicians and insurance companies for assessing risk of osteoarthritis (e.g., a clinician can target appropriate detection, prevention and therapeutic regimens to a patient after determining the patient's risk of osteoarthritis, and an insurance company can fine tune actuarial tables based upon population genotype assessments of osteoarthritis risk).
  • Risk of osteoarthritis sometimes is expressed as an odds ratio, which is the odds of a particular person having a genotype has or will develop osteoarthritis with respect to another genotype group (e.g., the most disease protective genotype or population average).
  • the determination is utilized to identify a subject at risk of osteoarthritis.
  • two or more polymorphic variations are detected in two or more regions in human genomic DNA associated with increased risk of osteoarthritis, such as a locus containing a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 or other locus referenced in Table B, for example.
  • a locus containing a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 or other locus referenced in Table B for example.
  • 3 or more, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100 or more polymorphic variants are detected in the sample.
  • polymorphic variants are detected in a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 region or other region referenced in Table B, for example.
  • polymorphic variants are detected at two or three positions in a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B.
  • polymorphic variants are detected at other genetic loci (e.g., the polymorphic variants can be detected in a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B in addition to other loci or only in other loci), where the other loci include but are not limited to those described in patent applications 60/559,011; 60/559,202; 60/559,203; 60/559,042; 60/559,275; 60/559,040 and 60/559,225, each of which is entitled “Methods for Identifying Risk of Osteoarthritis and Treatments Thereof," each of which was filed on 1 April 2004 and each of which is incorporated herein by reference in its entirety in jurisdictions allowing incorporation by reference.
  • Results from prognostic tests may be combined with other test results to diagnose osteoarthritis.
  • prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to osteoarthritis, the patient sample is analyzed, and the results of the analysis may be utilized to diagnose osteoarthritis.
  • osteoarthritis diagnostic method can be developed from studies used to generate prognostic methods in which populations are stratified into subpopulations having different progressions of osteoarthritis.
  • prognostic results may be gathered, a patient's risk factors for developing osteoarthritis (e.g., age, weight, occupational history, race, diet) analyzed, and a patient sample may be ordered based on a determined predisposition to osteoarthritis.
  • the nucleic acid sample typically is isolated from a biological sample obtained from a subject.
  • nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue.
  • the nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2.
  • the term "subject” refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms.
  • the nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.
  • the presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymo ⁇ hic variant in a sample.
  • primer extension methods For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,1 '4, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos.
  • RFLP restriction fragment length polymorphism
  • SSCP single strand conformation polymorphism detection
  • Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation.
  • PCR polymerase chain reaction
  • One oligonucleotide primer is complementary to a region 3' of the polymorphism and the other is complementary to a region 5' of the polymorphism.
  • a PCR primer pair may be used in methods disclosed in U.S. Pat. Nos.
  • PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP ® Systems available from Applied Biosystems.
  • oligonucleotide primers based upon a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B using knowledge available in the art.
  • PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B using knowledge available in the art.
  • an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation.
  • the term "adjacent" refers to the 3' end of the extension oligonucleotide being often 1 nucleotide from the 5' end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid.
  • the extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos.
  • a microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample.
  • a microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos.
  • the microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions.
  • the oligonucleotides may also be linked to the solid support directly or by a spacer molecule.
  • a microarray may comprise one or more oligonucleotides complementary to a polymorphic site set forth herein.
  • a kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample.
  • a kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B or a substantially identical sequence thereof, where the fragment includes a polymorphic site.
  • the kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664.
  • the kit often comprises an elongation oligonucleotide that hybridizes to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B in a nucleic acid sample adjacent to the polymorphic site.
  • the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide.
  • chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like.
  • the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides.
  • Kits optionally include buffers, vials, microtiter plates, and instructions for use.
  • a subject homozygous for an allele associated with an increased risk of osteoarthritis is at a comparatively high risk of osteoarthritis
  • a subject heterozygous for an allele associated with an increased risk of osteoarthritis is at a comparatively intermediate risk of osteoarthritis
  • a subject homozygous for an allele associated with a decreased risk of osteoarthritis is at a comparatively low risk of osteoarthritis.
  • a genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.
  • an epitope associated with increased risk of osteoarthritis in the polypeptide e.g. , an epitope comprising a valine at position 245 in an IR1LR1 polypeptide.
  • Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype as a particular treatment regimen may exert a differential effect depending upon the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects).
  • the following is an example of a pharmacogenomic embodiment.
  • a particular treatment regimen can exert a differential effect depending upon the subject's genotype.
  • a candidate therapeutic exTiibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction)
  • such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele.
  • a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but is comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele
  • the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.
  • the methods described herein are applicable to pharmacogenomic methods for preventing, alleviating or treating osteoarthritis.
  • a nucleic acid sample from an individual may be subjected to a prognostic test described herein.
  • a treatment or preventative regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing osteoarthritis assessed by the methods described herein.
  • certain embodiments are directed to a method for reducing osteoarthritis in a subject, which comprises: detecting the presence or absence of a polymorphic variant associated with osteoarthritis in a nucleotide sequence in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID
  • predisposition results may be utilized in combination with other test results to diagnose osteoarthritis .
  • Certain preventative treatments often are prescribed to subjects having a predisposition to ost oarthritis and where the subject is diagnosed with osteoarthritis or is diagnosed as having symptoms indicative of an early stage of osteoarthritis.
  • the treatment sometimes is preventative (e.g., is prescribed or administered to reduce the probability that osteoarthritis arises or progresses), sometimes is therapeutic, and sometimes delays, alleviates or halts the progression of osteoarthritis.
  • Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of osteoarthritis is prescribed and/or administered. For example, the treatment often is directed to decreasing pain and improving joint movement.
  • OA treatments include exercises to keep joints flexible and improve muscle strength.
  • Different medications to control pain including corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs, e.g., Voltaren); cyclooxygenase-2 (COX-2) inhibitors (e.g-. , Celebrex, Vioxx, Mobic, and Bextra); monoclonal antibodies (e.g., Remicade); tumor necrosis factor inhibitors (e.g., Enbrel); or injections of glucocorticoids, hyaluronic acid or chondrotin sulfate into joints that are inflamed and not responsive to NSAIDS.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • COX-2 cyclooxygenase-2
  • COX-2 cyclooxygenase-2
  • monoclonal antibodies e.g., Remicade
  • tumor necrosis factor inhibitors e.g., Enbrel
  • chondroitin sulfate also may be used as a therapeutic, as it may increase hyaluronic acid levels and viscosity of synovial fluid, and decrease collagenase levels in synovial fluid.
  • glucosamine can serve as an OA therapeutic as delivering it into joints may inhibit enzymes involved in cartilage degradation and enhance the production of hyaluronic acid.
  • acetaminophen may be used.
  • Other treatments include: heat/cold therapy for temporary pain relief; joint protection to prevent strain or stress on painful joints; surgery to relieve chronic pain in damaged joints; and weight control to prevent extra stress on weight-bearing joints.
  • osteoarthritis preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing osteoarthritis or those in an early stage of osteoarthritis), provided herein is a method for preventing or reducing the risk of developing osteoarthritis in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with osteoarthritis at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject with a predisposition to osteoarthritis, whereby the presence of the polymorphic variation is indicative of a predisposition to osteoarthritis in the subject; and (c) if such a predisposition is identified, providing the subject with information about methods or products to prevent or reduce osteoarthritis or to delay the onset of osteoarthritis.
  • Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition which comprises: (a) detecting the presence or absence of a polymorphic variation associated with osteoarthritis at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with osteoarthritis; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.
  • Pharmacogenomics methods also may be used to analyze and predict a response to osteoarthritis treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to osteoarthritis treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
  • the response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regimen (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen. [0116] The tests described herein also are applicable to clinical drug trials.
  • One or more polymorphic variants indicative of response to an agent for treating osteoarthritis or to side effects to an agent for treating osteoartliritis may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
  • another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug.
  • the polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (ii) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; (iii) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B; and (iv) a fragment of
  • step (c) optionally comprises administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
  • Also provided herein is a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product, which comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with osteoarthritis at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with osteoarthritis; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by t ie subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above.
  • compositions comprising a cell from a subject having osteoarthritis or at risk of osteoarthritis and one or more molecules specifically directed and targeted to a nucleic acid comprising a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence, other nucleotide sequence referenced in Table B, or an encoded amino acid sequence referenced herein.
  • a nucleic acid comprising a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence, other nucleotide sequence referenced in Table B, or an encoded amino acid sequence referenced herein.
  • Such directed molecules include, but are not limited to, a compound that binds to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence, or other nucleotide sequence referenced in Table B, or encoded amino acid sequence; a RNAi or siRNA molecule having a strand complementary or substantially complementary to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B (e.g., hybridizes to a PADI2, APOB, IL1JRL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nu
  • the antibody selectively binds to an epitope comprising an amino acid encoded by rsl367117, rsl041973 and rs398829.
  • the osteoarthritis directed molecule interacts with, a nucleic acid or polypeptide variant associated with osteoarthritis, such as variants referenced herein.
  • the osteoarthritis directed molecule interacts with a polypeptide involved ir a signal pathway of a polypeptide encoded by a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAA4TS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a nucleic acid comprising such a nucleotide sequence.
  • Compositions sometimes include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response.
  • Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or gamma- interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in- water emulsion (e.g: , an emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation); TitreMax; detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene (
  • compositions are useful for generating an immune response against osteoarthritis directed molecule (e.g., an " HLA-binding subsequence within a polypeptide encoded by a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence).
  • an immune response against osteoarthritis directed molecule e.g., an " HLA-binding subsequence within a polypeptide encoded by a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence.
  • a peptide having an amino acid subsequence of a polypeptide encoded by a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response.
  • the peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide.
  • the cell may be in a group of cells cultured in vitro or in a tissue maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human).
  • a composition comprises a component from a cell such as a nu leic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example.
  • a nu leic acid molecule e.g., genomic DNA
  • the aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in therapeutics described hereafter. Certain osteoarthritis directed molecules are described in greater detail below.
  • Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al., J. Med. Chem.37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound” library methods; and synthetic library methods using affinity chromatography selection..
  • Biolibrary and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)).
  • Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al., J. Med. Chem.
  • a compound sometimes alters expression and sometimes alters activity of a polypeptide target and may be a small molecule.
  • Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • peptides e.g., peptoids
  • amino acids amino acid analogs
  • polynucleotides polynucleotide analogs
  • nucleotides nucleotide analogs
  • an "antisense” nucleic acid refers to a nucleotide sequence complementary to a "sense" nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to a portion thereof or a substantially identical sequence thereof.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence (e.g., 5' and 3' untranslated regions in SEQ ID NO: 1-13 or a nucleotide sequence referenced in Table E5).
  • a nucleotide sequence e.g., 5' and 3' untranslated regions in SEQ ID NO: 1-13 or a nucleotide sequence referenced in Table E5
  • An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence (e.g., SEQ ID NO: 1-13, SEQ ID NO: 14-36 or a nucleotide sequence referenced in Table B), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • the antisense nucleic acids which include the ribozymes described hereafter, can be designed to target a PADI2, APOB, IL1JRL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence, often a variant associated with osteoarthritis, or a substantially identical sequence thereof.
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • Antisense nucleic acid also can be produced biologically using an expression vector into whic-h a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • antisense nucleic acids When utilized as therapeutics, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation.
  • antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.
  • Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules.
  • nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15: 6625-6641 (1987)).
  • Antisense nucleic acid molecules can also comprise a 2'-o- methylribonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 ( 1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)).
  • an antisense nucleic acid is a ribozyme.
  • a ribozyme having specificity for a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No.
  • a derivative of a Xetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S . Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5, 116,742).
  • target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261 : 1411-1418 (1993)).
  • Osteoarthritis directed molecules include in certain embodiments nucleic acids that can form triple helix structures with a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide.
  • Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a nucleotide sequence referenced herein or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992).
  • Potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3 ', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Osteoarthritis directed molecules include RNAi and siRNA nncleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. See, e.g., Fire et al., US Patent Number 6,506,559; Tuschl et al. PCT International Publication No .
  • RNA interference RNA interference
  • siRNA refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene.
  • siRNA refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the silRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence.
  • an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.
  • the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al,. Methods 26:199-213 (2002). Initially, 5' or 3' UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(N19)TT (N, an nucleotide), and regions with approximately 30%> to 70% G/C-content (often about 50% G/C-content) often are selected..
  • the sequence of the sense siRNA sometimes corresponds to (N19) TT or N21 (position 3 to 23 of the 23-nrt motif), respectively. In the latter case, the 3' end of the sense siRNA often is converted to TT. T ⁇ ie rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
  • the antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23-nt motif is not recognized sequence- specifically by the antisense siRNA, the 3'-most nucleotide residue of the antisense siRNA can be chosen deliberately.
  • the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) often is complementary to the targeted sequence.
  • TT often is utilized.
  • Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.
  • the sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof.
  • the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stra-nded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in lengtli.
  • the siRNA sometimes is about 21 nucleotides in length.
  • siRNA nucleic acids can be altered to form modified nucleic acid molecules.
  • the nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al, Bioorganic & Medicinal Chemistry 4 (1): 5-23 (1996)).
  • peptide nucleic acid refers to a nucleic acid mimic such as a DNA mim-ic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only trie four natural nucleobases are retained.
  • the neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al. , (1996) supra and Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93: 14670-675 (1996).
  • PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of " gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as "artificial restriction enzymes” when used in combination with other enzymes, (e.g., SI nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et al., (1996) supra; Perry-O'Keefe supra).
  • oligonucleotides may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lema tre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across cell membranes see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lema tre et al., Proc. Natl. Aca
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al., Bio-Techniques 6: 958-976 C 1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988) ).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross- linking agent, transport agent, or hybridization-triggered cleavage agent).
  • molecular beacon oligonucleotide primer and probe mole ules having one or more regions complementary to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, AD MTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B, or a substantially identical sequence thereof, two complementary regions, one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample.
  • Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Patent No. 5,854,033; Nazarenko et al., U.S. Patent No. 5,866,336, and Livak et al., U.S. Patent 5,876,930.
  • antibody refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • An antibody sometimes is a polyclonal, monoclonal, recombinant (e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody.
  • An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.
  • a full-length polypeptide or antigenic peptide fragment encoded by a nucleotide sequence referenced herein can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like.
  • An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence referenced herein, or substantially identical sequence thereof, and encompasses an epitope.
  • Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.
  • Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity.
  • an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production.
  • the antibody may bind an epitope on any domain or region on polypeptides described herein.
  • chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al U.S. Patent No. 4,816,567; Cabilly et al European Patent Application 125,023; Better et al., Science 240: 1041-1043 (1988); Liu et al., Proc. Natl.
  • An antibody can be a single chain antibody.
  • a single chain antibody (scFV) can be engineered (see, e.g., Colcher et al., Ann. N Y Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)).
  • Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.
  • Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor.
  • an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor (e.g., it has a mutagenized or deleted Fc receptor binding region).
  • an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • Antibody conjugates can be used for modifying a given biological response.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF gran
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, for example.
  • An antibody e.g., monoclonal antibody
  • An antibody can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling).
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and.
  • an antibody can be utilized as a test molecule for determining whether it can treat osteoarthritis, and as a therapeutic for administration to a subject for treating osteoarthritis.
  • An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue prepajrations, whole cells, preferably living cells, lysed cells, or cell fractions.
  • a "target molecule” as used herein refers to a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleotide sequence referenced in Table B, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing.
  • the methods also comprise determining the presence or absence off an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a cai didate osteoarthritis therapeutic.
  • Test molecules and candidate therapeutics include, but are not limited to, compounds. , antisense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a PALDI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleotide sequence or other nucleotide sequence referenced in Table B , or a substantially identical sequence or fragment thereof, and immunotherapeutics (e.g., antibodies and HLA-presented polypeptide fragments).
  • immunotherapeutics e.g., antibodies and HLA-presented polypeptide fragments.
  • a test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system.
  • a “modulator” may agonize (i.e., up-regulates) or antagonize (i.e., down-regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing (e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing (e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis of pro-polypeptides).
  • DNA processing e.g., DNA methylation or DNA repair
  • RNA transcription and/or RNA processing e.g., removal of intronic sequences
  • a modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of osteoarthritis).
  • catalysis e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization
  • an effect upon a cellular event e.g., effecting progression of osteoarthritis.
  • any modulator may be utilized, such as a peptidyl arginine deiminase modulator (e.g., PADI2 likely is a peptidyl arginine deiminase) described in WO-09851784 and WO0244360A2 or an apolipoprotein (e.g., APOB includes an apolipoprotein domain) modulatory compound (e.g., WO-2004017969, WO- 03002533, US 6,369,075, WO-02098839, WO-02098871, WO-00177077, WO-00153260, WO- 00105767), antibody (e.g., WO-9600903A1, US 6,309,844 and US 5,330,910) or antisense molecule (e.g., WO03011887A2 and WO03097662A1).
  • a peptidyl arginine deiminase modulator e.g.,
  • system refers to a cell free in vitro environment and a cell-based environment such as a collection of cells, a tissue, an organ, or an organism.
  • a system is "contacted-" with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal.
  • interaction refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.
  • titrametric, acidimetric, radiometric, NMR, mono layer, polarographic, spectrophotometric, fluorescent, and ESR assays probative of a target molecule interaction may be utilized.
  • G protein-coupled receptor assays are known, for example, and are described in WO-0242461 and WO-04013285.
  • ADAMTS2 activity and/or ADAMTS2 interactions can be detected and quantified using assays known in the art.
  • an immunoprecipitation assay or a kinase activity assay that employs a kinase-inactivated MEK can be utilized.
  • kinase inactivated MEKs are known in the art, such as a MEK that includes the mutation K97M.
  • mammalian cells e.g., COS or NIH-3T3
  • the cells are co- transfected with oncogenic RAS or SRC or both.
  • Oncogenic RAS or SRC activates ADAMTS2 kinase activity.
  • ADAMTS2 is immunoprecipitated from cell extracts using a monoclonal antibody (e.g., 9E 10) or a polyclonal antibody (e.g., from rabbit) specific for a unique peptide from ADAMTS2.
  • ADAMTS'2 is then resuspended in assay buffer containing GST-Mekl or GST-Mek2 and/or GST-ERK2.
  • [gamma 32 P] ATP can be added to detect and/or quantify phosphorylation activity. Samples are incubated for 5-30 minutes at 30°C, and then the reaction is terminated by addition of EDTA. TJhe samples are centrifuged and the supernatant fractions are collected.
  • Phosphorylation activity is detected using one of two methods: (i) activity of GST-ERK2 kinase can be measured using MBP (myelixi basic protein, a substrate for ERK) as substrate, or (ii) following incubation of immunoprecipitated ADAMTS2 in reaction buffer containing GST-ERK and [gamma 32 P] ATP, transfer of labeled ATP to kinase-dead ERK can be quantified by a phosphor-imager or densitometer following PAGE separation of polypeptide products (phosphorylated and non-phosphorylated forms).
  • MBP myelixi basic protein, a substrate for ERK
  • ADAMTS2 includes a domain having metalloprotease activity, and modulators of such activity are known.
  • modulators are set forth in WO03063762A2; WO-09937625; WO-09918076; WO-09838163; WO-09837877; WO9947550A1; WO0177092AJ; WO0040577A1; W09942436A1; W09838163A1; W09837877A1; WO04014379A1; WO03106381A2; WO03014098A1; WO03014092A1 and WO02096426A1.
  • Test molecule/target molecule interactions can be detected and/or quantified using assays known in the art.
  • an interaction can be determined by labeling the test molecule and/or the target molecule, where the label is covalently or non-covalently attached to the test molecule or target molecule.
  • the label is sometimes a radioactive molecule such as I, I, S or H, which can be detected by direct counting of radioemission or by scintillation counting.
  • enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product.
  • presence or absence of an interaction can be determined without labeling.
  • a microphysiometer e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • cells typically include a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleotide sequence referenced in Table B, an encoded polypeptide, or substantially identical nucleic acid or polypeptide thereof, and are often of mammalian origin, although the cell can be of any origin.
  • Whole cells, cell homogenates, and cell fractions e.g., cell membrane fractions
  • Wh-ere interactions between a test molecule with a target polypeptide are monitored, soluble and/or membrane bound forms of the polypeptide may be utilized.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether) n , 3-[(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate
  • test molecule 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl-N,N-dimethyl-3-ammonio-l-propane sulfonate.
  • FET fluorescence energy transfer
  • a fluorophore label on a first, "donor” molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, “acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the "donor” polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues.
  • Labels are chosen that emit different wavelengths of light, such that the "acceptor” molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal.
  • An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
  • determining the presence or absence of an interaction between a test molecule and a target molecule can be effected by monitoring surface plasmon resonance (see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et al, Curr. Opin. Struct. Biol 5: 699-705 (1995)).
  • “Surface plasmon resonance” or “biomolecular interaction analysis (BIA)” can be utilized to detect biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
  • the target molecule or test molecules are anchored to a solid phase, facilitating the detection of target molecule/test molecule complexes and separation of the complexes from free, uncomplexed molecules.
  • the target molecule or test molecule is immobilized to the solid support.
  • the target molecule is anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • the attachment between a test molecule and/or target molecule and the solid support may be covalent or non-covalent (see, e.g., U.S.
  • the solid support may be one or more surfaces of the system, such as one or more surfaces in each well of a microtiter plate, a surface of a silicon wafer, a surface of a bead (see, e.g., Lam, Nature 354: 82-84 (1991)) that is optionally linked to another solid support, or a channel in a microfluidic device, for example.
  • Types of solid supports, linker molecules for covalent and non-covalent attachments to solid supports, and methods for immobilizing nucleic acids and other molecules to solid supports are well known (see, e.g., U.S. Patent Nos.
  • target molecule may be immobilized to surfaces via biotin and streptavidin.
  • biotinylated target polypeptide can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • a target polypeptide can be prepared as a fusion polypeptide.
  • glutathione-S-transferase/target polypeptide fusion can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivitized microtiter plates, which are then combined with a test molecule under conditions conducive to complex formation (e.g. , at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, or the matrix is immobilized in the case of beads, and complex formation is determined directly or indirectly as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of target molecule binding or activity is determined using standard techniques.
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that a significant percentage of complexes formed will remain immobilized to the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of manners. Where the previously non-immobilized component is pre- labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect lab l can be used to detect complexes anchored on the surface, e.g., by adding a labeled antibody specific for the immobilized component, where the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody.
  • an assay is performed utilizing antibodies that specifically bind target molecule or test molecule but do not interfere with binding of the target molecule to the test molecule. Such antibodies can be derivitized to a solid support, and unbound target molecule may be immobilized by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Cell free assays also can be conducted in a liquid phase.
  • reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, e.g., Rivas, G, and Minton, Trends Biochem Sci Aug; 18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, eds. Current Protocols in Molecular Biology , J. Wiley: New York (1999)); and immunoprecipitation (see, e.g, Ausubel et al, eds., supra).
  • differential centrifugation see, e.g., Rivas, G, and Minton, Trends Biochem Sci Aug; 18(8): 284-7 (1993)
  • chromatography gel filtration chromatography, ion-exchange chromatography
  • electrophoresis see, e.g., Ausubel et al, eds
  • a cell or cell free mixture is contacted with a candidate compound and the expression of target mRNA or target polypeptide is evaluated relative to the level of expression of target mRNA o>r target polypeptide in the absence of the candidate compound.
  • the candidate compound is identified as an agonist of target mRNA or target polypeptide expression.
  • the candidate compound is identified as an antagonist or inhibitor of target mRNA or target polypeptide expression.
  • the level of target rnRNA or target polypeptide expression can be determined by methods described herein.
  • binding partners that interact with a target molecule are detected.
  • the target molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides in vivo, and these interacting molecules are referred to herein as "binding partners.”
  • Binding partners can agonize or antagonize target molecule biological activity.
  • test mo lecules that agonize or antagonize interactions between target molecules and binding partners can be useful as therapeutic molecules as they can up-regulate or down-regulated target molecule activity in vivo and thereby treat osteoarthritis.
  • Binding partners of target molecules can be identified by methods known in the art.
  • binding partners may be identified by lysing cells and analyzing cell lysates by electrophoretic techniques.
  • a two-hybrid assay or three-hybrid assay can be utilized (see, e.g., U.S. Patent No. 5,283,317; Zervos et al, Cell 72:223-232 (1993); Madura et al, J. Biol. Chem. 2&8: 12046- 12054 (1993); Bartel et al, Biotechniques 14: 920-924 (1993); Iwabuchi et al, Oncogene 8: 1693-1696 (1993); and Brent WO94/10300).
  • a two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay often utilizes two different DNA constructs.
  • a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B (sometimes referred to as the "bait") is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a known transcription factor e.g., GAL-4
  • a DNA sequence from a library of DNA sequences that encodes a potential binding partner (sometimes referred to as the "prey") is fused to a gene that encodes an activation domain of the known transcription factor.
  • a PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 or APOL3 nucleic acid or other nucleic acid referenced in Table B can be fused to the activation domain. If the "bait” and the "prey” molecules interact in vivo, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene ⁇ .
  • a reaction mixture containing the target molecule and the binding partner is prepared, under conditions and for a time sufficient to allow complex formation.
  • the reaction mixture often is provided in the presence or absence of the test molecule.
  • the test molecule can be included initially in the reaction mixture, or can be added at a time subsequent to the addition of the target molecule and its binding partner. Control reaction mixtures are incubated without the test molecule or with a placebo.
  • Formation of any complexes between t e target molecule and the binding partner then is detected. Decreased formation of a complex in the rea-ction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule antagonizes target molecule/binding partner complex formation. Alternatively, increased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule agonizes target molecule/binding partner complex formation. In another embodiment, complex formation of target molecule/binding partner can be compared to complex formation of mutant target molecule/binding partner (e.g., amino acid modifications in a target polypeptide).
  • mutant target molecule/binding partner e.g., amino acid modifications in a target polypeptide
  • the assays can be conducted in a heterogeneous or homogeneous format.
  • target molecule and/or the binding partner are immobilized to a solid phase, and complexes are detected on the solid phase at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase.
  • the order of addition of reactants can be varied to obtain different information about the molecules being tested. For example, test compounds that agonize target molecule/binding partner interactions can be identified by conducting the reaction in the presence of the test molecule in a competition format.
  • test molecules that agonize preformed complexes e.g., molecules with higher binding constants that displace one of the components from the complex
  • the target molecule or the binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly.
  • the anchored molecule can be immobilized by non-covalent or covalent attachments.
  • an immobilized antibody specific for the molecule to be anchored can be used to anchor the molecule to the solid surface. The partner of the immobilized species is exposed to the coated surface with or without the test molecule.
  • any complexes formed will remain immobilized on the solid surface.
  • the detection of label immobilized on the surface is indicative of complex.
  • an indirect label can be used to detect complexes anchored to t ie surface; e.g., by using a labeled antibody specific for the initially non-immobilized species.
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of test molecule, where the reaction products are separated from unreacted components, and the complexes are detected (e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes).
  • test compounds that inhibit complex or that disrupt preformed complexes can be identified.
  • a homogeneous assay can be utilized. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared.
  • One or both of the target molecule or binding partner is labeled, and the signal generated by the label(s) is quenched upon complex formation (e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays).
  • Addition of a test molecule that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target molecule/binding partner complexes can be identified.
  • Candidate therapeutics for treating osteoarthritis are identified from a group of test molecules that interact with a target molecule.
  • Test molecules are normally ranked according to the degree with which they modulate (e.g., agonize or antagonize) a function associated with the target molecule (e.g., DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or biological function/activity), and then top ranking modulators are selected. Also, pharmacogenomic information described herein can determine the rank of a modulator. The top 10% of ranked test molecules often are selected for further testing as candidate therapeutics, and sometimes the top 15%, 20%, or 25% of ranked test molecules are selected for further testing as candidate therapeutics.
  • Candidate therapeutics typically are formulated for administration to a subject.
  • Formulations and pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier one or more target molecule modulators.
  • the modulator often is a test molecule identified as having an interaction with a target molecule by a screening method described above.
  • the modulator may be a compound, an antisense nucleic acid, a ribozyme, an antibody, or a binding partner.
  • formulations may comprise a target polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier, where the polypeptide or fragment sometimes has an APOL3 biological activity (e.g., apolipoprotein activity), and sometimes includes all or part of an apolipoprotein domain.
  • APOL3 biological activity e.g., apolipoprotein activity
  • Formulations or pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier, a compound, an antisense nucleic acid, a ribozyme, an antibody, a binding partner that interacts with an ADAMTS2 polypeptide, ADAMTS2 nucleic acid, or a fragment thereof.
  • the formulated molecule may be one that is identified by a screening method described above.
  • formulations may comprise a ADAMTS2 polypeptide or fragment thereof, where the ADAMTS2 polypeptide contains an isoleucine at position 245 of SEQ ID NO: 44, and a pharmaceutically acceptable carrier.
  • formulations may comprise an active ADAMTS2 polypeptide or fragment thereof, where ADAMTS2 polypeptide fragments having activity are selected from amino acids 252- 1211, 253-1211, 254-1211, 255-1211, 256-1211, 257-1211, 258-1211, 259-1211 or 260-1211 of SEQ ID NO: 44, where it is understood that the active form of ADAMTS2 does not contain the propeptide domain.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • the term "pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. [0181] A pharmaceutical composition typically is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystaUine cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystaUine cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propeUant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propeUant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Molecules which exhibit high therapeutic indices are preferred. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such molecules lies preferably within a range of circulating concentrations that include the ED 5 0 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0.1 to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • polypeptide formulations featured herein is a method for treating osteoarthritis in a subject, which comprises contacting one or more cells in the subject with a first polypeptide, where the subject comprises a second polypeptide having one or more polymorphic variations associated with cancer, and where the first polypeptide comprises fewer polymorphic variations associated with cancer than the second polypeptide.
  • the first and second polypeptides are encoded by a nucleic acid which comprises a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence referenced in SEQ ID NO: 1-13 or referenced in Table B; a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-13 or referenced in Table B and a nucleotide sequence 90% or more identical to a nucleotide sequence in SEQ ID NO: 1-13 or referenced in Table B.
  • the subject often is a human.
  • a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14: 93 (1991).
  • Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a polypeptide such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al, (1994) Proc. Natl Acad. Sci. USA 97:3054-3057).
  • Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.
  • a therapeutic formulation described above can be administered to a subject in need of a therapeutic for inducing a desired biological response.
  • Therapeutic formulations can be administered by any of the paths described herein. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein.
  • treatment is defined as the application or administration of a therapeutic formulation to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect osteoarthritis, symptoms of osteoarthritis or a predisposition towards osteoarthritis.
  • a therapeutic formulation includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • Administration of a therapeutic formulation can occur prior to the manifestation of symptoms characteristic of osteoarthritis, such that osteoarthritis is prevented or delayed in its progression.
  • the appropriate therapeutic composition can be determined based on screening assays described herein. [0199] As discussed, successful treatment of osteoarthritis can be brought about by techniques that serve to agonize target molecule expression or function, or alternatively, antagonize target molecule expression or function.
  • modulators include, but are not limited to, small organic or inorganic molecules; antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof); and peptides, phosphopeptides, or polypeptides.
  • modulators include, but are not limited to, small organic or inorganic molecules; antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof); and peptides, phosphopeptides, or polypeptides.
  • antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level
  • Antisense, ribozyme and triple helix molecules are discussed above. It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method.
  • nucleic acid molecules may be utilized in treating or preventing osteoarthritis.
  • Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to ligands (see, e.g., Osborne, et al, Curr. Opin. Chem. Biol.1(1): 5-9 (1997); and Patel, D. J, Curr. Opin. Chem. Biol.
  • nucleic acid molecules for osteoarthritis treatment are gene therapy, which can also be referred to as allele therapy.
  • a gene therapy method for treating osteoarthritis in a subject which comprises contacting one or more cells in the subject or from the subject with a nucleic acid having a first nucleotide sequence (e.g., the first nucleotide sequence is identical to or substantially identical to a nucleotide sequence of SEQ ID NO: 1-13 or other nucleotide sequence referenced in Table B).
  • Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with osteoarthritis (e.g., the second nucleotide sequence is identical to or substantially identical to a nucleotide sequence of SEQ ID NO: 1-13 or other nucleotide sequence referenced in Table B).
  • the first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with osteoarthritis than the second nucleotide sequence.
  • the first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human.
  • Allele therapy methods often are utilized in conjunction with a method of first determining whether a subject has genomic DNA that includes polymorphic variants associated with osteoarthritis.
  • the method often comprises supplementing arthritis-associated ADAMTS2 polypeptide with a non-arthritis-associated ADAMTS2 polypeptide or fragment thereof, where the non-arthritis-associated form of ADAMTS2 contains an isoleucine at position 245 of SEQ ID NO: 44 having enzymatic activity.
  • the arthritis-associated ADAMTS2 polypeptide sometimes contains a valine at position 245 of SEQ ID NO: 44 having an altered enzymatic activity varying from the non- arthritis-associated polypeptide.
  • a method of increasing the synthesis of procollagen II comprising providing or administering to individuals in need of increasing levels of type II collagen the pharmaceutical or physiologically acceptable composition comprising active human ADAMTS2 protein or fragment thereof, where ADAMTS2 polypeptide fragments having activity are selected from amino acids 252-1211, 253-1211, 254-1211, 255-1211, 256-1211, 257-1211, 258-1211, 259-1211 or 260-1211 of SEQ ID NO: 44, where it is understood that the active form of ADAMTS2 does not contain the propeptide domain.
  • a method of increasing the synthesis of procollagen II comprising providing or administering to individuals in need of increasing levels of type II collagen the pharmaceutical or physiologically acceptable composition comprising an enzyme or molecule capable of cleaving ADAMTS2 propeptide, e.g., a furin-type endopeptidase or N- ethylmaleimide described herein [02 6]
  • a method which comprises contacting one or more cells in the subject or from the subject with a polypeptide encoded by a nucleic acid having a first nucleotide sequence e.g.
  • the first nucleotide sequence is identical to or substantially identical to the nucleotide sequence of SEQ ID NO: 1-13 or other nucleotide sequence referenced in Table B).
  • Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with osteoarthritis (e.g., the second nucleotide sequence is identical to or substantially identical to a nucleotide sequence of SEQ ID NO: 1-13 or other nucleotide sequence referenced in Table B).
  • the first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with osteoarthritis than the second nucleotide sequence.
  • the first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof.
  • the subject is often a human.
  • antibodies can be generated that are both specific for target molecules and that reduce target molecule activity. Such antibodies may be administered in instances where antagonizing a target molecule function is appropriate for the treatment of osteoarthritis.
  • stimulating antibody production in an animal or a human subject by injection with a target molecule is harmful to the subject, it is possible to generate an immune response against the target molecule by use of anti-idiotypic antibodies (see, e.g., Herlyn, Ann.
  • the smallest inhibitory fragment that binds to the target antigen is preferred.
  • peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used.
  • single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993)).
  • Modulators can be administered to a patient at therapeutically effective doses to treat osteoarthritis.
  • a therapeutically effective dose refers to an amount of the modulator sufficient to result in amelioration of symptoms of osteoarthritis.
  • Toxicity and therapeutic efficacy of modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50%> of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 0/ED 50 .
  • Modulators that exhibit large therapeutic indices are preferred. While modulators that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such molecules to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.
  • Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • Another example of effective dose determination for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. Molecules that modulate target molecule activity are used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions.
  • Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes readily can be assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC 50 .
  • An example of such a "biosensor” is discussed in Kriz et al, Analytical Chemistry 67: 2142-2144 (1995). [0213] The examples set forth below are intended to illustrate but not limit the invention.
  • SNPs proximal to the incident polymorphism in APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CASPR4 and APOL3 regions were identified and allelotyped in OA case and control pools.
  • Methods are described for producing target polypeptides encoded by the nucleic acids of Table B in vitro or in vivo, which can be utilized in methods that screen test molecules for those that interact with target polypeptides. Test molecules identified as interactors with target polypeptides can be screened further as osteoarthritis therapeutics.
  • Blood samples were collected from individuals diagnosed with knee osteoarthritis, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with knee osteoarthritis as gender and age-matched controls. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.
  • the solution was incubated at 37°C or room temperature if cell clumps were visible after mixing until the solution was homogeneous.
  • 2 ml of protein precipitation was added to the cell lysate.
  • the mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000 x g.
  • the supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100 ⁇ o isopropanol.
  • the samples were mixed by inverting the tubes gently until white threads of DNA were visible.
  • DNA was quantified by placing samples on a hematology mixer for at least 1 hour.
  • DNA was serially diluted (typically 1:80, 1 : 160, 1 :320, and 1 :640 dilutions) so that it would be within the measurable range of standards.
  • 125 ⁇ l of diluted DNA was transferred to a clear U-bottom microtitre plate, and 125 ⁇ l of IX TE buffer was transferred into each well using a multichannel pipette.
  • the DNA and IX TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 ⁇ l of diluted DNA was added to wells A5-H12 of a black flat bottom microtitre plate.
  • the plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 nm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/ ⁇ l were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/ ⁇ l or less were re-measured for confirmation.
  • a Fluoroskan Ascent Machine microplate fluorometer produced by Labsystems
  • samples for a pool were based upon the following criteria: the sample was derived from an individual characterized as Caucasian; the sample was derived from an individual of British paternal and maternal descent; case samples were derived from individuals diagnosed with specific knee osteoarthritis (OA) and were recruited from an OA knee replacement clinic. Control samples were derived from individuals free of OA, family history of OA, and rheumatoid arthritis. Also, sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study.
  • OA knee osteoarthritis
  • Phenotype information from each individual was collected and included age of the individual, gender, family history of OA, general medical information (e.g., height, weight, thyroid disease, diabetes, psoriasis, hysterectomy), joint history (previous and current symptoms, joint-related operations, age at onset of symptoms, date of primary diagnosis, age of individual as of primary diagnosis and order of involvement), and knee-related findings (crepitus, restricted passive movement, bony swelling/deformity). Additional knee information included knee history, current symptoms, any major knee injury, menisectomy, knee replacement surgery, age of surgery, and treatment history (including hormone replace therapy (HRT)). Samples that met these criteria were added to appropriate pools based on disease status. [0220] The selection process yielded the pools set forth in Table 1, which were used in the studies that follow:
  • a whole-genome screen was performed to identify particular SNPs associated with occurrence of osteoarthritis. As described in Example 1, two sets of samples were utilized, which included samples from female individuals having knee osteoarthritis (osteoarthritis cases), and samples from female individuals not having knee osteoarthritis (female controls). The initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another. Particular SNPs were considered as being associated with osteoarthritis when allele frequency differences calculated between case and control pools were statistically significant.
  • SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping then were analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p-value was calculated to determine whether the case and control groups had statistically significant differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level.
  • a whole-genome SNP screen began with an initial screen of approximately 25,000 SNPs over each set of disease and control samples using a pooling approach. The pools studied in the screen are described in Example 1. The SNPs analyzed in this study were part of a set of 25,488 SNPs confirmed as being statistically polymorphic as each is characterized as having a minor allele frequency of greater than 10%.
  • the SNPs in the set reside in genes or in close proximity to genes, and many reside in gene exons. Specifically, SNPs in the set are located in exons, introns, and within 5,000 base- pairs upstream of a transcription start site of a gene.
  • SNPs were selected according to the following criteria: they are located in ESTs; they are located in Locuslink or Ensembl genes; and they are located in Genomatix promoter predictions. SNPs in the set were also selected on the basis of even spacing across the genome, as depicted in Table 2. [0223] A case-control study design using a whole genome association strategy involving approximately 28,000 single nucleotide polymorphisms (SNPs) was employed. Approximately 25,000 SNPs were evenly spaced in gene-based regions of the human genome with a median inter-marker distance of about 40,000 base pairs. Additionally, approximately 3,000 SNPs causing amino acid substitutions in genes described in the literature as candidates for various diseases were used.
  • the case- control study samples were of female Caucasian origin (British paternal and maternal descent) 670 individuals were equally distributed in two groups: female confrols and female cases.
  • the whole genome association approach was first conducted on 2 DNA pools representing the 2 groups. Significant markers were confirmed by individual genotyping.
  • a MassARRAYTM system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hMETM or homogeneous MassEXTENDTM (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTENDTM primer), which is complementary to the amplified target up to but not inclu-ding the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.
  • hMETM or homogeneous MassEXTENDTM homogeneous, single-tube assay method
  • a third primer (the MassEXTENDTM primer), which is complementary to the amplified target up to but not inclu-ding the polymorphism, was then enzymatically extended one or a few bases through the polymorph
  • SpectroDESIGNERTM software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTENDTM primer which where used to genotype the polymorphism.
  • Other primer design software could be used or one of ordinary skill in the art could manually design primers based on his or her knowledge of the relevant factors and considerations in designing such primers.
  • Table 3 shows PCR primers and Table 4 shows extension primers used for analyzing polymorphisms.
  • the initial PCR amplification reaction was performed in a 5 ⁇ l total volume containing IX PCR buffer with 1.5 mM MgCl 2 (Qiagen), 200 ⁇ M each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest.
  • a primer extension reaction was initiated by adding a polymorphism-specific MassEXTENDTM primer cocktail to each sample.
  • Each MassEXTENDTM cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another.
  • ddNTPs dideoxynucleotides
  • dNTPs deoxynucleotides
  • the MassEXTENDTM reaction was performed in a total volume of 9 ⁇ l, with the addition of IX ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTENDTM primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP.
  • the deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon.
  • Samples were incubated at 94°C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72°C. [0230] Following incubation, samples were desalted by adding 16 ⁇ l of water (total reaction volume was 25 ⁇ l), 3 mg of SpectroCLEANTM sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation.
  • a second case control sample (replication sample #1) was created by using 100 Caucasian female cases from Chingford, UK, and 148 unrelated female cases from the St. Thomas twin study. Cases were defined as having Kellgren-Lawrence (KL) scores of at least 2 in at least one knee x-ray. In addition, 199 male knee replacement cases from Nottingham were included. (For a cohort description, see the Nottingham description provided in Example 1). The control pool was made up of unrelated female individuals from the St. Thomas twin study (England) with normal knee x-rays and without other indications of OA, regardless of anatomical location, as well as lacking family history of OA. The St.
  • KL Kellgren-Lawrence
  • Thomas twin study consists of Caucasian, female participants from the St. Thomas' Hospital, London, adult-twin registry, which is a voluntary registry of >4,000 twin pairs ranging from 18 to 76 years of age.
  • the replication sample 1 cohort was used to replicate the initial results.
  • Table 6 below summarizes the selected phenotype data collected from the case and control individuals.
  • a third case control sample was created by using individuals with symptoms of OA from Newfoundland, Canada. These individuals were recruited and examined by rheumatologists. Affected joints were x-rayed and a final diagnosis of definite or probable OA was made according to American College of Rheumatology criteria by a single rheumatologist to avoid any inter-examiner diagnosis variability. Controls were recruited from volunteers without any symptoms from the musculoskeletal system based on a normal joint exam performed by a rheumatologist. Only cases with a diagnosis of definite OA were included in the study. Only individuals of Caucasian origin were included. The cases consisted of 228 individuals with definite knee OA, 106 individuals with definite hip OA, and 74 individuals with hip OA.
  • Genonom, Inc. was utilized to perform SNP genotyping in a high-throughput fashion.
  • This genotyping platform was complemented by a homogeneous, single-tube assay method (hMETM or homogeneous MassEXTENDTM (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest.
  • a third primer (the MassEXTENDTM primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.
  • SpectroDESIGNERTM software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTENDTM primer which where used to genotype the polymorphism.
  • Other primer design software could be used or one of ordinary skill in the art could manually design primers based on his or her knowledge of the relevant factors and considerations in designing such primers.
  • Table 3 shows PCR primers and Table 4 shows extension probes used for analyzing (e.g., genotyping) polymorphisms in the replication cohorts.
  • the initial PCR amplification reaction was performed in a 5 ⁇ l total volume containing IX PCR buffer with 1.5 mM MgCl 2 (Qiagen), 200 ⁇ M each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest. [0239] Samples were incubated at 95°C for 15 minutes, followed by 45 cycles of 95°C for 20 seconds, 56°C for 30 seconds, and 72°C for 1 minute, finishing with a 3 minute final extension at 72°C.
  • Each MassEXTENDTM cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another. Methods for verifying, allelotyping and genotyping SNPs are disclosed, for example, in U.S. Pat. No. 6,258,538, the content of which is hereby incorporated by reference. In Table 1, ddNTPs are shown and the fourth nucleotide not shown is the dNTP. [0241] The MassEXTENDTM reaction was performed in a total volume of 9 ⁇ l, with the addition of
  • MassEXTENDTM primer 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP.
  • the deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at
  • MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpecfroTYPER RTTM software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.
  • Genotyping results for replication cohorts #1 and #2 are provided in Tables 8 and 9, respectively.
  • PADI2 is a peptidyl arginine deiminase enzyme, type II, that converts arginine residues within proteins to citrulline residues.
  • This gene is one of four known PADI genes that encode enzymes that catalyze conversion of arginine to citrulline in proteins.
  • Individuals with rheumatoid arthritis (RA) frequently have autoantibodies to citrullinated peptides, suggesting the involvement of the peptidylarginine deiminases citrullinating enzymes in RA (van Venrooij et al., Arthritis Res.;2(4):249-51. Epub 2000 May 24).
  • Pellino homo log 2 from Drosophila is a a member of the Pellino gene family, which are involved in Toll-like signalling pathways.
  • Pellino-2 associates with the pelle-like kinase/TL- lR-associated kinase protein to couple the pelle-like kinase/IL-lR-associated kinase protein to IL-1- or LPS-dependent signaling.
  • PELI2 may act as a downstream effector of " interleukin receptor signaling and may play a role in inflammation-mediated Osteoarthritis.
  • Pathway members downstream of PELI2 may be targetable ⁇ e.g., interleukin receptors).
  • G protein-coupled receptor 50 is a member of the G protein-coupled receptor family. GPR50 has significant homology to melatonin receptors and was isolated by PCR of human genomic DNA with degenerate primers based on conserved regions of melatonin receptors.
  • Example 4 APOB Proximal SNPs
  • rs 1367117 is associated with occurrence of osteoarthritis in subjects.
  • the polymorphic variant lies within the APOB gene and codes for a I98T amino acid change.
  • the guanine allele of SNP rsl367117 is associated with osteoarthritis (see Table 5) and codes for a threonine at position 98 (see, for example, amino acid sequence in SEQ ID NO: 38).
  • Apolipoprotein B is the main apolipoprotein of chylomicrons and low density lipoproteins (LDL,).
  • ApoB binds to sulfated proteoglycans, especially chondroitin and dermatan sulfate, that are components of cartilage (Camejo et. al., Atherosclerosis. 1998 Aug;139(2):205-22). This may contribute to inflammation/joint damage by lipoprotein oxidation products. In addition, increased levels of ApoB is seen as a risk factor for osteonecrosis (Miyanishi et. al., Ann Rheum Dis. 1999 Aug;58(8):514-6) . Lipoprotein deposition has been noted in inflammatory (rheumatoid) arthritis and may play a role in inflammation mediated osteoarthritis.
  • ApoB function can be modulated by addition of an antibody or a decoy receptor for ApoB.
  • antibodies and small molecules that specifically interact withApoB are described in U.S. Patent Nos. 6,107,045; 6,309,844; 5,330,910; and 6,369,075.
  • One hundred twenty-two additional allelic variants proximal to rs 1367117 were identified and subsequently allelotyped in osteoarthritis case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 10.
  • the chromosome positions provided in column four of Table 10 are based on Genome "Build 34" of NCBFs GenBan .
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IA for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure 1 A can be determined by consulting Table 13. For example, the left-most X on the left graph is at position 21188688.
  • Example 5 IL1RL2 Proximal SNPs [0257] It has been discovered that rsl024791, which lies within the IL1RL2 gene, is associated with occurrence of osteoarthritis in subjects. Interleukin-1 receptor-like 2 is a member of the interleukin 1 receptor family. IL1RL2 inhibits IL-1 activity and contains immmunoglobulin domains.
  • interleukin 1 receptor family genes including interleukin 1 receptor, type I (IL1R1), interleukin 1 receptor, type II (IL1R2), interleukin 1 receptor-like 1 (ILIRLI), and interleukin 18 receptor 1 (IL18R1), form a cytokine receptor gene cluster in a region mapped to chromosome 2ql2.
  • IL1RL2 may mediate inflammatory responses that can contribute to the development of OA.
  • IL1RL2 biological activity can be modulated by addition of an antibody, a recombinant binding partner, a binding agent, or a recombinant IL1RL2 protein or functional fragment thereof.
  • Allelotyping results from the discovery cohort are shown for cases and controls in Table 17.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for osteoarthritis case pools and control pools, respectively, where "AF" is allele frequency.
  • Some SNPs are labeled "untyped” because of failed assays.
  • the IL1RL2 proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 15 and 16.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 18 and 19, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IB for the discovery cohort. The position of each SNP on ttie chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base lO) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure IB can be determined by consulting Table 17. For example, the left-most X on the left graph is at position 102409525.
  • ILIRLI Interleukin 1 receptor-like 1 isoform 1
  • Interleukin 1 receptor-like 1 isoform 1 is a member of the interleukin 1 receptor family with no known ligand (orphan receptor).
  • ILIRLI exists in both a soluble and transmembrane form, suggesting that it may have ligand or liga_nd scavenging activity.
  • Studies of the similar gene in mouse suggested that this receptor can be induced by proinflammatory stimuli.
  • This gene and four other interleukin 1 receptor family genes form a cytokine receptor gene cluster.
  • interleukin 1 receptor type I (IL1R1)
  • interleukin 1 receptor type II (IL1R2)
  • interleukin 1 receptoir-like 2 IL1RL2
  • interleukin 18 receptor 1 IL18R1
  • the ILIRLI proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 21 and 22.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 24 and 25, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IC for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNTP designated by an X or other symbol on the graphs in Figure IC can be determined by consulting Table 23. For example, the left-most X on the left graph is at position 102527857.
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a lOkb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value. [0272] Finally, the exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3' end of each gene to show the direction of transcription.
  • Example 7 WASPIP Region Proximal SNPs [0273] It has been discovered that rsl465621 in the untranslated region (UTR) of the WASPIP gene is associated with occurrence of osteoarthritis in subjects. This gene encodes a protein that plays a role in actin cytoskeleton organization. The encoded protein binds to a region of Wiskott-Aldrich syndrome protein that is frequently mutated in Wiskott-Aldrich syndrome, an X-linked recessive disorder. Impairment of the interaction between these two proteins may contribute to the disease. Alternative transcript variants exist for this gene.
  • Biological activity of WASPIP or a pathway member downsfream of WASPIP can be modulated by addition of an antibody, a recombinant binding partner, a binding agent, or a recombinant WASPIP or downstream pathway member protein or functional fragment thereof.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for osteoarthritis case pools and control pools, respectively, where "AF" is allele frequency.
  • the WASPIP proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 27 and 28.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 30 and 31, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure ID for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure ID can be determined by consulting Table 29. For example, the left-most X on the left graph is at position 175603909.
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a lOkb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01 , to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p- values that were less than 10 "s were truncated at that value. [0280] Finally, the exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3' end of each gene to show the direction of transcription.
  • ADAMTS2 Region Proximal SNPs [0281] It has been discovered that SNP rs398829 is associated with occurrence of osteoarthritis in subjects. This gene encodes a disintegrin and metalloproteiixase with thrombospondin motifs-2 (ADAMTS2), which is a member of the ADAMTS protein family. Members of the family share several distinct protein modules, including a propeptide region, a metalloproteinase domain, a disintegrin-like domain, and a thrombospondin type 1 (TS) motif. ADAMT '-2 is involved in coUagens 1, 2 and 5 N-terminal processing, (type II collagen is the major form in cartilage).
  • ADAMT '-2 is involved in coUagens 1, 2 and 5 N-terminal processing, (type II collagen is the major form in cartilage).
  • the ADAMTS2 proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 33 and 34.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 36 and 37, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IE for the discovery cohort. The position of * each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure IE can be determined by consulting Table 35. For example, the left-most X on the left graph is at position 178695460.
  • BVES Proximal SNPs [0289] It was discovered that rs 1018810, an infronic SNP in the BVES gene, is associated with occurrence of osteoarthritis in subjects. BVES was identified as a blood vessel epicardial substance. Sequence analysis predicted 3 transmembrane helices with an extracellular C terminus. Northern blot analysis revealed that expression of an approximately 5.5-kb BVES transcript is restricted to skeletal muscle and adult and fetal heart. BVES is highly expressed in osteoarthritic cartilage according to EST database analysis, and may play a role in chondrocyte and/or bone cell development.
  • BVES biological activity may be modulated by addition of an antibody, a recombinant binding partner, a binding agent, or a recombinant BVES protein or functional fragment thereof.
  • allelic variants proximal to rs 1018810 were identified and subsequently allelotyped in osteoarthritis case and confrol sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 38.
  • the chromosome positions provided in column four of Table 38 are based on Genome "Build 34" of NCBI's GenBank.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for osteoarthritis case pools and control pools, respectively, where "AF" is allele frequency.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IF for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the confrol group.
  • the minor allele frequency of the confrol group for each SNP designated by an X or other symbol on the graphs in Figure IF can be determined by consulting Table 41. For example, the left-most X on the left graph is at position 105557091.
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a lOkb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value. [0295] Finally, the exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3' end of each gene to show the direction of transcription.
  • TM7SF3 Region Proximal SNPs [0296] It has been discovered that SNP rsl484086 in TM7SF3 is associated with occurrence of osteoarthritis in subjects.
  • TM7SF3 is an orphan receptor and is a member of the superfamily of 7- transmembrane domain proteins, one of the largest superfamilies of cell surface proteins. Members of this family include receptors for a variety of ligands, such as peptides, hormones, and ions, and for external sensory stimuli, such as odorants and light. Many 7-transmembrane molecules are able to recruit small G proteins, suggesting that they can transduce external signals to the cytoplasm.
  • the TM7SF3 proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 43 and 44.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 46 and 47, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IG for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis provides the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure IG can be determined by consulting Table 45. For example, the left-most X on the left graph is at position 27004780.
  • Example 11 LOXLl Region Proximal SNPs [0304] It has been discovered that rs8818 in the untranslated region (UTR) of the lysyl oxidase-like 1 (LOXLl) gene is associated with occurrence of osteoarthritis in subjects.
  • LOXLl is a Lysyl oxidase- like protein that catalyzes the cross-linking of collagen via lysine residues. Deficiency of the related protein, lysyl oxidase, causes a form of Ehlers-Danlos syndrome.
  • LOXLl likely is a secreted protein and its biological activity may be modulated by addition of an antibody, a recombinant binding partner, a binding agent, or a recombinant LOXLl protein or functional fragment thereof.
  • Fifty -eight additional allelic variants proximal to rs912428 were identified and subsequently allelotyped in osteoarthritis case and confrol sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 48.
  • the chromosome positions provided in column four of Table 48 are based on Genome "Build 34" of NCBI's GenBank.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for osteoarthritis case pools and confrol pools, respectively, where "AF" is allele frequency.
  • the LOXLl proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 49 and 50.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 52 and 53, respectively.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure IH for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p- value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figure IE can be determined by consulting Table 51. For example, the left-most X on the left graph is at position 71935363.
  • Example 12 CASPR4 Region Proximal SNPs
  • rs 1395486 in the cell recognition protein CASPR4 gene is associated with occurrence of osteoarthritis in subjects.
  • This gene product belongs to the neurexin family, members of which function in the nervous system as cell adhesion molecules and receptors.
  • C ⁇ SPR4 contains epidermal growth factor repeats and laminin G domains.
  • it includes an F5/8 type C domain, discoidin/neuropilin- and fibrinogen-li e domains, and thrombospondin N-terminal-like domains.
  • Alternative splicing of this gene results in 2 transcript variants encoding different isoforms.
  • CASPR4 biological activity can be modulated by addition of an antibody, a recombinant binding partner, a binding agent, or a recombinant CASPR4 protein or functional fragment thereof.
  • Fifty-six additional allelic variants proximal to rsl395486 were identified and subsequently allelotyped in osteoarthritis case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 54.
  • the chromosome positions provided in column four of Table 54 are based on Genome "Build 34" of NCBI's GenBank.
  • the CASPR4 proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 55 and 56.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 58 and 59, respectively.
  • allelotyping results were considered particularly significant witt a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated ira bold.
  • the allelotyping p- values were plotted in Figure II for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10 of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the confrol group for each SNP designated by an X or other symbol on the graplxs in Figure II can be determined by consulting Table 57.
  • the left-most X on the left graph is at position 76177855.
  • the allele frequency associated with each symbol shown can be determined.
  • multiple lines have been added to the graph.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the generally bottom-most curve is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models.
  • Th e black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a lOkb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • Example 13 APOL3 Region Proximal SNPs [0320] It has been discovered that SNP rsl32659 in APOL3 is associated with occurrence of osteoarthritis in subjects. APOL3 belongs to the high density lipoprotein family that plays a central role in cholesterol transport. The cholesterol content of membranes is important in cellular processes such as modulating gene transcription and signal transduction both in the adult brain and during neurodevelopment. It has been shown that the APOL1-APOL4 gene cluster on chromosome 22 exists only in primates (humans and African green monkeys) and not in dogs, pigs, or rodents, suggesting that this gene cluster has arisen recently in evolution (Monajemi et.
  • APOL3 is genetically linked to OA and may play a role in the pathophysiology of OA. brought about by inflammation. APOL3 is likely inhibited by a small molecule inhibitor or by specific antibodies.
  • APOL3 activity may be increased in a subject by administering APOL3 recombinant protein or a functional fragment thereof.
  • APOL3 recombinant protein or a functional fragment thereof Two hundred-nineteen additional allelic variants proximal to rsl 32659 were identified and subsequently allelotyped in osteoarthritis case and confrol sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 60.
  • the chromosome positions provided in column four of Table 60 are based on Genome "Build 34" of NCBI's GenBank. TABLE 60
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for osteoarthritis case pools and control pools, respectively, where "AF" is allele frequency.
  • the APOL3 proximal SNPs were also allelotyped in the replication cohorts using the methods described herein and the primers provided in Tables 61 and 62.
  • the replication allelotyping results for replication cohort #1 and replication cohort #2 are provided in Tables 64 and 65, respectively. TABLE 64
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p- values were plotted in Figure 1 J for the discovery cohort. The position of each SNP on the chromosome is presented on the x-axis. The y-axis provides the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the confrol group.
  • the minor allele frequency of the control group for each STSTP designated by an X or other symbol on the graph in Figure 1 J can be determined by consulting Table 63. For example, the left-most X on the left graph is at position 34781551.
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a lOkb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 -8 were truncated at that value.
  • Type II procollagen levels in osteoarthritic patients and autopsy controls is determined by radioimmunoassay (RIA) as previously described.
  • Allelic variations e.g., rs398829
  • genotyping See Examples 1 and 2.
  • Example 15 Effect of ADAMTS2 Polypeptides on Type II Collagen Processing Activity
  • recombinant polypeptides encompassing the ADAMTS2 variation of SEQ ID NO: 21 at position 733 and a wild-type ADAMTS2 polypeptide are expressed in cell lines such as chondrocytes.
  • ADAMTS2 pro-enzyme Since the allelic variation of ADAMTS2 at position 733 of SEQ ID: NO: 21 will prevent the conversion of the ADAMTS2 pro-enzyme to the catalytically active enzyme, processing of ADAMTS2 pro-enzyme is monitored by SDS-PAGE analysis followed by Western Blotting using antibodies to ADAMTS2 and methods common to someone skilled in the art. Reduced levels of pro- enzyme cleavage are apparent by the increased levels of immunopositive protein of higher molecular weight than of the cleaved active protein.
  • Example 16 Gene expression profiling in IL-1 beta and PMA stimulated SW1353 cells
  • the human chondrosarcoma cell line, SW1353, (ATCC HTB-94) was grown in L-15 media containing 10% FCS. Culture conditions were at 37 degrees with 0% CO2 with media changes every 2- 3 days.
  • SW1353 cells were grown to 80-90% confluence in 10 cm dishes and then stimulated with either lOng/ml IL-1 beta (human recombinant, Research Diagnostics) or with 200nm PMA (Sigma).
  • IL-1 beta stimulation was for 3 and 24 hours and PMA stimulation was for 3 and 24 hours. Control cells were grown and extracted in parallel with treated cells.
  • ILIRLI was seen to be upregulated by ILl-beta and by phorbol esters in a human chondrocyte cell line model (SW1353 monolayer cell line).
  • SW1353 monolayer cell line The expression profiling in IL-1 beta and PMA stimulated SW1353 cells grown in 3-D alginate cultures W1353 cells were cultured as above and then resupended in 1.2% alginate beads at a density of 4 millions cells/ml according to the manufacturer (Cambrex). Cells were grown for 2 weeks and an alginate bead was removed from culture and tested for the presence of proteoglycans by Alcian
  • ILIRLI was seen to be upregulated by ILl-beta and by phorbol esters in a human chondrocyte cell line model (SW1353 3-D alginate cell line).
  • RNA extraction and cDNA synthesis [0333] Cells from control chondrocytes and stimulated chondrocytes were isolated at the appropriate time period. mRNA "was isolated from total cell lysates using poly dT beads according to the manufacturer (Dynal). Isolated mRNA was used to generate cDNA using Superscript II reverse franscriptase according to the manufacturer (Invitrogen).
  • cDNA levels were normalized using the housekeeping gene, GAPDH.
  • Specific primers corresponding to MMP8 and MMP13 were used in semi-quantitative PCR as positive indicators of induction of an osteoarthritic phenotype. All specific primers used, including MMP8, MMP13, BVES, CHDCl and ILIRLI (transmembrane form, soluble form, soluble isoform 1 and soluble isoform 2) for semi-quantitative PCR and are listed in Table 66.
  • ILIRLI In a human chondrocyte cell line model, ILIRLI was seen to be upregulated by ILl-beta and by phorbol esters. ILIRLI has an unknown function, but it may possibly mediated inflammatory responses that can contribute to the development of OA. ILIRLI is druggable by antibodies or by protein agents.
  • cDNA is cloned into a pIVEX 2.3-MCS vector (Roche Biochem) using a directional cloning method.
  • a cDNA insert is prepared using PCR with forward and reverse primers having 5' resfriction site tags (in frame) and 5-6 additional nucleotides in addition to 3' gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length.
  • a Sal I resfriction site is infroduced by the forward primer and a Sma I restriction site is infroduced by the reverse primer.
  • PCR products are cut with the corresponding restriction enzymes (i.e., Sal I and Sma I) and the products are gel-purified.
  • the pIVEX 2.3-MCS vector is linearized using the same resfriction enzymes, and the fragment with the correct sized fragment is isolated by gel-purification.
  • Purified PCR product is ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production.
  • coli lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer.
  • 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution.
  • 0.75 ml of Reaction Mix, 50 ⁇ l of Energy Mix, and 10 ⁇ g of the template DNA is added to the E. coli lysate.
  • the reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 30°C for 24 hours with a stir bar speed of 150 rpm.
  • the pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the target polypeptide for the purpose of protein purification.
  • Target polypeptide is purified by contacting the contents of reaction device with resin modified with Ni 2+ ions.
  • Target polypeptide is eluted from the resin with a solution containing free Ni 2+ ions.
  • Example 18 Cellular Production of Target Polypeptides
  • Nucleic acids are cloned into DNA plasmids having phage recombination cites and target polypeptides are expressed therefrom in a variety of host cells.
  • Alpha phage genomic DNA contains short sequences known as attP sites
  • E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new art sites, L and R, which flank the inserted prophage DNA.
  • Phage excision from E. coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis.
  • DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target
  • a first step is to transfer the nucleic acid insert into a shuttle vector that contains attL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB.
  • a second method is to amplify the nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF.
  • PCR polymerase chain reaction
  • a third method is to utilize a topoisomerase- mediated process, in which the nucleic acid is amplified via PCR using gene-specific primers with the
  • the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.
  • the nucleic acid Once the nucleic acid is transferred into the shuttle vector, it can be cloned into an expression vector having attR sites.
  • vectors containing attR sites for expression of target polypeptide as a native polypeptide, N-fusion polypeptide, and C-fusion polypeptides are commercially available (e.g., pDEST (Invifrogen, Inc.)), and any vector can be converted into an expression vector for receiving a nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase).
  • the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37°C with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method.
  • expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N- terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the target polypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed a adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2.
  • the "Contig Position” provided in Table B corresponds to a nucleotide position set forth in the contig sequence (see “Contig Accession No.”), and designates the polymorphic site corresponding to the SNP reference number.
  • the sequence containing the polymorphisms also may be referenced by the "Nucleotide Accession No.” set forth in Table B.
  • the "Sequence Identification” corresponds to cDNA sequence that encodes associated target polypeptides (e.g., PADI2).
  • the position of the SNP within the cDNA sequence is provided in the "Sequence Position” column of Table B. If the SNP falls within an exon, the corresponding amino acid position (and amino acid change, if applicable) is provided as well. The amino acid found to be associated with OA is in bold. Also, the allelic variation at the polymorphic site and the allelic variant identified as associated with osteoarthritis is specified in Table B. All nucleotide and polypeptide sequences referenced and accessed by the parameters set forth in Table B are incorporated herein by reference.
  • Genomic nucleotide sequences for PADI2, APOB, IL1RL2, ILIRLI, WASPIP, ADAMTS2, BVES, TM7SF3, LOXLl, CNTNAP4 / CASPR4 andAPOL3 regions are set forth in SEQ ID NO: 1-13, respectively.
  • genomic nucleotide sequences for a PADI2 region (SEQ ID NO: 1), a APOB region (SEQ ID NO: 2), a IL1RL2 region (SEQ ID NO: 3), a ILIRLI region (SEQ ID NO: 4), a WASPIP region (SEQ D NO: 5), a ADAMTS2 region (SEQ ID NO: 6), a Rr ⁇ S region (SEQ ID NO: 7), a TM7SF3 region (SEQ ID NO: 8), a P- ⁇ X ⁇ region (SEQ ID NO: 9), a ZQZZJ region (SEQ ID NO: 10), a CNTNAP4 / CASPR4 region (SEQ ID NO: 11), a GPR50 region (SEQ ID NO: 12), and aAPOL3 region (SEQ ID NO: 13).
  • A or "a” is adenosine, adenine, or adenylic acid
  • C or “c” is cytidine, cytosine, or cytidylic acid
  • G or “g” is guanosine, guanine, or guanylic acid
  • T or “t” is thymidine, thymine, or thymidylic acid
  • I or “i” is inosine, hypoxanthine, or inosinic acid.
  • SNPs are designated by the following convention: “R” represents A or G, “M” represents A or C; “W” represents A or T; “Y” represents C or T; “S” represents C or G; "K” represents G or T; "V represents A, C or G; “H” represents A, C, or T; “D” represents A, G, or T; "B” represents C, G, or T; and "N” represents A, G, C, or T.
  • gaggcctccc cagtcatgct ttctgtactg cctgaggaac cataagctga ttaaacctct

Abstract

La présente invention a trait à des procédés permettant l'identification d'un risque d'ostéoarthrite chez un sujet, des réactifs et des trousses pour la mise en oeuvre des procédés, des procédés pour l'identification d'agents thérapeutiques candidats pour le traitement d'ostéoarthrite, et des procédés thérapeutiques et préventifs applicables à l'ostéoarthrite. Ces modes de réalisation reposent sur une analyse de variations polymorphes dans des séquences nucléotidiques au sein du génome humain.
EP05768810A 2004-04-01 2005-03-31 Procedes d'identification de risque d'osteoarthrite et traitements associes Withdrawn EP1756317A4 (fr)

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US55927504P 2004-04-01 2004-04-01
US55922504P 2004-04-01 2004-04-01
US55920304P 2004-04-01 2004-04-01
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US20030114410A1 (en) 2000-08-08 2003-06-19 Technion Research And Development Foundation Ltd. Pharmaceutical compositions and methods useful for modulating angiogenesis and inhibiting metastasis and tumor fibrosis
EP2537529B1 (fr) 2007-08-02 2018-10-17 Gilead Biologics, Inc. Anticorps inhibiteurs dirigés contre loxl2, et procédés d'utilisation associés
WO2010071405A1 (fr) * 2008-12-18 2010-06-24 Erasmus University Medical Center Rotterdam Marqueurs pour détecter une prédisposition au risque, une apparition et une progression d'ostéoarthrite
US9107935B2 (en) 2009-01-06 2015-08-18 Gilead Biologics, Inc. Chemotherapeutic methods and compositions
AU2010284036B2 (en) 2009-08-21 2014-12-18 Gilead Biologics, Inc. Catalytic domains from lysyl oxidase and LOXL2
AR091069A1 (es) 2012-05-18 2014-12-30 Amgen Inc Proteinas de union a antigeno dirigidas contra el receptor st2
WO2019210080A1 (fr) * 2018-04-25 2019-10-31 The Regents Of The University Of California Méthodes et compositions pour troubles du squelette et neurologiques
WO2019244934A1 (fr) * 2018-06-20 2019-12-26 株式会社ファーマフーズ Nouvel anticorps anti-pad2
WO2020018503A2 (fr) 2018-07-16 2020-01-23 Regeneron Pharmaceuticals, Inc. Anticorps anti-il36r

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