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US20120039900A1 - Biomarkers predictive of the responsiveness to tnfalpha inhibitors in autoimmune disorders - Google Patents

Biomarkers predictive of the responsiveness to tnfalpha inhibitors in autoimmune disorders Download PDF

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US20120039900A1
US20120039900A1 US13/282,850 US201113282850A US2012039900A1 US 20120039900 A1 US20120039900 A1 US 20120039900A1 US 201113282850 A US201113282850 A US 201113282850A US 2012039900 A1 US2012039900 A1 US 2012039900A1
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biomarkers
protein
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Bruno Stuhlmüller
Gerd-Reudiger Burmester
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AbbVie Inc
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Abbott Laboratories
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • rheumatoid arthritis is an autoimmune disease affecting more than two million people in the United States. RA causes chronic inflammation of the joints and typically is a progressive illness that has the potential to cause joint destruction and functional disability. The cause of rheumatoid arthritis is unknown, although genetic predisposition, infectious agents and environmental factors have all been implicated in the etiology of the disease. In active RA, symptoms can include fatigue, lack of appetite, low grade fever, muscle and joint aches and stiffness. Also during disease flare ups, joints frequently become red, swollen, painful and tender, due to inflammation of the synovium. Furthermore, since RA is a systemic disease, inflammation can affect organs and areas of the body other than the joints, including glands of the eyes and mouth, the lung lining, the pericardium, and blood vessels.
  • first line drugs Traditional treatments for the management of RA and other autoimmune disorders include fast acting “first line drugs” and slower acting “second line drugs.”
  • the first line drugs reduce pain and inflammation.
  • first line drugs include aspirin, naproxen, ibuprofen etodolac and other nonsteroidal anti-inflammatory drugs (NSAIDs), as well as corticosteroids, given orally or injected directly into tissues and joints.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • corticosteroids corticosteroids
  • second line drugs examples include gold, hydrochloroquine, azulfidine and immunosuppressive agents, such as methotrexate, azathioprine, cyclophosphamide, chlorambucil and cyclosporine. Many of these drugs, however, can have detrimental side-effects. Thus, additional therapies for rheumatoid arthritis and other autoimmune disorders have been sought.
  • TNF ⁇ inhibitors such as rheumatoid arthritis
  • REMICADETM infliximab
  • ENBRELTM etanercept
  • TNFR-Ig Fc fusion protein a TNFR-Ig Fc fusion protein
  • HUMIRATM adalimumab
  • TNF ⁇ inhibitors such as TNF ⁇ inhibitors typically is more expensive than traditional treatments and usually requires administration by injection, which, at least for certain agents, may require that the patient visit a medical office on a frequent basis.
  • TNF ⁇ inhibitors typically is more expensive than traditional treatments and usually requires administration by injection, which, at least for certain agents, may require that the patient visit a medical office on a frequent basis.
  • ways for predicting responsiveness to a TNF ⁇ inhibitor in patients having autoimmune disorders, such as rheumatoid arthritis patients are of particular interest.
  • This invention provides methods and compositions for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, such as rheumatoid arthritis, based on the discovery that the expression patterns of particular biomarkers in the subject correlate with responsiveness to a TNF ⁇ inhibitor.
  • a TNF ⁇ inhibitor in a subject having an autoimmune disorder, such as rheumatoid arthritis
  • a TNF ⁇ inhibitor Using microarray analysis of monocytes from representative rheumatoid arthritis (RA) patients treated with an anti-TNF ⁇ monoclonal antibody, 82 differentially expressed genes predictive of responsiveness to TNF ⁇ inhibitor treatment were identified by pairwise comparisons between future RA responders and future RA non-responders to anti-TNF ⁇ therapy.
  • hierarchical clustering and TaqMan®-PCR of RA responders/non-responders pre-treatment identified one gene of particular interest, CD11c, which was fully predictive of future response to anti-TNF ⁇ treatment.
  • the invention pertains to a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder (e.g., rheumatoid arthritis).
  • the method comprises: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to the sequences set forth in Table 9)
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (corresponding to sequences from Table 9 that are increased in ⁇ 80% of responders vs. non-responders).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (corresponding to sequences from Table 9 that are increased in ⁇ 90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44. (corresponding to CD11c, from Table 9, which is increased in 100% of responders vs. non-responders). In each of these embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81. (corresponding to sequences from Table 9 that are decreased in ⁇ 80% of responders vs. non-responders).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (corresponding to sequences from Table 9 that are decreased in ⁇ 90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (corresponding to sequences from Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is selected from the group consisting of Aquaporin 3 (Genbank Accession No. NM — 004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No.
  • Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM — 006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM — 000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM — 006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM — 002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No.
  • NM — 002823 Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM — 005896); Tumor protein D52 (Genbank Accession Nos. NM — 001025252, NM — 001025253, NM — 005079); Early growth response 1 (Genbank Accession No. NM — 001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM — 014888, NM — 001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No.
  • RNA binding protein 2 (Genbank Accession No. NM — 001025077, NM — 001025076, NM — 006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM — 006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM — 017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM — 001077269, NM — 003387); CD97 antigen (Genbank Accession Nos.
  • NM — 003916 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM — 014936); Desmocollin 2 (Genbank Accession Nos. NM — 024422, NM — 004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM — 002371, NM — 022438, NM — 022439, NM — 022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No.
  • NM — 005476 Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM — 001001437, NM — 021006); Carboxypeptidase A3 (Genbank Accession No. NM — 001870); Charcot-Leyden crystal protein (Genbank Accession No. NM — 001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM — 004545); Interleukin 8 receptor, beta (Genbank Accession No. NM — 001557); Platelet factor 4 variant 1 (Genbank Accession No.
  • NM — 002620 Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM — 006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM — 003786); Actinin, alpha 1 (Genbank Accession No. NM — 001102); NAD kinase (Genbank Accession No. NM — 023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM — 000442); Esterase D/formylglutathione hydrolase (Genbank Accession No.
  • NM — 001984 Chromosome 20 open reading frame 111 (Genbank Accession No. NM — 016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM — 001017369, NM — 006745); PIM-1 oncogene (Genbank Accession No. NM — 002648); GATA binding protein 2 (Genbank Accession No. NM — 032638); Cathepsin Z (Genbank Accession No. NM — 001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No.
  • NM — 000887 Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM — 006499, NM — 201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM — 006889, NM — 175862); Interleukin 8 (Genbank Accession No. NM — 000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM — 002001); Actin, gamma 1 (Genbank Accession No. NM — 001614); KIAA0746 protein (Genbank Accession No.
  • NM — 015187 Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID)
  • Genbank Accession No. NM — 002076 Glucosamine (N-acetyl)-6-sulfatase
  • Transcription factor 4 Genbank Accession Nos. BF592782, CR612521
  • Major histocompatibility complex, class II, DQ alpha 1 Genbank Accession Nos. NM — 002122, NM — 020056); Cell division cycle 2-like 6 (CDK8-like)
  • Major histocompatibility complex, class II, DQ beta 1 Genbank Accession No.
  • XM — 942240 Phospholipase C-like 2 (Genbank Accession No. NM — 015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM — 005242); TM2 domain containing 1 (Genbank Accession No. NM — 032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM — 012433); SUB1 homolog ( S. cerevisiae ) (Genbank Accession No. NM — 006713); MRNA; cDNA DKFZp564O0862 (Genbank Accession No.
  • Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM — 201413, NM — 000484, NM — 201414); Cytochrome b-5 (Genbank Accession Nos. NM — 001914, NM — 148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM — 183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM — 016645, NM — 001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No.
  • NM — 014184 Hypothetical protein FLJ21616 (Genbank Accession No. NM — 024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM — 018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM — 012336, NM — 001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM — 018538, NM — 001017922); LR8 protein (Genbank Accession No. NM — 014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No.
  • NM — 030915 Calmin (calponin-like, transmembrane) (Genbank Accession No. NM — 024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM — 031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM — 007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR — 003287) (corresponding to biomarkers listed in Table 9).
  • the one or more biomarkers is selected from the group consisting of Guanine nucleotide binding protein (G protein), beta polypeptide 1; Prothymosin, alpha (gene sequence 28); Early growth response 1; Homo spaiens leptin receptor gene-related protein (HS0BRGRP); Wiskott-Aldrich syndrome protein interacting protein; CD97 antigen; Crystallin, zeta (quinone reductase); Adaptor-related protein complex 1, sigma 2 subunit; Desmocollin 2; Chemokine (C—C motif) ligand 3; Interleukin 8 receptor, beta; ATP-binding cassette, sub-family C (CFTR/MRP), member 3; Actinin, alpha 1; NAD kinase; Platelet/endothelial cell adhesion molecule (CD31 antigen); Esterase
  • the one or more biomarkers is selected from the group consisting of Interleukin 8 receptor, beta; Platelet/endothelial cell adhesion molecule (CD31 antigen); Integrin alpha-X (antigen CD11c); Interleukin 8; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); and Kruppel-like factor 11 (corresponding to biomarkers from Table 9 that are decreased in ⁇ 80% of responders vs. non-responders).
  • the one or more biomarkers is Integrin alpha-X (antigen CD11c) (corresponding to a biomarker from Table 9 that is increased in 100% of responders vs. non-responders).
  • increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is selected from the group consisting of Aquaporin 3; Similar to ribosomal protein S24, clone MGC:8595; Transmembrane emp24 domain trafficking protein 2; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1; Calmodulin 1 (phosphorylase kinase, delta); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1); Tumor protein D52; Homo sapiens predicted osteoblast protein (GS3786); Cytochrome c oxidase subunit VIIb; CUG triplet repeat, RNA binding protein 2; Ubiquinol-cytochrome c reductase hinge protein; Glutamate-cysteine ligase, catalytic subunit; Rap guanine nucleot
  • Aquaporin 3 Similar to ribosomal protein S24, clone MGC:8595; Transmembrane emp24
  • the one or more biomarkers is selected from the group consisting of Homo sapiens predicted osteoblast protein (GS3786); Charcot-Leyden crystal protein; Neugrin, neurite outgrowth associated; Hypothetical protein FLJ21616; and Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers from Table 9 that are decreased in ⁇ 90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is Homo sapiens predicted osteoblast protein (GS3786) or Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers from Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for increased expression of a biomarker, which biomarker is CD11c, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on increased expression of CD11c in the subject.
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor, which TNF ⁇ inhibitor is adalimumab, in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to adalimumab in an autoimmune disorder, and (ii) predicting responsiveness of the subject to adalimumab based on expression of the one or more biomarkers in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to biomarkers set forth in Table 9).
  • a sample from the subject is assayed for expression of mRNA encoding the one or more biomarkers.
  • a sample from the subject is assayed for protein expression of the one or more biomarkers.
  • the methods of the invention further comprise selecting a treatment regimen with the TNF ⁇ inhibitor based upon expression of the one or more biomarkers in the subject. In another embodiment, the methods of the invention further comprise administering the TNF ⁇ inhibitor to the subject according to the treatment regimen such that autoimmune disorder is inhibited in the subject.
  • a preferred TNF ⁇ inhibitor of the invention is an anti-tumor necrosis factor-alpha (TNF ⁇ ) antibody, or antigen-binding portion thereof.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof can be, for example, a humanized antibody, a chimeric antibody or a multivalent antibody.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof can be infliximab or golimumab.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof is a human antibody.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof can be an isolated human antibody that dissociates from human TNF ⁇ with a K d of 1 ⁇ 10 ⁇ 8 M or less and a K off rate constant of 1 ⁇ 10 ⁇ 3 s ⁇ 1 or less, both determined by surface plasmon resonance, and neutralizes human TNF ⁇ cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 ⁇ 10 ⁇ 7 M or less.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof is an isolated human antibody with the following characteristics:
  • a) dissociates from human TNF ⁇ with a K off rate constant of 1 ⁇ 10 ⁇ 3 s ⁇ 1 or less, as determined by surface plasmon resonance;
  • b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
  • c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof is an isolated human antibody with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 304.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof is adalimumab.
  • a TNF ⁇ inhibitor is etanercept.
  • the subject is a human.
  • the invention pertains to a kit for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder (e.g., rheumatoid arthritis).
  • the kit comprises:
  • kits for use of the kit to predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70, even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (CD11c).
  • the instructions for use of the kit instruct that increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74, even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74.
  • the instructions for use of the kit instruct that decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor
  • the means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder comprises a nucleic acid preparation sufficient to detect expression of mRNA encoding the biomarker in a sample from the subject.
  • the means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder comprises an antibody preparation sufficient to detect protein expression of the biomarker in a sample from the subject.
  • the kit can further comprise a TNF ⁇ inhibitor for treating the autoimmune disorder in the subject.
  • the invention pertains to methods of monitoring an autoimmune disorder (e.g., RA) in a subject having the autoimmune disorder (e.g., RA).
  • RA rheumatoid arthritis
  • RA rheumatoid arthritis
  • PAM prediction analysis of microarrays
  • hierarchical clustering based on the comparison between RA responders and non-responders post-treatment.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (corresponding to biomarkers set forth in Table 3), thereby monitoring the autoimmune disorder in the subject.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (corresponding to biomarkers set forth in Table 4), thereby monitoring the autoimmune disorder in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-150, 112, 118 and 131 (upregulated biomarkers from Table 4), wherein expression of the one or more biomarkers is increased in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 151-177, 110 and 123 (downregulated biomarkers from Table 4), wherein expression of the one or more biomarkers is decreased in the subject.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (corresponding to biomarkers set forth in Table 5), thereby monitoring the autoimmune disorder in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (upregulated biomarkers from Table 5), wherein expression of the one or more biomarkers is increased in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233, 236, 237, 248, 251, 252, 254-261, 266, 267, 283, 284 and 289-292 (downregulated biomarkers from Table 5), wherein expression of the one or more biomarkers is decreased in the subject.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (corresponding to pre-treatment biomarkers set forth in Table 6), and wherein the subject is monitored prior to treatment with a TNF ⁇ inhibitor, thereby monitoring the autoimmune disorder in the subject.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (corresponding to post-treatment biomarkers set forth in Table 6), and wherein the subject is monitored after treatment with a TNF ⁇ inhibitor, thereby monitoring the autoimmune disorder in the subject.
  • the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (corresponding to biomarkers set forth in Table 7), thereby monitoring the autoimmune disorder in the subject.
  • the invention pertains to a method of building a database for use in selecting a subject having an autoimmune disorder (e.g., RA) for treatment with a TNF ⁇ inhibitor.
  • the method comprises: receiving, in a computer system, biomarker expression patterns from a plurality of subjects having an autoimmune disorder; and storing the biomarker expression pattern from each subject such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • the identifier of the subject can be, for example, a numerical identifier coded to an identity of the subject.
  • the method further comprises receiving, in the computer system, one or more treatment regimens for treatment of the autoimmune disorder in a subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • the invention also pertains to a computer program product containing executable instructions that when executed cause a processor to perform operations comprising: receiving, in a computer system, a biomarker expression pattern of a subject at one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder; and storing the biomarker expression pattern such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • the computer program can further cause the processor to perform an operation comprising: receiving, in the computer system, a treatment regimen for treatment of the autoimmune disorder in the subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • the invention pertains to a method of selecting an autoimmune disorder subject for treatment with a TNF ⁇ inhibitor, the method comprising: (i) identifying, in a database comprising a plurality of autoimmune disorder subjects, a subject whose database entry is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNF ⁇ inhibitor, and (ii) selecting the subject for treatment with a TNF ⁇ inhibitor, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • the method can further comprise selecting a treatment regimen by identifying, in the database, a treatment regimen that has been associated with the biomarker expression pattern of the subject and with an identifier of the subject.
  • the invention also pertains to a computer program product containing executable instructions that when executed cause a processor to perform operations comprising: (i) identifying, in a database including a plurality of autoimmune disorder subjects associated with biomarker expression patterns, a subject that is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNF ⁇ inhibitor; and (ii) outputting the identified subject as a subject to be treated with a TNF ⁇ inhibitor; wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • the computer program further causes the processor to perform an operation comprising outputting a treatment regimen that is associated with the subject to be treated with the TNF ⁇ inhibitor.
  • FIG. 1 is a bar graph showing the results of experiments validating the predictive gene CD11c by quantitative real-time PCR.
  • the expression of CD11c is expressed as the means ⁇ standard error of the mean normalized to that of the house keeping gene glycerol-aldehyd-3-phosphate dehydrogenase (GAPDH; % expression).
  • GPDH glycerol-aldehyd-3-phosphate dehydrogenase
  • the threshold level (40%) almost fully distinguished future responders from nonresponders (100% specificity, 94% sensitivity, and 96% power).
  • the threshold level to distinguish future responders from non-responders is indicated by a broken line (----); * P ⁇ 0.05, ** P ⁇ 0.01 as compared to normal donors; +++ P ⁇ 0.005 as compared to future responders to anti-TNF ⁇ therapy.
  • FIG. 2 is a graph showing the correlation between the mRNA expression of predictive CD11c in pre-treatment RA patients and their individual future ACR response (continuous ACR score).
  • the horizontal and vertical broken lines indicate the thresholds for the separation between RA-responders and RA-non-responders, both in terms of the pre-treatment CD11c mRNA levels (40%) and their ACR response (continuous ACR score ⁇ 30).
  • Responders continuously American College of Rheumatology [ACR] score ⁇ 40; clustered as ND
  • non-responders to anti-TNF therapy continuous ACR score ⁇ 30; clustered as RA
  • FIG. 3 is a graph showing the correlation between the mRNA expression of predictive CD11c in pre-treatment RA patients and their individual future strict ACR response in the following conventional used steps: ⁇ 0%, ⁇ 20%, ⁇ 50%, and ⁇ 70% (continuous ACR score).
  • This invention provides methods for predicting responsiveness to a TNF ⁇ inhibitor in a subject suffering from an autoimmune disorder, and methods for selecting a treatment regimen with a TNF ⁇ inhibitor, based on expression of particular biomarkers in the subject to be treated.
  • the invention is based, at least in part, on the observation that altered expression of particular biomarkers in a subject suffering from rheumatoid arthritis is associated with increased or decreased responsiveness to therapy with a TNF ⁇ inhibitor.
  • Microarray analysis, hierarchical clustering and TaqMan®-PCR analysis were used to examine normal donors (NA) and rheumatoid arthritis (RA) patients, who were categorized as being responsive to treatment with an anti-TNF ⁇ antibody (RA responders) or nonresponsive to treatment with an anti-TNF ⁇ antibody (RA nonresponders).
  • RA responders normal donors
  • RA nonresponders nonresponsive to treatment with an anti-TNF ⁇ antibody
  • a panel of 82 genes were identified whose expression was altered (upregulated or downregulated) in patients identified as either future RA responders or future RA nonresponders, demonstrating the ability of these genes to act as biomarkers for predicting responsiveness to TNF ⁇ inhibitor treatment.
  • one gene, encoding the antigen CD11c was identified as fully predicting the future response to anti-TNF ⁇ treatment.
  • the expression pattern of one or more biomarkers can be assessed in RA subjects for which TNF ⁇ inhibitor therapy is being considered, or subjects suffering from other autoimmune disorders amenable to TNF ⁇ inhibitor therapy, to thereby predict responsiveness of the subject to such therapy and/or to aid in the selection of an appropriate treatment regimen.
  • biomarker expression patterns described herein also can be using in monitoring an autoimmune disorder in a subject, e.g., monitoring the responsiveness of the subject to a particular therapy or assisting in the diagnosis or prognosis of the autoimmune disorder (e.g., RA) in the subject.
  • predicting responsiveness to a TNF ⁇ inhibitor is intended to refer to an ability to assess the likelihood that treatment of a subject with a TNF ⁇ inhibitor will or will not be effective in (e.g., provide a measurable benefit to) the subject.
  • an ability to assess the likelihood that treatment will or will not be effective typically is exercised before treatment with the TNF ⁇ inhibitor is begun in the subject.
  • an ability to assess the likelihood that treatment will or will not be effective can be exercised after treatment has begun but before an indicator of effectiveness (e.g., an indicator of measurable benefit) has been observed in the subject.
  • TNF ⁇ inhibitor as used herein is intended to encompass agents including proteins, antibodies, antibody fragments, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNF ⁇ antagonists and similar naturally- or nonnaturally-occurring molecules, and/or recombinant and/or engineered forms thereof, that, directly or indirectly, inhibits TNF ⁇ activity, such as by inhibiting interaction of TNF ⁇ with a cell surface receptor for TNF ⁇ , inhibiting TNF ⁇ protein production, inhibiting TNF ⁇ gene expression, inhibiting TNF ⁇ secretion from cells, inhibiting TNF ⁇ receptor signaling or any other means resulting in decreased TNF ⁇ activity in a subject.
  • TNF ⁇ inhibitor also includes agents which interfere with TNF ⁇ activity.
  • TNF ⁇ inhibitors include etanercept (ENBRELTM, Amgen), infliximab (REMICADETM, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRATM, Abbott Laboratories), CDP 571 (Celltech), and CDP 870 (Celltech), as well as other compounds which inhibit TNF ⁇ activity, such that when administered to a subject suffering from or at risk of suffering from a disorder in which TNF ⁇ activity is detrimental (e.g., RA), the disorder is treated.
  • a disorder in which TNF ⁇ activity is detrimental e.g., RA
  • the term also includes each of the anti-TNF ⁇ human antibodies and antibody portions described herein as well as those described in U.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S. patent application Ser. Nos. 09/801,185 (U.S. Publication No. 20030092059) and 10/302,356 (U.S. Publication No. 20030219438), each incorporated by reference herein.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody is also intended to encompass dual-specific antibodies and bispecific antibodies.
  • dual-specific antibody refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see e.g., PCT publication WO 02/02773). Accordingly a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bi-valent for each antigen it binds to.
  • bispecific antibody refers to full-length antibodies that are generated by quadroma technology (see Milstein, C. and A. C.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is mono-valent for each antigen it binds to.
  • a “bispecific” antibody of the invention has one binding arm that is specific for an epitope of TNF ⁇ and a second binding arm that is specific for a different antigen or epitope.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and C H1 domains
  • F(ab′) 2 fragment a bivalent fragment comprising two
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • chimeric antibody or “chimeric monoclonal antibody” are intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • Such “chimeric antibodies” can be prepared by standard recombinant technology well established in the art.
  • a nucleic acid encoding a V H region from a mouse antibody can be operatively linked to a nucleic acid encoding the heavy chain constant regions from a human antibody and, likewise, a nucleic acid encoding a V L region from a mouse antibody can be operatively linked to a nucleic acid encoding the light chain constant region from a human antibody.
  • humanized antibody or “humanized monoclonal antibody” are intended to refer to antibodies in which CDR sequences derived from the germline of a non-human mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • Such “humanized antibodies” can be prepared by standard recombinant technology well established in the art.
  • nucleic acids encoding the CDR1, CD2 and CDR3 regions from a V H region of a mouse antibody can be operatively linked to nucleic acids encoding the FR1, FR2, FR3 and FR4 regions of a human V H region, and the entire “CDR-grafted” V H region can be operatively linked to nucleic acid encoding the heavy chain constant regions from a human antibody.
  • nucleic acids encoding the CDR1, CD2 and CDR3 regions from a V L region of a mouse antibody can be operatively linked to nucleic acids encoding the FR1, FR2, FR3 and FR4 regions of a human V L region, and the entire “CDR-grafted” V L region can be operatively linked to nucleic acid encoding the light chain constant region from a human antibody.
  • human antibody is intended to refer to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • Human monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • human monoclonal antibody also includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant means such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma,
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • Such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Ig fusion protein and Fc fusion protein are intended to refer to a recombinant, composite protein comprising a polypeptide of interest operatively linked to a constant region portion of immunoglobulin, typically the hinge, CH 2 and CH 3 domains of heavy chain constant region, more typically the human IgG1 hinge, CH 2 and CH 3 domains.
  • the polypeptide of interest operatively linked to the Fc portion can be, for example, a full-length protein or only a portion of a full-length protein, such as one or more extracellular domains of a protein, e.g., one or more extracellular domains of a cell-surface protein.
  • Such “Ig fusion proteins” can be prepared by standard recombinant technology well established in the art.
  • a nucleic acid encoding the polypeptide of interest can be operatively linked to a nucleic acid encoding the hinge, CH 2 and CH 3 domains of a heavy chain constant region.
  • multivalent binding protein as a form of TNF ⁇ inhibitor, is used in this specification to denote a binding protein comprising two or more antigen binding sites.
  • multivalent binding proteins include dual variable domain (DVD) binding proteins.
  • the multivalent binding protein is preferably engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody.
  • a multivalent binding protein also can be a “multispecific binding protein.”
  • multispecific binding protein refers to a binding protein capable of binding two or more related or unrelated targets (wherein, with respect to this specification at least one of the targets is TNF ⁇ ).
  • Dual variable domain (DVD) binding proteins are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins.
  • DVDs may be monospecific, i.e., capable of binding one antigen (e.g., TNF ⁇ ) or multispecific, i.e., capable of binding two or more antigens (e.g., TNF ⁇ and one or more other antigens).
  • DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as “DVD Ig.”
  • Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites.
  • Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.
  • DVD binding proteins and methods of making DVD binding proteins are disclosed in US. Publication No. 20070071675, the entire contents of which are specifically incorporated herein by reference.
  • biomarker is intended to encompass a substance that is used as an indicator of a biologic state and includes genes (and nucleotide sequences of such genes), mRNAs (and nucleotide sequences of such mRNAs) and proteins (and amino acid sequences of such proteins).
  • a “biomarker expression pattern” is intended to refer to a quantitative or qualitative summary of the expression of one or more biomarkers in a subject, such as in comparison to a standard or a control.
  • CD11c refers to a protein having a full-length amino acid sequence as set forth at Genbank Accession No. NP — 000878 (also shown as SEQ ID NO: 302) and encoded by a full-length nucleotide sequence as set forth at Genbank Accession No. NM — 000887 (also shown as SEQ ID NO: 301).
  • CD11c is also known in the art as CD11C, CD11c antigen, Integrin alpha X, complement component 3 receptor 4 subunit, ITGAX, LeuM5, Integrin alpha X precursor, Leukocyte adhesion glycoprotein p150,p95 alpha chain, and Leukocyte adhesion receptor p150 subunit, which terms may be used interchangeably herein to refer to CD11c.
  • the term “Affymetrix ID” refers to a numerical identifier that corresponds to a sequence entry in an Affymetrix database, which entry includes the sequence as well as additional information relating to the sequence and corresponding protein.
  • the sequence entries, and additional information in the entries, for each Affymetrix ID are publicly available (e.g., by entering the Affymetrix ID number into the Affymetrix database search engine, e.g., at https://www.affymetrix.com/analysis/netaffx/index.affx). All sequence entries (such as Genbank Accession numbers), and additional information provided for each entry, corresponding to each of the Affymetrix ID numbers disclosed herein are hereby specifically incorporated by reference in their entirety.
  • the term “subject” includes humans, and non-human animals amenable to TNF ⁇ inhibitor therapy, e.g., preferably mammals, such as non-human primates, sheep, dogs, cats, horses and cows.
  • autoimmune disorder subject or “AD subject” is intended to refer to a subject (e.g., human patient) suffering from an autoimmune disorder.
  • rheumatoid arthritis subject or “RA subject” is intended to refer to a subject (e.g., human patient) suffering from rheumatoid arthritis.
  • treatment regimen is intended to refer to one or more parameters selected for the treatment of a subject, e.g., with a TNF ⁇ inhibitor, which parameters can include, but are not necessarily limited to, the type of agent chosen for administration, the dosage, the formulation, the route of administration and the frequency of administration.
  • the invention pertains to a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, such as rheumatoid arthritis.
  • the method comprises (i) assaying the subject for the expression of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on expression of the one or more biomarkers in the subject.
  • biomarkers is intended to mean that at least one biomarker in a disclosed list of biomarkers is assayed and, in various embodiments, more than one biomarker set forth in the list may be assayed, such as two, three, four, five, ten, twenty, thirty, forty, fifty, more than fifty, or all the biomarkers in the list may be assayed.
  • Predicting responsiveness of the subject to the TNF ⁇ inhibitor “based on expression of the one or more biomarkers in the subject” typically involves comparing the level, or pattern, of expression of the one or more biomarkers in the subject to a known standard or control (which known standard or control may be derived from, for example, a normal subject, a pre-established TNF ⁇ inhibitor responder or a pre-established TNF ⁇ inhibitor non-responder).
  • a known standard or control which known standard or control may be derived from, for example, a normal subject, a pre-established TNF ⁇ inhibitor responder or a pre-established TNF ⁇ inhibitor non-responder.
  • the level of expression of the biomarker(s) is measured in parallel with measurement of the level of expression of one or more “housekeeping” genes, such as GAPDH, whose expression level is not altered by the autoimmune disorder.
  • the level of expression of the biomarker(s) is determined to be “increased” or “decreased” relative to a constant basal level of expression of the housekeeping gene.
  • suitable housekeeping genes such as GAPDH, that can be used for comparison purposes are well known in the art.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to sequences of the biomarkers set forth in Table 9).
  • a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 is assayed and, in various embodiments, for example, two, three, four, five, ten, twenty, thirty, forty, fifty, more than fifty, or all the biomarkers in the list may be assayed.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (corresponding to sequences from Table 9 that are increased in ⁇ 80% of responders vs. non-responders).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (corresponding to sequences from Table 9 that are increased in ⁇ 90% of responders vs. non-responders).
  • at least one of the biomarkers to be assayed is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (corresponding to the biomarker CD11c, which, as set forth in Table 9, is increased in 100% of responders vs. non-responders).
  • increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor (e.g., increased expression relative to a standard or control level of expression, which standard or control level of expression can be based, for example, on the level of expression in previously established TNF ⁇ inhibitor non-responder RA subjects).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (corresponding to sequences from Table 9 that are decreased in ⁇ 80% of responders vs. non-responders).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (corresponding to sequences from Table 9 that are decreased in ⁇ 90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (corresponding to sequences from Table 9 that are decreased in 100% of responders vs. non-responders).
  • decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor (e.g., decreased expression relative to a standard or control level of expression, which standard or control level of expression can be based, for example, on the level of expression in previously established TNF ⁇ inhibitor non-responder RA subjects).
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in the autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is selected from the group consisting of Aquaporin 3 (Genbank Accession No. NM — 004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No.
  • Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM — 006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM — 000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM — 006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM — 002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No.
  • NM — 002823 Homo sapiens isocitrate dehydrogenase 1 (NADP + ) soluble (IDH1) (Genbank Accession No. NM — 005896); Tumor protein D52 (Genbank Accession Nos. NM — 001025252, NM — 001025253, NM — 005079); Early growth response 1 (Genbank Accession No. NM — 001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM — 014888, NM — 001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No.
  • RNA binding protein 2 (Genbank Accession No. NM — 001025077, NM — 001025076, NM — 006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM — 006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM — 017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM — 001077269, NM — 003387); CD97 antigen (Genbank Accession Nos.
  • NM — 003916 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM — 014936); Desmocollin 2 (Genbank Accession Nos. NM — 024422, NM — 004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM — 002371, NM — 022438, NM — 022439, NM — 022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No.
  • NM — 005476 Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM — 001001437, NM — 021006); Carboxypeptidase A3 (Genbank Accession No. NM — 001870); Charcot-Leyden crystal protein (Genbank Accession No. NM — 001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM — 004545); Interleukin 8 receptor, beta (Genbank Accession No. NM — 001557); Platelet factor 4 variant 1 (Genbank Accession No.
  • NM — 002620 Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM — 006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM — 003786); Actinin, alpha 1 (Genbank Accession No. NM — 001102); NAD kinase (Genbank Accession No. NM — 023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM — 000442); Esterase D/formylglutathione hydrolase (Genbank Accession No.
  • NM — 001984 Chromosome 20 open reading frame 111 (Genbank Accession No. NM — 016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM — 001017369, NM — 006745); PIM-1 oncogene (Genbank Accession No. NM — 002648); GATA binding protein 2 (Genbank Accession No. NM — 032638); Cathepsin Z (Genbank Accession No. NM — 001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No.
  • NM — 000887 Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM — 006499, NM — 201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM — 006889, NM — 175862); Interleukin 8 (Genbank Accession No. NM — 000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM — 002001); Actin, gamma 1 (Genbank Accession No. NM — 001614); KIAA0746 protein (Genbank Accession No.
  • NM — 015187 Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID)
  • Genbank Accession No. NM — 002076 Glucosamine (N-acetyl)-6-sulfatase
  • Transcription factor 4 Genbank Accession Nos. BF592782, CR612521
  • Major histocompatibility complex, class II, DQ alpha 1 Genbank Accession Nos. NM — 002122, NM — 020056); Cell division cycle 2-like 6 (CDK8-like)
  • Major histocompatibility complex, class II, DQ beta 1 Genbank Accession No.
  • XM — 942240 Phospholipase C-like 2 (Genbank Accession No. NM — 015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM — 005242); TM2 domain containing 1 (Genbank Accession No. NM — 032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM — 012433); SUB1 homolog ( S. cerevisiae ) (Genbank Accession No. NM — 006713); mRNA; cDNA DKFZp564O0862 (Genbank Accession No.
  • Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM — 201413, NM — 000484, NM — 201414); Cytochrome b-5 (Genbank Accession Nos. NM — 001914, NM — 148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM — 183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM — 016645, NM — 001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No.
  • NM — 014184 Hypothetical protein FLJ21616 (Genbank Accession No. NM — 024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM — 018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM — 012336, NM — 001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM — 018538, NM — 001017922); LR8 protein (Genbank Accession No. NM — 014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No.
  • NM — 030915 Calmin (calponin-like, transmembrane) (Genbank Accession No. NM — 024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM — 031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM — 007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR — 003287) (corresponding to biomarkers listed in Table 9).
  • the one or more biomarkers is selected from the group consisting of Guanine nucleotide binding protein (G protein), beta polypeptide 1; Prothymosin, alpha (gene sequence 28); Early growth response 1; Homo spaiens leptin receptor gene-related protein (HS0BRGRP); Wiskott-Aldrich syndrome protein interacting protein; CD97 antigen; Crystallin, zeta (quinone reductase); Adaptor-related protein complex 1, sigma 2 subunit; Desmocollin 2; Chemokine (C—C motif) ligand 3; Interleukin 8 receptor, beta; ATP-binding cassette, sub-family C (CFTR/MRP), member 3; Actinin, alpha 1; NAD kinase; Platelet/endothelial cell adhesion molecule (CD31 antigen); Esterase D/formylglutathione hydrolase; Chromosome 20 open reading frame 111; Cathepsin Z;
  • the one or more biomarkers is selected from the group consisting of Interleukin 8 receptor, beta; Platelet/endothelial cell adhesion molecule (CD31 antigen); Integrin alpha-X (antigen CD11c); Interleukin 8; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); and Kruppel-like factor 11 (corresponding to biomarkers listed in Table 9 that are increased in ⁇ 90% of responders vs. non-responders). Even more preferably, at least one of the biomarkers is Integrin alpha-X (antigen CD11c) (corresponding to a biomarker listed in Table 9 that is increased in 100% of responders vs. non-responders). In each of the embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is selected from the group consisting of Aquaporin 3; Similar to ribosomal protein S24, clone MGC:8595; Transmembrane emp24 domain trafficking protein 2; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1; Calmodulin 1 (phosphorylase kinase, delta); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1); Tumor protein D52; Homo sapiens predicted osteoblast protein (GS3786); Cytochrome c oxidase subunit VIIb; CUG triplet repeat, RNA binding protein 2; Ubiquinol-cytochrome c reductase hinge protein; Glutamate-cysteine ligase, catalytic subunit; Rap guanine nucleotide exchange factor (GEF) 2; Ataxin 1; Ectonucleotide pyrophosphatase/phosphorofug
  • the one or more biomarkers is selected from the group consisting of Homo sapiens predicted osteoblast protein (GS3786); Charcot-Leyden crystal protein; Neugrin, neurite outgrowth associated; Hypothetical protein FLJ21616; and Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers listed in Table 9 that are decreased in ⁇ 90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is Homo sapiens predicted osteoblast protein (GS3786) or Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers listed in Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder the method comprising: (i) assaying the subject for increased expression of a biomarker, which biomarker is CD11c, and (ii) predicting responsiveness of the subject to the TNF ⁇ inhibitor based on increased expression of CD11c in the subject.
  • the invention provides a method for predicting responsiveness to a TNF ⁇ inhibitor, which TNF ⁇ inhibitor is adalimumab, in a subject having an autoimmune disorder the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to adalimumab in the autoimmune disorder, and (ii) predicting responsiveness of the subject to adalimumab based on expression of the one or more biomarkers in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or selected from the group consisting of the biomarkers set forth in Table 9).
  • the subsets of sequences within SEQ ID NO: 1-82 that are either increased or decreased, as set forth in detail above, can be assayed.
  • the expression of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in the autoimmune disorder can be assayed in the subject using techniques well-established in the art.
  • the expression of the one or more biomarkers in the subject is assayed by obtaining an mRNA sample from the subject (e.g., isolated from peripheral blood mononuclear cells, by standard methods) and detecting the expression of mRNA(s) encoding the one or more biomarkers in the mRNA sample using standard molecular biology techniques, such as PCR analysis.
  • PCR analysis is revers transcriptase-polymerase chain reaction (RT-PCR).
  • RT-PCR revers transcriptase-polymerase chain reaction
  • Other suitable systems for mRNA sample analysis include microarray analysis (e.g., using Affymetrix's microarray system or Illumina's BeadArray Technology).
  • a detection reagent that detects the protein product encoded by the mRNA of the biomarker(s).
  • an antibody reagent is available that binds specifically to the biomarker protein product to be detected, and not to other proteins, then such an antibody reagent can be used to detect the expression of the biomarker of interest in a cellular sample from the subject, or a preparation derived from the cellular sample, using standard antibody-based techniques known in the art, such as FACS analysis, ELISA and the like.
  • the biomarkers described herein were originally identified in patients having rheumatoid arthritis (see the Examples) and thus a particularly preferred autoimmune disorder in which to apply the methods of the invention is rheumatoid arthritis.
  • the mechanism of action of the TNF ⁇ pathway is thought to be common to a large number of autoimmune disorders and TNF ⁇ inhibitors have been shown to be effective therapy in a variety of different autoimmune disorders. Accordingly, the method of the invention for predicting responsiveness to a TNF ⁇ inhibitor can be applied to essentially any autoimmune disorder in which TNF ⁇ inhibitor therapy is applied.
  • autoimmune disorders include Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, juvenile arthritis and ankylosing spondilitis
  • Other non-limiting examples of autoimmune disorders include autoimmune diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid spondylitis, gouty arthritis, allergy, autoimmune uveitis, nephrotic syndrome, multisystem autoimmune diseases, autoimmune hearing loss, adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathic interstitial lung disease, chronic obstructive pulmonary disease, asthma, restenosis, spondyloarthropathies, Reiter's syndrome, autoimmune hepatitis, inflammatory skin disorders, vasculitis of large vessels, medium vessels or small vessels, endometriosis, prostatitis and Sjogren's syndrome.
  • the invention provides methods of monitoring an autoimmune disorder in a subject having the autoimmune disorder based on biomarker expression patterns established using microarray analysis of, for example, RA subjects vs. normal donors, RA subjects vs. RA subjects treated with a TNF ⁇ inhibitor and/or RA subjects treated with a TNF ⁇ inhibitor vs. RA responders to TNF ⁇ inhibitors.
  • the subject is assayed for expression of one or more biomarkers (using techniques, for example, as described in the previous section), thereby monitoring the autoimmune disorder in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, namely v-maf musculoaponeurotic fibrosarcoma oncogene homolog F; Diphtheria toxin receptor (DTR); DEAH (Asp-Glu-Ala-His) box polypeptide 15; Ribonucleotide reductase M2 polypeptide; Solute carrier family 6, member 8; 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; Ferrochelatase (protoporphyria); Nuclear factor, interleukin 3 regulated; Thrombomodulin; Major histocompatibility complex, class II, DM beta; Forkhead box O3A; Hemoglobin, gamma A, gam
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, namely Uncharacterized hypothalamus protein HT007; Dual specificity phosphatase 22; Proteasome (prosome, macropain) subunit, beta type, 7; Membrane-spanning 4-domains, subfamily A, member 4; DKFZP434C171 protein; Protein phosphatase 1, catalytic subunit, beta isoform; Splicing factor, arginine/serine-rich 5; Sorting nexin 11; Farnesyltransferase, CAAX box, alpha; Peroxisomal D3,D2-enoyl-CoA isomerise; Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide;
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as being upregulated, namely Uncharacterized hypothalamus protein HT007; Dual specificity phosphatase 22; Proteasome (prosome, macropain) subunit, beta type, 7; Membrane-spanning 4-domains, subfamily A, member 4; DKEZP434C171 protein; Protein phosphatase 1, catalytic subunit, beta isoform; Splicing factor, arginine/serine-rich 5; Sorting nexin 11; Farnesyltransferase, CAAX box, alpha; Peroxisomal D3,D2-enoyl-CoA isomerise; Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide; UDP-N-acety
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as being downregulated, namely Tumor necrosis factor (ligand) superfamily, member 10; Chromosome 10 open reading frame 86; CD1D antigen, d polypeptide; Ewing sarcoma breakpoint region 1; Ribonuclease P 40 kDa subunit; PHD finger protein 20; Thioredoxin interacting protein; Ubiquinol-cytochrome c reductase core protein II; Hypothetical protein FLJ22662; Preimplantation protein 3; DKFZP564G2022 protein; Dipeptidase 2; Hemoglobin, alpha 1, alpha 2; Frequently rearranged in advanced T-cell lymphomas; DEAD (Asp-Glu-Ala-As
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, namely HLA-B associated transcript-1 (D6S81E); Interleukin enhancer binding factor 2, 45 kD (ILF2); Isolate Liv chaperone protein HSP90 beta (HSP90BETA) mRNA; Lysyl-tRNA synthetase mRNA, complete cds; nuclear gene for mitochondrial product; alternatively spliced; Tryptophanyl-tRNA synthetase (WARS); Profilin 1 (PFN1), mRNA; Seryl-tRNA synthetase (SARS); Similar to KIAA1007 protein, clone MGC:692, mRNA, complete cds; CD59 antigen p18-20 (antigen identified
  • Homoglobin alpha 1
  • Homo sapiens clone 24659 mRNA sequence/DEF Homo sapiens clone 24659 mRNA sequence
  • Calcium channel voltage-dependent, PQ type, alpha 1A subunit
  • H. sapiens SMAS mRNA H.
  • HEX gene encoding homeobox related protein
  • Mitogen-activated protein kinase kinase kinase 4 Serine palmitoyltransferase (LCB2) mRNA, partial cds
  • KIAA0971 protein/DEF Homo sapiens cDNA FLJ11495 fis, clone HEMBA1001950, highly similar to Homo sapiens mRNA for KIAA0971 protein; ESTs, Hs.97109; ESTs, Weakly similar to ALU7_HUMAN ALU; DEAD-box protein abs Consumer (ABS), mRNA; KIAA1513 protein (KIAA1513), mRNA; Cell division protein FtsJ (FJH1), mRNA; F-box only protein 3 (FBXO3), mRNA; Purinergic receptor (family A group 5) (P2Y5), mRNA; Integral inner nuclear membrane protein (MAN1), mRNA; Fanconi anemia, complementation group
  • polypeptide 5; PNAS-25 mRNA, complete cds.; Homo sapiens mRNA for FLJ00043 protein, partial cds; Homo sapiens cDNA: FLJ21737 fis, clone COLF3396; Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395/DEF Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395; Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F) LIKE protein; Heparin-binding EGF-like growth factor mRNA, complete cds; E.
  • MAFF v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as being upregulated, namely HLA-B associated transcript-1 (D6S81E); Interleukin enhancer binding factor 2, 45 kD (ILF2); Isolate Liv chaperone protein HSP90 beta (HSP90BETA) mRNA; Lysyl-tRNA synthetase mRNA, complete cds; nuclear gene for mitochondrial product; alternatively spliced; Tryptophanyl-tRNA synthetase (WARS);
  • Homoglobin alpha 1
  • Homo sapiens clone 24659 mRNA sequence/DEF Homo sapiens clone 24659 mRNA sequence
  • Calcium channel voltage-dependent, PQ type, alpha 1A subunit
  • H. sapiens SMA5 mRNA ESTs, Hs.97109
  • ESTs Weakly similar to ALU7_HUMAN ALU
  • Novel MAFF v-maf musculoaponeurotic
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, namely Solute carrier family 6; Amphiregulin; Keratin 1; Hemoglobin, alpha 1; MHC-II, DM beta; Purinergic receptor P2Y, G-protein coupled 5; Forkhead box O3A; Transferrin receptor (p90, CD71); Ring finger protein 10; Formin binding protein 4; and Hypothetical protein LOC54103), wherein the subject is monitored prior to treatment with a TNF ⁇ inhibitor.
  • SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 or the biomarkers set forth by name in Table 6 as pre-treatment bio
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, namely Diphteria toxin receptor; Heparin-binding EGF-like growth factor; Lipoyltransferase 1; APEX nuclease (multifunct.
  • DNA repair enzyme 1; GABA(A) receptor-associated protein like 1/3; Formin binding protein 4; Zinc finger protein 331; Ribonuclease P 40 kDa subunit; MHC class II, DM beta; v-maf fibrosarc. oncogene homolog F (avian); 2′,5′-oligoadenylate synthetase 1, 40/46 kDa; and Membrane-spanning 4-domains, subfam. A, memb. 4), wherein the subject is monitored after treatment with a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, namely Amphiregulin; Carbonic anhydrase 1; Charcot-Leyden crystal protein; Clusterin C; Tumor necrosis factor alpha induced protein 6; Thrombomodulin; Membrane-spanning 4-domains, subfamily A, member 4; Diptheria toxin receptor; S100 calcium binding protein A1; Uncharacterized hypothalamus protein HT007; MHC-class-II; HLA-DR alpha; Hypothetical protein L0054103; Tumor necrosis factor alpha; Interleukin 1 beta; Proteasome subunit beta type 7 precursor; and Protein
  • a preferred autoimmune disorder in which to apply the methods of the invention for monitoring an autoimmune disorder is rheumatoid arthritis.
  • the monitoring methods can be applied to essentially any autoimmune disorder in which TNF ⁇ inhibitor therapy is applied, including the autoimmune disorders listed in the previous section.
  • the above-described method for predicting the responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder further comprises selecting a treatment regimen with the TNF ⁇ inhibitor based upon expression of the one or more biomarkers in the subject.
  • the method still further comprises administering the TNF ⁇ inhibitor to the subject according to the treatment regimen such that the autoimmune disorder is inhibited in the subject.
  • the invention provides a method for selecting a treatment regimen for therapy with a TNF ⁇ inhibitor in a subject having an autoimmune disorder, the method comprising:
  • the invention provides a method of treating a subject having an autoimmune disorder with a TNF ⁇ inhibitor, the method comprising:
  • the treatment regimen that is selected typically includes at least one of the following parameters and more typically includes many or all of the following parameters: the type of agent chosen for administration, the dosage, the formulation, the route of administration and/or the frequency of administration.
  • TNF ⁇ inhibitors are biologic agents that have been approved by the FDA for use in humans in the treatment of rheumatoid arthritis, which agents include adalimumab (HUMIRATM), infliximab (REMICADETM) and etanercept (ENBRELTM), most preferably adalimumab (HUMIRATTM).
  • HUMIRATM adalimumab
  • REMICADETM infliximab
  • ENBRELTM etanercept
  • HUMIRATTM most preferably adalimumab
  • the TNF ⁇ inhibitor is an anti-tumor necrosis factor-alpha (TNF ⁇ ) antibody, or antigen-binding portion thereof.
  • TNF ⁇ anti-tumor necrosis factor-alpha
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof can be a humanized antibody, a chimeric antibody or a multivalent antibody.
  • the anti-TNF ⁇ antibody, or antigen-binding portion thereof is a human antibody, preferably a human antibody, or antigen-binding portion thereof, that binds to human TNF ⁇ with high affinity and a low off rate, and has a high neutralizing capacity.
  • the human antibodies are recombinant, neutralizing human anti-hTNF ⁇ antibodies.
  • adalimumab also referred to as HUMIRA® or D2E7
  • the amino acid sequence of the adalimumab VL region is shown in SEQ ID NO: 303; the amino acid sequence of the adalimumab VH region is shown in SEQ ID NO: 304.
  • D2E7 adalimumab; Humira®
  • the properties of D2E7 have been described in Salfeld et al., U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015, which are each incorporated by reference herein.
  • TNF ⁇ antibodies include chimeric and humanized murine anti-hTNF ⁇ antibodies which have undergone clinical testing for treatment of rheumatoid arthritis (see e.g., Elliott et al. (1994) Lancet 344:1125-1127; Elliot et al. (1994) Lancet 344:1105-1110; Rankin et al. (1995) Br. J. Rheumatol. 34:334-342).
  • the TNF ⁇ antibody used in the invention is infliximab (Remicade®, Johnson and Johnson; described in U.S. Pat. No.
  • CDP571 a humanized monoclonal anti-TNF-alpha IgG4 antibody
  • CDP 870 a humanized monoclonal anti-TNF-alpha antibody fragment
  • an anti-TNF dAb Peptech
  • CNTO 148 golimumab; Medarex and Centocor, see also WO 02/12502.
  • the TNF ⁇ inhibitors used in the methods of the invention include adalimumab antibodies and antibody portions, adalimumab-related antibodies and antibody portions, adalimumab-related DVD-Ig or dual specific antibodies, and other human antibodies and antibody portions with equivalent properties to adalimumab, such as high affinity binding to hTNF ⁇ with low dissociation kinetics and high neutralizing capacity.
  • a treatment regimen of the invention provides treatment with an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNF ⁇ with a K d of 1 ⁇ 10 ⁇ 8 M or less and a K off rate constant of 1 ⁇ 10 ⁇ 3 s ⁇ 1 or less, both determined by surface plasmon resonance, and neutralizes human TNF ⁇ cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 ⁇ 10 ⁇ 7 M or less.
  • the isolated human antibody, or antigen-binding portion thereof dissociates from human TNF ⁇ with a K off of 5 ⁇ 10 ⁇ 4 s ⁇ 1 or less, or even more preferably, with a K off of 1 ⁇ 10 ⁇ 4 s ⁇ 1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNF ⁇ cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 ⁇ 10 ⁇ 8 M or less, even more preferably with an IC 50 of 1 ⁇ 10 ⁇ 9 M or less and still more preferably with an IC 50 of 1 ⁇ 10 ⁇ 10 M or less.
  • the antibody is an isolated human recombinant antibody, or an antigen-binding portion thereof.
  • the TNF ⁇ inhibitor used in the treatment method of the invention is a human anti-TNF ⁇ antibody that has slow dissociation kinetics for association with hTNF ⁇ and that has light and heavy chain CDR3 domains that structurally are identical to or related to those of adalimumab.
  • Position 9 of the adalimumab VL CDR3 can be occupied by Ala or Thr without substantially affecting the K off .
  • a consensus motif for the adalimumab VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 305). Additionally, position 12 of the adalimumab VH CDR3 can be occupied by Tyr or Asn, without substantially affecting the K off . Accordingly, a consensus motif for the adalimumab VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 306). Moreover, as demonstrated in Example 2 of U.S. Pat. No.
  • the CDR3 domain of the adalimumab heavy and light chains is amenable to substitution with a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantially affecting the K off .
  • substitution of other amino acids within the CDR3 domains may be possible while still retaining the low off rate constant of the antibody, in particular substitutions with conservative amino acids.
  • no more than one to five conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. More preferably, no more than one to three conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. Additionally, conservative amino acid substitutions should not be made at amino acid positions critical for binding to hTNF ⁇ .
  • Positions 2 and 5 of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab VH CDR3 appear to be critical for interaction with hTNF ⁇ and thus, conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position 5 of the adalimumab VL CDR3 is acceptable, as described above) (see U.S. Pat. No. 6,090,382).
  • the antibody or antigen-binding portion thereof preferably contains the following characteristics:
  • a) dissociates from human TNF ⁇ with a K off rate constant of 1 ⁇ 10 ⁇ 3 s ⁇ 1 or less, as determined by surface plasmon resonance;
  • b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
  • c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
  • the antibody, or antigen-binding portion thereof dissociates from human TNF ⁇ with a K off of 5 ⁇ 10 ⁇ 4 s ⁇ 1 or less. Even more preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNF ⁇ with a K off of 1 ⁇ 10 ⁇ 4 s ⁇ 1 or less.
  • the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 307 (i.e., the adalimumab VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 308 (i.e., the adalimumab VH CDR2).
  • the LCVR further has CDR1 domain comprising the amino acid sequence of SEQ ID NO: 309 (i.e., the adalimumab VL CDR1) and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 310 (i.e., the adalimumab VH CDR1).
  • the framework regions for VL preferably are from the V ⁇ I human germline family, more preferably from the A20 human germline Vk gene and most preferably from the adalimumab VL framework sequences shown in FIGS. 1A and 1B of U.S. Pat. No. 6,090,382.
  • the framework regions for VH preferably are from the V H 3 human germline family, more preferably from the DP-31 human germline VH gene and most preferably from the adalimumab VH framework sequences shown in FIGS. 2A and 2B of U.S. Pat. No. 6,090,382.
  • the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 303 (i.e., the adalimumab VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 304 (i.e., the adalimumab VH).
  • the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.
  • the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region.
  • the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region.
  • the antibody comprises a kappa light chain constant region.
  • the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • the TNF ⁇ inhibitor of the invention is etanercept (described in WO 91/03553 and WO 09/406,476), infliximab (described in U.S. Pat. No. 5,656,272), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibody fragment), D2E7 (a human anti-TNF mAb), soluble TNF receptor Type I, or a pegylated soluble TNF receptor Type I (PEGs TNF-R1).
  • etanercept described in WO 91/03553 and WO 09/406,476
  • infliximab described in U.S. Pat. No. 5,656,272
  • CDP571 a humanized monoclonal anti-TNF-alpha IgG4 antibody
  • CDP 870 a humanized monoclonal anti-TNF-alpha antibody fragment
  • the TNF ⁇ antibody of the invention can be modified.
  • the TNF ⁇ antibody or antigen binding fragments thereof is chemically modified to provide a desired effect.
  • pegylation of antibodies and antibody fragments of the invention may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety).
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • a preferred water-soluble polymer for pegylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol.
  • Methods for preparing pegylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby the antibody or antibody fragment becomes attached to one or more PEG groups, and (b) obtaining the reaction products.
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • TNF ⁇ antibodies or fragments thereof can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody.
  • the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. Immunol. 147:2657-2662).
  • Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity.
  • an antibody or antibody portion of the invention can be derivatized or linked to another functional molecule (e.g., another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the anti-TNF ⁇ antibodies described herein, including immunoadhesion molecules.
  • an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody e.g., a bispecific antibody or a diabody
  • a detectable agent e.g., a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company, Rockford, Ill.
  • Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds.
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like.
  • An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
  • the detectable agent horseradish peroxidase when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable.
  • An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Selection of the particular parameters of the treatment regimen can be based on known treatment parameters for the TNF ⁇ inhibitor previously established in the art.
  • a treatment regimen for adalimumab HUMIRATM
  • a non-limiting example of a treatment regimen for etanercept ENBRELTM
  • a non-limiting example of a treatment regimen for infliximab REMICADETM
  • a treatment regimen can include administration of the TNF ⁇ inhibitor alone or can include combination of the TNF ⁇ inhibitor with other therapeutic agents, such as methotrexate (e.g., 10-20 mg/week) or prednisolone (e.g., 10 mg/week).
  • suitable treatment regimens for the TNF ⁇ inhibitors discussed herein will be readily apparent to the ordinarily skilled artisan based on prior studies of preferred administration parameters for the TNF ⁇ inhibitor.
  • a TNF ⁇ inhibitor typically is formulated into a pharmaceutical composition containing the TNF ⁇ inhibitor and a pharmaceutically acceptable carrier.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • Pharmaceutical compositions also can be administered in combination therapy, i.e., combined with other agents, such as other TNF ⁇ inhibitors and/or other therapeutic agents, such as traditional therapeutic agents for the treatment of autoimmune disorders, such as rheumatoid arthritis.
  • “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • compositions may include one or more pharmaceutically acceptable salts.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition also may include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents,
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a TNF ⁇ inhibitor of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • a preferred route of administration, particularly for antibody agents, is by intravenous injection or infusion.
  • Other preferred routes of administration include intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • a TNF ⁇ inhibitor of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the subject to be treated with the TNF ⁇ inhibitor is a human subject.
  • a preferred autoimmune disorder in which to apply the methods of the invention for selecting and using a treatment regimen is rheumatoid arthritis.
  • these methods can be applied to essentially any autoimmune disorder in which TNF ⁇ inhibitor therapy is applied, including the autoimmune disorders listed in the previous sections
  • kits for carrying out the methods of the invention pertains to kits for carrying out the methods of the invention.
  • the invention provides a kit for predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder.
  • the kit comprises:
  • kits for use of the kit to predicting responsiveness to a TNF ⁇ inhibitor in a subject having an autoimmune disorder, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above)
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (or the biomarkers set forth by name in Table 9 as being increased in ⁇ 80% of responders vs.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (or the biomarkers set forth by name in Table 9 as being increased in ⁇ 90% of responders vs. non-responders, as described above), and even more preferably at least one of the biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (or the CD11c biomarkers set forth by name in Table 9 as being increased in 100% of responders vs. non-responders).
  • the instructions for use of the kit instruct that increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (or the biomarkers set forth by name in Table 9 as being decreased in ⁇ 80% of responders vs.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (or the biomarkers set forth by name in Table 9 as being decreased in ⁇ 90% of responders vs. non-responders, as described above), and even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (or the biomarkers set forth by name in Table 9 as being decreased in 100% of responders vs. non-responders, as described above).
  • the instructions for use of the kit instruct that decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, as described above).
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, as described above), more preferably SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as being upregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as being downregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is decreased in the subject.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, as described above), more preferably SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as being upregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, as described above), wherein the instructions instruct that the subject is monitored prior to treatment with a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, as described above), wherein the instructions instruct that the subject is monitored after treatment with a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, as described above).
  • the means for measuring expression in the subject of the one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder comprises a nucleic acid preparation sufficient to detect expression of mRNA encoding the one or more biomarkers in a sample from the subject, such as a peripheral blood mononuclear cell sample from which mRNA is obtained by standard methods.
  • This nucleic acid preparation includes at least one, and may include more than one, nucleic acid probe or primer, the sequence(s) of which is designed such that the nucleic acid preparation can detect the expression of mRNA encoding the biomarker(s) of interest in the sample from the subject.
  • a preferred nucleic acid preparation includes two or more PCR primers that allow for PCR amplification of a segment of the mRNA encoding the biomarker(s) of interest.
  • the kit comprises a nucleic acid preparation sufficient to detect expression of CD11c mRNA in a sample from the subject.
  • the means for detecting expression in the subject of one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder can comprise a reagent that detects the gene product of the mRNA encoding the biomarker(s) of interest sufficient to distinguish it from other gene products in a sample from the subject.
  • a reagent is a monoclonal antibody preparation (comprising one or more monoclonal antibodies) sufficient to detect protein expression of the biomarker(s) of interest in a sample from the subject, such as a peripheral blood mononuclear cell sample.
  • the kit comprises a monoclonal antibody preparation sufficient to detect expression of CD11c protein in a sample from the subject.
  • the means for measuring expression of the one or more biomarkers can also include, for example, buffers or other reagents for use in an assay for evaluating biomarker expression (e.g., at either the mRNA or protein level).
  • the instructions can be, for example, printed instructions for performing the assay for evaluating the expression of the one or more biomarkers.
  • the means for measuring expression of at least one housekeeping gene comprises a nucleic acid preparation sufficient to detect expression of mRNA of the housekeeping gene (e.g., GAPDH) in a sample from the subject, such as a peripheral blood mononuclear cell sample from which mRNA is obtained by standard methods.
  • This nucleic acid preparation includes at least one, and may include more than one, nucleic acid probe or primer, the sequence(s) of which is designed such that the nucleic acid preparation can detect the expression of mRNA of the housekeeping gene(s) in the sample from the subject.
  • a preferred nucleic acid preparation includes two or more PCR primers that allow for PCR amplification of a segment of the mRNA of the housekeeping gene(s).
  • the means for detecting expression in the subject of at least one housekeeping gene can comprise a reagent that detects the gene product of housekeeping gene sufficient to distinguish it from other gene products in a sample from the subject.
  • a reagent is a monoclonal antibody preparation (comprising one or more monoclonal antibodies) sufficient to detect protein expression of housekeeping gene product in a sample from the subject, such as a peripheral blood mononuclear cell sample.
  • the means for isolating monocytes can comprise one or more reagents that can be used to separate monocytes from other cell types in a sample of peripheral blood mononuclear cells, for example by positive selection of the monocytes or by negative selection in which all other cell types other than monocytes are removed.
  • a reagent that binds CD14 on monocytes e.g., an anti-CD14 antibody
  • reagents such as those commercially available in the Monocyte Isolation Kit II (Miltenyi Biotec, Auburn, Calif.) can be used for negative selection, in which non-monocytes (T cells, B cells, NK cells, dendritic cells, basophils) are indirectly magnetically labeled using a cocktail of biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and CD235a (Glycophorin A), as well as anti-biotin MicroBeads, and then highly pure unlabeled monocytes are obtained by depletion of the magnetically labeled cells.
  • non-monocytes T cells, B cells, NK cells, dendritic cells, basophils
  • Glycophorin A biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and CD235a
  • anti-biotin MicroBeads anti-biotin MicroBeads
  • the kit can further comprise a TNF ⁇ inhibitor for treating an autoimmune disorder in the subject.
  • TNF ⁇ inhibitors for use in the kit include the TNF ⁇ inhibitors described in detail above with respect to treatment regimens, in particular anti-TNF ⁇ antibodies such as adalimumab, infliximab and/or golimumab, and/or Ig fusion proteins such as etanercept.
  • the kit is designed for use with a human subject.
  • the invention pertains to methods of building a database for use in selecting an autoimmune disorder subject for treatment with a TNF ⁇ inhibitor, or for use in selecting or monitoring a treatment regimen in an autoimmune disorder subject.
  • the method can comprise receiving, in a computer system, biomarker expression patterns from a plurality of subjects having an autoimmune disorder; and storing the biomarker expression pattern from each subject such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above).
  • the identifier of the subject can be, for example, the name of the subject or a numerical or symbolic identifier coded to the identity of the subject.
  • the method can further comprise receiving, in the computer system, one or more treatment regimens for treatment of an autoimmune disorder in a subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • a user can enter the subject's biomarker expression pattern, and optionally the subject's treatment regimen(s), into the computer system.
  • the subject's biomarker expression pattern can be received directly from equipment used in determining the expression of one or more biomarkers in a sample from the subject.
  • the invention provides a computer program product useful for building a database for use in selecting or monitoring an autoimmune disorder subject for treatment with a TNF ⁇ inhibitor.
  • the computer program can contain executable instructions that when executed cause a processor to perform operations comprising: receiving, in a computer system, a biomarker expression pattern of a subject at one or more biomarkers predictive of responsiveness to a TNF ⁇ inhibitor in an autoimmune disorder; and storing the biomarker expression pattern such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above).
  • the computer program can further cause the processor to perform an operation comprising: receiving, in the computer system, a treatment regimen for treatment of an autoimmune disorder in the subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • the invention provides a method of selecting an autoimmune disorder subject for a treatment with a TNF ⁇ inhibitor.
  • the method can comprise: (i) identifying, in a database comprising a plurality of autoimmune disorder subjects, a subject whose database entry is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNF ⁇ inhibitor, and (ii) selecting the subject for treatment with a TNF ⁇ inhibitor, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above).
  • the method can further comprise selecting a treatment regimen by identifying, in the database, a treatment regimen that has been associated with the biomarker expression pattern of the subject and with an identifier of the subject.
  • the invention provides a computer program product useful for identifying and/or selecting a subject for treatment with a TNF ⁇ inhibitor.
  • the computer program can contain executable instructions that when executed cause a processor to perform operations comprising: (i) identifying, in a database including a plurality of autoimmune disorder subjects associated with biomarker expression patterns, a subject that is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNF ⁇ inhibitor; and (ii) outputting the identified subject as a subject to be treated with a TNF ⁇ inhibitor; wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above).
  • the computer program can further cause the processor to perform an operation comprising outputting a treatment regimen that is associated with the subject to be treated with the TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (or the biomarkers set forth by name in Table 9 as being increased in ⁇ 80% of responders vs.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (or the biomarkers set forth by name in Table 9 as being increased in ⁇ 90% of responders vs. non-responders, as described above), and even more preferably at least one of the biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (or the biomarker, CD11c antigen, set forth by name in Table 9 as being increased in 100% of responders vs. non-responders, as described above).
  • increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (or the biomarkers set forth by name in Table 9 as being decreased in ⁇ 80% of responders vs.
  • the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (or the biomarkers set forth by name in Table 9 as being decreased in ⁇ 90% of responders vs. non-responders, as described above), and even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (or the biomarkers set forth by name in Table 9 as being decreased in 100% of responders vs. non-responders, as described above).
  • decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNF ⁇ inhibitor.
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, as described above).
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, as described above), more preferably SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as having increased expression, as described above), wherein expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as having decreased expression, as described above), wherein expression of the one or more biomarkers is decreased in the subject.
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, as described above), more preferably SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as having increased expression, as described above), wherein expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, as described above), wherein the subject is monitored prior to treatment with a TNF ⁇ inhibitor.
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, as described above), wherein the subject is monitored after treatment with a TNF ⁇ inhibitor.
  • the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, as described above).
  • Computer systems and database software well established in the art can be adapted for use in the methods and computer program products of the invention for building and searching a database for use in selecting or monitoring a treatment regimen for a subject having an autoimmune disorder or for selecting a particular autoimmune disorder subject for treatment with a TNF ⁇ inhibitor.
  • ACR American College of Rheumatology
  • ACR response evaluation was performed by applying the ACR criteria, but by defining 10% response steps instead of the usual 20, 50, and 70% steps. This was also done to allow more detailed analyses of the correlation between the ACR response and the mRNA expression levels of the predictive marker CD11c.
  • the mean time interval between the two MO samples was 9.4 ⁇ 1.8 months (mean ⁇ SEM; range 4-13.5 months), the mean age 50.3 ⁇ 3.9 years (range 39-70), and the mean disease duration 18.6 ⁇ 5.3 years (range 4-38).
  • RA patients RA1-RA84 were recruited from the Rheumatology Clinic of the Charotti University Clinic, the Rheumaklinik of the Charotti in Berlin-Buch, and from the Schlossparklinik in Berlin.
  • MO preparation was performed at 4° C. After purification, MO were lysed in RLT lysis buffer and total RNA was isolated using the RNeasy mini elute kit (Quiagen, Düsseldorf, Germany; yield 1.5-3.2 ⁇ g/sample). Quantification and quality control of RNA was performed at 260/280 nm using a Bioanalyzer 2100 unit (Agilent, Palo Alto, Calif., USA).
  • RNA samples 500 ng were amplified using the standard protocol of the manufacturer (Affymetrix®, Palo Alto, Calif., USA) and the Megascript kit (Ambion, Camhridgeshire, UK).
  • Biotinylated cRNA target was generated from amplified cRNAs using the Bioarray high-yield transcription kit (Enzo, New York, N.Y., USA). Samples were hybridized to Affymetrix® test and HG-U133A GeneChip arrays.
  • arrays were scanned twice at 3 ⁇ m resolution using a confocal scanner with an argon laser instrument (Agilent® G2500A GeneArray Scanner; Agilent, CA, USA).
  • RT-PC Real-time PCR
  • Applied Biosystems Foster City, Calif., USA
  • the primer Hs01015072_g1 commercially available from Applied Biosystems
  • Bio-Rad iQ real time PCR system Icycler; Bio-Rad; München, Germany.
  • the housekeeping gene GAPDH was used for normalization of the cDNA content.
  • the KEGG pathway analysis was performed using selected genes from the comparison of microarray data in responders and non-responders either pre- or post-treatment with anti-TNF ⁇ . Upregulated genes and downregulated genes within the illustrated pathways were color-coded in a gradient fashion (SLR 0.5 to ⁇ 1.5). A total of 4 pathways out of the 8 most highly ranked pathways were selected for illustration.
  • the non-parametric Mann-Whitney U test was applied to analyze differences between data from RA patients and normal donors, from untreated and anti-TNF ⁇ -treated RA patients, and from responders and non-responders to anti-TNF ⁇ -therapy. Correlation analyses between experimental and clinical/laboratory parameters of the patients were performed using the Pearson test and the software SPSS 13.0TM (SPSS Inc., Chicago, Ill., USA). For the U test, statistically significant differences were accepted for P ⁇ 0.05; for correlation analyses, the acceptance level was reduced to P ⁇ 0.01 to account for multiple comparisons.
  • RA4 and RA6 Two of the seven anti-TNF ⁇ -treated RA patients used for microarray analysis, i.e., patients RA4 and RA6, were non-responders to therapy according to the ACR improvement criteria ( ⁇ continuous ACR 30 score). In general, this was also reflected in the respective percent-reduction of other clinical parameters of local or systemic inflammation.
  • the group of seven RA patients employed for microarray analysis in the present study constituted a representative RA cohort, as demonstrated by well-known correlations among clinical parameters pre- and post-anti-TNF ⁇ treatment, as summarized in Table 2.
  • Hierarchical clustering with these genes resulted in precise (100%) classification of ND, RA patients pre-treatment, and clinically-defined responders ( ⁇ continuous ACR score 40; clustered as ND) or non-responders ( ⁇ continuous ACR score 30; clustered as RA; note RAantiTNF4 and RAantiTNF6).
  • PAM analysis prediction analysis of microarrays
  • Table 4 summarizes the results of the PAM analysis, showing 49 selected genes, five of them overlapping with genes listed in Table 3.
  • both non-responders (RAantiTNF4 and RAantiTNF6) were classified as RA patients, whereas the responders, RAantiTNF1-3, RAantiTNF5, and RAantiTNF7, were classified either as normals or as anti-TNF ⁇ -treated RA patients.
  • ND showed the lowest misclassification error at the threshold value (0.00; i.e., the highest similarity among individuals), followed by pre-treatment RA patients (0.14), and anti-TNF ⁇ -treated RA patients (0.42).
  • a number of the differentially-expressed genes showed highly significant correlations with clinical or laboratory parameters pre- and/or post-anti-TNF ⁇ treatment, indicating a potential clinical relevance of the genes and contributing to the selection of genes for validation with TaqMan® real-time RT-PCR. These genes are summarized in Table 6.
  • genes showed significantly differential expression (p ⁇ 0.000 for 7 genes; p ⁇ 0.041 for the remaining) in MO from RA patients responding to anti-TNF ⁇ therapy versus ND, including genes 10 upregulated in RA (Amphiregulin, Charcot-Leyden crystal protein, TNF ⁇ -induced protein 6, thrombomodulin, membrane-spanning 4-domains, subfamily A—member 4, S100 calcium binding protein A1, TNF ⁇ , IL-1 ⁇ , lipoyltransferase 1, and interferon regulatory protein 1), as well as 8 genes downregulated in RA (Uncharacterized hypothalamus protein HT007, MHC class II HLA-DR-alpha, hypothetical protein L0054103, proteasome subunit beta type 7 precursor, protein KIAA0174, microsomal signal peptidase 18 kDa subunit, ring zinc finger protein 361, and protein phosphatase 1, catalytic subunit, beta isoform). However, the expression for these genes showed no significant differences for the direct comparison between RA
  • the 18 genes showing significantly differential expression in MO from RA patients responding to anti-TNF ⁇ therapy versus ND included several genes with a likely pathogenetic importance in RA (e.g., amphiregulin, TNF ⁇ -induced protein 6, thrombomodulin, membrane-spanning 4-domains, subfamily A—member 4, S100 calcium binding protein A1, TNF ⁇ , IL-1 ⁇ , lipoyltransferase 1, interferon regulatory protein 1, MHC class II HLA-DR-alpha).
  • genes with a likely pathogenetic importance in RA e.g., amphiregulin, TNF ⁇ -induced protein 6, thrombomodulin, membrane-spanning 4-domains, subfamily A—member 4, S100 calcium binding protein A1, TNF ⁇ , IL-1 ⁇ , lipoyltransferase 1, interferon regulatory protein 1, MHC class II HLA-DR-alpha.
  • a threshold of ⁇ 80% for the pairwise comparisons between future RA responders and future RA non-responders prior to anti-TNF ⁇ therapy a total of 82 predictive genes was identified (11 genes for a threshold of ⁇ 90%; 3 genes for 100%). These genes are summarized in Table 9. The latter group (100%) consisted of 2 known proteins ( Homo sapiens predicted osteoblast protein (GS3786); integrin alpha-X (antigen CD11c) and an unknown protein ( Homo sapiens hypothetical protein FLJ10134).
  • the antigen CD11e appears highly interesting, since it is a surface molecule on human MO (and other cells of the myelomonocytic lineage), and since it has known inflammatory functions.
  • Hierarchical clustering with the selected genes resulted again in precise (100%) predictive classification of future responders and non-responders to therapy.
  • the results are summarized in the bar graph of FIG. 1 . This clear separation was lost in the case of combination therapy with anti-TNF ⁇ /MTX, possibly due to a differential importance of the CD11c mRNA expression for the anti-TNF ⁇ and MTX components.
  • the antigen CD11c appears of particular interest, since it is expressed on the surface of human MO (and other cells of the myelomonocytic lineage, e.g. dendritic cells), and since it has known functions in inflammatory reactions (e.g., as the complement receptor 4) and cell adhesion.
  • Pairwise comparison between RA responders and RA non-responders prior to treatment yielded a number of genes suitable for the prediction of a future response to anti-TNF ⁇ therapy (82 genes for a threshold of ⁇ 80%; 11 genes for ⁇ 90%; 3 genes for 100%; summarized in Table 9), resulting in exact classification of future responders and non-responders upon hierarchical clustering.
  • a threshold level of 70% 256 pre-treatment; 1295 post-treatment
  • Ingenuity® pathway analysis the differences between responders and non-responders either pre-treatment or post-treatment were concentrated in the functional gene ontology terms cellular movement, haematological system development, immune response, cell-to-cell signaling and interaction, as well as immunological disease.
  • pro-inflammatory cytokines e.g., interleukin-8 [IL8], chemokine (C—C motif) ligand 5 [CCL5], chemokine (C—X—C motif) ligand 5 [CXCL5], and chemokine (C—X—C motif) ligand 10 [CXCL10]
  • pro-destructive enzymes e.g., matrix metalloproteinase 9 [MMP9]
  • adhesion molecules and Fc ⁇ -receptors galectin-8 [LGALS8], integrin alpha-X [ITGAX] or CD11c, Fc-gamma receptor IIb [FCGR2B] or CD32, CD86 [CD86] and platelet/endothelial cell adhesion molecule 1 [PECAM1] or CD31
  • signal transduction molecules e.g., protein kinase B [AKT1]
  • TNF ⁇ in addition to its well-established pro-inflammatory properties, may also exhibit phase-dependent immunosuppressive properties (Kassiotis, G. et al. (2001) J. Exp. Med. 193:427-434).
  • KEGG pathway analysis Several interesting pathways with potential importance for the mechanisms underlying susceptibility to anti-TNF ⁇ -therapy were identified by KEGG pathway analysis; including Apotosis and the MAPK pathways.
  • RA29-aTNF/MTX 9 (53) 3.0 (72) n.d. 4.1 (13) 0 RA30-1110010101 52 42.6 n.d. 5.3 RA30-aTNF/MTX 17 (63) 10.1 (76) n.d. 2.6 (51) 50 RA31-1110004121 33 7.8 1.3 5.2 RA31-aTNF 18 (45) 5.9 (24) 0.9 (31) 3.4 (35) 40 RA32-1110008092 29 18.4 n.d. 6.8 RA32-aTNF 28 (3) 21.0 (0) n.d.
  • RA all) RA-aTNF vs. RA (responder) 36711_at 28.57 61.22 ⁇ 2.78 ⁇ 1.48 12.00 80.00 ⁇ 5.88 ⁇ 2.56 38037_at 12.24 73.47 ⁇ 2.59 ⁇ 1.37 — 84.00 ⁇ 3.70 ⁇ 1.89 201386_s_at 44.90 14.29 1.43 0.51 60.00 — 1.81 0.85 201890_at 2.04 69.39 ⁇ 3.86 ⁇ 1.95 4.00 64.00 ⁇ 3.88 ⁇ 1.96 202219_at 12.24 59.18 ⁇ 5.17 ⁇ 2.37 4.00 72.00 ⁇ 6.96 ⁇ 2.80 202464_s_at 12.24 69.39 ⁇ 2.18 ⁇ 1.12 — 84.00 ⁇ 3.01 ⁇ 1.59 203115_at 20.41 63.27 ⁇ 2.18 ⁇ 1.12 16.00 72.00 ⁇ 2.25 ⁇ 1.17 203574_at 10.20 53.06 ⁇ 1.54 ⁇ 0.62
  • /PROD 6- ⁇ 3.25 ⁇ 1.70 phosphofructo-2-kinase-fructose-2,6-biphosphatase 3 202545_at 100 0 KIAA0766 gene product (KIAA0766), mRNA 1.38 0.46 202838_at 100 0 N-acetylgalactosaminidase, alpha-(NAGA), mRNA 1.82 0.86 202896_s_at 0 100 Protein tyrosine phosphatase, non-receptor type substrate 1 (PTPNS1), mRNA ⁇ 2.00 ⁇ 1.00 202950_at 100 0 Crystallin, zeta (quinone reductase) (CRYZ), mRNA 2.51 1.33 203037_s_at 100 0 KIAA0429 gene product (KIAA0429), mRNA 2.41 1.27 203155_at 100 0 SET domain, bifurcated 1 (SETDB1), mRNA 4.23 2.08 203371_s
  • /PROD early growth response 3 ⁇ 6.54 ⁇ 2.71 206584_at 0 100 Homo sapiens MD-2 protein (MD-2), mRNA ⁇ 2.16 ⁇ 1.11 206877_at 0 100 Homo sapiens MAX dimerization protein (MAD), mRNA ⁇ 3.92 ⁇ 1.97 207170_s_at 100 0 DKFZP586A011 protein (DKFZP586A011), mRNA 2.20 1.14 208631_s_at 100 0 78 kDa gastrin-binding protein mRNA, complete cds 2.04 1.03 208691_at 0 100 Transferrin receptor (p90, CD71), clone MGC: 3151, mRNA, complete cds ⁇ 2.30 ⁇ 1.20 208868_s_at 0 100 Homo sapiens mRNA; cDNA DKFZp564N1272 (from clone DKFZp564N1272); ⁇
  • /PROD apurinic endonuclease 2.23 1.16 210053_at 100 0 TATA box binding protein (TBP)-associated factor, RNA polymerase II, D, 100 kD 2.23 1.16 210172_at 0 100 Human mRNA for ZFM1 protein alternatively spliced product, complete cds.
  • TBP TATA box binding protein
  • /PROD ZFM1 ⁇ 2.16 ⁇ 1.11 protein, alternatively spliced product 210766_s_at 100 0 Trachea cellular apoptosis susceptibility protein (CSE1) mRNA, complete cds 2.28 1.19 210949_s_at 100 0 Similar to eukaryotic translation initiation factor 3, subunit 8 (110 kD), clone MGC: 2.53 1.34 8693, mRNA, complete cds 211458_s_at 0 100 GABA-A receptor-associated protein mRNA, complete cds.
  • CSE1 Trachea cellular apoptosis susceptibility protein
  • /PROD GABA-A ⁇ 3.18 ⁇ 1.67 receptor-associated protein 211546_x_at 0 100 Human (clone 2-5) synuclein (NACP) mRNA, complete cds ⁇ 4.92 ⁇ 2.30 212199_at 100 0 Human putative ribosomal protein S1 mRNA 2.22 1.15 212224_at 100 0 Aldehyde dehydrogenase 1, soluble (ALDH1), mRNA.
  • /PROD aldehyde dehydrogenase 3.18 1.67 1, soluble 212388_at 100 0 Homo sapiens mRNA for KIAA1057 protein, partial cds 3.10 1.63 212591_at 100 0 RBP1-like protein 2.03 1.02 212696_s_at 100 0 Ring finger protein 4 3.05 1.61 212709_at 100 0 KIAA0197 protein 2.10 1.07 212714_at 100 0 Homo sapiens mRNA; cDNA DKFZp586F1323 (from clone DKFZp586F1323) 2.13 1.09 212893_at 100 0 Homo sapiens mRNA; cDNA DKFZp564I052 (from clone DKFZp564I052) 2.01 1.01 212989_at 100 0 Homo sapiens mRNA for Hmob33 protein, 3 untranslated region 2.25 1.17 213410_at 100 0 Homo sap
  • polypeptide 5 ⁇ 2.71 ⁇ 1.44 221652_s_at 100 0 PNAS-25 mRNA, complete cds. 2.55 1.35 221755_at 100 0 Homo sapiens mRNA for FLJ00043 protein, partial cds 3.44 1.09 221970_s_at 100 0 Homo sapiens cDNA: FLJ21737 fis, clone COLF3396 2.41 1.27 222127_s_at 0.1 0 Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395 /DEF Homo sapiens cDNA 2.31 1.21 FLJ13399 fis, clone PLACE1001395 36711_at 0 100 Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F) ⁇ 3.54 ⁇ 1.16 LIKE protein 38037_at 0 100 Heparin-binding EGF-like growth factor mRNA
  • oncogene homolog F (avian) 0.932 0.002** 205552 _s_at 2′,5′-oligoadenylate synthetase 1, 40/46 kDa ⁇ 0.951 0.001** DAS28/ 36711 v-maf fibrosarc.
  • oncogene homolog F (avian) 0.940 0.002** 205552 _s_at 2′,5′-oligoadenylate synthetase 1, 40/46 kDa ⁇ 0.898 0.006** Sw28/ 219607_s_at Membrane-spanning 4-domains, subfam. A, memb.
  • GPT glutamate pyruvic transferase
  • Hb haemoglobin
  • MoStiff Morning stiffness (minutes)
  • DisDur Disease duration (years)
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • BL TNFAIP6 Tumor necrosis factor alpha Hs00218482_m1 1.71 ⁇ 0.40 0.82 0.000 induced protein 6 BL THBD Thrombomodulin Hs00264920_s1 1.58 ⁇ 1.08 ⁇ 0.53 0.041 BL MS4A4A Membrane-spanning 4-domains, Hs00254770_m1 1.54 ⁇ 0.42 0.71 0.041 subfamily A, member 4 BL DTR Diptheria toxin receptor Hs00181813_m1 1.21 ⁇ 0.44 ⁇ 1.10 n.s.

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Abstract

The invention provides methods for predicting responsiveness to TNFα inhibitors in a subject suffering from an autoimmune disorder, such as rheumatoid arthritis. The methods involve assaying for expression of one or more biomarkers in the subject that are predictive of responsiveness to TNFα inhibitors. A preferred biomarker of the invention is CD11c. The methods can further comprise selecting a treatment regimen with a TNFα inhibitor in an autoimmune disorder subject based upon expression of the biomarker(s) in the subject. The methods can further comprise administering a TNFα inhibitor to the subject according to the selected treatment regimen. Kits that include means for measuring expression of one or more biomarkers that are predictive of responsiveness to TNFα inhibitors for an autoimmune disorder are also provided. Methods of preparing and using databases, and computer program products therefore, for selecting an autoimmune disorder subject for treatment with a TNFα inhibitor are also provided.

Description

    RELATED APPLICATIONS
  • This application is a continuation application of U.S. patent application Ser. No. 12/130,373, filed on May 30, 2008, which claims the benefit of priority to U.S. provisional patent application No. 60/932,888 filed on May 31, 2007. The contents of the above-mentioned priority application is hereby incorporated by reference in its entirety.
    • BACKGROUND OF THE INVENTION
  • Autoimmune disorders are a significant and widespread medical problem. For example, rheumatoid arthritis (RA) is an autoimmune disease affecting more than two million people in the United States. RA causes chronic inflammation of the joints and typically is a progressive illness that has the potential to cause joint destruction and functional disability. The cause of rheumatoid arthritis is unknown, although genetic predisposition, infectious agents and environmental factors have all been implicated in the etiology of the disease. In active RA, symptoms can include fatigue, lack of appetite, low grade fever, muscle and joint aches and stiffness. Also during disease flare ups, joints frequently become red, swollen, painful and tender, due to inflammation of the synovium. Furthermore, since RA is a systemic disease, inflammation can affect organs and areas of the body other than the joints, including glands of the eyes and mouth, the lung lining, the pericardium, and blood vessels.
  • Traditional treatments for the management of RA and other autoimmune disorders include fast acting “first line drugs” and slower acting “second line drugs.” The first line drugs reduce pain and inflammation. Example of such first line drugs include aspirin, naproxen, ibuprofen etodolac and other nonsteroidal anti-inflammatory drugs (NSAIDs), as well as corticosteroids, given orally or injected directly into tissues and joints. The second line drugs promote disease remission and prevent progressive joint destruction and are also referred to as disease-modifying anti-rheumatic drugs or DMARDs. Examples of second line drugs include gold, hydrochloroquine, azulfidine and immunosuppressive agents, such as methotrexate, azathioprine, cyclophosphamide, chlorambucil and cyclosporine. Many of these drugs, however, can have detrimental side-effects. Thus, additional therapies for rheumatoid arthritis and other autoimmune disorders have been sought.
  • More recently, biological therapies have been applied to the treatment of autoimmune disorders such as rheumatoid arthritis. For example, three TNFα inhibitors, REMICADE™ (infliximab), a chimeric anti-TNFα mAb, ENBREL™ (etanercept), a TNFR-Ig Fc fusion protein, and HUMIRA™ (adalimumab), a human anti-TNFα mAb, have been approved by the FDA for treatment of rheumatoid arthritis. While such biologic therapies have demonstrated success in the treatment of rheumatoid arthritis and other autoimmune disorders, not all subjects treated respond, or respond well, to a TNFα inhibitor. The use of TNFα inhibitors such as TNFα inhibitors typically is more expensive than traditional treatments and usually requires administration by injection, which, at least for certain agents, may require that the patient visit a medical office on a frequent basis. Thus, it would be very helpful to predict in advance of treatment whether a rheumatoid arthritis patient is likely to be responsive to treatment with a TNFα inhibitor. Accordingly, ways for predicting responsiveness to a TNFα inhibitor in patients having autoimmune disorders, such as rheumatoid arthritis patients, are of particular interest.
    • SUMMARY OF THE INVENTION
  • This invention provides methods and compositions for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, such as rheumatoid arthritis, based on the discovery that the expression patterns of particular biomarkers in the subject correlate with responsiveness to a TNFα inhibitor. Using microarray analysis of monocytes from representative rheumatoid arthritis (RA) patients treated with an anti-TNFα monoclonal antibody, 82 differentially expressed genes predictive of responsiveness to TNFα inhibitor treatment were identified by pairwise comparisons between future RA responders and future RA non-responders to anti-TNFα therapy. Furthermore, hierarchical clustering and TaqMan®-PCR of RA responders/non-responders pre-treatment identified one gene of particular interest, CD11c, which was fully predictive of future response to anti-TNFα treatment.
  • Accordingly, in one aspect, the invention pertains to a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder (e.g., rheumatoid arthritis). The method comprises: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to the sequences set forth in Table 9)
  • Within SEQ ID NOs: 1-82, certain genes were found to be upregulated in RA responders to TNFα inhibitors. Accordingly, in one embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (corresponding to sequences from Table 9 that are increased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (corresponding to sequences from Table 9 that are increased in ≧90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44. (corresponding to CD11c, from Table 9, which is increased in 100% of responders vs. non-responders). In each of these embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • Within SEQ ID NOs: 1-82, certain genes were found to be downregulated in RA responders to TNFα inhibitors. Accordingly, in one embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81. (corresponding to sequences from Table 9 that are decreased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (corresponding to sequences from Table 9 that are decreased in ≧90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (corresponding to sequences from Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another aspect, the invention provides a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is selected from the group consisting of Aquaporin 3 (Genbank Accession No. NM—004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No. NM—033022); Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM—006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM—000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM—006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM—002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No. NM—002823); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM—005896); Tumor protein D52 (Genbank Accession Nos. NM—001025252, NM—001025253, NM—005079); Early growth response 1 (Genbank Accession No. NM—001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM—014888, NM—001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No. NM—001866); CUG triplet repeat, RNA binding protein 2 (Genbank Accession No. NM—001025077, NM—001025076, NM—006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM—006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM—017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM—001077269, NM—003387); CD97 antigen (Genbank Accession Nos. NM—001025160, NM—001784, NM—078481); Glutamate-cysteine ligase, catalytic subunit (Genbank Accession No. NM—001498); Crystallin, zeta (quinone reductase) (Genbank Accession No. NM—001889); Rap guanine nucleotide exchange factor (GEF) 2 (Genbank Accession No. NM—014247); Ataxin 1 (Genbank Accession No. NM—000332); Adaptor-related protein complex 1, sigma 2 subunit (Genbank Accession No. NM—003916); Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM—014936); Desmocollin 2 (Genbank Accession Nos. NM—024422, NM—004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM—002371, NM—022438, NM—022439, NM—022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No. NM—005476); Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM—001001437, NM—021006); Carboxypeptidase A3 (Genbank Accession No. NM—001870); Charcot-Leyden crystal protein (Genbank Accession No. NM—001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM—004545); Interleukin 8 receptor, beta (Genbank Accession No. NM—001557); Platelet factor 4 variant 1 (Genbank Accession No. NM—002620); Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM—006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM—003786); Actinin, alpha 1 (Genbank Accession No. NM—001102); NAD kinase (Genbank Accession No. NM—023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM—000442); Esterase D/formylglutathione hydrolase (Genbank Accession No. NM—001984); Chromosome 20 open reading frame 111 (Genbank Accession No. NM—016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM—001017369, NM—006745); PIM-1 oncogene (Genbank Accession No. NM—002648); GATA binding protein 2 (Genbank Accession No. NM—032638); Cathepsin Z (Genbank Accession No. NM—001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No. NM—000887); Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM—006499, NM—201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM—006889, NM—175862); Interleukin 8 (Genbank Accession No. NM—000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM—002001); Actin, gamma 1 (Genbank Accession No. NM—001614); KIAA0746 protein (Genbank Accession No. NM—015187); Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (Genbank Accession No. NM—002076); Transcription factor 4 (Genbank Accession Nos. BF592782, CR612521); Major histocompatibility complex, class II, DQ alpha 1 (Genbank Accession Nos. NM—002122, NM—020056); Cell division cycle 2-like 6 (CDK8-like) (Genbank Accession No. NM—015076); Major histocompatibility complex, class II, DQ beta 1 (Genbank Accession No. XM—942240); Phospholipase C-like 2 (Genbank Accession No. NM—015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM—005242); TM2 domain containing 1 (Genbank Accession No. NM—032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM—012433); SUB1 homolog (S. cerevisiae) (Genbank Accession No. NM—006713); MRNA; cDNA DKFZp564O0862 (Genbank Accession No. AI278204); Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM—201413, NM—000484, NM—201414); Cytochrome b-5 (Genbank Accession Nos. NM—001914, NM—148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM—183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM—016645, NM—001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No. XM—941927); CCR4-NOT transcription complex, subunit 6 (Genbank Accession No. NM—015455); Ubiquinol-cytochrome c reductase complex (7.2 kD) (Genbank Accession Nos. NM—013387, NM—001003684); Hepatocellular carcinoma-associated antigen 112 (Genbank Accession No. NM—018487); Kruppel-like factor 11 (Genbank Accession No. XM—001129527); GGA binding partner (Genbank Accession No. NM—018318); Cornichon homolog 4 (Drosophila) (Genbank Accession No. NM—014184); Hypothetical protein FLJ21616 (Genbank Accession No. NM—024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM—018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM—012336, NM—001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM—018538, NM—001017922); LR8 protein (Genbank Accession No. NM—014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No. NM—030915); Calmin (calponin-like, transmembrane) (Genbank Accession No. NM—024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM—031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM—007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR—003287) (corresponding to biomarkers listed in Table 9).
  • Within the above-listed biomarkers, certain genes were found to be upregulated in RA responders to TNFα inhibitors. Accordingly, in one embodiment, the one or more biomarkers is selected from the group consisting of Guanine nucleotide binding protein (G protein), beta polypeptide 1; Prothymosin, alpha (gene sequence 28); Early growth response 1; Homo spaiens leptin receptor gene-related protein (HS0BRGRP); Wiskott-Aldrich syndrome protein interacting protein; CD97 antigen; Crystallin, zeta (quinone reductase); Adaptor-related protein complex 1, sigma 2 subunit; Desmocollin 2; Chemokine (C—C motif) ligand 3; Interleukin 8 receptor, beta; ATP-binding cassette, sub-family C (CFTR/MRP), member 3; Actinin, alpha 1; NAD kinase; Platelet/endothelial cell adhesion molecule (CD31 antigen); Esterase D/formylglutathione hydrolase; Chromosome 20 open reading frame 111; Cathepsin Z; Integrin alpha-X (antigen CD11c); Lectin, galactoside-binding, soluble, 8 (galectin 8); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen); Interleukin 8; Actin, gamma 1; Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID); Cell division cycle 2-like 6 (CDK8-like); Major histocompatibility complex, class II, DQ beta 1; Coagulation factor II (thrombin) receptor-like 1; Splicing factor 3b, subunit 1, 155 kDa; mRNA; cDNA DKEZp564O0862; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); Cold autoinflammatory syndrome 1; Kruppel-like factor 11; Nuclear prelamin A recognition factor; Calmin (calponin-like, transmembrane); and SRY (sex determining region Y)-box 18 (corresponding to biomarkers from Table 9 that are increased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is selected from the group consisting of Interleukin 8 receptor, beta; Platelet/endothelial cell adhesion molecule (CD31 antigen); Integrin alpha-X (antigen CD11c); Interleukin 8; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); and Kruppel-like factor 11 (corresponding to biomarkers from Table 9 that are decreased in ≧80% of responders vs. non-responders). Even more preferably, the one or more biomarkers is Integrin alpha-X (antigen CD11c) (corresponding to a biomarker from Table 9 that is increased in 100% of responders vs. non-responders). In each of these embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • Within the above-listed biomarkers, certain genes were found to be downregulated in RA responders to TNFα inhibitors. Accordingly, in one embodiment, the one or more biomarkers is selected from the group consisting of Aquaporin 3; Similar to ribosomal protein S24, clone MGC:8595; Transmembrane emp24 domain trafficking protein 2; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1; Calmodulin 1 (phosphorylase kinase, delta); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1); Tumor protein D52; Homo sapiens predicted osteoblast protein (GS3786); Cytochrome c oxidase subunit VIIb; CUG triplet repeat, RNA binding protein 2; Ubiquinol-cytochrome c reductase hinge protein; Glutamate-cysteine ligase, catalytic subunit; Rap guanine nucleotide exchange factor (GEF) 2; Ataxin 1; Ectonucleotide pyrophosphatase/phosphodiesterase 4; MAL, T-cell differentiation protein; Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase; Carboxypeptidase A3; Charcot-Leyden crystal protein; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa; Platelet factor 4 variant 1; Poly(A) binding protein interacting protein 1; Sterol-C4-methyl oxidase-like; PIM-1 oncogene; GATA binding protein 2; Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide; KIAA0746 protein; Transcription factor 4; Major histocompatibility complex, class II, DQ alpha 1; Phospholipase C-like 2; TM2 domain containing 1; SUB1 homolog (S. cerevisiae); Cytochrome b-5; Neugrin, neurite outgrowth associated; Ribosomal protein S26, 40S ribosomal protein; CCR4-NOT transcription complex, subunit 6; Ubiquinol-cytochrome c reductase complex (7.2 kD); Hepatocellular carcinoma-associated antigen 112; GGA binding partner; Cornichon homolog 4 (Drosophila); Hypothetical protein FLJ21616; Homo sapiens hypothetical protein FLJ10134; Erythroblast membrane-associated protein; LR8 protein; Likely ortholog of mouse limb-bud and heart gene (LBH); Chromosome 14 open reading frame 156; and Guanine nucleotide binding protein (G protein) alpha 12 (corresponding to biomarkers from Table 9 that are decreased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is selected from the group consisting of Homo sapiens predicted osteoblast protein (GS3786); Charcot-Leyden crystal protein; Neugrin, neurite outgrowth associated; Hypothetical protein FLJ21616; and Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers from Table 9 that are decreased in ≧90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is Homo sapiens predicted osteoblast protein (GS3786) or Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers from Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In yet another aspect, the invention provides a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for increased expression of a biomarker, which biomarker is CD11c, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on increased expression of CD11c in the subject.
  • In yet another aspect, the invention provides a method for predicting responsiveness to a TNFα inhibitor, which TNFα inhibitor is adalimumab, in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to adalimumab in an autoimmune disorder, and (ii) predicting responsiveness of the subject to adalimumab based on expression of the one or more biomarkers in the subject. Preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to biomarkers set forth in Table 9).
  • In one embodiment of the methods of the invention, a sample from the subject is assayed for expression of mRNA encoding the one or more biomarkers. In another embodiment of the methods of the invention, a sample from the subject is assayed for protein expression of the one or more biomarkers.
  • In one embodiment, the methods of the invention further comprise selecting a treatment regimen with the TNFα inhibitor based upon expression of the one or more biomarkers in the subject. In another embodiment, the methods of the invention further comprise administering the TNFα inhibitor to the subject according to the treatment regimen such that autoimmune disorder is inhibited in the subject.
  • A preferred TNFα inhibitor of the invention is an anti-tumor necrosis factor-alpha (TNFα) antibody, or antigen-binding portion thereof. The anti-TNFα antibody, or antigen-binding portion thereof, can be, for example, a humanized antibody, a chimeric antibody or a multivalent antibody. For example, the anti-TNFα antibody, or antigen-binding portion thereof, can be infliximab or golimumab. In another embodiment, the anti-TNFα antibody, or antigen-binding portion thereof, is a human antibody. For example, the anti-TNFα antibody, or antigen-binding portion thereof, can be an isolated human antibody that dissociates from human TNFα with a Kd of 1×10−8 M or less and a Koff rate constant of 1×10−3 s−1 or less, both determined by surface plasmon resonance, and neutralizes human TNFα cytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10−7 M or less. In another embodiment, the anti-TNFα antibody, or antigen-binding portion thereof, is an isolated human antibody with the following characteristics:
  • a) dissociates from human TNFα with a Koff rate constant of 1×10−3 s−1 or less, as determined by surface plasmon resonance;
  • b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
  • c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
  • In another embodiment, the anti-TNFα antibody, or antigen-binding portion thereof, is an isolated human antibody with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 304. In yet another embodiment, the anti-TNFα antibody, or antigen-binding portion thereof, is adalimumab. Yet another example of a TNFα inhibitor is etanercept.
  • Preferably, in the methods of the invention, the subject is a human.
  • In another aspect, the invention pertains to a kit for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder (e.g., rheumatoid arthritis). The kit comprises:
  • a) means for isolating monocytes;
  • b) means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder;
  • c) means for measuring expression of at least one housekeeping gene; and
  • d) instructions for use of the kit to predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
  • In one embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70, even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (CD11c). In each of these embodiments, the instructions for use of the kit instruct that increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74, even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74. In each of these embodiments, the instructions for use of the kit instruct that decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In one embodiment of the kit, the means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder comprises a nucleic acid preparation sufficient to detect expression of mRNA encoding the biomarker in a sample from the subject. In another embodiment of the kit, the means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder comprises an antibody preparation sufficient to detect protein expression of the biomarker in a sample from the subject. The kit can further comprise a TNFα inhibitor for treating the autoimmune disorder in the subject.
  • In another aspect, the invention pertains to methods of monitoring an autoimmune disorder (e.g., RA) in a subject having the autoimmune disorder (e.g., RA). These methods are based, at least in part, on microarray analysis of monocytes from representative rheumatoid arthritis (RA) patients treated with an anti-TNFα monoclonal antibody, including (i) hierarchical clustering with the genes resulting from a simultaneous comparison between RA versus ND and RA responders pre- versus post-anti-TNFα therapy, (ii) prediction analysis of microarrays (PAM); and (iii) hierarchical clustering based on the comparison between RA responders and non-responders post-treatment.
  • Accordingly, in another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (corresponding to biomarkers set forth in Table 3), thereby monitoring the autoimmune disorder in the subject.
  • In another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (corresponding to biomarkers set forth in Table 4), thereby monitoring the autoimmune disorder in the subject. In one embodiment, preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-150, 112, 118 and 131 (upregulated biomarkers from Table 4), wherein expression of the one or more biomarkers is increased in the subject. In another embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 151-177, 110 and 123 (downregulated biomarkers from Table 4), wherein expression of the one or more biomarkers is decreased in the subject.
  • In another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (corresponding to biomarkers set forth in Table 5), thereby monitoring the autoimmune disorder in the subject. In one embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (upregulated biomarkers from Table 5), wherein expression of the one or more biomarkers is increased in the subject. In another embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233, 236, 237, 248, 251, 252, 254-261, 266, 267, 283, 284 and 289-292 (downregulated biomarkers from Table 5), wherein expression of the one or more biomarkers is decreased in the subject.
  • In another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (corresponding to pre-treatment biomarkers set forth in Table 6), and wherein the subject is monitored prior to treatment with a TNFα inhibitor, thereby monitoring the autoimmune disorder in the subject.
  • In another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (corresponding to post-treatment biomarkers set forth in Table 6), and wherein the subject is monitored after treatment with a TNFα inhibitor, thereby monitoring the autoimmune disorder in the subject.
  • In yet another aspect, the invention provides a method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (corresponding to biomarkers set forth in Table 7), thereby monitoring the autoimmune disorder in the subject.
  • In yet another aspect, the invention pertains to a method of building a database for use in selecting a subject having an autoimmune disorder (e.g., RA) for treatment with a TNFα inhibitor. The method comprises: receiving, in a computer system, biomarker expression patterns from a plurality of subjects having an autoimmune disorder; and storing the biomarker expression pattern from each subject such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82. The identifier of the subject can be, for example, a numerical identifier coded to an identity of the subject. In a preferred embodiment, the method further comprises receiving, in the computer system, one or more treatment regimens for treatment of the autoimmune disorder in a subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • The invention also pertains to a computer program product containing executable instructions that when executed cause a processor to perform operations comprising: receiving, in a computer system, a biomarker expression pattern of a subject at one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder; and storing the biomarker expression pattern such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82. The computer program can further cause the processor to perform an operation comprising: receiving, in the computer system, a treatment regimen for treatment of the autoimmune disorder in the subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • In yet another aspect, the invention pertains to a method of selecting an autoimmune disorder subject for treatment with a TNFα inhibitor, the method comprising: (i) identifying, in a database comprising a plurality of autoimmune disorder subjects, a subject whose database entry is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNFα inhibitor, and (ii) selecting the subject for treatment with a TNFα inhibitor, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82. The method can further comprise selecting a treatment regimen by identifying, in the database, a treatment regimen that has been associated with the biomarker expression pattern of the subject and with an identifier of the subject.
  • The invention also pertains to a computer program product containing executable instructions that when executed cause a processor to perform operations comprising: (i) identifying, in a database including a plurality of autoimmune disorder subjects associated with biomarker expression patterns, a subject that is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNFα inhibitor; and (ii) outputting the identified subject as a subject to be treated with a TNFα inhibitor; wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82. In one embodiment, the computer program further causes the processor to perform an operation comprising outputting a treatment regimen that is associated with the subject to be treated with the TNFα inhibitor.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a bar graph showing the results of experiments validating the predictive gene CD11c by quantitative real-time PCR. mRNA expression was compared in monocytes from normal donors (n=16), as well as future responders and future non-responders to therapy with anti-TNFα (RA-ATNF; filled circles ; 15 responders and 12 non-responders) or to combination therapy with anti-TNFα/methotrexate (RA-αTNF/MTX; empty circles ∘; 7 responders and 9 non-responders) The expression of CD11c is expressed as the means±standard error of the mean normalized to that of the house keeping gene glycerol-aldehyd-3-phosphate dehydrogenase (GAPDH; % expression). Except for 1 RA patient with a borderline ACR response of 30 and a CD11c mRNA level directly at the distinction threshold (who was therefore classified as a false negative), the threshold level (40%) almost fully distinguished future responders from nonresponders (100% specificity, 94% sensitivity, and 96% power). The threshold level to distinguish future responders from non-responders is indicated by a broken line (----); * P≦0.05, ** P≦0.01 as compared to normal donors; +++ P≦0.005 as compared to future responders to anti-TNFα therapy.
  • FIG. 2 is a graph showing the correlation between the mRNA expression of predictive CD11c in pre-treatment RA patients and their individual future ACR response (continuous ACR score). The horizontal and vertical broken lines indicate the thresholds for the separation between RA-responders and RA-non-responders, both in terms of the pre-treatment CD11c mRNA levels (40%) and their ACR response (continuous ACR score ≦30). Responders (continuous American College of Rheumatology [ACR] score ≧40; clustered as ND) and non-responders to anti-TNF therapy (continuous ACR score ≦30; clustered as RA) were defined according to the criteria of the ACR.
  • FIG. 3 is a graph showing the correlation between the mRNA expression of predictive CD11c in pre-treatment RA patients and their individual future strict ACR response in the following conventional used steps: ≧0%, ≧20%, ≧50%, and ≧70% (continuous ACR score).
    •  DETAILED DESCRIPTION OF THE INVENTION
  • This invention provides methods for predicting responsiveness to a TNFα inhibitor in a subject suffering from an autoimmune disorder, and methods for selecting a treatment regimen with a TNFα inhibitor, based on expression of particular biomarkers in the subject to be treated. The invention is based, at least in part, on the observation that altered expression of particular biomarkers in a subject suffering from rheumatoid arthritis is associated with increased or decreased responsiveness to therapy with a TNFα inhibitor. Microarray analysis, hierarchical clustering and TaqMan®-PCR analysis were used to examine normal donors (NA) and rheumatoid arthritis (RA) patients, who were categorized as being responsive to treatment with an anti-TNFα antibody (RA responders) or nonresponsive to treatment with an anti-TNFα antibody (RA nonresponders). A panel of 82 genes were identified whose expression was altered (upregulated or downregulated) in patients identified as either future RA responders or future RA nonresponders, demonstrating the ability of these genes to act as biomarkers for predicting responsiveness to TNFα inhibitor treatment. In particular, one gene, encoding the antigen CD11c, was identified as fully predicting the future response to anti-TNFα treatment. Accordingly, the expression pattern of one or more biomarkers can be assessed in RA subjects for which TNFα inhibitor therapy is being considered, or subjects suffering from other autoimmune disorders amenable to TNFα inhibitor therapy, to thereby predict responsiveness of the subject to such therapy and/or to aid in the selection of an appropriate treatment regimen.
  • Furthermore, additional patterns of biomarker expression were identified by (i) hierarchical clustering with the genes resulting from a simultaneous comparison between RA versus ND and RA responders pre- versus post-anti-TNFα therapy; (ii) prediction analysis of microarrays; and (iii) hierarchical clustering based on the comparison between RA responders and non-responders post-treatment. Accordingly, the biomarker expression patterns described herein also can be using in monitoring an autoimmune disorder in a subject, e.g., monitoring the responsiveness of the subject to a particular therapy or assisting in the diagnosis or prognosis of the autoimmune disorder (e.g., RA) in the subject.
  • In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
  • The term “predicting responsiveness to a TNFα inhibitor”, as used herein, is intended to refer to an ability to assess the likelihood that treatment of a subject with a TNFα inhibitor will or will not be effective in (e.g., provide a measurable benefit to) the subject. In particular, such an ability to assess the likelihood that treatment will or will not be effective typically is exercised before treatment with the TNFα inhibitor is begun in the subject. However, it is also possible that such an ability to assess the likelihood that treatment will or will not be effective can be exercised after treatment has begun but before an indicator of effectiveness (e.g., an indicator of measurable benefit) has been observed in the subject.
  • The term “TNFα inhibitor” as used herein is intended to encompass agents including proteins, antibodies, antibody fragments, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNFα antagonists and similar naturally- or nonnaturally-occurring molecules, and/or recombinant and/or engineered forms thereof, that, directly or indirectly, inhibits TNFα activity, such as by inhibiting interaction of TNFα with a cell surface receptor for TNFα, inhibiting TNFα protein production, inhibiting TNFα gene expression, inhibiting TNFα secretion from cells, inhibiting TNFα receptor signaling or any other means resulting in decreased TNFα activity in a subject. The term “TNFα inhibitor” also includes agents which interfere with TNFα activity. Examples of TNFα inhibitors include etanercept (ENBREL™, Amgen), infliximab (REMICADE™, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA™, Abbott Laboratories), CDP 571 (Celltech), and CDP 870 (Celltech), as well as other compounds which inhibit TNFα activity, such that when administered to a subject suffering from or at risk of suffering from a disorder in which TNFα activity is detrimental (e.g., RA), the disorder is treated. The term also includes each of the anti-TNFα human antibodies and antibody portions described herein as well as those described in U.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S. patent application Ser. Nos. 09/801,185 (U.S. Publication No. 20030092059) and 10/302,356 (U.S. Publication No. 20030219438), each incorporated by reference herein.
  • The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • The term “antibody” is also intended to encompass dual-specific antibodies and bispecific antibodies. The term “dual-specific antibody”, as used herein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see e.g., PCT publication WO 02/02773). Accordingly a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bi-valent for each antigen it binds to. The term “bispecific antibody”, as used herein, refers to full-length antibodies that are generated by quadroma technology (see Milstein, C. and A. C. Cuello (1983) Nature, 305:537-40), by chemical conjugation of two different mAbs (see Staerz, U. D., et al. (1985) Nature 314:628-31), or by knob-into-hole or similar approaches which introduces mutations in the Fc region (see Holliger, P., T. Prospero, and G. Winter (1993) Proc. Natl. Acad. Sci. USA 90:6444-8), resulting in multiple different immunogloblin species of which only one is the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is mono-valent for each antigen it binds to. Thus, when used herein, a “bispecific” antibody of the invention has one binding arm that is specific for an epitope of TNFα and a second binding arm that is specific for a different antigen or epitope.
  • The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • The terms “chimeric antibody” or “chimeric monoclonal antibody” are intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Such “chimeric antibodies” can be prepared by standard recombinant technology well established in the art. For example, a nucleic acid encoding a VH region from a mouse antibody can be operatively linked to a nucleic acid encoding the heavy chain constant regions from a human antibody and, likewise, a nucleic acid encoding a VL region from a mouse antibody can be operatively linked to a nucleic acid encoding the light chain constant region from a human antibody.
  • The terms “humanized antibody” or “humanized monoclonal antibody” are intended to refer to antibodies in which CDR sequences derived from the germline of a non-human mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Such “humanized antibodies” can be prepared by standard recombinant technology well established in the art. For example, nucleic acids encoding the CDR1, CD2 and CDR3 regions from a VH region of a mouse antibody can be operatively linked to nucleic acids encoding the FR1, FR2, FR3 and FR4 regions of a human VH region, and the entire “CDR-grafted” VH region can be operatively linked to nucleic acid encoding the heavy chain constant regions from a human antibody. Likewise, nucleic acids encoding the CDR1, CD2 and CDR3 regions from a VL region of a mouse antibody can be operatively linked to nucleic acids encoding the FR1, FR2, FR3 and FR4 regions of a human VL region, and the entire “CDR-grafted” VL region can be operatively linked to nucleic acid encoding the light chain constant region from a human antibody.
  • The term “human antibody”, as used herein, is intended to refer to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. The term “human monoclonal antibody”, as used herein, also includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. Such recombinant human antibodies, however, can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • The terms “Ig fusion protein” and “Fc fusion protein” are intended to refer to a recombinant, composite protein comprising a polypeptide of interest operatively linked to a constant region portion of immunoglobulin, typically the hinge, CH2 and CH3 domains of heavy chain constant region, more typically the human IgG1 hinge, CH2 and CH3 domains. The polypeptide of interest operatively linked to the Fc portion can be, for example, a full-length protein or only a portion of a full-length protein, such as one or more extracellular domains of a protein, e.g., one or more extracellular domains of a cell-surface protein. Such “Ig fusion proteins” can be prepared by standard recombinant technology well established in the art. For example, a nucleic acid encoding the polypeptide of interest can be operatively linked to a nucleic acid encoding the hinge, CH2 and CH3 domains of a heavy chain constant region.
  • The term “multivalent binding protein”, as a form of TNFα inhibitor, is used in this specification to denote a binding protein comprising two or more antigen binding sites. Examples of multivalent binding proteins include dual variable domain (DVD) binding proteins. The multivalent binding protein is preferably engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody. A multivalent binding protein also can be a “multispecific binding protein.” The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets (wherein, with respect to this specification at least one of the targets is TNFα). Dual variable domain (DVD) binding proteins, as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e., capable of binding one antigen (e.g., TNFα) or multispecific, i.e., capable of binding two or more antigens (e.g., TNFα and one or more other antigens). DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as “DVD Ig.” Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in US. Publication No. 20070071675, the entire contents of which are specifically incorporated herein by reference.
  • As used herein, the term “biomarker” is intended to encompass a substance that is used as an indicator of a biologic state and includes genes (and nucleotide sequences of such genes), mRNAs (and nucleotide sequences of such mRNAs) and proteins (and amino acid sequences of such proteins). A “biomarker expression pattern” is intended to refer to a quantitative or qualitative summary of the expression of one or more biomarkers in a subject, such as in comparison to a standard or a control.
  • The terms “increased” or “increased expression” and “decreased” or “decreased expression”, with respect to the expression pattern of a biomarker(s), are used herein as meaning that the level of expression is increased or decreased relative to a constant basal level of expression of a household, or housekeeping, gene, whose expression level does not significantly vary under different conditions. A nonlimiting example of such a household, or housekeeping, gene is GAPDH. Other suitable household, or housekeeping, gene are well-established in the art.
  • As used herein, the term “CD11c” refers to a protein having a full-length amino acid sequence as set forth at Genbank Accession No. NP—000878 (also shown as SEQ ID NO: 302) and encoded by a full-length nucleotide sequence as set forth at Genbank Accession No. NM—000887 (also shown as SEQ ID NO: 301). CD11c is also known in the art as CD11C, CD11c antigen, Integrin alpha X, complement component 3 receptor 4 subunit, ITGAX, LeuM5, Integrin alpha X precursor, Leukocyte adhesion glycoprotein p150,p95 alpha chain, and Leukocyte adhesion receptor p150 subunit, which terms may be used interchangeably herein to refer to CD11c.
  • As used herein, the term “Affymetrix ID” refers to a numerical identifier that corresponds to a sequence entry in an Affymetrix database, which entry includes the sequence as well as additional information relating to the sequence and corresponding protein. The sequence entries, and additional information in the entries, for each Affymetrix ID are publicly available (e.g., by entering the Affymetrix ID number into the Affymetrix database search engine, e.g., at https://www.affymetrix.com/analysis/netaffx/index.affx). All sequence entries (such as Genbank Accession numbers), and additional information provided for each entry, corresponding to each of the Affymetrix ID numbers disclosed herein are hereby specifically incorporated by reference in their entirety.
  • As used herein, the term “subject” includes humans, and non-human animals amenable to TNFα inhibitor therapy, e.g., preferably mammals, such as non-human primates, sheep, dogs, cats, horses and cows.
  • As used herein, the term “autoimmune disorder subject” or “AD subject” is intended to refer to a subject (e.g., human patient) suffering from an autoimmune disorder.
  • As used herein, the term “rheumatoid arthritis subject” or “RA subject” is intended to refer to a subject (e.g., human patient) suffering from rheumatoid arthritis.
  • As used herein, the term “treatment regimen” is intended to refer to one or more parameters selected for the treatment of a subject, e.g., with a TNFα inhibitor, which parameters can include, but are not necessarily limited to, the type of agent chosen for administration, the dosage, the formulation, the route of administration and the frequency of administration.
  • Various aspects of the invention are described in further detail in the following subsections.
    • Prediction of Responsiveness to a TNFα Inhibitor for Autoimmune Disorders
  • In one aspect, the invention pertains to a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, such as rheumatoid arthritis. Typically, the method comprises (i) assaying the subject for the expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject. As used herein, the term “one or more biomarkers” is intended to mean that at least one biomarker in a disclosed list of biomarkers is assayed and, in various embodiments, more than one biomarker set forth in the list may be assayed, such as two, three, four, five, ten, twenty, thirty, forty, fifty, more than fifty, or all the biomarkers in the list may be assayed.
  • Predicting responsiveness of the subject to the TNFα inhibitor “based on expression of the one or more biomarkers in the subject” typically involves comparing the level, or pattern, of expression of the one or more biomarkers in the subject to a known standard or control (which known standard or control may be derived from, for example, a normal subject, a pre-established TNFα inhibitor responder or a pre-established TNFα inhibitor non-responder). In a preferred embodiment, the level of expression of the biomarker(s) is measured in parallel with measurement of the level of expression of one or more “housekeeping” genes, such as GAPDH, whose expression level is not altered by the autoimmune disorder. The level of expression of the biomarker(s) is determined to be “increased” or “decreased” relative to a constant basal level of expression of the housekeeping gene. Examples of suitable housekeeping genes, such as GAPDH, that can be used for comparison purposes are well known in the art.
  • Preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (corresponding to sequences of the biomarkers set forth in Table 9). Thus, at least one of the biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 is assayed and, in various embodiments, for example, two, three, four, five, ten, twenty, thirty, forty, fifty, more than fifty, or all the biomarkers in the list may be assayed.
  • In a preferred embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (corresponding to sequences from Table 9 that are increased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (corresponding to sequences from Table 9 that are increased in ≧90% of responders vs. non-responders). Even more preferably, at least one of the biomarkers to be assayed is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (corresponding to the biomarker CD11c, which, as set forth in Table 9, is increased in 100% of responders vs. non-responders). In each of these embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor (e.g., increased expression relative to a standard or control level of expression, which standard or control level of expression can be based, for example, on the level of expression in previously established TNFα inhibitor non-responder RA subjects).
  • In another preferred embodiment, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (corresponding to sequences from Table 9 that are decreased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (corresponding to sequences from Table 9 that are decreased in ≧90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (corresponding to sequences from Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor (e.g., decreased expression relative to a standard or control level of expression, which standard or control level of expression can be based, for example, on the level of expression in previously established TNFα inhibitor non-responder RA subjects).
  • In another aspect, the invention provides a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in the autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is selected from the group consisting of Aquaporin 3 (Genbank Accession No. NM—004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No. NM—033022); Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM—006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM—000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM—006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM—002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No. NM—002823); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM—005896); Tumor protein D52 (Genbank Accession Nos. NM—001025252, NM—001025253, NM—005079); Early growth response 1 (Genbank Accession No. NM—001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM—014888, NM—001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No. NM—001866); CUG triplet repeat, RNA binding protein 2 (Genbank Accession No. NM—001025077, NM—001025076, NM—006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM—006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM—017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM—001077269, NM—003387); CD97 antigen (Genbank Accession Nos. NM—001025160, NM—001784, NM—078481); Glutamate-cysteine ligase, catalytic subunit (Genbank Accession No. NM—001498); Crystallin, zeta (quinone reductase) (Genbank Accession No. NM—001889); Rap guanine nucleotide exchange factor (GEF) 2 (Genbank Accession No. NM—014247); Ataxin 1 (Genbank Accession No. NM—000332); Adaptor-related protein complex 1, sigma 2 subunit (Genbank Accession No. NM—003916); Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM—014936); Desmocollin 2 (Genbank Accession Nos. NM—024422, NM—004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM—002371, NM—022438, NM—022439, NM—022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No. NM—005476); Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM—001001437, NM—021006); Carboxypeptidase A3 (Genbank Accession No. NM—001870); Charcot-Leyden crystal protein (Genbank Accession No. NM—001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM—004545); Interleukin 8 receptor, beta (Genbank Accession No. NM—001557); Platelet factor 4 variant 1 (Genbank Accession No. NM—002620); Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM—006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM—003786); Actinin, alpha 1 (Genbank Accession No. NM—001102); NAD kinase (Genbank Accession No. NM—023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM—000442); Esterase D/formylglutathione hydrolase (Genbank Accession No. NM—001984); Chromosome 20 open reading frame 111 (Genbank Accession No. NM—016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM—001017369, NM—006745); PIM-1 oncogene (Genbank Accession No. NM—002648); GATA binding protein 2 (Genbank Accession No. NM—032638); Cathepsin Z (Genbank Accession No. NM—001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No. NM—000887); Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM—006499, NM—201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM—006889, NM—175862); Interleukin 8 (Genbank Accession No. NM—000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM—002001); Actin, gamma 1 (Genbank Accession No. NM—001614); KIAA0746 protein (Genbank Accession No. NM—015187); Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (Genbank Accession No. NM—002076); Transcription factor 4 (Genbank Accession Nos. BF592782, CR612521); Major histocompatibility complex, class II, DQ alpha 1 (Genbank Accession Nos. NM—002122, NM—020056); Cell division cycle 2-like 6 (CDK8-like) (Genbank Accession No. NM—015076); Major histocompatibility complex, class II, DQ beta 1 (Genbank Accession No. XM—942240); Phospholipase C-like 2 (Genbank Accession No. NM—015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM—005242); TM2 domain containing 1 (Genbank Accession No. NM—032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM—012433); SUB1 homolog (S. cerevisiae) (Genbank Accession No. NM—006713); mRNA; cDNA DKFZp564O0862 (Genbank Accession No. AI278204); Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM—201413, NM—000484, NM—201414); Cytochrome b-5 (Genbank Accession Nos. NM—001914, NM—148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM—183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM—016645, NM—001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No. XM—941927); CCR4-NOT transcription complex, subunit 6 (Genbank Accession No. NM—015455); Ubiquinol-cytochrome c reductase complex (7.2 kD) (Genbank Accession Nos. NM—013387, NM—001003684); Hepatocellular carcinoma-associated antigen 112 (Genbank Accession No. NM—018487); Kruppel-like factor 11 (Genbank Accession No. XM—001129527); GGA binding partner (Genbank Accession No. NM—018318); Cornichon homolog 4 (Drosophila) (Genbank Accession No. NM—014184); Hypothetical protein FLJ21616 (Genbank Accession No. NM—024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM—018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM—012336, NM—001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM—018538, NM—001017922); LR8 protein (Genbank Accession No. NM—014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No. NM—030915); Calmin (calponin-like, transmembrane) (Genbank Accession No. NM—024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM—031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM—007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR—003287) (corresponding to biomarkers listed in Table 9).
  • In a preferred embodiment, the one or more biomarkers is selected from the group consisting of Guanine nucleotide binding protein (G protein), beta polypeptide 1; Prothymosin, alpha (gene sequence 28); Early growth response 1; Homo spaiens leptin receptor gene-related protein (HS0BRGRP); Wiskott-Aldrich syndrome protein interacting protein; CD97 antigen; Crystallin, zeta (quinone reductase); Adaptor-related protein complex 1, sigma 2 subunit; Desmocollin 2; Chemokine (C—C motif) ligand 3; Interleukin 8 receptor, beta; ATP-binding cassette, sub-family C (CFTR/MRP), member 3; Actinin, alpha 1; NAD kinase; Platelet/endothelial cell adhesion molecule (CD31 antigen); Esterase D/formylglutathione hydrolase; Chromosome 20 open reading frame 111; Cathepsin Z; Integrin alpha-X (antigen CD11c); Lectin, galactoside-binding, soluble, 8 (galectin 8); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen); Interleukin 8; Actin, gamma 1; Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID); Cell division cycle 2-like 6 (CDK8-like); Major histocompatibility complex, class II, DQ beta 1; Coagulation factor II (thrombin) receptor-like 1; Splicing factor 3b, subunit 1, 155 kDa; mRNA; cDNA DKEZp564O0862; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); Cold autoinflammatory syndrome 1; Kruppel-like factor 11; Nuclear prelamin A recognition factor; Calmin (calponin-like, transmembrane); and SRY (sex determining region Y)-box 18 (corresponding to biomarkers listed in Table 9 that are increased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is selected from the group consisting of Interleukin 8 receptor, beta; Platelet/endothelial cell adhesion molecule (CD31 antigen); Integrin alpha-X (antigen CD11c); Interleukin 8; Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease); and Kruppel-like factor 11 (corresponding to biomarkers listed in Table 9 that are increased in ≧90% of responders vs. non-responders). Even more preferably, at least one of the biomarkers is Integrin alpha-X (antigen CD11c) (corresponding to a biomarker listed in Table 9 that is increased in 100% of responders vs. non-responders). In each of the embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another preferred embodiment, the one or more biomarkers is selected from the group consisting of Aquaporin 3; Similar to ribosomal protein S24, clone MGC:8595; Transmembrane emp24 domain trafficking protein 2; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1; Calmodulin 1 (phosphorylase kinase, delta); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1); Tumor protein D52; Homo sapiens predicted osteoblast protein (GS3786); Cytochrome c oxidase subunit VIIb; CUG triplet repeat, RNA binding protein 2; Ubiquinol-cytochrome c reductase hinge protein; Glutamate-cysteine ligase, catalytic subunit; Rap guanine nucleotide exchange factor (GEF) 2; Ataxin 1; Ectonucleotide pyrophosphatase/phosphodiesterase 4; MAL, T-cell differentiation protein; Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase; Carboxypeptidase A3; Charcot-Leyden crystal protein; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa; Platelet factor 4 variant 1; Poly(A) binding protein interacting protein 1; Sterol-C4-methyl oxidase-like; PIM-1 oncogene; GATA binding protein 2; Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide; KIAA0746 protein; Transcription factor 4; Major histocompatibility complex, class II, DQ alpha 1; Phospholipase C-like 2; TM2 domain containing 1; SUB1 homolog (S. cerevisiae); Cytochrome b-5; Neugrin, neurite outgrowth associated; Ribosomal protein S26, 40S ribosomal protein; CCR4-NOT transcription complex, subunit 6; Ubiquinol-cytochrome c reductase complex (7.2 kD); Hepatocellular carcinoma-associated antigen 112; GGA binding partner; Cornichon homolog 4 (Drosophila); Hypothetical protein FLJ21616; Homo sapiens hypothetical protein FLJ10134; Erythroblast membrane-associated protein; LR8 protein; Likely ortholog of mouse limb-bud and heart gene (LBH); Chromosome 14 open reading frame 156; and Guanine nucleotide binding protein (G protein) alpha 12 (corresponding to biomarkers listed in Table 9 that are decreased in ≧80% of responders vs. non-responders). More preferably, the one or more biomarkers is selected from the group consisting of Homo sapiens predicted osteoblast protein (GS3786); Charcot-Leyden crystal protein; Neugrin, neurite outgrowth associated; Hypothetical protein FLJ21616; and Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers listed in Table 9 that are decreased in ≧90% of responders vs. non-responders). Even more preferably, the one or more biomarkers is Homo sapiens predicted osteoblast protein (GS3786) or Homo sapiens hypothetical protein FLJ10134 (corresponding to biomarkers listed in Table 9 that are decreased in 100% of responders vs. non-responders). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In a particularly preferred aspect, the invention provides a method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder the method comprising: (i) assaying the subject for increased expression of a biomarker, which biomarker is CD11c, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on increased expression of CD11c in the subject.
  • In yet another particularly preferred embodiment, the invention provides a method for predicting responsiveness to a TNFα inhibitor, which TNFα inhibitor is adalimumab, in a subject having an autoimmune disorder the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to adalimumab in the autoimmune disorder, and (ii) predicting responsiveness of the subject to adalimumab based on expression of the one or more biomarkers in the subject. Preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or selected from the group consisting of the biomarkers set forth in Table 9). In more preferred embodiments, the subsets of sequences within SEQ ID NO: 1-82 that are either increased or decreased, as set forth in detail above, can be assayed.
  • In the methods of the invention for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in the autoimmune disorder can be assayed in the subject using techniques well-established in the art. In a preferred embodiment, the expression of the one or more biomarkers in the subject is assayed by obtaining an mRNA sample from the subject (e.g., isolated from peripheral blood mononuclear cells, by standard methods) and detecting the expression of mRNA(s) encoding the one or more biomarkers in the mRNA sample using standard molecular biology techniques, such as PCR analysis. A preferred method of PCR analysis is revers transcriptase-polymerase chain reaction (RT-PCR). Other suitable systems for mRNA sample analysis include microarray analysis (e.g., using Affymetrix's microarray system or Illumina's BeadArray Technology).
  • Additionally or alternatively, in certain situations it may be possible to assay for the expression of one or more biomarkers at the protein level, using a detection reagent that detects the protein product encoded by the mRNA of the biomarker(s). For example, if an antibody reagent is available that binds specifically to the biomarker protein product to be detected, and not to other proteins, then such an antibody reagent can be used to detect the expression of the biomarker of interest in a cellular sample from the subject, or a preparation derived from the cellular sample, using standard antibody-based techniques known in the art, such as FACS analysis, ELISA and the like.
  • It will be readily understood by the ordinarily skilled artisan that essentially any technical means established in the art for detecting biomarkers, at either the nucleic acid or protein level, can be adapted to detection of the biomarkers discussed herein and applied in the methods of the current invention for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder.
  • The biomarkers described herein were originally identified in patients having rheumatoid arthritis (see the Examples) and thus a particularly preferred autoimmune disorder in which to apply the methods of the invention is rheumatoid arthritis. The mechanism of action of the TNFα pathway, however, is thought to be common to a large number of autoimmune disorders and TNFα inhibitors have been shown to be effective therapy in a variety of different autoimmune disorders. Accordingly, the method of the invention for predicting responsiveness to a TNFα inhibitor can be applied to essentially any autoimmune disorder in which TNFα inhibitor therapy is applied. Other preferred autoimmune disorders include Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, juvenile arthritis and ankylosing spondilitis, Other non-limiting examples of autoimmune disorders include autoimmune diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid spondylitis, gouty arthritis, allergy, autoimmune uveitis, nephrotic syndrome, multisystem autoimmune diseases, autoimmune hearing loss, adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathic interstitial lung disease, chronic obstructive pulmonary disease, asthma, restenosis, spondyloarthropathies, Reiter's syndrome, autoimmune hepatitis, inflammatory skin disorders, vasculitis of large vessels, medium vessels or small vessels, endometriosis, prostatitis and Sjogren's syndrome.
    • Biomarkers for Monitoring an Autoimmune Disorder in a Subject
  • In another aspect, the invention provides methods of monitoring an autoimmune disorder in a subject having the autoimmune disorder based on biomarker expression patterns established using microarray analysis of, for example, RA subjects vs. normal donors, RA subjects vs. RA subjects treated with a TNFα inhibitor and/or RA subjects treated with a TNFα inhibitor vs. RA responders to TNFα inhibitors. In these monitoring methods, the subject is assayed for expression of one or more biomarkers (using techniques, for example, as described in the previous section), thereby monitoring the autoimmune disorder in the subject.
  • In one embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, namely v-maf musculoaponeurotic fibrosarcoma oncogene homolog F; Diphtheria toxin receptor (DTR); DEAH (Asp-Glu-Ala-His) box polypeptide 15; Ribonucleotide reductase M2 polypeptide; Solute carrier family 6, member 8; 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; Ferrochelatase (protoporphyria); Nuclear factor, interleukin 3 regulated; Thrombomodulin; Major histocompatibility complex, class II, DM beta; Forkhead box O3A; Hemoglobin, gamma A, gamma G; Synuclein, alpha (non A4 component of amyloid precursor); Hemoglobin, gamma A; Amphiregulin (schwannoma-derived growth factor); Lipoyltransferase 1; Solute carrier family 4, anion exchanger, member 1; S100 calcium binding protein A12 (calgranulin C); Keratin 1 (epidermolytic hyperkeratosis); Carbonic anhydrase I; CD1C antigen, c polypeptide; Tumor necrosis factor, alpha-induced protein 6; Ribonuclease, RNase A family, 2; Hemoglobin, delta; Transferrin receptor (p90, CD71); Ring finger protein 10; Chromosome 1 open reading frame 63; Hemoglobin, alpha 1, alpha 2; CD69 antigen (p60, early T-cell activation antigen; APEX nuclease (multifunctional DNA repair enzyme) 1; Membrane-spanning 4-domains, subfamily A, member 3; Heat shock 70 kDa protein 8; Hypothetical protein MGC12760; GABA(A) receptor-associated protein like 1, like 3; Aminolevulinate, delta-, synthase 2; Major histocompatibility complex, class II, DP alpha 1; Morf4 family associated protein 1-like 1; Aldehyde dehydrogenase 1 family, member A1; Formin binding protein 4; Zinc finger protein 24 (KOX 17); Hypothetical protein L0054103; Selenium binding protein 1; Hematopoietically expressed homeobox; Major histocompatibility complex, class II, DM alpha; Eukaryotic translation initiation factor 2-alpha kinase 1; Ankyrin repeat domain 49; Hypothetical protein FLJ20701; Zinc finger protein 331; Membrane-spanning 4-domains, subfamily A, member 4; Tensin 1; and Family with sequence similarity 46, member A, respectively).
  • In another embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, namely Uncharacterized hypothalamus protein HT007; Dual specificity phosphatase 22; Proteasome (prosome, macropain) subunit, beta type, 7; Membrane-spanning 4-domains, subfamily A, member 4; DKFZP434C171 protein; Protein phosphatase 1, catalytic subunit, beta isoform; Splicing factor, arginine/serine-rich 5; Sorting nexin 11; Farnesyltransferase, CAAX box, alpha; Peroxisomal D3,D2-enoyl-CoA isomerise; Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide; UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1; Hypothetical protein FLJ11259; APEX nuclease (multifunctional DNA repair enzyme) 1; Geranylgeranyl diphosphate synthase 1; Down syndrome critical region gene 5; Calpain 7; Major histocompatibility complex, class II, DP alpha 1; Brix domain containing 5; Chromosome 21 open reading frame 59; Tumor necrosis factor (ligand) superfamily, member 10; Chromosome 10 open reading frame 86; CD1D antigen, d polypeptide; Ewing sarcoma breakpoint region 1; Ribonuclease P 40 kDa subunit; PHD finger protein 20; Thioredoxin interacting protein; Ubiquinol-cytochrome c reductase core protein II; Hypothetical protein FLJ22662; Preimplantation protein 3; DKFZP564G2022 protein; Dipeptidase 2; Hemoglobin, alpha 1, alpha 2; Frequently rearranged in advanced T-cell lymphomas; DEAD (Asp-Glu-Ala-Asp) box polypeptide 48; Tumor necrosis factor, alpha-induced protein 2; Nucleophosmin (nucleolar phosphoprotein B23, numatrin); Interleukin 13 receptor, alpha 1; Leukocyte specific transcript 1, LST1; CGI-121 protein; RAS p21 protein activator 4/hypothetical protein FLJ21767; Cathepsin S; CD63 antigen (melanoma 1 antigen); JTV1 gene; KIAA0174; Thrombospondin 1; Hypothetical protein L0054103; Interferon regulatory factor 1; and SEC11-like 1 (S. cerevisiae)).
  • More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as being upregulated, namely Uncharacterized hypothalamus protein HT007; Dual specificity phosphatase 22; Proteasome (prosome, macropain) subunit, beta type, 7; Membrane-spanning 4-domains, subfamily A, member 4; DKEZP434C171 protein; Protein phosphatase 1, catalytic subunit, beta isoform; Splicing factor, arginine/serine-rich 5; Sorting nexin 11; Farnesyltransferase, CAAX box, alpha; Peroxisomal D3,D2-enoyl-CoA isomerise; Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide; UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1; Hypothetical protein FLJ11259; APEX nuclease (multifunctional DNA repair enzyme) 1; Geranylgeranyl diphosphate synthase 1; Down syndrome critical region gene 5; Calpain 7; Major histocompatibility complex, class II, DP alpha 1; Brix domain containing 5; and Chromosome 21 open reading frame 59), wherein expression of the one or more biomarkers is increased in the subject.
  • Additionally or alternatively, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as being downregulated, namely Tumor necrosis factor (ligand) superfamily, member 10; Chromosome 10 open reading frame 86; CD1D antigen, d polypeptide; Ewing sarcoma breakpoint region 1; Ribonuclease P 40 kDa subunit; PHD finger protein 20; Thioredoxin interacting protein; Ubiquinol-cytochrome c reductase core protein II; Hypothetical protein FLJ22662; Preimplantation protein 3; DKFZP564G2022 protein; Dipeptidase 2; Hemoglobin, alpha 1, alpha 2; Frequently rearranged in advanced T-cell lymphomas; DEAD (Asp-Glu-Ala-Asp) box polypeptide 48; Tumor necrosis factor, alpha-induced protein 2; Nucleophosmin (nucleolar phosphoprotein B23, numatrin); Interleukin 13 receptor, alpha 1; Leukocyte specific transcript 1, LST1; CGI-121 protein; RAS p21 protein activator 4/hypothetical protein FLJ21767; Cathepsin S; CD63 antigen (melanoma 1 antigen); JTV1 gene; KIAA0174; Thrombospondin 1; Hypothetical protein L0054103; Interferon regulatory factor 1; and SEC11-like 1 (S. cerevisiae), wherein expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, namely HLA-B associated transcript-1 (D6S81E); Interleukin enhancer binding factor 2, 45 kD (ILF2); Isolate Liv chaperone protein HSP90 beta (HSP90BETA) mRNA; Lysyl-tRNA synthetase mRNA, complete cds; nuclear gene for mitochondrial product; alternatively spliced; Tryptophanyl-tRNA synthetase (WARS); Profilin 1 (PFN1), mRNA; Seryl-tRNA synthetase (SARS); Similar to KIAA1007 protein, clone MGC:692, mRNA, complete cds; CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 and G344); Homo sapiens KIAA0064 gene product (KIAA0064), mRNA; Chromosome segregation 1 (yeast homolog)-like (CSE1L), mRNA; Protein phosphatase 1, regulatory subunit 7 (PPP1R7), mRNA; DEADH (Asp-Glu-Ala-AspHis) box polypeptide 1 (DDX1), mRNA; Threonyl-tRNA synthetase (TARS), mRNA; Dead box protein 15 mRNA, complete cds; SRY (sex determining region Y)-box 4/DEF=Human DNA sequence from clone RP3-322L4 on chromosome 6; Galactosidase, beta 1 (GLB1), mRNA; ATP-binding cassette, sub-family E (OABP), member 1; NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (14 kD, B14) (NDUFA6), mRNA; Pericentriolar material 1 (PCM1), mRNA; Excision repair cross-complementing rodent repair deficiency, complementation group 3 (ERCC3), mRNA; KIAA0907 protein (KIAA0907), mRNA; v-crk avian sarcoma virus CT10 oncogene homolog; 6-phosphofructo-2-kinasefructose-2,6-biphosphatase 3 (PFKFB3), mRNA./PROD=6-phosphofructo-2-kinase-fructose-2,6-biphosphatase 3; KIAA0766 gene product (KIAA0766), mRNA; N-acetylgalactosaminidase, alpha- (NAGA), mRNA; Protein tyrosine phosphatase, non-receptor type substrate 1 (PTPNS1), mRNA; Crystallin, zeta (quinone reductase) (CRYZ), mRNA; KIAA0429 gene product (KIAA0429), mRNA; SET domain, bifurcated 1 (SETDB1), mRNA; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3 (12 kD, B12) (NDUFB3), mRNA; Diphtheria toxin receptor (DTR); Thrombomodulin (THBD), mRNA; Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform (PPM1A), mRNA; CD37 antigen (CD37), mRNA; Hemoglobin, gamma G (HBG2), mRNA; Protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D), mRNA; Hematopoietically expressed homeobox (HHEX), mRNA; Amphiregulin (schwannoma-derived growth factor) (AREG), mRNA; Early growth response 2 (Krox-20 (Drosophila) homolog) (EGR2), mRNA; 2,5-oligoadenylate synthetase 1 (40-46 kD) (OAS1), transcript variant E16, mRNA; Early growth response 3 (EGR3), mRNA./PROD=early growth response 3; Homo sapiens MD-2 protein (MD-2), mRNA; Homo sapiens MAX dimerization protein (MAD), mRNA; DKEZP586A011 protein (DKEZP586A011), mRNA; 78 kDa gastrin-binding protein mRNA, complete cds; Transferrin receptor (p90, CD71), clone MGC:3151, mRNA, complete cds; Homo sapiens mRNA; cDNA DKFZp564N1272 (from clone DKFZp564N1272); complete cds; GABA-A receptor-associated protein like 1 (GABARAPL1) mRNA, complete cds; Translocation protein 1; Homo sapiens clone 016b03 My027 protein mRNA, complete cds; Protein kinase-related oncogene (PIM1) mRNA, complete cds; MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C); eIF-2-associated p67 homolog mRNA, complete cds; Human mRNA for hCREM (cyclic AMP-responsive element modulator) type 2 protein, complete cds; Apurinic endonuclease (APE) mRNA, complete cds./PROD=apurinic endonuclease; TATA box binding protein (TBP)-associated factor, RNA polymerase II, D, 100 kD; Human mRNA for ZFM1 protein alternatively spliced product, complete cds./PROD=ZFM1 protein, alternatively spliced product; Trachea cellular apoptosis susceptibility protein (CSE1) mRNA, complete cds; Similar to eukaryotic translation initiation factor 3, subunit 8 (110 kD), clone MGC:8693, mRNA, complete cds; GABA-A receptor-associated protein mRNA, complete cds./PROD=GABA-A receptor-associated protein; Human (clone 2-5) synuclein (NACP) mRNA, complete cds; Human putative ribosomal protein 51 mRNA; Aldehyde dehydrogenase 1, soluble (ALDH1), mRNA./PROD=aldehyde dehydrogenase 1, soluble; Homo sapiens mRNA for KIAA1057 protein, partial cds; RBP1-like protein; Ring finger protein 4; KIAA0197 protein; Homo sapiens mRNA; cDNA DKFZp586F1323 (from clone DKFZp586F1323); Homo sapiens mRNA; cDNA DKFZp5641052 (from clone DKFZp5641052); Homo sapiens mRNA for Hmob33 protein, 3 untranslated region; Homo sapiens mRNA; cDNA DKFZp586F1019 (from clone DKFZp586F1019); partial cds; Myosin, light polypeptide 4, alkali; atrial, embryonic; H. sapiens novel gene from PAC 117P20, chromosome 1; Homo sapiens clone 24582 mRNA sequence; Heterogeneous nuclear ribonucleoprotein H1 (H); Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide; Cyclin T2; cDNA: FLJ23227 fis, clone CAE00645, highly similar to AF052138; Hypothetical protein FLJ12619; C-terminal binding protein 1; SEC22, vesicle trafficking protein (S. cerevisiae)-like 1; Hemoglobin, alpha 1; Homo sapiens clone 24659 mRNA sequence/DEF=Homo sapiens clone 24659 mRNA sequence; Calcium channel, voltage-dependent, PQ type, alpha 1A subunit; H. sapiens SMAS mRNA; H. sapiens HEX gene encoding homeobox related protein; Mitogen-activated protein kinase kinase kinase 4; Serine palmitoyltransferase (LCB2) mRNA, partial cds; KIAA0971 protein/DEF=Homo sapiens cDNA FLJ11495 fis, clone HEMBA1001950, highly similar to Homo sapiens mRNA for KIAA0971 protein; ESTs, Hs.97109; ESTs, Weakly similar to ALU7_HUMAN ALU; DEAD-box protein abstrakt (ABS), mRNA; KIAA1513 protein (KIAA1513), mRNA; Cell division protein FtsJ (FJH1), mRNA; F-box only protein 3 (FBXO3), mRNA; Purinergic receptor (family A group 5) (P2Y5), mRNA; Integral inner nuclear membrane protein (MAN1), mRNA; Fanconi anemia, complementation group F (FANCF), mRNA; Hypothetical protein FLJ12820 (FLJ12820), mRNA; Hypothetical protein FLJ13119 (FLJ13119), mRNA; Hypothetical protein FLJ20189 (FLJ20189), mRNA; Hypothetical protein FLJ20701 (FLJ20701), mRNA; Homo sapiens chromosome 12 open reading frame 5 (C12ORF5), mRNA; Hypothetical protein FLJ22555 (FLJ22555), mRNA; Hypothetical protein FLJ11110 (FLJ11110), mRNA; CGI-12 protein (LOC51001), mRNA; Triggering receptor expressed on myeloid cells 1 (TREM1), mRNA; betaGlcNAc beta 1,4-galactosyltransferase. polypeptide 5; PNAS-25 mRNA, complete cds.; Homo sapiens mRNA for FLJ00043 protein, partial cds; Homo sapiens cDNA: FLJ21737 fis, clone COLF3396; Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395/DEF=Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395; Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F) LIKE protein; Heparin-binding EGF-like growth factor mRNA, complete cds; E. coli 7,8-diamino-pelargonic acid (bioA), biotin synthetase (bioB), 7-keto-8-amino-pelargonic acid synthetase (bioF), bioC protein, and dethiobiotin synthetase (bioD), complete cds; Escherichia coli/REF=J04423/DEF=E. coli bioC protein corresponding to nucleotides 4609-4883 of J04423/LEN=777 (−5 and −3 represent transcript regions 5 prime and 3 prime respectively)).
  • More preferably, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as being upregulated, namely HLA-B associated transcript-1 (D6S81E); Interleukin enhancer binding factor 2, 45 kD (ILF2); Isolate Liv chaperone protein HSP90 beta (HSP90BETA) mRNA; Lysyl-tRNA synthetase mRNA, complete cds; nuclear gene for mitochondrial product; alternatively spliced; Tryptophanyl-tRNA synthetase (WARS); Profilin 1 (PFN1), mRNA; Seryl-tRNA synthetase (SARS); Similar to KIAA1007 protein, clone MGC:692, mRNA, complete cds; Homo sapiens KIAA0064 gene product (KIAA0064), mRNA; Chromosome segregation 1 (yeast homolog)-like (CSE1L), mRNA; Protein phosphatase 1, regulatory subunit 7 (PPP1R7), mRNA; DEADH (Asp-Glu-Ala-AspHis) box polypeptide 1 (DDX1), mRNA; Threonyl-tRNA synthetase (TARS), mRNA; Dead box protein 15 mRNA, complete cds; SRY (sex determining region Y)-box 4/DEF=Human DNA sequence from clone RP3-322L4 on chromosome 6; Galactosidase, beta 1 (GLB1), mRNA; ATP-binding cassette, sub-family E (OABP), member 1; NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (14 kD, B14) (NDUFA6), mRNA; Pericentriolar material 1 (PCM1), mRNA; Excision repair cross-complementing rodent repair deficiency, complementation group 3 (ERCC3), mRNA; KIAA0907 protein (KIAA0907), mRNA; v-crk avian sarcoma virus CT10 oncogene homolog; KIAA0766 gene product (KIAA0766), mRNA; N-acetylgalactosaminidase, alpha- (NAGA), mRNA; Crystallin, zeta (quinone reductase) (CRYZ), mRNA; KIAA0429 gene product (KIAA0429), mRNA; SET domain, bifurcated 1 (SETDB1), mRNA; CD37 antigen (CD37), mRNA; Protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D), mRNA; Hematopoietically expressed homeobox (HHEX), mRNA; 2,5-oligoadenylate synthetase 1 (40-46 kD) (OAS1), transcript variant E16, mRNA; DKFZP586A011 protein (DKFZP586A011), mRNA; 78 kDa gastrin-binding protein mRNA, complete cds; Homo sapiens clone 016b03 My027 protein mRNA, complete cds; MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C); eIF-2-associated p67 homolog mRNA, complete cds; Apurinic endonuclease (APE) mRNA, complete cds./PROD=apurinic endonuclease; TATA box binding protein (TBP)-associated factor, RNA polymerase II, D, 100 kD; Trachea cellular apoptosis susceptibility protein (CSE1) mRNA, complete cds; Similar to eukaryotic translation initiation factor 3, subunit 8 (110 kD), clone MGC:8693, mRNA, complete cds; Human putative ribosomal protein 51 mRNA; Aldehyde dehydrogenase 1, soluble (ALDH1), mRNA./PROD=aldehyde dehydrogenase 1, soluble; Homo sapiens mRNA for KIAA1057 protein, partial cds; RBP1-like protein; Ring finger protein 4; KIAA0197 protein; Homo sapiens mRNA; cDNA DKFZp586F1323 (from clone DKFZp586F1323); Homo sapiens mRNA; cDNA DKFZp5641052 (from clone DKFZp5641052); Homo sapiens mRNA for Hmob33 protein, 3 untranslated region; Homo sapiens mRNA; cDNA DKFZp586F1019 (from clone DKFZp586F1019); partial cds; H. sapiens novel gene from PAC 117P20, chromosome 1; Homo sapiens clone 24582 mRNA sequence; Cyclin T2; H. sapiens HEX gene encoding homeobox related protein; Mitogen-activated protein kinase kinase kinase 4; Serine palmitoyltransferase (LCB2) mRNA, partial cds; KIAA0971 protein/DEF=Homo sapiens cDNA FLJ11495 fis, clone HEMBA1001950, highly similar to Homo sapiens mRNA for KIAA0971 protein; DEAD-box protein abstrakt (ABS), mRNA; KIAA1513 protein (KIAA1513), mRNA; Cell division protein FtsJ (FJH1), mRNA; F-box only protein 3 (FBXO3), mRNA; Purinergic receptor (family A group 5) (P2Y5), mRNA; Integral inner nuclear membrane protein (MAN1), mRNA; Fanconi anemia, complementation group F (FANCF), mRNA; Hypothetical protein FLJ12820 (FLJ12820), mRNA; Hypothetical protein FLJ13119 (FLJ13119), mRNA; Hypothetical protein FLJ20189 (FLJ20189), mRNA; Hypothetical protein FLJ20701 (FLJ20701), mRNA; Homo sapiens chromosome 12 open reading frame 5 (C12ORF5), mRNA; Hypothetical protein FLJ22555 (FLJ22555), mRNA; Hypothetical protein FLJ11110 (FLJ11110), mRNA; CGI-12 protein (LOC51001), mRNA; PNAS-25 mRNA, complete cds.; Homo sapiens mRNA for FLJ00043 protein, partial cds; Homo sapiens cDNA: FLJ21737 fis, clone COLF3396; Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395/DEF=Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395), wherein expression of the one or more biomarkers is increased in the subject.
  • Additionally or alternatively, more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233, 236, 237, 248, 251, 252, 254-261, 266, 267, 283, 284 and 289-292 (or the biomarkers set forth by name in Table 5 as being down-regulated, namely CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 and G344); 6-phosphofructo-2-kinasefructose-2,6-biphosphatase 3 (PFKFB3), mRNA./PROD=6-phosphofructo-2-kinase-fructose-2,6-biphosphatase 3; Protein tyrosine phosphatase, non-receptor type substrate 1 (PTPNS1), mRNA; NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3 (12 kD, B12) (NDUFB3), mRNA; Diphtheria toxin receptor (DTR); Thrombomodulin (THBD), mRNA; Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform (PPM1A), mRNA; Hemoglobin, gamma G (HBG2), mRNA; Amphiregulin (schwannoma-derived growth factor) (AREG), mRNA; Early growth response 2 (Krox-20 (Drosophila) homolog) (EGR2), mRNA; Early growth response 3 (EGR3), mRNA./PROD=early growth response 3; Homo sapiens MD-2 protein (MD-2), mRNA; Homo sapiens MAX dimerization protein (MAD), mRNA; Transferrin receptor (p90, CD71), clone MGC:3151, mRNA, complete cds; Homo sapiens mRNA; cDNA DKFZp564N1272 (from clone DKFZp564N1272); complete cds; GABA-A receptor-associated protein like 1 (GABARAPL1) mRNA, complete cds; Translocation protein 1; Protein kinase-related oncogene (PIM1) mRNA, complete cds; Human mRNA for hCREM (cyclic AMP-responsive element modulator) type 2 protein, complete cds; Human mRNA for ZFM1 protein alternatively spliced product, complete cds./PROD=ZFM1 protein, alternatively spliced product; GABA-A receptor-associated protein mRNA, complete cds./PROD=GABA-A receptor-associated protein; Human (clone 2-5) synuclein (NACP) mRNA, complete cds; Myosin, light polypeptide 4, alkali; atrial, embryonic; Heterogeneous nuclear ribonucleoprotein H1 (H); Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide; cDNA: FLJ23227 fis, clone CAE00645, highly similar to AF052138; Hypothetical protein FLJ12619; C-terminal binding protein 1; SEC22, vesicle trafficking protein (S. cerevisiae)-like 1; Hemoglobin, alpha 1; Homo sapiens clone 24659 mRNA sequence/DEF=Homo sapiens clone 24659 mRNA sequence; Calcium channel, voltage-dependent, PQ type, alpha 1A subunit; H. sapiens SMA5 mRNA; ESTs, Hs.97109; ESTs, Weakly similar to ALU7_HUMAN ALU; Triggering receptor expressed on myeloid cells 1 (TREM1), mRNA; betaGlcNAc beta 1,4-galactosyltransferase. polypeptide 5; Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F) LIKE protein; Heparin-binding EGF-like growth factor mRNA, complete cds; E. coli 7,8-diamino-pelargonic acid (bioA), biotin synthetase (bioB), 7-keto-8-amino-pelargonic acid synthetase (bioF), bioC protein, and dethiobiotin synthetase (bioD), complete cds; Escherichia coli/REF=J04423/DEF=E. coli bioC protein corresponding to nucleotides 4609-4883 of J04423/LEN=777 (−5 and −3 represent transcript regions 5 prime and 3 prime respectively)), wherein expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, namely Solute carrier family 6; Amphiregulin; Keratin 1; Hemoglobin, alpha 1; MHC-II, DM beta; Purinergic receptor P2Y, G-protein coupled 5; Forkhead box O3A; Transferrin receptor (p90, CD71); Ring finger protein 10; Formin binding protein 4; and Hypothetical protein LOC54103), wherein the subject is monitored prior to treatment with a TNFα inhibitor.
  • In yet another embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, namely Diphteria toxin receptor; Heparin-binding EGF-like growth factor; Lipoyltransferase 1; APEX nuclease (multifunct. DNA repair enzyme) 1; GABA(A) receptor-associated protein like 1/3; Formin binding protein 4; Zinc finger protein 331; Ribonuclease P 40 kDa subunit; MHC class II, DM beta; v-maf fibrosarc. oncogene homolog F (avian); 2′,5′-oligoadenylate synthetase 1, 40/46 kDa; and Membrane-spanning 4-domains, subfam. A, memb. 4), wherein the subject is monitored after treatment with a TNFα inhibitor.
  • In yet another embodiment of the monitoring method, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, namely Amphiregulin; Carbonic anhydrase 1; Charcot-Leyden crystal protein; Clusterin C; Tumor necrosis factor alpha induced protein 6; Thrombomodulin; Membrane-spanning 4-domains, subfamily A, member 4; Diptheria toxin receptor; S100 calcium binding protein A1; Uncharacterized hypothalamus protein HT007; MHC-class-II; HLA-DR alpha; Hypothetical protein L0054103; Tumor necrosis factor alpha; Interleukin 1 beta; Proteasome subunit beta type 7 precursor; and Protein KIAA0174; Microsomal signal peptidase 18 kDa subunit).
  • A preferred autoimmune disorder in which to apply the methods of the invention for monitoring an autoimmune disorder is rheumatoid arthritis. However, the monitoring methods can be applied to essentially any autoimmune disorder in which TNFα inhibitor therapy is applied, including the autoimmune disorders listed in the previous section.
  • Selection and Use of Treatment Regimens with TNFα Inhibitors
  • Given the observation that the expression pattern of particular biomarkers in an autoimmune disorder subject influences the responsiveness of the subject to a TNFα inhibitor, one can select an appropriate treatment regimen for the subject based on the expression of one or more biomarkers in the subject. Accordingly, in one embodiment, the above-described method for predicting the responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder further comprises selecting a treatment regimen with the TNFα inhibitor based upon expression of the one or more biomarkers in the subject. In another aspect, the method still further comprises administering the TNFα inhibitor to the subject according to the treatment regimen such that the autoimmune disorder is inhibited in the subject.
  • In another embodiment, the invention provides a method for selecting a treatment regimen for therapy with a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising:
  • assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor for treatment of the autoimmune disorder; and
  • selecting a treatment regimen with a TNFα inhibitor based upon expression of the one or more biomarkers in the subject.
  • In yet another embodiment, the invention provides a method of treating a subject having an autoimmune disorder with a TNFα inhibitor, the method comprising:
  • assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor for treatment of the autoimmune disorder;
  • selecting a treatment regimen with a TNFα inhibitor based upon expression of the one or more biomarkers in the subject; and
  • administering the TNFα inhibitor according to the treatment regimen such that the subject is treated for the autoimmune disorder.
  • The treatment regimen that is selected typically includes at least one of the following parameters and more typically includes many or all of the following parameters: the type of agent chosen for administration, the dosage, the formulation, the route of administration and/or the frequency of administration.
  • Particularly preferred TNFα inhibitors are biologic agents that have been approved by the FDA for use in humans in the treatment of rheumatoid arthritis, which agents include adalimumab (HUMIRA™), infliximab (REMICADE™) and etanercept (ENBREL™), most preferably adalimumab (HUMIRAT™).
  • In one embodiment, the TNFα inhibitor is an anti-tumor necrosis factor-alpha (TNFα) antibody, or antigen-binding portion thereof. For example, the anti-TNFα antibody, or antigen-binding portion thereof, can be a humanized antibody, a chimeric antibody or a multivalent antibody.
  • In another embodiment, the anti-TNFα antibody, or antigen-binding portion thereof, is a human antibody, preferably a human antibody, or antigen-binding portion thereof, that binds to human TNFα with high affinity and a low off rate, and has a high neutralizing capacity. Preferably, the human antibodies are recombinant, neutralizing human anti-hTNFα antibodies. The most preferred recombinant, neutralizing antibody used in the method of the invention is referred to herein as adalimumab, also referred to as HUMIRA® or D2E7 (the amino acid sequence of the adalimumab VL region is shown in SEQ ID NO: 303; the amino acid sequence of the adalimumab VH region is shown in SEQ ID NO: 304). The properties of D2E7 (adalimumab; Humira®) have been described in Salfeld et al., U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015, which are each incorporated by reference herein.
  • Other examples of TNFα antibodies include chimeric and humanized murine anti-hTNFα antibodies which have undergone clinical testing for treatment of rheumatoid arthritis (see e.g., Elliott et al. (1994) Lancet 344:1125-1127; Elliot et al. (1994) Lancet 344:1105-1110; Rankin et al. (1995) Br. J. Rheumatol. 34:334-342). In another embodiment, the TNFα antibody used in the invention is infliximab (Remicade®, Johnson and Johnson; described in U.S. Pat. No. 5,656,272, incorporated by reference herein), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech), and CNTO 148 (golimumab; Medarex and Centocor, see also WO 02/12502).
  • In one embodiment, the TNFα inhibitors used in the methods of the invention include adalimumab antibodies and antibody portions, adalimumab-related antibodies and antibody portions, adalimumab-related DVD-Ig or dual specific antibodies, and other human antibodies and antibody portions with equivalent properties to adalimumab, such as high affinity binding to hTNFα with low dissociation kinetics and high neutralizing capacity. In one embodiment, a treatment regimen of the invention provides treatment with an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFα with a Kd of 1×10−8 M or less and a Koff rate constant of 1×10−3 s−1 or less, both determined by surface plasmon resonance, and neutralizes human TNFα cytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10−7 M or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNFα with a Koff of 5×10−4 s−1 or less, or even more preferably, with a Koff of 1×10−4 s−1 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNFα cytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10−8 M or less, even more preferably with an IC50 of 1×10−9 M or less and still more preferably with an IC50 of 1×10−10 M or less. In a preferred embodiment, the antibody is an isolated human recombinant antibody, or an antigen-binding portion thereof.
  • It is well known in the art that antibody heavy and light chain CDR3 domains play an important role in the binding specificity/affinity of an antibody for an antigen. Accordingly, in another aspect, the TNFα inhibitor used in the treatment method of the invention is a human anti-TNFα antibody that has slow dissociation kinetics for association with hTNFα and that has light and heavy chain CDR3 domains that structurally are identical to or related to those of adalimumab. Position 9 of the adalimumab VL CDR3 can be occupied by Ala or Thr without substantially affecting the Koff. Accordingly, a consensus motif for the adalimumab VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 305). Additionally, position 12 of the adalimumab VH CDR3 can be occupied by Tyr or Asn, without substantially affecting the Koff. Accordingly, a consensus motif for the adalimumab VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 306). Moreover, as demonstrated in Example 2 of U.S. Pat. No. 6,090,382, the CDR3 domain of the adalimumab heavy and light chains is amenable to substitution with a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantially affecting the Koff. Still further, the skilled artisan will appreciate that, given the amenability of the adalimumab VL and VH CDR3 domains to substitutions by alanine, substitution of other amino acids within the CDR3 domains may be possible while still retaining the low off rate constant of the antibody, in particular substitutions with conservative amino acids. Preferably, no more than one to five conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. More preferably, no more than one to three conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. Additionally, conservative amino acid substitutions should not be made at amino acid positions critical for binding to hTNFα. Positions 2 and 5 of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab VH CDR3 appear to be critical for interaction with hTNFα and thus, conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position 5 of the adalimumab VL CDR3 is acceptable, as described above) (see U.S. Pat. No. 6,090,382).
  • Accordingly, in another embodiment, the antibody or antigen-binding portion thereof preferably contains the following characteristics:
  • a) dissociates from human TNFα with a Koff rate constant of 1×10−3 s−1 or less, as determined by surface plasmon resonance;
  • b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
  • c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
  • More preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFα with a Koff of 5×10−4 s−1 or less. Even more preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFα with a Koff of 1×10−4 s−1 or less.
  • In yet another embodiment, the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 307 (i.e., the adalimumab VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 308 (i.e., the adalimumab VH CDR2). Even more preferably, the LCVR further has CDR1 domain comprising the amino acid sequence of SEQ ID NO: 309 (i.e., the adalimumab VL CDR1) and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 310 (i.e., the adalimumab VH CDR1). The framework regions for VL preferably are from the VκI human germline family, more preferably from the A20 human germline Vk gene and most preferably from the adalimumab VL framework sequences shown in FIGS. 1A and 1B of U.S. Pat. No. 6,090,382. The framework regions for VH preferably are from the VH3 human germline family, more preferably from the DP-31 human germline VH gene and most preferably from the adalimumab VH framework sequences shown in FIGS. 2A and 2B of U.S. Pat. No. 6,090,382.
  • Accordingly, in another embodiment, the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 303 (i.e., the adalimumab VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 304 (i.e., the adalimumab VH). In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • In other embodiments, the TNFα inhibitor of the invention is etanercept (described in WO 91/03553 and WO 09/406,476), infliximab (described in U.S. Pat. No. 5,656,272), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibody fragment), D2E7 (a human anti-TNF mAb), soluble TNF receptor Type I, or a pegylated soluble TNF receptor Type I (PEGs TNF-R1).
  • The TNFα antibody of the invention can be modified. In some embodiments, the TNFα antibody or antigen binding fragments thereof, is chemically modified to provide a desired effect. For example, pegylation of antibodies and antibody fragments of the invention may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety). Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol.
  • Methods for preparing pegylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby the antibody or antibody fragment becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
  • In yet another embodiment of the invention, TNFα antibodies or fragments thereof can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody. To modify an antibody of the invention such that it exhibits reduced binding to the Fc receptor, the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. Immunol. 147:2657-2662). Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity.
  • An antibody or antibody portion of the invention can be derivatized or linked to another functional molecule (e.g., another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the anti-TNFα antibodies described herein, including immunoadhesion molecules. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.
  • Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Selection of the particular parameters of the treatment regimen can be based on known treatment parameters for the TNFα inhibitor previously established in the art. For example, a non-limiting example of a treatment regimen for adalimumab (HUMIRA™) is 40 mg every other week by subcutaneous injection. A non-limiting example of a treatment regimen for etanercept (ENBREL™) is 50 mg/week by subcutaneous injection. A non-limiting example of a treatment regimen for infliximab (REMICADE™) is 3 mg/kg by intravenous infusion at weeks 0, 2 and 6, then every 8 weeks. A treatment regimen can include administration of the TNFα inhibitor alone or can include combination of the TNFα inhibitor with other therapeutic agents, such as methotrexate (e.g., 10-20 mg/week) or prednisolone (e.g., 10 mg/week). Other suitable treatment regimens for the TNFα inhibitors discussed herein will be readily apparent to the ordinarily skilled artisan based on prior studies of preferred administration parameters for the TNFα inhibitor.
  • For administration to a subject, a TNFα inhibitor typically is formulated into a pharmaceutical composition containing the TNFα inhibitor and a pharmaceutically acceptable carrier. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions also can be administered in combination therapy, i.e., combined with other agents, such as other TNFα inhibitors and/or other therapeutic agents, such as traditional therapeutic agents for the treatment of autoimmune disorders, such as rheumatoid arthritis.
  • As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • The pharmaceutical compositions may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • A pharmaceutical composition also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • A TNFα inhibitor of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. A preferred route of administration, particularly for antibody agents, is by intravenous injection or infusion. Other preferred routes of administration include intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a TNFα inhibitor of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • In a preferred embodiment, the subject to be treated with the TNFα inhibitor is a human subject.
  • A preferred autoimmune disorder in which to apply the methods of the invention for selecting and using a treatment regimen is rheumatoid arthritis. However, these methods can be applied to essentially any autoimmune disorder in which TNFα inhibitor therapy is applied, including the autoimmune disorders listed in the previous sections
    • Kits of the Invention
  • In another aspect, the invention pertains to kits for carrying out the methods of the invention. For example, in one embodiment, the invention provides a kit for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder. In one embodiment, the kit comprises:
  • a) means for isolating monocytes;
  • b) means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder;
  • c) means for measuring expression of at least one housekeeping gene; and
  • d) instructions for use of the kit to predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above)
  • In one embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (or the biomarkers set forth by name in Table 9 as being increased in ≧80% of responders vs. non-responders, as described above), more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (or the biomarkers set forth by name in Table 9 as being increased in ≧90% of responders vs. non-responders, as described above), and even more preferably at least one of the biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (or the CD11c biomarkers set forth by name in Table 9 as being increased in 100% of responders vs. non-responders). In each of these embodiments, preferably the instructions for use of the kit instruct that increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (or the biomarkers set forth by name in Table 9 as being decreased in ≧80% of responders vs. non-responders, as described above), more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (or the biomarkers set forth by name in Table 9 as being decreased in ≧90% of responders vs. non-responders, as described above), and even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (or the biomarkers set forth by name in Table 9 as being decreased in 100% of responders vs. non-responders, as described above). In each of these embodiments, preferably the instructions for use of the kit instruct that decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, as described above).
  • In yet another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, as described above), more preferably SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as being upregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as being downregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, as described above), more preferably SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as being upregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233, 236, 237, 248, 251, 252, 254-261, 266, 267, 283, 284 and 289-292 (or the biomarkers set forth by name in Table 5 as being downregulated, as described above), wherein the instructions instruct that expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, as described above), wherein the instructions instruct that the subject is monitored prior to treatment with a TNFα inhibitor. In yet another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, as described above), wherein the instructions instruct that the subject is monitored after treatment with a TNFα inhibitor.
  • In yet another embodiment of the kit, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, as described above).
  • In a preferred embodiment, the means for measuring expression in the subject of the one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder comprises a nucleic acid preparation sufficient to detect expression of mRNA encoding the one or more biomarkers in a sample from the subject, such as a peripheral blood mononuclear cell sample from which mRNA is obtained by standard methods. This nucleic acid preparation includes at least one, and may include more than one, nucleic acid probe or primer, the sequence(s) of which is designed such that the nucleic acid preparation can detect the expression of mRNA encoding the biomarker(s) of interest in the sample from the subject. A preferred nucleic acid preparation includes two or more PCR primers that allow for PCR amplification of a segment of the mRNA encoding the biomarker(s) of interest. In a particularly preferred embodiment, the kit comprises a nucleic acid preparation sufficient to detect expression of CD11c mRNA in a sample from the subject.
  • Alternatively, the means for detecting expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder can comprise a reagent that detects the gene product of the mRNA encoding the biomarker(s) of interest sufficient to distinguish it from other gene products in a sample from the subject. A non-limiting example of such a reagent is a monoclonal antibody preparation (comprising one or more monoclonal antibodies) sufficient to detect protein expression of the biomarker(s) of interest in a sample from the subject, such as a peripheral blood mononuclear cell sample. In a particularly preferred embodiment, the kit comprises a monoclonal antibody preparation sufficient to detect expression of CD11c protein in a sample from the subject.
  • The means for measuring expression of the one or more biomarkers can also include, for example, buffers or other reagents for use in an assay for evaluating biomarker expression (e.g., at either the mRNA or protein level). The instructions can be, for example, printed instructions for performing the assay for evaluating the expression of the one or more biomarkers.
  • In a preferred embodiment, the means for measuring expression of at least one housekeeping gene comprises a nucleic acid preparation sufficient to detect expression of mRNA of the housekeeping gene (e.g., GAPDH) in a sample from the subject, such as a peripheral blood mononuclear cell sample from which mRNA is obtained by standard methods. This nucleic acid preparation includes at least one, and may include more than one, nucleic acid probe or primer, the sequence(s) of which is designed such that the nucleic acid preparation can detect the expression of mRNA of the housekeeping gene(s) in the sample from the subject. A preferred nucleic acid preparation includes two or more PCR primers that allow for PCR amplification of a segment of the mRNA of the housekeeping gene(s). Alternatively, the means for detecting expression in the subject of at least one housekeeping gene can comprise a reagent that detects the gene product of housekeeping gene sufficient to distinguish it from other gene products in a sample from the subject. A non-limiting example of such a reagent is a monoclonal antibody preparation (comprising one or more monoclonal antibodies) sufficient to detect protein expression of housekeeping gene product in a sample from the subject, such as a peripheral blood mononuclear cell sample.
  • The means for isolating monocytes can comprise one or more reagents that can be used to separate monocytes from other cell types in a sample of peripheral blood mononuclear cells, for example by positive selection of the monocytes or by negative selection in which all other cell types other than monocytes are removed. In one embodiment, a reagent that binds CD14 on monocytes (e.g., an anti-CD14 antibody) is included in the kit as means to isolate monocytes via positive selection. Alternatively, in another embodiment, reagents such as those commercially available in the Monocyte Isolation Kit II (Miltenyi Biotec, Auburn, Calif.) can be used for negative selection, in which non-monocytes (T cells, B cells, NK cells, dendritic cells, basophils) are indirectly magnetically labeled using a cocktail of biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and CD235a (Glycophorin A), as well as anti-biotin MicroBeads, and then highly pure unlabeled monocytes are obtained by depletion of the magnetically labeled cells.
  • In another embodiment, the kit can further comprise a TNFα inhibitor for treating an autoimmune disorder in the subject. Preferred TNFα inhibitors for use in the kit include the TNFα inhibitors described in detail above with respect to treatment regimens, in particular anti-TNFα antibodies such as adalimumab, infliximab and/or golimumab, and/or Ig fusion proteins such as etanercept.
  • Preferably, the kit is designed for use with a human subject.
    • Databases and Computer Programs
  • In another aspect, the invention pertains to methods of building a database for use in selecting an autoimmune disorder subject for treatment with a TNFα inhibitor, or for use in selecting or monitoring a treatment regimen in an autoimmune disorder subject. The method can comprise receiving, in a computer system, biomarker expression patterns from a plurality of subjects having an autoimmune disorder; and storing the biomarker expression pattern from each subject such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above). The identifier of the subject can be, for example, the name of the subject or a numerical or symbolic identifier coded to the identity of the subject. The method can further comprise receiving, in the computer system, one or more treatment regimens for treatment of an autoimmune disorder in a subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject. A user can enter the subject's biomarker expression pattern, and optionally the subject's treatment regimen(s), into the computer system. Alternatively, the subject's biomarker expression pattern can be received directly from equipment used in determining the expression of one or more biomarkers in a sample from the subject.
  • In another aspect, the invention provides a computer program product useful for building a database for use in selecting or monitoring an autoimmune disorder subject for treatment with a TNFα inhibitor. The computer program can contain executable instructions that when executed cause a processor to perform operations comprising: receiving, in a computer system, a biomarker expression pattern of a subject at one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder; and storing the biomarker expression pattern such that the biomarker expression pattern is associated with an identifier of the subject, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above). Optionally, the computer program can further cause the processor to perform an operation comprising: receiving, in the computer system, a treatment regimen for treatment of an autoimmune disorder in the subject such that the treatment regimen is associated with the biomarker expression pattern of the subject and the identifier of the subject.
  • In another aspect, the invention provides a method of selecting an autoimmune disorder subject for a treatment with a TNFα inhibitor. The method can comprise: (i) identifying, in a database comprising a plurality of autoimmune disorder subjects, a subject whose database entry is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNFα inhibitor, and (ii) selecting the subject for treatment with a TNFα inhibitor, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above). The method can further comprise selecting a treatment regimen by identifying, in the database, a treatment regimen that has been associated with the biomarker expression pattern of the subject and with an identifier of the subject.
  • In yet another aspect, the invention provides a computer program product useful for identifying and/or selecting a subject for treatment with a TNFα inhibitor. The computer program can contain executable instructions that when executed cause a processor to perform operations comprising: (i) identifying, in a database including a plurality of autoimmune disorder subjects associated with biomarker expression patterns, a subject that is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNFα inhibitor; and (ii) outputting the identified subject as a subject to be treated with a TNFα inhibitor; wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82 (or the biomarkers set forth by name in Table 9, as described above). The computer program can further cause the processor to perform an operation comprising outputting a treatment regimen that is associated with the subject to be treated with the TNFα inhibitor.
  • In one embodiment of the above-described methods and computer program, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 (or the biomarkers set forth by name in Table 9 as being increased in ≧80% of responders vs. non-responders, as described above), more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 37, 44, 47, 62 and 70 (or the biomarkers set forth by name in Table 9 as being increased in ≧90% of responders vs. non-responders, as described above), and even more preferably at least one of the biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44 (or the biomarker, CD11c antigen, set forth by name in Table 9 as being increased in 100% of responders vs. non-responders, as described above). In each of these embodiments, increased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another embodiment of the above-described methods and computer programs, the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 (or the biomarkers set forth by name in Table 9 as being decreased in ≧80% of responders vs. non-responders, as described above), more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 29, 65, 73 and 74 (or the biomarkers set forth by name in Table 9 as being decreased in ≧90% of responders vs. non-responders, as described above), and even more preferably the one or more biomarkers is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 74 (or the biomarkers set forth by name in Table 9 as being decreased in 100% of responders vs. non-responders, as described above). In each of these embodiments, decreased expression of the one or more biomarkers is predictive of responsiveness of the subject to a TNFα inhibitor.
  • In another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 83-133 (or the biomarkers set forth by name in Table 3, as described above).
  • In yet another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131 (or the biomarkers set forth by name in Table 4, as described above), more preferably SEQ ID NO: 134-150, 112, 118 (or the biomarkers set forth by name in Table 4 as having increased expression, as described above), wherein expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 151-177, 110 and 123 (or the biomarkers set forth by name in Table 4 as having decreased expression, as described above), wherein expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 178-292, 91 and 97 (or the biomarkers set forth by name in Table 5, as described above), more preferably SEQ ID NO: 178-185; 187-200, 202, 203, 205-207; 211, 213, 214, 216, 220, 221, 226, 228, 229, 231, 232, 234, 235, 238-247, 249, 250, 253, 262-265, 268-282 and 285-288 (or the biomarkers set forth by name in Table 5 as having increased expression, as described above), wherein expression of the one or more biomarkers is increased in the subject, and/or more preferably SEQ ID NO: 186, 201, 204, 208, 209, 91, 210, 212, 97, 215, 217-219, 222-225, 227, 230, 233, 236, 237, 248, 251, 252, 254-261, 266, 267, 283, 284 and 289-292 (or the biomarkers set forth by name in Table 5 as having decreased expression, as described above), wherein expression of the one or more biomarkers is decreased in the subject.
  • In yet another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123 (or the biomarkers set forth by name in Table 6 as pre-treatment biomarkers, as described above), wherein the subject is monitored prior to treatment with a TNFα inhibitor. In yet another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131 (or the biomarkers set forth by name in Table 6 as post-treatment biomarkers, as described above), wherein the subject is monitored after treatment with a TNFα inhibitor.
  • In yet another embodiment of the above-described methods and/or computer programs, the biomarker expression pattern(s) can be for one or more biomarkers selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300 (or the biomarkers set forth by name in Table 7, as described above).
  • Computer systems and database software well established in the art can be adapted for use in the methods and computer program products of the invention for building and searching a database for use in selecting or monitoring a treatment regimen for a subject having an autoimmune disorder or for selecting a particular autoimmune disorder subject for treatment with a TNFα inhibitor.
  • The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference in their entirety.
    • EXAMPLES Example 1 Materials and Methodologies
  • In this example, the materials and methodologies used in the subsequent Examples are described.
    • Patients
  • Purified monocytes (MO) derived from a total of 84 RA patients were used. The clinical data for the RA patients, pre- and post-anti-TNFα treatment, is summarized in Table 1. All patients fulfilled the revised American College of Rheumatology (ACR) criteria (Arnett, F. C. et al. (1988) Arthritis Rheum. 31:315-324). Patients were defined as responders (≧continuous ACR score 40) or non-responders (≦continuous ACR score 30) to anti-TNFα monotherapy, MTX monotherapy or combination therapy. In order to account for a gradual transition from clinical responders to non-responders to therapy, a continuous ACR response evaluation was performed by applying the ACR criteria, but by defining 10% response steps instead of the usual 20, 50, and 70% steps. This was also done to allow more detailed analyses of the correlation between the ACR response and the mRNA expression levels of the predictive marker CD11c.
  • For Affymetrix® microarray analysis, MO samples from 7 RA patients (RA1-RA7; all females) undergoing the Abbott DE011 trial were used (ReACT; monoclonal antibody Adalimumab®). In this multicenter, double-blinded study patients received Adalimumab® (20 mg weekly or 40 mg biweekly) in the outpatient Rheumatology Clinic of the Charite University Hospital in Berlin, Germany, for a total of 2 years. Non-steroidal anti-inflammatory drugs and 10 mg prednisolone-equivalent per week were allowed in addition to the anti-TNFα therapy. For TaqMan® real-time PCR, 26 patients (RA8-RA84 from the Abbott DE013 trial were used (ATTRACT). In this study Adalimumab®, methotrexate (MTX; 10-20 mg/week) or a combination of both therapeutics was applied.
  • The mean time interval between the two MO samples (blood sampling before and during therapy) was 9.4±1.8 months (mean±SEM; range 4-13.5 months), the mean age 50.3±3.9 years (range 39-70), and the mean disease duration 18.6±5.3 years (range 4-38). As controls, healthy individuals were recruited (n=7; 6 females, 1 male; mean age 36.1±7.4 years). RA patients RA1-RA84 were recruited from the Rheumatology Clinic of the Charité University Clinic, the Rheumaklinik of the Charité in Berlin-Buch, and from the Schlossparklinik in Berlin.
    • Separation of Peripheral Blood Mononuclear Cells, Purification of Monocytes, RNA Isolation
  • Peripheral blood (30 to 35 ml) was obtained by venopuncture, immediately stored in heparin-containing vacutainers (Beckton-Dickinson, Rutherford, N.J., USA), and cooled to 4° C. Blood samples were subjected to a Ficoll-Hypaque gradient (d=1.077 g/ml; Biochrom, Berlin, Germany). To enrich MO, negative selection magnetic cell sorting (MACS; Miltenyi; Bergisch Gladbach, Germany) was subsequently applied. Successful purification of MO (purity of 83-90%; 1.3-3.2×106 MO/patient) was validated by FACS analysis using CD14 and CD45 antibodies (Beckman-Coulter, Krefeld, Germany). In all cases, purified MO showed >98% vitality using propidiumiodide staining (Pharmingen, San-Diego, Calif., USA). MO preparation was performed at 4° C. After purification, MO were lysed in RLT lysis buffer and total RNA was isolated using the RNeasy mini elute kit (Quiagen, Düsseldorf, Germany; yield 1.5-3.2 μg/sample). Quantification and quality control of RNA was performed at 260/280 nm using a Bioanalyzer 2100 unit (Agilent, Palo Alto, Calif., USA).
    • RNA Amplification and Labeling
  • For target synthesis, 500 ng of total RNA were amplified using the standard protocol of the manufacturer (Affymetrix®, Palo Alto, Calif., USA) and the Megascript kit (Ambion, Camhridgeshire, UK).
  • Biotin-Labeling of cRNA and Gene-Chip Hybridization
  • Biotinylated cRNA target was generated from amplified cRNAs using the Bioarray high-yield transcription kit (Enzo, New York, N.Y., USA). Samples were hybridized to Affymetrix® test and HG-U133A GeneChip arrays.
  • Following washing and staining, arrays were scanned twice at 3 μm resolution using a confocal scanner with an argon laser instrument (Agilent® G2500A GeneArray Scanner; Agilent, CA, USA).
    • Bioinformatic Analysis of Differentially Expressed Genes in ND and RA Patients Pre- and Post Anti-TNFα Treatment
  • All GeneChips were analyzed for signal calculation and pairwise comparisons using the GCOS 1.4 software package with standard settings provided by Affymetrix®. Scaling was performed to a target value of 150 and normalization was set to “1”. Pairwise comparison data were grouped to generate a percentage level of increased and decreased comparisons. Fold-changes were calculated from the mean of the SLR values in pairwise comparisons. Filtering was performed on the basis of “increased/decreased” comparisons with a percentage cutoff as indicated in the results. For hierarchical clustering, the software tool “Gene Expression Similarity Investigation Suite” (Genesis; Sturn, A. et al. (2002) Bioinformatics 18:207-208; http://genome.tugraz.at/Software/GenesisCenter.html) was applied using normalized signal intensities, Pearson distance correlation, and complete linkage clustering. Prediction analysis was performed using the PAM software (http://www.bioconductor.org; Khan, J. et al. (2001) Nat. Med. 7:673-679).
    • Data Sets of Publication
  • The complete ASCI-file datasets have been deposited in the microarray GEO database (http://www.ncbi.nlm.nih.gov/geo/).
    • TaqMan® Real Time PCR
  • Real-time PCR (RT-PC) was performed using a TaqMan® 7500 system and pre-designed TaqMan® low density gene expression primers (Applied Biosystems; Foster City, Calif., USA) or, in the case of CD11c, the primer Hs01015072_g1 (commercially available from Applied Biosystems) in a Bio-Rad iQ real time PCR system (Icycler; Bio-Rad; München, Germany). The housekeeping gene GAPDH was used for normalization of the cDNA content. Quantification was performed using the SDS 2.2.0 software (Applied Biosystems); results were expressed as relative quantities of the logarithm of the ΔΔCT values (log RQ), as the relative quantity of expression (RQ; fold-change in comparison to normal donor expression), or as the % expression as normalized to GAPDH.
    • Literature-Associated Pathway Analysis Using Ingenuity
  • Gene ontology and gene interaction analyses were executed using the Ingenuity® Pathway analysis tool v.4.0 (Ingenuity, Redwood City, Calif., USA; Jenssen, T. K. et al. (2001) Nat. Genet. 28:21-28; http://www.ingenuity.com). Highest scoring neighborhood analysis of literature-based gene connections was performed by comparing up- and downregulated genes in anti-TNFα responders and non-responders (pre- and post-treatment). Significantly regulated genes in both comparisons were merged from the networks 1 to 5, complemented by transcription factors and finally overlayed with their relative expression values.
    • Expression-Based Pathway Analysis Using Kyoto Encyclopedia of Genes and Genomes (KEGG)
  • The KEGG pathway analysis (Kanehisa, M. and Bork, P. (2003) Nat. Genet. 33:305-310) was performed using selected genes from the comparison of microarray data in responders and non-responders either pre- or post-treatment with anti-TNFα. Upregulated genes and downregulated genes within the illustrated pathways were color-coded in a gradient fashion (SLR 0.5 to ≧1.5). A total of 4 pathways out of the 8 most highly ranked pathways were selected for illustration.
    • Statistical Analysis
  • The non-parametric Mann-Whitney U test was applied to analyze differences between data from RA patients and normal donors, from untreated and anti-TNFα-treated RA patients, and from responders and non-responders to anti-TNFα-therapy. Correlation analyses between experimental and clinical/laboratory parameters of the patients were performed using the Pearson test and the software SPSS 13.0™ (SPSS Inc., Chicago, Ill., USA). For the U test, statistically significant differences were accepted for P≦0.05; for correlation analyses, the acceptance level was reduced to P≦0.01 to account for multiple comparisons.
    • Example 2 Clinical and Laboratory Assessments for RA Patients and Normal Donors
  • Two of the seven anti-TNFα-treated RA patients used for microarray analysis, i.e., patients RA4 and RA6, were non-responders to therapy according to the ACR improvement criteria (≦continuous ACR 30 score). In general, this was also reflected in the respective percent-reduction of other clinical parameters of local or systemic inflammation. The group of seven RA patients employed for microarray analysis in the present study constituted a representative RA cohort, as demonstrated by well-known correlations among clinical parameters pre- and post-anti-TNFα treatment, as summarized in Table 2.
  • The identification of patients RA4 and RA6 as non-responders was also confirmed by hierarchical clustering of clinical parameters.
    • Example 3 Gene Expression Profiling and Analysis Differential Gene Expression in Responders Versus Non-Responders to Anti-TNFα-Therapy
  • A total of 119 differentially-expressed genes was identified by comparing RA and normal donors (ND; n=7 each; total of 49 comparisons). Hierarchical clustering of ND, as well as RA patients pre- and post-treatment with these genes also identified 5 responders and 2 non-responders (RAantiTNF4 and RAantiTNF6).
  • In order to select therapeutically relevant genes, a subpopulation of 51 differentially-expressed genes was then identified by the simultaneous comparison between RA versus normal donors (ND; total of 49 comparisons) and RA responders (n=5) pre- versus post-anti-TNFα therapy (25 comparisons). These genes showed an increase or decrease of the signal log ratio (SLR; between −4.36 and 4.61) in >70% of the pair-wise comparisons between RA and ND. These genes are summarized in Table 3.
  • Hierarchical clustering with these genes resulted in precise (100%) classification of ND, RA patients pre-treatment, and clinically-defined responders (≧continuous ACR score 40; clustered as ND) or non-responders (≦continuous ACR score 30; clustered as RA; note RAantiTNF4 and RAantiTNF6). This was confirmed by supervised pattern discovery using prediction analysis of microarrays (PAM analysis) at a threshold value of 4.3 in order to minimize the misclassification error. Table 4 summarizes the results of the PAM analysis, showing 49 selected genes, five of them overlapping with genes listed in Table 3. In this case, both non-responders (RAantiTNF4 and RAantiTNF6) were classified as RA patients, whereas the responders, RAantiTNF1-3, RAantiTNF5, and RAantiTNF7, were classified either as normals or as anti-TNFα-treated RA patients. Notably, ND showed the lowest misclassification error at the threshold value (0.00; i.e., the highest similarity among individuals), followed by pre-treatment RA patients (0.14), and anti-TNFα-treated RA patients (0.42).
  • Identification of responders/non-responders was also confirmed by hierarchical clustering of RA patients post-treatment. A total of 117 genes differentially expressed in RA responders versus RA non-responders was identified in post-anti-TNFα therapy samples, which showed an increase or decrease in 100% of the respective pair-wise comparisons (total of 10 comparisons). The 117 genes identified in this analysis are summarized in Table 5, three of which genes overlap with the genes summarized in Table 3. Hierarchical clustering with these genes resulted in precise (100%) classification of responders and non-responders to therapy.
  • A number of the differentially-expressed genes showed highly significant correlations with clinical or laboratory parameters pre- and/or post-anti-TNFα treatment, indicating a potential clinical relevance of the genes and contributing to the selection of genes for validation with TaqMan® real-time RT-PCR. These genes are summarized in Table 6.
    • Example 4 Real-Time RT-PCR Validation of Genes Differentiating Responders and Non-Responders
  • Sixteen genes with a likely pathogenetic importance in RA (and 6 control genes) from Affymetrix® gene expression profiling were selected for validation by TaqMan® real-time RT-PCR (n=10 anti-TNFα-treated RA patients prior to therapy; n=14 ND). The RT-PCR results confirmed the results of Affymetrix® gene expression profiling for 17 of 22 genes (approx. 77%), summarized in Table 7. This applied to genes regarded as differentially expressed in RA versus ND by Affymetrix® analysis (decreased: <−70%; increased: >70%) and to equally expressed control genes. By real-time RT-PCR, 18 genes showed significantly differential expression (p≦0.000 for 7 genes; p≦0.041 for the remaining) in MO from RA patients responding to anti-TNFα therapy versus ND, including genes 10 upregulated in RA (Amphiregulin, Charcot-Leyden crystal protein, TNFα-induced protein 6, thrombomodulin, membrane-spanning 4-domains, subfamily A—member 4, S100 calcium binding protein A1, TNFα, IL-1β, lipoyltransferase 1, and interferon regulatory protein 1), as well as 8 genes downregulated in RA (Uncharacterized hypothalamus protein HT007, MHC class II HLA-DR-alpha, hypothetical protein L0054103, proteasome subunit beta type 7 precursor, protein KIAA0174, microsomal signal peptidase 18 kDa subunit, ring zinc finger protein 361, and protein phosphatase 1, catalytic subunit, beta isoform). However, the expression for these genes showed no significant differences for the direct comparison between RA responders and non-responders to anti-TNFα therapy.
  • The potential clinical relevance of some of the differentially expressed genes is underlined by a significant correlation with the ACR response at different time points during anti-TNFα therapy, as summarized in Table 8.
  • Validation of the 22 selected genes by TaqMan® real-time RT-PCR confirmed the results of Affymetrix® gene expression profiling for 17 of 22 genes (approx. 77% true positives or negatives), in accordance with the rates reported in other gene expression studies and therefore underlining the validity of the present data. The 18 genes showing significantly differential expression in MO from RA patients responding to anti-TNFα therapy versus ND included several genes with a likely pathogenetic importance in RA (e.g., amphiregulin, TNFα-induced protein 6, thrombomodulin, membrane-spanning 4-domains, subfamily A—member 4, S100 calcium binding protein A1, TNFα, IL-1β, lipoyltransferase 1, interferon regulatory protein 1, MHC class II HLA-DR-alpha).
    • Example 5 Gene Expression Profiling and Analysis Differential Gene Expression in Responders Versus Non-Responders Prior to Anti-TNFα-Therapy (Predictive Genes)
  • Using a threshold of ≧80% for the pairwise comparisons between future RA responders and future RA non-responders prior to anti-TNFα therapy, a total of 82 predictive genes was identified (11 genes for a threshold of ≧90%; 3 genes for 100%). These genes are summarized in Table 9. The latter group (100%) consisted of 2 known proteins (Homo sapiens predicted osteoblast protein (GS3786); integrin alpha-X (antigen CD11c) and an unknown protein (Homo sapiens hypothetical protein FLJ10134). In particular the antigen CD11e appears highly interesting, since it is a surface molecule on human MO (and other cells of the myelomonocytic lineage), and since it has known inflammatory functions. Hierarchical clustering with the selected genes resulted again in precise (100%) predictive classification of future responders and non-responders to therapy. Marginal co-clustering of the patient RA5 with the non-responders RA4 and RA6 at the threshold values 80% and 100% possibly identified RA5 as a ‘weak’ responder, as also indicated by a marginal position in the hierarchical clustering of clinical parameters.
  • Interestingly, future responders to anti-TNFα-therapy (when directly compared to non-responders) showed a pattern shift from the recently described ‘inflammatory’ MO to ‘resident’ MO subsets (Gordon, S. et al. (2005) Nat. Immunol. 5:953-963) both prior to therapy and post-treatment. Although most of the individual molecules showed an identical pattern shift in both pre-treatment and post-treatment comparisons, in particular the activating (CD16a,b) and inhibiting Fcγ-receptors (CD32) displayed an opposite behavior. This pattern shift was observed despite the fact that all pre-treatment or post-treatment comparisons between RA patients (all, future responders, future non-responders) and ND indicated a dominance of ‘inflammatory’ MO in the respective RA groups. Post-treatment, strikingly, responders to anti-TNFα-therapy became barely distinguishable from ND in contrast to non-responders, which still showed a clear ‘inflammatory’ predominance. These results indicate that successful anti-TNFα-therapy acts by blocking TNFα signaling via TNFα-receptors 1 and 2 and by subsequent induction of a major change in the composition of pro-inflammatory and other MO subsets.
    • Example 6 Real-Time RT-PCR Validation of Predictive CD11c
  • TaqMan® real-time RT-PCR confirmed the discrimination of future RA responders (n=15) from future nonresponders (n=12) to anti-TNF monotherapy (at the level of continuous ACR score 30) on the basis of their CD11c mRNA expression in monocytes.
  • TaqMan® real-time RT-PCR confirmed the separation of future RA responders n=55 from future non-responders n=12 to anti-TNFα monotherapy on the basis of their CD11c mRNA expression in MO. The results are summarized in the bar graph of FIG. 1. This clear separation was lost in the case of combination therapy with anti-TNFα/MTX, possibly due to a differential importance of the CD11c mRNA expression for the anti-TNFα and MTX components.
  • Of the 3 genes identified by pairwise comparison between RA responders and RA non-responders prior to treatment at the level of 100% (and confirmed by TaqMan® real-time RT-PCR), the antigen CD11c appears of particular interest, since it is expressed on the surface of human MO (and other cells of the myelomonocytic lineage, e.g. dendritic cells), and since it has known functions in inflammatory reactions (e.g., as the complement receptor 4) and cell adhesion.
  • The validity of CD11c as a predictive biomarker is further underlined by a significant correlation (r=0.651, P=<0.0001, n=27) between the CD11c expression prior to therapy and the future percentage of the ACR response, illustrated in the graph of FIG. 2.
  • Except for 1 RA patient with a borderline ACR response of 30 and a CD11c mRNA level directly at the distinction threshold (who was therefore classified as a false negative), the threshold level (40%) almost fully distinguished future responders from nonresponders (100% specificity, 94% sensitivity, and 96% power). This clear separation was lost in the case of combination therapy with anti-TNF/methotrexate (MTX), possibly owing to a differential importance of the CD11c mRNA expression for the anti-TNF and MTX treatment components. This was further underlined by the fact that: i) future responders to MTX monotherapy did not significantly differ in their CD11c mRNA expression level from future nonresponders to MTX monotherapy; and ii) there was no significant correlation between the future ACR response of RA patients treated with MTX monotherapy and their CD11c mRNA expression (data not shown).
  • Strikingly, this was true not only for the continuous ACR score, but also for the clinically applied, discrete ACR criteria.
  • A significant correlation (r=0.656, P=<0.0001, n=27) was observed between CD11c expression and the future percentage of strict ACR response, illustrated in the graph in FIG. 3. This finding complements and expands previous reports on the identification of molecules capable of predicting a future response to anti-TNFα therapy (Lequerre, T. et al. (2006) Arthritis Res. Ther. 8:R105; Toh, M. L. et al. (2006) Arthritis Rheum. 54:2109-2118).
    • Example 7 Ingenuity® Pathway Analysis
  • Ingenuity® pathway analysis of the genes in Tables 3 and 5 indicated a direct or indirect influence of anti-TNFα therapy on several molecules thought to be relevant for the pathogenesis and/or severity of RA, e.g., HLA-DMA/B (Morel, J. et al. (2004) Ann. Rheum. Dis. 63:1581-1586), CD69 (Marzio, R. et al. (1999) Immunopharmacol. Immunotoxicol. 2:565-582) thrombomodulin (Cobankara, V. et al. (2004) Clin. Rheumatol. 23:430-434), membrane-spanning 4-domains, subfamily A—member 4 (Fujikado, N. et al. (2006) Arthritis Res. Ther. 8:1-13), and forkhead box 03a (Jonsson, H. et al. (2005) Nat. Med. 11:666-671). The present approach, therefore, represents a powerful tool to identify gene regulation patterns applicable for diagnosis, as well as for therapy stratification and monitoring in rheumatic diseases, in particular in view of the fact that blood MO are much more easily available than synovial tissue samples. This is further supported by the fact that a high number of individual genes show significant correlations with clinical parameters in RA patients pre- and/or post-anti-TNFα treatment (see Table 6).
  • Pairwise comparison between RA responders and RA non-responders prior to treatment yielded a number of genes suitable for the prediction of a future response to anti-TNFα therapy (82 genes for a threshold of ≧80%; 11 genes for ≧90%; 3 genes for 100%; summarized in Table 9), resulting in exact classification of future responders and non-responders upon hierarchical clustering. Using all genes differentially expressed in the above comparison at a threshold level of 70% (256 pre-treatment; 1295 post-treatment) for Ingenuity® pathway analysis, the differences between responders and non-responders either pre-treatment or post-treatment were concentrated in the functional gene ontology terms cellular movement, haematological system development, immune response, cell-to-cell signaling and interaction, as well as immunological disease.
  • In particular, numerous relevant mediators were identified: i) pro-inflammatory cytokines (e.g., interleukin-8 [IL8], chemokine (C—C motif) ligand 5 [CCL5], chemokine (C—X—C motif) ligand 5 [CXCL5], and chemokine (C—X—C motif) ligand 10 [CXCL10]); ii) pro-destructive enzymes (e.g., matrix metalloproteinase 9 [MMP9]); iii) adhesion molecules and Fcγ-receptors (galectin-8 [LGALS8], integrin alpha-X [ITGAX] or CD11c, Fc-gamma receptor IIb [FCGR2B] or CD32, CD86 [CD86] and platelet/endothelial cell adhesion molecule 1 [PECAM1] or CD31); iv) signal transduction molecules (e.g., protein kinase B [AKT1], apoptosis regulator Bel-2 [BCL2], p21-activated protein kinase 1 [PAK1]); and v) transcription factors (e.g., Mad-related protein 2 [SMAD2], interferon regulatory factor 1 [IRF1], c-myb [MYB], early growth response protein 1 [EGR1], signal transducer and activator of transcription 1 [STAT1], and nuclear factor NF-κB 1 [NFKB1]; for the remaining abbreviations see the respective gene cards [http://www.genecards.org/index.shtml]). These molecules were differentially expressed between RA responders and RA non-responders either pre-treatment or post-treatment and in some cases even inverted their expression upon anti-TNFα-therapy (see interleukin-8 receptor beta [IL8RB], Amyloid-beta A4 precursor [APP]; overexpressed in RA responders pre-treatment and underexpressed post-treatment). Similar opposite variations in transcript levels between RA responders and RA non-responders have recently been reported when comparing baseline to 3-month results (Lequerre, T. et al. (2006) Arthritis Res. Ther. 8:R105).
  • Most strikingly, the transcription of TNFα itself, as well the transcription of members of the subsequent NFKB-pathway (NFKB1 and inhibitor of NF-κB [IKBKB]), was upregulated in RA responders post-treatment. This previously unreported finding at first sight questions the central pro-inflammatory role of TNFα in RA. However, several caveats should be considered: i) The mRNA expression levels of TNFα measured in the present study may not be proportional to the levels of circulating TNFα protein and/or bioactivity, the latter apparently predictive of the clinical response to TNFα inhibition (Marotte, H. et al. (2005) Arthritis Res. Ther. 7:R149-155); ii) TNFα, in addition to its well-established pro-inflammatory properties, may also exhibit phase-dependent immunosuppressive properties (Kassiotis, G. et al. (2001) J. Exp. Med. 193:427-434).
  • Several interesting pathways with potential importance for the mechanisms underlying susceptibility to anti-TNFα-therapy were identified by KEGG pathway analysis; including Apotosis and the MAPK pathways.
  • TABLE 1
    Clinical parameters of patients before and after anti-TNF treatment
    Methods Duration
    Affymetrix (A) of Disease Morning Swollen joint Painful joint
    real-time treatment, duration, Age, stiffness, min count 68 count 68
    Patient (R/P) mo y y Gender RF (% reduction) (% reduction) (% reduction)
    RA1-1110006211 A/P 0 4 52 F + 320  10 19
    RA1-aTNF A 13.5 15 (95) 3 (70)  8 (58)
    RA2-1110005181 A/P 0 9 53 F + 180  16 50
    RA2-aTNF A 6  0 (100) 1 (94) 19 (62)
    RA3-1110005291 A/P 0 18 39 F +  0 10 28
    RA3-aTNF A 5.5 0 (0) 2 (80)  8 (29)
    RA4-1110005031 A/P 0 12 47 F + 720  20 43
    RA4-aTNF A 17 60 (92) 19 (5)  35 (19)
    RA5-1110004141 A/P 0 38 70 F + 120  11 56
    RA5-aTNF A 15 120 (0)  2 (82) 61 (0) 
    RA6-1110004131 A/P 0 38 51 F + 60 12 27
    RA6-aTNF A 4 60 (0)  4 (67) 28 (0) 
    RA7-1110006221 A/P 0 11 40 F + 150  18 44
    RA7-aTNF A 4.5 10 (93) 5 (72) 25 (43)
    RA8-06507 R 0 <1 28 F − 120  14 17
    RA8-aTNF/MTX R 6  0 (100)  0 (100)  3 (82)
    RA9-06513 R 0 1 53 F − 120  16 20
    RA9-aTNF/MTX R 4.5 15 (87) 12 (25)  16 (20)
    RA10-05602 R 0 1 48 F + 180   8 40
    RA10-MTX R 3.5 240 (0)  10 (0)  29 (27)
    RA11-05610 R/P 0 <1 38 F − 360  14 22
    RA11-aTNF R 6 30 (92) 3 (79)  9 (59)
    RA12-05609 R/P 0 1 63 F + 60 17 29
    RA12-aTNF R 6 10 (83) 5 (71)  9 (69)
    RA13-05608 R 0 2 44 F + 45 12 24
    RA13-aTNF/MTX R 6  0 (100)  0 (100)  1 (96)
    RA14-05612 R/P 0 1 29 F + 60  8 11
    RA14-aTNF/MTX R 6  0 (100)  0 (100)  0 (100)
    RA15-06504 R/P 0 2 47 F + 60 10 23
    RA15-aTNF R 6 20 (67) 2 (80)  3 (87)
    RA16-06308 R/P 0 <1 60 F + 120  17 29
    RA16-aTNF/MTX R 6 15 (87) 1 (94) 16 (45)
    RA17-06516 R 0 1 36 F + 30 17 17
    RA11-aTNF R/P 6 15 (50) 11 (35)   0 (100)
    RA18-06309 R 0 1 83 F − 30 11 27
    RA18-aTNF/MTX R 6  0 (100)  0 (100)  0 (100)
    RA19-05613 R/P 0 2 63 F + 120  25 43
    RA19-aTNF R 6 15 (87) 9 (64) 17 (60)
    RA20-06304 R/P 0 <1 50 F − 60 12 29
    RA20-aTNF R 6  0 (100) 2 (83)  5 (83)
    RA21-06514 R/P 0 2 56 F − 30 21 52
    RA21-aTNF/MTX R 6 30 (0)  15 (29)  16 (69)
    RA22-05606 R/P 0 <1 36 F + 60 12 22
    RA22-aTNF/MTX R 6  0 (100) 2 (83)  9 (83)
    RA23-06306 R/P 0 1 34 F + 30 13 27
    RA23-aTNF R 3 30 (0)  18 (0)  27 (0) 
    RA24-06305 R 0 n.d. 18 F + 30 14 20
    RA24-MTX R 6 60 (0)  2 (86)  8 (60)
    RA25-06515 R/P 0 <1 54 M − 45 22 43
    RA25-aTNF R 6 20 (66) 7 (68) 28 (35)
    RA26-06506 R/P 0 1 50 F − 60  9 23
    RA26-aTNF R 6 120 (0)  20 (0)  45 (0) 
    RA27-05601 R/P 0 1 42 F + 60  8 24
    RA27-aTNF R 6.5 60 (0)  7 (12) 30 (0) 
    RA28-1110004171 P 0 n.d. 34 F + 60 20 47
    RA28-aTNF 3.5  0 (100) 4 (80)  4 (91)
    RA29-1110011271 P 0 11 62 F + 120   8 35
    RA29-aTNF/MTX 0.5 120 (0)  9 (0)  44 (0) 
    RA30-1110010101 P 0 7 35 F + 120   8 12
    RA30-aTNF/MTX 10  0 (100) 2 (75)  7 (42)
    RA31-1110004121 P 0 13 33 F + 30 11 27
    RA31-aTNF 6 20 (67) 2 (82)  7 (74)
    RA32-1110008092 P 0 20 43 F + 60 14 38
    RA32-aTNF 6.5  0 (100) 6 (57)  7 (72)
    RA33-1110005121 P 0 8 61 F − 180  19 59
    RA33-aTNF 17.5 120 (66)  2 (89) 12 (81)
    RA34-1110111281 P 0 12 66 F + 90 10  5
    RA34-MTX 12 120 (0)  8 (20) 9 (0)
    RA35-1110112121 P 0 6 63 F + 180   8 20
    RA35-MTX 9  0 3 (63)  4 (80)
    RA36-1110201231 P 0 10 36 F 0 60 30 19
    RA36-MTX 4 30 (50)  0 (100)  0 (100)
    RA37-1110211051 P 0 1 59 F + 180   2 21
    RA37-MTX 3 15 (92)  0 (100)  4 (81)
    RA38-1110309012 P 0 <1 32 F + 180   7 29
    RA38-MTX 3  0 (100) 1 (85)  3 (90)
    RA39-1110309122 P 0 1 26 F + 180   6 23
    RA39-MTX 3 15 (92) 1 (83)  9 (41)
    RA40-1110310302 P 0 1 45 F + 10 10 17
    RA40-MTX 4  0 (100)  0 (100)  2 (88)
    RA41-1110411251 P 0 <1 58 F 0 180  6 16
    RA41-MTX 3  0 (100)  0 (100)  2 (88)
    RA42-1110409071 P 0 1 52 F 0  0  9  4
    RA42-MTX 3 0 (0)  0 (100)  0 (100)
    RA43-1110409141 P 0 <1 58 M + 10  8 22
    RA43-MTX 3 10 (0)   0 (100)  5 (77)
    RA44-1110409223 P 0 1 67 F 0 60 12 20
    RA44-MTX 3  0 (100)  0 (100)  4 (80)
    RA45-1110411291 P 0 <1 44 F 0 30 25 43
    RA45-MTX 3  0 (100) 9 (64) 17 (60)
    RA46-1110602212 P 0 10 45 F + 120   3 13
    RA46-aTNF/MTX 6 120 (0)   0 (100) 21 (0) 
    RA47-1110503081 P 0 21 57 F + 360   8 10
    RA47-TNF 6 180 (50)  7 (14) 11 (0) 
    RA48-1110703082 P 0 7 60 F + 240   0 10
    RA48-TNF 6 120 (50)  0 (0)   5 (50)
    RA49-1110309122 P 0 1 26 F + 180   6 24
    RA49-MTX 9 15 (92) 1 (83)  9 (62)
    RA50-1100308202 P 0 6 63 F +  0  0 18
    RA50-MTX 3 0 (0) 1 (0)   2 (89)
    RA51-1110309111 P 0 2 45 M + 180  16 30
    RA51-MTX 3 120 (33)  9 (44) 24 (20)
    RA52-1110401081 P 0 1 62 F +  0  0 18
    RA52-MTX 5.5 0 (0) 0 (0)   0 (100)
    RA53-1110406011 P 0 1 61 F + 30 10 33
    RA53-MTX 3  5 (83)  0 (100)  1 (97)
    RA54-1110406112 P 0 3 61 M + 10  6 22
    RA54-MTX 3  0 (100)  0 (100)  5 (77)
    RA55-1110406111 P 0 <1 57 F + 60  2 12
    RA55-MTX 3  0 (100)  0 (100)  2 (83)
    RA56-1110407131 P 0 1 63 F + 30  9 30
    RA56-MTX 3  0 (100)  0 (100)  6 (80)
    RA57-1110407281 P 0 2 56 F +  0  7 19
    RA57-MTX 3 0 (0)  0 (100)  2 (89)
    RA58-1110409201 P 0 1 61 F + 10  5 16
    RA58-MTX 3  0 (100)  0 (100)  4 (75)
    RA59-1110410271 P 0 6 35 M + 15  0  5
    RA59-MTX 3  0 (100) 0 (0)   2 (60)
    RA60-1110503231 P 0 1 71 F + 30  6 23
    RA60-MTX 3  0 (100)  0 (100)  5 (78)
    RA61-1110504061 P 0 1 51 F + 45 12 27
    RA61-MTX 3  0 (100)  0 (100)  0 (100)
    RA62-1110505301 P 0 1 59 F 0 120   2  9
    RA62-MTX 3  0 (100)  0 (100) 12 (0) 
    RA63-1110507281 P 0 2 43 F + 30 10 17
    RA63-MTX 3  0 (100) 6 (40) 15 (12)
    RA64-1110511281 P 0 <1 69 F + 90  7 21
    RA64-MTX 3  0 (100) 1 (86)  2 (90)
    RA65-1110405273 P 0 5 55 F +  0  0  2
    RA65-aTNF/MTX 4 10 (0)  6 (0)  16 (0) 
    RA66-1110502091 P 0 36 62 F + 30 13 14
    RA66-aTNF/MTX 30 15 (50) 2 (85) 14 (0) 
    RA67-1110507261 P 0 25 43 F +  0  4  5
    RA67-aTNF/MTX 25 0 (0)  0 (100)  1 (80)
    RA68-1110603141 P 0 12 72 F + 30 7  7
    RA68-aTNF 12 60 (0)   0 (100)  0 (100)
    RA69-1110510181 P 0 2 33 F + 50 6  6
    RA69-aTNF/MTX 12  0 (100)  0 (100)  0 (100)
    RA70-06303 P 0 1 53 F + 30 14 17
    RA70-aTNF/MTX 6 10 (66)  0 (100)  0 (100)
    RA71-06516 P 0 3 79 F + 30 17 17
    RA71-TNF 6 15 (50)  0 (100) 11 (35)
    RA72-06510 P 0 3 68 F + 10 25 34
    RA72-TNF 6 10 (0)  2 (92)  0 (100)
    RA73-06309 P 0 1 36 F 0 45 11 27
    RA73-TNF/MTX 6  0 (100)  0 (100)  0 (100)
    RA74-06310 P 0 3 48 F 0 360  14 22
    RA74-TNF 12 15 6 (57) 14 (36)
    RA75-06307 P 0 2 51 F + 360  16 22
    RA75-MTX 6  0 (100) 2 (87)  3 (86)
    RA76-06311 P 0 3 43 F + 120  14 36
    RA76-TNF 3  0 (100)  0 (100)  0 (100)
    RA77-06513 P 0 1 53 F 0 120  16 20
    RA77-TNF/MTX 4.5 15 (87) 12 (25)  16 (20)
    RA78-06512 P 0 1 83 F 0 30 15 53
    RA78-MTX 5.5 15 (50) 27 (0)  63 (0) 
    RA79-06514 P 0 2 56 F 0 30 21 52
    RA79-TNF/MTX 6 30 (0)  15 (29)  16 (69)
    RA80-1110505241 P 0 4 47 F + 24  5  9
    RA80-TNF/MTX 3.5 36 (0)  4 (20)  4 (56)
    RA81-06305 P 0 1 18 F + 30  8 20
    RA81-MTX 6 60 (0)  2 (75) 14 (70)
    RA82-05602 P 0 1 48 F + 180  29 40
    RA82-MTX 3.5 240 (0)  10 (66)   8 (20)
    RA83-05607 P 0 3 61 M + 120  25 40
    RA83-MTX 8  0 (100) 3 (88)  3 (92)
    RA84-06302 P 0 3 42 F + 60 16 39
    RA84-MTX 6 10 (83) 6 (62) 18 (51)
    ACR
    ESR, CRP, improvement
    mm/hr mg/L HAQ score DAS28 (% continuous
    Patient (% reduction) (% reduction) (% reduction) (% reduction) score)
    RA1-1110006211 69 57.5 2.0 6.0
    RA1-aTNF  8 (88) 2.0 (97) 1.3 (35) 3.1 (48) 50
    RA2-1110005181 46 52.2 1.6 6.3
    RA2-aTNF 12 (74) 2.8 (94) 1.0 (37) 2.9 (54) 60
    RA3-1110005291 72 61.1 1.5 6.1
    RA3-aTNF 16 (78) 3.9 (93) 1.0 (33) 4.1 (33) 60
    RA4-1110005031 58 85.3 2.1 6.9
    RA4-aTNF 40 (31) 20.0 (77)  1.3 (38) 6.5 (6)  0
    RA5-1110004141 80 41.5 2.1 6.3
    RA5-aTNF 11 (86) 4.5 (88) 1.4 (33) 4.0 (37) 50
    RA6-1110004131 46 36.0 1.6 5.9
    RA6-aTNF 32 (30) 22.3 (39)  1.6 (0)  5.1 (14) 10
    RA7-1110006221 30 17.3 1.9 6.0
    RA1-aTNF 14 (53) 11.3 (35)  1.3 (32) 4.1 (32) 40
    RA8-06507 30 <3.5 1.6 6.1
    RA8-aTNF/MTX  4 (87) <3.5 (0)     0.0 (100) 2.4 (61) 70
    RA9-06513 34 <3.5 1.3 6.2
    RA9-aTNF/MTX 14 (59) <3.5 (0)    1.4 (0)  5.2 (26) 20
    RA10-05602 31 <3.5 2.2 7.1
    RA10-MTX 39 (0)  <3.5 (0)    2.3 (0)  6.8 (4)  0
    RA11-05610 40 39.2 1.4 6.5
    RA11-aTNF 45 (0)  36.4 (7)  0.3 (79) 4.3 (34) 50
    RA12-05609 22 14.3 0.5 6.6
    RA12-aTNF 30 (0)  <3.5 (100) 0.3 (40) 4.3 (35) 50
    RA13-05608 32  5.1 0.8 6.1
    RA13-aTNF/MTX 28 (12) <3.5 (100)  0.0 (100) 2.7 (56) 90
    RA14-05612 35 13.1 1.0 5.0
    RA14-aTNF/MTX  8 (77) <3.5 (100)  0.0 (100) 1.7 (66) 100
    RA15-06504 28 <3.5 1.8 6.3
    RA15-aTNF 24 (14) 4.1 (0)  1.3 (28) 3.5 (44) 50
    RA16-06308 56 <3.5 1.8 7.3
    RA16-aTNF/MTX 28 (50) <3.5 (0)    1.3 (28) 4.8 (44) 40
    RA17-06516 80 22.8 1.8 6.5
    RA17-aTNF 24 (30) 27.5 (0)  0.6 (67) 3.2 (51) 30
    RA18-06309 24 39.4 1.3 6.5
    RA18-aTNF/MTX  8 (67) 8.5 (78) 0.6 (54) 1.9 (71) 70
    RA19-05613 53 31.5 2.3 8.5
    RA19-aTNF 85 (0)  40.0 (0)  1.3 (43) 5.6 (34) 60
    RA20-06304 24 10.0 1.0 6.6
    RA20-aTNF 16 (33) 10.0 (0)  1.0 (0)  3.8 (42) 30
    RA21-06514 34 <3.5 1.4 8.3
    RA21-aTNF/MTX  6 (82) <3.5 (0)    0.9 (36) 5.4 (35) 20
    RA22-05606 40 23.1 2.4 6.2
    RA22-aTNF/MTX 30 (25) 7.3 (68) 0.8 (67) 4.2 (32) 50
    RA23-06306 90 61.1 2.1 7.1
    RA23-aTNF 62 (33) 53.4 (23)  2.0 (5)  7.2 (0)  0
    RA24-06305 22 29.1 0.3 4.7
    RA24-MTX 20 (10) 31.3 (0)  0.1 (33) 4.1 (13) 20
    RA25-06515 34 <3.5 1.0 8.0
    RA25-aTNF  9 (74) <3.5 (0)    1.6 (0)  5.8 (27) 0
    RA26-06506 16 <3.5 1.5 5.9
    RA26-aTNF 50 (0)  <3.5 (0)    1.9 (0)  8.3 (0)  0
    RA27-05601 30  6.8 1.1 5.8
    RA27-aTNF 58 (0)  16.9 (0)  1.5 (0)  6.7 (0)  0
    RA28-1110004171 30  7.0 1.8 6.2
    RA28-aTNF 16 (47) 6.9 (1)  0.4 (78) 3.6 (42) 80
    RA29-1110011271 19 16.5 n.d. 4.3
    RA29-aTNF/MTX  9 (53) 3.0 (72) n.d. 4.1 (13) 0
    RA30-1110010101 52 42.6 n.d. 5.3
    RA30-aTNF/MTX 17 (63) 10.1 (76)  n.d. 2.6 (51) 50
    RA31-1110004121 33  7.8 1.3 5.2
    RA31-aTNF 18 (45) 5.9 (24) 0.9 (31) 3.4 (35) 40
    RA32-1110008092 29 18.4 n.d. 6.8
    RA32-aTNF 28 (3)  21.0 (0)  n.d. 3.4 (50) 50
    RA33-1110005121 28 22.4 1.8 6.3
    RA33-aTNF 26 (7)  14.0 (36)  1.9 (0)  4.1 (35) 20
    RA34-1110111281 18  9.0  1.43  4.29
    RA34-MTX 13 (28) 12.0 (0)  0.74 (49)  5.10 (0)  0
    RA35-1110112121 17 66.0  2.67 6.3
    RA35-MTX 12 (29) <3.5 (100) 0.67 (76)  3.5 (45) 60
    RA36-1110201231 38 45.0  2.33 6.3
    RA36-MTX 17 (55) 5.0 (89) 0.33 (86)  2.1 (67) 80
    RA37-1110211051 27 11.0  2.44 5.5
    RA37-MTX  8 (70) 5.0 (55) 1.44 (41)  2.9 (47) 40
    RA38-1110309012 20  8.1  1.11 5.4
    RA38-MTX 10 (50) 1.2 (85) 0.44 (60)  2.5 (54) 70
    RA39-1110309122 101  33.0  2.67 6.5
    RA39-MTX 81 (20) 44.5 (0)  2.10 (21)  5.6 (14) 20
    RA40-1110310302 72 77.6 2.1 6.8
    RA40-MTX 18 (75) 6.9 (91) 1.1 (49) 3.4 (50) 50
    RA41-1110411251 17 <3.5 2.4 5.4
    RA41-MTX 12 (29) <3.5 (0)      0 (100) 3.1 (43) 80
    RA42-1110409071 37 22.0 2.1 5.7
    RA42-MTX  5 (87) 6.6 (70) 0.4 (81) 1.1 (81) 80
    RA43-1110409141 27 32.0 2.3 5.9
    RA43-MTX  3 (89) 4.6 (86) 2.0 (13) 1.7 (71) 70
    RA44-1110409223 55 62.6  1.33  3.13
    RA44-MTX 12 (78) 3.80 (94)  1.40 (0)  4.93 (0)  70
    RA45-1110411291 34 18.0 1.4 4.9
    RA45-MTX 20 (41) 12.6 (30)    0 (100) 2.2 (65) 60
    RA46-1110602212 74 17.8 1.7 5.8
    RA46-aTNF/MTX 20 (63) 2.7 (85) 1.1 (35) 4.4 (24) 30
    RA47-1110503081 74 <3.5 2.0 6.5
    RA47-TNF 20 (0)  <3.5 (0)    2.8 (0)  6.7 (0)  0
    RA48-1110703082 28 106.0  1.1 6.2
    RA48-TNF 120 (12)  83.6 (21)  1.0 (9)  4.1 (34) 0
    RA49-1110309122 40 33.0 0.9 6.6
    RA49-MTX 40 (0)  34.5 (0)  0.7 (22) 5.7 (14) 10
    RA50-1100308202 28  9.8 1.4 4.6
    RA50-MTX 21 (25) 9.9 (0)  1.7 (0)  4.0 (13) 0
    RA51-1110309111 10  4.0 2.2 6.1
    RA51-MTX  5 (50) 1.3 (77) 2.1 (5)  5.1 (16) 0
    RA52-1110401081 30  6.5 1.1 4.4
    RA52-MTX 25 (17) 4.8 (26) 0.1 (91) 2.3 (48) 90
    RA53-1110406011 10 23.0 1.78 6.4
    RA53-MTX  0 (100) 3.1 (87) 0.3 (83) 2.4 (62) 80
    RA54-1110406112 10  9.0 0.7 5.2
    RA54-MTX 20 (0)  16.7 (0)  1.9 (0)  3.3 (37) 40
    RA55-1110406111 25 24.8 1.9 5.4
    RA55-MTX 12 (52) 6.7 (73) 0.3 (84) 2.4 (56) 80
    RA56-1110407131 55 37.8 1.67 8.8
    RA56-MTX 14 (75) 1.0 (97) 1.10 (34)  3.7 (58) 30
    RA57-1110407281 21  3.9 1.6 5.5
    RA57-MTX 10 (52) <3.5 (100) 0.2 (87) 2.5 (55) 70
    RA58-1110409201 20 <3.5 2.2 4.3
    RA58-MTX 14 (30) <3.5 (0)    0.4 (82) 2.0 (53) 70
    RA59-1110410271 12 <3.5 0.8 2.6
    RA59-MTX 25 (0)  7.9 (0)  0.8 (0)  2.7 (0)  30
    RA60-1110503231 14  5.6 2 5.9
    RA60-MTX  8 (43) 4.9 (12) 0.9 (55) 3.1 (47) 40
    RA61-1110504061 16 <3.5 2.5 7.0
    RA61-MTX  4 (75) <3.5 (0)    0.4 (84) 1.0 (86) 70
    RA62-1110505301 10 <3.5 2.1 4.7
    RA62-MTX  6 (40) <3.5 (0)    1.1 (48) 3.2 (26) 40
    RA63-1110507281 68 42.0 1.8 6.0
    RA63-MTX 20 (71) 33.0 (21)  1.1 (39) 4.0 (33) 30
    RA64-1110511281 25 14.5 1.2 5.8
    RA64-MTX 10 (60) 27.0 (0)  0.9 (25) 3.0 (48) 20
    RA65-1110405273  6 <3.5 1.2 3.0
    RA65-aTNF/MTX 12 (0)  <3.5 (0)    0.8 (33) 4.7 (0)  0
    RA66-1110502091 10 <3.5 1.9 4.2
    RA66-aTNF/MTX 12 (0)  4.2 (0)    0 (100) 4.1 (2)  40
    RA67-1110507261 32 11.4 1.7 4.4
    RA67-aTNF/MTX 16 (50) 7.7 (32) 1.1 (35) 2.8 (36) 30
    RA68-1110603141 72  4.8 1.3 4.7
    RA68-aTNF  4 (94) <3.5 (100)  0.0 (100) 2.4 (49) 30
    RA69-1110510181 12  9.0 3.0 4.7
    RA69-aTNF/MTX 26 (0)  <3.5 (100) 1.1 (63) 2.4 (49) 80
    RA70-06303 26 31.9 1.3 5.8
    RA70-aTNF/MTX  6 (77)   0 (100) 0.1 (92) 1.6 (73) 70
    RA71-06516 80 22.8 1.8 6.5
    RA71-TNF 24 (70) 7.5 (67) 0.6 (67) 3.2 (49) 30
    RA72-06510 80 41.6 2.1 8.2
    RA72-TNF 58 (27) 21.4 (49)  1.5 (29) 2.6 (68) 70
    RA73-06309 24 39.4 1.3 6.5
    RA73-TNF/MTX  8 (75) 8.5 (78) 0.6 (54) 1.9 (71) 70
    RA74-06310 30 21.9 3.0 8.1
    RA74-TNF 13 (57) 14.3 (35)  2.3 (23) 2.9 (64) 50
    RA75-06307 64 83.4 2.6 7.0
    RA75-MTX 22 (66) 11.5 (86)  0.8 (69) 2.2 (69) 70
    RA76-06311 32 53.1 0.8 7.0
    RA76-TNF  8 (75) 4.9 (91) 0.1 (87) 1.7 (75) 80
    RA77-06513 34 <3.5 1.3 6.2
    RA77-TNF/MTX 14 (59) <3.5 (0)    1.4 (0)  5.3 (15) 20
    RA78-06512 33  7.4 2.4 6.7
    RA78-MTX  8 (76) 4.9 (34) 3.0 (0)  8.3 (0)  0
    RA79-06514 34 <3.5 1.3 8.3
    RA79-TNF/MTX  6 (82) <3.5 (0)    0.9 (31) 5.4 (35) 20
    RA80-1110505241 24 10.2 1.3 4.4
    RA80-TNF/MTX 36 (0)  <3.5 (100) 1.1 (15) 4.1 (7)  20
    RA81-06305 22 29.1 0.3 6.0
    RA81-MTX 20 (9)  31.3 (0)  0.1 (33) 4.1 (32) 20
    RA82-05602 31 <3.5 2.1 4.0
    RA82-MTX 39 (0)  <3.5 (0)    2.3 (0)  6.5 (0)  0
    RA83-05607 29  4.6 2.5 7.8
    RA83-MTX 12 (59)   0 (100) 1.3 (52) 3.1 (60) 60
    RA84-06302 30  9.6 2.5 7.4
    RA84-MTX 24 ( )  <3.5 (100) 0.8 (68) 5.3 (28) 60
    A = Affymetrix; R = real-time RT-PCR (validation of gene expression); P = real-time RT-PCR (validation of predictive gene); RF = rheumatoid factor;; ESR = erythrocyte sedimentation rate; CRP = C-reactive protein concentration, HAQ = Health Assessment Questionnaire; DAS28 = disease activity score (28 joints);
    + = positive; − = negative; n.d. = not determined; aTNF = anti-TNF monoclonal antibody therapy; MTX = methotrexate therapy
  • TABLE 2
    Correlations among clinical parameters (Pearson correlation test;
    pre- and post-anti-TNFα treatment)
    r-value p-value
    Morning stiffness/Swollen joint count 28 0.718 0.004
    Morning stiffness/Swollen joint count 68 0.664 0.010
    Swollen joint count 28/Swollen joint count 68 0.990 0.000
    Painful joint count 28/Painful joint count 68 0.794 0.001
    ESR/CRP 0.854 0.000
    ESR/VAS (physician) 0.764 0.001
    ESR/DAS 28 0.813 0.000
    CRP/Morning stiffness 0.733 0.003
    CRP/Swollen joint count 28 0.762 0.002
    CRP/Swollen joint count 68 0.728 0.003
    CRP/DAS 28 0.786 0.001
    DAS 28/Swollen joint count 28 0.891 0.000
    DAS 28/Swollen joint count 68 0.878 0.000
    DAS 28/Painful joint count 28 0.660 0.010
    VAS (physician)/Swollen joint count 68 0.672 0.008
    VAS (physician)/Painful joint count 28 0.697 0.006
    VAS (physician)/DAS 28 0.790 0.001
    Different clinical parameters showed correlations in RA patients pre- and post anti-TNFα treatment (n = 14 in all cases).
    ESR = erythrocyte sedimentation rate;
    CRP = C-reactive protein;
    DAS 28 = disease activity score (28 joints); and
    VAS (physician) = visual analogue score (physican's global assessment)
  • TABLE 3
    Genes differentially regulated in peripheral blood monocytes of both RA
    patients versus normal donors and RA patients pre-versus post-anti-TNFα
    Increased Decreased Fold
    Affymetrix (%) (%) change SLR
    ID Gene name RA vs. ND
    36711_at v-maf musculoaponeurotic fibrosarcoma 71.43 14.29 3.44 1.78
    oncogene homolog F
    38037_at Diphtheria toxin receptor (DTR) 71.43 10.20 2.87 1.52
    201386_s_at DEAH (Asp-Glu-Ala-His) box polypeptide 15 2.04 73.47 −1.66 −0.73
    201890_at Ribonucleotide reductase M2 polypeptide 81.63 8.16 4.39 2.13
    202219_at Solute carrier family 6, member 8 71.43 8.16 8.44 3.08
    202464_s_at 6-phosphofructo-2-kinase/fructose-2,6- 81.63 — 2.74 1.46
    biphosphatase 3
    203115_at Ferrochelatase (protoporphyria) 83.67 8.16 3.87 1.95
    203574_at Nuclear factor, interleukin 3 regulated 77.55 — 2.16 1.11
    203887_s_at Thrombomodulin 71.43 16.33 2.49 1.32
    203932_at Major histocompatibility complex, class II, 2.04 75.51 −1.82 −0.87
    DM beta
    204131_s_at Forkhead box O3A 77.55 4.08 2.21 1.14
    204419_x_at Hemoglobin, gamma A, gamma G 75.51 6.12 2.88 1.52
    204467_s_at Synuclein, alpha (non A4 component of 71.43 4.08 3.60 1.85
    amyloid precursor)
    204848_x_at Hemoglobin, gamma A 75.51 6.12 3.83 1.94
    205239_at Amphiregulin (schwannoma-derived growth 81.63 4.08 6.13 2.62
    factor)
    205571_at Lipoyltransferase 1 — 79.59 −1.90 −0.93
    205592_at Solute carrier family 4, anion exchanger, 77.55 10.20 4.83 2.27
    member 1
    205863_at S100 calcium binding protein A12 71.43 4.08 1.69 0.76
    (calgranulin C)
    205900_at Keratin 1 (epidermolytic hyperkeratosis) 75.51 6.12 4.03 2.01
    205950_s_at Carbonic anhydrase I 81.63 4.08 5.88 2.56
    205987_at CD1C antigen, c polypeptide — 87.76 −2.93 −1.55
    206025_s_at Tumor necrosis factor, alpha-induced protein 6 71.43 — 3.85 1.94
    206111_at Ribonuclease, RNase A family, 2 73.47 — 1.95 0.97
    206834_at Hemoglobin, delta 83.67 10.20 4.08 2.03
    207332_s_at Transferrin receptor (p90, CD71) 81.63 2.04 2.35 1.23
    208632_at Ring finger protein 10 71.43 6.12 2.06 1.04
    209007_s_at Chromosome 1 open reading frame 63 4.08 73.47 −2.14 −1.10
    209458_x_at Hemoglobin, alpha 1, alpha 2 85.71 2.04 3.00 1.59
    209795_at CD69 antigen (p60, early T-cell activation 71.43 12.24 2.95 1.56
    antigen
    210027_s_at APEX nuclease (multifunctional DNA repair — 75.51 −1.93 −0.94
    enzyme) 1
    210254_at Membrane-spanning 4-domains, subfamily A, 73.47 16.33 3.41 1.77
    member 3
    210338_s_at Heat shock 70 kDa protein 8 6.12 71.43 −2.09 −1.06
    211038_s_at Hypothetical protein MGC12760 — 71.43 −2.07 −1.05
    211458_s_at GABA(A) receptor-associated protein like 1, 71.43 — 1.95 0.96
    like 3
    211560_s_at Aminolevulinate, delta-, synthase 2 87.76 4.08 24.35 4.61
    211991_s_at Major histocompatibility complex, class II, DP 4.08 79.59 −3.01 −1.59
    alpha 1
    212199_at Morf4 family associated protein 1-like 1 2.04 75.51 −1.94 −0.96
    212224_at Aldehyde dehydrogenase 1 family, member A1 4.08 75.51 −2.56 −1.36
    212232_at Formin binding protein 4 — 77.55 −1.83 −0.87
    212534_at Zinc finger protein 24 (KOX 17) 6.12 71.43 −1.49 −0.58
    213142_x_at Hypothetical protein LOC54103 4.08 73.47 −1.77 −0.82
    214433_s_at Selenium binding protein 1 71.43 10.20 4.59 2.20
    215933_s_at Hematopoietically expressed homeobox — 75.51 −1.83 −0.87
    217478_s_at Major histocompatibility complex, class II, 4.08 73.47 −2.02 −1.01
    DM alpha
    217736_s_at Eukaryotic translation initiation factor 2-alpha 75.51 — 1.97 0.98
    kinase 1
    219069_at Ankyrin repeat domain 49 — 73.47 −1.59 −0.67
    219093_at Hypothetical protein FLJ20701 — 85.71 −3.32 −1.73
    219228_at Zinc finger protein 331 77.55 8.16 3.94 1.98
    219607_s_at Membrane-spanning 4-domains, subfamily A, 91.84 — 2.96 1.57
    member 4
    221748_s_at Tensin 1 81.63 4.08 4.29 2.10
    221766_s_at Family with sequence similarity 46, member A — 71.43 −1.71 −0.77
    Increased Decreased Fold Increased Decreased Fold
    Affymetrix (%) (%) change SLR (%) (%) change SLR
    ID RA-aTNF vs. RA (all) RA-aTNF vs. RA (responder)
    36711_at 28.57 61.22 −2.78 −1.48 12.00 80.00 −5.88 −2.56
    38037_at 12.24 73.47 −2.59 −1.37 — 84.00 −3.70 −1.89
    201386_s_at 44.90 14.29 1.43 0.51 60.00 — 1.81 0.85
    201890_at 2.04 69.39 −3.86 −1.95 4.00 64.00 −3.88 −1.96
    202219_at 12.24 59.18 −5.17 −2.37 4.00 72.00 −6.96 −2.80
    202464_s_at 12.24 69.39 −2.18 −1.12 — 84.00 −3.01 −1.59
    203115_at 20.41 63.27 −2.18 −1.12 16.00 72.00 −2.25 −1.17
    203574_at 10.20 53.06 −1.54 −0.62 — 76.00 −2.06 −1.04
    203887_s_at 12.24 65.31 −2.34 −1.23 — 76.00 −3.14 −1.65
    203932_at 63.27 4.08 1.64 0.71 84.00 — 1.68 0.75
    204131_s_at 18.37 55.10 −1.42 −0.50 4.00 64.00 −1.41 −0.50
    204419_x_at 14.29 75.51 −3.18 −1.67 8.00 92.00 −4.16 −2.04
    204467_s_at 24.49 65.31 −2.26 −1.18 16.00 80.00 −2.93 −1.55
    204848_x_at 16.33 65.31 −3.22 −1.69 4.00 80.00 −4.63 −2.21
    205239_at 20.41 71.43 −3.36 −1.75 — 96.00 −5.91 −2.56
    205571_at 51.02 12.24 1.35 0.44 72.00 — 1.61 0.69
    205592_at 16.33 65.31 −3.81 −1.93 8.00 76.00 −5.63 −2.49
    205863_at 14.29 55.10 −1.49 −0.58 8.00 56.00 −1.56 −0.64
    205900_at 20.41 67.35 −2.31 −1.21 16.00 84.00 −3.18 −1.67
    205950_s_at 22.45 67.35 −2.87 −1.52 16.00 84.00 −4.14 −2.05
    205987_at 63.27 2.04 1.72 0.79 68.00 — 1.72 0.78
    206025_s_at 12.24 59.18 −1.97 −0.98 4.00 72.00 −2.74 −1.46
    206111_at 8.16 67.35 −1.59 −0.67 — 72.00 −1.68 −0.75
    206834_at 20.41 65.31 −3.13 −1.64 16.00 80.00 −4.59 −2.20
    207332_s_at 2.04 79.59 −2.45 −1.29 — 84.00 −2.57 −1.36
    208632_at 12.24 59.18 −1.71 −0.78 4.00 68.00 −1.69 −0.76
    209007_s_at 57.14 14.29 1.54 0.62 76.00 4.00 1.87 0.90
    209458_x_at 8.16 63.27 −2.09 −1.06 4.00 76.00 −2.60 −1.38
    209795_at 14.29 75.51 −3.18 −1.67 16.00 76.00 −3.77 −1.92
    210027_s_at 67.35 10.20 1.64 0.72 84.00 — 2.02 1.02
    210254_at 14.29 79.59 −4.56 −2.19 20.00 76.00 −3.69 −1.88
    210338_s_at 55.10 18.37 1.57 0.66 68.00 4.00 2.04 1.03
    211038_s_at 42.86 16.33 1.29 0.37 56.00 4.00 1.46 0.55
    211458_s_at 16.33 65.31 −1.92 −0.94 — 84.00 −2.75 −1.46
    211560_s_at 16.33 75.51 −10.81 −3.43 8.00 80.00 −20.53 −4.36
    211991_s_at 75.51 8.16 2.37 1.24 80.00 4.00 2.54 1.34
    212199_at 59.18 10.20 1.56 0.64 80.00 — 2.11 1.08
    212224_at 71.43 12.24 2.28 1.19 92.00 — 2.74 1.45
    212232_at 48.98 14.29 1.27 0.35 72.00 — 1.57 0.65
    212534_at 53.06 4.08 1.35 0.43 76.00 — 1.45 0.53
    213142_x_at 51.02 12.24 1.25 0.32 72.00 — 1.51 0.60
    214433_s_at 18.37 65.31 −3.08 −1.62 12.00 68.00 −3.55 −1.83
    215933_s_at 55.10 6.12 1.51 0.59 76.00 0.00 1.85 0.88
    217478_s_at 61.22 12.24 1.68 0.75 64.00 8.00 1.85 0.88
    217736_s_at 14.29 51.02 −1.46 −0.54 8.00 60.00 −1.41 −0.50
    219069_at 57.14 12.24 1.30 0.38 84.00 — 1.58 0.66
    219093_at 71.43 8.16 2.43 1.28 84.00 — 2.59 1.37
    219228_at 10.20 71.43 −3.26 −1.70 12.00 80.00 −4.62 −2.21
    219607_s_at 6.12 73.47 −1.95 −0.96 — 80.00 −2.06 −1.04
    221748_s_at 24.49 65.31 −2.12 −1.08 16.00 80.00 −2.42 −1.28
    221766_s_at 53.06 10.20 1.30 0.38 80.00 — 1.66 0.73
    The table presents the percentage of pairwise comparisons between the respective groups showing an increase or decrease for the fold-change/Signal Log Ratio (SLR; 51 candidate genes were selected); n = 7 each for the comparisons RA vs. ND and RA-αTNF vs. RA (all); n = 5 for the comparison RA-αTNF vs. RA (responder).
    The pairwise comparison RA-αTNF vs. RA (all) is shown in the central 4 columns to assess the effect of treatment on differential gene expression in all TNFα-treated RA patients.
    RA = rheumatoid arthritis (pre-anti-TNFα treatment); ND = normal donor; RA-αTNF = rheumatoid arthritis (post-anti-TNFα treatment)
  • TABLE 4
    Genes differentially regulated in peripheral blood monocytes of both RA patients versus normal donors and RA
    patients pre-versus post-anti-TNFα therapy (PAM analysis)
    Affymetrix ID Gene title RA score
    221622_s_at Uncharacterized hypothalamus protein HT007 0.7405
    218845_at Dual specificity phosphatase 22 0.6694
    200786_at Proteasome (prosome, macropain) subunit, beta type, 7 0.5297
    219607_s_at Membrane-spanning 4-domains, subfamily A, member 4 0.4449
    212886_at DKFZP434C171 protein 0.2690
    201407_s_at Protein phosphatase 1, catalytic subunit, beta isoform 0.2653
    212266_s_at Splicing factor, arginine/serine-rich 5 0.1771
    53912_at Sorting nexin 11 0.1277
    200090_at Farnesyltransferase, CAAX box, alpha 0.1167
    218025_s_at Peroxisomal D3,D2-enoyl-CoA isomerase 0.1032
    204232_at Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide 0.0993
    201722_s_at UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 0.0942
    218627_at Hypothetical protein FLJ11259 0.0841
    210027_s_at APEX   nuclease   (multifunctional   DNA   repair   enzyme)   1 0.0817
    202322_s_at Geranylgeranyl diphosphate synthase 1 0.0646
    221689_s_at Down syndrome critical region gene 5 0.0549
    203356_at Calpain 7 0.0535
    211991_s_at Major histocompatibility complex, class II, DP alpha 1 0.0515
    218462_at Brix domain containing 5 0.0378
    218123_at Chromosome 21 open reading frame 59 0.0173
    214329_x_at Tumor necrosis factor (ligand) superfamily, member 10 −0.0025
    219067_s_at Chromosome 10 open reading frame 86 −0.0046
    205789_at CD1D antigen, d polypeptide −0.0104
    209214_s_at Ewing sarcoma breakpoint region 1 −0.0111
    213427_at Ribonuclease P 40 kDa subunit −0.0471
    209422_at/206567_s_at PHD finger protein 20 −0.0480/−0.0698
    201010_s_at Thioredoxin interacting protein −0.0520
    200883_at Ubiquinol-cytochrome c reductase core protein II −0.0521
    218454_at Hypothetical protein FLJ22662 −0.0544
    202918_s_at Preimplantation protein 3 −0.0574
    212204_at DKFZP564G2022 protein −0.0698
    219452_at Dipeptidase 2 −0.0800
    209458_x_at Hemoglobin,   alpha   1,   alpha   2 −0.0809
    219889_at Frequently rearranged in advanced T-cell lymphomas −0.0832
    201303_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 48 −0.0836
    202510_s_at Tumor necrosis factor, alpha-induced protein 2 −0.0872
    221923_s_at Nucleophosmin (nucleolar phosphoprotein B23, numatrin) −0.0902
    201887_at Interleukin 13 receptor, alpha 1 −0.1075
    211582_x_at Leukocyte specific transcript 1, LST1 −0.1193
    219030_at CGI-121 protein −0.1259
    212706_at RAS p21 protein activator 4/hypothetical protein FLJ21767 −0.1393
    202902_s_at Cathepsin S −0.1644
    200663_at CD63 antigen (melanoma 1 antigen) −0.1764
    202138_x_at/209971_x_at JTV1 gene −0.1950/−0.2868
    200851_s_at KIAA0174 −0.2019
    201109_s_at Thrombospondin 1 −0.2079
    213142_x_at Hypothetical   protein   LOC54103 −0.2285
    202531_at Interferon regulatory factor 1 −0.2904
    216274_s_at SEC11-like 1 (S. cerevisiae) −0.6951
    Positive RA scores indicate genes overexpressed in RA,
    negative RA scores indicate those underexpressed in RA.
    PAM = Prediction analysis of microarrays;
    bold and underlined letters indicate genes overlapping with the 51 genes identified by Affymetrix ® gene expression profiling and analysis (see Table 3).
  • TABLE 5
    Genes differentially regulated in RA-anti-TNFα responders versus RA-anti-TNFα non-responders (post anti-TNFα therapy)
    In- De-
    creased creased Fold
    Affymetrix ID (%) (%) Gene title change SLR
    200041_s_at 100 0 HLA-B associated transcript-1 (D6S81E) 2.53 1.34
    200052_s_at 100 0 Interleukin enhancer binding factor 2, 45 kD (ILF2) 3.73 1.90
    200064_at 100 0 Isolate Liv chaperone protein HSP90 beta (HSP90BETA) mRNA 2.66 1.41
    200079_s_at 100 0 Lysyl-tRNA synthetase mRNA, complete cds; nuclear gene for mitochondrial product; 2.16 1.11
    alternatively spliced
    200629_at 100 0 Tryptophanyl-tRNA synthetase (WARS) 2.14 1.10
    200634_at 100 0 Profilin 1 (PFN1), mRNA 2.16 1.11
    200802_at 100 0 Seryl-tRNA synthetase (SARS) 2.20 1.14
    200860_s_at 100 0 Similar to KIAA1007 protein, clone MGC: 692, mRNA, complete cds 2.11 1.08
    200983_x_at 0 100 CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, −2.43 −1.28
    EL32 and G344)
    200991_s_at 100 0 Homo sapiens KIAA0064 gene product (KIAA0064), mRNA 2.71 1.44
    201112_s_at 100 0 Chromosome segregation 1 (yeast homolog)-like (CSE1L), mRNA 2.01 1.01
    201214_s_at 100 0 Protein phosphatase 1, regulatory subunit 7 (PPP1R7), mRNA 2.17 1.12
    201241_at 100 0 DEADH (Asp-Glu-Ala-AspHis) box polypeptide 1 (DDX1), mRNA 2.30 1.20
    201263_at 100 0 Threonyl-tRNA synthetase (TARS), mRNA 2.14 1.10
    201386_s_at 100 0 Dead box protein 15 mRNA, complete cds 2.22 1.15
    201417_at 100 0 SRY (sex determining region Y)-box 4 /DEF = Human DNA sequence from 3.18 1.67
    clone RP3-322L4 on chromosome 6
    201576_s_at 100 0 Galactosidase, beta 1 (GLB1), mRNA 2.00 1.00
    201872_s_at 100 0 ATP-binding cassette, sub-family E (OABP), member 1 2.28 1.19
    201892_s_at 100 0 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (14 kD, B14) (NDUFA6), mRNA 2.19 1.13
    202174_s_at 100 0 Pericentriolar material 1 (PCM1), mRNA 2.08 1.06
    202176_at 100 0 Excision repair cross-complementing rodent repair deficiency, complementation 2.93 1.55
    group 3 (ERCC3), mRNA
    202220_at 100 0 KIAA0907 protein (KIAA0907), mRNA 2.39 1.26
    202225_at 100 0 v-crk avian sarcoma virus CT10 oncogene homolog 2.10 1.07
    202464_s_at 0 100 6-phosphofructo-2-kinasefructose-2,6-biphosphatase 3 (PFKFB3), mRNA. /PROD = 6- −3.25 −1.70
    phosphofructo-2-kinase-fructose-2,6-biphosphatase 3
    202545_at 100 0 KIAA0766 gene product (KIAA0766), mRNA 1.38 0.46
    202838_at 100 0 N-acetylgalactosaminidase, alpha-(NAGA), mRNA 1.82 0.86
    202896_s_at 0 100 Protein tyrosine phosphatase, non-receptor type substrate 1 (PTPNS1), mRNA −2.00 −1.00
    202950_at 100 0 Crystallin, zeta (quinone reductase) (CRYZ), mRNA 2.51 1.33
    203037_s_at 100 0 KIAA0429 gene product (KIAA0429), mRNA 2.41 1.27
    203155_at 100 0 SET domain, bifurcated 1 (SETDB1), mRNA 4.23 2.08
    203371_s_at 0 100 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3 (12 kD, B12) (NDUFB3), mRNA −2.13 −1.09
    203821_at 0 100 Diphtheria   toxin   receptor   (DTR) −3.63 −1.86
    203887_s_at 0 100 Thrombomodulin (THBD), mRNA −2.75 −1.46
    203966_s_at 0 100 Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform −2.73 −1.45
    (PPM1A), mRNA
    204192_at 100 0 CD37 antigen (CD37), mRNA 3.66 1.87
    204419_x_at 0 100 Hemoglobin, gamma G (HBG2), mRNA −5.50 −2.46
    204566_at 100 0 Protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D), mRNA 2.00 1.00
    204689_at 100 0 Hematopoietically expressed homeobox (HHEX), mRNA 2.04 1.03
    205239_at 0 100 Amphiregulin (schwannoma-derived growth factor) (AREG), mRNA −3.38 −1.04
    205249_at 0 100 Early growth response 2 (Krox-20 (Drosophila) homolog) (EGR2), mRNA −2.55 −1.35
    205552_s_at 100 0 2,5-oligoadenylate synthetase 1 (40-46 kD) (OAS1), transcript variant E16, mRNA 2.08 1.06
    206115_at 0 100 Early growth response 3 (EGR3), mRNA. /PROD = early growth response 3 −6.54 −2.71
    206584_at 0 100 Homo sapiens MD-2 protein (MD-2), mRNA −2.16 −1.11
    206877_at 0 100 Homo sapiens MAX dimerization protein (MAD), mRNA −3.92 −1.97
    207170_s_at 100 0 DKFZP586A011 protein (DKFZP586A011), mRNA 2.20 1.14
    208631_s_at 100 0 78 kDa gastrin-binding protein mRNA, complete cds 2.04 1.03
    208691_at 0 100 Transferrin receptor (p90, CD71), clone MGC: 3151, mRNA, complete cds −2.30 −1.20
    208868_s_at 0 100 Homo sapiens mRNA; cDNA DKFZp564N1272 (from clone DKFZp564N1272); −3.18 −1.67
    complete cds
    208869_s_at 0 100 GABA-A receptor-associated protein like 1 (GABARAPL1) mRNA, complete cds −3.29 −1.72
    208942_s_at 0 100 Translocation protein 1 −2.93 −1.55
    209092_s_at 100 0 Homo sapiens clone 016b03 My027 protein mRNA, complete cds 2.07 1.05
    209193_at 0 100 Protein kinase-related oncogene (PIM1) mRNA, complete cds −2.51 −1.33
    209200_at 100 0 MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) 2.48 1.31
    209861_s_at 100 0 eIF-2-associated p67 homolog mRNA, complete cds 2.55 1.35
    209967_s_at 0 100 Human mRNA for hCREM (cyclic AMP-responsive element modulator) type 2 protein, −3.07 −1.62
    complete cds
    210027_s_at 100 0 Apurinic endonuclease (APE) mRNA, complete cds. /PROD = apurinic endonuclease 2.23 1.16
    210053_at 100 0 TATA box binding protein (TBP)-associated factor, RNA polymerase II, D, 100 kD 2.23 1.16
    210172_at 0 100 Human mRNA for ZFM1 protein alternatively spliced product, complete cds. /PROD = ZFM1 −2.16 −1.11
    protein, alternatively spliced product
    210766_s_at
    100 0 Trachea cellular apoptosis susceptibility protein (CSE1) mRNA, complete cds 2.28 1.19
    210949_s_at 100 0 Similar to eukaryotic translation initiation factor 3, subunit 8 (110 kD), clone MGC: 2.53 1.34
    8693, mRNA, complete cds
    211458_s_at 0 100 GABA-A receptor-associated protein mRNA, complete cds. /PROD = GABA-A −3.18 −1.67
    receptor-associated protein
    211546_x_at
    0 100 Human (clone 2-5) synuclein (NACP) mRNA, complete cds −4.92 −2.30
    212199_at 100 0 Human putative ribosomal protein S1 mRNA 2.22 1.15
    212224_at 100 0 Aldehyde dehydrogenase 1, soluble (ALDH1), mRNA. /PROD = aldehyde dehydrogenase 3.18 1.67
    1, soluble
    212388_at 100 0 Homo sapiens mRNA for KIAA1057 protein, partial cds 3.10 1.63
    212591_at 100 0 RBP1-like protein 2.03 1.02
    212696_s_at 100 0 Ring finger protein 4 3.05 1.61
    212709_at 100 0 KIAA0197 protein 2.10 1.07
    212714_at 100 0 Homo sapiens mRNA; cDNA DKFZp586F1323 (from clone DKFZp586F1323) 2.13 1.09
    212893_at 100 0 Homo sapiens mRNA; cDNA DKFZp564I052 (from clone DKFZp564I052) 2.01 1.01
    212989_at 100 0 Homo sapiens mRNA for Hmob33 protein, 3 untranslated region 2.25 1.17
    213410_at 100 0 Homo sapiens mRNA; cDNA DKFZp586F1019 (from clone DKFZp586F1019); partial cds 2.53 1.34
    213515_x_at 0 100 Myosin, light polypeptide 4, alkali; atrial, embryonic −4.69 −2.23
    213528_at 100 0 H. sapiens novel gene from PAC 117P20, chromosome 1 2.87 1.52
    213604_at 100 0 Homo sapiens clone 24582 mRNA sequence 2.07 1.05
    213619_at 0 100 Heterogeneous nuclear ribonucleoprotein H1 (H) −2.48 −1.31
    213655_at 0 100 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, −3.03 −1.60
    epsilon polypeptide
    213743_at 100 0 Cyclin T2 2.08 1.06
    213788_s_at 0 100 cDNA: FLJ23227 fis, clone CAE00645, highly similar to AF052138 −2.20 −1.14
    213872_at 0 100 Hypothetical protein FLJ12619 −4.29 −2.1
    213979_s_at 0 100 C-terminal binding protein 1 −4.41 −2.14
    214257_s_at 0 100 SEC22, vesicle trafficking protein (S. cerevisiae)-like 1 −2.97 −1.57
    214414_x_at 0 100 Hemoglobin, alpha 1 −2.04 −1.03
    214696_at 0 100 Homo sapiens clone 24659 mRNA sequence /DEF = Homo sapiens clone 24659 −2.93 −1.55
    mRNA sequence
    214933_at 0 100 Calcium channel, voltage-dependent, PQ type, alpha 1A subunit −2.17 −1.12
    215043_s_at 0 100 H. sapiens SMA5 mRNA −4.08 −2.03
    215933_s_at 100 0 H. sapiens HEX gene encoding homeobox related protein 2.17 1.12
    216199_s_at 100 0 Mitogen-activated protein kinase kinase kinase 4 2.77 1.47
    216202_s_at 100 0 Serine palmitoyltransferase (LCB2) mRNA, partial cds 2.75 1.46
    216996_s_at 100 0 KIAA0971 protein /DEF = Homo sapiens cDNA FLJ11495 fis, clone HEMBA1001950, 2.20 1.14
    highly similar to Homo sapiens mRNA for KIAA0971 protein
    217554_at 0 100 ESTs, Hs.97109 −3.63 −1.86
    217682_at 0 100 ESTs, Weakly similar to ALU7_HUMAN ALU −3.01 −1.59
    217840_at 100 0 DEAD-box protein abstrakt (ABS), mRNA 2.39 1.26
    218229_s_at 100 0 KIAA1513 protein (KIAA1513), mRNA 2.07 1.05
    218356_at 100 0 Cell division protein FtsJ (FJH1), mRNA 2.20 1.14
    218432_at 100 0 F-box only protein 3 (FBXO3), mRNA 2.16 1.11
    218589_at 100 0 Purinergic receptor (family A group 5) (P2Y5), mRNA 2.68 1.42
    218604_at 100 0 Integral inner nuclear membrane protein (MAN1), mRNA 2.04 1.03
    218689_at 100 0 Fanconi anemia, complementation group F (FANCF), mRNA 8.94 3.16
    218889_at 100 0 Hypothetical protein FLJ12820 (FLJ12820), mRNA 2.01 1.01
    218973_at 100 0 Hypothetical protein FLJ13119 (FLJ13119), mRNA 2.07 1.05
    219069_at 100 0 Hypothetical protein FLJ20189 (FLJ20189), mRNA 2.23 1.16
    219093_at 100 0 Hypothetical protein FLJ20701 (FLJ20701), mRNA 3.32 1.73
    219099_at 100 0 Homo sapiens chromosome 12 open reading frame 5 (C12ORF5), mRNA 2.13 1.09
    219176_at 100 0 Hypothetical protein FLJ22555 (FLJ22555), mRNA 2.13 1.09
    219243_at 100 0 Hypothetical protein FLJ11110 (FLJ11110), mRNA 2.07 1.05
    219363_s_at 100 0 CGI-12 protein (LOC51001), mRNA 2.31 1.21
    219434_at 0 100 Triggering receptor expressed on myeloid cells 1 (TREM1), mRNA −4.35 −2.12
    221485_at 0 100 betaGlcNAc beta 1,4-galactosyltransferase. polypeptide 5 −2.71 −1.44
    221652_s_at 100 0 PNAS-25 mRNA, complete cds. 2.55 1.35
    221755_at 100 0 Homo sapiens mRNA for FLJ00043 protein, partial cds 3.44 1.09
    221970_s_at 100 0 Homo sapiens cDNA: FLJ21737 fis, clone COLF3396 2.41 1.27
    222127_s_at 0.1 0 Homo sapiens cDNA FLJ13399 fis, clone PLACE1001395 /DEF = Homo sapiens cDNA 2.31 1.21
    FLJ13399 fis, clone PLACE1001395
    36711_at 0 100 Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein F) −3.54 −1.16
    LIKE protein
    38037_at 0 100 Heparin-binding EGF-like growth factor mRNA, complete cds −3.63 −1.86
    AFFX-BioB-M_at 0 100 E. coli 7,8-diamino-pelargonic acid (bioA), biotin synthetase (bioB), 7-keto-8-amino- −4.69 −2.58
    pelargonic acid synthetase (bioF), bioC protein, and dethiobiotin synthetase (bioD),
    complete cds
    AFFX-r2-Ec-bioC- 0 100 Escherichia coli /REF = J04423 /DEF = E coli bioC protein corresponding to −4.22 −1.86
    3_at nucleotides 4609-4883 of J04423 /LEN = 777 (−5 and −3 represent transcript
    regions 5 prime and 3 prime respectively)
    The listed genes (n = 117) show differential expression in 100% of the pairwise comparisons;
    bold and underlined letters indicate genes overlapping with the 51 genes identified by Affymetrix ® gene expression profiling and analysis (see Table 3)
  • TABLE 6
    Correlations between clinical parameters and differentially expressed genes (Pearson correlation
    test; pre- and post-anti-TNFα treatment; n = 7 for all; bold letters identify genes occurring twice, bold and
    italics letters identify genes occurring ≧3 times)
    Affymetrix_ID Gene title r-value p-value
    Pre-anti-TNFα treatment
    Leuko/ 202219_at Solute carrier family 6 0.901 0.006**
    205239_at Amphiregulin 0.879 0.009**
    Thromb/ 205900_at Keratin 1 0.923 0.003**
    Mono/ 204018_x_at Hemoglobin, alpha 1 −0.908 0.005**
    GPT/ 203932_at MHC-II, DM beta −0.912 0.004**
    218589_s_at Purinergic receptor P2Y, G-protein coupled 5 −0.919 0.003**
    Hb/ 205900_at Keratin 1 −0.944 0.001**
    MoStiff/ 202219_at Solute carrier family 6 0.893 0.007**
    203932_at MHC-II, DM beta −0.898 0.006**
    204131_s_at Forkhead box O3A 0.917 0.004**
    205239_at Amphiregulin 0.954 0.001**
    207332_s_at Transferrin receptor (p90, CD71) 0.926 0.003**
    208632_at Ring finger protein 10 0.913 0.004**
    DisDur/ 212232_at Formin binding protein 4 0.955 0.001**
    213142_x_at Hypothetical protein LOC54103 0.877 0.009**
    Post-anti-TNFα treatment
    ESR/
    Figure US20120039900A1-20120216-P00001
    Figure US20120039900A1-20120216-P00002
    Figure US20120039900A1-20120216-P00003
    Figure US20120039900A1-20120216-P00004
    Figure US20120039900A1-20120216-P00005
    Figure US20120039900A1-20120216-P00006
    Figure US20120039900A1-20120216-P00003
    Figure US20120039900A1-20120216-P00004
    Figure US20120039900A1-20120216-P00007
    Figure US20120039900A1-20120216-P00008
    Figure US20120039900A1-20120216-P00009
    Figure US20120039900A1-20120216-P00010
    Figure US20120039900A1-20120216-P00011
    Figure US20120039900A1-20120216-P00012
    Figure US20120039900A1-20120216-P00013
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00015
    Figure US20120039900A1-20120216-P00016
    Figure US20120039900A1-20120216-P00017
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00018
    Figure US20120039900A1-20120216-P00019
    Figure US20120039900A1-20120216-P00020
    Figure US20120039900A1-20120216-P00021
    219228_at Zinc finger protein 331 0.894 0.007**
    CRP/
    Figure US20120039900A1-20120216-P00001
    Figure US20120039900A1-20120216-P00002
    Figure US20120039900A1-20120216-P00022
    Figure US20120039900A1-20120216-P00023
    Figure US20120039900A1-20120216-P00005
    Figure US20120039900A1-20120216-P00006
    Figure US20120039900A1-20120216-P00024
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00007
    Figure US20120039900A1-20120216-P00008
    Figure US20120039900A1-20120216-P00025
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00011
    Figure US20120039900A1-20120216-P00012
    Figure US20120039900A1-20120216-P00026
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00015
    Figure US20120039900A1-20120216-P00016
    Figure US20120039900A1-20120216-P00027
    Figure US20120039900A1-20120216-P00014
    Figure US20120039900A1-20120216-P00018
    Figure US20120039900A1-20120216-P00019
    Figure US20120039900A1-20120216-P00028
    Figure US20120039900A1-20120216-P00029
    Thromb/
    Figure US20120039900A1-20120216-P00007
    Figure US20120039900A1-20120216-P00008
    Figure US20120039900A1-20120216-P00030
    Figure US20120039900A1-20120216-P00031
    Figure US20120039900A1-20120216-P00018
    Figure US20120039900A1-20120216-P00019
    Figure US20120039900A1-20120216-P00032
    Figure US20120039900A1-20120216-P00004
    Granulo/ 213427_at Ribonuclease P 40 kDa subunit −0.875 0.010**
    Hb/
    Figure US20120039900A1-20120216-P00001
    Figure US20120039900A1-20120216-P00002
    Figure US20120039900A1-20120216-P00033
    Figure US20120039900A1-20120216-P00031
    Figure US20120039900A1-20120216-P00005
    Figure US20120039900A1-20120216-P00034
     factor
    Figure US20120039900A1-20120216-P00035
    Figure US20120039900A1-20120216-P00014
    203932_at MHC class II, DM beta 0.885 0.008**
    Figure US20120039900A1-20120216-P00011
    Figure US20120039900A1-20120216-P00012
    Figure US20120039900A1-20120216-P00036
    Figure US20120039900A1-20120216-P00023
    Figure US20120039900A1-20120216-P00015
    Figure US20120039900A1-20120216-P00016
    Figure US20120039900A1-20120216-P00037
    Figure US20120039900A1-20120216-P00014
    Dis_act/ 36711_at v-maf fibrosarc. oncogene homolog F (avian) 0.932 0.002**
    205552 _s_at 2′,5′-oligoadenylate synthetase 1, 40/46 kDa −0.951 0.001**
    Figure US20120039900A1-20120216-P00015
    Figure US20120039900A1-20120216-P00016
    Figure US20120039900A1-20120216-P00038
    Figure US20120039900A1-20120216-P00010
    Figure US20120039900A1-20120216-P00018
    Figure US20120039900A1-20120216-P00019
    Figure US20120039900A1-20120216-P00009
    Figure US20120039900A1-20120216-P00010
    DAS28/ 36711 v-maf fibrosarc. oncogene homolog F (avian) 0.940 0.002**
    205552 _s_at 2′,5′-oligoadenylate synthetase 1, 40/46 kDa −0.898  0.006**
    Figure US20120039900A1-20120216-P00007
    Figure US20120039900A1-20120216-P00008
    Figure US20120039900A1-20120216-P00039
    Figure US20120039900A1-20120216-P00031
    Figure US20120039900A1-20120216-P00015
    Figure US20120039900A1-20120216-P00016
    Figure US20120039900A1-20120216-P00040
    Figure US20120039900A1-20120216-P00010
    Figure US20120039900A1-20120216-P00018
    Figure US20120039900A1-20120216-P00019
    Figure US20120039900A1-20120216-P00041
    Figure US20120039900A1-20120216-P00023
    Sw28/ 219607_s_at Membrane-spanning 4-domains, subfam. A, memb. 4 0.931 0.002**
    GPT = glutamate pyruvic transferase;
    Hb = haemoglobin;
    MoStiff = Morning stiffness (minutes);
    DisDur = Disease duration (years);
    ESR = erythrocyte sedimentation rate;
    CRP = C-reactive protein;
    DAS 28 = disease activity score (28 joints);
    Sw = number of swollen joints (28 joints)
  • TABLE 7
    TaqMan PCR validation in anti-TNFα-treated RA patients and normal donors
    PCR
    RA-αTNF
    (responder)
    Time Gene versus ND Max −ΔRQ p value
    point Description Gene title Applera ID (RQ; Means ± SEM) (Therapy) (U-Test)
    Validated genes
    BL AREG Amphiregulin Hs00155832_m1 10.23 ± 2.93  −7.60 0.000
    BL CA-1 Carbonic anhydrase 1 Hs00266139_m1 6.83 ± 3.49 −5.93 n.s.
    BL CLC Charcot-Leyden crystal protein Hs00171342_m1 5.36 ± 4.82 −3.26 0.041
    BL CLU Clusterin C Hs00156548_m1 3.94 ± 2.87 −2.77 n.s.
    BL TNFAIP6 Tumor necrosis factor alpha Hs00218482_m1 1.71 ± 0.40 0.82 0.000
    induced protein 6
    BL THBD Thrombomodulin Hs00264920_s1 1.58 ± 1.08 −0.53 0.041
    BL MS4A4A Membrane-spanning 4-domains, Hs00254770_m1 1.54 ± 0.42 0.71 0.041
    subfamily A, member 4
    BL DTR Diptheria toxin receptor Hs00181813_m1 1.21 ± 0.44 −1.10 n.s.
    BL S100A12 S100 calcium binding protein A1 Hs00194525_m1 1.07 ± 0.21 0.11 0.041
    BL HT007 Uncharacterized hypothalamus Hs00218482_m1   0.51 ± −0.09 0.01 0.000
    protein HT007
    BL HLA-DR MHC-class-II; HLA-DR alpha Hs00219578_m1   0.59 ± −0.15 0.09 0.000
    BL LOC54103 Hypothetical protein LOC54103 Hs00367929_m1   0.90 ± −0.21 −0.24 0.041
    BL TNF Tumor necrosis factor alpha Hs00174128_m1 1.22 ± 0.53 −0.84 0.041
    BL IL1B Interleukin 1 beta Hs00174097_m1 1.14 ± 0.42 −0.28 0.041
    BL PSMB7 Proteasome subunit beta type 7 Hs00160607_m1   0.72 ± −0.19 0.16 0.041
    precursor
    BL KIAA0174 Protein KIAA0174 Hs00796085_sh 0.89 ± 0.30 0.08 0.041
    BL SPC18 Microsomal signal peptidase Hs00819308_m1 0.96 ± 0.11 −0.25 0.041
    18 kDa subunit
    Discrepancies between Affymetrix and TaqMan PCR
    BL LIPT1 Lipoyltransferase 1 Hs00376962_m1 2.17 ± 0.54 −0.68 0.000
    BL ZNF361 Ring zinc finger protein 361 Hs00367929_m1   0.55 ± −0.11 −0.14 0.000
    BL THBS1 Thrombospondin-1 Hs00170236_m1 0.88 ± 0.25 −0.34 n.s.
    BL IRF1 Interferon regulatory protein 1 Hs00233698_m1 4.95 ± 0.18 3.37 0.000
    BL PPP1CB Protein phosphatase 1, catalytic Hs00160349_m1 0.93 ± 0.09 −0.28 0.041
    subunit, beta isoform
    Affymetrix
    RA-αTNF FC RA-αTNF FC RA-αTNF
    (responder) (responder) RA-αTNF all all
    Time Affymetrix versus ND versus ND versus ND versus ND
    point ID (% of comp.) (Means) (% of comp.) (Means)
    Validated genes
    BL 205239_at 82.86 5.48 81.63 6.13
    BL 205950_s_at 88.57 4.92 81.63 5.88
    BL 206207_at 62.86 2.46 73.47 3.97
    BL 222043_at 68.57 2.57 75.51 2.68
    BL 206026_s_at 62.86 2.07 73.27 2.24
    BL 203887_s_at 74.29 2.64 71.43 2.49
    BL 219607_s_at 88.57 2.66 91.84 2.96
    BL 38037_at 68.57 2.91 71.43 2.87
    BL 205863_at 62.86 1.50 71.43 1.69
    BL 221622_s_at −88.57 −1.74 −85.71 −1.66
    BL 208894_at −65.71 −1.77 −75.51 −2.15
    BL 222150_s_at −80.00 −1.84 −73.47 −1.71
    BL 207113_s_at 34.29 1.03 −46.94 −1.28
    BL 205067_at 57.14 1.62 57.02 1.44
    BL 200786_at −54.29 −1.27 −57.74 −1.27
    BL 200851_s_at −42.86 −1.17 −46.94 −1.23
    BL 201290_at −17.14 −1.04 −72.24 −1.03
    Discrepancies between Affymetrix and TaqMan PCR
    BL 205571_at −74.29 −1.74 −79.59 −1.90
    BL 219228_at 77.14 4.19 77.55 3.94
    BL 201110_s_at 85.71 16.35 77.55 12.60
    BL 202531_at −40.00 −1.30 −55.70 −1.44
    BL 201409_s_at 60.00 1.63 57.14 1.69
    Validation of 22 selected genes from Affymetrix ® gene expression profiling by Taqman real-time RT-PCR (10 anti-TNFα-treated RA patients prior to therapy; 14 normal donors − ND); genes differentially expressed (decreased: <−70%; increased: >70%) in RA versus ND according to the Affymetrix ® analysis are shown in normal type, equally-expressed genes are displayed in italics; RQ = relative quantity; FC = fold-change; BL = baseline (pre-antiTNFα therapy)
  • TABLE 8
    Correlations between TaqMan PCR results and the
    ACR response in anti-TNFα-treated RA patients
    Gene Time
    description point Gene title Applera ID r-value p n
    CA1 w 12 Carbonic anhydrase 1 Hs00266139_m1 0.642 0.045 10
    CA1 w 26 0.962 0.000 9
    CLC w 4 Charcot-Leyden crystal protein Hs00171342_m1 0.766 0.027 8
    THBD w 4 Thrombomodulin Hs00264920_s1 0.766 0.027 8
    THBS1 w 26 Thrombospondin 1 Hs00170236_m1 −0.710 0.032 9
    IRF1 w 4 Interferon regulatory factor 1 Hs00233698 m1 0.778 0.023 8
  • TABLE 9
    Genes differentially regulated in RA-anti-TNFα responders versus RA-anti-TNFα non-responders (pre-anti-TNFα therapy;
    predictive genes)
    Increased Decreased Fold-
    Affymetrix ID Gene title (%) (%) Change SLR
    39248_at Aquaporin 3 10 80 −4.6 −2.19
    200061_s_at Similar to ribosomal protein S24, clone MGC: 8595 0 80 −1.39 −0.47
    200087_s_at Transmembrane emp24 domain trafficking protein 2 0 80 −1.6 −0.63
    200642_at Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 0 80 −1.8 −0.84
    200655_s_at Calmodulin 1 (phosphorylase kinase, delta) 0 80 −1.5 −0.59
    200745_s_at Guanine nucleotide binding protein (G protein), beta polypeptide 1 80 0 1.6 0.66
    200772_x_at Prothymosin, alpha (gene sequence 28) 80 0 1.7 0.75
    201193_at Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) 0 80 −1.4 −0.49
    201690_s_at Tumor protein D52 0 80 −3.3 −1.73
    201693_s_at Early growth response 1 80 0 3.1 1.61
    201889_at
    Figure US20120039900A1-20120216-P00042
    		 0
    Figure US20120039900A1-20120216-P00043
    		 −1.7 −0.74
    202110_at Cytochrome c oxidase subunit VIIb 0 80 −1.6 −0.64
    202157_s_at CUG triplet repeat, RNA binding protein 2 0 80 −1.6 −0.64
    202233_s_at Ubiquinol-cytochrome c reductase hinge protein 0 80 −1.7 −0.74
    202378_s_at Homos spaiens leptin receptor gene-related protein (HS0BRGRP) 80 20 1.15 0.20
    202664_at Wiskott-Aldrich syndrome protein interacting protein 80 0 1.9 0.91
    202910_s_at CD97 antigen 80 10 1.6 0.70
    202922_at Glutamate-cysteine ligase, catalytic subunit 0 80 −2.3 −1.17
    202950_at Crystallin, zeta (quinone reductase) 80 0 2.2 1.16
    203097_s_at Rap guanine nucleotide exchange factor (GEF) 2 20 80 −1.3 −0.38
    203231_s_at Ataxin 1 0 80 −8.2 −3.03
    203300_x_at Adaptor-related protein complex 1, sigma 2 subunit 80 20 1.9 0.89
    204160_s_at Ectonucleotide pyrophosphatase/phosphodiesterase 4 10 80 −2.4 −1.27
    204750_s_at Desmocollin 2 80 0 12.9 3.69
    204777_s_at MAL, T-cell differentiation protein 10 80 −2.6 −1.39
    205042_at Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase 0 80 −1.9 −0.95
    205114_s_at Chemokine (C—C motif) ligand 3 80 0 2.0 0.96
    205624_at Carboxypeptidase A3 0 80 −4.0 −2.00
    206207_at Charcot-Leyden crystal protein 0
    Figure US20120039900A1-20120216-P00044
    		 −4.9 −2.29
    206790_s_at NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa 0 80 −1.5 −0.61
    207008_at Interleukin 8 receptor, beta
    Figure US20120039900A1-20120216-P00044
    		 0 4.0 2.00
    207815_at Platelet factor 4 variant 1 20 80 −2.4 −1.28
    208051_s_at Poly(A) binding protein interacting protein 1 0 80 −1.6 −0.68
    208161_s_at ATP-binding cassette, sub-family C (CFTR/MRP), member 3 80 0 4.3 2.10
    208637_x_at Actinin, alpha 1 80 0 2.4 1.24
    208918_s_at NAD kinase 80 0 1.5 0.59
    208982_at Platelet/endothelial cell adhesion molecule (CD31 antigen)
    Figure US20120039900A1-20120216-P00044
    		 0 1.6 0.71
    209009_at Esterase D/formylglutathione hydrolase 80 20 1.5 0.54
    209020_at Chromosome 20 open reading frame 111 80 20 1.5 0.54
    209146_at Sterol-C4-methyl oxidase-like 0 80 −1.9 −0.89
    209193_at PIM-1 oncogene 10 80 −2.3 −1.19
    209710_at GATA binding protein 2 20 80 −3.5 −1.81
    210042_s_at Cathepsin Z 80 20 1.9 0.94
    210184_at
    Figure US20120039900A1-20120216-P00045
    Figure US20120039900A1-20120216-P00043
    		 0 2.3 1.22
    210732_s_at Lectin, galactoside-binding, soluble, 8 (galectin 8) 80 0 2.4 1.23
    210895_s_at CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) 80 10 1.9 0.92
    211506_s_at Interleukin 8
    Figure US20120039900A1-20120216-P00044
    		 0 3.2 1.67
    211734_s_at Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide 0 80 −5.0 −2.31
    211995_x_at Actin, gamma 1 80 0 1.4 0.46
    212314_at KIAA0746 protein 10 80 −1.7 −0.73
    212335_at Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) 80 0 1.5 0.54
    212386_at Transcription factor 4 0 80 −2.5 −1.29
    212671_s_at Major histocompatibility complex, class II, DQ alpha 1 10 80 −3.3 −1.72
    212897_at Cell division cycle 2-like 6 (CDK8-like) 80 0 1.8 0.83
    212999_x_at Major histocompatibility complex, class II, DQ beta 1 80 20 1.8 0.88
    213309_at Phospholipase C-like 2 0 80 −1.9 −0.9
    213506_at Coagulation factor II (thrombin) receptor-like 1 80 0 2.4 1.23
    213883_s_at TM2 domain containing 1 10 80 −1.4 −0.46
    214305_s_at Splicing factor 3b, subunit 1, 155 kDa 80 20 1.5 0.62
    214512_s_at SUB1 homolog (S. cerevisiae) 0 80 −1.7 −0.76
    214807_at MRNA; cDNA DKFZp564O0862 80 10 1.7 0.79
    214953_s_at Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer
    Figure US20120039900A1-20120216-P00044
    		 0 1.9 0.93
    disease)
    215726_s_at Cytochrome b-5 0 80 −2.1 −1.08
    216016_at Cold autoinflammatory syndrome 1 80 0 6.1 2.60
    217722_s_at Neugrin, neurite outgrowth associated 0
    Figure US20120039900A1-20120216-P00044
    		 −1.9 −0.91
    217753_s_at Ribosomal protein S26, 40S ribosomal protein 10 80 −2.9 −1.51
    217970_s_at CCR4-NOT transcription complex, subunit 6 0 80 −1.9 −0.91
    218190_s_at Ubiquinol-cytochrome c reductase complex (7.2 kD) 0 80 −2.0 −1.03
    218345_at Hepatocellular carcinoma-associated antigen 112 10 80 −3.9 −1.95
    218486_at Kruppel-like factor 11
    Figure US20120039900A1-20120216-P00044
    		 0 2.1 1.06
    218545_at GGA binding partner 0 80 −1.6 −0.67
    218728_s_at Cornichon homolog 4 (Drosophila) 0 80 −1.8 −0.84
    219269_at Hypothetical protein FLJ21616 0 90 −2.1 −1.06
    219410_at
    Figure US20120039900A1-20120216-P00046
    		 0
    Figure US20120039900A1-20120216-P00043
    		 −4.7 −2.33
    219862_s_at Nuclear prelamin A recognition factor 80 0 1.5 0.62
    219905_at Erythroblast membrane-associated protein 0 80 −1.8 −0.85
    220532_s_at LR8 protein 10 80 −5.2 −2.39
    221011_s_at Likely ortholog of mouse limb-bud and heart gene (LBH) 0 80 −2.8 −1.47
    221042_s_at Calmin (calponin-like, transmembrane) 80 0 1.9 0.89
    221434_s_at Chromosome 14 open reading frame 156 0 80 −1.7 −0.72
    221737_at Guanine nucleotide binding protein (G protein) alpha 12 10 80 −2.0 −1.96
    AFFX-M27830_5_at SRY (sex determining region Y)-box 18 80 0 4.3 2.09
    The criterion for gene selection (total of 82) was the percentage of pairwise comparisons between future RA responders and future RA non-responders to anti-TNFα therapy showing an increase or decrease for the fold-change/Signal Log Ratio prior to therapy; bold letters indicate a percentage of 80% for the pairwise comparisons (total of 10), bold and italic letters 
    Figure US20120039900A1-20120216-P00047
     , bold, italic, and underlined letters 
    Figure US20120039900A1-20120216-P00048
     in the latter case, also the gene names are shown in bold, italic, and underlined letters.
  • SUMMARY OF SEQUENCE LISTING
    SEQ ID NO: AffymetrixID Sequence
    1 39248_at cttctacaggcttttgggaagtagggtggatgtg
    ggtagggctgggaggagggggccacagcttaggt
    ttggagctctggatgtacatacataagtaggagc
    agtgggacgtgtttctgtcataatgcaggcatga
    agggtggagtgaagtcaggtcataagtttcatgt
    ttgcttttgttttgttttgtttttaatgtatgta
    gcagatgttacagtcttagggatccgggatggga
    gaccccactttagaaagggtcgtcactcctttaa
    tcctcta
    2 200061_s_at agatcgccatcatgaacgacaccgtaactatccgcactag
    aaagttcatgaccaaccgactacttcagaggaaacaaatg
    gtcattgatgtccttcaccccgggaaggcgacagtgccta
    agacagaaattcgggaaaaactagccaaaatgtacaagac
    cacaccggatgtcatctttgtatttggattcagaactcat
    tttggtggtggcaagacaactggctttggcatgatttatg
    attccctggattatgcaaagaaaaatgaacccaaacatag
    acttgcaagacatggcctgtatgagaagaaaaagacctca
    agaaagcaacgaaaggaacgcaagaacagaatgaagaaag
    tcagggggactgcaaaggccaatgttggtgctggcaaaaa
    gtgagctggagattggatcacagccgaaggagtaaaggtg
    ctgcaatgatgttagctgtggccactgtggatttttcgca
    aga
    3 200087_s_at gggtactgaccatcagtgtcagcattagggtttt
    ggtttttgtttcttttgggtatttcttttttggc
    acatgtgaatcttgttttgtgtaaaatgaaatta
    ctttctcttgttctctgatgatgggtttaaaatt
    aaaagagcatccggttttggtatggggatgatcc
    aggattatgttgtgactgatacatattagttact
    tgtgctttttttttttttttttnggatctttgca
    agggcaaaactacaagtaacgagttttatataat
    taatttaaatttgttacaggttttcatgttcagg
    ataaaccatacttccaccttgggtgagaacactt
    gcaacagtttattaatgaggtgactttcacctta
    ggacaactgttgcatgccaagttttttgtgtgtg
    tgaaacacttcaaaactgatttaaaagatgtaaa
    tttaaaattggttgtatctaatatgccccagg
    4 200642_at ctgaagggcgacggcccagtgcagggcatcatcaatttcg
    agcagaaggaaagtaatggaccagtgaaggtgtggggaag
    cattaaaggactgactgaaggcctgcatggattccatgtt
    catgagtttggagataatacggcaggctgtaccagtgcag
    gtcctcactttaatcctctatccagaaaacacggtgggcc
    aaaggatgaagagaggcatgttggagacttgggcaatgtg
    actgctgacaaagatggtgtggccgatgtgtctattgaag
    attctgtgatctcactctcaggagaccattgcatcattgg
    ccgcacactggtggtccatgaaaaagcagatgacttgggc
    aaaggtggaaatgaagaaagtacaaagacaggaaacgctg
    gaagtcgtttggcttgtggtgtaattgggatcgcccaata
    aacattcccttg
    5 200655_s_at acctattcaaatgggttctagttcaatttgttta
    gtataaattgtcatagctggtttactgaaaacaa
    acacatttaaaattggtttacctcaggatgacgt
    gcagaaaaatgggtgaaggataaaccgttgagac
    gtggccccactggtaggatggtcctcttgtactt
    cgtgtgctccgacccatggtgacgatgacacacc
    ctggtggcatgcccgtgtatgttggtttagcgtt
    gtctgcattgttctagagtgaaacaggtgtcagg
    ctgtcactgttcacacaaatttttaataagaaac
    atttaccaagggagcatctttggactctctgttt
    ttaaaaccttctgaaccatgacttggagccggca
    gagtaggctgtggctgtggacttcagcacaacca
    tcaacattgctgttcaaagaaattacagtttacg
    tccattccaagttgt
    6 200745_s_at gcgtgatcagagaatccttcaccttatgctgaaaagtgag
    ctcagatccagcaccaatgttcctcctgacccatcctgtc
    tatcttctcagttgagtttttaatctcactttgggtttcc
    ttgtgaagttggagggaagtttataatagcctaacactac
    cccacccccaactaggaggaacctctgttttcaagagaga
    tgcctgtcctgtgcttggatagtcagtcaattatttgtgt
    atgaaacaatgtacaaatcaatgttttgaaaataatgatc
    tcagactttctaagttaaagttttaaaaattttgattgtt
    tgccatattgggtgggtttactcttagaatcgcatgctgt
    agaaaatgctcaaaagtgcatatgggactcagtccttagg
    tgttctttttcttttaagaaataacctcttacagttgtaa
    ccattgcggctctgtccacttctcgttgctgctctgtggc
    acatatcggaagcagtacagcgcgcgg
    7 200772_x_at gaaaagccatctttgcattgttcctcatccgncc
    tccttgctcngccgcagccgcctccgccgcgcgc
    ctcctccgccgccgcggactnccggcagctttat
    cgccagagtccctgaactctcgctttctttttaa
    tccccntgcatcggatcaccggcgtgccccacca
    tgtcagacgcagccgtagacaccagctccgaaat
    caccaccaaggacttaaaggagaagaaggaagtt
    gtggaagaggcagaaanatggaagaganncgccc
    ctgctnaacgggnaatgctnnananantgaggaa
    naatggggnagcaggaggctngacaatngaggta
    gacgaangaaganggaagananggtggggangga
    angagganggagganagaangaaggtgantggtg
    anggaangaggantggangantgaangatgagga
    agctgagtcagctacgggcaagcgggcagctgaa
    ngatgatgaggatgacgatgtcgataccaangaa
    gcagaagaccgacgaggatgactagacagc
    8 201193_at tagccacagtattgctccctaaaatatgcataaagtagaa
    attcactgccttcccctcctgtccatgaccttgggcacag
    ggaagttctggtgtcatagatatcccgttttgtgaggtag
    agctgtgcattaaacttgcacatgactggaacgaagtatg
    agtgcaactcaaatgtgttgaagatactgcagtcattttt
    gtaaagaccttgctgaatgtttccaatagactaaatactg
    tttaggccgcaggagagtttggaatccggaataaatacta
    cctggagtcttgtcctctccatttttctctttctcctcct
    ggcctggcctgaatattatactactctaaatagcatattt
    catccaagtgcaataatgtaagctgaatcttttttggact
    tctgctggcctgttttatttcttttatataaatgtgattt
    ctcagaaattgatattaaacactatcttatcttctcctga
    actgttgatttt
    9 201690_s_at atgatactgtagaacctgtctcctactttgaaaa
    ctgaatgtcagggctgagtgaatcaaagtgtcta
    gacatatttgcatagaggccaaggtattctattc
    taataactgcttactcaacactaccaccttttcc
    ttatactgtatatgattatggcctacaatgttgt
    atttgttatttattaaattgtgattgttttatta
    ttgtttatgccaaatgttaactgccaagcttgga
    gtgacctaaagcattttttaaaagcatggctaga
    tttacttcagtataaattatcttatgaaaaccaa
    attttaaaagccacaggtgttgattgttataaaa
    taacatgctgccattcttgattgctagagttttt
    gttagtactttggatgc
    10 201693_s_at gggctttcggacatgacagcaaccttttctccca
    ggacaattgaaatttgctaaagggaaaggggaaa
    gaaagggaaaagggagaaaaagaaacacaagaga
    cttaaaggacaggaggaggagatggccataggag
    aggagggttcctcttaggtcagatggaggttctc
    agagccaagtcctccctctctactggagtggaag
    gtctattggccaacaatcctttctgcccacttcc
    ccttccccaattactattccctttgacttcagct
    gcctgaaacagccatgtccaagttcttcacctct
    atccaaagaacttgatttgcatggattttggata
    aatcatttcagtatcatctccatcatatgcctga
    ccccttgctcccttcaatgctagaaaatcgagtt
    ggcaaaatggggtttgggcccctcagagccctgc
    cctgcacccttgtacagtgtctgtgccatggatt
    tcgtttttcttggggtactcttgatt
    11 201889_at Ttgaccccaaatgactttatacccaattctacataaaaat
    atagaagatctatctttttttgttaccttcagatgttcac
    taaataactcagtttttaagcagaagttttcagggcatta
    aatatatgttgtgtatgaagtatctcaaactggaacataa
    atttagtgatcaaactgccattcacagtgtaaggcagcac
    ttaaatttcgaacctaaagtttagatgcattgtataaaaa
    aacctaaaagcagtatctgttatttagctgtaaaccaagt
    tggaagctattcggataatttcttaaatattgatgaactt
    tggagtactgtttcttccttcaaactgaatgtaattaatt
    catgaataaatgcaccttatatgtttaaacaatctttgta
    tacttttgggatttttggtgcttatatgctaaatcacatt
    cagcatgtgtattttgacatttaaaatacttccctcaatt
    ctgtaaatt
    12 202110_at tcagctcacttcaagggtacctgaagcgaattgg
    caccaaagcagcagctgtattgccgcagttctag
    cttcaccttcacgatgtttcccttggtcaaaagc
    gcactaaatcgtctccaagttcgaagcattcagc
    aaacaatggcaaggcagagccaccagaaacgtac
    acctgattttcatgacaaatacggtaatgctgta
    ttagctagtggagccactttctgtattgttacat
    ggacatatgtagcaacacaagtcggaatagaatg
    gaacctgtcccctgttggcagagttaccccaaag
    gaatggaggaatcagtaatcatcccagctggtgt
    aataatgaattgtttaaaaaacagctcataattg
    atgccaaattaaagcactgtgtacccattaagat
    a
    13 202157_s_at tgtggcaaaaacgtttctttcttattttttttcttttcct
    aaaacagacttgaaagtattatacagggattggcattctt
    cccggtcactggtaacaatagcaatatgtgtccagggaca
    cagaatgttggtttctaacagactacttccaaaaacagtt
    tgagaaaaaaactgtctgattttaagtctctagaggtctg
    taatagtttttacatttttcaggcagtgtaaagttttttg
    ataaggccattttaggtggctcactttctcattaagatat
    atatatagaaccactttttgtagattagtataagaaaaat
    atttaccctgttttggggcaaatgctacctatttgtgtca
    ccttttgctgaactcacagttagacaatccatggtttaat
    gcacatgaaattacctatattttatactgtttcaatgtac
    aggagaaaggttactgtaaactgtgttatgttggtgcttc
    tgtgaattaagttgtggtttcatcatgagtc
    14 202233_s_at gctcgtgttgaatctagaaccgtagccagacatgggactg
    gaggacgagcaaaagatgcttaccgaatccggagatcctg
    aggaggaggaagaggaagaggaggaattagtggatcccct
    aacaacagtgagagagcaatgcgagcagttggagaaatgt
    gtaaaggcccgggagcggctagagctctgtgatgagcgtg
    attcctctcgatcacatacagaagaggattgcacggagga
    gctctttgacttcttgcatgcgagggaccattgcgtggcc
    cacaaactctttaacaacttgaaataaatgtgtggactta
    agttgcaccccagtcttcatcatctgggcatcagaatatt
    tccttatggttttggatgtaccatttgtttcttatttgtg
    taactgtaagttcacatcaacctcatgggtttggcttgag
    gctggtagcttctatgtaattcgcaatgattccatctaaa
    15 202378_s_at acatgtgcacatgcggcattttactatgaaatttaatatg
    ctgggttttttaatacctttatatatcatgttcactttaa
    gaaagacttcataagtaggagatgagttttattctcagca
    aatagacctgtcaaatttagattatgttactcaaattatg
    ttacttgtttggctgttcatgtagtcacggtgctctcaga
    aaatatattaacgcagtcttgtaggcagctgccaccttat
    gcagtgcatcgaaaccttttgcttggggatgtgcttggag
    aggcagataacgctgaagcaggcctctcatgacccaggaa
    ggccggggtggatccctctttgtgttgtagtccatgctat
    taaaagtgtggcccacagaccaagagcctcaacatttcct
    agagccttattagaaatgcagaatctgaagccccactctg
    gacccaggacattttgatgagatccaaaggagttgtatgc
    acatgaaagtttgagaagcatcatcatagagaagtaaaca
    tcacacccaacttccttatctttccagtggctaaac
    16 202664_at gaaatcagagcttacatgtgtgtttttttataacattttc
    agataaatgtattcaacatgtaatacagtattttnacatt
    cacctcttattttatattgaaatgtattacagtattaaaa
    ctcagtgttcagtatttatttcactatgcattttatttag
    taaaagccaggagaaatgtttaatccaatggtgccttact
    ttgtgatttaaaagaaatcaacttttttttatgtctaagt
    agtagattatttgcatatttgtaaaaactgttaggtcttt
    atattttaaagtgtaataccagttttgttattttagtagc
    agaaatgggatgattgttaaagttccccaaaaatgttggc
    atgaaattaatttttccctccttatagtcaaggaccgtag
    aggaagaaaaactttttttttcatatcatgcactatgtaaa
    cagacacattttgctatctgtgtcatcaggatagtgtaag
    tggtagggtagagactaccctagacatctgcatctttgta
    agttagcca
    17 202910_s_at ctgtggccacagcagctttgtacacgaagaccatccatcc
    tcccttcgtccaccactctactccctccaccctccctccc
    tgatcccgtgtgccaccaggagggagtggcagctatagtc
    tggcaccaaagtccaggacacccagtggggtggagtcgga
    gccactggtcctgctgctggctgcctctctgctccacctt
    gtgacccagggtggggacaggggctggcccagggctgcaa
    tgcagcatgttgccctggcacctgtggccagtactcggga
    cagactaagggcgcttgtcccatcctggacttttcctctc
    atgtctttgctgcagaactgaagagactaggcgctggggc
    tcagcttccctcttaagctaagactgatgtcagaggcccc
    atggcgaggccccttggggccactgcctgaggctcacggt
    acagaggcctgccctgcctggccgggcaggaggttctc
    18 202922_at aaaaatggcgttcttctcttgtggcctgttattc
    tgattgctgctgtatacagtttngtcactcttta
    gtttttagttaagcatactgatagactttcctct
    aaaagccattcactccagattttacctggggaat
    attctacatactgcttactttctctataaaactc
    atcaataaatcatgaaaggcactgagttttgtaa
    atcaggaccctaaatgtttaattgtaaataagtt
    tcagataattattatagctttgcgttgaagtttg
    ttgt
    19 202950_at taacatgttagttgtcatttggcatgagtgtgcattccag
    taattcttaattgatatttgattaattccatacctttgat
    taaaacatgctagttcaaaataagactgctcagtttccaa
    gggttttcaagcctacttacctttataaaggttctctagt
    ctctgattagccatgactgtattggactttgaacattttc
    tgaactaaaaacctctattctaaactaatctcatttggat
    gtgtaagtcttttgtaaaggcaagaataaataatatccag
    gacaatttattagttttctcagtattttcccaaatattag
    aatatttacttcattattggttggctgccaatgaccccat
    atgttctgtgagaatagtagctttatctttgatataatac
    atagtctccaaataggtaatacttcgcaattgattagatt
    ttcagagtagatttagagttatctgtttttctggtgaggg
    tcaaat
    20 203097_s_at tttccattaaattcagctgatcatattgatcagt
    agataaacgtaaatagcttcaaattttaaaagtg
    gaattgcagtgttttttcactgtatcaaacaatg
    tcagtgctttatttaataattctcttctgtatca
    tggcatttgtctacttgcttattacattgtcaat
    tatgcatttgtaattttacatgtaatatgcatta
    tttgccagttttattatataggctatggacctca
    tgtgcatatagaaagacagaaatctagctctacc
    acaagttgcacaaatgttatctaagcattaagta
    attgtagaacataggactgctaatctcagttcgc
    tctgtgatgtcaagtgcagaatgtacaattaact
    ggtgatttcctcatacttttgatactacttgtac
    ctgtatgtcttttagaaagacattggtggagtct
    gtatcccttttgtatttttaatacaataattgta
    catattggttatatttttgttgaagatggtagaa
    atgtactatgtttatgcttctacatccag
    21 203231_s_at tgcaccaggtgaaaacagggccagcagactccatggccca
    attcggtttcttcggtggtgatgtgaaaggagagaattac
    actttttttttttttaagtggcgtggaggcctttgcttcc
    acatttgtttttaacccagaatttctgaaatagagaattt
    aagaacacatcaagtaataaatatacagagaatatacttt
    tttataaagcacatgcatctgctattgtgttgggttggtt
    tcctctcttttccacggacagtgttgtgtttctggcatag
    ggaaactccaaacaacttgcacacctctactccggagctg
    agatttcttttacatagatgacctcgcttcaaatacgtta
    ccttactgatgataggatcttttcttgtagcactatacct
    tgtgggaatttttttttaaatgtacacctgatttgagaag
    ctga
    22 203300_x_at gaacttgttcagaccgttttagcacggaaaccta
    aaatgtgcagcttccttgagtggcgagatctgaa
    gattgtttacaaaagatatgctagtctgtatttt
    tgctgtgctattgaggatcaggacaatgaactaa
    ttaccctggaaataattcatcgttatgtggaatt
    acttgacaagtatttcggcagtgtctgtgaacta
    gatatcatctttaattttgagaaggcttatttta
    ttttggatgagtttcttttgggaggggaagttca
    ggaaacatccaagaaaaatgtccttaaagcaatt
    gagcaggctgatctactgcaggaggaagctgaaa
    ccccacgtagtgttcttgaagaaattggactgac
    ataactctcctcccttgttgatgacttcttgtgg
    catttcacacactgtagatggtcactcccttcat
    gtccatgttagctcatggtgtaagatgatgtctt
    gtcagtattactgttttgctaagccgcttcattc
    atgcctacacaatttt
    23 204160_s_at ttgtggttgttgagaggcattttcaaaccctgta
    taaataatccatgctgttggtcataagttaactg
    tattaagaacagtaaaataaataaaaaccaatag
    tactaatttngctttaaaaaaatttctaattttt
    ttcacataaaacaattatcctaaaggttaatagt
    tgatcgaaacagaataatagaaaaattctncttt
    aatttccattaaaaagcaaatagcattgacacat
    ttaaagcttttcatttaaagtagtggatgttttt
    gaagtatctaaaatagtagcagaatattttatac
    ttggtccttgcaatggtgtgagttttaatgattg
    cattatcgtgattggtggttatgagtttcagaaa
    tctatacttggcatccaactcatgagtggatttt
    atataggatggaacaggaaggtatgtcctgtcag
    tatcttaaccctttcaacaagacatttacctatt
    tgtctttccttacgttctcaaaatat
    24 204750_s_at gaccctcgcgatcttaatatttgccagtgatgcctgcaaa
    aatgtgacattacatgttccctccaaactagatgccgaga
    aacttgttggtagagttaacctgaaagagtgctttacagc
    tgcaaatctaattcattcaagtgatcctgacttccaaatt
    ttggaggatggttcagtctatacaacaaatactattctat
    tgtcctcggagaagagaagttttaccatattactttccaa
    cact
    25 204777_s_at ggaagtcttcataaagccgcagtagaacttgagc
    tgaaaacccagatggtgttaactggccgccccac
    tttccggcataactttttagaaaacagaaatgcc
    cttgatggtggaaaaaagaaaacaaccacccccc
    cactgcccaaaaaaaaaagccctgccctgttgct
    cgtgggtgctgtgtttactctcccgtgtgccttc
    gcgtccgggttgggagcttgctgtgtctaacctc
    caactgctgtgctgtctgctagggtcacctcctg
    tttgtgaaaggggaccttcttgttcgggggtggg
    aagtggcgaccgtgacctgagaaggaaagaaaga
    tcctctgctgacccctggagcagctctcgagaac
    tacctgttggtattgtccacaagctctcccgagc
    gccccatcttgtgccatgttttaagtcttcatgg
    atgttctgcatgtcatggggactaaaactcaccc
    aacagatctttccagaggtccatggtgg
    26 205042_at ttacatttgaactatatccttcctagtgggttagtgtgaa
    aaagagtttggctgattcctaaaactctgccagccctgca
    gtaatctccaggcctggttattgttcagacattccatggt
    gattcctgggaaggaagcttggctgctcagtttctgagtc
    tggggtgagataatgttctggaaggacatctgttctttgg
    tgtaatctctcatggtgaaatctgctctgtacatcagaca
    attgcattgctaccaagtttcataccaa
    27 205114_s_at gattccacagaatttcatagctgactactttgagacgagc
    agccagtgctccaagcccggtgtcatcttcctaaccaagc
    gaagccggcaggtctgtgctgaccccagtgaggagtgggt
    ccagaaatatgtcagcgacctagagctgagtgcctgaggg
    gtccagaagcttcgaggcccagcgacctcggtgggccagt
    ggggaggagcaggagcctgagccttgggaaacatgcgtgt
    gacctccacagctacctcttctatggactggttgttgcca
    aacagccacactgtgggactcttcttaacttaaattttaa
    tttatttatactatttagtttttgtaatttattttcgatt
    tcacagtgtgtttgtgattgtttgctctgagagttcccct
    gtcccctcccccttccctcacaccgcgtctggtgacaacc
    gagtggctgtcatcagcctgtgtaggcagtcatggcacca
    aagccaccagactgacaaatgtgtatc
    28 205624_at tatgaaacccgctacatctatggcccaatagaatcaacaa
    tttacccgatatcaggttcttctttagactgggcttatga
    cctgggcatcaaacacacatttgcctttgagctccgagat
    aaaggcaaatttggttttctccttccagaatcccggataa
    agccaacgtgcagagagaccatgctagctgtcaaatttat
    tgccaagtatatcctcaagcatacttcctaaagaactgcc
    ctctgtttggaataagccaattaatccttttttgtgcctt
    tcatcagaaagtcaatcttcagttatccccaaatgcagct
    tctatttcacctgaatccttctcttgctcatttaagtccc
    atgttactgctgtttgcttttacttactttcagtagcacc
    ataacgaagtagctttaagtgaaaccttttaactaccttt
    ctttgctccaagtgaagtttggacccagcagaaagcatta
    ttttgaaaggtgatatacagtggggcacagaaaacaaatg
    aaaaccctcagtttctcacagattttcaccatgtggcttc
    atcaa
    29 206207_at aggagacaacaatgtccctgctacccgtgccatacacaga
    ggctgcctctttgtctactggttctactgtgacaatcaaa
    gggcgaccacttgtctgtttcttgaatgaaccatatctgc
    aggtggatttccacactgagatgaaggaggaatcagacat
    tgtcttccatttccaagtgtgctttggtcgtcgtgtggtc
    atgaacagccgtgagtatggggcctggaagcagcaggtgg
    aatccaagaacatgccctttcaggatggccaagaatttga
    actgagcatctcagtgctgccagataagtaccaggtaatg
    gtcaatggccaatcctcttacacctttgaccatagaatca
    agcctgaggctgtgaagatggtgcaagtgtggagagatat
    ctccctgaccaaatttaatgtcagctatttaaagagataa
    ccagacttcatgttgccaaggaatccctgtctctacgtga
    acttgggattcca
    30 206790_s_at gaacttacttcagattgtgcgggaccactgggttcatgtt
    cttgtccctatgggatttgtcattggatgttatttagaca
    gaaagagtgatgaacggctaactgccttccggaacaagag
    tatgttatttaaaagggaattgcaacccagtgaagaagtt
    acctggaagtaaagactggctagattatcgaatgttcaca
    ttttaaagttctgagaga
    31 207008_at acctaacgaagtatccttcagcctgaaagaggaatgaagt
    actcatacatgttacaacacggacgaaccttgaaaacttt
    atgctaagtgaaataagccagacatcaacagataaatagt
    ttatgattccacctacatgaggtactgagagtgaacaaat
    ttacagagacagaaagcagaacagtgattaccagggactg
    aggggaggggagcatgggaagtgacggtttaatgggcaca
    gggtttatgtttaggatgttgaaaaagttctgcagataaa
    cagtagtgatagttgtaccgcaatgtgacttaatgccact
    aaattgacacttaaaaatggtttaaatggtcaattttgtt
    atgtatattttatatcaatttaaaaaaaaacctgagcccc
    aaaaggtattttaatcaccaaggctgattaaaccaaggct
    agaaccacctgcc
    32 207815_at caagccctgctgtacaagaaaatcattaaggaacatttgg
    agagttagctactagctgcctaagtgtgcactttcaatct
    aactgtgaaagaatcttctgatgtttgtattatccttctt
    atattatattaacaaaataaatcaagttgtggtatagtca
    atctatttcttaataatactgcaaaaataatgctgacaca
    tcacaatttcatattttaaaatttccagaattttaagcaa
    aaagcattatgaaggaaggcttggtttaataaagactgat
    tttgttcagtgttatatgttagctgatacatatttgttca
    tttatgtgattgcagtactttatagctacatatttacctt
    gaatgttacaattagcttgccaata
    33 208051_s_at gaactcccagctaaacaaccaagacttcactgaagattta
    ttccaattctagaattgttctttttttattttttattttt
    tcaactgactaacttcattaccttaaagcctagaacatta
    ttctgctttatttatatggctttctcaattttattttgta
    gcatgggttgaatcgaactttttactagagaattttacta
    gatatttgtcattcaagttttcatctgctttataattgat
    acaccttgagggtcacttttctaatacttttacctataat
    gtggtacccacctcagcccctaataaataatatttttacc
    ctaatgtcaaatctttttcccaggctaactaaaaactgtg
    tacaaaaggattgcttgtaaatatgcatgtaaatagttct
    gttaataacccactgttttacatttggtacatctgtgtct
    gctaatacagttagctttctcacttttctgcttgtttgtt
    cagtctgaatt
    34 208161_s_at gatagcaaacactgggggcaccttaagattttgcacctgt
    aaagtgccttacagggtaactgtgctgaatgctttagatg
    aggaaatgatccccaagtggtgaatgacacgcctaaggtc
    acagctagtttgagccagttagactagtcccccggtctcc
    cgattcccaactgagtgttatttgcacactgcactgtttt
    caaataacgattttatgaaatgacctctgtcctccctctg
    atttttcatattttcctaaagtttcgtttctgttttttaa
    taaaaagctttttcctcctggaacagaagacagctgctgg
    gtcaggccacccctaggaactcagtcctgtactctggggt
    gctgcctgaatccattaaaaat
    35 208637_x_at ggtcccgaggagttcaaagcctgcctcatcagcttgggtt
    atgatattggcaacgacccccagggagaagcagaatttgc
    ccgcatcatgagcattgtggaccccaaccgcctgggggta
    gtgacattccaggccttcattgacttcatgtcccgcgaga
    cagccgacacagatacagcagaccaagtcatggcttcctt
    caagatcctggctggggacaagaactacattaccatggac
    gagctgcgccgcgagctgccacccgaccaggctgagtact
    gcatcgcgcggatggccccctacaccggccccgactccgt
    gccaggtgctctggactacatgtccttctccacggcgctg
    tacggcgagagtgacctctaatccaccccgcccggccgcc
    ctcgtcttgtgcgccgtgccctgccttgcacctccgccgt
    cgcccatctcctgcctgggttcggtttcagctcccagcct
    ccacccgggtgagctggggcccacgtggcatcgatcctcc
    ctgcccgcgaagtgacagtttac
    36 208918_s_at gaaatgggctgggagtgcttctgtcctgctgacaccccgc
    ggtgggtccctggagcgcggcctccagctgccgcaatttc
    catgccaggatatttttccgcaaatcagtcggttgaaatt
    cagaggagtcagaatgactcgacctgtccttcaatgttga
    taata
    37 208982_at attgcttgctaaagaagtggtctcctgaggtcttaagaca
    ttcctgacagtgtcttgagtgggtgggagagaggntgctg
    tcattgcgctgtggaatttcacagatgagaaccacgccta
    gccaaaatcacttttcctgtttgcctcagtgacacagctg
    cagggaccctcgtggatgttgtattaaat
    38 209009_at gagcttccccaactcataaatgccaattttccagtggatc
    cccaaaggatgtctatttttggccactccatgggaggtca
    tggagctctgatctgtgctttgaaaaatcctggaaaatac
    aaatctgtgtcagcatttgctccaatttgcaaccctgtac
    tctgtccctggggcaaaaaagcctttagtggatatttggg
    aacagatcaaagtaaatggaaggcttatgatgctacccac
    cttgtgaaatcctatccaggatctcagctggacatactaa
    ttgatcaagggaaagatgaccagtttcttttagatggaca
    gttactccctgataacttcatagctgcctgtacag
    39 209020_at caggggcagtcgttgagcctttgagaacttctgttccaag
    gctcccatcagagagtaagaaggaagactcctctgacgct
    acccaagtcccccaagcaagtctcaaagccagtgatctct
    ctgactttcaatcagtttccaagctaaaccaggggcaagc
    catgcacatgcataggcaaggaatgccagtgtaagagatg
    gcatgatatggaagtgtattccttttcaggcctgcagagt
    gtccctcccttggctccagaacgaagatccacacttgagg
    actactctcagtcgctgcacgccagaactctgtctggctc
    tccccgatcctgttctgagcaagctcgagtcttcgtggat
    gatgtgaccattgaggacctgtcaggctacatggagtatt
    acttgtatattcccaagaaaatgtcccacatgggcagaaa
    tgatgtacacctgatagcaagaagctaattcatatgcttt
    aaaccaatgaaggcttgtcaaagagatttagttaatggca
    gaccttgtggccactttgtgtgagaagacatctctttctg
    ctca
    40 209146_at gaacctcatcaattgatagcagtgagtgactgaagcttcc
    aaatcaagaaaagccggcaccaagaacttccattctaatc
    tagagctgaccagtttgagctgattctctctttgaagagt
    ccttcttgattgcagtgcagtactggcatttctgaatgga
    tgtaagtggagtattttagtctaaaggcttttcaaattac
    ttgaatttttttaaaaattgaggagctttatttctattta
    cccttccatttttgtatatcaaatttccattgtcattaaa
    aactgtatcttgaaactttgtgaactgacttgctgtattt
    gcactttgagctcttgaaa
    41 209193_at gattgtagtggatctaatttttcagaaattttgcctttaa
    gttattttacctgtttttgtttcttgttttgaaagatgcg
    cattctaacctggaggtcaatgttatgtatttatttattt
    atttatttggttcccttcctattccaagcttccatagctg
    ctgccctagttttctttcctcctttcctcctctgacttgg
    ggaccttttgggggagggctgcgacgcttgctctgtttgt
    ggggtgacgggactcaggcgggacagtgctgcagctccct
    ggcttctgtggggcccctcacctacttacccaggtgggtc
    ccggctctgtgggtgatggggaggggcattgctgactgtg
    tatataggataattatgaaaagcagttctggatggtgtgc
    cttccagatcctctctggggctgtgttttgagcagcaggt
    agcctggctggttttatctgagtga
    42 209710_at gatcccacagccctagtatgaaagctgggggtgg
    ggaggggcctttgctgcccttggtttctgggggc
    tggttggcatttgctggcctggcagggggtgaag
    gcaggagttgggggcaggtcaggaccaggaccca
    gggagaggctgtgtccctgctggggtctcaggtc
    cagctttactgtggctgtctggatccttcccaag
    gtacagctgtatataaacgtgtcccgagcttaga
    ttctgtatgcggtgacggcggggtgtggtggcct
    gtgaggggcccctggcccaggaggaggattgtgc
    tgatgtagtgaccaagtgcaatatgggcgggcag
    tcgctgcagggagcaccacggccagaagtaactt
    attttgtactagtgtccgcataagaaaaagaatc
    ggcagtattttctgtttttatgttttatttggct
    tgttttattttggattagtgaactaagttattgt
    taattatgtacaacatttatatattgtctgtaaa
    aaatgtatgctatcctcttattcct
    43 210042_s_at gaaatctacgcaaatggtcccatcagctgtggaataatgg
    caacagaaagactggcaaactacaccggaggcatctatgc
    cgaataccaggacaccacatatataaaccatgtcgtttcc
    gtggctgggtggggcatcagtgatgggactgagtactgga
    ttgtccggaattcatggggtgaaccatggggcgagagagg
    ctggctgaggatcgtgaccagcacctataaggatgggaag
    ggcgccagatacaaccttgccatcgaggagcactgtacat
    ttggggaccccatcgtttaaggccatgtcactagaagcgc
    agtttaagaaaaggcatggtgacccatgaccagaggggat
    cctatggttatgtgtgccaggctggctggcaggaactggg
    gtggctatcaatattggatggcgaggacagcgtggtactg
    gctgcgagtgttcctgagagttgaaagtgggatgacttat
    gacacttgcacagcatggctctgcctcacaatgatgcagt
    cagccac
    44 210184_at cagttctgaatatgctgctcatccccacctgtct
    tcaacagctccccattaccctcaggacaatgtct
    gaactctccagcttcgcgtgagaagtccccttcc
    atcccagagggtgggcttcagggcgcacagcatg
    agagcctctgtgcccccatcaccctcgtttccag
    tgaattagtgtcatgtcagcatcagctcagggct
    tcatcgtggggctctcagttccgattccccaggc
    tgaattgggagtgagatgcctgcatgctgggttc
    tgcacagctggcctcccgcggttgggtcaacatt
    gctggcctggaagggaggagcgccctctagggag
    ggacatggccccggtgcggctgcagctcaccagc
    cccaggggcagaagagacccaaccacttcctatt
    ttttgaggctatgaatatagtacctgaaaaaatg
    ccaagcactagattatttttttaaaaagcgtact
    ttaaatgtttgtgttaatacacattaaaacatcg
    cacaaaaacgatgcatctaccgctcc
    45 210732_s_at cccagcttcctagtaatagaggaggagacatttctaaaat
    cgcacccagaactgtctacaccaagagcaaagattcgact
    gtcaatcacactttgacttgcaccaaaataccacctatga
    actatgtgtcaa
    46 210895_s_at gtgtacataaatttgacctgctcatctatacacggttacc
    cagaacctaagaagatgagtgttttgctaagaaccaagaa
    ttcaactatcgagtatgatggtattatgcagaaatctcaa
    gataatgtcacagaactgtacgacgtttccatcagcttgt
    ctgtttcattccctgatgttacgagcaatatgaccatctt
    ctgtattctggaaactgacaagacgcggcttttatcttca
    cctttctctatagagcttgaggaccctcagcctcccccag
    accacattccttggattacagctgtacttccaacagttat
    tatatgtgtgatggttttctgtctaattctatggaaatgg
    aagaagaagaagcggcctcgcaactcttataaatgtgg
    47 211506_s_at gtgtgaaggtgcagttttgccaaggagtgctaaa
    gaacttagatgtcagtgcataaagacatactcca
    aacctttccaccccaaatttatcaaagaactgag
    agtgattgagagtggaccacactgcgccaacaca
    gaaattattgtaaagctttctgatggaagagagc
    tctgtctggaccccaaggaaaactgggtgcagag
    ggttgtggagaa
    48 211734_s_at acatctccattacaaatgccacagttgaagacag
    tggaacctactactgtacgggcaaagtgtggcag
    ctggactatgagtctgagcccctcaacattactg
    taataaaagctccgcgtgagaagtactggctaca
    attttttatcccattgttggtggtgattctgttt
    gctgtggacacaggattatttatctcaactcagc
    agcaggtcacatttctcttgaagattaagagaac
    caggaaaggcttcagacttctgaacccacatcct
    aagccaaaccccaaaaacaactgatataattact
    caagaaatatttgcaacattagtttttttccagc
    atcagcaattactactcaattgtcaaacacagct
    tgca
    49 211995_x_at agtactcggtgtggatcggtggctccatcctggcctcact
    gtccaccttccagcagatgtggattagcaagcaggagtac
    gacgagtcgggcccctccatcgtccaccgcaaatgcttct
    aaacggactcagcagatgcgtagcatttgctgcatgggtt
    aattgagaatagaaatttgcccctggcaaatgcacacacc
    tcatgctagcctcacgaaactggaataagccttcgaaaag
    aaattgtccttgaagcttgtatctgatatcagcactggat
    tgtagaacttgttgctgattttgaccttgtattgaagtta
    actgttccccttggtatttgtttaataccctgtacatatc
    tttgagttcaacctttagtacgtgtggcttggtcacttcg
    tggctaaggtaagaacgtgcttgtggaagacaagtctgtg
    gcttggtgagtctgtgtggccagcagcctctgatctgtgc
    agggtattaacgtgtcagggctgagtgttctgggatttct
    ctagaggct
    50 212314_at tattttggtacctgtgcttgccacagccctgttcctcaaa
    gctgaattgatagatttctctttgacttccaagacctagc
    agttataaggcaccttgaaataaattgtttgtgcctggaa
    atgcagggagggcaatagctttgtaaattggtttacattt
    ttctccttgaatttttctagggtcctagtgcttccgaatc
    atttaatggcattgtcggatatcttttacatttcaattgc
    aatccatgaaattacatttagaagattcttagtacttaac
    tgtagtcttctccatgaattacacgttagaatagactggc
    agcaacngaatatgcagcaagtaagcctctagcttatagt
    ttcatccctacccctcatgcctgcgtgagtctgtacaggg
    atatgtgtgtgtgtgtgtgtgtgtgtgtgttagagaggaa
    gaggaagagcagaatgtctgtatactacatgctgctaagg
    tagtgaataaatcagtaatgcaatattgtgggtccaaact
    actctttgcactact
    51 212335_at accacgcttggctaagaacacctaaatttttatg
    tttcttggctcaaaaaccagttccatttctaatg
    ttgtcctcacaagaaggctaattggtggtgagac
    agcaggggaggaggaagagctgtggtttgtaact
    tgttcaactcaggcaataagcgattttagcttta
    tttaaagtcttctgtccagctttaagcactttgt
    aagacatggctgaaagtagcttttctatcagaat
    tgcagatagtcatgttgggctaacagtcaattgg
    atatattcctttacctcacatgaccccagcaact
    gtggtggtatctagaggtgaaacaggcaagtgaa
    atggacacctctgctgtgaatgttttagagaagg
    aaattcaaaaaatgttgtaactgaaagcactgtt
    gaatatgggtatcggctttctttttcactttgac
    tcttaacattatcagtcaacttccacattaatg
    52 212386_at gagatttaccatgtatcagtgcctggctttttgt
    tataaagctttgtttgtctagtgctcttttgcta
    taaaatagactgtagtacaccctagtaggaaaaa
    aaaaaaactaaatttaaaaataaaaaatatattt
    ggcttatttttcgcaggagcaatccttttatacc
    atgaatatt
    53 212671_s_at accaatgaggttcctgaggtcacagtgttttccaagtctc
    ccgtgacactgggtcagcccaacaccctcatctgtcttgt
    ggacaacatctttcctcctgtggtcaacatcacntggctg
    agcaatgggcactcagtcacagaaggtgtttctgagacca
    gcttcctctccaagagtgatcattccttcttcaagatcag
    ttacctcaccttcctcccttctgntgatgagatttatgac
    tgcaaggtggagcactggggcctggatgagcctcttctga
    aacactgggagcctg
    54 212897_at aaggagacctttgaagctttttgagggcaaactttacctt
    tgtggtccccaaatgatggcatttctctttgaaatttatt
    agatactgttatgtcccccaagggtacaggaggggcatcc
    ctcagcctatgggnaacacccaaactaggaggggttattg
    acaggaaggaatgaatccaagtgaaggctttctgctcttc
    gtgttacaaaccagtttcagagttagctttctggggaggt
    gtgtgtttgtgaaaggaattcaagtgttgcaggacagatg
    agctcaaggtaaggtagctttggcagcagggctgatacta
    tgaggctgaaacaatccttgtgatgaagtagatcatgcag
    tgacatacaaagaccaaggattatgtatatttttatatct
    ctgtggttttgaaactttagtacttagaattttggccttc
    tgcactactcttttgctcttacgaacataatggactctta
    agaatggaaagggatgacatttacctatgtgtgctgcctc
    attcctggtgaagcaactgctacttgttctctatgcctct
    aaaat
    55 212999_x_at cactgcagaatgaaggaacatcccttgaggtgac
    ccagccaacctgtggccagaaggaggnttgtacc
    ttgaaaagacactgaaagcattttggngtgtnaa
    gtaagggtgggcagaggaggtagaaaatcaattc
    aattgtcgcatcattcatggttctttaatattga
    tgctcagtgcantggcctnagaatatcccagcct
    ctcttctggtttgntgagtgctntntaagtaagc
    atggtngaattgtttggggncanatatagtganc
    cttggtcactggtgtttcaaacattctggnaagt
    cacatcnatcaagaatantttttanttttaagaa
    agcataaccagcaataaa
    56 213309_at gtatgtagcaatcctgcgtgtgaaggcaaataaactcttt
    aacaggcaattatattgctggccaaaatatgctatatttg
    tatacaaagacattctaactcagttccagtatgaagaaag
    attattcactctagctccactgagaaacattttcctaagt
    gaaaacaatttcttaagatggaaatggattggattgtcaa
    attattatttattggagaaaaaaacctgatctacacattt
    ttacttatatggggttgccagagtctctgggttctagatg
    attttggtggcatgcttgctgagccataattactaaagag
    aatgtaagtggacgggttccctgaatccccggggtccttg
    gagagccatcgaggagaatgtgcaattggactgaagctcc
    ctggctgaagatacatgccgagtcagcacatgggtagaga
    t
    57 213506_at gcacacagagatttgagaaccattgttctgaatgctgctt
    ccatttgacaaagtgccgtgataatttttgaaaagagaag
    caaacaatggtgtctcttttatgttcagcttataatgaaa
    tctgtttgttgacttattaggactttgaattatttcttta
    ttaaccctctgagttttngtatgtattattattaaagaaa
    aatgcaatcaggattttaaacatgtaaatacaaattttgt
    ataacttttgatgacttcagtgaaattttcaggtagtctg
    agtaatagattgttttgccacttagaatagcatttgccac
    ttagtattttaaaaaataattgttggagtatttattgtca
    gttttgttcacttgttatctaatacaaaattataaagcct
    tcagagggtttggaccacatctctttggaaa
    58 213883_s_at agagaacttctagtgtatggatttaaagatttctcttttt
    cattcatataccattttatgagttctgtataattttttgt
    ggtttttgttttgttgagttaaagtatattattgtgagat
    ttatttaataggacttcctttgaaagctgtataatagtgt
    ttctcgggcttctgtctctatgagagatagcttattactc
    tgatactctttaatcttttacaaaggcaagttgccacttg
    tcatttttgtt
    59 214305_s_at gttctgacccctggaaagacaccaattggcacaccagcca
    tgaacatggctacccctactccaggtcacataatgagtat
    gactcctgaacagcttcaggcttggcggtgggaaagagaa
    attgatgagagaaatcgcccactttctgatgaggaattag
    atgctatgttcccagaaggatataaggtacttcctcctcc
    agctaattatgttcctattcgaactccagctcgaaagctg
    acagctactccaacacctttgggtggtatgactggtttcc
    acatgcaaactgaagatcgaactatgaaaagtgttaatga
    ccagccatctgg
    60 214512_s_at gaccaagagggtgttcgactgctagagccgancgaagcga
    tgcctaaatcaaaggaacttgtttcttcaagctcttctgg
    cagtgattctgacagtgaggttgacaaaaagttaaagagg
    aaaaagcaagttgctccagaaaaacctgtaaagaaacaaa
    agacaggtgagacttcgagagccctgtcatcttctaaaca
    gagcagcagcagcagagatgataacatgtttcagattggg
    a
    61 214807_at tcttcattccacgagaattttgatttttaacagcagtctc
    tctttttctcagcattgcaaatatatatgtatatatacat
    tcatgaccaaagtatcgcttactgaccatgcagctgtaaa
    ccttctgtgcctatcaaacaaatacatagcatgaanctaa
    ttttagaagtttcatgggggaattttaggggaaagtataa
    acctaagagtgagtgaatggagatgattcatggaaaaaaa
    aaanaaaaannnanatgtgctatnaggcagagttattaac
    ttcttttagttgttgtttgagatngngttctgctcttgtt
    ncccaggctggagtgcantggcgtgatctcgtctcnctgc
    aacctccgcctcccaggttcaagcgattctcctgccccag
    ctactttggaggctgaagtgtaagagttgcttgagc
    62 214953_s_at atagattctctcctgattatttatcacatagccccttagc
    cagttgtatattattcttgtggtttgtgacccaattaagt
    cctactttacatatgctttaagaatcgatgggggatgctt
    catgtgaacgtgggagttcagctgcttctcttgcctaagt
    attcctttcctgatcactatgcattttaaagttaaacatt
    tttaagtatttcagatgctttagagagattttttttccat
    gactgcattttactgtacagattgctgcttctgctatatt
    tgtgatataggaattaagaggatacacacgtttgtttctt
    cgtgcctgttttatgtgcacacattaggcattgagacttc
    aagcttttctttttttgtccacgtatctttgggtctttga
    taaagaaaagaatccctgttcattgtaagcacttttacgg
    ggcgggtg
    63 215726_s_at gacagaccaaagttaaacaagcctccggaaactcttatca
    ctactattgattctagttccagttggtggaccaactgggt
    gatccctgccatctctgcagtggccgtcgccttgatgtat
    cgcctatacatggcagaggactgaacacctcctcagaagt
    cagcgcaggaagagcctgctttggacacgggagaaaagaa
    gccattgctaactacttcaactgacagaaaccttcacttg
    aaaacaatgattttaatatatctctttctttttcttccga
    cattagaaacaaaacaaaaagaactgtcctttctgcgcnc
    aaatttttcgagtgtgcctttttattcatctactttattt
    tgatgtttccttaatgtgtaatt
    64 216016_at gccaatgctaaaaagctgcagatccagcccagccagctgg
    aattgttctactgtttgtacgagatgcaggaggaggactt
    cgtgcaaagggccatggactatttccccaagattgagatc
    aatctctccaccagaatggaccacatggtttcttcctttt
    gcattgagaactgtcatcgggtggagtcactgtccctggg
    gtttctccataacatgcccaaggaggaagaggaggaggaa
    aaggaaggccgacaccttgatatggtgcagtgtgtcctcc
    caagctcctctcatgctgcctgttctcatgggtaaggaaa
    ctcggcttccaggtgcttcctcctgcttcctcgccagctt
    cttcttggcacctgcctcctctcatctcttttcaactatc
    ttccaaatactgttgccacagctacatcataatgccacca
    ctgtctgtttgagactccttcatgagcaaagattgatgta
    tggtaggtggat
    65 217722_s_at ggagatgcctcgtgaaacacagctgggcaagtattaatgt
    atatggaacagcctggatttctgcatatggataagccacc
    ttggaataggaagaggtgttgagcctggactgtgggagga
    aagagctgcgtggatagattcaaacttcctgtggtagtgc
    tcccagtctgacctctgtagaccttcagtactcactcttc
    ttgcttaggctctctgtgtgttgaaagccatcccgtgttg
    catgtgttgttacaattttctgtgatacttgcaat
    66 217753_s_at tggtcgtgccaaaaagggccgcggccacgtgcagcctatt
    cgctgcactaactgtgcccgatgcgtgcccaaggacaagg
    ccattaagaaattcgtcattcgaaacatagtggaggccgc
    agcagtcagggacatttctgaagcgagcgtcttcgatgcc
    tatgtgcttcccaagctgtatgtgaagctacattactgtg
    tgagttgtgcaattcacagcaaagtagtcaggaatcgatc
    tcgtgaagcccgcaaggaccgaacacccccaccccgattt
    agacctgcgggtgctgccccagctcccccaccaaagccca
    tgtaaggagctgagttctt
    67 217970_s_at tgagctggagctggttggactaatgagactgagg
    aagcagcttttcctacgatctgcattatgtaatc
    acaggtccagagagctttatggaagcgggagagg
    aggagcacttactcatgttgtatttgttaatgga
    ggatgtcatcttttcatagatgctagaactagag
    tgcacttgttagatgctaaaggtttgagctttac
    acaaaatgtcttcatctgtatttgttattgtcta
    caatatatttgaatttggggcagcatattaagat
    gtaatggcctgttatgtcttgaaaatacttgttt
    tgcctcttccaggcatactgcattctgtggatca
    gtttgaacagcttctccaccttatttggacagtg
    ataaattgaaccaagagtgtagatttacaagtgt
    aaccttcaaaagaggaagaactatttggggtctg
    taggtaatgaacagtcacaccaaaatagactatg
    atgcttttgttaagaaaggtttcatgttttagat
    attttccgtgtcctaaataatt
    68 218190_s_at tgaagaaacatggcggccgcgacgttgacttcgaaattgt
    actccctgctgttccgcaggacctccaccttcgccctcac
    catcatcgtgggcgtcatgttcttcgagcgcgccttcgat
    caaggcgcggacgctatctacgaccacatcaacgagggga
    agctgtggaaacacatcaagcacaagtatgagaacaagta
    gttccttggaggcccccatccaggccagaaggaccaggtc
    cacccagcagctgtttgcccagagctggagcctcagcttg
    aagatgatgctcaaggtactcttcatggaccaccattcgc
    tgttggcaagaaacggctttacttacaaaacagactcttt
    accttctgctgtgtttgaa
    69 218345_at gatttccgatatggctactcttattacaacagtgcctgcc
    gcatctccagctcgagtgactggaacactccagcccccac
    tcagagtccagaagaagtcagaaggctacacctatgtacc
    tccttcatggacatgctgaaggccttgttcagaacccttc
    aggccatgctcttgggtgtttggattctgctgcttctggc
    atctctggcccctctgtggctgtactgctggagaatgttc
    ccaaccaaagggaaaagagaccagaaggaaatgttggaag
    tgagtggaatctagccatgcctctcctgattattagtgcc
    tggtgcttctgcaccgggcgtccctgcatctgactgctgg
    aagaagaaccagactgaggaaaagaggcttttcaacagcc
    ccagttatcctggccccatgaccgtggccacagccctgct
    ccagcagcacttgcccattccttacaccccttccccatcc
    tgctccgcttcatgtcccctcctgagtagtcatgtgataa
    taaactctcatgttattgttcccag
    70 218486_at gactgtacacactataaatggcatcaaatttggatatttt
    tcttaattatgacatgcaaagtaatgtgagtcctgccagt
    attctggtggataaggtcttntgagtatttggttgcttgt
    cacaacattctccaagcagtgatatttctaaagaggagat
    acatgttgaaaacggttttaatttacacttccatttcctg
    attacatttggaaatactttgtgtaaaccatcccccttcc
    acctccatttgtctgttgaaagattttaagttggaaacag
    ttcctgtctgaaaactcttctgagaaccacaaaccttgtg
    tatggattcggcatggagccctcagctggcggctctgggt
    gctgacggccgctggagaggtgggctcccctcgtgcactt
    tattgcctgggcagttttgcttgatcttttgtgactttga
    gccttttaagtagtttgaatgataagacttaaaatgtttc
    ataattatgttttatgtaacagactttgacattatttaaa
    cgagcatgtgtaatgtaacttttctctttgaatcata
    71 218545_at agcatggatttcaacatcacttatttatctgtataattgg
    aaataaaacaccgatatgatagagaatcattccggcatta
    cctaacctcttctgcagttggatctatgtattttcattgg
    tctactgaaaacgaacaatacaattaaaagcactaaagat
    tattatattaattcaactttgatctgatatatcacttaaa
    ctaaaggggtgtgtgtggtgtatgcttgtttcctatttct
    gctctttaaagatactttgaatcaataaaaccattagtct
    acaaatcaaattgtgaacttaatctctagaaagagaatat
    aactcagccatttataggaatttaggttcaagtacaggat
    atatgaaatcttttcccagtatttcagaatgtacttaatt
    cacaggcaggatgcttcaatgcaaaatcatgaatattttt
    aattcaaaactaaaatgtcattaatatgtatgtatgcaaa
    tgttttatcttattttctgaaatgcatctactttcatggg
    ctttgtacgtttctgagattt
    72 218728_s_at atatcgatacattatggtgccgagtggtaacatgggagtg
    tttgatccaacagaaatacacaatcgagggcagctgaagt
    cacacatgaaagaagccatgatcaagcttggtttccactt
    gctctgcttcttcatgtatctttatagtatgatcttagct
    73 219269_at aggctagaaaatcttgctgctccgtcttagcattccaaga
    gagtgcttccaggtatttagatagccctcagttctcaaat
    attagactacgtgtaaaatcttgggtacctttagattctt
    gtaacactagtctgtactcccttttccttccccaagactg
    ataggatgcaagctgaggtcgtggcacaggaatgacagac
    accatttggggagtatccacagagtcaaaggaacactaga
    atccccacctcagcgtgaggataattgatttccagctgca
    ataagccgtgcctcattatagccacactgtggctagatta
    tacttctttgggtgctgtgctaagaatgtcaatggaaaaa
    gccgatctcagattttgtttgaagttaacatgcctgacac
    agacatcctttcctctcacaagctgtgtgacttagtagat
    aaaatactgccttctgccttt
    74 219410_at tgtttctcaccatatgcttttgttggcattatgcagtaac
    cattgtcatcgttggaatgaattatgctttcattacctgg
    ttggttaaatctagacttaagaggctctgctcctcagaag
    ttggacttctgaaaaatgctgaacgagaacaagaatcaga
    agaagaaatgtgactttgatgagcttccagtttttctaga
    taaaccttttcttttttacattgttcttggttttgtttct
    cgatcttttgtttggagaacagctggctaaggatgactct
    aagtgtactgtttgcatttccaatttggttaaagtatttg
    aatttaaatattttctttttagctttgaaaatattttggg
    tgatactttcattttgcacatcatgcacatcatggtattc
    aggggctagagtgatttttttccagattatctaaagttgg
    atgcccacactatgaaagaaatatttgttttatttgcctt
    atagatatgctcaaggttactgggcttgctactatttgta
    actccttgaccatggaattatacttgtttatcttgttgct
    gca
    75 219862_s_at cggccaaggagctgttcaacgaggatgtggagga
    ggtcacttaccgagccctgagaaacaaagacttc
    caagaggtcacccttgagaagaacggagaggtgg
    tgttacgctttgctgcagcctatggctttcgaaa
    catccagaacatgatcctgaagcttaagaagggc
    aagttcccattccactttgtggaggtcctcgcct
    gtgctggaggatgcttaaatggcagaggccaagc
    ccagactccagacggacatgcggataaggccctg
    ctgcggcagatggaaggcatttacgctgacatcc
    ctgtgcggcgtccggagtccagtgcacacgtgca
    ggagctgtaccaggagtggctggaggggatcaac
    tcccccaaggcccgagaggtgctgcataccacgt
    accagagccaggagcgtggcacacacagcctgga
    catcaagtggtgaagtcaggccagggcctt
    76 219905_at ggctcacgtgattatggaggcggttaaatcccaa
    gatctgcaggataagtcagcaagatggagtccca
    tgagagctgatggtttagttccagtctgatggca
    gcaggcttgacgccaaggaagagatgatgtttaa
    ttcaagtccgaaggcaaggaaaaagctgatggtc
    ctgtccaaaggctattaggcaggaagaattctct
    tagggcagagttagctcttttgttctattcaggc
    cttcaactgattcagcaaggcccgcccacatttg
    ggaggacagtctgcattactcagtctactgattt
    gaatgttaatgtcattgcgaaacaccctcacagg
    aacactcagaataatgtttgaccaaatagctggg
    catcttgtgacccagttaagtg
    77 220532_s_at atctggcaaactgaaccctttttatacatcgacactgtgt
    gtgatcgctcagaccctgtcttccctaccactgggtacag
    atggatgcggcgaagtcaagagaaccaatggcagaaggag
    gagtgtagagcttacatgcagatgctgaggaagttgttca
    cagcaatccgtgccctgttcctggctgtctgtgtcttgaa
    ggtcattgtgtccttggtttccttgggagtaggtcttcga
    aacttgtgtggccagagctcccagcccctgaatgaggaag
    gatcagagaagaggctactgggggagaattcagtgccccc
    ttcgccctctagggagcagacctccactgccattgtcctg
    tgagccgccaaagaccccacggggtgcccgcatgtccctg
    tctagggcagcccagggcccccactcctggctcctcacac
    ttgcctcccctatggccgctctccagaccctcctcctttc
    ttctccccacatccgcacctgctgttcccactctggggtt
    ctcaagtccatga
    78 221011_s_at gagtggttcatccatactctcattccctcgcctccccttg
    tggacgggggtcttgccttttcaattcctgtgttttggtg
    tcttcccttatctgctaccctgaatcacctgtcctggtct
    tgctgtgtgatgggaacatgcttgtaaactgcgtaacaaa
    tctactttgtgtatgtgtctgtttatgggggtggtttatt
    atttttgctggtccctagaccactttgtatgaccgtttgc
    agtctgagcaggccaggggctgacagctaatgtcaggacc
    ctcagcggtggagcctgctggggggacccagctgctcttg
    gacaagtggctgagctcctatctggcctcctctttttttt
    ttttttcaagtaatttgtgtgtatttctaactgattgtat
    tgaaaaaattcctagtatttcagtaaaaatgcctgttgtg
    agatgaacctcctgtaacttctatctgttcttttttgagg
    ctcaggga
    79 221042_s_at tgaccccatggacagccatcagtcccaggaatcc
    ccaaacctggaaaacatagcaaaccccctagaag
    aaaatgtaacgaaagaatcaatcagtagtaaaaa
    aaaggaaaaaaggaaacatgtggaccacgtagaa
    agttcactatttgtagcaccaggaagtgttcaat
    cctcagatgacctagaagaagacagtagcgacta
    cagcattccttccaggactagtcacagtgactcc
    agcatttaccttcgacgacatactcataggtctt
    cggaatcggatcattttagctatgttcagttgag
    gaacgcagcagatctggatgacagaagaaaccga
    atattaaccaggaaggccaacagctcaggagaag
    ccatgtcactggggagccacagcccgcagagtga
    ctccctgacacagcttgtccagcagccggatatg
    atgtattttattctcttcctgtggctcctggtgt
    actgcttgctgctcttcccacaactggatgttag
    caggctctgatacgtg
    80 221434_s_at cctcagcagcgcgaggtgctgcggcgctgcgtagaagtat
    caatcagccggttgcttttgtgagaagaattccttggact
    gcggcgtcgagtcagctgaaagaacactttgcacagttcg
    gccatgtcagaaggtgcattttaccttttgacaaggagac
    tggctttcacagaggtttgggttgggttcagttttcttca
    gaagaaggacttcggaatgcactacaacag
    81 221737_at gaaatctatttttaactcttatgttcgtagagaaattgtt
    tcaaggattttgagtcataggtctgtaatttatagagatc
    tctagaattcttattgtaattttcctacttctttgataaa
    agaaaaataagtcagattgttaactccaagattgaaaaaa
    aaaactcttgaaagaagattattagttgtaactaatttgg
    gggttctgggcacagacatctaacctggtattgtaaggca
    gaggctcccattggaatggtagtggtccgggtcagttgtt
    catggtgtaagctttgcacagtgtattaacattgggaggg
    tctggcttgaaaatttggccaccctcagcc
    82 AFFX- tagttttaccctactgatgatgtgttgttgccat
    M27830_5_at ggtaatcctgctcagtacgagaggaaccgcaggt
    tcagacatttggtgtatgtgcttggctga
    83 36711_at ttgcacggatctaagttattctccccagccagag
    cccgngctnnctgctcccngggaaaagntggcgt
    antggncctgagctgggntttatattttatatct
    gcaaataaatnacattttatcntanatttaggga
    aagccngagagnaacaacaaaaaatgtttaagcc
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnntattgcccggc
    tcctagaatttatttatttcctgacttacagcaagcgagttatcgtcttc
    tgtattttg
    84 38037_at ccactctatgagttggacttcagtcttgcctaggcgattt
    tgtctaccatttgtgttttgaaagcccaaggtgctgatgt
    caaagtgtaacagatatcagtgtctccccgtgtcctctcc
    ctgncaagtctcagaagaggttgggcttccatgcctgtag
    ctttcctggtccctcacccccatngccccaggcccacagc
    gtgggaactcactttcccttgtgtcaagacatttctnnnn
    nnnnnnnnnnnnnnnnnnnnnnactccatgcaggggtcag
    tgcagcagaggacagtctggagaaggtattagcaaagcaa
    aaggctgagaaggaacagggaacattggagctgactgttc
    ttggtaactgattacctgccaattgctaccgagaaggttg
    gaggtggggaaggctttgtataatcccacccacctcacca
    aaacgannnnnnnnnnnnnnnnnngtcctttctggaagtt
    tctggtgccatttc
    85 201386_s_at Ggagatcatctgacactgctgaacgtctaccatgctttta
    aacaaaatcatgaatcggttcagtggtgttatgacaactt
    cattaactacaggtccctgatgtccgcagacaatgtacgc
    cagcagctatctcgaattatggacagatttaatttgcctc
    gtcgaagtactgactttacaagcagggactattatattaa
    tataagaaaagctttggttactgggtattttatgcaggtg
    gcacatttagaacgaacagggcattacttaactgtgaaag
    ataaccaggtggttcagttgcatccctctactgttcttga
    ccacaaacctgaatgggtgctttataatgagtttgttcta
    acaacaaagaattacatccggacatgtacagatatcaagc
    cagaatggttggtgaaaattgcccctcaatattatgacat
    gagcaatttcccacagtgtgaagcaaagagacagttggac
    cgcatcattgcc
    86 201890_at gctactttgaattaatctgcctttatgtttgggagaagaa
    agctgagacattgcatgaaagatgatgagagataaatgtt
    gatcttttggccccatttgttaattgtattcagtatttga
    acgtcgtcctgtttattgttagttttcttcatcatttatt
    gtatagacaatttttaaatctctgtaatatgatacatttt
    cctatcttttaagttattgttacctaaagttaatccagat
    tatatggtccttatatgtgtacaacattaaaatgaaaggc
    tttgtcttgcattgtgaggtacaggcggaagttggaatca
    ggttttaggattctgtctctcattagctgaataatgtgag
    gattaacttctgccagct
    87 202219_at aagcttcgagctgttgcgtgtgtgagtctgttgtgtggat
    gtgcgtgtgtggtccccagccccagactggattggaaaag
    tgcatggtgggggcctcggggctgtccccacgctgtccct
    ttgccacaagtctatggggcaagaggctgcaatattccgt
    cctgggtgtctgggctgctaacctggcctgctcaggcttc
    ccaccctgtgcggggcacacccccaggaagggaccctgga
    cacggctcccacgtccaggcttaaggtggatgcacttccc
    gcacctccagtcttctgtgtagcagctttaacccacgttt
    gtctgtcacgtccagtcccgagacggctgagtgaccccaa
    gaaaggcttccccgacacccagacagaggctgcagggctg
    gggctgggtgagggtggcgggcctgcggggacattctact
    gtgctaaa
    88 202464_s_at tattctgtcctgagaccacgggcaaagctcttccattttg
    agagagaagaaaaactgtttggaaccacaccaatgatatt
    tttctttgtaatacttgaaatttatttttttattattttg
    atagcagatgtgctatttatttatttaatatatgtataag
    gagtcctaaacaatagaaagctgtagaagctgtagagata
    ggcttcagttgttaattggtttggagcctcctatgtgtga
    cttatgactctctgtgttctgtgtatttgtctgaattaat
    gacctgggatataaagctatgctagctttcaaacaggaga
    tgccttcagaaagctttgtatattttgcagttgccagacc
    aataaaatacc
    89 203115_at aactccaattcaggagcccttgcgagtatatctgaagcac
    ttatttgctaaggaaacctgaattgatancagtactgtgc
    tgtctggaataatgtccttgatactgagttgggaccagac
    tggcttttatagtgacaggcaaagaggaatttattgagat
    cactgctcatggcatttgttgctgtaagaagtgttgcctt
    tgattgttactaaccacggatgggtaacggtcatacatta
    ggctagtgtttggtaggacaaaatctttttagagctttga
    gaattgtcatcctgttggtcaactttgaaatacaaatgtt
    tgccctggtaattagcaatgaactgctggcagtttcttca
    gctgtgtatatacggatctggcttttaattgatgaatcaa
    cttctacagaaacttttgcagggacagtgttgatgaggca
    gtttagcttgccagggtgatgataaagcccaggtccctgc
    atgtatagtgctcttctaaagaatatgcattcttgaacta
    cttaactttt
    90 203574_at gtatggtacattcttctcttactcctttctcagtgcaagt
    gactaacattcaagattggtctctcaaatcggagcactgg
    catcaaaaagaactgagtggcaaaactcagaatagtttca
    aaactggagttgttgaaatgaaagacagtggctacaaagt
    ttctgacccagagaacttgtatttgaagcaggggatagca
    aacttatctgcagaggttgtctcactcaagagacttatag
    ccacacaaccaatctctgcttcagactctgggtaaattac
    tactgagtaagagctgggcatttagaaagatgtcatttgc
    aatagagcagtccattttgtattatgctgaattttcactg
    gacctgtgatgtcatttcactgtgatgtgcacatgttgtc
    tgtttggtgtctttttgtgcacagattatgatgaagatta
    gattgtgttatcactctgcctgtgtatagtcagatagtcc
    atgcgaaggctgtatat
    91 203887_s_at cagggggtgtgtctgctcagtaatttgaggacaaccattc
    cagactgcttccaattttctggaatacatgaaatatagat
    cagttataagtagcaggccaagtcaggcccttattttcaa
    gaaactgaggaattttctttgtgtagctttgctctttggt
    agaaaaggctaggtacacagctctagacactgccacacag
    ggtctgcaaggtctttggttcagctaagctaggaatgaaa
    tcctgcttcagtgtatggaaataaatgtatcatagaaatg
    taacttttgtaagacaaaggttttcctcttctattttgta
    aactca
    92 203932_at tcttacggggacacttacacctgtgtggtagagcacattg
    gggctcctgagcccatccttcgggactggacacctgggct
    gtcccccatgcagaccctgaaggtttctgtgtctgcagtg
    actctgggcctgggcctcatcatcttctctcttggtgtga
    tcagctggcggagagctggccactctagttacactcctct
    tcctgggtccaattattcagaaggatggcacatttcctag
    aggcagaatcctacaacttccactccaagtgagaaggaga
    ttcaaactcaatgatgctaccatgcctctccaacatcttc
    aaccccctgacattatcttggatcctatggtttctccatc
    caattctttgaatttcccagtctcccctatgtaaaactta
    gcaacttgggggacctcattcctgggactatgctgtaacc
    aaattattgtccaaggctatatttctgggatgaatataat
    ctgaggaagggagttaaagaccctcctggggctctcagtg
    tgccatagaggac
    93 204131_s_at ttaccattccgggttcgagcatcacaagcttttgagcgca
    tggaactccataaactaacaaattacataaactaaagggg
    gattttctttcttcttttgtttggtagaaaattatccttt
    tctaaaaactgaacaatggcacaattgtttgctatgtgca
    cccgtccaggacagaaccgtgcataggcaaaaggagtgga
    gcacagcgtccggcccagtgtgtttccggttctgagtcag
    ggtgatctgtggacgggaccccagcaccaagtctacgggt
    gccagatcagtagggcctgtgatttcctgtcagtgtcctc
    agctaatgtgaacagtgttggtctgctggttagaaactag
    aatattgatattttcaggaaagaaatcagctcagctctcc
    actcattgccaaatgtcac
    94 204419_x_at acactcgcttctggaacgtctgaggttatcaataagctcc
    tagtccagacgccatgggtcatttcacagaggaggacaag
    gctactatcacaagcctgtggggcaaggtgaatgtggaag
    atgctggaggagaaaccctgggaaggctcctggttgtcta
    cccatggacccagaggttctttgacagctttggcaacctg
    tcctctgcctctgccatcatgggcaaccccaaagtcaagg
    cacatggcaagaaggtgctgacttccttgggagatgccat
    aaagcacctggatgatctcaagggcacctttgcccagctg
    agtgaactgcactgtgacaagctgcatgtggatcctgaga
    acttcaagctcctgggaaatgtgctggtgaccgttttggc
    aatccatttcggcaaagaattcacccctgaggtgcaggct
    tcctggcagaagatggtgactggagtggccaatgccctgt
    cctccagataccactgagctcactgcc
    95 204467_s_at ctcggaattccctgaagcaacactgccagaagtgtgtttt
    ggtatgcactggttccttaagtggctgtgattaattattg
    aaagtggggtgttgaagaccccaactactattgtagagta
    gtctatttctcccttcaatcctgtcaatgtttgctttatg
    tattttggggaactgttgtttgatgtgtatgtgtttataa
    ttgttatacatttttaattgagccttttattaacatatat
    tgttatttttgtctcgaaataattttttagttaaaatcta
    ttttgtctgatattggtgtgaatgctgta
    96 204848_x_at acactcgcttctggaacgtctgagattatcaataagctcc
    tagtccagacgccatgggtcatttcacagaggaggacaag
    gctactatcacaagcctgtggggcaaggtgaatgtggaag
    atgctggaggagaaaccctgggaaggctcctggttgtcta
    cccatggacccagaggttctttgacagctttggcaacctg
    tcctctgcctctgccatcatgggcaaccccaaagtcaagg
    cacatggcaagaaggtgctgacttccttgggagatgccat
    aaagcacctggatgatctcaagggcacctttgcccagctg
    agtgaactgcactgtgacaagctgcatgtggatcctgaga
    acttcaagctcctgggaaatgtgctggtgaccgttttggc
    aatccatttcggcaaagaattcacccctgaggtgcaggct
    tcctggcagaagatggtgactgcagtggccagtgccctgt
    cctccagataccactgagcctcttgcccatgattcagagc
    t
    97 205239_at atttcaaaatttctgcattcacggagaatgcaaatatata
    gagcacctggaagcagtaacatgcaaatgtcagcaagaat
    atttcggtgaacggtgtggggaaaagtccatgaaaactca
    cagcatgattgacagtagtttatcaaaaattgcattagca
    gccatagctgcctttatgtctgctgtgatcctcacagctg
    ttgctgttattacagtccagcttagaagacaatacgtcag
    gaaatatgaaggagaagctgaggaacgaaagaaacttcga
    caagagaatggaaatgtacatgctatagcataactgaaga
    taaaattacaggatatcacattggagtcactgccaagtca
    tagccataaatgatgactcggtcctctttccagtggatca
    taagacaatggaccctttttgttatgatggttttaaactt
    tcaattgtcactttttatgctatttctgtata
    98 205571_at gatcaggagcaatgccactgctagcataccttccttagtg
    aaaaatcttttggaaaaggatcccactctgacctgtgaag
    tactaatgaatgctgttgctacagagtatgctgcctatca
    tcaaattgataatcacattcacctaataaacccaacggat
    gagacactgtttcctggaataaatagcaaagccaaagaac
    tgcaaacttgggagtggatatatggcaaaactccaaagtt
    tagtataaatacttcctttcatgtgttatatgaacagtca
    cacttggaaattaaagtattcatagacataaagaatggaa
    gaattgaaatttgtaatattgaagcacctgatcattggtt
    gccattggaaatacgtgacaaattaaattcaagtcttatt
    ggcagtaagttttgcccaactgaaactaccatgctaacaa
    atatattacttagaacatgtccacaagaccacaaact
    99 205592_at tgggggccacagactcaacatgtgtgtgtggtggggttcc
    agcccaacatagagtaacattatttgtacctcccaggcta
    gctcagtccatgggaggctctcctgtccctgaaagctgac
    acccacctttcaccacttcgcccatgctacagttcagttt
    cctcgtctgtaaaatggggatgataatggtacctaccttg
    cagtgttgttataaggattaaaggagacagtgcaagaaaa
    ggccttggttggtgaagagcccaacctcggaggggagctg
    ctgggatcctccttatcttgactgggatgtccctgtctcc
    ccctccccttgctccttgaacatggccaaggaaagtgaaa
    aacaaaaattattcactctgctagcacccttccccttgat
    gcctgggaataggttttgccaa
    100 205863_at tagctccacattcctgtgcattgaggggttaacattaggc
    tgggaagatgacaaaacttgaagagcatctggagggaatt
    gtcaatatcttccaccaatactcagttcggaaggggcatt
    ttgacaccctctctaagggtgagctgaagcagctgcttac
    aaaggagcttgcaaacaccatcaagaatatcaaagataaa
    gctgtcattgatgaaatattccaaggcctggatgctaatc
    aagatgaacaggtcgactttcaagaattcatatccctggt
    agccattgcgctgaaggctgcccattaccacacccacaaa
    gagtaggtagctctctgaaggctttttacccagcaatgtc
    ct
    101 205900_at tcaagtcctctggtggcagttccagcgtgaggtttgtttc
    taccacttattccggagtaaccagataaagagatgccctc
    tgtttcattagctctagttctcccccagcatcactaacaa
    atatgcttggcaagaccgaggtcgatttgtcccagcctta
    ccggagaaaagagctatggttagttacactagctcatcct
    attcccccagctctttcttttctgctgtttcccaatgaag
    ttttcagatcagtggcaatctcagttcccttgctatgacc
    ctgctttgttctttcccgagaaacagttcagcagtgacca
    ccacccacatgacatttcaagcaccaccttaagccagcca
    gagtaggaccagttagacctagggtgtggacagctccttg
    catcttaacactgtgc
    102 205950_s_at gagccccattcacaaattttgacccctctactctccttcc
    ttcatccctggatttctggacctaccctggctctctgact
    catcctcctctttatgagagtgtaacttggatcatctgta
    aggagagcatcagtgtcagctcagagcagctggcacaatt
    ccgcagccttctatcaaatgttgaaggtgataacgctgtc
    cccatgcagcacaacaaccgcccaacccaacctctgaagg
    gcagaacagtgagagcttcattttgatgattctgagaaga
    aacttgtccttcctcaagaacacagccctgcttctgacat
    aatccagttaaaataataatttttaagaaataaatttatt
    tcaatattagcaagacagcatgccttcaaatcaatctgta
    aaactaagaaacttaaattttagttcttactgcttaattc
    aaataataattagtaagctagcaaatagtaatctgtaagc
    ataagcttatcttaaattcaagtttagtttgaggaattct
    ttaaaattacaactaagtgatttgtatgtctatttttttc
    103 205987_at ccagctgttgctggtttgtcatgcctccggcttctaccca
    aagcctgtttgggtgacatggatgcggaatgaacaggagc
    aactgggcactaaacatggtgatattcttcctaatgctga
    tgggacatggtatcttcaggtgatcctggaggtggcatct
    gaggagcctgctggcctgtcttgtcgagtgagacacagca
    gtctaggaggccaggacatcatcctctactggggacacca
    ctcttccatgaattggattgccttggtagtgatagtgccc
    ttggtgattctaatagtccttgtgttatggtttaagaagc
    actgctcatatcaggacatcctgtgagactcttccccctg
    actcccccattgtgttaagaacccagcaacccaggagcct
    agtacaatatagtgatgccatcccgtcgactctccattta
    aattgtt
    104 206025_s_at ggataccccattgtgaagccagggcccaactgtggatttg
    gaaaaactggcattattgattatggaatccgtctcaatag
    gagtgaaagatgggatgcctattgctacaacccacacgca
    aaggagtgtggtggcgtctttacagatccaaagcaaattt
    ttaaatctccaggcttcccaaatgagtacgaagataacca
    aatctgctactggcacattagactcaagtatggtcagcgt
    attcacctgagttttttagattttgaccttgaagatgacc
    caggttgcttggctgattatgttgaaatatatgacagtta
    cgatgatgtccatggctttgtgggaagatactgtggagat
    gagcttccagatgacatcatcagtacaggaaatgtcatga
    ccttgaagtttctaagtgatgcttcagtgacagctggagg
    tttccaaatcaaatatgttgcaatggatcctgtatccaaa
    tccagtcaag
    105 206111_at Gtttacctgggctcaatggtttgaaacccagcac
    atcaatatgacctcccagcaatgcaccaatgcaa
    tgcaggtcattaacaattatcaacggcgatgcaa
    aaaccaaaatactttccttcttacaacttttgct
    aacgtagttaatgtttgtggtaacccaaatatga
    cctgtcctagtaacaaaactcgcaaaaattgtca
    ccacagtggaagccaggtgcctttaatccactgt
    aacctcacaactccaagtccacagaatatttcaa
    actgcaggtatgcgcagacaccagcaaacatgtt
    ctatatagttgcatgtgacaacagagatcaacga
    cgagaccctccacagtatccggtggttccagttc
    acctggatagaatcatctaagctcctgtatcagc
    actcctcatcatcactcatctgccaagctcctcaatcatagcca
    agatcccatctctccatatactttggg
    106 206834_at Ggagaagactgctgtcaatgccctgtggggcaaa
    gtgaacgtggatgcagttggtggtgaggccctgg
    gcagattactggtggtctacccttggacccagag
    gttctttgagtcctttggggatctgtcctctcct
    gatgctgttatgggcaaccctaaggtgaaggctc
    atggcaagaaggtgctaggtgcctttagtgatgg
    cctggctcacctggacaacctcaagggcactttt
    tctcagctgagtgagctgcactgtgacaagctgc
    acgtggatcctgagaacttcaggctcttgggcaa
    tgtgctggtgtgtgtgctggcccgcaactttggc
    aaggaattcaccccacaaatgcaggctgcctatc
    agaaggtggtggctggtgtggctaatgccctggc
    tcacaagtaccattgagatcctggactgtttcct
    gataaccataagaagaccctatttccctagattc
    tattttctgaacttgggaacacaatgcctacttcaagggtatggcttc
    107 207332_s_at aaggagagtcccctgaaggtctgacacgtctgcctaccca
    ttcgtggtgatcaattaaatgtaggtatgaataagttcga
    agctccgtgagtgaaccatcatataaacgtgtagtacagc
    tgtttgtcatagggcagttggaaacggcctcctagggaaa
    agttcatagggtctcttcaggttcttagtgtcacttacct
    agatttacagcctcacttgaatgtgtcactactcacagtc
    tctttaatcttcagttttatctttaatctcctcttttatc
    ttggactgacatttagcgtagctaagtgaaaaggtcatag
    ctgagattcctggttcgggtgttacgcacacgtacttaaa
    tgaaagcatgtggcatgttcatcgtataacacaatatgaa
    tacagggcatgcattttgcagcagtgagtctcttcagaaa
    acccttttctacagttagggttgagttacttcctatcaag
    ccagtacgtgctaacaggctcaatattcctgaatgaaata
    tcagactagtgacaagctcctggtcttgagatgtcttctc
    108 208632_at aacccagaatggcacacactgctctgctgtagcatcatgt
    cagggcttcctggactcagtacacctctcagtttgtcttt
    taaaaaacagctgaatctttactacctatttagttctcct
    tgttaaagaaacaggggtgggaataaaatggatttaggna
    cacccagtttgaattgcagtttttttttttctgacacatg
    gccaggctgtggtgccagcttaatggagtaggctgtcctt
    ggcacttgcatgtgtgaaaggagggttttgcctcttcttg
    agcatggcttgagttggtaaggaaagctgtaactcacgaa
    gccctgagacctgctacccctaagatcgagcttgttttca
    gtgactggcttgagtcataggaggaggagtctggtacagc
    tgcaggagagcagggccatctgaagcggtagcattgccac
    catctccctctcatctagagcagttttcttatgccttggt
    tt
    109 209007_s_at gaagatggaactcgaaatcccaatgaaaaacctacccagc
    aaagaagcatagcttttagctctaataattctgtagcaaa
    gccaatacaaaaatcagctaaagctgccacagaagaggca
    tcttcaagatcaccaaaaatagatcagaaaaaaagtccat
    atggactgtggatacctatctaaaagaagaaaactgatgg
    ctaagtttgcatgaaaactgcactttattgcaagttagtg
    tttctagcattatcccatccctttgagccattcaggggta
    cttgtgcatttaaaaaccaacacaaaaagatgtaaatact
    taacactcaaatattaacattttaggtttctcttgcagat
    atgagagatagcacagatggaccaaaggttatgcacaggt
    gggagtcttttgtatatagttgtaaatattgtcttggtta
    tgtaaaaatgaaattttttagacacagtaattgaactgta
    ttcctgttttgtat
    110 209458_x_at agagaacccaccatggtgctgtctcctgccgacaagacca
    acgtcaaggccgcctggggtaaggtcggcgcgcacgctgg
    cgagtatggtgcggaggccctggagaggatgttcctgtcc
    ttccccaccaccaagacctacttcccgcacttcgacctga
    gccacggctctgcccaggttaagggccacggcaagaaggt
    ggccgacgcgctgaccaacgccgtggcgcacgtggacgac
    atgcccaacgcgctgtccgccctgagcgacctgcacgcgc
    acaagcttcgggtggacccggtcaacttcaagctcctaag
    ccactgcctgctggtgaccctggccgcccacctccccgcc
    gagttcacccctgcggtgcacgcctccctggacaagttcc
    tggcttctgtgagcaccgtgctgacctccaaataccgtta
    agctg
    111 209795_at tagtctaattgaatcccttaaactcagggagcatttataa
    atggcaaatgcttatgaaactaagatttgtaatatttctc
    tctttttagagaaatttgccaatttactttgttatttttc
    cccaaaaagaatgggatgatcgtgtatttatttttttact
    tcctcagctgtagacaggtccttttcgatggtacatattt
    ctttgcctttataatcttttatacagtgtcttacagagaa
    aagacataagcaaagactatgaggaatatttgcaagacat
    agaatagtgttggaaaatgtgcaatatgtgatgtggcaaa
    tctctattaggaaatattctgtaatcttcagacctagaat
    aatactagtcttataataggtttgtgactttcctaaatca
    attctattacgtgcaatacttcaatacttcat
    112 210027_s_at Tgggatgaagcctttcgcaagttcctgaagggcc
    tggcttcccgaaagccccttgtgctgtgtggaga
    cctcaatgtggcacatgaagaaattgaccttcgc
    aaccccaaggggaacaaaaagaatgctggcttca
    cgccacaagagcgccaaggcttcggggaattact
    gcaggctgtgccactggctgacagctttaggcac
    ctctaccccaacacaccctatgcctacacctttt
    ggacttatatgatgaatgctcgatccaagaatgt
    tggttggcgccttgattactttttgttgtcccac
    tctctgttacctgcattgtgtgacagcaagatcc
    gttccaaggccctcgcgagtgatcactgtcctat
    caccctatacctagcactgtgacaccacccctaa
    atcactttgagcctgggaaataagccccctcaac
    taccattccttctttaaacactcttcagagaaat
    ctgcattctatttctcatgtataaaacgaggaatcctccaaccaggct
    cctgtgataga
    113 210254_at Atacaccagcagggcaattagcagcttggggaagttggaa
    gtctcgatgttgtgatagtagaccacggaggtgacagcag
    ccatgaacgccatcccggctggcatgtacaggtgcagatg
    gtgggattcggtcaccccatcagacaggatgccctctgca
    atctcacacaccaggacgaagagcagcatgaaggtcagga
    tccaccgcaggttgtgcccngggaaatgaagccatgtgct
    gtggtggatgtgcaccttggagctctgacttccccatcca
    atgaagaggatggggaaggtgatgaagagtaggaagacgt
    gcggcaccacgttgagcgcgtccacaaagcagccgttgtt
    gaggaccc
    114 210338_s_at gaactgaatgctgacctgttccgtggcaccctggacccag
    tagagaaagcccttcgagatgccaaactagacaagtcaca
    gattcatgatattgtcctggttggtggttctactcgtatc
    cccaagattcagaagcttctccaagacttcttcaatggaa
    aagaactgaataagagcatcaaccctgatgaagctgttgc
    ttatggtgcagctgtccaggcagccatcttgtctggagac
    aagtctgagaatgttcaagatttgctgctcttggatgtca
    ctcctctttcccttggtattgaaactgctggtggagtcat
    gactgtcctcatcaagcgtaataccaccattcctaccaag
    cagacacagaccttcactacctattctgacaaccagcctg
    gtgtgcttattcag
    115 211038_s_at tggtgttccttgaatgcctatcttccttttgtgcctcgga
    acctctcacgcctgccacaagttactcttttccttggtag
    ttctaaactttaaataaggtaatgcctgtaagaatgccat
    aaatgctcaataattgtcatctgttattattttcatcagt
    aacatcatctgaatcatcagtattgtctgcttttaacagc
    tgcatttttcattgtccaaatatagtcacatacatttgac
    cattttataattattgaataataaattcgttctgctattt
    tacaatgaaaaataatgctgcagagagcatttttgcacat
    gtatcgtggcagatgtaggccagaggctcttctttatcca
    tcctatggccaacctatgaatgtatacacgtttaatgaga
    ttttgccagcaatcaaagccttcagggaaaatgtccctag
    ctctttactacatcagatcaagaactctggataattggca
    taacatcctggaatagctgaaacagagatattattctctg
    ctgtcctctgttgtctttgtcttttcacgtcttaata
    116 211458_s_at aaatagcattaaactggaattgacagagtgagttgagcat
    ctctgtctaacctgctctttctctctggtgctcctcatct
    cacccctaccttggaatttaataagcttcaggcatttcca
    attgcagactaaaaccacttctaccatctcctctagtatt
    ttccatgtatcaggacagagatgtcttatgtagggaaggg
    gcaggtatgaagtaaggtagattatctatacctctcactc
    attcaggattctcgctcccatgctgctgtcccttcattct
    cacactcacaggaatgctatgtgatggccagctgcttccc
    ttcttggttatccactgcagctgctagttagaaaggtttg
    cagggatgacttttagtaaatcatggggattttattgatt
    tattatcacttataggattttgtggggtgggagtggggag
    caggaattgcactcagacatgacatttcaattcatctctg
    caaatgaaaagggttcttcctcttgggggaaatctgtgtg
    tcagttctgtcagctgcaagttctt
    117 211560_s_at tccccatccgggtgggcaatgcagcactcaacagcaagct
    ctgtgatctcctgctctccaagcatggcatctatgtgcag
    gccatcaactacccaactgtcccccggggtgaagagctcc
    tgcgcttggcaccctccccccaccacagccctcagatgat
    ggaagattttgtggagaagctgctgctggcttggactgcg
    gtggggctgcccctccaggatgtgtctgtggctgcctgca
    atttctgtcgccgtcctgtacactttgagctcatgagtga
    gtgggaacgttcctacttcgggaacatggggccccagtat
    gtcaccacctatgcctgagaagccagctgcctaggattca
    caccccacctgcgcttcacttgggtccaggcctactcctg
    tcttctgctttgttgtgtgcctctagctgaattgagccta
    a
    118 211991_s_at Gcactgggaggcccaagagccaatccagatgcct
    gagacaacggagactgtgctctgtgccctgggcc
    tggtgctgggcctagtnggcatcatcgtgggcac
    cgtcctcatcataaagtctctgcgttctg
    119 212199_at attttgctgttacctttgtgacctgattgttttttggaac
    acgtcaagacgtgggatcagaatcttccaactttagaggt
    gcaatggaagacactacgctacttggttgagcctggtgaa
    gaatgtattaatgagactgctttgcataaaactgggaaga
    aagagaagacagttggagatggaagatggttttgtatata
    ttttggaactttagttcctctgtgagacgaaagaggagag
    ctatgttttgtgtcacattgtctgatatatattgtgtaac
    ctgtcaggtgagttgatttagacaacatagctgacctttt
    atgacaaggcagtttgaatagggactattgtaataccctc
    acacattatcggggcancagagaatggcatggaagagaca
    gtctacagagagctttaagaggccggagaaaggaaaagac
    attatcagggcctggaaagtctcttccagttcatcagggt
    ag
    120 212224_at Acagtgttctctaatgttacagatgagatgcgca
    ttgccaaagaggagatttttggaccagtgcagca
    aatcatgaagtttaaatctttagatgacgtgatc
    aaaagagcaaacaatactttctatggcttatcag
    caggagtgtttaccaaagacattgataaagccat
    aacaatctcctctgctctgcaggcaggaacagtg
    tgggtgaattgctatggcgtggtaagtgcccagt
    gcccctttggntgggattcaagatgtctggaaat
    ggaagagaactgggagagtacggtttccatgaat
    atacagaggtcaaaacagtcacagtgaaaatctc
    tcagaagaactcataaagaaaatacaagagtggagaga
    agctcttcaatagctaagcatctccttacagtcactaatat
    121 212232_at agaaggcagctctgcattctaccttgcttgactggaattg
    tctgaagctttttctggcctcttttctctagtcggccacc
    cctgaagtgctgaggtctaagtggtttacctcgtgctgat
    agatggccacactctttagagtagttctcataagttctag
    aactggtagctcggtcgtttcgcacactaggtggcataca
    ggcagcagcaggtgttcatatccttgattttgagaatttc
    ccctcaagtatgtggcagtaaatacaacaagacactctat
    gtattaatgtctccattgtcttaaccctgttccaaaacaa
    aattcacctcctttctttatgtg
    122 212534_at gtggattgtttgtatcccttacctgctttctattgggtta
    tgtgtggatatattgtttttatttgttcagcatctccttc
    cccatcttctggtaacacaacctttatttatttgtgggga
    acctattccctgtggcttaggtgagcatgtgaccaggcct
    ggcctcctgagtcccacagcttcctagccacagtgataaa
    agaatgggntatataacttaagccaggctaaggaaagccc
    ttaacagaacttctgctggaactactggaaagaaggcttt
    atggagatcccaggaaccaaggaccatgtaagcctgaatt
    tgtgccatgtggagagagtctgtctgaggagaaactcgga
    tgctagcagaaatggaaagagaactaagttctgatgtcat
    ttttctggaggccctagatccagctgt
    123 213142_x_at ctgctgccattttaatcttgctcattaaccttactccttt
    gagaattctttaacaatatttaaaattggtaacaaaaata
    gtttagccataattgtttagccatgtgagtttcaggttgg
    tacacgttcagacagaactgctgtatcacattccaatttt
    gaatagccagtgagcaatcaagtgtagagaaatgataaat
    ggcctaagaaggcatacagtggcataaacgatgctcttcc
    tagtagcttaataggccacaagctagtttctgttgcactc
    tgaaataaaatatgctttaaaaatgtagggaacagtgctt
    agaaaagcaaaaactaggtgtgtcattgaaataataggca
    taaaaattaaatgttacataagaacactatttggaaagag
    ggtccttttaaaaactgaatttgtactaaatcagatttgc
    catgtccagtacagaataatttgtacttagtatttgcagc
    agggtttgtctttgtga
    124 214433_s_at cttcctcggaggcagcattgttaagggaggccctgtgcaa
    gtgctggaggacgaggaactaaagtcccagccagagcccc
    tagtggtcaagggaaaacgggtggctggaggccctcagat
    gatccagctcagcctggatgggaagcgcctctacatcacc
    acgtcgctgtacagtgcctgggacaagcagttttaccctg
    atctcatcagggaaggctctgtgatgctgcaggttgatgt
    agacacagtaaaaggagggctgaagttgaaccccaacttc
    ctggtggacttcgggaaggagccccttggcccagcccttg
    cccnatgagctccgctaccctgggggcgattgtagctctg
    acatctggatttgaaggctccaccctcatcacccacactc
    cctattttgggccctcacttccttggggacctggcttcat
    tctgctctctcttggcacccgacccttggcagcatgtacc
    acacagccaagctgagactgtggcaatgtgttgagtcata
    tacatttactgaccactgttgct
    125 215933_s_at ggacagttcctgtgatcagaggcaagatttgcccagngaa
    cagaataaaggtgcttctttggatagctctcaatgttcgc
    cctcccctgcctcccaggaagaccttgaatcagagatttc
    agaggattctgatcaggaagtggacattgagggcgataaa
    agctattttaatgctggatgatgaccactggcattggcat
    gttcagaaaactggatttaggaataatgttttgctacaga
    aaatcttcatagaagaactggaaggctatataagaaaggg
    aatcaattctctggtattctggaaacctaaaaatatttgg
    tgcactgctcaattaacaaacctacatggagaccttaatt
    ttgacttaacaaatagtttatgtactgctcttaggttgtt
    ttgataaagtgacattatagtgattaaattctttccnctt
    taaaaaaacagntagtggttttcactatttataaatagga
    ccttcttgaacgacttttctg
    126 217478_s_at ctgttttgtcagtaatctcttcccacccatgctgacagtg
    aactggcagcatcattccgtccctgtggaaggatttgggc
    ctacttttgtctcagctgtcgatggactcagcttccaggc
    cttttcttacttaaacttcacaccagaaccttctgacatt
    ttctcctgcattgtgactcacgaaattgaccgctacacag
    caattgcctattgggtaccccggaacgcactgccctcaga
    tctgctggagaatgtgctgtgtggcgtggcctttggcctg
    ggtgtgctgggcatcatcgtgggcattgttctcatcatct
    acttccggaagccttgctcaggtgactgattcttccagac
    cagaatttgatgccagcagcttcagccatccaaacagagg
    atgctcagatttctcacatcctgc
    127 217736_s_at tctgtctggtcttcctctagaaggttctaccgcagaaatt
    gatgtgtgctccctgccctcgtcactgcccaagcccgggc
    ctgcacatactcactggactgttccagttttgacagctgc
    cagtcttcctgcccctttcacactgcagctgaagttcatt
    acctgaaggacgcctcatcatttcattccttggctccaaa
    ccttctgctgcctctaagataaaagctcaacttcttaaca
    gtgtacagtgtgcaacttccaacctttttatctgttctct
    ccaccttcagtttagcgtcattccaaaaccacacccttgc
    aaagctttgtactccgcaccccagatgatctccaggcagc
    tcagatctctttcctgcctttgccctgcactgttccccgg
    tacttcctcctttattgtagcactcagctccccagccaat
    ctgtacatccctcagaggcagcgatctgatgaattggttt
    ttgaatcccagaaacggtctgccatagagttcccagtcat
    cacggtagat
    128 219069_at gaatattgtatactgcatcccctaccacaatttacacaat
    cctgtggatagtcctacctcaccctggtcaacctacatga
    tccttaagctaatggcgaatcacgatgaccttgtagacat
    gcacacaactatacctttgtccaacagatcataatatatc
    tgctatccaactggttttacctgcctaatcctactgattt
    gggcactgcttgtatagtctctcaagttcacaggaaatgt
    tgattttctaaggtcctcattt
    129 219093_at tagagacccatgtcatcttaacctaaagggaaatcttatt
    gcgttatcataaaattgatgatatcttagggtcagaattg
    cccttttttttattttgaatgggaagctctcactaaaaca
    atcctgagatttcttaatttcatggttctttaaatattat
    aaacacagagtcaacatagaatgaaattgtatttgttaaa
    atacacacattggaggacaagagcagatgactacttttcg
    aagtaatgctgctccttcctaaaagtctgttttcaatcct
    ggtaatattaggggcactgcggcacctaagaagccttaaa
    tgagagctaatccaatttagagagcgatggtgtcagcatt
    tcggtctgcatatctgtgtgtccgtatctgcgtttgtgtg
    cgtgtacgtgtgcccctgtgtgtgggcccagttttcaggc
    atgtagaataagcatggagtcatattgaggaggactcact
    tcttgaagat
    130 219228_at ggggttcgagcctcgttaagcacgagaggatacatacggg
    cgagaagccgtacaagtgcacagaatgtgggaaggccttc
    aattgtggctatcacctcactcagcacgagagaatccaca
    caggcgaaaccccgtataaatgtaaggagtgtgggaaggc
    tttcatttatggatcgagcctcgtgaaacatgagagaatt
    cataccggggtgaaaccctatgggtgtacagaatgtggga
    agagctttagtcacggccatcagcttacacaacatcagaa
    aacgcacagtggggcgaaatcctacgaatgtaaggagtgc
    gggaaggcatgtaaccacctaaaccatctccgagaacatc
    agaggatccacaacagttgaagagccttttgaacgcagta
    gcccgctcgtatctatggtttcgctttccacagtttgtta
    cctgca
    131 219607_s_at atcaacacatttagcttggcgttttattcattccatcacc
    cttactgtaactactatggcaactcaaataattgtcatgg
    gactatgtccatcttaatgggtctggatggcatggtgctc
    ctcttaagtgtgctggaattctgcattgctgtgtccctct
    ctgcctttggatgtaaagtgctctgttgtacccctggtgg
    ggttgtgttaattctgccatcacattctcacatggcagaa
    acagcatctcccacaccacttaatgaggtttgaggccacc
    caaagatcaacagacaaatgctccagaaatctatgctgac
    tgtgacacaag
    132 221748_s_at atattttgtatcatcgtgcctatagccgctgccaccgtgt
    ataaatcctggtgtntgctccttatcctggacatgaatgt
    attgtacactgacgcgtccccactcctgtacagctgcttt
    gtttctttgcaatgcattgtatggcttta
    133 221766_s_at gatttccaggaagcctttgatcacctttgtaacaagatca
    ttgccaccaggaacccagaggaaatccgagggggaggcct
    gcttaagtactgcaacctcttggtgaggggctttaggccn
    gcctctgatgaaatcaagacccttcaaaggtatatgtgtt
    ccaggtttttcatcgacttctcagacattggagagcagca
    gagaaaactggagtcctatttgcagaaccactttgtggga
    ttggaagaccgcaagtatgagtatctcatgacccttcatg
    gagtggtaaatgagagcacagtgtgccntgatgggacatg
    aaagaagacagactttaaaccttatcaccatgctggctat
    ccgggtgttagctgaccaaaatgtcattcctaatgtggct
    aatgtcacttgctattaccagccagccccctatgtagcag
    atgccaactttagcaattactacattgcacaggttcagcc
    agtattcacgtgccagcaacagacctactccacttggcta
    ccctgcaattaaga
    134 221622_s_at atgcatcattggctacacttccatttttgtctactgttgt
    tactgacaagctttttgtaattgatgctttgtattcagat
    aatataagcaaggaaaactgtgttttcagaagctcactga
    ttggcatagtttgtggtgttttctatcccagttctttggc
    ttttactaaaaatggacgcctggcaaccaagtatcatacc
    gttccactgccaccaaaaggaagggttttaatccattgga
    tgacgctttgtcaaacacaaatgaaattaattgcgattcc
    tctagtctttcagattatgtttggaatattaaatggctat
    accattatgcaagtatttgaaagagacacttgagaaaact
    atacatgaagagtaaccaaaaaaatgaatggttgctaact
    tagcaaaatgaagtttctataaagaggactcaggcattgc
    tgaaagagttaaaagtaactgtgaacaaataatttgttct
    gtgccttttgcctggtatatagc
    135 218845_at aaaacattctggccgctccaggaattctgaagttctgggc
    ctttctcagaagactgtaatgtacctgaagtttctgaaat
    attgcaaacccacagagtttaggctggtgctgccaaaaag
    aaaagcaacatagagtttaagtatccagtagtgatttgta
    aacttgtttttcatttgaagctgaatatatacgtagtcat
    gtttatgttgagaactaaggatattctttagcaagagaaa
    atattttccccttatccccactgctgtggaggtttctgta
    cctcgcttggatgcctgtaaggatcccgggagccttgccg
    cactgccttgtgggtggcttggcgctcgtgattgcttcct
    gtgaacgcctcccaaggacgagcccagtgtagttgtgtgg
    gcgtgaactctgcccgtgtgttctcaaattccccagcttg
    ggaaatagcccttggtgtgggttttatctctggtttgtgt
    tctccgtggtggaattgaccgaaagctctatgttt
    136 200786_at gttacattggtgcagccctagttttagggggagtagatgt
    tactggacctcacctctacagcatctatcctcatggatca
    actgataagttgccttatgtcaccatgggttctggctcct
    tggcagcaatggctgtatttgaagataagtttaggccaga
    catggaggaggaggaagccaagaatctggtgagcgaagcc
    atcgcagctggcatcttcaacgacctgggctccggaagca
    acattgacctctgcgtcatcagcaagaacaagctggattt
    tctccgcccatacacagtgcccaacaagaaggggaccagg
    cttggccggtacaggtgtgagaaagggactactgcagtcc
    tcactgagaaaatcactcctctggagattgaggtgctgga
    agaaacagtccaaacaatggacacttcctgaatggcatca
    gtgggtggctggccgcggttctggaaggtggtgagcattg
    aggcccagtaagacactcatgtgg
    137 212886_at acaggcacatcaggctgcagaatgcgctttagaaagcatt
    gttttagtccaggcacagtggctcacgcctgtaatcccag
    cactttgggaggccgaggtgggtggatcacaaggttggga
    gattgagaccatcctggctaacacagtgaaaccctgtctc
    tactaaaaaaatacaaaaaattagcttggcgtggtggtgg
    gcgcctgtagtcccagcagcttgggaggctgaggctggag
    aatggtgtgaacccaggaggcggagcttgcagtgagccaa
    gatcgcgccactgcactccagcccgggtgacagagcaaga
    ctccgtctcaaaaaaaagaaaagaaaaaagaaagcattgt
    tttaattgagaggggcagggctggagaaggagcaagttgt
    ggggagccaggcttccctcacgcagcctgtggtggatgtg
    ggaaggagatcaacttctcctcactctgggacagacgatg
    tatggaaactaa
    138 201407_s_at ggatgtgattctaaaagcttttattgagcattgtcaaatt
    tgtaagcttcatagggatggacatcatatctataatgccc
    ttctatatgtgctaccatagatgtgacatttttgacctta
    atatcgtctttgaaaatgttaaattgagaaacctgttaac
    ttacattttatgnattggcacattgtattacttactgcaa
    gagatatttcattttcagcacagtgcaaaagttctttaaa
    atgcatatgtctttttttctaattccgttttgttttaaag
    cacattttaaatgtagttttctcatttagtaaaagt
    139 212266_s_at gttgagtttgcctcttatggtgacttaaagaatgctattg
    aaaaactttctggaaaggaaataaatgggagaaaaataaa
    attaattgaaggcagcaaaaggcacagtaggtcaagaagc
    aggtctcgatcccggaccagaagttcctctaggtctcgta
    gccgatcccgttcccgtagtcgcaaatcttacagccggtc
    aagaagcaggagcaggagccggagccggagcaagtcccgt
    tctgttagtaggtctcccgtgcctgagaagagccagaaac
    gtggttcttcaagtagatctaagtctccagcatctgtgga
    tcgccagaggtcccggtcccgatcaaggtccagatcagtt
    gacagtggcaattaaactgtaaataacttgccctgggggc
    ctttttttaaaaaacaaaaaccacaaaaattcccaaacca
    tacttgctaaaaattctggtaagtatgtgcttttctgtgg
    gggtgggatttggaaggggggttgggttgggctggatatc
    tttgt
    140 53912_at acaatgaggcattctgtcctcctgctgccattcttcatct
    ccactgagagccagagctggtaggagccgagnnnccacag
    gcattctgcattgctctactcttaggtttgtgtgtgtgat
    ccttcccctccctgtcgcccactcctccctcctctggcta
    tcctaccctgtctgtgggctcttttactaccagcctatgc
    tgtgggactgtcatggcatttagttcagagtggaggggnn
    nnnnnnnnnnnnnaaatgcaagtatttnannnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnaanattgttgttgca
    atttgtgtctaacaagctgtagcagagaaggagggagtga
    gcgctggcagtatttcctttcataaatcatgaatttatca
    gtgtggaaataatgcttcagaactgtgctctgtagccctc
    ctgcannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnngcgtgaaccttgttaggtatactttacctgatgc
    tgcttccatcctcgcagtctg
    141 200090_at tccctttgcctgtggtgtaaaagtgcatcacacaggtatt
    gctttttaacaagaactgatgctccttgggtgctgctgct
    actcagactagctctaagtaatgtgattcttctaaagcaa
    agtcattggatgggaggaggaagaaaaagtcccataaagg
    aacttttgtagtcttatcaacatataatctaatcccttag
    catcagctcctccctcagtggtacatgcgtcaagatttgt
    agcagtaataactgcaggtcacttgtatgtaatggatgtg
    aggtagccgaagtttggttcagtaagcagggaatacagtc
    gttccatcagagctggtctgcacactcacattatcttgct
    atcactgtaaccaacta
    142 218025_s_at ctccttgggctattcgatgccgtgtatgcatctgacaggg
    caacatttcatacaccatttagtcacctaggccaaagtcc
    ggaaggatgctcctcttacacttttccgaagataatgagc
    ccagccaaggcaacagagatgcttatttttggaaagaagt
    taacagcgggagaggcatgtgctcaaggacttgttactga
    agttttccctgatagcacttttcagaaagaagtctggacc
    aggctgaaggcatttgcaaagcttcccccaaatgccttga
    gaatttcaaaagaggtaatcaggaaaagagagagagaaaa
    actacacgctgttaatgctgaagaatgcaatgtccttcag
    ggaagatggctatcagatgaatgcacaaatgctgtggtga
    acttcttatccagaaaatcaaaactgtgatgaccactaca
    gcagagtaaagcatgtccaaggaaggatgtgctgttacct
    ctgatttccagtac
    143 204232_at cgatctccagcccaagatgattccagcagtggtcttgctc
    ttactccttttggttgaacaagcagcggccctgggagagc
    ctcagctctgctatatcctggatgccatcctgtttctgta
    tggaattgtcctcaccctcctctactgtcgactgaagatc
    caagtgcgaaaggcagctataaccagctatgagaaatcag
    atggtgtttacacgggcctgagcaccaggaaccaggagac
    ttacgagactctgaagcatgagaaaccaccacagtagctt
    tagaatagatgcggtcatattcttctttggcttctggttc
    ttccagccctcatggttggcatcacatatgcctgcatgcc
    attaacaccagctggccctacccctataatgatcctgtgt
    cctaaattaatatacaccagtggttcctcctccctgttaa
    agactaatgctcagatgctgtttacggatatttatattct
    agtctcactctcttgtcccacccttcttctcttccccatt
    cccaactccagctaaaatat
    144 201722_s_at attttgttttcatctgtgatagtcatggatgcttttattt
    tccttggggtgctgaaattgagctgaaaaaaaaaggctct
    ttgaatatagttttaatttctctctacagttttttttgtt
    tggtttgtgggctgttggaattgtaatttttaattgcctt
    ctaaaaaatggaaatttaacaatgtctgatctcagctgaa
    caaattagatgtttcagttgctcttgggtcaactggctta
    cagatttacatgtgcacacacacacaaatttcttatcaca
    ttttcgacttcttcacttgacctaactgattatgcgaaat
    acccaagattcatgctactgtaccacagatttgttttcac
    agcaataaatcttcagttctgttgtttatgattccactta
    acaaaaggcctgcagaagtgatttattatttgggtatttg
    gagataatacatttgatggttttttggaaaacctttttca
    ctccatactcagatatgcttcattgtcaaatgcatattta
    gattagattattgaattgtaatgtttatctgctgctttt
    145 218627_at taatcatttctgggttcactgcgactcactgtagtgctgg
    ggatcccccttgtaacactggaactgaaaggcagtgatga
    aagctatgtcaagcattcattattctgaagaggaggagaa
    atgccacatacctttcccatgggacctgtggtggaatgaa
    tccatacttctgcctcacttcgagcagacttttgttctcg
    gcgctcctcacgatggagtttcatgcttcattttcacatc
    tctctgcacaattagattgggagctccttgagggcagagt
    acgtgccttaatctttatctttgtaatgccaccatgaaca
    gagtgcctcctggtacactgtaggagcttaagaaatactc
    actgaatgcatgaatgaatgaatgaacaaatgaaggaatg
    actaaggatgtttgtagtgctataatatagaatgggattt
    actctgctttacc
    146 202322_s_at gttctcatttcctactattcatgctatttggtcaaggcct
    gaaagcacccaggtgcagaatatcttgcgccagagaacag
    aaaacatagatataaaaaaatactgtgtacattatcttga
    ggatgtaggttcttttgaatacactcgtaatacccttaaa
    gagcttgaagctaaagcctataaacagattgatgcacgtg
    gtgggaaccctgagctagtagccttagtaaaacacttaag
    taagatgttcaaagaagaaaatgaataatgttaagccatt
    cttgattggacctcatagcttattttagttaatctttttt
    ttgtcttttagccttaccaccttttaaaaaatttgttatt
    ctccagaaacagtaaataggtgagtaggggtggtgcaagt
    gaattcgttttcatttagaagcccctctgtacagataatc
    aaaattcaaagttgaaagaatcaaaagcagccacagttat
    gtaggtctgatttgaatgtcataattgcagtgacaggaca
    ttgccaccaactctatcctactaccatcaatgt
    147 221689_s_at Accgtcgccattgccagaaagagcgatttatggctttgtt
    cttttcttaagctcccaatttggcttcatactttacctcg
    tgtgggcctttattcctgaatcttggctaaactctttagg
    tttaacctattggcctcaaaaatattgggcagttgcatta
    cctgtctacctccttattgctatagtaattggctacgtgc
    tcttgtttgggattaacatgatgagtacctctccactcga
    ctccatccatacaatcacagataactatgcaaaaaatcaa
    cagcagaagaaataccaagaggaggccattccagccttaa
    gagatatttctattagtgaagtaaaccaaatgttctttct
    tgcagccaaagaactttacaccaaaaactgaactgtgtgt
    aaccatagtaacaccaagcacgtatttatttataagtttt
    tgccattataattttgaccataaattaatttaaccatctc
    tcttattaatagagaagtaaaaaatgtaagttgaccttct
    cttagattat
    148 203356_at tatacatcttgggcaactagttaccaaatgaattgtgcca
    ccataactgattttaattttgcattatttatgattttaaa
    atatttgttgcccaggtgttatgaaagaataaagctttta
    agtatagactaccttagcatgaagatgctcatgcctaaga
    atgaaaattgttgaggttatctcccattcaatcatgtagc
    aagaacttaaagaaattcactactgcagtttttattttta
    aaaaacagtaattgagatattgaagacattacaatttagt
    ttgtgtggtctttttttaaattgctgtatcgttcagtctc
    ttgtggcaatagcactttgaagaaaatagagaatttaata
    tatggtgattgggatatgtagcattcaaaaaaagtgaatt
    gccaagatactggtgtcatgtaaattcccactttacataa
    aaacccatcaggacagaatgatgctcaatattttaaaatt
    ctaaaaatagggtgggatttttcattgtctctactttata
    attatcaaaacttattttgtattgctactaccttaaattg
    aaa
    149 218462_at aaatgagcagtgttcgtcttcgtaaagaaattaagagaag
    aggcaaggaccccacagaacacatacctgaaataattctg
    aataattttacaacacggctgggtcattcaattggacgta
    tgtttgcatctctctttcctcataatcctcaatttatcgg
    aaggcaggttgccacattccacaatcaacgggattacata
    ttcttcagatttcacagatacatattcaggagtgaaaaga
    aagtgggaattcaggaacttggaccacgttttaccttaaa
    attaaggtctcttcagaaaggaacctttgattctaaatat
    ggagagtatgaatgggtccataagccccgggaaatggata
    caagtagaagaaaattccatttataaagtactgagagaat
    gatattggattttgctgaacaggcctatcttgaactttgg
    taaattatttttgacagaatactcttttcaaaatggcatt
    tgctgatttcataaacctttcacgtctggacgaattacca
    aatgccatgaattgccactgtgtg
    150 218123_at gtggaagccggtgtctgtgttaccatggagatggtgaaag
    atgccttggaccagcttcgaggcgcggtgatgattgttta
    ccccatggggttgccaccgtatgatcccatccgcatggag
    tttgaaaataaggaagacttgtcgggaacacaggcagggc
    tcaacgtcattaaagaggcagaggcgcagctgtggtgggc
    agccaaggagctgagaagaacgaagaagctttcagactac
    gtggggaagaatgaaaaaaccaaaattatcgccaagattc
    agcaaaggggacagggagctccagcccgagagcctattat
    tagcagtgaggagcagaagcagctgatgctgtactatcac
    agaagacaagaggagctcaagagattggaagaaaatgatg
    atgatgcctatttaaactcaccatgggcggataacactgc
    tttgaaaagacattttcatggagtgaaagacataaagtgg
    agaccaagatgaagttcaccagctgatgacacttccaaa
    151 214329_x_at aggttgcagtgtggtgagatcatgccactacactccagcc
    tggcgacagagcgagacttggtttcnaaaaaaaaaaaaaa
    aaaaacttcagtaagtacgtgttatttttttcaataaaat
    tctattacagtatgtcatgtttgctgtagtgctcatattt
    attgttgtttttgttttagtactcacttgtttcataatat
    caagattactaaaaatgggggaaaggacttctaatctttt
    tttcataatatctttgacacatattacagaag
    152 219067_s_at cgagaagatcctgataccccaatgtccttctttgactttg
    tggttgatcctcattctttcccccgtacagtggaaaacat
    ctttcatgtttccttcattatacgggatggttttgcaaga
    ataagacttgaccaagaccgactgccagtaatagagcctg
    ttagtattaatgaagaaaatgagggatttgaacataacac
    acaagttagaaatcaaggaattatagctttgagttaccgt
    gactgggaggagattgtgaagacctttgagatttcagagc
    ctgtgattactccaagtcagaggcagcagaagccaagtgc
    ttgatgctagctgaaggactcaaatggatagtgaagtcca
    aaacggaaagcggcatgtattgtaca
    153 205789_at gtcatgaggcagctttcatcacacccttttaacatttatc
    taaaagaatttaaattctttttcaaaaattacactacaag
    tttataagcccaaatggctctgtgaaatcagaagtgcaaa
    ggtgtgcaaacttgtatctgaagacctaccagggacaagc
    aggtaagagctgatgtgagtgtgtgtgatgggatctgtaa
    ggaactggaacacacatgtcctatccaaaggaatcagctg
    cagctgcttgttgtcaagtataaagtcaggacctggcttg
    gctttaaccgtttttcaagaaaactggaaatctggatttt
    cagcgaacatgcctgattttaaaaggttgactcaagtttt
    tacaaaatactatgtgggacacctcaaatacatacctact
    gactgatgacaaacccaggagtttgtgtgtcttttataaa
    aagtttgccctggatgtcatat
    154 209214_s_at cagtgtcccaatccgggttgtggaaaccagaacttcgcct
    ggagaacagagtgcaaccagtgtaaggccccaaagcctga
    aggcttcctcccgccaccctttccgcccccgggtggtgat
    cgtggcagaggtggccctggtggcatgcggggaggaagag
    gtggcctcatggatcgtggtggtcccggtggaatgttcag
    aggtggccgtggtggagacagaggtggcttccgtggtggc
    cggggcatggaccgaggtggctttggtggaggaagacgag
    gtggccctggggggccccctggacctttgatggaacagat
    gggaggaagaagaggaggacgtggaggacctggaaaaatg
    gataaaggcgagcaccgtcaggagcgcagagatcggccct
    actagatgcagagaccccgcagagctgcattgactaccag
    atttattttttaaaccagaaaatgttttaaatttataatt
    ccatatttataatgttggccacaacattatgattattcct
    tgtctgtactttagtatttttcacca
    155 213427_at cgttgagagatctccagtgcccagtgctgcagagcagcga
    gctggagggaacgccagaggtgtcctgccgggctctggag
    ctcttcgactggctcggcgccgtcttcagtaatgtcgacc
    taaataatgagcctaataatttcatatcaacctattgctg
    tcctgagccaagcacagtggtggcaaaagcttatttgtgt
    acaatcactggcttcatacttccagagaagatctgtctcc
    tattggaacatctctgtcactactttgatgaaccgaagtt
    agctccatgggttacactgtccgttcaaggctttgcagac
    agccctgtttcttgggaaaaaaatgaacatggttttcgaa
    aaggaggagaacatttatataactttgtgatttttaataa
    tcaggactattggcttcagatggctgtt
    156 209422_at Cagctcgggcgacaactgcaagactccatctcaaaaaaat
    aaaaataaaagaaaagaaagaaatatgtgcactacctaag
    ttttgtctttagaaaaactatccacctataaaaaattacc
    ttgacaaaaatagttccggtttgactaatcattttgtttc
    tttaagtggtaagtgtatgcaaggtggatccttgatgagc
    caacattgcactgtggatacatatctatgtttacgcgcta
    ttagaacagaaggcgctgtatatagaaatgttgctttgaa
    gcaatatttgcaaaacacgcagacttctgtatctg
    157 201010_s_at gtgttctcctactgcaaatattttcatatgggaggatggt
    tttctcttcatgtaagtccttggaattgattctaaggtga
    tgttcttagcactttaattcctgtcaaattttttgttctc
    cccttctgccatcttaaatgtaagctgaaactggtctact
    gtgtctctagggttaagccaaaagacaaaaaaaattttac
    tacttttgagattgccccaatgtacagaattatataattc
    taacgcttaaatcatgtgaaagggttgctgctgtcagcct
    tgcccactgtgacttcaaacccaaggaggaactcttgatc
    aagatgcccaaccctgtgatcagaacctccaaatactgcc
    atgagaaactagagggcaggtgttcataaaagccctttga
    acccccttcctgccctgtgttaggagatagggatattggc
    ccctcactgcagctgccagcacttggtcagtcactctcag
    ccatagcactttgttcactgtcctgtgtcagagcactgag
    ctccacccttttctgagagttat
    158 200883_at ttgccaaggcaactcagcagccatttgatgtttctgcatt
    taatgccagttactcagattctggactctttgggatttat
    actatctcccaggccacagctgctggagatgttatcaagg
    ctgcctataatcaagtaaaaagaatagctcaaggaaacct
    ttccaacacagatgtccaagc
    159 218454_at gtggctatccactgttagttcagaagctgggcttggacta
    ctcttatgatttagctccacgagccaaaattttccggcgt
    gaccaagggaaagtgactgatacggcatccatgaaatata
    tcatgcgatacaacaattataagaaggatccttacagtag
    aggtgacccctgtaataccatctgctgccgtgaggacctg
    aactcacctaacccaagtcctggaggttgttatgacacaa
    aggtggcagatatctacctagcatctcagtacacatccta
    tgccataagtggtcccacagtacaaggtggcctccctgtt
    tttcgctgggaccgtttcaacaaaactctacatcagggca
    tgccagaggtctacaactttgattttattaccatgaaacc
    aattttgaaacttgatataaaatgaaggagggagatgacg
    gactagaagactgtaaataagataccaaaggcactatttt
    agctatgtttttcccatcagaat
    160 202918_s_at gagtgtcctgctatagactatactagacacacacttgatg
    gtgctgcatgtcttctgaatagcaataaatattttcccag
    cagggttagcataaaggaatcatctgtagcgaaactagga
    tcagtatgccgtaggatttacagaatattttcacatgctt
    attttcatcatcggcagatatttgatgaatatgaaaatga
    aacatttttgtgtcatcggtttactaagtttgtgatgaaa
    tacaatttgatgtccaaggataacctgattgtaccaattt
    tagaagaggaagtacagaattcagtttctggggaaagtga
    agcatgaagggaatcataggaaaaatgtactgatcatata
    attaacattatgtactgtatatatcattttagacacatca
    atcatgtatccatattatagcttctttgtttagtataggt
    ttttgtatgctgggtttgccttttaaaatgggaaatactt
    tttaagttattcataagctgtatattcaccagtgtggcac
    tcatggtttt
    161 212204_at gcttaactcttttgacatctgctattgtgacacatcccat
    tgctggcaatgtggtgcacactccgaaacttttaactact
    gttttgtaagcctccaagggtggcattgcagggtccttag
    gcaatgttttgtttgcctttatgcagagaggtgctccaag
    tgctgtgattgagcaccgtgctagaggaactgtaatgctt
    cagaagttgtagcttatacaaaggaaacaggtcctgctgg
    cttaatttaaacagttattgcatgaagtagcgtggaggcc
    ctggactgctgctcgttctttaggatggactgttctggta
    tctggtattggtttagagactgttaataagggacatcaca
    aggtgatgggattcatttgaagcactctatttctgtttta
    atg
    162 219452_at atcacttcgaccacatcaaggctgtcattggatccaagtt
    catcgggattggtggagattatgatggggccggcaaattc
    cctcaggggctggaagacgtgtccacatacccggtcctga
    tagaggagttgctgagtcgtggctggagtgaggaagagct
    tcagggtgtccttcgtggaaacctgctgcgggtcttcaga
    caagtggaaaaggtacaggaagaaaacaaatggcaaagcc
    ccttggaggacaagttcccggatgagcagctgagcagttc
    ctgccactccgacctctcacgtctgcgtcagagacagagt
    ctgacttcaggccaggaactcactgagattcccatacact
    ggacagccaagttaccagccaagtggtcagtctcagagtc
    ctccccccacatggccccagtccttgcagttgtggccacc
    ttcccagtccttattctgtggctctgatgacccagttagt
    cctgccagatgtcactgta
    163 219889_at tactcaagcgggtggctctgggatcctgggggcctgggtt
    gggggctagggagacgccatgtgatggacactccagggac
    acacagcctagcacagcagcttataatgggctctccgggg
    ccatttgcaataacagctgcaattccctggatagacgagt
    tgatttcctccctctgcccctcccccagccataccagctg
    gcctttgtaagtgcaggaaaccgagtagaaaatgtgaccc
    tccaaatggagaagctgccagctttgccattgtgaaccat
    ggtgaagtgcttggaacatactgttcactcactctaaagg
    cgctgagactgtgctgttgttctcgtttttatagtcaatg
    gcttgttcatcatccagatgtggctactgacatatctaca
    cttcgcaccggagtgtctggaatt
    164 201303_at caacttcactgtatcctcaatgcatggagacatgccccag
    aaagagcgggagtccatcatgaaggagttccggtcgggcg
    ccagccgagtgcttatttctacagatgtctgggccagggg
    gttggatgtccctcaggtgtccctcatcattaactatgat
    ctccctaataacagagaattgtacatacacagaattggga
    gatcaggtcgatacggccggaagggtgtggccattaactt
    tgtaaagaatgacgacatccgcatcctcagagatatcgag
    cagtactattccactcagattgatgagatgccgatgaacg
    ttgctgatcttatctgaagcagcagatcagtgggatgagg
    gagactgttcacctgctgtgtactcctgtttggaagtatt
    tagatccagattctacttaatggggtttatatggactttc
    ttctcataaatggcctgccgtctc
    165 202510_s_at gtcgtctttctattttcaggtcagctgattagccacctta
    gttccatctgcaactttagttcccactggctgtgtaacct
    aacatagtcacaggctctggggactgtcacgtggacatct
    ttgggaggccgttattctgcccaccgcaccctccgttcat
    cccctgccctgccgggcacctcgctctaccccaggaaaat
    gtgagctcgttttcctgctcggcatgtgctccccctaagg
    ctctgctcctccctgggcctgaaagttccttctcagcctg
    agagggggcccttcgatctcaggcatgactcagcccggct
    gatgcctctgcagtgctgagtcaggatttggggccggctc
    tcttgggtctgtccccttttcccaggtactgccttacaaa
    gctgtggccaggaagtggccggtataaaggatgcccaagg
    tctttgtacgtgtg
    166 221923_s_at tagtccatactgagtgtcatcaacaatccagactgaagtc
    ttctattttaatctcaatccccttttctgatttgccaccc
    atgcctcttcaggctggaaacaatctcttggttccctaaa
    gcactttcttctgactgctgtgattcagtgaaccttgccc
    tttgctttctattacttgtgcatttgcctcacctgacaat
    gttttaaatcgcctttgtatctccttagctgctcaataa
    167 201887_at ctgggaagcaaaacccatgcctccccctagccatttttac
    tgttatcctatttagatggccatgaagaggatgctgtgaa
    attcccaacaaacattgatgctgacagtcatgcagtctgg
    gagtggggaagtgatcttttgttcccatcctcttctttta
    gcagtaaaatagctgagggaaaagggagggaaaaggaagt
    tatgggaatacctgtggtggttgtgatccctaggtcttgg
    gagctcttggaggtgtctgtatcagtggatttcccatccc
    ctgtgggaaattagtaggctcatttactgttttaggtcta
    gcctatgtggattttttcctaacatacctaagcaaaccca
    gtgtcaggatggtaattcttattctttcgttcagttaagt
    ttttcccttcatctgggcactgaagggatatgtgaaacaa
    tgttaacatttttggtagtcttcaaccagggattgtt
    168 211582_x_at agcccacctgctggatgaggaacttgaggcaagtcaccag
    cccctgatcatttcgcctaaaagagcaaggactagagttc
    ctgacctccaggccagtccctgatccctgacctaatgtta
    tcgcggaatgatgatatatgtatctacgggggcctggggc
    tgggcgggctcctgcttctggcagtggtccttctgtccgc
    ctgcctgtgttggctgcatcgaagagcaccttctgtcctg
    gtcccaggcccagggctcctcagagcaggaactccactat
    gcatctctgcagaggctgccagtgcccagcagtgagggac
    ctgacctcaggggcagagacaagagaggcaccaaggagga
    tccaagagctgactatgcctgcattgctgagaacaaaccc
    acctgagcaccccagacaccttcctcaacccaggcgggtg
    gaca
    169 219030_at agttaacacatcagctggacctatttcccgaatgcagggt
    aacccttctgttatttaaagatgtaaaaaatgcgggagac
    ttgagaagaaaggccatggaaggcaccatcgatggatcac
    tgataaatcctacagtgattgttgatccatttcagatact
    tgtggcagcaaacaaagcagttcacctctacaaactggga
    aaaatgaagacaagaactctatctactgaaattattttca
    acctttccccaaataacaatatttcagaggctttgaaaaa
    atttggtatctcagcaaatgacacttcaattctaattgtt
    tacattgaagagggagaaaaacaaataaatcaagaatacc
    taatatctcaagtagaaggtcatcaggtttctctgaaaaa
    tcttcctgaaataatgaatattacagaagtcaaaaagata
    tataaactctcttcacaagaagaaagtatcgggacattat
    tggatgctat
    170 212706_at agatatttaggtgacttacagcaaccaatngcaacanaac
    aaaatgttaagaaatgatctttntatgaggcaatnggaaa
    tttgaacactgatcaactataggatgattggaattattaa
    tttttaaaggtgtgataagatactgcacttggctgggcac
    agtggcacatgcctgtaatcccagctacttggcaggctga
    ggtgggagaatcgcttaagctcaggagttcgagaccagcc
    tgggcaacgtggcgaaatccccgtctttacaaaaacaaac
    aaacaaacaaaaaagatattgcagttgtgttgtaagcgtc
    cttatctttcagagctacatagtggaatgtttatggaata
    tttaggataaatgatataggcatttgggatttgctgcaaa
    atgacccagagg
    171 202902_s_at caaaatatcgtgctgccacatgttcaaagtacactgaact
    tccttatgggagagaagatgtcctgaaagaagctgtggcc
    aataaaggcccagtgtctgttggtgtagatgcgcgtcatc
    cttctttcttcctctacagaagtggtgtctactatgaacc
    atcctgtactcagaatgtgaatcatggtgtacttgtggtt
    ggctatggtgatcttaatgggaaagaatactggcttgtga
    aaaacagctggggccacaactttggtgaagaaggatatat
    tcggatggcaagaaataaaggaaatcattgtgggattgct
    agctttccctcttacccagaaatctagaggatctctcctt
    tttataacaaatcaagaaatatgaagcactttctcttaac
    ttaatttttcctgctgtat
    172 200663_at tgtcttatgatcacgtttgccatctttctgtctcttatca
    tgttggtggaggtggccgcagccattgctggctatgtgtt
    tagagataaggtgatgtcagagtttaataacaacttccgg
    cagcagatggagaattacccgaaaaacaaccacactgctt
    cgatcctggacaggatgcaggcagattttaagtgctgtgg
    ggctgctaactacacagattgggagaaaatcccttccatg
    tcgaagaaccgagtccccgactcctgctgcattaatgtta
    ctgtgggctgtgggattaatttcaacgagaaggcgatcca
    taaggagggctgtgtggagaagattgggggctggctgagg
    aaaaatgtgctggtggtagctgcagcagcccttggaattg
    cttttgtcgaggttttgggaattgtctttgcctgctgcct
    cgtgaagagtat
    173 202138_x_at cagaataaaaaacagccccgccaagactatcagctgggat
    tcactttaatttggaagaatgtgccgaagacgcagatgaa
    attcagcatccagacgatgtgccccatcgaaggcgaaggg
    aacattgcacgtttcttgttctctctgtttggccagaagc
    ataatgctgtcaacgcaacccttatagatagctgggtaga
    tattgcgatttttcagttaaaagagggaagcagtaaagaa
    aaagccgctgttttccgctccatgaactctgctcttggga
    agagcccttggctcgctgggaatgaactcaccgtagcaga
    cgtggtgctgtggtctgtactccagcagatcggaggctgc
    agtgtgacagtgccagccaatgtgcagaggtggatgaggt
    cttgtgaaaacctggctccttttaacacggccctcaagct
    ccttaagtgaattgccgtaactgattttaaagggtttaga
    ttttaagaatggtgctctttcatgcctattatcagta
    174 200851_s_at gttgaaggcctcgcttagttgtactggattctcagggagc
    cctctgtggccttttgctttgcgtgctgtttcccttgtac
    cagagggcggcaccgtggaaattctgttttccctgtagca
    tattgtgttggattgcattactggcagagaaaggacaagg
    tgccattcaagtcctagggtgggcttccagctgccttaat
    agaagtactcaagtcttttgggtagtgagctggaaagcct
    acaggaaaagaggggtacctgttttcatttgaaaactttg
    attcatggaacctttaaaactaatctcagaaaaatttttg
    gtgcccatgcagctgtagttgttcactgctttcctggatg
    gatgggactcttatgtcataacttctgttactcctttggc
    ccatagctaaggtcatccttccccacaggggtggctttgg
    gattggatgatacagcttttgcttctgtgtagtatacctg
    tacatacttgtttcaggcagcctttc
    175 201109_s_at tcatgatgctgactggcgttagctgattaacccatgtaaa
    taggcacttaaatagaagcaggaaagggagacaaagactg
    gcttctggacttcctccctgatccccacccttactcatca
    cctngcagtggccagaattagggaatcagaatcaaaccag
    tgtaaggcagtgctggctgccattgcctggtcacattgaa
    attggtggcttcattctagatgtagcttgtgcagatgtag
    caggaaaataggaaaacctaccatctcagtgagcaccagc
    tgcctcccaaaggaggggcagccgtgcttatatttttatg
    gttacaatggcacaaaattattatcaacctaactaaaaca
    ttccttttctcttttttcctgaattatcatggagttttct
    aattctctcttttggaatgtangattttttttaaatgctt
    tacgatgtaaaatatttattttttacttattctggaagat
    ctggctgaaggattattcatggaacaggaagaagcgtaaa
    gactatccatgtcatctttgttgagagtcttcgtgact
    176 202531_at acaggagtcagtgtctggctttttcctctgagcccagctg
    cctggagagggtctcgctgtcactggctggctcctagggg
    aacagaccagtgaccccagaaaagcataacaccaatccca
    gggctggctctgcactaagcgaaaattgcactaaatgaat
    ctcgttccaaagaactaccccttttcagctgagccctggg
    gactgttccaaagccagtgaatgtgaaggaaactcccctc
    cttcggggcaatgctccctcagcctcagaggagctctacc
    ctgctccctgctttggctgaggggcttgggaaaaaaactt
    ggcactttttcgtgtggatcttgccacatttctgatcaga
    ggtgtacactaacatttcccccgagctcttggcctttgca
    tttatttatacagtgccttgctcggggcccaccaccccct
    caagccccagcagccctcaacaggcccagggagggaagtg
    tgagcgccttggtatgacttaa
    177 216274_s_at ggaatgattgtctcatcggcactaatgatctggaaggggt
    taatggtaataactggaagtgaaagtccgattgtagtggt
    gctcagtggcagcatggaacctgcatttcatagaggagat
    cttctctttctaacaaatcgagttgaagatcccatacgag
    tgggagaaattgttgttttcaggatagaaggaagagagat
    tcctatagttcaccgagtcttgaagattcatgaaaagcaa
    aatgggcatatcaagtttttgaccaaaggagataataatg
    cggttgatgaccgaggcctctataaacaaggacaacattg
    gctagagaaaaaagatgttgtggggagagccaggggattt
    gttccttatattggaatngtgacgatcctcatgaatgact
    atcctaaatttangtatgcagttctctttttgctgggttt
    attcgtgctggtncatcgtgagtaagaagcctgccttgct
    gttcctgggaagatgccatagttttcgttactg
    178 200041_s_at agaggctttctcggtatcagcagtttaaagattttcaacg
    acgaattcttgtggctaccaacctatttggccgaggcatg
    gacatcgagcgggtgaacattgcttttaattatgacatgc
    ctgaggattctgacacctacctgcatcgggtggccagagc
    aggccggtttggcaccaagggcttggctatcacatttgtg
    tccgatgagaatgatgccaagatcctcaatgatgtgcagg
    atcgctttgaggtcaatattagtgagctgcctgatgagat
    agacatctcctcctacattgaacagacacggtagaagact
    cgcccattttggaatgtg
    179 200052_s_at ggacatggtctgctatacagctcagactctcgtccgaatc
    ctctcacatggtggctttaggaagatccttggccaggagg
    gtgatgccagctatcttgcttctgaaatatctacctggga
    tggagtgatagtaacaccttcagaaaaggcttatgagaag
    ccaccagagaagaaggaaggagaggaagaagaggagaata
    cagaaagaaccacctcaaggagaggaagaagaaagcatgg
    aaactcaggagtgacattcccttcactccttttcctaccc
    aagggaaagactggagcctaagctgcctgctactggcttt
    acatggtgacagacattccgtggataggaagatagcagga
    gaaagtaactccatagagtgtcattccactggttgatatt
    ggcttagctgccagtctcccatttgtgacctatgccatcc
    atctataatggaggataccaacatttcttcctaatattct
    ataatctccaactcctga
    180 200064_at aatagacttgtgtcttcaccttgctgcattgtgaccagca
    cctacggctggacagccaatatggagcggatcatgaaagc
    ccaggcacttcgggacaactccaccatgggctatatgatg
    gccaaaaagcacctggagatcaaccctgaccaccccattg
    tggagacgctgcggcagaaggctgaggccgacaagaatga
    taaggcagttaaggacctggtggtgctgctgtttgaaacc
    gccctgctatcttctggcttttcccttgaggatccccaga
    cccactccaaccgcatctatcgcatgatcaagctaggtct
    aggtattgatgaagatgaagtggcagcagaggaacccaat
    gctgcagttcctgatgagatcccccctctcgagggcgatg
    aggatgcgtctcgcatggaagaagtcgattaggttaggag
    ttcatagttggaaaacttgtgcccttgtatagtgtccc
    181 200079_s_at agctgccagaaacgaacctctttgaaactgaagaaactcg
    caaaattcttgatgatatctgtgtggcaaaagctgttgaa
    tgccctccacctcggaccacagccaggctccttgacaagc
    ttgttggggagttcctggaagtgacttgcatcaatcctac
    attcatctgtgatcacccacagataatgagccctttggct
    aaatggcaccgctctaaagagggtctgactgagcgctttg
    agctgtttgtcatgaagaaagagatatgcaatgcgtatac
    tgagctgaatgatcccatgcggcagcggcagctttttgaa
    gaacaggccaaggccaaggctgcaggtgatgatgaggcca
    tgttcatagatgaaaacttctgtactgccctggaatatgg
    gctgccccccacagctggctggggcatgggcattgatcga
    gtcgccatgtttctcacggactccaacaacatcaaggaag
    tacttctgtttcctgccatgaaacccga
    182 200629_at tccagtttactgaactccagaccatgcatgtagtccactc
    cagaaatcatgctcgcttcccttggcacaccagtgttctc
    ctgccaaatgaccctagaccctctgtcctgcagagtcagg
    gtggcttttcccctgactgtgtccgatgccaaggagtcct
    ggcctccgcagatgcttcattttgacccttggctgcagtg
    gaagtcagcacagagcagtgccctggctgtgtcctggacg
    ggtggacttagctagggagaaagtcgaggcagcagccctc
    gaggccctcacagatgtctaggcaggcctcatttcatcac
    gcagcatgtgcaggcctggaagagcaaagccaaatctcag
    ggaagtccttggttgatgtatctgggtctcctc
    183 200634_at gaaaacgttcgtcaacatcacgccagctgaggtgggtgtc
    ctggttggcaaagaccggtcaagtttttacgtgaatgggc
    tgacacttgggggccagaaatgttcggtgatccgggactc
    actgctgcaggatggggaatttagcatggatcttcgtacc
    aagagcaccggtggggcccccaccttcaatgtcactgtca
    ccaagactgacaagacgctagtcctgctgatgggcaaaga
    aggtgtccacggtggtttgatcaacaagaaatgttatgaa
    atggcctcccaccttcggcgttcccagtactgacctcgtc
    tgtcccttccccttcaccgctccccacagctttgcacccc
    tttcctccccatacacacacaaaccattttattttttggg
    ccattaccccataccccttattgctgccaaaaccacatg
    184 200802_at taattgcacggattaccaggctcgccggcttcgaatccga
    tatgggcaaaccaagaagatgatggacaaggtggagtttg
    tccatatgctcaatgctaccatgtgcgccactacccgtac
    catctgcgccatcctggagaactaccagacagagaagggc
    atcactgtgcctgagaaattgaaggagttcatgccgccag
    gactgcaagaactgatcccctttgtgaagcctgcgcccat
    tgagcaggagccatcaaagaagcagaagaagcaacatgag
    ggcagcaaaaagaaagcagcagcaagagacgtcaccctag
    aaaacaggctgcagaacatggaggtcaccgatgcttgaac
    attcctgcctccctatttgccaggctttcatttctgtctg
    ctgagatctcagagcctgcccaacagcagggaagccaagc
    acccattcatccccctgcccccatctgactgcgtagctga
    gaggggaacagtgccatgtaccacacagatgttcctgtct
    cctcgcatgggcatagggacc
    185 200860_s_at acttcagttgcaccatgctgtacctttttgcagaggccaa
    tacggaagccatccaagaacagatcacaagagttctcttg
    gaacggttgattgtaaataggccacatccttggggtcttc
    ttattaccttcattgagctgattaaaaacccagcgtttaa
    gttctggaaccatgaatttgtacactgtgccccagaaatc
    gaaaagttattccagtcggtcgcacagtgctgcatgggac
    agaagcaggcccagcaagtaatggaagggacaggtgccag
    ttagacgaaactgcatctctgttgtacgtgtcagtctaga
    ggtctcactgcaccgagttcataaactgactgaagaatcc
    tttcagctcttcctgactttcccagccctttggtttgtgg
    gtatctgccccaactactgttgggatcagcctcctgtctt
    atgtgggcacgttcca
    186 200983_x_at aggagttgagacctacttcacagtagttctgtggacaatc
    acaatgggaatccaaggagggtctgtcctgttcgggctgc
    tgctcgtcctggctgtcttctgccattcaggtcatagcct
    gcagtgctacaactgtcctaacccaactgctgactgcaaa
    acagccgtcaattgttcatctgattttgatgcgtgtctca
    ttaccaaagctgggttacaagtgtataacaagtgttggaa
    gtttgagcattgcaatttcaacgacgtcacaacccgcttg
    agggaaaatgagctaacgtactactgctgcaagaaggacc
    tgtgtaactttaacgaacagcttgaaaatggtgggacatc
    cttatcagagaaaacagttcttctgctggtgactccattt
    ctggcagcagcctggagccttcatccctaagtcaacacca
    ggagagcttctcccaaactccccgttcctgcgtagtccgc
    tttctcttgctgccacattctaaagg
    187 200991_s_at tgccacccgggagtctatggtcaaactctcaagtaagctg
    agtgccgtgagcttgcggggaattggcagtcccagcacag
    atgccagtgccagtgatgtccacggcaatttcgccttcga
    gggcattggagatgaggatctgtaatctccactgcttgga
    tgtctgccctctaccccagaggaatttacagaaacttgcc
    ctgtgcctgtgtcccccatgctaggggcggaggggtcttt
    tccttcttctttcctacctaccccttttctcttggccagg
    ggcctcgtatcctacctttccttgtcccctgggctggctg
    cacagaggattgccccttctcttttcagagctggccctcg
    atgccaaattagcat
    188 201112_s_at agatctgtgcggttggcataaccaacttactaacagaatg
    tcccccaatgatggacactgagtataccaaactgtggact
    ccattattacagtctttgattggtctttttgagttacccg
    aagatgataccattcctgatgaggaacattttattgacat
    agaagatacaccaggatatcagactgccttctcacagttg
    gcatttgctgggaaaaaagagcatgatcctgtaggtcaaa
    tggtgaataaccccaaaattcacctggcacagtcacttca
    catgttgtctaccgcctgtccaggaagggttc
    189 201214_s_at agagctgcaagagttctggatgaacgacaatctccttgag
    agctggagcgacctcgacgagctgaagggagccaggagcc
    tggagacagtgtacctggagcggaaccccttgcagaagga
    cccccagtaccggcggaaggtcatgctcgccctcccctcc
    gtgcggcagatcgatgccacgttcgtcaggttctgagtcc
    ttcttggctcctcatgtggtccctctcctcggaagaactg
    cccagccacgggtttttaacccacctgttgctcctgaggt
    cgtcactatatcaacagtcacaaacccaatggcaataaag
    gcactgacgatagctggc
    190 201241_at aggatgggtctggcaatttccctggtggcaacagaaaaag
    aaaaggtttggtaccatgtatgtagcagccgtggaaaagg
    gtgttataacacaagactcaaggaagatggaggctgtacc
    atatggtacaacgagatgcagttactatctgagatagaag
    aacacctgaactgtaccatttctcaggttgagccggatat
    aaaggtaccagtggatgaatttgatgggaaagttacctac
    ggtcagaaaagggctgctggtggtggaagctataaaggcc
    atgtggatattttggcacctactgttcaagagttggctgc
    ccttgaaaaggaggcgcagacatctttcctgcatcttggc
    taccttcctaaccagctgttcagaa
    191 201263_at gatctggatccaggctgtacattgaataaaaagattcgaa
    atgcacagttagcacagtataacttcattttagttgttgg
    tgaaaaagagaaaatcactggcactgttaatatccgcaca
    agagacaataaggtccacggggaacgcaccatttctgaaa
    ctatcgagcggctacagcagctcaaagagttccgcagcaa
    acaggcagaagaagaattttaatgaaaaaattacccagat
    tggctccatggaaaaggaggaacagcgtttccgtaaaatt
    gactttgtactcgaaaacgtcaatttatattgaacttgga
    ggaggagtttggcaaagtctgaaataggtcaacctgcagg
    cgtaactattt
    192 201386_s_at ggagatcatctgacactgctgaacgtctaccatgctttta
    aacaaaatcatgaatcggttcagtggtgttatgacaactt
    cattaactacaggtccctgatgtccgcagacaatgtacgc
    cagcagctatctcgaattatggacagatttaatttgcctc
    gtcgaagtactgactttacaagcagggactattatattaa
    tataagaaaagctttggttactgggtattttatgcaggtg
    gcacatttagaacgaacagggcattacttaactgtgaaag
    ataaccaggtggttcagttgcatccctctactgttcttga
    ccacaaacctgaatgggtgctttataatgagtttgttcta
    acaacaaagaattacatccggacatgtacagatatcaagc
    cagaatggttggtgaaaattgcccctcaatattatgacat
    gagcaatttcccacagtgtgaagcaaagagacagttggac
    cgcatcattgcc
    193 201417_at gtaaaccacatcttttttgcactttttttataagcaaaaa
    cgtgccgtttaaaccactggatctatctaaatgccgattt
    gagttcgcgacactatgtactgcgtttttcattcttgtat
    ttgactatttaatcctttctacttgtcgctaaatataatt
    gttttagtcttatggcatgatgatagcatatgtgttcagg
    tttatagctgttgtgtttaaaaattgaaaaaagtggaaaa
    catctttgtacatttaagtctgtattataataagcaaaaa
    gattgtgtgtatgtatgtttaatataacatgacaggcact
    aggacgtctgcctttttaaggcagttccgttaagggtttt
    tgtttttaaacttttttttgccatccatcctgtgcaatat
    gccgtgta
    194 201576_s_at aatggctttaaccttggccgctattggccagcccggggcc
    ctcagttgaccttgtttgtgccccagcacatcctgatgac
    ctcggccccaaacaccatcaccgtgctggaactggagtgg
    gcaccctgcagcagtgatgatccagaactatgtgctgtga
    cgttcgtggacaggccagttattggctcatctgtgaccta
    cgatcatccctccaaacctgttgaaaaaagactcatgccc
    ccacccccgcaaaaaaacaaagattcatggctggaccatg
    tatgatgatgaaagcctgtgtctttgagggattctaccct
    gaacatacctcacagatcctccctgtcatgccacatttca
    ctgattggaatgtggaaatggaaaaggaatttaggatgtg
    cattttcacctgaggtttccct
    195 201872_s_at gagatccaaacaactataggccacgaataaacaaacttaa
    ttcaattaaggatgtagaacaaaagaagagtggaaactac
    tttttcttggatgattagactgactctgagaatattgata
    agccatttattaaaaggagtatttactagaattttttgtc
    atataaaacttgaatcaggattttatgccccacatactct
    ggaacttgaagtataatatacttaatataacataaaaagc
    cagttgggttctaaattgtagttgaaacacagaaaatgcc
    acttttctgttcctgaagaggctcttttgtgcataatatt
    ctaaaatgaagacatttcaagctatacaaattacttccaa
    gttttcatgatgtatgggaagattttcagtaggtgtatta
    tattcacggtaccaaatgctgaccagtgttgctccatttt
    ttaaatcttgaaaagggtttctgtacttacctggtttgcc
    aagtatgccagtgtaatgaaactgcccttattttaaaagc
    cagtcaaagattccactgattgacatttgat
    196 201892_s_at tgtcagagtatgcacggcgctttggtgttccggtcattgc
    tgatggaggaatccaaaatgtgggtcatattgcgaaagcc
    ttggcccttggggcctccacagtcatgatgggctctctcc
    tggctgccaccactgaggcccctggtgaatacttcttttc
    cgatgggatccggctaaagaaatatcgcggtatgggttct
    ctcgatgccatggacaagcacctcagcagccagaacagat
    atttcagtgaagctgacaaaatcaaagtggcccagggagt
    gtctggtgctgtgcaggacaaagggtcaatccacaaattt
    gtcccttacctgattgctggcatccaacactcatgccagg
    acattggtgccaagagcttgacccaagtccgagccatgat
    gtactctggggagcttaagtttgagaagagaacgtcctca
    gcccaggtggaaggtggcgtccatagcctccattcgtatg
    agaagcggcttttctgaaaagggatccagcacacctcct
    197 202174_s_at caacctttaaatagtgctgcccataaggagtcacctccta
    ctgttgattcaactcaacagcctaaccctttgccgttacg
    tttacctgaaatggaacccttagtgcctagagtcaaagaa
    gttaaatctgctcaggaaactcctgaaagctctctggctg
    gaagtcctgatactgaatctccagtgttagtgaatgacta
    tgaagcagaatctggtaatataagtcaaaagtctgatgaa
    gaagattttgtaaaagttgaagatttaccactgaaactga
    caa
    198 202176_at ggtgatcacgaaactcgctggcatggaggaggaagacttg
    gcgttttcgacaaaagaagagcaacagcagctcttacaga
    aagtcctggcagccactgacctggatgccgaggaggaggt
    ggtggctggggaatttggctccagatccagccaggcatct
    cggcgctttggcaccatgagttctatgtctggggccgacg
    acactgtgtacatggagtaccactcatcgcggagcaaggc
    gcccagcaaacatgtacacccgctcttcaagcgctttagg
    aaatgatgcttaggcagggtacttcgttcaagaccggcgc
    ttggcacccttgttggaaagggattttcagcataacattt
    tccttccacctctttgaccttccctccagcgttggccaaa
    ttgtgctgaggaagatgcatcaagggcttggctgtgcctt
    cataggtcatctagggttttataaaggaggaggagacaat
    attttttcaaactttttggggagtggggtcatttctgtat
    at
    199 202220_at tgatccaaagctggcaccttcaggcacattggtctcatag
    ccattactgtttttattgcccttctaagatcctgtcttca
    gctgggtcagagaaaacttcttgactaaaactggtcagaa
    ctcatcacagaaatgaaatacagtggtctctctctcccag
    aactggttgcagctaaaacagagagatctgactgctggct
    ataggattttggacttaatgactgaaattgcaaattgtcc
    tttttcttggcattacagattttgccaaaataactttttg
    tatcaaatattgatgtgtgaaagtgaaggagctagtctgc
    tgaaccaggaatagtttgagatattgaactgtcatttttg
    cacatttgaatactttgcaggctggctttgtataaactta
    tcctctggtttcctatatgttgt
    200 202225_at gatactgtaaagtccacacacacattaaatcttgttttcc
    tgaaagtatggcatcaaaaatacttgtagaaaaaccttgt
    cacaactgatttgaatgttcctattntnnnnnnctttgac
    tttgatattggcttgtaatgtctcttttcatcatatgtaa
    tatcagtggaacaggcagcgctactcaagtcctaaggatt
    cctcagtgatcagtgatccagggccgttcatgaaccactg
    ggctggatttgactgttgagtgtggcagttaatgcccctc
    aagaaatcaaaggatgtcttataagtgtcttccaaaaaaa
    agcaaatgctgaaatcctattggc
    201 202464_s_at tattctgtcctgagaccacgggcaaagctcttccattttg
    agagagaagaaaaactgtttggaaccacaccaatgatatt
    tttctttgtaatacttgaaatttatttttttattattttg
    atagcagatgtgctatttatttatttaatatatgtataag
    gagtcctaaacaatagaaagctgtagaagctgtagagata
    ggcttcagttgttaattggtttggagcctcctatgtgtga
    cttatgactctctgtgttctgtgtatttgtctgaattaat
    gacctgggatataaagctatgctagctttcaaacaggaga
    tgccttcagaaagctttgtatattttgcagttgccagacc
    aataaaatacc
    202 202545_at atcgcccctgagatcctacagggcctgaagtacacattct
    ctgtggactggtggtctttcggggtccttctgtacgagat
    gctcattggccagtcccccttccatggtgatgatgaggat
    gaactcttcgagtccatccgtgtggacacgccacattatc
    cccgctggatcaccaaggagtccaaggacatcctggagaa
    gctctttgaaagggaaccaaccaagaggctgggaatgacg
    ggaaacatcaaaatccaccccttcttcaagaccataaact
    ggactctgctggaaaagcggaggttggagccacccttcag
    gcccaaagtgaagtcacccagagactacagtaactttgac
    caggagttcctgaacgagaaggcgcgcctctcctacagcg
    acaagaacctcatcgactccatggaccagtctgcattcgc
    tggct
    203 202838_at agaaagaggcgctgctcactgttttcctgcttcagttttt
    ctcttatagtaccatcactataatcaacgaacttctcttc
    tccacccagagatggcttttccaacacattttaattaaag
    gaactgagtacattaccctgatgtctaaatggaccaaaga
    tctgagatccattgtgattatatctgtatcaggtcagcag
    aagaaggaactgagcagttgaactctgagttcatcaattc
    taatatttggaaattatctacaatggaatcttccctctgt
    tctctgataacctacttgcttactcaatgcctttaagcca
    agtcaccctgttgcctatgggaggaggtggaaggatttgg
    caagctcaaccacatgctatttagttagcatcagttgtca
    ccaacagtctttctgcaaagggcaggagagctttggggga
    aaggaaaaggcttaccaggctgctatggtcaactcttcag
    aa
    204 202896_s_at ccaccacccaactggggctagagtggggaagatttcccct
    ttagatcaaactgccccttccatggaaaagctggaaaaaa
    actctggaacccatatccaggcttggtgaggttgctgcca
    acagtcctggcctcccccatccctaggcaaagagccatga
    gtcctggaggaggagaggacccctcccaaaggactggaag
    caaaaccctctgcttccttgggtccctccaagactccctg
    gggcccaactgtgttgctccacccggacccatctctccct
    tctagacctgagcttgcccctccagctagcactaagcaac
    atctcgctgtaagcgcctgtaaattactgtgaaatgtgaa
    acgtgcaatcttgaaactgaggtgtt
    205 202950_at taacatgttagttgtcatttggcatgagtgtgcattccag
    taattcttaattgatatttgattaattccatacctttgat
    taaaacatgctagttcaaaataagactgctcagtttccaa
    gggttttcaagcctacttacctttataaaggttctctagt
    ctctgattagccatgactgtattggactttgaacattttc
    tgaactaaaaacctctattctaaactaatctcatttggat
    gtgtaagtcttttgtaaaggcaagaataaataatatccag
    gacaatttattagttttctcagtattttcccaaatattag
    aatatttacttcattattggttggctgccaatgaccccat
    atgttctgtgagaatagtagctttatctttgatataatac
    atagtctccaaataggtaatacttcgcaattgattagatt
    ttcagagtagatttagagttatctgtttttctggtgaggg
    tcaaat
    206 203037_s_at Agtagtgcctgtggtttagcccaccaatcttgatgactaa
    aagtagctgatgcattgtgcatatgatgcttgagatggtt
    tttgcaaaagcagaaatcgctgcaaggtaatcacaataga
    taaaagtggtattttaaacctttgaaataaatggatgtaa
    ctgtaccttggtacagcttttcacttgtttagtttttaaa
    cgttagtataatctgaataaataaaatgttgccaaattca
    atgtagaaagaatgtgacaacacaccttgggtagttctgc
    ttgtgtttttgcatattgtaaaagcagtgtcacagctaaa
    aagaaagaaatcgtttctaacagtaaattattgtgcttta
    gttgctagtttgtactgagagttgacctctccctgtgcag
    ttttttgttctaaacttgtataaataacaattgtgtaatg
    tgtctccctcctacattgtaacaatt
    207 203155_at gtcttcgtggatacccatgatcttcgcttcccctgggtgg
    ccttctttgccagcaaaagaatccgggctgggacagaact
    tacttgggactacaactacgaggtgggcagtgtggaaggc
    aaggagctactctgttgctgtggggccattgaatgcagag
    gacgtcttctttagaggacagccttcttcccaacccttct
    tgaactgtcgtttcctcaggaactgggtcttcctgattgt
    tgaaccctgacccgaagtctctgggctagctactcccccc
    agctcctagttgatagaaatgggggttctggaccagatga
    tcccttccaatgtggtgctagcaggcaggatcccttctcc
    acctccaaaggccctaaagggtggggagagatcaccactc
    taacctcggcctgacatccctcccatcccatatttgtcca
    agtgttcctgcttctaacagactttgttcttagaatggag
    cctgtgtatctactatc
    208 203371_s_at Ctttcctgccgtgagaaactcgaggtgaccaacctccgtt
    tccggttggctccggttgcagagttgagtgtcctgagagg
    tcagattgctgtcagacatggcccatgaacatggacatga
    gcatggacatcataaaatggaacttccagattatagacaa
    tggaagatagaagggacaccattagaaactatccagaaga
    agctggctgcaaaagggctaagggatccatggggccgcaa
    tgaagcttggagatacatgggtggctttgcaaagagtgtt
    tccttttctgatgtattctttaaaggattcaaatggggat
    ttgctgcatttgtggtagctgtaggagctgaatattacct
    ggagtccctgaataaagataagaagcatcactgaagataa
    tacctggaagcatcatagtggtttcttaactctccaaaat
    aagatttcttctctgtagcctacttgtctggtttatcc
    209 203821_at gagccactctatgagttggacttcagtcttgcctaggcga
    ttttgtctaccatttgtgttttgaaagcccaaggtgctga
    tgtcaaagtgtaacagatatcagtgtctccccgtgtcctc
    tccctgccaagtctcagaagaggttgggcttccatgcctg
    tagctttcctggtccctcacccccatggccccaggccaca
    gcgtgggaactcactttcccttgtgtcaagacatttctct
    aactcctgccattcttctggtgctactccatgcaggggtc
    agtgcagcagaggacagtctggagaaggtattagcaaagc
    aaaaggctgagaaggaacagggaacattggagctgactgt
    tcttggtaactgattacctgccaattgctaccgagaaggt
    tggaggtggggaaggctttgtataatcccacccacctcac
    caaaacgatgaaggtatgctgtcatggtcctttctggaag
    tttctggtgccatttctgaactgttacaac
    210 203966_s_at gatgcaacagatatatagccctttcaagtcatgttgtgtt
    tggacttggggttggaacagggagagcagcagccatgtca
    gctacacgctcaaatgtgcagatgattatggaaaataacc
    tcaaaatcttacaaagctgaacatccaaggagttattgaa
    aactatcttaaatgttcttggtaggggagttggcattgtt
    gataaagccagtcccttcatttaactgtctttcaggatgt
    tccttcgttgtttccatgagtattgcaggtaataatacag
    tgtgttccataagaatctcaatcttggggctaaatgcctt
    gtttctttgcacctcttttcaagtccttacatttaattac
    taattgataagcagcagcttcctacatatagtaggaaact
    gccacatttttgctatcat
    211 204192_at tacccgcaggactggttccaagtcctcatcctgagaggta
    acgggtcggaggcgcaccgcgtgccctgctcctgctacaa
    cttgtcggcgaccaacgactccacaatcctagataaggtg
    atcttgccccagctcagcaggcttggacacctggcgcggt
    ccagacacagtgcagacatctgcgctgtccctgcagagag
    ccacatctaccgcgagggctgcgcgcagggcctccagaag
    tggctgcacaacaaccttatttccatagtgggcatttgcc
    tgggcgtcggcctactcgagctcgggttcatgacgctctc
    gatattcctgtgcagaaacctggaccacgtctacaa
    212 204419_x_at acactcgcttctggaacgtctgaggttatcaataagctcc
    tagtccagacgccatgggtcatttcacagaggaggacaag
    gctactatcacaagcctgtggggcaaggtgaatgtggaag
    atgctggaggagaaaccctgggaaggctcctggttgtcta
    cccatggacccagaggttctttgacagctttggcaacctg
    tcctctgcctctgccatcatgggcaaccccaaagtcaagg
    cacatggcaagaaggtgctgacttccttgggagatgccat
    aaagcacctggatgatctcaagggcacctttgcccagctg
    agtgaactgcactgtgacaagctgcatgtggatcctgaga
    acttcaagctcctgggaaatgtgctggtgaccgttttggc
    aatccatttcggcaaagaattcacccctgaggtgcaggct
    tcctggcagaagatggtgactggagtggccagtgccctgt
    cctccagataccactgagctcactgcc
    213 204566_at gccacttgtcttgaaaactgtgcaactttttaaagtaaat
    tattaagcagactggaaaagtgatgtattttcatagtgac
    ctgtgtttcacttaatgtttcttagagccaagtgtctttt
    aaacattattttttatttctgatttcataattcagaacta
    aatttttcatagaagtgttgagccatgctacagttagtct
    tgtcccaattaaaatactatgcagtatctcttacatcagt
    agcatttttctaaaaccttagtcatcagatatgcttacta
    aatcttcagcatagaaggaagtgtgtttgcctaaaacaat
    ctaaaacaattcccttctttttcatcccagaccaatggca
    ttattaggtcttaaagtagttactcccttctcgtgtttgc
    ttaaaatatgtgaagttttccttgctatttcaataacaga
    tggtgctgctaattcccaacatt
    214 204689_at cttttctgtaatctgtttatctcccacttaatggaaaggc
    aaaggggtaccccaaatccagaggtgcctacatttcaggc
    agccttggagtattttaaaaggaaaacattctttactttt
    atatgacattcttatactgctgtctcaaatcctttttcat
    ttcagagctcttgtctcagagatgtgtgttctttttgtca
    gagatatggttgatgagaatcttaaatgcttgttttgcac
    tatcacttagtacctgtttgaccaaggtgttaagggatag
    tacctcccatcagcagagaaactg
    215 205249_at ggaacgtagcaatatctgctccttttcgagttgtttgaga
    aatgtaggctattttttcagtgtatatccactcagatttt
    gtgtatttttgatgtacccacactgttctctaaattctga
    atctttgggaaaaaatgtaaagcatttatgatctcagagg
    ttaacttatttaagggggatgtacatattctctgaaacta
    ggatgcatgcaattgtgttggaagtgtccttggtcgcctt
    gtgtgatgtagacaaatgttacaaggctgcatgtaaatgg
    gttgccttattatggagaaaaaaatcactccctgagttta
    gtatggctgtatatttatgcctattaatatttggaatttt
    ttttagaaagtatatttttgtatgctttgttttgtgactt
    aaaagtgttacctttgtagtcaaatttcagataagaatgt
    acataatgttaccggagctgatttgtttggtcattagctc
    ttaatagttgtgaaaaaataaatctattctaacgcaaaac
    cact
    216 205552_s_at gctcctgacggtctatgcttgggagcgagggagcatgaaa
    acacatttcaacacagcccaggaatttcggacggtcttgg
    aattagtcataaactaccagcaactctgcatctactggac
    aaagtattatgactttaaaaaccccattattgaaaagtac
    ctgagaaggcagctcacgaaacccacgcctgtgatcctgg
    acccggcggaccctacaggaaacttgggtggtggagaccc
    aaagcgttggaggcagctggcacaagaggctgaggcctgg
    ctgaattacccatgctttaagaattgg
    217 206115_at ctcgctccttctggtatatgcatgtcactgcatgataatt
    gagttttcctttgttttaataaaactgttctcagacatta
    agctaaactaagagaaaaataactttgttgccaaaaggtt
    gtgctatccagattttttatatgtctgcatgtttaaaaaa
    aaaaaagcaacaaaagaaaatgcactctaacttatgtgaa
    ctgagagaaaaaaatcaggttttaaacaggaaaacctatg
    gggaatgatattttttgaaagacttttgtataaagttgag
    tacttagaaaaaagacaaaccagatgtaatatattttgtg
    gatgtttttatttcttggatttatagtaccttatactaag
    gttaaaaaaatatgcttgatattgtgaaaaggtgaaattc
    ttcaccaacatttcatttgctcctttgtcatattgtaatg
    ccaatataatatagttaatgaaaacagcatttttaaaaac
    cgaaatattgaaatggtgtaatgttgtaccatttgcactg
    tgagc
    218 206584_at tgatgattagttactgatcctctttgcatttgtaaagctt
    tggagatattgaatcatgttaccatttctgtttttttcca
    ccctgttttcttccatatttactgaagctcagaagcagta
    ttgggtctgcaactcatccgatgcaagtatttcatacacc
    tactgtgataaaatgcaatacccaatttcaattaatgtta
    acccctgtatagaattgaaaggatccaaaggattattgca
    cattttctacattccaaggagagatttaaagcaattatat
    ttcaatctctatataactgtcaacaccatgaatcttccaa
    agcgcaaagaagttatttgccgaggatctgatgacgatta
    ctctttttgcagagctctgaagggagagactgtgaataca
    acaatatcattctccttcaagggaataaaattttctaagg
    gaaaatacaaatgtgttgttgaagctatttctgggagccc
    agaagaaatgctcttttgcttggagtttg
    219 206877_at gaaaagccgttcaccaaatcgaccagcttcagcgagagca
    gcgacacctgaagaggcagctggagaagctgggcattgag
    aggatccggatggacagcatcggctccaccgtctcctcgg
    agcgctccgactccgacagggaagaaatagacgttgacgt
    ggagagcacggactatctcacaggtgatctggactggagc
    agcagcagtgtgagcgactctgacgagcggggcagcatgc
    agagcctcggcagtgatgagggctattccagcaccagcat
    caagagaataaagctgcaggacagtcacaaggcgtgtctt
    ggtctctaagagagtgggcactgcggctgtctccttgaag
    gttctccctgttggttctgattaggtaacgtattggacct
    gcccacaactcccttgcacgtaaacttcagt
    220 207170_s_at aaaacagcactcctttggctggagcacttgtgtccgtgca
    tgtacttgggtgtttccctccatcctttctgatatgacca
    aaaatcaagttgttttgttttttgtcaccttcactggcat
    gggctaaccacttctttttcaaaccctctgaacacctttt
    tctgatgggtaacttgcaggaatattctattggaaaagat
    aacaggaagtacaagtgcttcttgaccccttcctcaatgt
    ttctagccttcactctccattgtcttttctgggctgtatt
    acagccctctgtggatcttcaactctgctgcctccactgt
    gatgcagcagtccaactgtaactgacagtggctgccttct
    ctgggccatggatcaca
    221 208631_s_at Ccagaactgctgacacagatggtgtccaagggcttcctag
    gtcgtaaatctgggaagggcttttacatctatcaggaggg
    tgtgaagaggaaggatttgaattctgacatggatagtatt
    ttagcgagtctgaagctgcctcctaagtctgaagtctcat
    cagacgaagacatccagttccgcctggtgacaagatttgt
    gaatgaggcagtcatgtgcctgcaagaggggatcttggcc
    acacctgcagagggagacatcggagccgtctttgggcttg
    gcttcccgccttgtctgggagggcctttccgctttgtgga
    tctgtatggcgcccagaagatagtggaccggctcaagaaa
    tatgaagctgcctatggaaaacagttcaccccatgccagc
    tgctagctgaccatgctaacagccctaacaagaagttcta
    ccagtgagcaggcctcatgcctcgctcagtcagtgcacta
    accccagctgccggcagtgctgattctccaacagagtg
    222 208691_at gagttctgtcatgattcactattctagaacttacatgacc
    tttactgtgttagctctttgaatgttcttgaaattttaga
    ctttctttgtaaacaaataatatgtccttatcattgtata
    aaagctgttatgtgcaacagtgtggagattccttgtctga
    tttaataa
    223 208868_s_at acatctagaaacattacaccacacacaccgtcatcacatt
    ttcacatgctcaattgatattttttgctgcttcctcggcc
    cagggagaaagcatgtcaggacagagctgttggattggct
    ttgatagaggaatggggatgatgtaagtttacagtattcc
    tggggtttaattgttgtgcagtttcatagatgggtcagga
    ggtggacaagttggggccagagatgatggcagtccagcag
    caactccctgtgctcccttctctttgggcagagattctat
    ttttgacatttgcacaagacaggtagggaaaggggacttg
    tggtagtggaccatacctggggaccaaaagagacccactg
    taattgatgcattgtggcccctgatcttccctgtctcaca
    cttcttttctcccatcccggttgcaatctcactcagacat
    cacagtaccaccccaggggtggcagtagacaacaacccag
    aaatttagacagggatctcttacctttggaaaataggggt
    taggcatgaaggtggttgtgattaagaagatggtt
    224 208869_s_at Gactaaaaccactcttagcatctcctctagtattttccat
    gtatcaggacagaggtgtcttatgtagggagggggcaagt
    atgaagtaaggtaattatatactactctcattcaggattc
    ttgctcccatgctgctgtcccttcaggctcacatgcacag
    gaatgctacatgatggccagctgcttccctccttggttat
    catccactgcagctgctagttagaaaggtttggagggatg
    acttttagtaaatcatggggattttattgatttattttca
    cttttgggattttgtggggtgggagtggggagcaggaatt
    gcactcagacatgacatttcaattcatctctgctaatgaa
    aagggttctttctcttgggggaaatgtgtgtgtcagttct
    gtcagctgcaagttcttgtataatgaagtcaatgccatca
    ggccaaggaaataaaa
    225 208942_s_at caacatggcggaacgcaggagacacaagaagcggatccag
    gaagttggtgaaccatctaaagaagagaaggctgtggcca
    agtatcttcgattcaactgtccaacaaagtccaccaatat
    gatgggtcaccgggttgattattttattgcttcaaaagca
    gtggactgtcttttggattcaaagtgggcaaaggccaaga
    aaggagaggaagctttatttacaaccagggagtctgtggt
    tgactactgcaacaggcttttaaagaagcagttttttcac
    cgagccctaaaagtaatg
    226 209092_s_at aaatccaatcatgagtccaggtagagaacgcctgctgtaa
    tctacactgttgctgggactgcgcattctgtatataactg
    tgttggatgagtgacagatgattgtccagactaggacagc
    ggcatgaacatgactttggttgggattgcggatagttagg
    gttacctctgaatcgtgtagcttttatgagagcagctgtg
    caagtgaatccacattaatgccttgtcgtggtgccattcc
    cagcgcctgacgatacgctcttctattgtcttattctggc
    aggttttgacgttttaaattttttaaagaaattttattcc
    ttggaccaaaaggtttggttaaccacccccctcttacttg
    ctttcacattttgagtgtccagaggaaacagaaaggaatg
    agtgtgtgacgttgctgcacgcctgactctgtgcgagctt
    ctttctgtgtatat
    227 209193_at gattgtagtggatctaatttttcagaaattttgcctttaa
    gttattttacctgtttttgtttcttgttttgaaagatgcg
    cattctaacctggaggtcaatgttatgtatttatttattt
    atttatttggttcccttcctattccaagcttccatagctg
    ctgccctagttttctttcctcctttcctcctctgacttgg
    ggaccttttgggggagggctgcgacgcttgctctgtttgt
    ggggtgacgggactcaggcgggacagtgctgcagctccct
    ggcttctgtggggcccctcacctacttacccaggtgggtc
    ccggctctgtgggtgatggggaggggcattgctgactgtg
    tatataggataattatgaaaagcagttctggatggtgtgc
    cttccagatcctctctggggctgtgttttgagcagcaggt
    agcctggctggttttatctgagtga
    228 209200_at ggagcaatccaagccacatatcttctacatcaaatttttc
    cattttggttattttcataatctggtattgcattttgcct
    tccctgttcatacctcaaattgattcatacctcagtttaa
    ttcagagaggtcagttaagtgacggattctgttgtggttt
    gaatgcagtaccagtgttctcttcgagcaaagtagacctg
    ggtcactgtaggcataggacttggattgcttcagatggtt
    tgctgtatcatttttcttctttttcttttcctggggactt
    gtttccattaaatgagagtaattaaaatcgcttgtaaatg
    agggcatacaagcatttgcaacaaatattcaaatagaggc
    tcacagcggcataagctggactttgtcgccactagatgac
    aagatgttataactaagttaaaccacatctgtgtatctca
    agggact
    229 209861_s_at aagtaaatacttgatggctctgaagaatctgtgtgacttg
    ggcattgtagatccatatccaccattatgtgacattaaag
    gatcatatacagcgcaatttgaacataccatcctgttgcg
    tccaacatgtaaagaagttgtcagcagaggagatgactat
    taaacttagtccaaagccacctcaacacctttattttctg
    agctttgttggaaaacatgataccagaattaatttgccac
    atgttgtctgttttaacagtggacccatgtaatactttta
    tccatgtttaaaaagaaggaatttggacaaaggcaaaccg
    tctaatgtaattaacca
    230 209967_s_at ttcctaaaaatgcttcactgtacgtagttaagtcgtagct
    ataacttcaaattttttaaaagggacaaactgtaaaaaat
    gtgtgtattcttaaaatgcaatatttgtaaggcttgttcc
    aatgccacatacttgcagctcccattctatgtgtcatcaa
    tagtgtcctatgcaataaattatttgcaggtcttta
    231 210027_s_at tgggatgaagcctttcgcaagttcctgaagggcctggctt
    cccgaaagccccttgtgctgtgtggagacctcaatgtggc
    acatgaagaaattgaccttcgcaaccccaaggggaacaaa
    aagaatgctggcttcacgccacaagagcgccaaggcttcg
    gggaattactgcaggctgtgccactggctgacagctttag
    gcacctctaccccaacacaccctatgcctacaccttttgg
    acttatatgatgaatgctcgatccaagaatgttggttggc
    gccttgattactttttgttgtcccactctctgttacctgc
    attgtgtgacagcaagatccgttccaaggccctcgcgagt
    gatcactgtcctatcaccctatacctagcactgtgacacc
    acccctaaatcactttgagcctgggaaataagccccctca
    actaccattccttctttaaacactcttcagagaaatctgc
    attctatttctcatgtataaaacgaggaatcctccaacca
    ggctcctgtgataga
    232 210053_at aacgtagttactgtatggcactcaaaaactatgttaaatg
    atccactaactttttttttcttggcccatgattaatggaa
    tgtatgtaactaggtagggttcctttcttagatctagagg
    aagtacagccacccactgacatctgaatttatatacctgt
    tgagttttgagtgcacccaaacactcgataaaccaggtga
    agaaatttagcttccatgttctacttcagctaaaacagct
    acatacaacctagtacacttgaagtcagacagacatttca
    gttgcttacctccagtactgagccttgctttgggaaacta
    aaagatttagaccaagtcactgccagtttttgcctttg
    233 210172_at Ggtaaatatacgttctgcatccaaatgcactttgagattg
    ttacgatttattctgagcaagatctgcattttttgaaagt
    ttgagattgtagttatcttttttgagatgagatactttca
    cgactttggtatcatctgtcagtttttgcccagtgagttc
    tgcatttgcagctccttgttttggtctgttcgtaactgca
    gtgttctccatgagtatcaggaaggtagggtttcacttag
    gagtaagaacagtccccagtccagcagccacccttttcag
    ctgctgttcattgccagttgatgaggtgagtgtcatctgc
    ctctctggacaggccccagtggagaacaccgcaggtactg
    taaaccaagtacttttcacagcgtggccttt
    234 210766_s_at Ggttccatcaatggtgagcaccagcctgaatgcagaagcg
    ctccagtatctccaagggtaccttcaggcagccagtgtga
    cactgctttaaactgcatttttctnaatgggctaaaccca
    gatggtttcctaggaaatcacaggcttctgagcacagctg
    catt
    235 210949_s_at acaaagtccgcaccatgctggttaggaagatccaggaaga
    gtcactgaggacctacctcttcacctacagcagtgtctat
    gactccatcagcatggagacgctgtcagacatgtttgagc
    tggatctgcccactgtgcactccatcatcagcaaaatgat
    cattaatgaggagctgatggcctccctggaccagccaaca
    cagacagtggtgatgcaccgcactgagcccactgcccagc
    agaacctggctctgcagctggccgagaagctgggcagcct
    ggtggagaacaacgaacgggtgtttgaccacaagcagggc
    acctacgggggctacttccgagaccagaaggacggctacc
    gcaaaaacgagggctacatgc
    236 211458_s_at aaatagcattaaactggaattgacagagtgagttgagcat
    ctctgtctaacctgctctttctctctggtgctcctcatct
    cacccctaccttggaatttaataagcttcaggcatttcca
    attgcagactaaaaccacttctaccatctcctctagtatt
    ttccatgtatcaggacagagatgtcttatgtagggaaggg
    gcaggtatgaagtgaggtagattatctatacctctcactc
    attcaggattctcgctcccatgctgctgtcccttcattct
    cacactcacaggaatgctatgtgatggccagctgcttccc
    ttcttggttatccactgcagctgctagttagaaaggtttg
    cagggatgacttttagtaaatcatggggattttattgatt
    tattatcacttataggattttgtggggtgggagtggggag
    caggaattgcactcagacatgacatttcaattcatctctg
    caaatgaaaagggttcttcctcttgggggaaatctgtgtg
    tcagttctgtcagctgcaagttctt
    237 211546_x_at gggagttgtggctgctgctgagaaaaccaaacagggtgtg
    gcagaagcagcaggaaagacaaaagagggtgttctctatg
    tagtggctgagaagaccaaagagcaagtgacaaatgttgg
    aggagcagtggtgacgggtgtgacagcagtagcccagaag
    acagtggagggagcagggagcattgcagcagccactggct
    ttgtcaaaaaggaccagttgggcaagaatgaagaaggagc
    cccacaggaaggaattctggaagatatgcctgtggatcct
    gacaatgaggcttatgaaatgccttctgagg
    238 212199_at attttgctgttacctttgtgacctgattgttttttggaac
    acgtcaagacgtgggatcagaatcttccaactttagaggt
    gcaatggaagacactacgctacttggttgagcctggtgaa
    gaatgtattaatgagactgctttgcataaaactgggaaga
    aagagaagacagttggagatggaagatggttttgtatata
    ttttggaactttagttcctctgtgagacgaaagaggagag
    ctatgttttgtgtcacattgtctgatatatattgtgtaac
    ctgtcaggtgagttgatttagacaacatagctgacctttt
    atgacaaggcagtttgaatagggactattgtaataccctc
    acacattataggggcancagagaatggcatggaagagaca
    gtctacagagagctttaagaggccggagaaaggaaaagac
    attatcagggcctggaaagtctcttccagttcatcagggt
    ag
    239 212224_at acagtgttctctaatgttacagatgagatgcgcattgcca
    aagaggagatttttggaccagtgcagcaaatcatgaagtt
    taaatctttagatgacgtgatcaaaagagcaaacaatact
    ttctatggcttatcagcaggagtgtttaccaaagacattg
    ataaagccataacaatctcctctgctctgcaggcaggaac
    agtgtgggtgaattgctatggcgtggtaagtgcccagtgc
    ccctttggntgggattcaagatgtctggaaatggaagaga
    actgggagagtacggtttccatgaatatacagaggtcaaa
    acagtcacagtgaaaatctctcagaagaactcataaagaa
    aatacaagagtggagagaagctcttcaatagctaagcatc
    tccttacagtcactaatat
    240 212388_at gtgacagttatgacaggcttaccttggaagagttgtcatt
    tttactgccaattttttggatgaagatgtttttataaacc
    tttcaaaatggtctgcaaacagagcaggaattgcacaatt
    aactcaataatgctgtgtgttctcaagaagctcccttagt
    gaggccgatcttaagatggccgattctgcccgttgaaggc
    atncctgggaaagaaaacaagcatcccagcgggcatctca
    ccacgacttctcctggagtcctcacacggtcactgacaac
    tacagtcagttttaggaactagagtgccgtatcatcagac
    ttaccctgtcctgccccaccttccctgctaacatcgaggt
    gtgtgcagttaccttctgagcttggaacaagcagactgga
    attttcctctgctacctcttgtgtataaaatcttgttt
    241 212591_at ggcccagattgcagatggatttcgtattagagttgatctc
    gcatctgagacctcatctagagacaagagatcggtttttg
    tggggaatctcccttataaagttgaagaatctgccattga
    gaagcactttctggactgtggaagtatcatggccgtgagg
    attgtgagagacaaaatgacaggcatcggcaaagggtttg
    gctatgtgctctttgagaatacagattctgttcatcttgc
    tctgaaattaaataattctgaactcatggggagaaaactc
    agagtcatgcgttctgttaataaagaaaaatttaaacaac
    aaaattcaaatccacgattgaagaatgtcagtaaacctaa
    gcagggacttaattttacttccaaaactgcagaaggacat
    cctaaaagcttatttattggagaaaaagctgttctcctta
    aaacgaagaagaaaggacagaagaaaagtggacgccctaa
    gaaacagagaaaacagaaataacaaccaggaactgctttt
    tcttttcctgctgagtactgctaata
    242 212696_s_at ctgaaggagtgtccctcctctatgtgaaaagaaaattgtt
    ttattcttncattctgactttttaancngttnggctcact
    tnccagttagtttgaatgaaaataataattttctacttgg
    nagttgaagagggcagaatccgcagctctcatcattgtga
    tgtgtagcatgtctgccctctgactggacatcattgccat
    taactttcttctgggcatcacggcaatgtcacgatgccca
    gacttggagcaaggcaaccttggagtcagtccactcataa
    aatatggtaacacccattttaaaatttaagttttgtcctt
    aaagacaacttcagtggttaattataaaagttgtgttact
    tcgtcctaaattaaattgatagaaagatttaaaaatgtgt
    tttgtttctactattcagaaactgcgaactagggaaaggt
    tggtatgaaaaaatgtctttccttttttcaatgtacatag
    ttcaactctttctttgttacatttaaactatatccatgga
    tatcagtctgctttggactcctctgctagtgttacagatg
    243 212709_at tgcaaattaagactcaccttcactttccaaaatagctgag
    ggttgtnggcttgttgtagctgaccaccaaaagcagtcac
    tgcaaatcttttaattcttccctatcaccttttgtatttt
    aatgcaattattttggtccagaactgacctgtattttctg
    tattgtacacaaaagctaataattttgtgtactttttatt
    tattttggaggttttatatgatcttcaattgagtattaaa
    taatttgcctagattaagcctaaaatgatgaccagctaat
    taaagaagatattttgaatctggttctgagctaaagttga
    gtaaattcttagctaagaaaaaattggaaatccatcatct
    atattagcaacagattctcagagtaaattgttaacttcta
    tgatttatgataatcaagctggacttgatcatacaagtta
    gtctcataatgtattggaccaaaatgtaaacttcattggt
    cagatttagaagcattcatgctcacaagttttggga
    244 212714_at aactttaggcttttgggcatatgctagtctgagcttccga
    aaagatacatatatgtttcccttttcattagctgaatgag
    gatattttaagaagttgaaagagaatttattttcaagttg
    tgagtaaatcctcctttgaaattcacctgattattagata
    acttaaagtttatttttaaaagctgacaactttttatgaa
    tcttcgagttgacagttcctaaaagcgtaactcagatatt
    aatgggctgtgtattaaatggttttattttcagttttgca
    gcacagaacactgttgaaatatccatatcaacttgatttt
    tttaacctaattcaggtgtcctttgcatctcttaaatgtt
    gggggtgggggtcagagccagttatccggcttctgttttg
    tcgattgcttagatttgttcctgttgtc
    245 212893_at aattgtccttaattaccaacagtgaagcactacaggaggc
    aactgtggcattgcttccttaaccagctcatggtgtgtga
    atgttataaaattgtcactcagatatattttttaaatgta
    atgttatataagatgatcatgtgatgtgtacaaactatgg
    tgaaaagtgccagtggtagtaactgtgtaaagtttctaat
    tcacaacattaattcctttaaaatacacagccttctgcct
    ctgtatttggagttgtcagtacaactcatcaaagaaaact
    gcctaatataaaaatcatatatatggtaataatttccctc
    ttttgtagtctgcacaagatccataaaagattgtattttt
    attactatttaaacaagtgattaaatttagtctgcacagt
    gagcaaaggttcacatgcattcttttatactgctggattt
    tgttgtgcatcatttaaaacattttgtatgtttcttctta
    tctgtatat
    246 212989_at gtcagagcctgttcacatattgtgatcaggtaacaatgac
    atgtaccacttaaattattttatcgtcgatttggtagttc
    nattttaactactcaatgaaaacagccatgaatatctttt
    cttaaagagagttttgaaaatgatcacttacctaaaactt
    gaaagctatgaattagttatccatactctcatgacaattt
    tgttggtgaacaacaaaaaagagatctatttctttaaaag
    atatttgtgcagaaactgcatgtaactctaagttttactc
    ctaacatacatatgtttggggaagtattctattctatact
    tgccaatgtggagaacaaaatagttttttaagaatgaaga
    agtatatatatccattctgtattttacgtgcagcagaatt
    atcttccgtaggatttt
    247 213410_at gacaaattacggttgagttctgtggcttcttcacttgaag
    tgctaacatcagaatcaaacttaaagcttccactatttat
    gtctttgagaagtatgtagtacctcggtattaacagacct
    gctgtgatgcagttacactttcacgtatttttgaagtatg
    tcaagctacacgggtctaagatatgattattttggataaa
    atgttactttggtcaagagaacttttatccagatgacatt
    acaggttcaagtgggttaaggagacctcctgtacatctac
    agtgtttccttttaaattgtccagaaaaaaggtgtgttct
    tcataaacttcagtgcaggatttttcaaagacgagctgtt
    gtgcaatttgctgtatttaatgcatgttctgaaaggattc
    acttttgactttatatgacagttgatcaagaacaggtact
    accccttt
    248 213515_x_at ggatgatctcaagggcacctttgcccagctgagtgaactg
    cactgtgacaagctgcatgtggatcctgagaacttcaagc
    nncctgggaaatgtgctggtgaccgttttggcaatccatt
    tcggcaaagaattcacccctgaggtgcaggcttcctggca
    gaagatggtgactggagtggccagtgccctgtcctccaga
    taccactgagctcactgcccatgatgcagagctt
    249 213528_at Aactgcattcaagggagggtccaaagaaattcactttcaa
    gattataacagtatggtgattgatgaagtaaccttaccta
    atgtagtagctaactccactttggaagatgaagaaaatga
    tgtaaatgagccagatgtgaaaagatgcaggaaaccaaaa
    gtaacacaactatataaatgccgatttttttctggtgagt
    ggtctgagttttgtaagcttgtactaagtagtgaaaaact
    ttttgtaaaatatgatctcattctcacctcagaaaccatt
    tacaacccagattattatagtaatttgcaccagactttcc
    ttagactgttaagtaaaaatggacgtgtacttttggccag
    caaagcacattattttggtgtaggtggaggtgttcatctc
    tttcaga
    250 213604_at gcaatgtcagtatccattttggcacataaagatttttgat
    gagccctgtttgcatagagccagatgttttcccctccccc
    aagagtatctacatcagggatgtgacttggtgcgaagagt
    caggggaaagaggaaaaacccaatttctaaatgacctcct
    tgcccaacttactaaaatggctgcagagcagacacaggat
    gaatttgaacctgacacaggatgaatttgaacctttggtc
    tcatttatggaaaaacttgtgcaattttttttctgtgcta
    cactacatacaaatcaccaaattacaaattacccttttgt
    gatccttggtgtactgagcagtttctttggggctttttct
    ttctgggaagcgggagggaaaggagcaaggtgtcatcctg
    ctcttc
    251 213619_at aatcaaggcctcagaatttcatacaaacaccaagaccaaa
    atcctaagtattggtattgcgtctcaaatttttcccatta
    acttnaaaaaaaaaaaaagcttaaacttacgtgccttaca
    ggttattaaatgaaactagaattaacaaacatgccaaaat
    gtttcacttttaatagtagacacagctcctatattgtttt
    acaaaaaaataaaagcatgtctttcaacatgcatccaaaa
    cagtgttcaatttaacgtggcaaagggcaacatttaacat
    aattcaactgcttttacctaaatacgcttactgcttaagt
    acatcctataactaacttgagaaaagctggaacttaagtt
    taacagttatagtttactcagcttcactgttacatcctag
    atgagtattgtattcaaaaatactgggccttaagtcttca
    taacaatcctgatttccacttagagt
    252 213655_at cttcaacgctaacctgcttcagtgggagagtaaagtaggc
    aagaatgagcagccacggattgttgaactgttaccagcac
    catgcttttcagcaacatttcagcggagttggaaacattt
    tttacagcaaaaccattacaacgaagtccctccccaaacc
    acctttaaccatctcaagctaacacccaattacttgcaaa
    cactggtataaaacacagtttaaacaattagaaaaatgaa
    aactgataccacttatgcctctatagtgtgattaacctct
    ctcttagatgcttgcatcacctataagtctaatggctttc
    aaatgtaatttccatttgctaatggtgatcttgccacatc
    tggcacggagacgacacagtaatgctgaaaaagcctctat
    gtagtcctgttagtgtcttaaagaacctaaaagctgggac
    cagtaaaatccacagaaattcactcttgccttta
    253 213743_at ttgtgtgagctattcaaactcttcaacccctgaacagggt
    attaagcttccaaaataatgatggggataaatatggaaat
    cctttttaagttgtatttccattaaacaaaaacccttata
    attcatactatcatgaatttgctttatccatctcatttgc
    ataacagttcatctgtctggtcccattaggctctaccaaa
    gaaagactctgatgagtggacattattactgtgactcttg
    taagtagccataaataaaccaaaatagtatcaaatttagg
    tatgaaattccacatgtgcaaa
    254 213788_s_at gtttcagtcatttccggactaactgtgacaacgcgtgagc
    agggagcaccgtgcgagtctccgggagggaatcctcctgg
    ggcccagagactcctccacccctggggagggcagagaggc
    tcgggagggcctggccaggccactggaggctggcagggag
    caggcatgtccacccgcaagcctgggaggctaactctggc
    attcctggccggagccgccatgctcattggtgggccagtt
    tgggacatccccgtactcaaagaccatatggcaggctctg
    ggaaaacaaaaccaaaacatcaccttctattaaactctgt
    atattattattttttacaatagaaagttaaaaatcaagac
    ttagatttactatacattttttctctcagattacaaagtt
    tatattatataactggggttccctaaattgatttctttta
    aaacagtcttaaagagaccagaagtgaatacaaaagaact
    aaacaaaataaaaaattagaatatgctgtagctgaaagct
    gtctatacctgtaagcctccaagtttcat
    255 213872_at aaactggtcagctagagattcttttttctttctaagctga
    gcacgtagtctgttcagagcttgtttcttccgggagtttg
    ggtcccccattttgaaatncnggtggtactaaagcctttg
    gaaattgtcactaaactatgggcactttttcttaagactc
    aagtacaacagaaacaagtcattttttttcctgctaatat
    gattgattagcgaaaatcacgactataacccaaaaactgc
    accttctgtcaatattagcagactgtcatattacagggtc
    aagaaacaaaagctgctgtccagtcatgtttggacaataa
    cgtttggggtcagacgggaaaaagggaggaaagaaaggaa
    agaaagaggagaaaataactaactttctggaaaacacatt
    tggcttaactgccaaaataaaggctttgcggagaaatgaa
    aagcctataatcaggatttaggtgtgcaataaaacacagc
    tgacaccagaccaatccctaaaatccatccggattttccc
    ccctttttagaaaagggattaaggaacagggagggggaag
    tgtgatccttgctttc
    256 213979_s_at aaatggttacagtcacaaacatgattttaaccaaaatatt
    gctagcctaccacatcagcaggacggcactggtgcaggca
    gggacgctgccaccctccacgtccccaggacagacgtcga
    tgggcagcgggcacgctcggcaccgcccaagcttttcctt
    ttggagctgcttcgtgatgccgtcttcatttggaacaagg
    gggggttcatgccaaaattaggaaaaacagcctttgtttt
    gtttttctaaattattctaaaaataagacaagcaggtaga
    aaaaacaatgcactgtgtggcataaaaagaaaaaggggaa
    ggattcattgtcctgagaagtttgccaactgcctcattct
    ggggcacgttccaacataca
    257 214257_s_at attataacctgggcctgcctttgttctcatgaagccagga
    gcctctccatatcctatacttgctcttacgctaataacaa
    accaaatgctgcaaaataaaagtaataatgacccaaacta
    atttaagtcttttgtttaaggagtaaatgagagaaacatt
    ttagcttcttaatcaaggagtgctataatttcaaggcatc
    ttaatataattcacttaccctaaagcaattgtgcaataag
    caaattataaaaggaaaacaacaaaggttaactttctaca
    ggggccaatagacaagatctgtggagcacagcaattaacc
    ttcacatactggagtcttgtttaaaaggcgatcaaaaact
    cagattactga
    258 214414_x_at tcaacttcaagctcctaagccactgcctgctggtgaccct
    ggccgcccacctccccgccgagttcaacccctgnggtgca
    cgcctccctgganaagttcctggcttctgtgagcaccgtg
    ctgacctccaaataccgttaagctggagcctcggtagccg
    ttcnnnnnncnngctnggcnntccaacgggccctcctccc
    ctccttgcaccggcccttcctggtctttgaataaagtctg
    agtg
    259 214696_at gagtatacatcggtgcaggcttcctggatgacagttgggt
    gatatgtgtcatgtggcctaaaagcctccatgtcatttga
    cctacgaattctatctttgggaatttatcctaagaaanta
    cttanggatttanttngtgataagatgttcatcccagcat
    tgcaatggagaaaaatgggaagcaatggtttggttgggaa
    tttattccttttctgctgtaacgaaagtttgcaatagggg
    attgcttaagtaaattattgtatctccatccagatggtgg
    agtaccgcgcagacattaaaagtcatgtaaaagaacatct
    gactgaaagaaaaatgctccttgaatattaaaaggttgta
    aaaatagtgcatgttatgtgatttcaattttgttttttaa
    aatatgggtgtatgcttgtatacgtagagcagataaaaaa
    gacggaaggcatactaaaaaatgttgagtggttatctttg
    tatggtggaacaaagtcactgtaattttcatctttggtt
    260 214933_at aatctttcctttccctgatgaagacagctggtggccgagt
    gcggnaangaagccagaaggaaccagaatcccagngccct
    acacccaccaccagacacactcacacccacacacgttctc
    agacacacacaagagtgcttgccggttataccaaacccta
    ctattactgcctgcagaaatcaatttaaaaaaataataat
    aacaataaacaattttaaaaaggacaaaaaaattaatgat
    tgagaaaagaggcatttttttctgacatttggtcctgctt
    gaaacaacaaaagaagaagaaaaacccaccatcaccaccg
    attcctttgcttcttttttccttttttcctaccttgtttg
    aaaaccgtgggcttgggactgtgaattattgcatgacat
    261 215043_s_at Cagtctttcccagggaactccgatgaagtgttccaacaaa
    atgagcgagtgaaccaagaagaggatgacattagatccag
    gagatacaacagaggagataatctccaggatgcctgtgaa
    gaaagatccctggatcccaggatgattataggacaagttg
    ttcataatccagcaggccagaagacttccagggaaactca
    tt
    262 215933_s_at ggacagttcctgtgatcagaggcaagatttgcccagngaa
    cagaataaaggtgcttctttggatagctctcaatgttcgc
    cctcccctgcctcccaggaagaccttgaatcagagatttc
    agaggattctgatcaggaagtggacattgagggcgataaa
    agctattttaatgctggatgatgaccactggcattggcat
    gttcagaaaactggatttaggaataatgttttgctacaga
    aaatcttcatagaagaactggaaggctatataagaaaggg
    aatcaattctctggtattctggaaacctaaaaatatttgg
    tgcactgctcaattaacaaacctacatggagaccttaatt
    ttgacttaacaaatagtttatgtactgctcttaggttgtt
    ttgataaagtgacattatagtgattaaattctttccnctt
    taaaaaaacagntagtggttttcactatttataaatagga
    ccttcttgaacgacttttctg
    263 216199_s_at ctggagtctggggtatgttgtcatagagatgatgactggc
    aaggtttgcacagatgaagaatgaagcctagtagaatatg
    gacttggaaaattctcttaatcactactgtatgtaatatt
    tacataaagactgtgctgagaagcagtataagccttttta
    accttccaagactgaagactgcacaggtgacaagcgtcac
    ttctcctgctgctcctgtttgtctgatgtggcaaaaggcc
    ctctggagggctggtggccacgaggttaaagaagctgcat
    gttaagtgccattactactgtacacggaccatcgcctctg
    tctcctccgtgtctcgcgcgactgagaaccgtgacatcag
    cgtagtgttttgacctttctaggttcaaaagaagttgtag
    tgttatcaggcgtcccataccttgtttttaatctcctgtt
    tgttgagtgcactgactgtgaaacctttaccttttttgtt
    gttgttggcaagctgcaggttt
    264 216202_s_at gttcacaaagagttttggtgcttctggaggatatattgga
    ggcaagaaggagctgatagactacctgcgaacacattctc
    atagtgcagtgtatgccacgtcattgtcacctcctgtagt
    ggagcagatcatcacctccatgaagtgcatcatggggc
    265 216996_s_at gctgcatactttggatacttgtctaaaacttgatgatact
    gtctatctgagggacatagccttgtcactcccacagctgc
    cgcgggagctgccatcgtcacatacaaatgcaaaggtggc
    agaggtgctgagcagccttctgggaggtgaaggacacttc
    tcaaaggatgtgcacttgccacacaattatcatattgatt
    ttgaaatcagaatggacactaacaggaatcaagtgctacc
    actttctgatgtgatacaacttctgctacagatattcaaa
    gagtagctgtgctatgtgtttccagatctgcttattgttt
    gggttcaagccaccccagaggattccttgctatgaaaatg
    cggcatttgaatgcaatgggttttcatgtgatcttggtca
    ataactgggagatggacaaactagagatggaagatgcagt
    cacatttttgaagactaaaatctattcagtagaagctctt
    cctgttgctg
    266 217554_at aatgctcaccaaattcacaggaaagaagtgcctaggtgat
    catttactgctatttactgcttcctggtatggagcaaaat
    tgccctctggtaggtaactacatttctaattagtatgaaa
    ggttctactattttcctttcttttgcttatttccccttaa
    gcacaatttgcagaccctacttctatttaactgatcatac
    ccttttaaatgccttgtcatcatttttcatagttcctgca
    tcctagaaaaaataaaaaatcatttaaaatattccttggt
    gtctagaaaagaaaatttctctaacaatagagatnatcat
    tttgctttctcacttaagctgatctgaatgatgatttggt
    actgcctttatgag
    267 217682_at cacgaggtcaatttatggttatcaaataggtttttttttt
    tttttttgagactgagtgtcgctctatagcccaggctgga
    gtgtagtgctcactgcgacttccgcctcctgggttcaant
    gattctcgtgcctcagtctcccgagtagctgggattgtag
    gcgcctgccaccacgcccagctaatttttgtatttgtagt
    agnnatggggtttcatcatgttggcgaagctggtcttgaa
    cacctgacctcaagtgatctgccttagcctcactgcttgc
    ccctangtggtgggattgcaggtgtgagccactgtgctgg
    cctcaaatagtttttatcaaagctacttcaattagtggtt
    agcaaggcttaaagactaattcagtgctttattttgacac
    ttgttcgcaacatgttgctttttttcctgtgtcctatggg
    acctagtcatctgtatgttaga
    268 217840_at gccatccagcacgtcatcaattatgacatgccagaggaga
    ttgagaactatgtacaccggattggccgcaccgggcgctc
    gggaaacacaggcatcgccactaccttcatcaacaaagcg
    tgtgatgagtcagtgctgatggacctcaaagcgctgctgc
    tagaagccaagcagaaggtgccgcccgtgctacaggtgct
    gcattgcggggatgagtccatgctggacattggaggagag
    cgcggctgtgccttctgcgggggcctgggtcatcggatca
    ctgactgccccaaactcgaggctatgcagaccaagcaggt
    cagcaacatcggtcgcaaggactacctggcccacagctcc
    atggacttctgagccgacagtcttcccttctctccaagag
    gcctcagtccccaagactgccaccagtctacacatacagc
    agccccctggacagaatcagcatttcagctcagctggcct
    gg
    269 218229_s_at gacctcacagacaaagccattgctagaaatgtcattccaa
    tgatcagatctggaaacaggctgccataaccacttttcct
    tcttgtagactcagctcacctgtatatttaaactgttctt
    ggcatcttgaaacacctatttctactcaggtactcattgt
    cctgttactgattcacctttctgatccttttcaaccagtt
    ttcccccaaggggggaaattttacttaacctctagtattt
    gaacaactcaatatttgaattgttgccccatttgctttta
    cctgtactgtattcttggtcatctcaatggcgtctaaac
    ccagctactttgcattccagaagtttccattccctccaat
    tccacctaatttttcatctgtcctagttactggctctttc
    ttcatgtcttatttctcttgctttgggagcttaaaagatt
    ttacaagacctaattttgggttccttccttggagccatag
    ttaccctgccaa
    270 218356_at Ctgaaatttccatctggggattaacttctgtctttctggt
    gaacaatatagcaattcacgcattcttcaagcagcaaaag
    ttcccggaacaattagggaagacgtatggtctgaatttat
    ccaggcagtgggtctgctttggtttttgctggaaatttat
    atcagtgtctgggctcc
    271 218432_at gaaactatgtgactcattctgtgaaaagacttcttgcagt
    tgtgagttatttagaaatgatcaaaatttgtaattaggct
    aatccatttagtgattcctaatattttgtactcacagaga
    actaattgactaaacaacgaacgctagtggtttgtcct
    tagacaatctgtctttgaatttaaagtctttatcgctaag
    accttgactttaaatttttcatcactacaaccttgaattt
    aatttcaggtcttcaacatgatgaccttggatttaattta
    aagtcttcaacactatgcgctttatcatattattcacaga
    tgcatttttgaaatgtagtatgtaaaagtatgtaacgtgc
    tgtttattaacaaaagattgttcacaacatctcatgtagt
    ttaaatttgtaaatactgcttctgttttgtttctccttta
    tacacttgactgt
    272 218589_at gggtaacaatgcctcagaagcctgctttgaaaattttcca
    gaagccacatggaaaacatatctctcaaggattgtaattt
    tcatcgaaatagtgggattttttattcctctaattttaaa
    tgtaacttgttctagtatggtgctaaaaactttaaccaaa
    ccagttacattaagtagaagcaaaataaacaaaactaagg
    ttttaaaaatgatttttgtacatttgatcatattctgttt
    ctgttttgttccttacaatatcaatcttattttatattct
    cttgtgagaacacaaacatttgttaattgctcagtagtgg
    cagcagtaaggacaatgtacccaatcactctctgtattgc
    tgtttccaactgttgttttgaccctatagtttactacttt
    acatcggacacaattcagaattcaataaaaatgaaaaact
    ggtctgtcaggagaagtgacttcagattctctgaagttca
    tggtgcagagaattttattcagcataacctacagacctta
    aaaagtaagatatttgacaatgaatctgctgcctgaa
    273 218604_at tttgttttattgtatgcgttgggtttgcagcatgaacttg
    cacagataatgcacgttttctggttaagtaaacatgatgc
    acactattctgtaacagaaagccctattgtgccttacctg
    tgtgctttgtgggcaccttgtttatgaagaataaaaaat
    gatttgttatctgaagagaataaattttaaattctcagtt
    tatgtctcagatgctaacgtgtgaaaatataaatatatat
    aatatataaagtaaccagtcttcctgtattttatgtgcat
    catagtgatttatctgagcttagtgacccccatcttgtaa
    cctgttgcaagagtgaatgtaaaaaatagttgtggcattt
    taaaaggtcgcctttgatgcagatgcatctttttcttgct
    tctaaaacatatttcatgtaaacattgtacatttattatt
    gtaatatatactattatgcagcttattttacctgaaactg
    ttaagccgaccaagatccct
    274 218689_at cctgggtgttccacagctgatagtgattgccttgaataaa
    ttcaagggcaatttattcatttttactagggagatagacc
    tttacagcaatcaagatatttttgtccatatccaggttag
    ctggtaagaggatttttttggagaaaaaaaatgatattta
    gaaagttaatttctaattccggaatggaataaaaacaata
    tgagtagtgtaatcttgtagaaaaagagttgtataatctt
    gtagaatttctcattctgtggtacaacccaggggtaaact
    attattccagtagtcagtacacttttctagataaatcttg
    agtgaaaaccagcaatttctttttccttgtggtctgattc
    ctttttctaatccatgaaggccatcttgtagattacattt
    atcattaatgcaagaataaagacaattcctcctgtcagtt
    gcgtgaattttttttaagaaacaacccagtgaagagttct
    accatagcaaggcctaatgttagctttagctttagaaaat
    aacagtttgtgaacttacttccctatatttgcagctgt
    275 218889_at gcttgtattcaggttcattggcttttgctggatgatccac
    ctaaagaagttacctaatttggccttttaaaaaaggtgtt
    agtgtttattatagctactttcaaggaaagtttgaatatg
    attctagtctctaaagttcttcacgttttctgacattccc
    tggagggtgactggggaagaattgctccagggtagaagaa
    ccaggcccaagactttaccattctgatctagagacaaagg
    atactcaatgaggagcttttttcccctcttggaacaggta
    aaatgctttttcttattaatataattataaaacagtattt
    tatgtaacagctattcccatattctaggagtggcctaaga
    aatgcgtgtttcagtgactagatta
    276 218973_at ctgcaagtgaaacctcagcgcctgatggcagctatgtaca
    catgtgacatcatggccactggtgatgttctcggtcgagt
    ctatgctgtcttgtcaaagagagaaggtcgggtacttcaa
    gaagaaatgaaagaagggacagacatgttcatcatcaagg
    ctgtgctgcctgttgctgaaagctttggttttgctgatga
    aatcaggaagaggacaagtggcctggccagcccacaacta
    gtattcagccattgggagatcattcccagtgaccccttct
    gggtgccaactactgaggaggaatacttgcactttgggga
    gaaggctgactctgagaaccaagcccggaagtacatgaac
    gcagtacgaaagcggaaggggctttatgtggaagaaaaga
    ttgtggagcatgcagaaaagcagaggacactcagcaaaaa
    taagtagctacctactactggtggattcttttccttatag
    tgaatttaaaagtatcatcaagggtttaatattgggaaaa
    tttctttttgccacattatctctgtttattcact
    277 219069_at gaatattgtatactgcatcccctaccacaatttacacaat
    cctgtggatagtcctacctcaccctggtcaacctacatga
    tccttaagctaatggcgaatcacgatgaccttgtagacat
    gcacacaactatacctttgtccaacagatcataatatatc
    tgctatccaactggttttacctgcctaatcctactgattt
    gggcactgcttgtatagtctctcaagttcacaggaaatgt
    tgattttctaaggtcctcattt
    278 219093_at tagagacccatgtcatcttaacctaaagggaaatcttatt
    gcgttatcataaaattgatgatatcttagggtcagaattg
    cccttttttttattttgaatgggaagctctcactaaaaca
    atcctgagatttcttaatttcatggttctttaaatattat
    aaacacagagtcaacatagaataaaattgtatttgttaaa
    atacacacattggaagacaagagcagatgactacttttcg
    aagtaatgctgctccttcctaaaagtctgttttcaatcct
    ggtaatattaggggcactgcggcacctaagaagccttaaa
    tgagagctaatccaatttagagagcgatggtgtcagcatt
    tcggtctgcatatctgtgtgtccgtatctgcgtttgtgtg
    cgtgtacgtgtgcccctgtgtgtgggcccagttttcaggc
    atgtagaataagcatggagtcatattgaggaggctcact
    tcttgaagat
    279 219099_at aatggactgactgaaactcgctaaggttaaatctgcatca
    aaatctaaccaatttgagcctctgaagggagtgccattgg
    ctttatttacttctctcctctgctagtcctgatttggaaa
    cagttaaaagccaatttttagctccagtggaaccatagcc
    acataaaactttaatggacaaccatatagaattaacttat
    tttgtccaagtacagttggcattttccagaataattttac
    caccctgctagatgtcatctctggattgcacatggatgat
    gaaggaactcagcattgaaagttgggggattagtaacctt
    gttacaacggtttctttttcattttagcctattttaatgg
    ctattggtaagatactgtatgtttttagtatctcatccag
    tgcttagaagaaagaatggtttataattcccagtacatgt
    tt
    280 219176_at Caacaggaggcatctccatttactatgatgagaaaggaag
    gaagtttgttaacatcctgatgtgcttttggtatctaacc
    agtgccaacatccccagtgaaactttaagaggagccagtg
    tattccaggttaagttggggaatcagaatgtggaaactaa
    acaacttcttagtgcaagctatgagtttcagagggagttc
    acacaaggagtaaagcctgactggaccattgcacggattg
    aacactcaaaattattagaataattttcttggaaaaatca
    gcttatggactttagcagttgctgtgaaaaactaaggaag
    aaaaattttggggtcatttgatcttcacttaatctaagtc
    tgtgaattacttttatattattttgaaatactccttgcag
    tatattggcatgatacagtaaaagcattttccacagattg
    ttatcaccttcttt
    281 219243_at tcttctagattctctctatgttggcagataatctcccctt
    gtagcttccactcacttattcttgcattcagagtcacaat
    gatcatcttacccatgtggtttttgagaaagaaagatcaa
    ttctttgtttgcagtgggtaatcttagagatggagatgat
    tgtagaattattcctagatgagtgtcaatttatttaattc
    cattgtcatataaggagtcaaattgtttcttatcatttgt
    tcattgaagaacagagacctgtctggaaaatcgatctcta
    caaattcaattaaataatgatccccaaatgctgaaaaagt
    gaaatacagcaattcaacagataatagagcaatgtttagt
    atattcagctgtatctgtagaaactctttgacgaacctca
    atttaaccaatttgatgaatacccagttctcttcttttct
    agagaaagatagttgcaacctcacctccctcactcaacac
    tttgaatacttattgtttggcaggtcatccacacact
    282 219363_s_at tggtcagaaaagcaccatttttgctgaacttttcagtgga
    aagactggataaccagattggattttttcagaaggaactg
    gaacttagtgtgaagaagactagagatctggtagttcgtc
    tcccaaggctgctaactggaagtctggaacccgtgaagga
    aaatatgaaggtttatcgtcttgaacttggttttaaacat
    aacgaaattcaacatatgatcaccagaatcccaaagatgt
    taactgcaaataaaatgaaacttaccgagacgtttgattt
    tgtgcacaatgtgatgagcattccccaccacatcattgtca
    agttcccacaggtatttaatacaaggctgtttaaggtca
    aagaaagacacttgtttcttacctatttaggaagagcaca
    gtatgatccagcaaaacctaactacatctctttggacaaa
    ctagtatctattcctgatgaaatattttgtgaagagattg
    ccaaagcatcagtacaggact
    283 219434_at ccatgatcatggtttactgcgcgtccgaatggtcaacctt
    caagtggaagattctggactgtatcagtgtgtgatctacc
    agcctcccaaggagcctcacatgctgttcgatcgcatccg
    cttggtggtgaccaagggtttttcagggacccctggctcc
    aatgagaattctacccagaatgtgtataagattcctccta
    ccaccactaaggccttgtgcccactctataccagccccag
    aactgtgacccaagctccacccaagtcaactgccgatgtc
    tccactcctgactctgaaatcaaccttacaaatgtgacag
    atatcatcagggttccggtgttcaacattgtcattctcct
    ggctggtggattcctgagtaagagcctggtcttctctgtc
    ctgtttgctgtcacgctgaggtcatttgtaccctaggccc
    acgaacccacgagaatgtcctctgact
    284 221485_at aatgtgaccttgtgcatatattggtagctgaaaatcttca
    aggctactgatgggtggccccttaatcttgtctttgattg
    ctgtgtgcagggaaaggtgtccccgtttgttcatgctgtt
    ttggggggtgggggggtatttgcaagaatactcattttga
    cataataggtcctcttgtcagagatcctctaccacagaca
    ttaatagctgagcaggagccacatggattgattgtatcca
    ctcaccattgacgatggcattgagcgtagctagcttattt
    ccatcactacgtgtttttgagcttgctcttacgttttaag
    aggtgccaggggtacatttttgcactgaaatcta
    285 221652_s_at ggccagtgtcgttattatccttgtggagaatagaatcaat
    actgccaattccagaaaacatcaggaatttgctggacgtt
    tgaactctgttaataacagagctgaactatatcaacatct
    taaagaggaaaatgggatggagacaacagaaaatggaaaa
    gccagccggcagtgaagagtgacttgaagaactaaattta
    gcatattgcaaaaatattttgtgcggaattcgatataagt
    acttttacagcaagatggtatagttatgttgcctggactg
    gtttttacatttttaaaatatttcagctgtcatttttgta
    ctaattataaaattggcacataattcaaaaatatacattt
    gagatgatttgtcctcccaaattatacaagtttattttat
    ggtataaagtgttctctctggaaatgtttttaaaaaaatt
    cttaggcttctctttgcgaaataaa
    286 221755_at Ggagcctcatggagtcaggtgccaacaagctgcaggagga
    ggtgctgatccaggagtggttcaccctggtcaacaagaag
    nacgctctcatcnnnanncannnccngcnncnnnnnnnca
    tggaggagcaggacttggagcgaaggttcgagctgctgag
    ccgcgagctgcgggccatgctggccatcgaagactggcag
    aaaacgtccgctcagcagcaccgagagcagatcctactgg
    aggagctggtgtcgctggtgaaccagcgcgatgagctagt
    ccgggacctggaccacaaggagcggatcgccctggaggag
    gacgagcgcctggagcgcggcctggaacagcggcgccgca
    agctgagccggcagttgagccggcgggagcgc
    287 221970_s_at gcgaaaatgctactatgactcttcctggaatacacccacc
    taccttgaaccagattatggattggatatgtctacttctg
    gatgcgaattttactgttgtngtaatgatgccagaagcaa
    agaggctactgataaatctttacaagcttgtaaaatctca
    gatatctgtttattctgagctcaacaagattgaagtaagt
    tttcgggagctacagaaattaaatcaagaaaagaataata
    gaggattatattcaattgaagtgctggagctcttctgata
    ttatcaattctccttcatagacattttataaagctctttt
    atgtgaactcttgcttcatccaggcaagaacggtgttttg
    tttgcgaccatctcagtgtcaagagaaacgtgtcagtgag
    tacctggaccatcacttaactgatgctccggggtaggact
    gcaggtttcacatgaacctgttntaggctgtggacattgg
    tgtggaga
    288 222127_s_at ctcgctgttatcctggatctggtgttacaatggaattcac
    tattcaggacattctggattattgttccagcattgcacag
    tcccactaaaccttgtgaaagaagaaaagataactgaatg
    aagcatttgagtataacagacactataccaaaataccaag
    caactgttttgagaacccagacttaaaattttatgtatta
    ttaaatgttagataaatgggtagtaccatactacaaatat
    ttaaatgcaaaattaccaacctatatagcagttttatttg
    ccctataggttgcatactaacttaagcattcatgtcacca
    taaaatgcctttagcatttctcaatgactggatgggaaat
    tttcctttattgcctagctgcttgtgtttgagtggttgtc
    ctatgagcaatgcatttggagttcttcagctttcactact
    tctctgttgcttgctaatcatg
    289 36711_at ttgcacggatctaagttattctccccagccagagcccgng
    ctnnctgctcccngggaaaagntggcgtantggncctgag
    ctgggntttatattttatatctgcaaataaatnacatttt
    atcntanatttagggaaagccngagagnaacaacaaaaaa
    tgtttaagccnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnntattgcccggctcct
    agaatttatttatttcctgacttacagcaagcgagttatc
    gtcttctgtattttg
    290 38037_at ccactctatgagttggacttcagtcttgcctaggcgattt
    tgtctaccatttgtgttttgaaagcccaaggtgctgatgt
    caaagtgtaacagatatcagtgtctccccgtgtcctctcc
    ctgncaagtctcagaagaggttgggcttccatgcctgtag
    ctttcctggtccctcacccccatngccccaggcccacagc
    gtgggaactcactttcccttgtgtcaagacatttctnnnn
    nnnnnnnnnnnnnnnnnnnnnnactccatgcaggggtcag
    tgcagcagaggacagtctggagaaggtattagcaaagcaa
    aaggctgagaaggaacagggaacattggagctgactgttc
    ttggtaactgattacctgccaattgctaccgagaaggttg
    gaggtggggaaggctttgtataatcccacccacctcacca
    aaacgannnnnnnnnnnnnnnnnngtcctttctggaagtt
    tctggtgccatttc
    291 AFFX-BioB-M_at gccggagttttacggcaatatcatcaccacacgcacttat
    caggaacgcctcgatacgctggaaaaagtgcgcgatgccg
    ggatcaaagtctgttctggcggcattgtgggcttaggcga
    aacggtaaaagatcgcgccggattattgctgcaactggca
    aacctgccgacgccgccggaaagcgtgccaatcaacatgc
    tggtgaaggtgaaaggcacgccgcttgccgataacgatga
    tgtcgatgcctttgattt
    292 AFFX-r2-Ec-bioC- cgctggtgcagggatcgttacccgaacgtcatcaggcgtg
    3_at gcaggcggtggacgagcgtccgcatgctaatcgcttttta
    ccgccagatgaaatcgaacagtcgctgaacggcgtgcatt
    atcaacatcatattcagcccatcacgctgtggtttgatga
    tgcgctcagtgccatgcgttcgctgaaaggcatcggtgcc
    acgcatcttcatgaagggcgcgacccgcgaatattaacgc
    gttcgcagttgcagcgattgcaactggcctggccgc
    293 204018_x_at ggtccccacagactcagagagaacccaccatggtgctgtc
    tcctgccgacaagaccaacgtcaaggccgcctggggtaag
    gtcggcgcgcacgctggcgagtatggtgcggaggccctgg
    agaggatgttcctgtccttccccaccaccaagacctactt
    cccgcacttcgacctgagccacggctctgcccaggttaag
    ggccacggcaagaaggtggccgacgccctgaccaacgccg
    tggcgcacgtggacgacatgcccaacgcgctgtccgccct
    gagcgacctgcacgcgcacaagcttcgggtggacccggtc
    aacttcaagctcctaagccactgcctgctggtgaccctgg
    ccgcccacctccccgccgagttcacccctgcggtgcacgc
    ctccctggacaagttcctggcttctgtgagcaccgtgctg
    acctccaaataccgttaagctg
    294 222043_at tcgactctgctgctcatgggaagaacagaattgctcctgc
    atgcaactaattcaataaaactgtcttgtgagctgatcgc
    ttggagggtcctctttttatgttgagttgctgcttcccgg
    catgccttcattttgctatggggggcaggcaggggggatg
    gaaaataagtagaaacaaaaaagcagtggctaagatggta
    tagggactgtcataccagtgaagaataaaagggtgaagaa
    taaaagggatatgatgacaaggttgatccacttcaagaat
    tgcttgctttcaggaagagagatgtgtttcaacaagccaa
    cta
    295 206026_s_at Tacaagtactacttctactggaaataaaaactttttagct
    ggaagatttagccacttataaaaaaaaaaaaggatgatca
    aaacacacagtgtttatgttggaatcttttggaactcctt
    tgatctcactgttattattaacatttatttattatttttc
    taaatgtgaaagaaatacataatttagggaaaattggaaa
    atataggaaactttaaacgagaaaatgaaacctctcataa
    tcccactgcatagaaataacaagcgttaacattttcatat
    ttttttctttcagtcatttttgtatttgtggtatatgtat
    atatgtacctatatgtatttgcatttgaaattttggaatc
    ctgctctatgtacagttttgtattatactttttaaatctt
    gaactttatgaacattttctgaaatcattgattattctac
    aaaaacatgattttaaacagctgtaaaatattctatgata
    tgaatgttttatgcattatttaagcctgtctctattgttg
    gaatttcaggtcattttcat
    296 208894_at cgatcaccaatgtacctccagaggtaactgtgctcacgaa
    cagccctgtggaactgagagagcccaacgtcctcatctgt
    ttcatagacaagttcacccca
    297 222150_s_at taatcttgctcattaaccttactcctttgagaattcttta
    acaatatttaaaattggtaacaaaaatagtttagccataa
    ttgtttagccatgtgagtttcaggttggtacacgttcaga
    cagaactgctgtatcacattccaattttgaatagccagtg
    agcaatcaagtgtagagaaatgataaatggcctaagaagg
    catacagtggcataaacgatgctcttcctagtagcttaat
    aggccacaagctagtttctgttgccctctgaaataaaata
    tgctttaaaaatgtagggaccagtgcttagaaaagcaaaa
    actaggtgtgtcattgaaataataggcataaaaattaaat
    gttacataagacccctatttggaaaaagggtccttttaaa
    aactgaatttgtactaaatcagatttgccatgtccagtac
    agaataatttgtacttagtatttgcagcagggtttgtctt
    tgtgaattcag
    298 207113_s_at ccagaactcactggggcctacagctttgatccctgacatc
    tggaatctggagaccagggagcctttggttctggccagaa
    tgctgcaggacttgagaagacctcacctagaaattgacac
    aagtggaccttaggccttcctctctccagatgtttccaga
    cttccttgagacacggagcccagccctccccatggagcca
    gctccctctatttatgtttgcacttgtgattatttattat
    ttatttattatttatttatttacagatgaatgtatttatt
    tgggagaccggggtatcctgggggacccaatgtaggagct
    gccttggctcagacatgttttccgtgaaaacggaggctga
    acaataggctgttcccatgtagccccctggcctctgtgcc
    ttcttttgattatgttttttaaaatattatctgattaagt
    tgtctaaacaatgctgatttggtgaccaactgtcactcat
    tgctg
    299 205067_at agctgtacccagagagtcctgtgctgaatgtggactcaat
    ccctagggctggcagaaagggaacagaaaggtttttgagt
    acggctatagcctggactttcctgttgtctacaccaatgc
    ccaactgcctgccttagggtagtgctaagaggatctcctg
    tccatcagccaggacagtcagctctctcctttcagggcca
    atccccagcccttttgttgagccaggcctctctcacctct
    cctactcacttaaagcccgcctgacagaaaccacggccac
    atttggttctaagaaaccctctgtcattcgctcccacatt
    ctgatgagcaaccgcttccctatttatttatttatttgtt
    tgtttgttttattcattggtctaatttattcaaagggggc
    aagaagtagcagtgtctgtaaaagagcctagtttttaata
    gctatggaatcaattcaatttggactggtgtgctctcttt
    aaatcaagtcc
    300 201290_at gaacacacgtgttggtgcttctgggtagcactggtttgca
    ttagtttatgtttccatgccagagtttgtgtgggccgggcg
    catgtgcaccacagagtgcactcgaggggactttcagtca
    caggatttcataattgtcattgtcacactttcaaattttt
    gtacatcagtgaatttttttatattaaaaggttgagccaa
    aaagcccccagtgtttgtattttgaagccaagcttcactt
    ctaaagtgcctacagagacttgtaaatgaaaatgcagctc
    tgcacgactttgaaaccgtcatacctccttctattaggaa
    tggcatatactgaggtggtcgtaagtcttaacttct
    301 agtaccttggtccagctcttcctgcaacggcccaggagct
    CD11c cagagctccacatctgaccttctagtcatgaccaggacca
    full-length n.t. gggcagcactcctcctgttcacagccttagcaacttctct
    aggtttcaacttggacacagaggagctgacagccttccgt
    gtggacagcgctgggtttggagacagcgtggtccagtatg
    ccaactcctgggtggtggttggagccccccaaaagataac
    agctgccaaccaaacgggtggcctctaccagtgtggctac
    agcactggtgcctgtgagcccatcggcctgcaggtgcccc
    cggaggccgtgaacatgtccctgggcctgtccctggcgtc
    taccaccagcccttcccagctgctggcctgcggccccacc
    gtgcaccacgagtgcgggaggaacatgtacctcaccggac
    tctgcttcctcctgggccccacccagctcacccagaggct
    cccggtgtccaggcaggagtgcccaagacaggagcaggac
    attgtgttcctgatcgatggctcaggcagcatctcctccc
    gcaactttgccacgatgatgaacttcgtgagagctgtgat
    aagccagttccagagacccagcacccagttttccctgatg
    cagttctccaacaaattccaaacacacttcactttcgagg
    aattcaggcgcagctcaaaccccctcagcctgttggcttc
    tgttcaccagctgcaagggtttacatacacggccaccgcc
    atccaaaatgtcgtgcaccgattgttccatgcctcatatg
    gggcccgtagggatgccgccaaaattctcattgtcatcac
    tgatgggaagaaagaaggcgacagcctggattataaggat
    gtcatccccatggctgatgcagcaggcatcatccgctatg
    caattggggttggattagcttttcaaaacagaaattcttg
    gaaagaattaaatgacattgcatcgaagccctcccaggaa
    cacatatttaaagtggaggactttgatgctctgaaagata
    ttcaaaaccaactgaaggagaagatctttgccattgaggg
    tacggagaccacaagcagtagctccttcgaattggagatg
    gcacaggagggcttcagcgctgtgttcacacctgatggcc
    ccgttctgggggctgtggggagcttcacctggtctggagg
    tgccttcctgtaccccccaaatatgagccctaccttcatc
    aacatgtctcaggagaatgtggacatgagggactcttacc
    tgggttactccaccgagctggccctctggaaaggggtgca
    gagcctggtcctgggggccccccgctaccagcacaccggg
    aaggctgtcatcttcacccaggtgtccaggcaatggagga
    tgaaggccgaagtcacggggactcagatcggctcctactt
    cggggcctccctctgctccgtggacgtagacagcgacggc
    agcaccgacctggtcctcatcggggccccccattactacg
    agcagacccgagggggccaggtgtctgtgtgtcccttgcc
    cagggggtggagaaggtggtggtgtgatgctgttctctac
    ggggagcagggccacccctggggtcgctttggggcggctc
    tgacagtgctgggggatgtgaatggggacaagctgacaga
    cgtggtcatcggggccccaggagaggaggagaaccggggt
    gctgtctacctgtttcacggagtcttgggacccagcatca
    gcccctcccacagccagcggatcgcgggctcccagctctc
    ctccaggctgcagtattttgggcaggcactgagcgggggt
    caagacctcacccaggatggactggtggacctggctgtgg
    gggcccggggccaggtgctcctgctcaggaccagacctgt
    gctctgggtgggggtgagcatgcagttcatacctgccgag
    atccccaggtctgcgtttgagtgtcgggagcaggtggtct
    ctgagcagaccctggtacagtccaacatctgcctttacat
    tgacaaacgttctaagaacctgcttgggagccgtgacctc
    caaagctctgtgaccttggacctggccctcgaccctggcc
    gcctgagtccccgtgccaccttccaggaaacaaagaaccg
    gagtctgagccgagtccgagtcctcgggctgaaggcacac
    tgtgaaaacttcaacctgctgctcccgagctgcgtggagg
    actctgtgacccccattaccttgcgtctgaacttcacgct
    ggtgggcaagcccctccttgccttcagaaacctgcggcct
    atgctggccgccgatgctcagagatacttcacggcctccc
    taccctttgagaagaactgtggagccgaccatatctgcca
    ggacaatctcggcatctccttcagcttcccaggcttgaag
    tccctgctggtggggagtaacctggagctgaacgcagaag
    tgatggtgtggaatgacggggaagactcctacggaaccac
    catcaccttctcccaccccgcaggactgtcctaccgctac
    gtggcagagggccagaaacaagggcagctgcgttccctgc
    acctgacatgtgacagcgccccagttgggagccagggcac
    ctggagcaccagctgcagaatcaaccacctcatcttccgt
    ggcggcgcccagatcaccttcttggctacctttgacgtct
    cccccaaggctgtcctgggagaccggctgcttctgacagc
    caatgtgagcagtgagaacaacactcccaggaccagcaag
    accaccttccagctggagctcccggtgaagtatgctgtct
    acactgtggttagcagccacgaacaattcaccaaatacct
    caacttctcagagtctgaggagaaggaaagccatgtggcc
    atgcacagataccaggtcaataacctgggacagagggacc
    tgcctgtcagcatcaacttctgggtgcctgtggagctgaa
    ccaggaggctgtgtggatggatgtggaggtctcccacccc
    cagaacccatcccttcggtgctcctcagagaaaatcgcac
    ccccagcatctgacttcctggcgcacattcagaagaatcc
    cgtgctggactgctccattgctggctgcctgcggttccgc
    tgtgacgtcccctccttcagcgtccaggaggagctggatt
    tcaccctgaagggcaacctcagctttggctgggtccgcca
    gatattgcagaagaaggtgtcggtcgtgagtgtggctgaa
    attacgttcgacacatccgtgtactcccagcttccaggac
    aggaggcatttatgagagctcagacgacaacggtgctgga
    gaagtacaaggtccacaaccccacccccctcatcgtaggc
    agctccattgggggtctgttgctgctggcactcatcacag
    cggtactgtacaaagttggcttcttcaagcgtcagtacaa
    ggaaatgatggaggaggcaaatggacaaattgccccagaa
    aacgggacacagacccccagcccgcccagtgagaaatgat
    cccctctttgccttggacttcttctcccccgcgagttttc
    cccacttacttaccctcacctgtcaggcctgacggggagg
    aaccactgcaccaccgagagaggctgggatgggcctgctt
    cctgtctttgggagaaaacgtcttgcttgggaaggggcct
    ttgtcttgtcaaggttccaactggaaacccttaggacagg
    gtccctgctgtgttccccaaaggacttgacttgcaatttc
    tacctagaaatacatggacaatacccccaggcctcagtct
    cccttctcccatgaggcacgaatgatctttctttcctttc
    tttttttttttttttcttttcttttttttttttttgagac
    ggagtctcgctctgtcacccaggctggagtgcaatggcgt
    gatctcggctcactgcaacctccgcctcccgggttcaagt
    aattctgctgtctcagcctcctgagtagctgggactacag
    gcacacgccacctcgcccggcccgatctttctaaaataca
    gttctgaatatgctgctcatccccacctgtcttcaacagc
    tccccattaccctcaggacaatgtctgaactctccagctt
    cgcgtgagaagtccccttccatcccagagggtgggcttca
    gggcgcacagcatgagaggctctgtgcccccatcaccctc
    gtttccagtgaattagtgtcatgtcagcatcagctcaggg
    cttcatcgtggggctctcagttccgatttcccaggctgaa
    ttgggagtgagatgcctgcatgctgggttctgcacagctg
    gcctcccgcgttgggcaacattgctggctggaagggagga
    gcgccctctagggagggacatggccccggtgcggctgcag
    ctcacccagccccaggggcagaagagacccaaccacttct
    attttttgaggctatgaatatagtacctgaaaaaatgcca
    agacatgattatttttttaaaaagcgtactttaaatgttt
    gtgttaataaattaaaacatgcacaaaaagatgcatctac
    cgctcttgggaaatatgtcaaaggtctaaaaataaaaaag
    ccttctgtgaaaaaaaaaaaaaaaaa
    302 mtrtraalllftalatslgfnldteeltafrvds
    CD11c agfgdsvvqyanswvvvgapqkitaanqtgglyq
    full-length a.a. cgystgacepiglqvppeavnmslglslasttsp
    sqllacgptvhhecgrnmyltglcfllgptqltq
    rlpvsrqecprqeqdivflidgsgsissrnfatm
    mnfvravisqfqrpstqfslmqfsnkfqthftfe
    efrrssnplsllasvhqlqgftytataiqnvvhr
    lfhasygarrdaakilivitdgkkegdsldykdv
    ipmadaagiiryaigvglafqnrnswkelndias
    kpsqehifkvedfdalkdiqnqlkekifaiegte
    ttssssfelemaqegfsavftpdgpvlgavgsft
    wsggaflyppnmsptfinmsqenvdmrdsylgys
    telalwkgvqslvlgapryqhtgkaviftqvsrq
    wrmkaevtgtqigsyfgaslcsvdvdsdgstdlv
    ligaphyyeqtrggqvsvcplprgwrrwwcdavl
    ygeqghpwgrfgaaltvlgdvngdkltdvvigap
    geeenrgavylfhgvlgpsispshsqriagsqls
    srlqyfgqalsggqdltqdglvdlavgargqvll
    lrtrpvlwvgvsmqfipaeiprsafecreqvvse
    qtlvqsniclyidkrsknllgsrdlqssvtldla
    ldpgrlspratfqetknrslsrvrvlglkahcen
    fnlllpscvedsvtpitlrlnftlvgkpllafrn
    lrpmlaadaqryftaslpfekncgadhicqdnlg
    isfsfpglksllvgsnlelnaevmvwndgedsyg
    ttitfshpaglsyryvaegqkqgqlrslhltcds
    apvgsqgtwstscrinhlifrggaqitflatfdv
    spkavlgdrllltanvssenntprtskttfqlel
    pvkyavytvvssheqftkylnfseseekeshvam
    hryqvnnlgqrdlpvsinfwvpvelnqeavwmdv
    evshpqnpslrcssekiappasdflahiqknpvl
    dcsiagclrfrcdvpsfsvqeeldftlkgnlsfg
    wvrqilqkkvsvvsvaeitfdtsvysqlpgqeaf
    mraqtttvlekykvhnptplivgssiggllllal
    itavlykvgffkrqykemmeeangqiapengtqt
    psppsek
    303 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
    Adalimumab VL Leu Ser Ala Ser Val Gly Asp Arg Val Thr
    Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
    Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
    Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala
    Ala Ser Thr Leu Gln Ser Gly Val Pro Ser
    Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
    Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
    Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg
    Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln
    Gly Thr Lys Val Glu Ile Lys
    304 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
    Adalimumab VH Leu Val Gln Pro Gly Arg Ser Leu Arg Leu
    Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp
    Asp Tyr Ala Met His Trp Val Arg Gln Ala
    Pro Gly Lys Gly Leu Glu Trp Val Ser Ala
    Ile Thr Trp Asn Ser Gly His Ile Asp Tyr
    Ala Asp Ser Val Glu Gly Arg Phe Thr Ile
    Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
    Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser
    Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr
    Trp Gly Gln Gly Thr Leu Val Thr Val Ser
    Ser
    305 Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa
    Adalimumab Xaa = Thr or Ala
    VL CDR3
    306 Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu
    Adalimumab Asp Xaa
    VH CDR3 Xaa = Tyr or Asn
    307 Ala Ala Ser Thr Leu Gln Ser
    Adalimumab
    VL CDR2
    308 Ala Ile Thr Trp Asn Ser Gly His Ile Asp
    Adalimumab Tyr Ala Asp Ser Val Glu Gly
    VH CDR2
    309 Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu
    Adalimumab Ala
    VL CDR1
    310 Asp Tyr Ala Met His
    Adalimumab
    VH CDR1

Claims (22)

What is claimed is:
1. A method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising: (i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder, and (ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-10, 12-28, 30-43, 45-61, 63, 64, 66-73, and 75-82,
wherein decreased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 12-14, 18, 20, 21, 23, 25, 26, 28, 30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 66-69, 71-73, 76-78, 80 and 81 is predictive of responsiveness of the subject to a TNFα inhibitor; and/or
wherein increased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43, 45-47, 49, 51, 54, 55, 57, 59, 61, 64, 70, 75, 79 and 82 is predictive of responsive of the subject to a TNFα inhibitor.
2. A method for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the method comprising:
(i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder, and
(ii) predicting responsiveness of the subject to the TNFα inhibitor based on expression of the one or more biomarkers in the subject, wherein the one or more biomarkers is selected from the group consisting of Aquaporin 3 (Genbank Accession No. NM—004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No. NM—033022); Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM—006815); Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM—000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM—006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM—002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No. NM—002823); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM—005896); Tumor protein D52 (Genbank Accession Nos. NM—001025252, NM—001025253, NM—005079); Early growth response 1 (Genbank Accession No. NM—001964); Cytochrome c oxidase subunit VIIb (Genbank Accession No. NM—001866); CUG triplet repeat, RNA binding protein 2 (Genbank Accession No. NM—001025077, NM—001025076, NM—006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM—006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM—017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM—001077269, NM—003387); CD97 antigen (Genbank Accession Nos. NM—001025160, NM—001784, NM—078481); Glutamate-cysteine ligase, catalytic subunit (Genbank Accession No. NM—001498); Crystallin, zeta (quinone reductase) (Genbank Accession No. NM—001889); Rap guanine nucleotide exchange factor (GEF) 2 (Genbank Accession No. NM—014247); Ataxin 1 (Genbank Accession No. NM—000332); Adaptor-related protein complex 1, sigma 2 subunit (Genbank Accession No. NM—003916); Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM—014936); Desmocollin 2 (Genbank Accession Nos. NM—024422, NM—004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM—002371, NM—022438, NM—022439, NM—022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No. NM—005476); Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM—001001437, NM—021006); Carboxypeptidase A3 (Genbank Accession No. NM—001870); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM—004545); Interleukin 8 receptor, beta (Genbank Accession No. NM—001557); Platelet factor 4 variant 1 (Genbank Accession No. NM—002620); Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM—006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM—003786); Actinin, alpha 1 (Genbank Accession No. NM—001102); NAD kinase (Genbank Accession No. NM—023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM—000442); Esterase D/formylglutathione hydrolase (Genbank Accession No. NM—001984); Chromosome 20 open reading frame 111 (Genbank Accession No. NM—016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM—001017369, NM—006745); PIM-1 oncogene (Genbank Accession No. NM—002648); GATA binding protein 2 (Genbank Accession No. NM—032638); Cathepsin Z (Genbank Accession No. NM—001336); Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM—006499, NM—201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM—006889, NM—175862); Interleukin 8 (Genbank Accession No. NM—000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM—002001); Actin, gamma 1 (Genbank Accession No. NM—001614); KIAA0746 protein (Genbank Accession No. NM—015187); Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (Genbank Accession No. NM—002076); Transcription factor 4 (Genbank Accession Nos. BF592782, CR612521); Major histocompatibility complex, class II, DQ alpha 1 (Genbank Accession Nos. NM—002122, NM—020056); Cell division cycle 2-like 6 (CDK8-like) (Genbank Accession No. NM—015076); Major histocompatibility complex, class II, DQ beta 1 (Genbank Accession No. XM—942240); Phospholipase C-like 2 (Genbank Accession No. NM—015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM—005242); TM2 domain containing 1 (Genbank Accession No. NM—032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM—012433); SUB1 homolog (S. cerevisiae) (Genbank Accession No. NM—006713); MRNA, cDNA DKFZp564O0862 (Genbank Accession No. AI278204); Cytochrome b-5 (Genbank Accession Nos. NM—001914, NM—148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM—183395); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No. XM—941927); CCR4-NOT transcription complex, subunit 6 (Genbank Accession No. NM—015455); Ubiquinol-cytochrome c reductase complex (7.2 kD) (Genbank Accession Nos. NM—013387, NM—001003684); Hepatocellular carcinoma-associated antigen 112 (Genbank Accession No. NM—018487); Kruppel-like factor 11 (Genbank Accession No. XM—001129527); GGA binding partner (Genbank Accession No. NM—018318); Cornichon homolog 4 (Drosophila) (Genbank Accession No. NM—014184); Hypothetical protein FLJ21616 (Genbank Accession No. NM—024567); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM—012336, NM—001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM—018538, NM—001017922); LR8 protein (Genbank Accession No. NM—014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No. NM—030915); Calmin (calponin-like, transmembrane) (Genbank Accession No. NM—024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM—031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM—007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR—003287).
3. A method for predicting responsiveness to a TNFα inhibitor, which TNFα inhibitor is adalimumab, in a subject having an autoimmune disorder, the method comprising:
(i) assaying the subject for expression of one or more biomarkers predictive of responsiveness to adalimumab in an autoimmune disorder, and
(ii) predicting responsiveness of the subject to adalimumab based on expression of the one or more biomarkers in the subject,
wherein decreased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 12-14, 18, 20, 21, 23, 25, 26, 28, 30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 66-69, 71-73, 76-78, 80 and 81 is predictive of responsiveness of the subject to a TNFα inhibitor; and/or
wherein increased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43, 45-47, 49, 51, 54, 55, 57, 59, 61, 64, 70, 75, 79 and 82 is predictive of responsive of the subject to a TNFα inhibitor.
4. The method of any of claims 1-3, wherein a sample from the subject is assayed for expression of mRNA encoding the one or more biomarkers.
5. The method of any of claims 1-3, wherein a sample from the subject is assayed for protein expression of the one or more biomarkers.
6. The method of any of claims 1-3, which further comprises selecting a treatment regimen with the TNFα inhibitor based upon expression of the one or more biomarkers in the subject.
7. The method of claim 6, which further comprises administering the TNFα inhibitor to the subject according to the treatment regimen such that the autoimmune disorder is inhibited in the subject.
8. The method of any of claims 1-3, wherein the TNFα inhibitor is an anti-tumor necrosis factor-alpha (TNFα) antibody, or antigen-binding portion thereof.
9. The method of claim 8, wherein the anti-TNFα antibody, or antigen-binding portion thereof, is a human antibody, a humanized antibody, a chimeric antibody or a multivalent antibody.
10. The method of claim 8, wherein the anti-TNFα antibody, or antigen-binding portion thereof, is adalimumab.
11. The method of claim 9, wherein the anti-TNFα antibody, or antigen-binding portion thereof, is selected from the group consisting of
i) an isolated human antibody that dissociates from human TNFα with a Kd of 1×10−8 M or less and a Koff rate constant of 1×10−3 s−1 or less, both determined by surface plasmon resonance, and neutralizes human TNFα cytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10−7 M or less;
ii) an isolated human antibody with the following characteristics:
a) dissociates from human TNFα with a Koff rate constant of 1×10-3 s-1 or less, as determined by surface plasmon resonance;
b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 305, or modified from SEQ ID NO: 305 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 306, or modified from SEQ ID NO: 306 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12; and
iii) an isolated human antibody with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 304.
12. The method of claim 1, wherein the TNFα inhibitor is etanercept.
13. The method of any one of claims 1-3, wherein the subject is a human.
14. The method of any one of claims 1-3, wherein the autoimmune disorder is rheumatoid arthritis.
15. A kit for predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, the kit comprising:
a) means for isolating monocytes;
b) means for measuring expression in the subject of one or more biomarkers predictive of responsiveness to a TNFα inhibitor in an autoimmune disorder;
c) means for measuring expression of at least one housekeeping gene; and
d) instructions for use of the kit to predicting responsiveness to a TNFα inhibitor in a subject having an autoimmune disorder, wherein decreased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 8, 9, 11-14, 18, 20, 21, 23, 25, 26, 28-30, 32, 33, 40-42, 48, 50, 52, 53, 56, 58, 60, 63, 65-69, 71-74, 76-78, 80 and 81 is predictive of responsiveness of the subject to a TNFα inhibitor and/or wherein increased expression of the one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 10, 15-17, 19, 22, 24, 27, 31, 34-39, 43, 44-47, 49, 51, 54, 55, 57, 59, 61, 62, 64, 70, 75, 79 and 82 is predictive of responsive of the subject to a TNFα inhibitor.
16. A method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 83-133, thereby monitoring the autoimmune disorder in the subject.
17. A method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 134-177, 110, 112, 118, 123 and 131, thereby monitoring the autoimmune disorder in the subject.
18. A method of monitoring an autoimmune disorder in a subject having an autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 178-292, 91 and 97, thereby monitoring the autoimmune disorder in the subject.
19. A method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 87, 97, 101, 293, 92, 272, 93, 107, 108, 121 and 123, and wherein the subject is monitored prior to treatment with a TNFα inhibitor, thereby monitoring the autoimmune disorder in the subject.
20. A method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 290, 209, 98, 112, 116, 121, 130, 155, 92, 289, 216 and 131, and wherein the subject is monitored after treatment with a TNFα inhibitor, thereby monitoring the autoimmune disorder in the subject.
21. A method of monitoring an autoimmune disorder in a subject having the autoimmune disorder, the method comprising: assaying the subject for expression of one or more biomarkers, wherein the one or more biomarkers is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, 102, 29, 294, 295, 91, 131, 290, 100, 134, 296, 297, 298, 299, 136, 174 and 300, thereby monitoring the autoimmune disorder in the subject.
22. A method of selecting an autoimmune disorder subject for a treatment with a TNFα inhibitor, the method comprising:
(i) identifying, in a database comprising a plurality of autoimmune disorder subjects, a subject whose database entry is associated with a biomarker expression pattern that is predictive of responsiveness to treatment with a TNFα inhibitor, and
(ii) selecting the subject for treatment with a TNFα inhibitor, wherein the biomarker expression pattern reports expression in the subject of one or more biomarkers encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-82.
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