BIOMARKERS PREDICTIVE OF THE RESPONSIVENESS TO TNFALPHA INHIBITORS IN AUTOIMMUNE DISORDERS

- Abbott Laboratories

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.

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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. NM004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No. NM033022); Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No. NM002823); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM005896); Tumor protein D52 (Genbank Accession Nos. NM001025252, NM001025253, NM005079); Early growth response 1 (Genbank Accession No. NM001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM014888, NM001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No. NM001866); CUG triplet repeat, RNA binding protein 2 (Genbank Accession No. NM001025077, NM001025076, NM006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM001077269, NM003387); CD97 antigen (Genbank Accession Nos. NM001025160, NM001784, NM078481); Glutamate-cysteine ligase, catalytic subunit (Genbank Accession No. NM001498); Crystallin, zeta (quinone reductase) (Genbank Accession No. NM001889); Rap guanine nucleotide exchange factor (GEF) 2 (Genbank Accession No. NM014247); Ataxin 1 (Genbank Accession No. NM000332); Adaptor-related protein complex 1, sigma 2 subunit (Genbank Accession No. NM003916); Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM014936); Desmocollin 2 (Genbank Accession Nos. NM024422, NM004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM002371, NM022438, NM022439, NM022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No. NM005476); Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM001001437, NM021006); Carboxypeptidase A3 (Genbank Accession No. NM001870); Charcot-Leyden crystal protein (Genbank Accession No. NM001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM004545); Interleukin 8 receptor, beta (Genbank Accession No. NM001557); Platelet factor 4 variant 1 (Genbank Accession No. NM002620); Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM003786); Actinin, alpha 1 (Genbank Accession No. NM001102); NAD kinase (Genbank Accession No. NM023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM000442); Esterase D/formylglutathione hydrolase (Genbank Accession No. NM001984); Chromosome 20 open reading frame 111 (Genbank Accession No. NM016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM001017369, NM006745); PIM-1 oncogene (Genbank Accession No. NM002648); GATA binding protein 2 (Genbank Accession No. NM032638); Cathepsin Z (Genbank Accession No. NM001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No. NM000887); Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM006499, NM201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM006889, NM175862); Interleukin 8 (Genbank Accession No. NM000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM002001); Actin, gamma 1 (Genbank Accession No. NM001614); KIAA0746 protein (Genbank Accession No. NM015187); Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (Genbank Accession No. NM002076); Transcription factor 4 (Genbank Accession Nos. BF592782, CR612521); Major histocompatibility complex, class II, DQ alpha 1 (Genbank Accession Nos. NM002122, NM020056); Cell division cycle 2-like 6 (CDK8-like) (Genbank Accession No. NM015076); Major histocompatibility complex, class II, DQ beta 1 (Genbank Accession No. XM942240); Phospholipase C-like 2 (Genbank Accession No. NM015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM005242); TM2 domain containing 1 (Genbank Accession No. NM032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM012433); SUB1 homolog (S. cerevisiae) (Genbank Accession No. NM006713); MRNA; cDNA DKFZp564O0862 (Genbank Accession No. AI278204); Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM201413, NM000484, NM201414); Cytochrome b-5 (Genbank Accession Nos. NM001914, NM148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM016645, NM001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No. XM941927); CCR4-NOT transcription complex, subunit 6 (Genbank Accession No. NM015455); Ubiquinol-cytochrome c reductase complex (7.2 kD) (Genbank Accession Nos. NM013387, NM001003684); Hepatocellular carcinoma-associated antigen 112 (Genbank Accession No. NM018487); Kruppel-like factor 11 (Genbank Accession No. XM001129527); GGA binding partner (Genbank Accession No. NM018318); Cornichon homolog 4 (Drosophila) (Genbank Accession No. NM014184); Hypothetical protein FLJ21616 (Genbank Accession No. NM024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM012336, NM001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM018538, NM001017922); LR8 protein (Genbank Accession No. NM014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No. NM030915); Calmin (calponin-like, transmembrane) (Genbank Accession No. NM024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR003287) (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. NP000878 (also shown as SEQ ID NO: 302) and encoded by a full-length nucleotide sequence as set forth at Genbank Accession No. NM000887 (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. NM004925); Similar to ribosomal protein S24, clone MGC:8595 (Genbank Accession No. NM033022); Transmembrane emp24 domain trafficking protein 2 (Genbank Accession No. NM006815; Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (Genbank Accession No. NM000454); Calmodulin 1 (phosphorylase kinase, delta) (Genbank Accession No. NM006888); Guanine nucleotide binding protein (G protein), beta polypeptide 1 (Genbank Accession No. NM002074); Prothymosin, alpha (gene sequence 28) (Genbank Accession No. NM002823); Homo sapiens isocitrate dehydrogenase 1 (NADP+) soluble (IDH1) (Genbank Accession No. NM005896); Tumor protein D52 (Genbank Accession Nos. NM001025252, NM001025253, NM005079); Early growth response 1 (Genbank Accession No. NM001964); Homo sapiens predicted osteoblast protein (GS3786) (Genbank Accession Nos. NM014888, NM001040020); Cytochrome c oxidase subunit VIIb (Genbank Accession No. NM001866); CUG triplet repeat, RNA binding protein 2 (Genbank Accession No. NM001025077, NM001025076, NM006561); Ubiquinol-cytochrome c reductase hinge protein (Genbank Accession No. NM006004); Homo sapiens leptin receptor gene-related protein (HS0BRGRP) (Genbank Accession No. NM017526); Wiskott-Aldrich syndrome protein interacting protein (Genbank Accession Nos. NM001077269, NM003387); CD97 antigen (Genbank Accession Nos. NM001025160, NM001784, NM078481); Glutamate-cysteine ligase, catalytic subunit (Genbank Accession No. NM001498); Crystallin, zeta (quinone reductase) (Genbank Accession No. NM001889); Rap guanine nucleotide exchange factor (GEF) 2 (Genbank Accession No. NM014247); Ataxin 1 (Genbank Accession No. NM000332); Adaptor-related protein complex 1, sigma 2 subunit (Genbank Accession No. NM003916); Ectonucleotide pyrophosphatase/phosphodiesterase 4 (Genbank Accession No. NM014936); Desmocollin 2 (Genbank Accession Nos. NM024422, NM004949); MAL, T-cell differentiation protein (Genbank Accession Nos. NM002371, NM022438, NM022439, NM022440); Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Genbank Accession No. NM005476); Chemokine (C—C motif) ligand 3 (Genbank Accession Nos. NM001001437, NM021006); Carboxypeptidase A3 (Genbank Accession No. NM001870); Charcot-Leyden crystal protein (Genbank Accession No. NM001828); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7 kDa (Genbank Accession No. NM004545); Interleukin 8 receptor, beta (Genbank Accession No. NM001557); Platelet factor 4 variant 1 (Genbank Accession No. NM002620); Poly(A) binding protein interacting protein 1 (Genbank Accession No. NM006451); ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (Genbank Accession No. NM003786); Actinin, alpha 1 (Genbank Accession No. NM001102); NAD kinase (Genbank Accession No. NM023018); Platelet/endothelial cell adhesion molecule (CD31 antigen) (Genbank Accession No. NM000442); Esterase D/formylglutathione hydrolase (Genbank Accession No. NM001984); Chromosome 20 open reading frame 111 (Genbank Accession No. NM016470); Sterol-C4-methyl oxidase-like (Genbank Accession Nos. NM001017369, NM006745); PIM-1 oncogene (Genbank Accession No. NM002648); GATA binding protein 2 (Genbank Accession No. NM032638); Cathepsin Z (Genbank Accession No. NM001336); Integrin alpha-X (antigen CD11c) (Genbank Accession No. NM000887); Lectin, galactoside-binding, soluble, 8 (galectin 8) (Genbank Accession Nos. NM006499, NM201545); CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (Genbank Accession Nos. NM006889, NM175862); Interleukin 8 (Genbank Accession No. NM000584); Fc fragment of IgE, high affinity I, receptor for alpha polypeptide (Genbank Accession No. NM002001); Actin, gamma 1 (Genbank Accession No. NM001614); KIAA0746 protein (Genbank Accession No. NM015187); Glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (Genbank Accession No. NM002076); Transcription factor 4 (Genbank Accession Nos. BF592782, CR612521); Major histocompatibility complex, class II, DQ alpha 1 (Genbank Accession Nos. NM002122, NM020056); Cell division cycle 2-like 6 (CDK8-like) (Genbank Accession No. NM015076); Major histocompatibility complex, class II, DQ beta 1 (Genbank Accession No. XM942240); Phospholipase C-like 2 (Genbank Accession No. NM015184); Coagulation factor II (thrombin) receptor-like 1 (Genbank Accession No. NM005242); TM2 domain containing 1 (Genbank Accession No. NM032027); Splicing factor 3b, subunit 1, 155 kDa (Genbank Accession No. NM012433); SUB1 homolog (S. cerevisiae) (Genbank Accession No. NM006713); mRNA; cDNA DKFZp564O0862 (Genbank Accession No. AI278204); Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) (Genbank Accession Nos. NM201413, NM000484, NM201414); Cytochrome b-5 (Genbank Accession Nos. NM001914, NM148923); Cold autoinflammatory syndrome 1 (Genbank Accession No. NM183395); Neugrin, neurite outgrowth associated (Genbank Accession Nos. NM016645, NM001033088); Ribosomal protein S26, 40S ribosomal protein (Genbank Accession No. XM941927); CCR4-NOT transcription complex, subunit 6 (Genbank Accession No. NM015455); Ubiquinol-cytochrome c reductase complex (7.2 kD) (Genbank Accession Nos. NM013387, NM001003684); Hepatocellular carcinoma-associated antigen 112 (Genbank Accession No. NM018487); Kruppel-like factor 11 (Genbank Accession No. XM001129527); GGA binding partner (Genbank Accession No. NM018318); Cornichon homolog 4 (Drosophila) (Genbank Accession No. NM014184); Hypothetical protein FLJ21616 (Genbank Accession No. NM024567); Homo sapiens hypothetical protein FLJ10134 (Genbank Accession No. NM018004); Nuclear prelamin A recognition factor (Genbank Accession Nos. NM012336, NM001038618); Erythroblast membrane-associated protein (Genbank Accession Nos. NM018538, NM001017922); LR8 protein (Genbank Accession No. NM014020); Likely ortholog of mouse limb-bud and heart gene (LBH) (Genbank Accession No. NM030915); Calmin (calponin-like, transmembrane) (Genbank Accession No. NM024734); Chromosome 14 open reading frame 156 (Genbank Accession No. NM031210); Guanine nucleotide binding protein (G protein) alpha 12 (Genbank Accession No. NM007353); and SRY (sex determining region Y)-box 18 (Genbank Accession No. NR003287) (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 APEXnuclease(multifunctionalDNArepairenzyme)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,alpha1,alpha2 −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 HypotheticalproteinLOC54103 −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 Diphtheriatoxinreceptor(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/ 219228_at Zinc finger protein 331 0.894 0.007** CRP/ Thromb/ Granulo/ 213427_at Ribonuclease P 40 kDa subunit −0.875 0.010** Hb/  factor 203932_at MHC class II, DM beta 0.885 0.008** 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** 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. 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 0 −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 −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 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) 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 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 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 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 −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 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 0 −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   , bold, italic, and underlined letters   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

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.

Patent History

Publication number: 20120039900
Type: Application
Filed: Oct 27, 2011
Publication Date: Feb 16, 2012
Applicant: Abbott Laboratories (Abbott Park, IL)
Inventors: Bruno Stuhlmüller (Berlin), Gerd-Reudiger Burmester (Berlin)
Application Number: 13/282,850