WO2005086872A2 - Ptpn22 polymorphisms in diagnosis and therapy - Google Patents

Abstract

The invention provides 1) methods and compositions for detecting polymorphisms of the PTPN22 genomic DNA; 2) methods for associating polymorphisms of the PTPN22 gene with the occurrence of an immune disorder, inflammatory disorder or cell proliferation disorder; 3) methods for identifying subjects at risk of an immune disorder, inflammatory disorder or cell proliferation disorder by determining if they have a polymorphism of the PTPN22 gene and treating such subjects with a tyrosine kinase inhibitor to prevent or delay the progression of such diseases; 4) methods for identifying subjects having an immune disorder, inflammatory disorder or cell proliferation disorder who are promising candidates for therapy with a tyrosine kinase inhibitor by determining if such subjects have a polymorphism of the PTPN22 gene; and 5) methods of treating subjects having an immune disorder, inflammatory disorder or cell proliferation disorder mediated by a polymorphism of the PTPN22 gene by administering to such subjects a tyrosine kinase inhibitor.

Description

PTPN22 POLYMORPHISMS IN DIAGNOSIS AND THERAPY

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] NOT APPLICABLE

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] NOT APPLICABLE

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDLX SUBMITTED ON A COMPACT DISK. [0003] NOT APPLICABLE FIELD OF THE INVENTION [0004] This invention relates to polymorphisms of the tyrosine phosphatase PTPN22 gene, their association with cell proliferation, immune, and inflammatory disorders; methods and compositions used to detect the polymoφhisms; and the use of modulators (e.g., inhibitors of protein tyrosine kinase) of the regulatory protein phosphorylation/dephosphorylation cycle to treat subjects having such disorders or polymorphisms. BACKGROUND OF THE INVENTION [0005] Protein kinases and protein phosphatases are important in the regulation of many cell processes. For instance, kinases and phosphatases respectively phosphorylate and dephosphorylate proteins involved in cell signaling, differentiation, and proliferation. This phosphorylation and dephosphorylation can greatly modify the structure and activity of such regulatory protems to modulate cellular transformation, proliferation, and growth; hormone and cytokine signalling; inflammation; T and B cell co-stimulation; and apoptosis.

[0006] Protein kinases catalyze protein phosphorylation by transfer of a phosphate group from ATP to the substrate protein. Kinases can be divided between those specific for phosphorylating serine and threonine residues, and those specific for phosphorylating tyrosine residues and are termed protein serine/threonine kinases and tyrosine protein kinase, respectively. Many protein kinases autophosporylate to modify their own activity. Well over 1000 kinases have been identified.

[0007] Protein phosphatases catalyze the removal of the phosphate group from a phosphorylated protein or peptide. More than 100 phosphatases have been identified. A protein phosphatase can also be classified according to its specificity for hydrolyzing a phosphate group attached to Ser, Thr or Tyr. A phosphatase able to dephosphorylate tyrosine is a Protein Tyrosine Phosphatase (PTP). Protein tyrosine phosphatases have a catalytic domain of approximately 200 to 300 amino acids and can be divided into membrane-bound receptors or cytoplasmic phosphatases. The intracellular location of cytoplasmic phosphatases depends on the primary amino acid sequences outside the catalytic domain.

[0008] The ability of specific kinases and phosphatases to interact in a protein phosphorylation/dephosphorylation cycle first requires that they be expressed in the same cell. Other factors, such as, for example, their substrate specificities, also determine their ability to interact. Kinases and phosphatases, for instance, each have varied and multiple binding domains outside their catalytic domain which are specific for other proteins which promote their subcellular localization. [0009] One of the best characterized examples of the role of protein kinases and phosphatases in health and disease is chronic myeloid leukemia. The Bcr-Abl protein kinase plays an important role in the development of this disease which is characterized by the Philadelphia chromosome abnormality. In this abnormality, a cross-over transposes the abl gene on chromosome 9 to chromosome 22 in the break cluster region (Bcr) gene. The Abl gene encodes a protein tyrosine kinase. The Bcr-Abl fusion protein is a constitutively expressed protein with protein kinase activity and disturbs the balance of protein phosphorylation/dephosphorylation in the cell. Numerous studies show that this disturbance leads to cellular transformation and chronic myeloid leukemia (CML). Administration of an inhibitor of the Bcr-Abl tyrosine kinase (e.g., imatinib mesylate (Gleevec™ or STI571)) inhibits tumor cell growth in vivo.

[0010] Platelet-derived growth factor (PDGF) is a major cell proliferation signal for cells of mesenchymal origin. PDGF is very active during ontogeny and in some proliferative disorders, including cancer and atherosclerosis. The binding of PDGF to the PDGF receptor (PDGFR) activates the PDFGR tyrosine kinase to cause an increased rate of cellular proliferation. Treatment with a protein tyrosine kinase inhibitor can counter the action of PDGF. Imatinib mesylate inhibits kinases such as, for example, abl-related gene (ARG), c- khyand PDGFR in addition to the Bcr-Abl cancer causing fusion protein,. Of particular interest is the recent report that imatinib mesylate can be used on another disease state, a rare cancer referred to as hypereosinophila syndrome (HES). Subsequently, it was found that the mechanism of HES disease is the fusion of the PDGF gene to a growth hormone gene. [0011 ] PTPN22, also known as Lyp (see, WlPO/International Patent Publication WO99/36548 (22 July 1999)), regulates the function of Cbl and its associated protein kinases via its affect on the tyrosine protein kinase signaling pathways. PTNP22 is an intracellular protein of about 105-kD and has a single tyrosine phosphatase catalytic domain. Four proline-rich potential SH3 domain binding sites are located in the noncatalytic domain of PTNP22. The PTPN22 noncatalytic domain includes an NXXY motif that may become tyrosine phosphorylated and is a potential cognate site for a phosphotyrosine binding domain. PTNP22 is localized to chromosome lpl3. PTPN22 has an alternative spliced form, Lyp2. Lyp2 is a 85-kD protein having a different 7-amino acid C-terminus.

[0012] PTPN22 is expressed in a number of cell types involved in the immune response and inflammation. PTNP22 is highly expressed in lymphoid tissues and cells, including both mature B and T cells and thymocytes. Phytohemagglutinin induces PTPN22 expression in peripheral T lymphocytes.

[0013] PTNP22 is also constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Cbl, which is a protein substrate of PTPN22, is critical in the regulation of diverse processes in a many cells and tissues. Cbl is a negative regulator of several receptor protein tyrosine kinase signaling pathways. Cbl is also an adaptor protein in tyrosine phosphorylation-dependent signaling. Cbl-interacting proteins are active in cellular signaling for stimuli such as, for example, cytokines, antigens, growth factors and cellular adhesion. Cbl proteins help to regulate cell proliferation, cell differentiation, and cell motility and structure. The disruption of Cbl signaling pathways can lead to disease. And, conversely, the modulation of Cbl signaling networks can be of therapeutic benefit in such diseases. In particular, Cbl proteins are thought to play a role in such diseases as cancer, autoimmunity, and inflammation. See, Dikic I, et al, Cell Mol Life Sci. 60(9): 1805-27 (2003). Phosphorylation of the Cbl adaptor protein is reduced when PTNP22 is overexpressed (see, Cohen S, et al., Blood 15;93(6):2013-24 (1999)). This finding indicates that the protein is a probable physiological substrate for the enzyme. [0014] PTPN22 is expressed in myeloid cell lines as well as normal granulocytes and monocytes. Recent studies also point to the involvement of PTPN22 in CML. Erythroid and myeloid leukemic cell lines have distinct expression patterns of tyrosine phosphatases. In particular, the phosphorylation of multiple proteins in KCL22 chronic myeloid leukemia blast cells (e.g., Cbl, Bcr-Abl, Erkl/2, and CrkL PTPN22I is reduced by PTPN22 overexpression. Additionally, the phosphorylation of Bcr-Abl, Grb2, and Myc is reduced in Cos-7 cells co- expressing PTPN22 and Bcr-Abl. Further, anchorage-independent clonal growth of KCL22 cells is suppressed by PTPN22 overexpression. A negative regulatory role for Lyp in T-cell signaling is indicated by these interactions between Lyp and the adaptor Grb2. Overall, the ability of PTPN22 activity to reduce signaling by Bcr-Abl indicates PTPN22 is a potential tumor suppressor gene (Chien et al., J Biol Chem. 278: 27413-27420 (2003); and Hill, R. j. et al., Exp. Hemat. 30: 237-244 (2002)). [0015] Genomic polymoφhisms and, more particularly, single nucleotide polymoφhisms (SNPs) can provide a fast and efficient means for elucidating the etiology of diseases and for identifying individuals whose genetic make-up puts them at increased risk of disease. Single nucleotide polymoφhisms (SNPs) are especially suited for the identification of genotypes because 1) SNPs are the single most frequent type of genotypic variation; 2) SNPs located in genes can directly affect protein structure or expression and thereby identify the disease pathways and strategies for therapeutic intervention; and 3) SNPs are suitable for high throughput genotyping systems used to screen large populations of subjects.

[0016] Given the importance of PTPN22 in the regulation of cellular processes, there is an unmet need 1) to identify diseases or conditions mediated or modulated by PTPN22 which are suited to therapy with modulators of the regulatory protein phosphorylation/ dephosphorylation cycle and 2) to identify subjects having altered PTPN22 activity who may then be identified as being at risk of such diseases and/or treated with suitable tyrosine kinase/phosphatase modulators. The invention provides for these and other needs by providing new methods and compositions useful in the diagnosis, prevention and treatment of diseases associated with abnormal or unwanted protein phosphorylation. BRIEF SUMMARY OF THE INVENTION [0017] This invention relates to the discovery that polymoφhisms of the intracellular tyrosine phosphatase PTPN22 are associated with cellular proliferative, immune system and inflammatory disorders in humans.

[0018] In a first aspect, the present invention provides methods for determining whether a human subject is at increased risk of developing a cellular proliferative disorder (e.g., CML), inflammatory disorder (e.g., asthma, atherosclerosis, Alzheimer's disease), or immune system disorder (e.g., rheumatoid arthritis) by determining the PTPN22 genotype or phenotype of the subject. For example, a single nucleotide polymoφhism (SNP) of the PTPN22 gene is determined in a nucleic acid sample obtained from the subject. The presence of the nucleotide occurrence is associated with reduced PTPN22 tyrosine phosphatase activity and altered phosphorylation of regulatory proteins and an increased incidence of cellular proliferative disorders (e.g., cancer, CML), autoimmunity (e.g., rheumatoid arthritis), and inflammation (e.g., Alzheimer's disease, artherosclerosis). In another example, a sample of tissue from the subject can be assayed for PTPN22 tyrosine phosphatase activity and the amount of such activity can serve to determine whether the subject would have an increased risk for developing a cellular proliferative disorder (e.g., cancer, CML), autoimmunity (rheumatoid arthritis), and inflammation (e.g., Alzheimer's disease, artherosclerosis). In one embodiment, the presence or absence of the SNP-1 polymoφhism or other polymoφhism set forth in Tables 1-5 is detected. In some embodiments, the polymoφhism can be any one or more of SNP-1 through SNP-13 as set forth in Tables 1-5.

[0019] In a second aspect, the invention provides methods for screening one or more subjects as candidates for treatment with a protein tyrosine kinase inhibitor by determining the PTPN22 genotype or phenotype of the subject(s). In this aspect, the invention also provides methods for treating individuals who have, or at risk of developing, diseases or disorders associated with disregulation of the tyrosine kinase/ tyrosine phosphatase phosphorylation/dephosphorylation cycle (e.g., a cellular proliferative disorder (e.g., CML), inflammatory disorder (e.g., asthma, artherosclerosis, Alzheimer's disease), or immune system disorder (e.g., rheumatoid arthritis)). Individuals having a PTPN22 genotype or phenotype characterized by a reduced intracellular tyrosine phosphatase PTPN22 activity are more likely to have positive response to therapy with a tyrosine kinase inhibitor than individuals lacking such a PTPN22 polymoφhism. Thus, the subject invention includes tests that allow for individualization or personalization of tyrosine kinase inhibitor drug therapy for such individuals by determining the PTPN22 genotype or phenotype of the individual. Individuals having a variant PTPN22 genotype or phenotype are promising candidates for therapy with a therapeutically effective amount of a tyrosine kinase modulator. Individuals with a genotype or phenotype providing a reduced PTPN22 tyrosine phosphatase activity can be treated, for example, with an Abl or PDGF receptor tyrosine kinase inhibitor. In one embodiment, the inhibitor is a specific Abl tyrosine kinase inhibitor. By such methods, individuals who are candidates for therapy with modulators of tyrosine kinases and/or tyrosine phosphatases can be identified. In some embodiments, the SNP to be detected is SNP-1 or another SNP of Tables 1-5. In yet other embodiments, the SNP to be detected is selected from the group of SNPs 1-13 as set forth in Tables 1-5. For instance, an individual with rheumatoid arthritis who is identified as having a SNP-1 polymoφhism can be targeted for therapy with a tyrosine kinase inhibitor (e.g., imatinib mesylate).

[0020] In one embodiment, therefore, the invention provides a method for treating, preventing or reducing the risk of developing a cellular proliferative disorder (e.g., CML), inflammatory disorder (e.g., asthma, artherosclerosis, Alzheimer's disease) or immune system disorder (e.g., rheumatoid arthritis) by first determining the presence of a variant PTPN22 tyrosine phosphatase gene of a subject and then administering to said subject a therapeutically or prophylactically effective amount of one or more tyrosine kinase inhibitors. In one embodiment, the inhibitor is an Abl or PDGFR tyrosine kinase inhibitor. In one embodiment, the tyrosine kinase inhibitor is imatinib mesylate. In another embodiment, the variant is the PTPN22 polymoφhism SNP-1 or another polymoφhism (e.g., SNPs 1-13) as set forth in Tables 1 -5.

[0021] In still another embodiment, the invention provides a method of treating a human by first determining the presence of a variant PTPN22 gene in the subject, and then administering a tyrosine kinase inhibitor to the subject. In yet another embodiment, the variant is the PTPN22 polymoφhism SNP-1 or another polymoφhism (e.g., any one of SNPs 1-13) set forth in Tables 1-5. In a still further embodiment, the variant comprises SNP-1 and the inhibitor is imatinib mesylate.

[0022] In a third aspect, the invention provides methods of treating a non-cancerous inflammatory or immune system disorder other than rheumatoid arthritis by administration to a subject having the disorder a tyrosine kinase inhibitor, hi one embodiment, the inhibitor is an Abl or PDGFR tyrosine kinase inhibitor. In one embodiment, the tyrosine kinase inhibitor is imatinib. In one embodiment, the subject has the SNP-1 or another polymoφhism (e.g., SNPs 1-13) of the PTPN22 gene as set forth in Tables 1 -5.

[0023] In one embodiment, the invention provides a method for treating, preventing or reducing the risk of a cellular proliferative disorder (e.g., CML), inflammatory disorder (e.g., asthma, artherosclerosis, Alzheimer's disease) or immune system disorder (e.g., rheumatoid arthritis) in a human subject harboring or having a variant PTPN22 gene by administering a therapeutically or prophylactic ally effective amount of one or more tyrosine kinase inhibitors (e.g., imatinib mesylate). In one embodiment, the inhibitor is an Abl or PDGFR tyrosine kinase inhibitor. In a further embodiment, the variant tyrosine phosphatase gene comprising the SNP-1 polymoφhism wherein a C is substituted by a T and the tyrosine kinase inhibitor is imatinib mesylate. In yet another embodiment, the variant gene comprises one or more polymoφhisms (e.g., SNPs 1-13) as set forth in Tables 1- 5.

[0024] The polymoφhisms (e.g., SNP-1 and any of SNPs 1-13) of the invention are also useful for conducting clinical trials of drug candidates for cellular proliferative disorders, immune disorders, and inflammatory disorders, Such trials are performed on treated or control populations having similar or identical polymoφhic profiles at a defined collection of polymoφhic sites, including, but not limited to, those set forth in Tables 1- 5. Use of genetically matched populations eliminates or reduces variation in treatment outcome due to genetic factors, leading to a more accurate assessment of the efficacy of a potential drug, h some embodiments, the drug is a tyrosine kinase inhibitor. In further embodiments, the inhibitor is an Abl or PDGFR tyrosine kinase inhibitor. In still another embodiment, the inhibitor is imatinib.

[0025] In a fourth aspect, the invention provides methods and compositions for detecting one or more single nucleotide polymoφhisms of the PTPN22 gene set forth in Tables 1-5. SNP-1 is a single nucleotide polymoφhism of the PTPN22 gene and represents a missense mutation in which the nucleotide at position 1970 of SEQ ID NO. 2 is a T rather than a C and codes for a tryptophan rather than an arginine in the PTPN22 protein at position 620 of SEQ ID NO:l. In one embodiment, the SNP-1 or other SNP of Tables 1-5 is detected in a sequence of genomic DNA obtained in a sample from a human. In another embodiment, the SNP-1 or other SNP of Tables 1-5 is detected in a sequence of messenger RNA obtained in a sample from a human. In yet other embodiments, the SNP is any one of SNP1 through SNP20 as set forth in Tables 1-5.

[0026] In some aspects, the invention provides nucleic acids of between 10 and 100 bases comprising at least 10 contiguous nucleotides including a SNP site of Tables 1-5 or a complement thereof. The nucleic acids can be DNA or RNA and labeled or unlabeled. In some embodiments, the nucleic acids may be between 10 and 50 bases or between 20 and 50 bases long. The base occupying the SNP polymoφhic site in such nucleic acids may be either the base occurring with greater frequency (e.g., wild type) or the base occurring with lesser frequency (e.g., the variant) in the sampled population. For instance, the base occupying the SNP-1 polymoφhic site in such nucleic acids may be either C or a T. In still other embodiments, the polymoφhism corresponds to any of SNP-1 through SNP-20. In yet further embodiments, the base at the polymoφhic site is the variant base or its complement.

[0027] The invention further provides allele-specific oligonucleotides that hybridize to a nucleic acid segment of PTPN22 genomic DNA or its complement in which the oligonucleotide comprises a polymoφhic site set forth in Tables 1-5. Such oligonucleotides are useful as probes or primers. These allele-specific oligonucleotides include the SNP-1 polymoφhic site or another polymoφhic site (e.g., SNPs 1-13) as set forth in Tables 1-5. The base at the SNP-1 site may be C or T.

[0028] The invention further provides methods of analyzing a nucleic acid sequences. Such methods entail obtaining the nucleic acid from an individual; and determining a base occupying the SNP-1 polymoφhic site or other polymoφhic site set forth in Tables 1 - 5. In some methods, the nucleic acid is obtained from a plurality of individuals, and a base occupying any one or more of the polymoφhic position of SNP-1 or Tables 1- 5 is determined in each of the individuals. Each individual is then tested for the presence of a immune or inflammatory disease or disorder, and the presence of the disease is associated or correlated with the occurrence of one or more polymoφhisms of the PTPN22 gene (e.g., those set forth in Tables 1-5, including SNPs 1-13).

[0029] The invention further provides kits for identifying persons who 1) have an increased likelihood of responding to treatment (e.g., preventive or therapeutic treatment) with a tyrosine kinase inhibitor and/or 2) who are at an increased risk of developing an immune system, inflammatory, or cellular proliferation disorder. Often, the kits contain instructions as to how to inteφret the results with respect the likelihood of the person or subject responding to administration of a tyrosine kinase inhibitor or the likelihood of a person developing such disorders. The kits may also contain one or more pairs of allele- specific oligonucleotides hybridizing to different forms of a polymoφhism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymoφhisms shown in Tables 1-5. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kit also contains instructions for carrying out the methods for the detection of a SNP in a sample. [0030] In a fifth aspect, the invention provides a variant form of the PTPN22 protein as set forth in Tables 1-5 or encoded by a nucleotide represented therein. In one embodiment, the PTPN22 protein has a tryptophan residue rather than an arginine residue at position 620 of SEQ ID NO:l. In another aspect, the invention provides methods of detecting the variant protein of SNP-1 or another variant form set forth in Tables 1-5 or encoded by a nucleotide represented therein.

[0031] The invention yet still further provides a method for identifying a person at an increased risk of developing a cell proliferation, inflammatory or immune disorder by determining the PTPN22 phenotype of the subject by obtaining a sample of the PTPN22 protein and measuring or determining its phosphatase activity, substrate binding properties, or antibody binding properties (e.g., using one or more antibodies which can distinguish between PTPN22 protein variants). In some embodiments, the PTPN22 phenotype is identified using antibody or protein arrays, enzyme-linked immunosorbent assays, radioimmunoassay, spatial immobilization (e.g., 2D-PAGE and scintillation counting), high performance liquid chromatography or mass spectrophotometry. In some embodiments, the PTPN22 variant corresponds to a SNP of Tables 1-5.

[0032] The invention further provides methods of diagnosing a PTPN22 variant phenotype which places a subject at increased risk of developing an inflammatory, immune system, or cellular proliferative disorder. Such methods entail determining which polymoφhic form(s) are present in a sample from a subject at one or more polymoφhic sites shown in Tables 1-5 and diagnosing the presence of a disease or disorder phenotype correlated with the form(s) in the subject. In some embodiments, the polymoφhism is SNP-1 or a SNP from the SNPs set forth in Tables 1-5. [0033] In another aspect, the invention further provides methods of analyzing a nucleic acid sequence and identifying additional polymoφhic forms in disequilibrium linkage with a polymoφhic form of the PTPN22 gene which affects health or disease. For instance, such methods can entail obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymoφhic sites shown in Tables 1-5 or other polymoφhic sites in equilibrium dislinkage therewith. Some methods determine a set of bases occupying a set of the polymoφhic sites shown in Tables 1-5. In some methods, the nucleic acid is obtained from a plurality of individuals, and abase occupying one of the polymoφhic positions is determined in each of the individuals. Each individual is also assessed for the presence or absence of an immune, inflammation, cell proliferation disease or disorder phenotype (e.g., rheumatoid arthritis) and the presence or absence of the disease or disorder phenotype is correlated with the base of the polymoφhism.

[0034] The invention also provides methods of screening polymoφhic sites of PTPN22 DNA linked to polymoφhic sites shown in Tables 1-5 for suitability for diagnosing a inflammatory disorder, immune system disorder, or cellular proliferation disorder phenotype. For instance, such methods can entail identifying a polymoφhic site linked to a polymoφhic site shown in Tables 1-5 wherein a polymoφhic form of the polymoφhic site shown in Tables 1-5 has been correlated with a phenotype. One then determines haplotypes in a population of individuals to indicate whether the linked polymoφhic site has a polymoφhic form in disequlibrium linkage with the polymoφhic form correlated with the phenotype.

[0035] In another of its aspects, the invention is drawn to methods of testing PTPN22 SNPs or other PTPN22 polymoφhisms for their association with cellular proliferation disorders, immune system disorders, or inflammatory disorders (e.g., RA) by use of prospective or retrospective epidemiological comparisons of the incidence or frequency of the subject polymoφhism between a control population and a population having a cellular proliferation disorder, immune system disorder, or inflammatory disorder (e.g., RA).

DESCRIPTION OF THE TABLES

[0036] Table 1 sets forth the amino acid sequence of a PTPN22 protein (Table 1 A); the transcript nucleic acid sequence corresponding to the PTPN22 protein with indications of the polymoφhisms therein (Table IB), and various polymoφhisms of the PTPN22 transcript nucleic acid sequence (Table IC).

[0037] Table 2 sets forth the nucleic acid sequence of a PTPN22 DNA transcript with polymoφhisms indicated therein (Table 2 A); the amino acid sequence of the PTPN22 protein encoded by the transcript (Table 2B), and various polymoφhisms of the PTPN22 transcript nucleic acid sequence (Table 2C).

[0038] Table 3 sets forth the nucleic acid sequence of a PTPN22 DNA transcript with polymoφhisms indicated therein (Table 3 A); the amino acid sequence of the PTPN22 protein encoded by the transcript (Table 3B), and various polymoφhisms of the PTPN22 transcript nucleic acid sequence (Table 3C). [0039] Table 4 sets forth the nucleic acid sequence of a PTPN22 DNA transcript with polymoφhisms indicated therein (Table 4A); the amino acid sequence of the PTPN22 protein encoded by the transcript (Table 4B), and various polymoφhisms of the PTPN22 transcript nucleic acid sequence (Table 4C).

[0040] Table 5 sets forth the nucleic acid sequence of a PTPN22 genomic DNA with polymoφhisms indicated therein (Table 5 A) and various polymoφhisms of the PTPN22 transcript nucleic acid sequence (Table 5B).

[0041] Table 6 sets forth the odds ratio for the SNP-1 polymoφhism for subjects with rheumatoid arthritis as compared to well-matched controls in an original study (Table 6A) and a replicate study (Table 6B).

[0042] Table 7 sets forth published PTPN22 genomic, cDNA, and protein sequences which in some embodiments may be used as reference sequences. See NCBI entry BC017785 and Strausberg, R et al., Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002)

[0043] Tables 1 -5 disclose SNP and associated gene/transcript/protein information of the PTPN22 gene. For each gene, these tables provide a header containing gene/transcript/protein information, followed by a transcript and protein sequence (in Tables 1-4) or a genomic sequence (in Table 5), and then SNP information regarding each SNP found in that gene/transcript. [0044] SNPs may be included in any of Tables 1-5. Tables 1-4 present SNPs relative to their transcript sequences and encoded protein sequences. Table 5 presents the SNPs relative to their genomic sequences (in some instances Table 5 may also include, after the last gene sequence, genomic sequences of one or more intergenic regions, as well as SNP context sequences and other SNP information for any SNPs that lie within these intergenic regions). SNPs can readily be cross-referenced between Tables based on their hCV identification numbers.

[0045] The gene/transcript/protein information includes: - a public Genbank accession number (e.g., RefSeq NM number) for the transcript (Tables 1-4 only) - a public Genbank accession number (e.g., RefSeq NP number) for the protein (Tables 1 -4 only) - an art-known gene symbol - an art-known gene/protein name - the chromosome number of the chromosome on which the gene is located - an OMIM (Online Mendelian Inheritance in Man; Johns Hopkins University/NCBI) public reference number for obtaining further information regarding the medical significance of each gene - alternative gene/protein name(s) and/or symbol(s) in the OMIM entry

[0046] Due to the presence of alternative splice forms, multiple transcript/protein entries can be provided for a single gene entry in Tables 1-4; i.e., for a single Gene Number, multiple entries may be provided in series that differ in their transcript/protein information and sequences.

[0047] Following the gene/transcript/protein information is a transcript sequence and protein sequence (in Tables 1-4), or a genomic sequence (in Table 5), for each gene, as follows: - transcript sequence (Tables 1-4 only) with SNPs identified by their IUB codes (transcript sequences can include 5' UTR, protein coding, and 3' UTR regions). If there are differences between the nucleotide sequence of the hCT transcript and the corresponding public transcript sequence identified by the Genbank accession number, the hCT transcript sequence (and encoded protein) is provided, unless the public sequence is a RefSeq transcript sequence identified by an NM number, in which case the RefSeq NM transcript sequence (and encoded protein) is provided. However, whether the hCT transcript or RefSeq NM transcript is used as the transcript sequence, the disclosed SNPs are represented by their IUB codes within the transcript.) - the encoded protein sequence (Tables 1-4 only) - the genomic sequence of the gene (Table 5 only), including 6kb on each side of the gene boundaries (i.e., 6kb on the 5' side of the gene plus 6kb on the 3' side of the gene).

[0048] After the last gene sequence, Table 5 may include additional genomic sequences of intergenic regions (in such instances, these sequences are identified as "Intergenic region:" followed by a numerical identification number), as well as SNP context sequences and other SNP information for any SNPs that lie within each intergenic region (and such SNPs are identified as "INTERGENIC" for SNP type). The SNP information includes: - context sequence (taken from the transcript sequence in Tables 1-4, and taken from the genomic sequence in Table 2) with the SNP represented by its IUB code, including 100 bp upstream (5') of the SNP position plus 100 bp downstream (3') of the SNP position.. - Alternative hCV internal identification number for the SNP - SNP position [position of the SNP within the given transcript sequence (Tables 1-4) or within the given genomic sequence (Table 5)] - Population/allele/allele count information in the format of [populationl(allelel,count|allele2,count) population2(allelel,count|allele2,count) total (allele 1, total count|allele2,total count)]. The information in this field includes populations/ethnic groups in which particular SNP alleles have been observed ("cau" = Caucasian, "his" = Hispanic, "chn" = Chinese, and "afr" = African- American, "jpn" = Japanese, "ind" = Indian, "mex" = Mexican, "ain" = "American Indian, "era" = Celera donor, "no__pop" = no population information available), identified SNP alleles, and observed allele counts (within each population group and total allele counts), where available ["-" in the allele field represents a deletion allele of an insertion/deletion ("indel") polymoφhism (in which case the corresponding insertion allele, which may be comprised of one or more nucleotides, is indicated in the allele field on the opposite side of the "I"); "-"in the count field indicates that allele count information is not available].

[0049] For some SNPs genes/regulatory regions of 39 individuals (20 Caucasians and 19 African Americans) were re-sequenced and, since each SNP position is represented by two chromosomes in each individual (with the exception of SNPs on X and Y chromosomes in males, for which each SNP position is represented by a single chromosome), up to 78 chromosomes were genotyped for each SNP position. Thus, the sum of the African- American ("afr") allele counts is up to 38, the sum of the Caucasian allele counts ("cau") is up to 40, and the total sum of all allele counts is up to 78. - SNP type (e.g., location within gene/transcript and/or predicted functional effect) ["MIS-SENSE MUTATION" = SNP causes a change in the encoded amino acid (i.e., a non-synonymous coding SNP); "SILENT MUTATION" = SNP does not cause a change in the encoded amino acid (i.e., a synonymous coding SNP); "STOP CODON MUTATION" = SNP is located in a stop codon; "NONSENSE MUTATION" = SNP creates or destroys a stop codon; "UTR 5" = SNP is located in a 5' UTR of a transcript; "UTR 3" = SNP is located in a 3' UTR of a transcript; "PUTATIVE UTR 5" = SNP is located in a putative 5' UTR; "PUTATIVE UTR 3" = SNP is located in a putative 3' UTR; "DONOR SPLICE SITE" = SNP is located in a donor splice site (5' intron boundary); "ACCEPTOR SPLICE SITE" = SNP is located in an acceptor splice site (3' intron boundary); "CODING REGION" = SNP is located in a protein-coding region of the transcript; "EXON" = SNP is located in an exon; "INTRON" = SNP is located in an intron; "UNKNOWN" = SNP type is not defined; "INTERGENIC" = SNP is intergenic, i.e., outside of any gene boundary] - Protein coding information (Tables 1-4 only), where relevant, in the format of [protein SEQ ID NO:#, amino acid position, (amino acid-1, codonl) (amino acid-2, codon2)]. The information in this field includes SEQ ID NO of the encoded protein sequence, position of the amino acid residue within the protein identified by the SEQ ID NO that is encoded by the codon containing the SNP, amino acids (represented by one- letter amino acid codes) that are encoded by the alternative SNP alleles (in the case of stop codons, "X" is used for the one-letter amino acid code), and alternative codons containing the alternative SNP nucleotides which encode the amino acid residues (thus, for example, for missense mutation-type SNPs, at least two different amino acids and at least two different codons are generally indicated; for silent mutation-type SNPs, one amino acid and at least two different codons are generally indicated, etc.). In instances where the SNP is located outside of a protein-coding region (e.g., in a UTR region), "None" is indicated following the protein SEQ ID NO.

DETAILED DESCRIPTION OF THE INVENTION [0050] The present invention relates to the discovery reported herein that PTPN22 polymoφhisms are associated with human diseases or disorders of the immune system, cellular proliferation, and inflammation. The inventors screened over 12,000 SNPs from throughout the entire genome in the discovery sample set cohort which lead to the discovery that polymoφhisms of the PTPN22 are statistically associated with the occurrence of rheumatoid arthritis in humans. [0051] The discovery utilized the SNP-1 polymoφhism of the PTPN22 gene in assessing the potential importance of the PTPN22 polymoφhisms in rheumatoid arthritis (RA). RA is an exemplary model for cellular proliferative, immune system, and inflammatory disorders. SNP-1 represents a missense mutation at position 1970 of SEQ ID NOs. 29 or 32 of PTPN22 transcript DNA (see Table 1 or 4, respectively ) wherein the nucleotide residue is a T rather than a C. The SNP-1 polymoφhism codes for a tryptophan rather than an arginine in PTPN22 protein at position 620 of SEQ ID NO:l. The inventors discovered that the SNP-1 polymoφhism occurs much more often (about 15%) in a population having rheumatoid arthritis as compared to a control population. The odds ratio is about 1.7 and indicates that the SNP-1 polymoφhism occurs nearly twice as frequently in patients with rheumatoid arthritis than in well-matched controls (see Table 6).

[0052] The specific mutation represented by SNP-1 is a non-conservative one and illustrates how SNPs can affect the expression or function of the PTPN22 gene or protein. SNP-1 substitutes an aromatic tryptophan for a highly charged arginine. This mutation is located within a binding domain known to play a critical role in binding other proteins. The specificity of binding to other proteins (e.g., adaptor proteins including GRB2 and CSK) is important to the specificity, activity, and subcellular localization of PTPN22. PTPN22 tyrosine phosphatase activity plays a role in the phosphorylation/dephosphorylation cycle for such important regulatory proteins as Cbl, Bcr-Abl, Erkl/2, and CrkL. PTPN22 is also highly expressed in lymphoid tissues. [0053] Thus, PTPN22 polymoφhisms are a risk factor and/or etiological factor in cellular proliferative disorders, cancer, immune system disorders, and inflammatory disorders.

[0054] The present invention therefore provides methods for determining whether a human subject is at increased risk of developing a cellular proliferative disorder, immune system disorder, or inflammatory disorder by determining the PTPN22 genotype or phenotype of the subject. For example, in a nucleic acid sample from the subject, a nucleotide occurrence of a single nucleotide polymoφhism (SNP) of the PTPN22 gene is determined. The presence of the nucleotide occurrence is associated with reduced PTPN22 tyrosine phosphatase activity and altered phosphorylation of regulatory proteins. In another example, a sample of tissue from the subject can be assayed for PTPN22 tyrosine phosphatase activity and the amount of such activity can serve to determine whether the subject would have an increased risk for developing a cellular proliferative disorder, immune system disorder, or inflammatory disorder. In some embodiments, the polymoφhism is selected from those of Tables 1-5, including, but not limited to, non-silent SNPs of SNPs 1-13. In other embodiments, the polymoφhism is a non-silent, exonic polymoφhism of Tables 1-5.

[0055] The invention also provides methods for identifying subjects to be treated with a therapeutically effective amount of a tyrosine kinase modulator/inhibitor by determining the PTPN22 genotype and/or phenotype of the subject. For example, in a nucleic acid sample from the subject, a nucleotide occurrence of a single nucleotide polymoφhism (SNP) of the PTPN22 gene is determined. The presence of the nucleotide occurrence is associated with reduced PTPN22 tyrosine phosphatase activity and altered phosphorylation of regulatory proteins (e.g., Cbl, Bcr-Abl, Erkl/2, and CrkL). In another example, a sample of tissue from the subject can be assayed for PTPN22 activity and the amount of such activity can serve to determine the PTPN22 genotype and/or phenotype of the subject. The puφose of the treatment can be prophylactic (e.g., prevent disease or reduce risk of progressing to disease) or therapeutic (e.g., to treat a manifested disease). Such diseases include cellular proliferative disorders, immune system disorders, and inflammatory disorders. Such diseases also include those mediated or exacerbated by reduced tyrosine phosphatase (e.g., PTPN22) activity or mediated by increased phosphorylated forms of PTPN22 substrates (e.g., Cbl, Bcr-Abl, Erkl/2, and CrkL). The methods of the present invention, in some embodiments, identify whether a subject has a PTPN22 polymoφhism of Tables 1-5, including SNP-1. [0056] In other embodiments, the invention identifies whether the subject is heterozygous or homozygous at a position corresponding to nucleotide 1970 of SEQ ID NO: 29. In some embodiments, the methods of the present invention thus identify whether an individual is homozygous or heterozygous for the PTPN22 SNP-1 allele. That is, the method identifies whether a diploid pair of nucleotide occurrences at a position corresponding to nucleotide 1970 of SEQ ID NO: 29 (SNP-1) is a thymidine residue or not or a cytosine residue or not. The magnitude of the risk of developing the subject disorders is typically influenced by whether a subject is homozygous or heterozygous for a missense mutation (e.g., SNP-1). The results presented herein identify PTPN22 as a susceptibility allele of some penetration. SNP- 1 homozygous individuals are therefore expected to be at even greater risk of developing cellular proliferative disorders (e.g., cancers (e.g., CML), autoimmune disorders (e.g., rheumatoid arthritis), and inflammation disorders (e.g., Alzheimer's disease, atherosclerosis)). Homozygous individuals are also more likely expected to prophylactically or therapeutically benefit from administration of a tyrosine kinase inhibitor. [0057] Detection of the SNPs of the present invention also has obvious additional utilities in the fields of forensics and paternity testing where comparisons of nucleic acid sequences can be useful in determining whether a sample of a subject's genomic DNA matches that of another sample of interest. See, for instance, U.S. Patent Application No. 20030170699 which is incoφorate.

[0058] In so far as the effects of PTPN22 modulation of other enzymes (e.g., tyrosine kinase activity) can influence serine/threonine kinase activity, serine/threonine kinase inhibitors may also be administered, alone or in conjunction with a tyrosine kinase inhibitor, to treat the above described conditions or persons having a PTPN22 polymoφhism, particularly, for instance such persons whose PTPN22 polymoφhism is associated with the occurrence of such conditions, ed herein by reference.

DEFINITIONS

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] All publications, patents, patent applications, databases and other references cited in this application are herein incoφorated by reference in their entirety as if each individual publication, patent, patent application, database or other reference was specifically and individually indicated to be incoφorated by reference to the extent that each is not inconsistent with the present disclosure.

[0061] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0062] As used herein nucleic acid, polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric (e.g., 2 or more monomers) of nucleotides of any length. A nucleic acid can be DNA, RNA, mRNA, or cDNA, and be single- or double- stranded. Oligonucleotides can be naturally occurring nucleotides or synthetic nucleotides, but are typically prepared by synthetic means. Preferred nucleic acids of the invention include segments of DNA or their complements including a nucleotide having a sequence identical or completely complementary to the sequence of SEQ ID NO:29 about the SNP-1 site or including sequences identical or completely complementary to a sequence of Tables 1- 5 in which the sequences include a SNP site set forth therein. The segments are usually between 10 and 100 contiguous bases, and often range from about 12 to 30, 15 to 30, or 20 to 30 nucleotides or from about 20 to about 50 nucleotides. The nucleic acid bases are typically selected from G, C, T, U, and A. In the some nucleic acids, the polymoφhic site is occupied by a base that correlates with an immune or inflammatory disorder or cellular proliferation disorder or susceptibility thereto. Some nucleic acids contain one or a plurality of polymoφhic sites and have one, or two or more polymoφhic sites giving rise two or more different amino acids specified by the polymoφhic codons of the polymoφhic sites.

[0063] Although nucleotides are usually joined by phosphodiester linkages, the term also includes polymeric nucleotides containing neutral amide backbone linkages composed of aminoethyl glycine units. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), those containing pendant moieties, including, for example, proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide. Polynucleotides include both sense and antisense strands.

[0064] Sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

[0065] A complementary nucleotide sequence is one which allows binding to the reference nucleotide sequence in a sequence specific manner under stringent conditions. A complementary sequence is usually at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence which would be completely complementary. Complementary sequences include completely complementary sequences as determined by application of the Watson-Crick base pairing rules such that the bases G, A, and T of the first nucleic acid are respectively and consistently paired with the bases C, U, and A of the second or reference nucleic acid (e.g., 5'-A-G-T-C-3' base pairs with 3'-T-C-A-G-5'). [0066] As used herein, the term isolated, in the context of nucleic acid molecules, refers to a nucleic acid molecule which is separated from other nucleic acid molecules or cellular materials or other chemical reagents when such are present in the source of the nucleic acid molecule. An "isolated" nucleic acid molecule, for example, a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a preferred embodiment, a nucleic acid molecule encoding a single nucleotide polymoφhism of the invention or including the position of a single nucleotide polymoφhism of the invention is isolated. In another preferred embodiment, the SNP of the isolated nucleotide is SNP-1.

[0067] Linkage disequilibrium or allelic association denotes a preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for the particular allelic frequencies in the population. To illustrate, let locus Y have alleles yi and y₂, which occur equally frequently. Let Y be linked locus Z having alleles Z and z₂, which occur equally frequently. The haplotype yiZj ought to have a frequency of 0.25 in the population. If yjzi occurs more frequently, then alleles yi and z_\ are in linkage disequilibrium. Linkage disequilibrium may result from natural selection or because an allele is too new to have achieved equilibrium with the linked allele. [0068] Linkage disequilibrium markers can be used to detect susceptibility to a disease (or other phenotype) even when the marker itself does not cause the disease. To illustrate, a marker (Y) that is not a cause of a disease, but which is in linkage disequilibrium with a gene(Z) causing the disease, can be used to detect or to indicate susceptibility to the disease even when the gene Z may not have been identified or detected. Newer alleles (i.e., arising from mutation relatively recently) are expected to have a larger genomic sequencement in linkage disequilibrium. The age of an allele can be determined by comparing its occurrence between ethnic human groups and/or between humans and related species.

[0069] Hybridization probes are capable of binding in a base-specific manner to a completely complementary strand of nucleic acid. Such probes include nucleic acids and peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991). Hybridizations are usually performed under stringent hybridization conditions. Stringent hybridization conditions typically refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30°C below the T_m. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents (e.g., formamide). For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. [0070] The term primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (e.g., DNA or RNA polymerase or reverse transcriptase)) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer. Primers typically range from about 15 to about 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair means a set of primers including a 5' upstream primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3', downstream primer that hybridizes with the complement of the 3' end of the sequence to be amplified. In some embodiments, a primer is completely complementary to its reference sequence. In other embodiments, the primer may be at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence or an exact complement thereof. A suitable primer for a SNP-1 polymoφhism is AATGATTCAGGTGTCC.

[0071] A label refers to any visible or radioactive moiety than can be attached to or incoφorated into a cDNA or protein. Visible labels include, but are not limited to, anthocyanins, green fluorescent protein (GFP), j8-glucuronidase, luciferase, Cy3 and Cy5, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like

[0072] Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymoφhic marker or site is the locus at which divergence occurs. A polymoφhic locus may be as small as one base pair (e.g., a SNP). The allelic form occurring most frequently in a selected population is sometimes referred to herein as the wildtype form. Diploid organisms may be homozygous or heterozygous for an allelic form. A diallelic polymoφhism has two forms. Polymoφhism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.

[0073] A single nucleotide polymorphism (SNP) occurs at a polymoφhic site occupied by a single nucleotide, which is the site of variation between allelic sequences.. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100) or even 1/1000 members of the populations). A single nucleotide polymoφhism typically arises due to substitution of one nucleotide for another at the polymoφhic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymoφhisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.

[0074] Variant refers to molecules that are recognized variations of the most common or standard reference form of a protein or polynucleotide that encodes it. For instance, the reference sequence may be a sequence set forth in Table 7. Or the reference sequence may be a most common sequence of Tables 1-5. Allelic variants typically have a high percent identity to the cDNAs and may differ by about three bases per one hundred bases.

[0075] Percent(sequence) identity with respect to the sequences identified herein is defined as the percentage of residues in a candidate sequence that are identical with the residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for pmposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences being compared. For puφoses herein, percent identity values can also be obtained, for instance, using the sequence comparison computer program, ALIGN-2, the source code of which has been filed with user documentation in the US Copyright Office, Washington, D.C., 20559, registered under the US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. For instance, two nucleic acid sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence

[0076] The terms "identical" or percent "identity," in the context of two or more nucleic acids refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. A window may be the size of the full length of the shorter nucleic acid to be compared. [0077] For sequence comparison, typically one sequence acts as a reference sequence, to which the other or test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. The reference sequence may be specified according to the sequence about a SNP as set forth in Tables 1-5, the most commonly occurring sequence or in Tables 1-5 or in the studied population(s) for any subject comparison window, or a sequence from Table 7 of over any chosen comparison window.

[0078] A comparison window, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150. in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The window often is the size of the shortest nucleic acid of interest to be compared. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection.

[0079] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Shaφ, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

[0080] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0081] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat ' Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0082] Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

[0083] The term modulate means to induce any change including increasing or decreasing. For instance, the term modulator includes both inhibitors and activators of an enzyme. A preferred modulator inhibitor of the protein tyrosine kinase is Gleevec™.

[0084] The term treatment relates to both prevention or prophylaxis of disease or a disorder as well as to a reduction, improvement, or slowed progression of an existing or manifested disease or disorder. The improvement may be objective (e.g., improved movement, increased activity) or subjective (e.g., decreased pain). A preventive or prophylactic treatment pertains to a treatment of a subject who does not exhibit signs or symptoms of the disease or condition to be prevented. As used herein, the term prophylactically effective amount refers to that amount of a therapeutic agent sufficient to prevent or delay the development of the signs or symptoms of a disease or condition. A therapeutic treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, wherein treatment is administered for the puφose of diminishing or eliminating or slowing the progression of those pathological signs or symptoms. As used herein, the term therapeutically effective amount refers to that amount of a therapeutic agent sufficient to result in amelioration of one or more symptoms of a subject disease or condition. An effective amount of an agent or a protein tyrosine kinase inhibitor is an amount sufficient to prevent or retard the development of the subject disease or condition or to ameliorate or reduce the severity or progression of the disease or condition.

[0085] A cellular proliferation/proliferative disorder includes those diseases that affect cellular proliferation, growth, apoptosis, and differentiation. Such disorders can be mediated by increases in cell number, size or content; programmed cell death, by development of a specialized set of characteristics which differ from that of a precursor cell. Examples of cellular proliferation disorders include cancer, e.g., prostate cancer, pancreatic cancer, melanoma, breast cancer, colon cancer, lung cancer, ovarian cancer, as well as other types of carcinomas, sarcomas, lymphomas, and/or leukemias; tumor angiogenesis and metastasis; and hematopoietic and/or myeloproliferative disorders. Other examples of disorders characterized by aberrant regulation of apoptosis include stroke-associated cell death and neurodegenerative disorders including, but not limited to, Alzheimer's disease, dementias related to Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, senile dementia, and Huntington's disease.

[0086] Proliferative disorders include, but are not limited to, cancer, and particularly to cancer of cells mediating the immune response. In addition, the invention is particularly useful in treating or preventing infl-immatory diseases associated with cellular proliferation. Inflammatory diseases include inflammatory diseases associated with cellular proliferation. An inflammatory disease associated with cellular proliferation is a disease in wliich non- cancerous lymphoproliferation contributes to tissue or organ damage leading to disease. For instance, excessive T cell proliferation at the site of a tissue or organ will cause damage to the tissue or organ, mflammatory diseases are well known in the art and have been described extensively in medical textbooks (See, e.g., Harrison's Principles of Experimental Medicine, 13th Edition, McGraw-Hill, Inc., N.Y.).

[0087] Cellular proliferative disorders include those inflammatory/inflammation diseases or disorders associated with cellular proliferation. Such disorders include, but are not limited to, proliferative glomerulonephritis; lupus erythematosus; scleroderma; temporal arteritis; thromboangiitis obliterans; mucocutaneous lymph node syndrome; asthma; host versus graft; inflammatory bowel disease; multiple sclerosis; rheumatoid arthritis; thyroiditis; Grave's disease; antigen-induced airway hyperactivity; pulmonary eosinophilia; Guillain-Barre syndrome; allergic rhinitis; myasthenia gravis; human T-lymphotrophic virus type 1- associated myelopathy; heφes simplex encephalitis; inflammatory myopathies; atherosclerosis; and Goodpasture's syndrome, hi some embodiments, the cellular proliferation disorder is an immune cell (e.g., lymphocytes, moncytes, neurtrophils, etc.) proliferation disorder.

[0088] By inflammatory/inflammation disorder is meant any disease or disorder characterized by an abnormal and/or excessive inflammatory response. Inflammatory disorders include, without limitation, coronary artery disease, rheumatoid arthritis, osteoporosis, nephropathy in diabetes mellitus, alopecia areata, Graves' disease, systemic lupus erythramatosus, lichen sclerosis, ulcerative colitis, periodontal disease, juvenile chronic arthritis, chronic iridocyclitis, psoriases, insulin dependent diabetes, diabetic complications, diabetic retinopathy, atherosclerosis, Crohn's disease, osteoarthritis, congestive heart failure, neurodegenerative diseases, and any other typically non-infectious diseases with an inflammatory component.

[0089] An inflammatory response includes, but is not limited to, any of the activation of the complement cascade, the recruitment of inflammatory cells (including monocytes, macrophages and neutrophils), the release of inflammatory cytokines (including IL-1, IL-6 and TNF), mast cell activities, the release of free oxygen radicals and lysosomal enzymes into the tissue fluid, clotting and vasoconstriction. The inflammatory response also includes the local and systemic effects of IL-1 and TNF. Inflammation can include, but not be limited to, a monocytic inflammatory response wherein the inflammatory response is initiated primarily by monocyte/macrophage activation. The monocytic inflammatory response can be particularly contrasted to an antibody response where a foreign substance that has previously been contacted with the subject is recognized by antibodies, stimulating memory B cells and leading to the rapid production of antibodies that can then activate an inflammatory response. [0090] An immune/immune system disorder is a mediated by an increased activity of the immune system. Immune disorders may be an autoimmune disorder (e.g., rheumatoid arthritis) or an immune response directed toward a non-self antigen (e.g., asthma) or an immune response associated with inflammation. Such disorders include, without exclusion, non-infectious conditions, including but not limited to, adult respiratory distress syndrome, arthrosclerosis, asthma, atherosclerosis, cholecystitis, cirrhosis, Crohn's disease, diabetes mellitus, emphysema, hypereosinophilia, inflammation, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, rheumatoid arthritis, scleroderma, and ulcerative colitis. Immune disorders include harmful or unwanted inflammatory responses and harmful or unwanted antigen-specific responses of an individual. Typically, the disorders are mediated at least in part by proliferation of cells of the immune system.

[0091] Sample is used in its broadest sense and may comprise a bodily fluid including, but not limited to, ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like. [0092] Imatinib mesylate or imatinib is also known as Gleevec™. Imatinib mesylate is designated chemically as 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3- pyridinyl)-2-pyrimidinyl] amino] -phenyl]benzamide methanesulfonate.

[0093] PTPN22 is also known as Lyp (see, WIPO Patent Publication WO99/36548 (22 July 1999)) and see also Cohen et al. hnmunbiology 93(6): 2013-2024 (1999)). PTPN22 regulates the function of Cbl and its associated protein kinases. PTPN22 is an intracellular protein of about 105-kD and has a single tyrosine phosphatase catalytic domain. Four proline-rich potential SH3 domain binding sites are located in the noncatalytic domain of PTPN22. The PTPN22 noncatalytic domain includes an NXXY motif that may become tyrosine phosphorylated and is a potential cognate site for a phosphotyrosine binding domain. PTPN22 is localized to chromosome lpl3. PTPN22 has an alternative spliced isoform, Lyp2. Lyp2 is a 85-kD protein having a different 7-amino acid C-terminus. PTPN22 is highly expressed in lymphoid tissues and cells, including both mature B and T cells and thymocytes.

PTPN22 Polymorphisms [0094] PTPN22 genomic, transcript nucleic acid sequences and PTPN22 protein amino acid sequences are set forth in Tables 1-5, and 7. Exemplary polymoφhisms of the PTPN22 gene and protein are set forth in Tables 1-5. These polymoφhisms are also disclosed in U.S. Provisional Application No. 60/495,115, filed August 15, 2003 and assigned to the same assignee as the present application and specifically incoφorated by reference herein in its entirety. The numerous polymoφhisms shown in Tables 1-5 were identified by sequencing of target sequences from unrelated individuals of diverse ethnic and geographic backgrounds.

[0095] Methods of identifying and detecting SNPs are well known in the art. See for instance, U.S. Patent No. 6,632,606. See also U.S. Patent Application No. 20030099942 published May 29, 2003 and assigned to the same assignee as the present application and herein specifically incoφorated by reference in its entirety.

[0096] Preferred polymoφhisms have at least two alleles, each occurring at frequency of greater than 0.1%, 1%, and more preferably greater than 10% or 20% of a selected population or the general population. A polymoφhic locus may be as small as one base pair. Polymoφhic markers include restriction fragment length polymoφhisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements (e.g., Alu).

[0097] The first identified allelic form is arbitrarily designated, unless otherwise indicated, as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A dialletic polymoφhism has two forms. A triallelic polymoφhism has three forms.

[0098] In some embodiments of the invention, the polymoφhism is SNP-1. In other embodiments, the polymoφhism is any one of SNPs 1-13, preferably a non-silent SNP. In some embodiments, the SNP occurs in an intronic genomic sequence which affect the expression of the PTPN22 gene (e.g., a control or regulatory element). In other embodiments, the polymoφhism is an exonic polymoφhism of Tables 1 -5 which results in an amino acid substitution in the PTPN22 protein. In some embodiments, the polymoφhism is a transition, a transversion, or a deletion of a nucleotide. In other embodiments, the polymoφhism affects the catalytic or binding site of the PTPN22 protein. In other embodiments, the polymoφhism is non-conservative and involves the substitution of a charged amino acid residue for a neutral amino acid residue (or vice versa) or a positive amino acid for a negative amino acid, or a hydrophobic amino acid for a hydrophilic amino acid. In further such embodiments, the substitution occurs at a catalytic or binding site of the PTPN22 protein. Such sites are known in the art (see, for instance, Nager et al., Cell 112: 859-871 (2003); Schindler et al., Science 289:1938-1942 (2000); and Cohen et al., Blood 93(6):2013-2024 (1999)) which are incoφorated herein by reference.

[0099] In some embodiments the SNP type is a missense mutation or non-synonymous coding SNP; a silent mutation which does not not cause a change in the encoded amino acid (i.e., a synonymous coding SNP); a stop codon mutation located in a stop codon; a nonsense mutatation creating or destroying a stop codon; a "UTR 5" SNP located in a 5' UTR of a transcript; a "UTR 3" SNP located in a 3 ' UTR of a transcript; a putative UTR 5" SNP located in a putative 5' UTR; a putative UTR 3 SNP located in a putative 3' UTR; a donor splice site SNP located in a donor splice site (5' intron boundary); an acceptor splice site SNP located in an acceptor splice site (3' intron boundary); a coding region SNP located in a protein-coding region of the transcript; and exon SNP is located in an exon; an intron SNP located in an intron; or an intergenic SNP, i.e., outside of any gene boundary. [0100] In some embodiments, the PTPN22 SNP results in an absence of detectable PTPN22 protein or substantially reduced levels (e.g, at least a 25%, 50%, or 75% reduction) of PTPN22 protein (e.g., functional PTPN22 proteins, total PTPN22 protein) in a cell or sample of a subject. In other embodiments the SNP results in a PTPN22 protein having no or substantially reduced (e.g, at least a 25%, 50%, or 75% reduction) in enzymatic activity as compared to the reference protein (e.g., a protein of the most commonly occurring sequence, or first identified sequence, or the amino acid sequence of Table 7B). Methods for characterizing PTPN22 proteins are known to one of ordinary skill in the art. See, also, U.S. Patent Application No.20030099942 published May 29, 2003 and assigned to the same assignee and incoφorated by reference in its entirety.

[0101] In some embodiments, the SNP is confirmed to be statistically associated, with a probability of the association of being due to chance of less than 1 in 10, or more preferably, 1 in 20, with a cellular proliferation disorder, immune system disorder, or inflammatory disorder. Methods for confirming these associations are known to one of ordinary skill in the art and exemplified in Example 1. In one of its aspects, the invention is drawn to methods of testing PTPN22 SNPs for their association with such disorders by use of prospective or retrospective comparisons of the incidence or frequency of SNP polymoφhism in PTPN22 nucleic acids between a control population and a population having a cellular proliferation disorder, immune system disorder, or inflammatory disorder (e.g., RA).

[0102] The increased risk of an individual to develop a disorder or to have an increased likelihood of responding to a therapy is with reference to a population of individuals who do not harbor the polymoφhic form associated with the increased risk or increased likelihood. Generally, the increased risk can be assessed in terms of an odds ratio which compares the frequency of a polymoφhism in a population having a disorder to a well-matched control population not having the disorder. Odds ratios that are greater than 1 are generally indicative of an increased risk being associated with a polymoφhism. The greater the odds ratio the greater the risk. In some embodiments, the SNP is associated with odds ratios of at least 1.4, 1.5, 1.8, 2, 3, or 5 for an inflammatory disorder, immune system disorder or a cell proliferation disorder. Such SNPs can be particularly useful in assessing the risk of developing such a disorder or the increased likelihood of a person having such a SNP responding therapeutic or prophylactic treatment with a tyrosine kinase inhibitor. Analysis of PTPN22 Polymorphisms [0103] PTPN22 genomic or transcript nucleic acid or PTPN22 protein amino acid sequences may be used to identify subjects having a PTPN22 polymoφhism. Preferred methods for determining a PTPN22 genotype or phenotype analyze a subject's nucleic acids, but a subject's PTPN22 protein amino acid sequence may also be analyzed to determine the genotype or phenotype. Methods for analyzing a subject's phenotype using proteins are well known in the art. For instance, see U.S. Patent Application No. 20030099942, filed April 2, 2001, and which is assigned to the same assignee as the present application and is incoφorated herein by reference. For instance, the polymoφhism may be detected in a method that comprises contacting a polynucleotide or protein sample from an individual with a specific binding agent for the polymoφhism and determining whether the agent binds to the polynucleotide or protein, where the binding indicates that the polymoφhism is present.

Methods of Nucleic Acid Analysis

[0104] A method according to the present invention can identify a nucleotide occurrence for either strand of DNA, typically genomic DNA. Accordingly, it will be recognized that for embodiments in which a nucleotide occurrence at a position corresponding to nucleotide 1970 (SNP-1) is identified, the method can identify a nucleotide in the opposite strand to that listed in SEQ ED NO: 29 or 561. For these embodiments, the method determines the risk of developing a subject disease based on a complementary nucleotide. For example, where the opposite strand to that corresponding to SEQ ID NO: 29 is analyzed, an adenosine occurrence at a nucleotide corresponding to nucleotide 1970 of SEQ ID NO: 29 in an opposite or complementary strand, will provide an identification of a SNP-1 allele.

[0105] As indicated above, the sequence listings of Tables 1 -5 provide the nucleotide flanking sequences for SNP-1 and other SNPs of the invention. As is well known in the field of genetics, nucleotide and amino acid sequences obtained from different sources for the same gene may vary both in the numbering scheme and in the precise sequence. Such differences may be due to inherent sequence variability within the gene and or to sequencing errors. Accordingly, reference herein to a particular polymoφhic site by number (e.g., PTPN22 polymoφhic site 1970 of SEQ ID NOS: 29 and 561) will be understood by those of skill in the art to include those polymoφhic sites that correspond in sequence and location within the gene, even where different numbering nomenclature schemes are used to describe them. [0106] It will be recognized that the 5' and 3' flanking sequences exemplified herein, provide sufficient information to identify the SNP location within the human PTPN22 gene.

[0107] Polymoφhic alleles can be detected by determining the DNA polynucleotide sequence, or by detecting the corresponding sequence in RNA transcripts from the polymoφhic gene, or where the nucleic acid polymoφhism results in a change in an encoded protein by detecting such amino acid sequence changes in encoded proteins; using any suitable technique as is known in the art. Polynucleotides utilized for genotyping are typically genomic DNA, or a polynucleotide fragment derived from a genomic polynucleotide sequence, (e.g., as in a library made using genomic material from the individual (e.g., a cDNA library)). For instance, the polymoφhism may be detected in a method that comprises contacting a polynucleotide sample from an individual with a specific binding agent for the polymoφhism and determining whether the agent binds to the polynucleotide, where the binding indicates that the polymoφhism is present.

Preparation of Samples

[0108] Polymoφhisms are detected in a target nucleic acid from an individual being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed.

[0109] Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See, generally, PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202 (each of which is incoφorated by reference for all piuposes).

[0110] Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

Analysis or Detection of Polymorphisms in Target DNA [0111 ] Numerous methods exist for the detection of polymoφhisms and SNPs within a nucleotide sequence. A review of such is found in, for example, Landegren et al., Genome Res., 8:169-116, 1998. SNPs can readily be detected by restriction fragment length polymoφhism (RFLP)(see, U.S. Pat. Nos. 5,324,631; 5,645,995). RFLP analysis requires, however, the SNP either creates or destroys a restriction enzyme cleavage site and for an available enzyme. SNPs can detected by direct sequencing of nucleotide sequences. Hybridization assays have also been developed to detect SNPs. Mismatch distinction by polymerases and ligases can also detect SNPs. U.S. Patent Application Publication No. 20030099942 which discloses many such methods of sampling and nucleic acid analysis is incoφorated by reference herein in its entirety.

Allele-Specific Probes

[0112] The use of allele-specific probes for detecting polymoφhisms is well known to one of ordinary skill in the art. (see Saiki et al., Nature 324, 163-166 (1986); European Patent No. 235,726; and International Patent Publication No. WO 89/11548. Allele-specific probes hybridize to only one polymoφhic form of target DNA but not another. Hybridization conditions for allele specific probes are kept sufficiently stringent to insure a different hybridization intensity between alleles, preferably a binary response differential wherein a probe hybridizes to only one of the alleles. Many such probes hybridize to a segment of target DNA and align with a central polymoφhic position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position). Such probes can discriminate between different allelic forms.

[0113] Allele-specific probes usually are used in pairs, wherein one pair member has a perfect match to a reference form of a target nucleic acid sequence and the other member has a perfect match to a variant form. Several such pairs of probes can be fixed on the same support to provide a simultaneous analysis of multiple polymoφhisms within the same target sequence. [0114] The TaqMan™ assay (U.S. Pat. No. 5,962,233; Livak et al., Nature Genet., 9:341- 342, 1995) is very commonly used to detect SNPs. TaqMan™ employs allele specific probes having a donor dye on one end and an acceptor dye on the other end. The dye pairs interact via fluorescence resonance energy transfer (FRET). A target sequence is amplified using PCR with labeled allele specific probes. The PCR conditions are optimally set so that a single nucleotide difference affects probe binding. The 5' nuclease activity of the Taq polymerase enzyme cleaves a perfectly complementary probe during PCR while leaving a probe with a single mismatched base is not cleaved. Probe cleavage dissociates the donor dye from the quenching acceptor dye to yield a greatly increased donor fluorescence.

[0115] Molecular beacons assays can also be used to detect SNPs (U.S. Pat. No. 5,925,517; Tyagi et al., Nature Biotech., 16:49-53, 1998). In these assays, the allele specific probes contain complementary sequences flanking the target specific species to form a haiφin structure. The loop of the haiφin is complimentary to the target sequence. Each arm of the haiφin contains either donor or acceptor dyes. When not hybridized to a target, the haiφin structure serves to bring the donor and acceptor dye closer together and thereby extinguishes the donor fluorescence. When hybridized to target, the donor and acceptor dyes separate an increase fluorescence by up to 900-fold. Molecular beacons often are used with amplification of the target sequence by PCR to provide real time detection of the presence of target sequences.

Allele-Specific Primers

[0116] An allele-specific primer hybridizes to a site on target DNA overlapping a polymoφhism. The allele specific primer only primes amplification of an allelic form having complete complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). Allele specific primers are used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the operation of the two primers and requires that the particular allelic form be present. A second pair of primers serves as a control. In the second set, one of which shows a single base mismatch at the polymoφhic site and the other primer exhibits perfect complementarily at a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The 3'-most position of the oligonucleotide aligned with the polymoφhism is most destabilizing to elongation from the primer. The method works best when the mismatch is at the 3'-most position. See, e.g., International Patent Publication No. WO 93/22456. Direct-Sequencing

[0117] Dideoxy-chain termination method or Maxam-Gilbert methods can be used to analyze directly the sequence of polymoφhisms of the present invention (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

Denaturing Gradient Gel Electrophoresis [0118] Denaturing gradient gel electrophoresis can also be used to analuze the amplification products generated using the polymerase chain reaction. Alleles can be identified and distinguished based on their sequence-dependent melting and electrophoretic migration properties. Erlich, ed., VCR Technology, Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7. Microtiter Array Diagonal Gel Electrophoresis can allow for a high throughput screening for SNPs that affect restriction sites ( see Day and Humphries, Anal. Biochem., 222:389-395, 1994).

Single-Strand Conformation Polymorphism Analysis

[0119] Single-strand conformation polymoφhism analysis can be used to distinguish target sequences. This method identifies polymoφhisms via electrophoretic migration alterations of the corresponding single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989). Amplified PCR products can be heated or otherwise denatured, to form single stranded amplification products which subsequently refold or form secondary structures which are depend on their sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to nucleotide sequence differences between alleles.

[0120] Multiplexed allele-specific diagnostic assay (MASDA) can also be used to identify alleles (U.S. Pat. No. 5,834,181; Shuber et al., Hum. Molec. Genet., 6:337-347, 1997). Further methods of identifying SNPs and other nucleic acid polymoφhisms are taught in U.S. Patent Application Publication No. 20030170674, which is incoφorated by reference herein. [0121] A wide variety of labels and methods of conjugating them to primers or probes are known to those of ordinary skill in the art. These labels may be used in various nucleic acid detection methods. Synthesis of labeled molecules may be achieved using commercially available kits (Promega, Madison Wis.) (e.g., ³²P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.). Nucleotides may be directly labeled with fluorescent, chemiluminescent, or chromogenic agents, and the like. Such labels are often conjugated via amine, thiol, or other groups present in the molecules using reagents (e.g., BIODIPY or FITC (Molecular Probes, Eugene Oregon.).

Methods of Associating a PTPN22 Polymorphism with an Inflammatory, Immune System, or Cell Proliferation Disorder. [0122] A PTPN22 polymoφhism can be associated with the occurrence of an inflammatory, immune system, or cell proliferation disorder by comparing the rates of occurrence or frequency of the polymoφhism in a population having the disorder to that of a population lacking the disorder. Such comparisons typically use control populations which well-match the disorder population in terms of age, sex, race, national origin, health practices and/or stratify the analysis of the populations according to such potential confounders as age, race, sex, socioeconomic status, etc. Statistical analysis is conducted, as well known to one of ordinary skill in the art to assess the likelihood of an associate occurring by chance.

Typically, a 5% probability cut-off is arbitrarily chosen to establish a non-random difference between populations. Additional confidence in the finding of an association can be accomplished by repeating the study with wholly different populations and/or comparing the results for various analytical strata of the studied populations. Example 1 and Tables 6 and 7 related to such methods.

Tyrosine Kinase Inhibitors

[0123] An exemplary tyrosine kinase inhibitor is imatinib mesylate (see, International Patent Application Publication No. WO 99/03854). Another example of a suitable tyrosine kinase inhibitor is PD 1739555 (see, Nagar et al. Cancer Research 62:4236-4243 (2002)). PD1739555 inhibits Abl. Other protein tyrosine kinase inhibitors include include AG 1295, AF490, AG 1517, AG957. See, Sun et al., Blood 97(7):2008-15 (2001) and Levitzki, Pharmacol Ther. 82(2-3) :231-9 (1999). Many compounds inhibit the proliferation of cells by way of inhibition of either the Abl or other protein tyrosine kinase (e.g., the PDGF receptor tyrosine kinase, the Kit receptor tyrosine kinase). These inhibitors are also suitable for use according to the invention. An exemplary inhibitor is specific (e.g., reduces the activity of a subject kinase by 50% at a concentration wliich is at least 5-fold less (or in some embodiments, at least 10-fold less thai- or 50-fold less than) than the concentration which produces a 50% inhibition of the reference tyrosine kinase (e.g., the Src tyrosine kinase, erbB-2 kinase) for the Abl tyrosine kinase, PDGF receptor, Kit, or Bcr-Abl tyrosine kinase (e.g., iminatib mesylate). See, Nagar et al., Cell 112:859-71 (2003). Such comparisons are made under standard experimental conditions approximating physiological pH and ionic strengths as would be known to one of ordinary skill in the art. hi some embodiments, the tyrosine kinase inhibitor is specific for the PDGFR or the Kit tyrosine kinase.

[0124] International Application No. WO 97/02266, International Patent Publication Application WO98/35958 and especially European Patent Application EP 0 564409-A, as well as International Application Publication No. WO99/03854, all of which are incoφorated by reference herewith, mention compounds that are inhibitors of at least one of the tyrosine kinases mentioned above. Further compounds that are of interest are Tyφhostin AG957 (see Kaur et al., Anticancer Drugs 5, 213-222 (1994), Herbimycin A (Okabe and Uehara, Leukemia and Lymphoma 12, 2156-2162 (1994), Blood 80, 1330-1338 (1994) and Leuk. Res. 18, 213-220 (1994), as well as Rioran et al., Oncogene 16, 133-1542 (1998)); Tyφhostins AG 1295, AG 1296 (see Kovalenko et al., Cancer Res. 54, 6106-6114 (1994), Lipson et al., Pharmacol & Exp-Therap. 285, 844-852 (1998), Krystal et al., Cancer Res. 57, 2203-2208 (1997)); SU 101 (Leflunomide), as well as its metabolite (see Shawer et al., Clin. Cancer Res. 3, 1167-1177 (1997), Mattar et al., FEBSLett. 334, 161-164 (1993), Cherwinskyi et al., Inflamm. Res. 3, 1167-1177 (1997), and Strawn et al., Exp. Opin. Invest Drugs 7, 533-573 (1998)); and Pyridopyrimidines (see e.g. Hamby et al., J. Med. Chem. 40, 2296-2303 (1997), Dahring et al., J. Pharmacol. Exp. Ther. 281, 1446-1456 (1997), Klutcho et al., Life Sci. 62, 143-150 (1998), Panek et al, J Pharmacol. Exp. Ther. 283, 1433-1444 (1997), Boschelli et al., J. Med. Chem. 41, 4365-4377 (1998)). Other tyrosine kinase inhibitors are taught in U.S. Patent Application Publication No. 20030166615 published on September 4, 2003; 2) U.S. Patent Application Publication No.20030186977 published on October 2, 2003; 3) U.S. Patent Application Publication No. 20030060515 published on March 27, 2003. Additional tyrosine kinase inhibitors are taught in U.S. Patents Nos. 6,265,403; 6,207,669; 6,162,804; 5,905,149; 5,648,378; 5,409,949; and 5,821,246. All the patents, patent applications and references mentioned above are specifically incoφorated herein by reference. [0125] Methods for administering pharmaceuticals applicable to tyrosine kinase inhibitors are well known in the art. Methods for determining dosage regimens (e.g., frequency, routes of administration, formulations, and dosages) are also well known in the art. (see, Gennaro AR et al., Remington 's Pharmaceutical Sciences 20th ed (2000) and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Lippincott Williams & Wilkins Publishers, January 2000)). These references are specifically incoφorated herein by reference,

[0126] Administration of an appropriate amounts of an inhibitor may be by any means known in the art, including but not limited to oral or rectal, parenteral, intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular. In some embodiments, administration is transdermal. An appropriate amount or dose of the inhibitor may be determined empirically as is known in the art. An appropriate or therapeutic amount is an amount sufficient to treat a condition of interest (e.g., inflammation, rheumatoid arthritis, arthrosclerosis, a disorder or disease of the immune system, etc.). The candidate compound can be administered as often as required to effect the desired result, for example, hourly, every six, eight, twelve, or eighteen hours, daily, or weekly

[0127] The inhibitors for use according to the invention, for instance, may comprise the active inhibitor or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient. An exemplary route of administration is the oral route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

[0128] In practical use, the inhibitors for use according to the invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, including but not limited to water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as, for example, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

[0129] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. The active compounds can also be administered intranasally as, for example, liquid drops or spray. [0130] In some embodiments, the dosage of the tyrosine kinase inhibitor is from about 0.01 mg/kg to 1000 mg/kg of body weight per day or from 1 mg to 100 mg/kg per day. The optimal dosage of the tyrosine kinase inhibitor will vary, depending on factors such as, for example, type and extent of progression of the condition, the overall health status of the patient, the potency of the tyrosine kinase inhibitor, and route of administration. Optimization of the tyrosine kinase dosage is within ordinary skill in the art.

[0131] Imatinib mesylate or Gleevec™ may be given in oral dosages effective for prophylaxis. These dosages can be in amounts from about 50 to 200 mg/day, from about 100 to about 800 mg/day; from about 200 to about 500 mg/day or from about 300 to about 700 mg/day. Imatinib may be given in oral dosages effective for preventive or therapeutic treatment of cellular proliferative disorders, inflammation disorders, or immune system disorders. Suitable dosages for such treatments include, but are not limited to, about 50 to 200 mg/day, about 100 to about 800 mg/day; about 200 to about 500 mg day; and about 300 to about 700 mg/day.

[0132] In some embodiments, a subject is treated with an effective amount of a combination of two tyrosine kinase inhibitors (e.g., imatinib mesylate and AG490) which may be administered together or separately.

Serine/Threonine Kinase Inhibitors

[0133] Inhibitors of serine/threonine kinases are also known to one of ordinary skill in the art. See, for instance, U.S. Patent Nos. 6,383,790; 5,741,689; and U.S. Patent Publication No.20020052386, published May 2, 2002; U.S. Patent Publication No.20030004174, published January 2, 2003; U.S. Patent Publication No. 20030199534, published October 23, 2003, U.S. Patent Publication No. 20030144337 published July 31, 2003. Such inhibitors include 6-dimethylaminopurine (DMAP). [0134] In so far as PTPN22 modulation of tyrosine kinase activity can directly or indirectly modulate serine/threonine kinase activity, serine/threonine kinase inhibitors may also be administered, alone or in conjuction with a tyrosine kinase inhibitor, to treat the above described conditions or persons having a PTPN22 polymoφhism, particularly, for instance such persons whose PTPN22 polymoφhism is associated with the occurrence of such conditions. [0135] Methods for administering pharmaceuticals applicable to these kinase inhibitors are well known in the art. Methods for determining dosage regimens (e.g., frequency, routes of administration, formulations, and dosages) are also well known in the art. (see, Gennaro AR et al., Remington 's Pharmaceutical Sciences 20th ed (2000) and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Lippincott Williams & Wilkins Publishers, January 2000)). These references are specifically incoφorated herein by reference. [0136] Administration of an appropriate amounts of an inhibitor may be by any means known in the art, including but not limited to oral or rectal, parenteral, intraperitoneal, intravenous, subcutaneous, subdermal, intranasal, or intramuscular. In some embodiments, administration is transdermal. An appropriate amount or dose of the inhibitor may be determined empirically as is known in the art. An appropriate or therapeutic amount is an amount sufficient to treat a condition of interest (e.g., inflammation, rheumatoid arthritis, arthrosclerosis, a disorder or disease of the immune system, etc.). The candidate compound can be administered as often as required to effect the desired result, for example, hourly, every six, eight, twelve, or eighteen hours, daily, or weekly

[0137] The inhibitors for use according to the invention, for instance, may comprise the active inhibitor or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient. An exemplary route of administration is the oral route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

[0138] In practical use, the inhibitors for use according to the invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media maybe employed, including but not limited to water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as, for example, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. [0139] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

[0140] In some embodiments, the dosage of the kinase inhibitor is from about 0.01 mg/kg to 1000 mg/kg of body weight per day or from 1 mg to 100 mg/kg per day. The optimal dosage of the kinase inhibitor will vary, depending on factors such as, for example, type and extent of progression of the condition, the overall health status of the patient, the potency of the kinase inhibitor, and route of administration. Optimization of the kinase dosage is within ordinary skill in the art.

Example 1.

[0141] Case-control studies to determine the association of SNPs in the human genome with Rheumatoid Arthritis (RA) have been carried out using genomic DNA extracted from two cohorts, a discovery sample set cohort of 475 cases and 475 controls, and a replication sample set cohort of 844 cases and 926 controls. In both cohorts, the cases and controls were matched on the basis of grandparental-origin, gender, and age. [0142] > 12,000 SNPS from throughout the entire genome were analyzed in the discovery sample set cohort. All patients in this cohort are Rheumatoid Factor positive (RF+). Ninety- six of these SNPS were assayed on individual DNAs while the remainder were assayed using pooled DNAs that had been stratified according to their case/control status, gender (male vs female), age of onset (<38 vs 38-55 vs >55), and HLA-DRBl genotype. Summary statistics for demographic and environmental traits, HLA-DRBl genotypic frequencies, and SNP allele frequencies were obtained and compared between cases and controls.

[0143] For the 96 individually genotyped SNPs, several tests of association were calculated for both non-stratified and strata-specific settings: 1) Fisher's exact test of allelic association (Fisher, R. A. 1954. Statistical Methods for Research Workers. 12^th ed. Oliver & Boyd, Edinburgh), 2) William' s-corrected likelihood ratio G-test of genotypic association (Sokal, R. R. and F. J. Rohlf. 1995. Biometry: the Principles and Practice of Statistics in Biological Research. 3rd edition. W. H. Freeman and Co.: New York), and 3) Exact test of Hardy- Weinberg Equilibrium for cases and controls (Nielsen et al. 1999. Am. J. Hum. Genet. 63:1531-1540). Additionally, the false negative rate was addressed by calculating power to replicate a given finding in the discovery sample set cohort in the replication sample set cohort. Effect sizes were estimated through allelic odds ratios and odds ratios for the heterozygote and homozygote with 95% confidence intervals.

[0144] A SNP was considered for replication if a) it exhibited a p-value below 0.05 in any of the previously mentioned tests of association, b) the power to replicate the finding in replication sample set cohort was above 0.50, or c) if the effect size was significantly greater than 1.0 for the predisposing allele. As significant deviations from Hardy- Weinberg Equilibrium in controls can indicate genotyping errors, SNPs with Hardy- Weinberg Equilibrium tests significant at the 0.01 level in controls were excluded from replication consideration.

[0145] For SNPs analyzed on pooled DNAs, allelic association tests were performed using Fisher's exact test, allelic odds ratios were calculated, and power to replicate with allelic association was estimated. SNPs with exact p-values below 0.05 or SNPs with high power to replicate were considered for replication. Genes with multiple significant SNPs were also considered for replication. Analysis of the distribution of p-values across all pooled assays demonstrated that genome-wide sampling bias between cases and controls, which could potentially increase the rate of spurious results, was unlikely. [0146] Based on the criteria outlined above, 95 SNPs were chosen for replication in the replication sample set cohort using individual genotyping. Of these 95, 9 were replicated with a p-value of <0.05 (see Table 7. A replicated hit indicates that changes in the gene, including, but not limited to, the marker(s) listed, are associated with RA. Because nearby markers tend to be inherited together (linkage disequilibrium), markers within 50 kb of the replicated hit (including, but not limited to, markers within the same gene) (Reich et al. 2001. Nature 411:199-204.), which can also be associated with RA, are also included.

[0147] Table 6 provides results of statistical analyses for a SNP disclosed in Tables 1-5 (SNPs can be cross-referenced between Tables based on their hCV identification numbers. The association data was obtained via the above-described methods. The statistical results shown in Table 6 provide support for the association of such PTPΝ22 SNPs with rheumatoid arthritis. The statistical results provided in Table 6 show that the association of these SNPs with rheumatoid arthritis is supported by, for example, different allele frequencies in cases compared with controls, and p-values < 0.05 in an allelic association test. Moreover, Table 6 provides results of replication studies, in addition to initial discovery results, which further verify the association of the SNPs, in this case, SNP-1, with rheumatoid arthritis. The common and allele-specfic PCR primers suitable for detecting SNP-1 or the wild-type PTPN22 allele at the SNP-1 site are as set forth in Table 6.

[0148] The column labeled "Strata" in Table 6 indicate if the analysis of the dataset for each SNP was stratified based on a particular characteristic such as, for example, age of rheumatoid arthritis onset, sex, HLA genotype, etc. (these characteristics, and the associated entries in Table 6, are described below), or if the dataset was analyzed independent of any stratification (SNPs for which the dataset was analyzed independent of any stratification are indicated as "ALL" in the Strata column); the column labeled "Allele" identifies each allele for which the associated statistics are based on; the frequency of this allele in cases and controls is listed in the next two columns (in the columns headed "case frq" and "control frq", respectively); the column labeled "allelicAsc p" provides p-values derived from allelic association tests; "OR" provides the Odds Ratio; and the columns labeled "C1-" and "C1+" provide 95% confidence intervals. These last six columns ("case frq", "control frq", "allelicAsc p", "OR", "C1-", and "C1+") provide statistical results for both a discovery sample set and a replication sample set. [0149] The entries in the Strata column, which identify particular characteristics for which SNP analyses were stratified, are as follows for HLA DRB1 alleles and genotypes [other Strata include male/female sex and age (in years) of rheumatoid arthritis onset (e.g., "onset<38", "onset38-55", and "onset>55")]:

HLA DRB1 Alleles DRB1 Coded Allele Actual DRB1 Allele* 01 R DRB 1*01 allele with an arginine at amino acid 70 (e.g. DRB1*0101) 04K DRB1*04 allle with a lysine at amino acid 70 (e.g. DRB1*0401 ) 04R DRB1 *04 allele with an arginine at amino acid 70 (e.g. DRB1 *0404) 10 DRB1*1001 X Any DRB 1 allele except 01 R, 04K, 04R or 10

HLA DRB 1 Genotypes HLA Derived Genotype HLA DRB1 STRATA HLA-DRB1 STRATA ABBREVIATION 01R.01R HIGH RISK -2 SHARED EPITOPES HR-2SE 01R.04K HIGH RISK -2 SHARED EPITOPES HR-2SE 01R.04R HIGH RISK -2 SHARED EPITOPES HR-2SE 01R.10 HIGH RISK -2 SHARED EPITOPES HR-2SE

04K,04K HIGH RISK -2 SHARED EPITOPES HR-2SE

04K.04R HIGH RISK -2 SHARED EPITOPES HR-2SE

04K.10 HIGH RISK -2 SHARED EPITOPES HR-2SE

04R.04R HIGH RISK -2 SHARED EPITOPES HR-2SE

04R.10 HIGH RISK -2 SHARED EPITOPES HR-2SE

10,10 HIGH RISK -2 SHARED EPITOPES HR-2SE

04K.X HIGH RISK -1 SHARED EPITOPE HR-1SE

04R.X HIGH RISK -1 SHARED EPITOPE HR-1SE

01R.X LOW RISK -1 SHARED EPITOPE LR-1SE

10,X LOW RISK -1 SHARED EPITOPE LR-1SE

X,X LOW RISK -0 SHARED EPITOPES LR-0SE

Example 2.

[0150] A blood sample is obtained from a human patient without signs or symptoms of an immune or inflammatory disorder (e.g., rheumatoid arthritis) but optionally with a family history of the disorder. Using techniques well known to one of ordinary skill in the art, the genomic DNA is isolated from the blood sample and amplified using PCR and the presence or absence of a SNP from Tables 1-5 (e.g., SNP-1) is determined with an oligonucleotide specific probes which is specific for the SNP (e.g. a T or a C at position 1970 of SEQ ID NO: 29 for SNP-1). If the patient has the polymoφhism, the patient is determined to be at an increased risk of an immune or inflammatory disorder and is treated prophylactically with a tyrosine kinase inhibitor (e.g., imatinib mesylate).

Example 3 [0151] A blood sample is obtained from a human patient without signs or symptoms of a cellular proliferative disorder (e.g., cancer or CML). Using techniques well known to one of ordinary skill in the art, the genomic DNA is isolated from the blood sample and amplified using PCR and the presence or absence of a SNP polymoφhism (e.g., SNP-1) according to Tables 1-5 is determined using a oligonucleotide specific probes (e.g., a probe for SNP-1 is specific for a nucleic acid having one of either T or C at position 1970 of SEQ ID NO: 29). If the patient has the SNP polymoφhism, the patient is determined to be at an increased risk of an immune or inflammatory disorder and is treated prophylactically with a tyrosine kinase inhibitor (e.g., imatinib mesylate).

Example 4

[0152] A clinical study is being conducted on the efficacy of a tyrosine kinase inhibitor (e.g., imatinib mesylate) in the treatment of an immune or inflammatory disorder, the presence or absence of a SNP polymoφhism of Tables 1-5 (e.g., SNP-1) is determined for each subject as set forth in Example 1. The study results are stratified according to the absence or presence of the polymoφhism in the subject populations. The adjustment for an otherwise unrecognized source of variation in responsiveness increases the power of the study to detect an effect and helps to avoid any confounding of results which might result from uncontrolled or random differences in the SNP frequency between the study populations.

Example 5

[0153] A blood sample is obtained from a human patient with signs or symptoms of an immune or inflammatory disorder (e.g., rheumatoid arthritis). Using techniques well known to one of ordinary skill in the art, the genomic DNA is isolated from the blood sample and amplified using PCR and the presence or absence of the SNP polymoφhism set forth in Tables 1-5 is determined with oligonucleotide specific probes (e.g., a SNP-1 specific probe is specific for one of either a T or a C at position 1970 of SEQ ID NO: 29). If the patient has the SNP polymoφhism, the patient is determined to have an increased likelihood of being responsive to therapy with a tyrosine kinase inhibitor and the inhibitor (e.g., imatinib mesylate) is administered to treat the signs and symptoms.

[0154] Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating, preventing or reducing the risk of rheumatoid arthritis or other immune system or inflammatory disease or disorder in a human having a variant form of the PTPN22 tyrosine phosphatase gene, which method comprises administering to said human an effective amount of one or more protein kinase inhibitors wherein the inhibitors are selected from the group consisting of tyrosine kinase inhibitors and serine/threonine kinase inhibitors.

2. The method of claim 1 , wherein the variant tyrosine phosphatase gene encodes a variant form of Lyp 1 or Lyp2.

3. The method of claim 2, wherein the gene encodes a transcript having the nucleotide sequence of SEQ ID NOs: 29, 30, 31, or 32 or a single nucleotide polymoφhism of any one thereof.

4. The method of claim 3, wherein the polymoφhism is a polymoφh of Tables Tables 1, 11, III, or IN.

5. The method of claim 4, wherein the polymoφh is the SΝP- 1 polymoφh.

6. The method of claim 1, wherein the tyrosine kinase inhibitor is imatinib mesylate.

7. The method of claim 6, wherein the variant comprises a single nucleotide polymoφhism selected from the group consisting of SΝPs 1 through 13.

8. The method of claim 1 , wherein the tyrosine kinase inhibitor is a non-specific tyrosine kinase inhibitor.

9. The method of claim 1 , wherein the tyrosine kinase inhibitor is an inhibitor of Abl or PDGF receptor tyrosine kinase.

10. The method of claim 1, wherein the disease or disorder is not cancer and is not rheumatoid arthritis.

11. A method for treating, preventing or reducing the risk of a non-cancerous immune system or inflammatory disorder other than rheumatoid arthritis in a human, said method comprising administering to said human an effective amount of one or more tyrosine kinase inhibitors or one or more serine/threonine kinase inhibitors, or a combination thereof.

12. The method of claim 11 , wherein the human has a variant PTPN22 tyrosine phosphatase gene,

13. The method of claim 12, wherein the variant tyrosine phosphatase gene encodes a variant of Lyp 1 or Lyp2.

14. The method of claim 13, wherein the gene encodes a transcript having the full nucleotide sequence of SEQ ID NOs: 29, 30, 31, or 32 or a single nucleotide polymoφhism of any one thereof.

15. The method of claim 14, wherein the polymoφhism is a polymoφh of Tables Tables 1, II, III, or IN.

16. The method of claim 15, wherein the polymoφh is SΝP-1.

17. The method of claim 11, wherein the tyrosine kinase inhibitor is imatinib mesylate.

18. The method of claim 11, wherein the polymoφhism comprises a SNP selected from the group consisting of SNPs 1 through 13.

19. The method of claim 11, wherein the tyrosine kinase inhibitor is a non-specific tyrosine kinase inhibitor.

20. The method of claim 11 , wherein the tyrosine kinase inhibitor is an inhibitor of Abl or PDGF receptor tyrosine kinase.

21. A method for treating, preventing or reducing the risk of rheumatoid arthritis or another immune system or inflammatory disorder in a human, which method comprises:

(i) determining the presence of a variant PTPN22 tyrosine phosphatase gene of the human and (ii) administering to said subject a therapeutically or prophylactically effective amount of one or more tyrosine kinase inhibitors.

22. The method of claim 21, wherein the inhibitor is PD1739555.

23. The method of claim 21 , wherein the gene encodes a transcript having the full nucleotide sequence of SEQ ID NOs: 29, 30, 31, or 32 or a single nucleotide polymoφhism of any one thereof.

24. The method of claim 23 , wherem the polymoφhism is a polymoφh of Tables Tables 1, 11, in, IV or V.

25. The method of claim 23, wherein the polymoφh is SNP-1.

26. The method of claim 21 , wherein the tyrosine kinase inhibitor is imatinib mesylate.

27. The method of claim 21 , wherein the tyrosine kinase inhibitor is a non-specific tyrosine kinase inhibitor.

28. The method of claim 21 , wherein the tyrosine kinase inhibitor is an inhibitor of Abl or PDGF receptor tyrosine kinase.

29. A method of treating a human at risk of developing an immune or inflammatory disorder, said method comprising: (i) determining the presence of a variant PTPN22 tyrosine phosphatase gene in the human, and (ii) administering a tyrosine kinase inhibitor or serine/threonine kinase inhibitor to the subject.

30. The method of claim 29, wherein the variant comprises SNP-1.

31. A method of screening polymoφhic sites of PTPN22 DNA linked to polymoφhic sites shown in Tables 1-5 for suitability for diagnosing a phenotype, said method comprising: (i) identifying a polymoφhic site linked to a polymoφhic site shown in Tables 1- 5 wherein a polymoφhic form of the polymoφhic site shown in Tables 1-5 has been correlated with an immune or inflammatory disorder phenotype; and (ii) determining haplotypes in a population of individuals to indicate whether the linked polymoφhic site has a polymoφhic form in disequlibrium linkage with the polymoφhic form correlated with the phenotype.

32. A method of determining or diagnosing a PTPN22 phenotype, said method comprising: (i) determining which polymoφhic form(s) are present in a sample from a subject at one or more of the polymoφhic sites shown in Tables 1-5; and (ii) determining or diagnosing the presence of a phenotype correlated with the form(s) in the subject; wherein the phenotype is an immune or inflammation or cellular proliferation disorder phenotype.

33. A method of identifying PTPN22 polymoφhs associated with a disease or disorder, said method comprising: (i) obtaining PTPN22 genomic or transcript nucleic acids samples from a plurality of individuals from a first population, wherein the members of the first population do not have the disorder; (ii) obtaining PTPN22 genomic or transcript nucleic acids samples from a plurality of individuals from a second population, wherein the members of the second population population have the disorder; (iii) comparing the frequency of occurrence of a genomic or transcript PTPN22 polymoφhism between the first and second populations to identify a polymoφhism associated with the disorder; wherein the disorder is selected from the group consisting of cellular proliferation, immune system and inflammation disorders.

34. The method of claim 33, wherein the polymoφhism is a polymoφhism set forth in Tables 1-5.

35. The method of claim 33, wherein the disorder is rheumatoid arthritis.