HK1209459B - Methods and compositions for identifying and treating lupus - Google Patents

Description

Methods and compositions for identifying and treating lupus

This application is a divisional application of the invention application with the application date of May 21, 2008, Chinese application number 200880025560.5, and invention name “Methods and compositions for identifying and treating lupus”.

Cross-reference to related applications

This patent application claims priority to U.S. Patent Application No. 60/939,156, filed May 21, 2007, and U.S. Patent Application No. 61/013,283, filed December 12, 2007. The contents of these patent applications and all references contained therein are hereby incorporated by reference in their entirety.

Field of the Invention

Generally speaking, the present invention relates to a unique set of genetic polymorphisms associated with lupus, and methods for assessing the risk of developing lupus and for diagnosing and treating lupus.

Background of the Invention

Lupus is an autoimmune disease involving antibodies that attack connective tissue. It is estimated that the disease affects nearly 1 million Americans, primarily women between the ages of 20 and 40. The main form of lupus is a systemic form (systemic lupus erythematosus; SLE). Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with strong genetic and environmental factors (see, for example, Hochberg MC, Dubois' Lupus Erythematosus. 5th ed., Wallace DJ, Hahn BH eds. Baltimore: Williams and Wilkins (1997); Wakeland EK et al., Immunity 2001; 15(3): 397-408; Nath SK et al., Curr. Opin. Immunol. 2004; 16(6): 794-800). Autoantibodies play an important role in the pathogenesis of SLE, and the diverse clinical manifestations of the disease are due to the deposition of immune complexes containing antibodies in blood vessels (this causes inflammation in the kidney, brain and skin), and the direct pathogenic effects of autoantibodies that contribute to hemolytic anemia and thrombocytopenia. In general, SLE is characterized as an autoimmune connective tissue disorder with extensive clinical features, which mainly affects women, especially those from certain ethnic groups (D'Cruz et al., Lancet (2007), 369:587-596). SLE is related to the generation of antinuclear antibodies, the immune complexes in the circulation, and the activation of the complement system. The incidence of SLE is about 1 in 700 women aged between 20 and 60. SLE can affect any organ system and can cause serious tissue damage. There are many autoantibodies with different specificities in SLE. SLE patients often produce autoantibodies with anti-DNA, anti-Ro, and anti-platelet specificity and can initiate clinical features of the disease (such as glomerulonephritis, arthritis, serositis, complete heart block in neonates, and hematological abnormalities). These autoantibodies may also be involved in central nervous system disorders. Arbuckle et al. documented the formation of autoantibodies before the clinical onset of SLE (Arbuckle et al. N. Engl. J. Med. 349 (16): 1526-1533 (2003)). The diagnosis of lupus (including SLE) is not easy, which has led clinicians to resort to a classification method based on multifactorial signs and symptoms (Gill et al., American Family Physician (2003), 68 (11): 2179-2186).

Lupus can be fatal if left untreated because it progresses from attacking the skin and joints to internal organs, including the lungs, heart, and kidneys (with kidney disease being a primary concern), making early and accurate diagnosis and/or assessment of the risk of developing lupus extremely important. Lupus manifests primarily as a series of flare-ups with intervening periods of little or no disease manifestation. Kidney damage (measured by the amount of protein in the urine) is one of the most dramatic areas of damage in SLE related to pathogenicity and accounts for at least 50% of the mortality and morbidity from the disease.

One of the most difficult challenges in the clinical management of complex autoimmune diseases such as lupus is the accurate and early identification of the disease in patients. In the past few years, many linkage and candidate gene studies have been performed to identify genetic factors that contribute to SLE susceptibility. Haplotypes carrying the HLA class II alleles DRB1*0301 and DRB1*1501 are clearly associated with the disease and the presence of antibodies to nuclear autoantigens (see, for example, Goldberg MA et al., Arthritis Rheum 1976; 19(2): 129-32; Graham RR et al., Am J Hum Genet 2002; 71(3): 543-53; and Graham RR et al., Eur J Hum Genet 2007; 15(8): 823-30). More recently, variants of interferon regulatory factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT4) have been found to be important risk factors for SLE (see, e.g., Sigurdsson S et al., Am J Hum Genet 2005; 76(3):528-37; Graham RR et al., Nat Genet 2006; 38(5):550-55; Graham RR et al., Proc Natl Acad Sci U S A 2007; 104(16):6758-63; and Remmers EF et al., N Engl J Med 2007; 357(10):977-86). The identification of IRF5 and STAT4 as SLE risk genes provides support for the concept that the type I interferon pathway is important for disease pathogenesis (see, e.g., Ronnblom L et al., J Exp Med 2001; 194(12):F59-63; Baechler EC et al., Curr Opin Immunol 2004; 16(6):801-07; Banchereau J et al., Immunity 2006; 25(3):383-92; Miyagi T et al., J Exp Med 2007; Epub 2007; Sep 10).

For this purpose, it would be highly advantageous to have a molecular-based diagnostic method that can be used to objectively identify the presence of a disease in a patient and/or to classify the disease in a patient. Genetic variation or polymorphism is a genetic variation present in the genome of an organism. Polymorphisms include single nucleotide polymorphisms (SNPs) (see, e.g., Carlson et al., Nature 2004; 429: 446-452; Bell, Nature 2004; 429: 453-463; Evans and Relling, Nature 2004; 429: 464-468). SNPs have been firmly linked to the risk and/or presence of serious diseases, such as diabetes (Sladek et al., Nature 2007; 445: 881-828; Zeggini et al., Science 2007; Apr 26; Scott et al., Science 2007; Apr 26; and Saxena et al., Science 2007; Apr 26); Crohn's disease (e.g., Hampe et al., Nat. Genet. 2007; Feb; 39(2): 207-11); rheumatoid arthritis (e.g., U.S. Patent Publication No. 2007/0031848); and other inflammatory immune diseases (e.g., U.S. Patent 6,900,016; U.S. Patent 7,205,106).

Until recently, it was not possible to comprehensively examine the genome for variants that alter the risk for complex diseases such as lupus. However, the generation of extensive catalogs of common human variation coupled with technological advances that allow cost-effective and accurate genotyping of hundreds of thousands of variants (see, e.g., Nature 2005; 437(7063): 1299-320) has fueled a revolution in human genomics. For the first time, it has been possible to conduct well-powered genome-wide association scans to more comprehensively test the hypothesis that common variants influence risk. This technology has been highly demonstrated in the past two years (see, for example, Dewan A et al., Science 2006; 314(5801):989-92; Nature 2007; 447(7145):661-78, Matarin M et al., Lancet neurology 2007; 6(5):414-20; Moffatt MF et al., Nature 2007; 448(7152):470-73; Plenge RM et al., N Engl J Med 2007; Saxena R et al., Science 2007; 316(5829):1331-36; Scott LJ et al., Science 2007; 316(5829):1341-45; Scuteri A et al., PLoS Genet 2007; 3(7):e115). The identified risk loci provide new insights into molecular pathways that are dysregulated in human disease.

However, there is still a significant lack of reliable information about the association of SNPs with complex diseases (such as lupus), so it is clear that there is a continuing need to identify polymorphisms associated with such diseases. Such associations would be of great benefit in identifying the presence of lupus in a patient or determining the susceptibility to the disease. Additionally, statistically and biologically significant and reproducible information about the association of SNPs with complex diseases (such as lupus) can be utilized in a holistic form to focus on identifying specific patient subsets that are expected to benefit significantly from the treatment carried out with a specific therapeutic agent, such as one that has shown or has shown in clinical studies to be therapeutically beneficial in such specific lupus patient subpopulations.

The invention described herein satisfies the above-identified needs and provides other advantages.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides accurate, simple, and rapid methods and compositions for identifying lupus and for assessing the risk of developing lupus, which are based at least in part on the identification of one or more genetic variations (e.g., SNPs) that are associated with the presence, subtype, and/or patient subpopulations of lupus with high statistical and biological significance. More specifically, the present invention relates to the identification of unique sets of SNPs associated with lupus and its subtypes, unique combinations of such SNPs, and linkage disequilibrium regions, and the identification of patient subpopulations suffering from lupus and its subtypes.

In some aspects, the SNPs are related to genes. In other aspects, the SNPs are related to genes and can be located in intergenic or intragenic regions, and more specifically, can be located in coding regions or non-coding regions. The genes related to the SNPs of the present invention can be related to unknown genes, or can be related to known genes (e.g., ITGAM or BLK).

The SNPs identified herein provide targets for the development of therapeutic agents for the diagnosis and treatment of genetically identified lupus patients, including the diagnosis and targeted treatment of a subpopulation of lupus patients that exhibit a unique genetic signature comprising one or more of the SNPs of the present invention. For example, in one aspect, genes containing the genetic variations identified herein, nucleic acids (e.g., DNA or RNA) associated with these genes, and proteins encoded by these genes can be used as targets for the development of therapeutic agents (e.g., small molecule compounds, antibodies, antisense/RNAi agents, etc.) or directly as therapeutic agents (e.g., therapeutic proteins, etc.) for the treatment of lupus.

Thus, in one aspect, the present invention provides a collection of one or more SNPs that form a unique genetic signature for assessing the risk of developing lupus. In one aspect, the unique genetic signature comprises about 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

On the one hand, the unique genetic signature comprises one or more SNPs, two or more SNPs, three or more SNPs, four or more SNPs, five or more SNPs, six or more SNPs, seven or more SNPs, eight or more SNPs, nine or more SNPs, ten or more SNPs, eleven or more SNPs, twelve or more SNPs, thirteen or more SNPs, fourteen or more SNPs, fifteen or more SNPs, sixteen or more SNPs, seventeen or more SNPs, eighteen or more SNPs, nineteen or more SNPs or twenty or more SNPs selected from any SNP listed in Figures 1-17 and Tables 1-10. On the one hand, the SNPs of the genetic signature are selected from Table 6. In another aspect, the SNP is selected from rs9888739, rs13277113, rs7574865, rs2269368, rs6889239, rs2391592 and rs21177770. In another aspect, the SNP is selected from rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

In another aspect, the present invention provides a method for assessing whether a subject is at risk for developing lupus by detecting the presence of a genetic signature indicative of risk for developing lupus in a biological sample obtained from the subject, wherein the genetic signature comprises a set of one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the set of SNPs comprises about 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. On the other hand, the SNP set comprises 2 or more SNPs, 3 or more SNPs, 4 or more SNPs, 5 or more SNPs, 6 or more SNPs, 7 or more SNPs, 8 or more SNPs, 9 or more SNPs, 10 or more SNPs, 11 or more SNPs, 12 or more SNPs, 13 or more SNPs, 14 or more SNPs, 15 or more SNPs, 16 or more SNPs, 17 or more SNPs, 18 or more SNPs, 19 or more SNPs, or 20 or more SNPs selected from any SNP listed in Figures 1-17 and Tables 1-10. On the other hand, the SNP set comprises 1-19 species of SNPs selected from Table 6. On the other hand, the SNP set comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10. On the other hand, the SNP set comprises an ITGAMSNP selected from any ITGAM SNP listed in Tables 7-10. In another aspect, the SNP set further comprises a BLK SNP selected from any of the BLK SNPs listed in Tables 7 to 10. In another aspect, the SNP set comprises one or more SNPs selected from the group consisting of rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

In another aspect, the present invention provides a method for diagnosing lupus in a subject by detecting the presence of a genetic signature indicative of lupus in a biological sample obtained from the subject, wherein the genetic signature comprises a set of one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

On the other hand, the invention provides a kind of isolated polynucleotide or its fragment, its length is at least about 10 Nucleotide, wherein said polynucleotide or its fragment comprise: a) with the genetic variation of the nucleotide position corresponding to the position of the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10, or (b) the complement of (a).On the one hand, the isolated polynucleotide is a genomic DNA, which comprises the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10.On the other hand, the isolated polynucleotide is an RNA, which comprises the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10.

In one aspect, the present invention provides an isolated PRO-associated polynucleotide or fragment thereof having a length of at least about 10 nucleotides, wherein the PRO-associated polynucleotide or fragment thereof comprises: a) a genetic variation at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, or (b) the complement of (a). In one aspect, the isolated polynucleotide is genomic DNA encoding a gene (and/or a regulatory region of the gene) comprising a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In another aspect, the SNP is in a region of a chromosome that does not encode a gene. In another aspect, the SNP is in an intergenic region of a chromosome. In another aspect, the isolated polynucleotide is a primer. In another aspect, the isolated polynucleotide is an oligonucleotide.

On the other hand, the present invention provides an oligonucleotide, which is (a) an allele-specific oligonucleotide that hybridizes to a region of a genetic variation at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, or (b) the complement of (a). On the one hand, the SNP is in a PRO-associated polynucleotide that encodes a gene (or its regulatory region) comprising a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. On the other hand, the SNP is in genomic DNA that encodes a gene (or its regulatory region) comprising a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. On the other hand, the SNP is in a non-coding region of the gene. On the other hand, the SNP is in the coding region of the gene. On the other hand, the allele-specific oligonucleotide is an allele-specific primer.

In another aspect, the present invention provides a kit comprising any of the oligonucleotides described above and, optionally, at least one enzyme. In one aspect, the at least one enzyme is a polymerase. In another aspect, the at least one enzyme is a ligase.

In another aspect, the present invention provides a microarray comprising any of the above oligonucleotides.

In another aspect, the present invention provides a method for detecting the presence of a variation in a polynucleotide at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10, the method comprising (a) contacting a nucleic acid suspected of containing the variation with an allele-specific oligonucleotide specific for the variation under conditions suitable for hybridization of the allele-specific oligonucleotide to the nucleic acid; and (b) detecting the presence of allele-specific hybridization. In one aspect, the variation contains a SNP as listed in Figures 1-17 and Tables 1-10. In one aspect, the polynucleotide is a PRO-associated polynucleotide.

In another aspect, the present invention provides a method for amplifying a nucleic acid comprising a variation at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, the method comprising (a) contacting the nucleic acid with a primer that hybridizes to the nucleic acid at the 3' sequence of the variation, and (b) extending the primer to generate an amplification product comprising the variation. In one aspect, the polynucleotide is a PRO-associated polynucleotide.

On the other hand, the invention provides a method for measuring the genotype of a biological sample from a mammal, the method comprising detecting in a nucleic acid material derived from the biological sample whether there is a variation at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10 in a polynucleotide, wherein the polynucleotide is a PRO associated polynucleotide.

On the other hand, the biological sample is known or suspected to comprise a polynucleotide of the present invention, wherein the polynucleotide comprises a variation at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) selected from any SNP listed in Figures 1-17 and Tables 1-10. On the other hand, the biological sample is a disease tissue. On the other hand, the detection comprises implementing a method selected from the group consisting of: primer extension assays; allele-specific primer extension assays; allele-specific nucleotide incorporation assays; allele-specific oligonucleotide hybridization assays; 5' nuclease assays; an assay using a molecular beacon; and oligonucleotide ligation assays.

In another aspect, the present invention provides a method for subclassifying lupus in a mammal, the method comprising detecting the presence of one or more SNPs listed in Figures 1-17 and Tables 1-10 in a biological sample derived from the mammal, wherein the biological sample is known or suspected to contain at least one polynucleotide comprising a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the polynucleotide is a PRO-associated polynucleotide.

In another aspect, the detection comprises performing a method selected from the group consisting of: a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; a 5' nuclease assay; an assay using a molecular beacon; and an oligonucleotide ligation assay.

In another aspect, the present invention provides a method for predicting whether a subject with lupus will respond to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in a polynucleotide at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, wherein the presence of the variation indicates that the subject will respond to the therapeutic agent. In one aspect, the polynucleotide is a PRO-associated polynucleotide.

In another aspect, the present invention provides a method for diagnosing or prognosing lupus in a subject, the method comprising detecting the presence of a variation in a polynucleotide derived from a biological sample obtained from the subject, wherein: (a) the biological sample is known or suspected to contain a polynucleotide comprising the variation; (b) the variation comprises a SNP selected from any SNP listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP selected from any SNP listed in Figures 1-17 and Tables 1-10; and (c) the presence of the variation is diagnostic or prognostic of lupus in the subject.

In another aspect, the present invention provides a method for diagnosing or prognosing lupus in a subject, the method comprising detecting the presence of a variation in a PRO or PRO-associated polynucleotide derived from a biological sample obtained from the subject, wherein: (a) the biological sample is known or suspected to contain a PRO or PRO-associated polynucleotide comprising the variation; (b) the variation comprises a SNP listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP listed in Figures 1-17 and Tables 1-10; and (c) the presence of the variation is diagnostic or prognostic of lupus in the subject.

In another aspect, the present invention provides a method for aiding in the diagnosis or prognosis of lupus in a subject, the method comprising detecting the presence of a variation in a polynucleotide derived from a biological sample obtained from the subject, wherein: (a) the biological sample is known or suspected to contain a polynucleotide comprising the variation; (b) the variation comprises a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10; and (c) the presence of the variation is diagnostic or prognostic of the condition or symptom of lupus in the subject.

In another aspect, the present invention provides a method for aiding in the diagnosis or prognosis of lupus in a subject, the method comprising detecting the presence of a variation in a PRO or PRO-associated polynucleotide derived from a biological sample obtained from the subject, wherein: (a) the biological sample is known or suspected to contain a PRO or PRO-associated polynucleotide comprising the variation; (b) the variation comprises a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10; and (c) the presence of the variation is diagnostic or prognostic of a condition or symptom of lupus in the subject.

In another aspect, the polynucleotide comprises a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one aspect, the variation is in genomic DNA comprising a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the SNP is in a chromosomal region that does not encode a gene. In another aspect, the SNP is in an intergenic region.

In another aspect, the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one aspect, the variation is in the genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the SNP is in a non-coding region of the gene. In another aspect, the SNP is in a coding region of the gene.

In another aspect, the present invention provides a method for identifying a therapeutic agent that is effective in treating lupus in a subpopulation of patients, the method comprising correlating the efficacy of the agent with the presence of one or more SNPs in the patient selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, thereby identifying the agent as effective in treating lupus in the subpopulation of patients.

In another aspect, the present invention provides a method for identifying a therapeutic agent that is effective in treating lupus in a patient subpopulation, the method comprising correlating the efficacy of the agent with the presence of a combination of SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, thereby identifying the agent as effective in treating lupus in the patient subpopulation.

In another aspect, the present invention provides a method of treating a lupus condition in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent effective to treat the condition.

In another aspect, the present invention provides a method of treating a subject having a lupus condition, the method comprising administering to the subject a therapeutic agent effective to treat the condition in a subject having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

In another aspect, the present invention provides a method for treating a subject with a lupus condition, the method comprising administering to the subject a therapeutic agent shown in at least one clinical study to be effective in treating the condition, wherein the agent is administered to at least five human subjects in the clinical study, each of the human subjects having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the at least five subjects have a total of two or more different SNPs across the group of at least five subjects. In another aspect, the at least five subjects have the same SNP across the entire group of at least five subjects.

In another aspect, the present invention provides a method for treating lupus in a subject comprising a specific subpopulation of lupus patients, wherein the subpopulation is characterized at least in part by an association with a genetic variation at a nucleotide position corresponding to a SNP selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, and wherein the method comprises administering to the subject an effective amount of a therapeutic agent approved as a therapeutic for the subpopulation. In one aspect, the subpopulation has lupus nephritis. In another aspect, the subpopulation is female. In another aspect, the subpopulation is of European descent.

In another aspect, the present invention provides a method comprising manufacturing a lupus therapeutic agent and packaging the agent and instructions for administering the agent to a subject having or suspected of having lupus and having a genetic variation at a position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

In another aspect, the present invention provides a method for detailing a therapeutic agent for use in a subpopulation of lupus patients, the method comprising providing instructions for administering the therapeutic agent to the subpopulation of patients, wherein the subpopulation of patients is characterized by a genetic variation at a position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

In another aspect, the present invention provides a method for marketing a therapeutic agent for use in a subpopulation of lupus patients, the method comprising informing a target audience about the use of the therapeutic agent for treating the subpopulation of patients, the subpopulation of patients being characterized by the presence of a genetic variation in such subpopulation patients at a position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

In another aspect, the present invention provides a method for modulating signaling through a B cell receptor in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) selected from any of the SNPs listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively modulates signaling through a B cell receptor.

On the other hand, the present invention provides a method for regulating Th17 cell differentiation in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) selected from any SNP listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively regulates Th17 cell differentiation.

In another aspect, the present invention provides a set of SNPs comprising a genetic signature indicating a risk of developing lupus, wherein the set of SNPs comprises one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In one aspect, the set of SNPs comprises about 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10. In another aspect, the set of SNPs comprises one or more SNPs selected from the group consisting of rs9888739, rs13277113, rs7574865, rs2269368, rs6889239, rs2391592, and rs21177770. On the other hand, the SNP set comprises 2 or more SNPs, 3 or more SNPs, 4 or more SNPs, 5 or more SNPs, 6 or more SNPs, 7 or more SNPs, 8 or more SNPs, 9 or more SNPs, 10 or more SNPs, 11 or more SNPs, 12 or more SNPs, 13 or more SNPs, 14 or more SNPs, 15 or more SNPs, 16 or more SNPs, 17 or more SNPs, 18 or more SNPs, 19 or more SNPs, or 20 or more SNPs selected from any SNP listed in Figures 1-17 and Tables 1-10. On the other hand, the SNP set comprises 1-19 species of SNPs selected from Table 6. On the other hand, the SNP set comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10. On the other hand, the SNP set comprises an ITGAM SNP selected from any ITGAM SNP listed in Tables 7-10. In another aspect, the SNP set further comprises a BLK SNP selected from any of the BLK SNPs listed in Tables 7 to 10. In another aspect, the SNP set comprises one or more SNPs selected from the group consisting of rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

In another aspect, the present invention provides a set of SNPs comprising a genetic signature indicative of lupus, wherein the set of SNPs comprises one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

The present invention relates to the following items.

1. A method for assessing whether a subject is at risk for developing lupus, the method comprising: detecting the presence of a genetic signature indicating risk for developing lupus in a biological sample obtained from the subject, wherein the genetic signature comprises a set of one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

2. The method of claim 1 , wherein the set of SNPs comprises approximately 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

3. The method of claim 1 , wherein the set of SNPs comprises 2 or more SNPs, 3 or more SNPs, 4 or more SNPs, 5 or more SNPs, 6 or more SNPs, 7 or more SNPs, 8 or more SNPs, 9 or more SNPs, 10 or more SNPs, 11 or more SNPs, 12 or more SNPs, 13 or more SNPs, 14 or more SNPs, 15 or more SNPs, 16 or more SNPs, 17 or more SNPs, 18 or more SNPs, 19 or more SNPs, or 20 or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

4. The method of claim 1 , wherein the SNP set comprises 1-19 SNPs selected from Table 6.

5. The method of item 1, wherein the SNP set comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10.

6. The method of claim 1 , wherein the set of SNPs comprises an ITGAMSNP selected from any of the ITGAM SNPs listed in Tables 7-10.

7. The method of claim 6, wherein the SNP set further comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10.

8. The method of claim 1 , wherein the set of SNPs comprises one or more SNPs selected from the group consisting of rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

9. A method for diagnosing lupus in a subject, the method comprising: detecting the presence of a genetic signature indicative of lupus in a biological sample obtained from the subject, wherein the genetic signature comprises a set of one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

10. An isolated polynucleotide comprising (a) a PRO-associated polynucleotide or a fragment thereof having a length of at least about 10 nucleotides, wherein the PRO-associated polynucleotide or fragment thereof comprises a genetic variation at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, or (b) the complement of (a).

11. The isolated polynucleotide of claim 10, wherein the genetic variation is in genomic DNA encoding a gene (or its regulatory region) comprising a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

12. The isolated polynucleotide of claim 11, wherein the SNP is in a non-coding region of the gene.

13. The isolated polynucleotide of claim 11, wherein the SNP is in the coding region of the gene.

14. The isolated polynucleotide of claim 10, wherein the isolated polynucleotide is a primer.

15. The isolated polynucleotide of claim 10, wherein the isolated polynucleotide is an oligonucleotide.

16. An oligonucleotide that is (a) an allele-specific oligonucleotide that hybridizes to a region of a PRO-associated polynucleotide that contains a genetic variation at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, or (b) the complement of (a).

17. The oligonucleotide of item 16, wherein the SNP is in genomic DNA encoding a gene (or its regulatory region) comprising a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

18. The oligonucleotide of claim 17, wherein the SNP is in a non-coding region of the gene.

19. The oligonucleotide of claim 17, wherein the SNP is in the coding region of the gene.

20. The oligonucleotide of claim 16, wherein the allele-specific oligonucleotide is an allele-specific primer.

21. A kit comprising the oligonucleotide of item 16 and optionally at least one enzyme.

22. The kit of claim 21 , wherein the at least one enzyme is a polymerase.

23. The kit of claim 21 , wherein the at least one enzyme is a ligase.

24. A microarray comprising the oligonucleotides of item 16.

25. A method for detecting the presence of a variation in a PRO-associated polynucleotide at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10, the method comprising (a) contacting a nucleic acid suspected of containing the variation with an allele-specific oligonucleotide specific for the variation under conditions suitable for hybridization of the allele-specific oligonucleotide to the nucleic acid; and (b) detecting the presence of allele-specific hybridization.

26. The method of claim 25, wherein the variation comprises a SNP as listed in Figures 1-17 and Tables 1-10.

27. A method for amplifying a nucleic acid comprising a variation in a PRO-associated polynucleotide at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10, the method comprising (a) contacting the nucleic acid with a primer that hybridizes to the nucleic acid at the 3' sequence of the variation, and (b) extending the primer to generate an amplification product comprising the variation.

28. The method of claim 27, wherein the variation comprises a SNP as listed in Figure 1-17 and Table 1-10.

29. A method for determining the genotype of a biological sample from a mammal, the method comprising detecting in nucleic acid material derived from the biological sample the presence or absence of a variation in a PRO-associated polynucleotide at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10.

30. The method of claim 29, wherein the variation comprises a SNP as listed in Figure 1-17 and Table 1-10.

31. The method of claim 29, wherein the biological sample is known or suspected to contain a PRO or PRO-related polynucleotide comprising the variation.

32. The method of claim 29, wherein the biological sample is diseased tissue.

33. The method of claim 29, wherein the detection comprises performing a method selected from the group consisting of: a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; a 5' nuclease assay; an assay using a molecular beacon; and an oligonucleotide ligation assay.

34. A method for subclassifying lupus in a mammal, the method comprising detecting in a biological sample derived from the mammal the presence of a variation in a PRO-associated polynucleotide at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10, wherein the biological sample is known or suspected to contain a PRO or PRO-associated polynucleotide comprising the variation.

35. The method of claim 34, wherein the variation is a genetic variation.

36. The method of claim 35, wherein the variation comprises a SNP as listed in Figures 1-17 and Tables 1-10.

37. The method of claim 34, wherein the detection comprises performing a method selected from the group consisting of: a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; a 5' nuclease assay; an assay using a molecular beacon; and an oligonucleotide ligation assay.

38. A method for predicting whether a subject with lupus will respond to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in a PRO-associated polynucleotide at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP) as listed in Figures 1-17 and Tables 1-10, wherein the presence of the variation indicates that the subject will respond to the therapeutic agent.

39. The method of claim 38, wherein the variation is a genetic variation.

40. The method of claim 39, wherein the variation comprises a SNP as listed in Figure 1-17 and Table 1-10.

41. A method of diagnosing or prognosing lupus in a subject, the method comprising detecting the presence of a variation in a PRO or PRO-associated polynucleotide derived from a biological sample obtained from the subject, wherein:

(a) the biological sample is known or suspected to contain a PRO or PRO-related polynucleotide comprising the variation;

(b) the variation comprises a SNP listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP listed in Figures 1-17 and Tables 1-10; and

(c) the presence of the variant is diagnostic or prognostic for lupus in the subject.

42. A method of aiding in the diagnosis or prognosis of lupus in a subject, the method comprising detecting the presence of a variation in a PRO or PRO-associated polynucleotide derived from a biological sample obtained from the subject, wherein:

(a) the biological sample is known or suspected to contain a PRO or PRO-related polynucleotide comprising the variation;

(b) the variation comprises a SNP listed in Figures 1-17 and Tables 1-10, or is located at a nucleotide position corresponding to a SNP listed in Figures 1-17 and Tables 1-10; and

(c) the presence of the variant is diagnostic or prognostic of a lupus condition or symptom in the subject.

43. The method of item 41 or 42, wherein the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region.

44. The method of claim 43, wherein the linkage disequilibrium region is one of the regions listed in Figures 1-17 and Tables 1-10.

45. The method of item 41 or 42, wherein the variation is in genomic DNA encoding a gene or its regulatory region, and wherein each of the genes or its regulatory region comprises a SNP listed in Figures 1-17 and Tables 1-10.

46. The method of claim 45, wherein the SNP is in a non-coding region of the gene.

47. The method of claim 45, wherein the SNP is in the coding region of the gene.

48. A method for identifying a therapeutic agent that is effective in treating lupus in a subpopulation of patients, the method comprising correlating the efficacy of the agent with the presence of a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) in the subpopulation of patients, wherein the SNP is one of the SNPs listed in Figures 1-17 and Tables 1-10, thereby identifying the agent as effective in treating lupus in the subpopulation of patients.

49. A method of treating a lupus condition in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent effective to treat the condition.

50. A method of treating a subject having a lupus condition, the method comprising administering to the subject a therapeutic agent effective to treat the condition in a subject having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

51. A method of treating a subject having a lupus condition, the method comprising administering to the subject a therapeutic agent shown in at least one clinical study to be effective in treating the condition, wherein the agent is administered to at least 5 human subjects in the clinical study, each of the human subjects having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

52. The method of claim 51, wherein the at least 5 subjects have a total of 2 or more different SNPs across the group of at least 5 subjects.

53. The method of claim 51 , wherein the at least 5 subjects have the same SNP across the entire group of at least 5 subjects.

54. A method of treating a lupus subject from a specific subpopulation of lupus patients, wherein the subpopulation is characterized at least in part by association with a genetic variation at a nucleotide position corresponding to a SNP listed in Figures 1-17 and Tables 1-10, and wherein the method comprises administering to the subject an effective amount of a therapeutic agent approved as a therapeutic agent for the subpopulation.

55. The method of claim 54, wherein the subpopulation suffers from lupus nephritis.

56. The method of claim 54, wherein the subpopulation is female.

57. The method of claim 54, wherein the subpopulation is of European descent.

58. A method comprising manufacturing a lupus therapeutic and packaging the agent with instructions for administering the agent to a subject having or suspected of having lupus and having a genetic variation at a position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

59. A method of detailing a therapeutic agent for use in a subpopulation of lupus patients, the method comprising providing instructions for administering the therapeutic agent to the subpopulation of patients characterized by genetic variation at positions corresponding to single nucleotide polymorphisms (SNPs) listed in Figures 1-17 and Tables 1-10.

60. A method for marketing a therapeutic agent for use in a subpopulation of lupus patients, the method comprising informing a target audience about the use of the therapeutic agent for treating the subpopulation of patients, wherein the subpopulation of patients is characterized by the presence of a genetic variation in such subpopulation of patients at a position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10.

61. A method for regulating signaling through a B cell receptor in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively regulates signaling through the B cell receptor.

62. A method for regulating Th17 cell differentiation in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively regulates Th17 cell differentiation.

63. A set of SNPs comprising a genetic signature indicative of risk for developing lupus, wherein the set of SNPs comprises one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

64. The SNP set of claim 63, wherein the SNP set comprises approximately 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

65. The SNP set of claim 63, wherein the SNP set comprises one or more SNPs selected from the group consisting of rs9888739, rs13277113, rs7574865, rs2269368, rs6889239, rs2391592, and rs21177770.

66. The SNP set of claim 63, wherein the SNP set comprises 2 or more SNPs, 3 or more SNPs, 4 or more SNPs, 5 or more SNPs, 6 or more SNPs, 7 or more SNPs, 8 or more SNPs, 9 or more SNPs, 10 or more SNPs, 11 or more SNPs, 12 or more SNPs, 13 or more SNPs, 14 or more SNPs, 15 or more SNPs, 16 or more SNPs, 17 or more SNPs, 18 or more SNPs, 19 or more SNPs, or 20 or more SNPs selected from any SNP listed in Figures 1-17 and Tables 1-10.

67. The SNP set of claim 63, wherein the SNP set comprises 1-19 SNPs selected from Table 6.

68. The SNP set of item 63, wherein the SNP set comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10.

69. The SNP set of item 63, wherein the SNP set comprises an ITGAM SNP selected from any ITGAM SNP listed in Tables 7-10.

70. The SNP set of item 69, wherein the SNP set further comprises a BLK SNP selected from any BLK SNP listed in Tables 7-10.

71. The SNP set of claim 63, wherein the SNP set comprises one or more SNPs selected from the group consisting of rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

72. A set of SNPs comprising a genetic signature indicative of lupus, wherein the set of SNPs comprises one or more SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the results of the genome-wide association scan from SLE, identifying 5 major genes. The data represent 502,033 SNP variants typed in 3 sample series (a total of 1311 SLE cases and 3340 controls). Panel A shows a quantile-quantile plot of the observed P value distribution versus the expected null P value distribution. Diamonds represent all P values, while circles represent P values after excluding HLA, IRF5, and STAT4 region variants. Panel B is a graphical representation of -log ₁₀ P values from a combined analysis of chromosomal organization. Panel B does not show other HLA region variants (N=34) with P< ^1x10-13 .

Figure 2 shows that variants of interest from the BLK/C8orf13 region are associated with expression levels in transformed B cells. A) Shows -log ₁₀ P values from the BLK/C8orf13 region. The color of the diamond indicates the r ² association with rs13277113. All RefSeq genes in this region are shown above the plot, which shows the LD within the region, as determined by analysis of a control chromosome. Notably, this region of interest on chromosome 8 is located within a common polymorphic 4.2 Mb intrachromosomal inversion (see, e.g., Giglio et al. Am J Hum Genet 2001;68(4):874-83 and Sugawara et al. Genomics 2003;82(2):238-44) and is associated with a rare low level of extended LD across this region, as shown. However, the association of BLK/C8orf13 with SLE is independent of this inversion. Shown is the expression of BLK (B) and C8orf13 (C) in transformed B cell lines from 210 unrelated healthy CEU HapMap founders stratified by genotype at rs13277113. The significance of differential expression was determined using an unpaired Student's T test.

Figure 3 shows that variants within the ITGAM/ITGAX locus are associated with SLE. Panel A shows the -log ₁₀ P values from the ITGAM/ITGAX region. The color of the diamond indicates the r ² association with rs11574637. All RefSeq genes in the region are shown above the plot, which shows the LD in the region, as determined by the control chromosome studied. Panel B depicts the genomic structure of ITGAM, the conserved major protein domain, and the relationship between rs11574637 and the two non-synonymous alleles of ITGAM.

Figure 4 depicts the frequency of clinical features in SLE series 1–3 and Swedish cases.

FIG5 depicts the top 50 loci associated with SLE in a genome-wide scan in 1311 cases and 3340 controls.

FIG6 depicts the expression levels of BLK, C8orf1, and control genes in 210 transformed B cell lines from HapMap individuals.

FIG7 depicts the expression of BLK in transformed B cells from a HapMap population.

Figure 8 depicts the association of variants in the C8orf13/BLK and ITGAM/ITGAX regions with SLE using case/control series.

Figure 9 depicts the association of C8orf13/BLK and ITGAM/ITGAX variants with 11 ACR clinical criteria in SLE series 1-3.

Figure 10 depicts the association of C8orf13/BLK and ITGAM/ITGAX variants with 11 ACR clinical criteria in 521 Swedish SLE cases. In the Swedish sample, 521 cases were examined for association with ACR criteria. Statistical significance was assessed by 2x2 contingency tables and chi-square tests. Calculated P values were not adjusted for multiple testing because the ACR criteria are known to be associated and a simple Bonferroni correction of x = 0.05/11 = 0.0045 may be overly conservative.

FIG11 depicts the formula for combining modified Z scores weighted for series size and adjusted for the residual genomic control inflation factor (λgc). The variance (σ2) was calculated for each series, where p = allele frequency in cases and controls. The combined Z score (Z*) for the three SLE series was calculated, where Z1, Z2,

and Z3 equal the Z-score of the EIGENSTRAT-corrected chi-square based on the association of variants from each series with SLE, and where λ1, λ2, and λ3 are the residual genomic control inflation factors (λgc) after EIGENSTRAT correction for each series.

The following keywords apply to the titles in Figures 12A-17:

Figures 12A and 12B together depict an analysis of a subset of lupus nephritis patients, showing 11 regions containing 20 candidate SNPs that are considered likely to contain at least one risk allele for lupus nephritis. Figures 12C and 12D together provide further characterization of linkage disequilibrium regions, the identities of certain genes within these regions, and the criteria for identifying such genes.

Figure 13A depicts an analysis of a subset of women, showing six individual regions containing nine candidate SNPs that were considered likely to contain at least one risk allele. Figure 13B provides further characterization of linkage disequilibrium regions, the identities of certain genes within these regions, and the criteria used to identify such genes.

Figure 14A depicts the analysis of the main panel, showing 6 individual regions containing 8 candidate SNPs that were considered likely to contain at least 1 risk allele. Figure 14B provides further characterization of linkage disequilibrium regions, certain genes within these regions, and criteria for identifying such genes.

Figure 15 depicts a depiction of linkage disequilibrium regions and SNPs contained therein based on certain data from Figures 12A-12D.

FIG. 16 depicts a depiction of linkage disequilibrium regions and SNPs contained therein based on certain data from FIGs. 13A-13B .

FIG. 17 depicts a depiction of linkage disequilibrium regions and SNPs contained therein based on certain data from FIG. 14A-14B .

Detailed Description of the Invention

The present invention provides accurate, simple, and rapid methods and compositions for identifying lupus and for assessing the risk of developing lupus, which are based at least in part on the identification of one or more genetic variations (e.g., SNPs) that are associated with the presence, subtype, and/or patient subpopulations of lupus with high statistical and biological significance. More specifically, the present invention relates to the identification of unique sets of SNPs associated with lupus and its subtypes, unique combinations of such SNPs, and linkage disequilibrium regions, and the identification of patient subpopulations suffering from lupus and its subtypes.

In some embodiments, the SNPs are related to genes, and may be located in regions between genes or within genes, and more specifically, may be located in coding regions or non-coding regions. Genes associated with the SNPs of the present invention may be associated with unknown genes, or may be associated with known genes (e.g., ITGAM or BLK).

The SNPs identified herein provide targets for the development of therapeutic agents for the diagnosis and treatment of genetically identified lupus patients, including the diagnosis and targeted treatment of a subpopulation of lupus patients that exhibit a unique genetic signature comprising one or more of the SNPs of the present invention. For example, in one embodiment, genes containing the genetic variations identified herein, nucleic acids associated with these genes (e.g., DNA or RNA), and proteins encoded by these genes can be used as targets for the development of therapeutic agents (e.g., small molecule compounds, antibodies, antisense/RNAi agents, etc.) or directly as therapeutic agents for the treatment of lupus (e.g., therapeutic proteins, etc.).

General Technology

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J.Gait, ed., 1984); "Animal Cell Culture" (R.I.Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., ed., 1987, and periodic updates thereof); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994).

The primers, oligonucleotides and polynucleotides used in the present invention can be generated using standard techniques known in the art.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. The following documents provide general guidance for many of the terms used in this application to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd edition, J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th edition, John Wiley & Sons (New York, N.Y. 1992).

I. Definition

For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural, and vice versa. In the event of a conflict between any definitions set forth below and any document incorporated herein by reference, the definitions set forth below shall control.

As used herein, "lupus" or "lupus condition" refers to an autoimmune disease or condition that generally involves antibodies that attack connective tissue. The main form of lupus is a systemic one, systemic lupus erythematosus (SLE), including cutaneous SLE and subacute cutaneous SLE, as well as other types of lupus (including nephritic, extrarenal, encephalitis, pediatric, nonrenal, discoid, and alopecia lupus). Generally speaking, see D'Cruz et al., supra.

The terms "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to nucleotide polymers of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by coupling with a labeling component. Other types of modifications include, for example, "caps," replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as, for example, proteins (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 alkylating agents, those with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. Additionally, any hydroxyl group typically present in a carbohydrate can be replaced with, for example, a phosphonate group, a phosphate group, protected with standard protecting groups, or activated to prepare additional linkages to other nucleotides, or can be coupled to a solid support. The 5' and 3' terminal OH groups can be phosphorylated or replaced with amine or organic capping group modules of 1-20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides can also contain analog forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-oxy-methyl, 2'-oxy-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomers, epimers such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl ribonucleoside. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), wherein R or R' is independently H or a substituted or unsubstituted hydrocarbon group (1-20 carbon atoms), optionally containing an ether (-O-) linkage, an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an araldyl group. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" refers to a short single-stranded polynucleotide having a length of at least about 7 nucleotides and less than about 250 nucleotides. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equal and fully applicable to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of a complementary nucleic acid, typically providing a free 3'-OH group.

The term "PRO" refers to a polypeptide encoded by any gene encoded by a nucleic acid sequence located within a linkage disequilibrium region (LD region), wherein the LD region is determined according to the information listed in Figures 1-17 and Tables 1-10. In one embodiment, the PROs of the present invention do not include polypeptides known in the art to cause lupus. In one embodiment, the PROs of the present invention do not include polypeptides known in the art to be associated with lupus, such as IRF5, or any polypeptide encoded by the genes specified in Tables 5-9 of WO2007/019219. The term "PRO-associated polynucleotide" or "nucleic acid associated with PRO" refers to a nucleic acid molecule comprising a contiguous sequence, wherein the contiguous sequence comprises a position identified herein as exhibiting genetic variation. In one embodiment, the position exhibiting genetic variation is located at the 5' or 3' end of the contiguous sequence. In one embodiment, the position exhibiting genetic variation in the contiguous sequence (in its 5' and/or 3' region) is flanked by one or more nucleotides constituting naturally occurring flanking sequences of the position. In one embodiment, the position exhibiting genetic variation is a position corresponding to a SNP specified in any of Figures 1-17 and Tables 1-10.

The term "genetic variation" or "nucleotide variation" refers to a change in the nucleotide sequence (e.g., an insertion, deletion, inversion, or substitution of one or more nucleotides, such as a single nucleotide polymorphism (SNP)) relative to a reference sequence (e.g., a frequently found and/or wild-type sequence, and/or a major allele sequence). Unless otherwise indicated, the term also encompasses corresponding changes in the complement of the nucleotide sequence. In one embodiment, the genetic variation is a somatic polymorphism. In one embodiment, the genetic variation is a germline polymorphism.

"Single nucleotide polymorphism" or "SNP" refers to a single base position in an RNA or DNA molecule (e.g., a polynucleotide) at which different alleles or variable nucleotides are present in a population. A SNP position (interchangeably referred to herein as SNP, SNP site, SNP locus) is typically preceded or followed by a highly conserved allele sequence (e.g., a sequence that varies in less than 1/100 or 1/1000 members of a population). An individual can be homozygous or heterozygous for the allele at each SNP position.

The term "amino acid variation" refers to a change in the amino acid sequence relative to a reference sequence (eg, an insertion, substitution, or deletion of one or more amino acids, such as an internal deletion or N-terminal or C-terminal truncation).

The term "variation" refers to a nucleotide variation or an amino acid variation.

The terms "genetic variation at a nucleotide position corresponding to a SNP," "nucleotide variation at a nucleotide position corresponding to a SNP," and grammatical variations thereof refer to nucleotide variations in a polynucleotide sequence at the relative corresponding nucleotide position occupied by the SNP in the genome. Unless otherwise indicated, the term also encompasses corresponding variations in the complement of the nucleotide sequence. In some embodiments, the nucleotide variation is at the relative corresponding nucleotide position occupied by the SNP in the genome in a PRO-associated polynucleotide sequence.

The term "linkage disequilibrium region SNP" or "LD region SNP" refers to a SNP present in a specific DNA region, and such region is described with suitable nucleic acid/genomic markers (e.g., coordinates or SNPs). In one embodiment, the LD region is described with a first coordinate (e.g., coordinate A) and a second coordinate (e.g., coordinate B), and both coordinates refer to the same chromosome. In one embodiment, the LD region is described with a first SNP (e.g., SNP A) and a second SNP (e.g., SNP B). Thus, in one embodiment, an LD region SNP refers to a SNP located in a nucleic acid region (e.g., genomic region) ranging from a first coordinate to a second coordinate or from a first SNP to a second SNP. Examples of such LD regions and LD region SNPs are shown in Figures 1-17 and Tables 1-10.

The term "array" or "microarray" refers to an ordered arrangement of hybridizable array elements (preferably polynucleotide probes, such as oligonucleotides) on a substrate. The substrate can be a solid substrate (such as a glass slide) or a semi-solid substrate (such as a nitrocellulose membrane).

The term "amplification" refers to the process of generating one or more copies of a reference nucleic acid sequence or its complement. Amplification can be linear or exponential (e.g., PCR). A "copy" does not necessarily mean perfect sequence complementarity or identity relative to the template sequence. For example, a copy can include nucleotide analogs such as deoxyinosine, deliberately introduced sequence changes (such as those introduced via primers containing sequences that can hybridize to the template but are not completely complementary), and/or sequence errors that occur during the amplification process.

The term "allele-specific oligonucleotide" refers to an oligonucleotide that hybridizes to a region of a target nucleic acid that contains a nucleotide variation (generally, a substitution). "Allele-specific hybridization" means that when the allele-specific oligonucleotide hybridizes to its target nucleic acid, the nucleotides in the allele-specific oligonucleotide specifically base pair with the nucleotide variation. An allele-specific oligonucleotide that is capable of allele-specific hybridization with respect to a particular nucleotide variation is said to be "specific" for that variation.

The term "allele-specific primer" means that the allele-specific oligonucleotide is a primer.

The term "primer extension assay" refers to an assay in which nucleotides are added to a nucleic acid, generating a longer nucleic acid or "extension product" that is detected directly or indirectly. Nucleotides can be added to extend the 5' or 3' end of the nucleic acid.

The term "allele-specific nucleotide incorporation assay" refers to a primer extension assay in which a primer (a) hybridizes to a target nucleic acid at a region 3' or 5' to a nucleotide variation and (b) is extended by a polymerase, thereby incorporating a nucleotide complementary to the nucleotide variation into the extension product.

The term "allele-specific primer extension assay" refers to a primer extension assay in which an allele-specific primer is hybridized to a target nucleic acid and extended.

The term "allele-specific oligonucleotide hybridization assay" refers to an assay in which (a) an allele-specific oligonucleotide hybridizes to a target nucleic acid and (b) the hybridization is detected directly or indirectly.

The term "5' nuclease assay" refers to an assay in which hybridization of an allele-specific oligonucleotide to a target nucleic acid allows for nucleolytic cleavage of the hybridized probe, generating a detectable signal.

The term "assay employing a molecular beacon" refers to an assay in which hybridization of an allele-specific oligonucleotide to a target nucleic acid results in a detectable signal at a level that is higher than the level of detectable signal emitted by the free oligonucleotide.

The term "oligonucleotide ligation assay" refers to an assay in which an allele-specific oligonucleotide and a second oligonucleotide are hybridized adjacent to each other on a target nucleic acid and ligated together (directly or indirectly via intervening nucleotides), and the ligation product is detected directly or indirectly.

Generally, the terms "target sequence," "target nucleic acid," or "target nucleic acid sequence" refer to a polynucleotide sequence of interest in which nucleotide variations are suspected or known to exist, including copies of such target nucleic acids generated by amplification.

As used herein, a subject that is "at risk" of forming lupus may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms before treatment methods described herein. "At risk" means that a subject has one or more risk factors as described herein and known in the art, which are measurable parameters associated with lupus formation. A subject with one or more of these risk factors has a higher probability of forming lupus than a subject without one or more of these risk factors. For example, in some embodiments, a subject that is "at risk" of forming lupus has a genetic tag comprising one or more SNPs listed in Figures 1-17 and Tables 1-10. In another embodiment, a subject that is "at risk" of forming lupus has a genetic tag comprising one or more SNPs listed in Table 6.

The term "detecting" includes any means of detection, including direct and indirect detection.

The term "diagnosis" as used herein refers to the identification or classification of a molecular or pathological state, disease, or condition. For example, "diagnosis" can refer to the identification of a particular type of lupus condition, such as SLE. "Diagnosis" can also refer to the classification of a particular subtype of lupus, for example, by tissue/organ involvement (e.g., lupus nephritis), by molecular signatures (e.g., a subpopulation of patients characterized by genetic variations in a particular gene or nucleic acid region).

The term "aiding diagnosis" is used herein to refer to a method that aids in making a clinical determination of the presence, extent, or other properties of a particular type of lupus symptom or condition. For example, a method that aids in making a lupus diagnosis may include measuring the amount of one or more SNPs or detecting the presence of one or more SNPs in a biological sample from an individual. In another example, a method that aids in making a lupus diagnosis may include measuring the amount of one or more SNPs or detecting the presence of one or more SNPs in a biological sample from an individual.

The term "prognosis" is used herein to refer to the possibility of predicting the symptoms of a disease attributable to an autoimmune disorder, including, for example, recurrence, flaring, and drug resistance of an autoimmune disease such as lupus. The term "prediction" is used herein to refer to the possibility that a patient will have a good or bad response to a drug or a group of drugs. In one embodiment, the prediction relates to the extent of those responses. In one embodiment, the prediction relates to whether the patient will survive or improve after treatment (e.g., treatment with a specific therapeutic agent) and not relapse for a certain period of time and/or the probability of surviving or improving after treatment (e.g., treatment with a specific therapeutic agent) and not relapse for a certain period of time. The prediction methods of the present invention can be used clinically to make treatment decisions and select the most appropriate treatment form for any particular patient. The prediction methods of the present invention are valuable tools for predicting whether a patient is likely to have a good response to a treatment regimen, such as a given treatment regimen, including, for example, administering a given therapeutic agent or combination, surgical intervention, steroid therapy, etc., or for predicting whether a patient is likely to survive long-term after a treatment regimen. The diagnosis of SLE can be based on current American College of Rheumatology (ACR) standards. Active disease can be defined by one British Isles Lupus Activity Group (BILAG) "A" criterion or two BILAG "B" criteria. Some signs, symptoms, or other indicators adapted from: Tan et al. "The Revised Criteria for the Classification of SLE" Arth Rheum 25 (1982) for diagnosing SLE may be malar rash such as a rash on the cheek, a discoid rash, or red raised spots, photosensitivity such as a reaction to sunlight (causing the formation or increase of a rash), oral ulcers such as ulcers in the nose or mouth (usually painless), arthritis such as non-erosive arthritis (arthritis in which the bone around the joints is not destroyed) involving two or more surrounding joints, serositis, pleuritis, or pericarditis, renal disorders such as excessive protein in the urine (greater than 0.5 gm/day or 3+ on a test stick) and/or cellular casts. lupus) (abnormal elements derived from urine and/or leukocytes and/or renal tubular cells), neurological signs, symptoms, or other indicators, (epileptic) seizures (convulsions), and/or psychosis in the absence of medications or metabolic disorders known to cause such effects, and hematological signs, symptoms, or other indicators such as hemolytic anemia or leucopenia (leukocyte count less than 4,000 cells per cubic millimeter) or lymphopenia (less than 1,500 lymphocytes per cubic millimeter) or thrombocytopenia (less than 100,000 platelets per cubic millimeter). Generally, leucopenia and lymphopenia must be detected on two or more occasions. Generally, thrombocytopenia must be detected in the absence of medications known to induce thrombocytopenia. The present invention is not limited to these signs, symptoms, or other indicators of lupus.

As used herein, "treatment" or "treating" refers to clinical intervention that attempts to alter the natural course of the treated individual or treated cells and can be performed before or during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of a disease or its condition or symptoms, alleviating a condition or symptom of a disease, diminishing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, ameliorating or palliating the disease state, and achieving immunity or an improved prognosis. In some embodiments, the methods and compositions of the present invention can be used to attempt to delay the development of a disease or condition.

An "effective amount" refers to an amount effective at the dosage and duration necessary to achieve the desired therapeutic or prophylactic effect. A "therapeutically effective amount" of a therapeutic agent may vary depending on factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount also refers to an amount in which the therapeutic beneficial effects outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount effective at the dosage and duration necessary to achieve the desired prophylactic effect. Typically, but not necessarily, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is administered to a subject prior to the onset of disease or at an early stage of disease.

An "individual," "subject," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In certain embodiments, the mammal is a human.

As used herein, "patient subpopulation" and grammatical variations thereof refer to a subset of patients characterized by one or more unique, measurable and/or identifiable characteristics that distinguish the subset of patients from other subsets in the broader disease category to which it belongs. Such characteristics include disease subcategory (e.g., SLE, lupus nephritis), gender, lifestyle, health history, involved organs/tissues, treatment history, etc. In one embodiment, a patient subpopulation is characterized by a genetic signature, including genetic variations (such as SNPs) in specific nucleotide positions and/or regions.

As used herein, the term "sample" refers to a composition obtained or derived from a subject of interest that contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" and its various variations refer to any sample obtained from a subject of interest that is expected or known to contain cells and/or molecular entities to be characterized.

"Tissue or cell sample" refers to a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or puncture sample; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, or tissue obtained from a known source or believed to be unaffected by the disease or condition for which the methods or compositions of the present invention are being used to identify. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy part of the body of the same subject or patient in whom the disease or condition is being identified using a composition or method of the invention. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy part of the body of an individual who is not the subject or patient in whom the disease or condition is being identified using a composition or method of the invention.

For purposes herein, a "section" of a tissue sample refers to a piece or slice of a tissue sample, such as a thin slice of tissue or cells cut from a tissue sample. It should be understood that multiple sections of a tissue sample can be prepared and analyzed according to the present invention, provided that it is understood that the present invention encompasses methods for using the same section of a tissue sample for both morphological and molecular analysis, or for both protein and nucleic acid analysis.

"Correlate" or "associate" means comparing the performance and/or results of a first analysis or protocol to the performance and/or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol can be used to implement a second analysis or protocol, and/or the results of a first analysis or protocol can be used to decide whether the second analysis or protocol should be implemented. With respect to embodiments of gene expression analysis or protocols, the results of a gene expression analysis or protocol can be used to decide whether a particular treatment protocol should be implemented.

The word "label" as used herein refers to a compound or composition that is coupled or fused, directly or indirectly, to a reagent, such as a nucleic acid probe or antibody, to facilitate detection of the reagent to which it is coupled or fused. The label can be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze chemical alteration of a detectable substrate compound or composition.

"Drug" or "pharmaceutical agent" refers to an active pharmaceutical agent used to treat a disease, disorder, and/or condition. In one embodiment, the disease, disorder, and/or condition is lupus or a symptom or side effect thereof.

As used in accordance with the present invention, the term "increased resistance" to a particular therapeutic agent or treatment option refers to a decreased response to a standard dose of the drug or to a standard treatment regimen.

As used in accordance with the present invention, the term "reduced sensitivity" to a particular therapeutic agent or treatment option refers to a decreased response to a standard dose of the agent or to a standard treatment regimen, wherein the decreased response can be (at least partially) compensated for by increasing the dose of the agent, or increasing the intensity of the treatment.

"Patient response" can be assessed using any endpoint that indicates a benefit to the patient, including but not limited to: (1) inhibition of disease progression to some extent, including slowing and complete arrest; (2) reduction in the number of disease events and/or symptoms; (3) reduction in lesion size; (4) inhibition (i.e., reduction, slowing, or complete arrest) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing, or complete arrest) of disease spread; (6) alleviation of the autoimmune response, which may but need not result in regression or ablation of disease lesions; (7) alleviation to some extent of one or more symptoms associated with the disorder; (8) prolongation of the length of time after treatment in which the patient remains disease-free; and/or (9) reduction in mortality at a given time point after treatment.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely inhibits or neutralizes the biological activity of a polypeptide (such as a PRO), or partially or completely inhibits the transcription or translation of a nucleic acid encoding the polypeptide. Exemplary antagonist molecules include, but are not limited to, antagonistic antibodies, polypeptide fragments, oligopeptides, organic molecules (including small molecules), and antisense nucleic acids.

The term "agonist" is used in the broadest sense and includes any molecule that partially or completely mimics the biological activity of a polypeptide (such as a PRO), or increases the transcription or translation of a nucleic acid encoding the polypeptide. Exemplary agonist molecules include, but are not limited to, agonistic antibodies, polypeptide fragments, oligopeptides, organic molecules (including small molecules), PRO-related polynucleotides, PRO polypeptides, and PRO-Fc fusions.

A "therapeutic agent targeting PRO or PRO-related polynucleotides" refers to any agent that affects the expression and/or activity of PRO or PRO-related polynucleotides, including but not limited to any PRO agonist or antagonist described herein, including such therapeutic agents already known in the art and those developed later.

As used herein, "lupus therapeutic agent," "therapeutic agent effective for treating lupus," and grammatical variations thereof refer to agents that are known, clinically shown, or expected by clinicians to provide a therapeutic benefit in subjects with lupus when provided in an effective amount. In one embodiment, the phrase includes any agent that is marketed by a manufacturer or otherwise used by practicing clinicians as a clinically accepted agent that is expected to provide a therapeutic effect in subjects with lupus when provided in an effective amount. In one embodiment, the lupus therapeutic agent comprises a nonsteroidal anti-inflammatory drug (NSAID), which includes acetylsalicylic acid (e.g., aspirin), ibuprofen (Motrin), naproxen (Naprosyn), indomethacin (Indocin), nabumetone (Relafen), tolmetin (Tolectin), and any other embodiment comprising therapeutically equivalent active ingredients and formulations thereof. In one embodiment, lupus therapeutic agents include acetaminophen (e.g., Tylenol), corticosteroids, or antimalarials (e.g., chloroquine, hydroxychloroquine). In one embodiment, lupus therapeutic agents include immunomodulatory drugs (e.g., azathioprine, cyclophosphamide, methotrexate, cyclosporine). In one embodiment, the lupus therapeutic agent is an anti-B cell agent (e.g., anti-CD20 (e.g., rituximab), anti-CD22), an anti-cytokine agent (e.g., anti-tumor necrosis factor alpha, anti-interleukin 1 receptor (e.g., anakinra), anti-interleukin 10, anti-interleukin 6 receptor, anti-interferon alpha, anti-B lymphocyte stimulator), an inhibitor of costimulation (e.g., anti-CD154, CTLA4-Ig (e.g., abatacept), a regulator of B cell anergy (e.g., LJP 394 (e.g., abetimus). In one embodiment, lupus therapeutics include hormone therapy (e.g., DHEA), and anti-hormonal therapy (e.g., the anti-prolactin agent bromocriptine). In one embodiment, lupus therapeutics are agents that provide immunoadsorption, such as anti-complement factors (e.g., anti-C5a), T cell vaccination, cell transfection with the T cell receptor ζ chain, or peptide therapy (e.g., edratide targeting anti-DNA idiotype).

As used herein, a therapeutic agent that has "marketing authorization" or has been "approved as a therapeutic agent," or grammatical variations thereof, refers to an agent (e.g., in the form of a pharmaceutical formulation, a drug) that is approved, licensed, registered, or authorized by a relevant governmental entity (e.g., a federal, state, or local regulatory agency, department, or bureau) for sale by and/or through and/or under the name of a commercial entity (e.g., a for-profit entity) for the treatment of a particular condition (e.g., lupus) or a subpopulation of patients (e.g., patients with lupus nephritis, patients of a particular race, gender, lifestyle, disease risk profile, etc.). Relevant governmental entities include, for example, the Food and Drug Administration (FDA), the European Medicines Evaluation Agency (EMEA), and their equivalents.

"Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins with similar structural features. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. The latter class of polypeptides is produced at low levels, for example, by the lymphatic system and at elevated levels by myelomas.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). Antibodies can be chimeric, human, humanized and/or affinity matured.

The term "anti-PRO antibody" or "antibody that binds to PRO" refers to an antibody that can bind to PRO with sufficient affinity to make the antibody useful as a diagnostic and/or therapeutic agent targeting PRO. Preferably, the degree of binding of the anti-PRO antibody to an unrelated, non-PRO protein is less than about 10% of the binding of the antibody to PRO, as measured by, for example, radioimmunoassay (RIA). In certain embodiments, the antibody that binds to PRO has a dissociation constant (Kd) of less than or equal to 1 μM, less than or equal to 100 nM, less than or equal to 10 nM, less than or equal to 1 nM, or less than or equal to 0.1 nM. In certain embodiments, the anti-PRO antibody binds to an epitope in PRO that is conserved between PROs from different species.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. The term specifically refers to an antibody whose heavy chains include an Fc region.

"Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, the two-chain Fv species consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. The six CDRs of Fv together give the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the complete binding site.

Fab fragments contain the variable domains of the heavy and light chains, and also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Herein, Fab'-SH is the designation for Fab' in which the cysteine residues of the constant domains bear free thiol groups. F(ab') ₂ antibody fragments were originally generated as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present on a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which contains a heavy chain variable domain (VH) and a light chain variable domain (VL) connected in the same polypeptide chain (VH-VL). By using a linker that is too short to prevent pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404,097; WO 93/1161; Hudson et al. (2003) Nat. Med. 9: 129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9: 129-134.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical, except for possible mutations (e.g., naturally occurring mutations) that may be present in very small amounts. Thus, the modifier "monoclonal" indicates that the antibody is not a feature of a mixture of different antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence is obtained by a process comprising selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be selecting a unique clone from a collection of numerous clones (such as hybridoma clones, phage clones, or recombinant DNA clones). It should be understood that the selected target binding sequence can be further changed, for example, in order to improve affinity for the target, humanize the target binding sequence, increase its yield in cell culture, reduce its immunogenicity in vivo, create multispecific antibodies, etc., and antibodies comprising the altered target binding sequence are also monoclonal antibodies of the present invention. Different from typical polyclonal antibody preparations that comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Beyond their specificity, the advantage of monoclonal antibody preparations is that they are usually not contaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1988; Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, 563-681, Elsevier, N.Y., 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, 563-681, Elsevier, N.Y., 1981), 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6855-9855 (1984)).

The "humanized" form of non-human (e.g., mouse) antibodies refers to a chimeric antibody that minimally comprises a sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody refers to an immunoglobulin in which the hypervariable region residues in the human immunoglobulin (recipient antibody) are replaced with hypervariable region residues of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate with desired specificity, affinity, and/or ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may be included in residues not found in the recipient antibody or the donor antibody. These modifications can be made to further improve the performance of the antibody. Generally speaking, a humanized antibody will comprise at least one, typically two, substantially entire variable domains, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following reviews and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); and Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

"Human antibody" refers to an antibody that comprises an amino acid sequence corresponding to that of an antibody produced by a human and/or has been produced using any of the techniques disclosed herein for producing human antibodies. Such techniques include screening human-derived combinatorial libraries, such as phage display libraries (see, e.g., Marks et al., J. Mol. Biol., 222:581-597 (1991) and Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991)); using human myeloma and mouse-human heteromyeloma cell lines to produce human monoclonal antibodies (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 55-93 (Marcel Dekker, Inc., New York, NY)). York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)); and the production of monoclonal antibodies in transgenic animals (e.g., mice) that are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). This definition of a human antibody specifically excludes humanized antibodies that comprise antigen-binding residues from non-human animals.

An "affinity matured" antibody is one that has one or more alterations in one or more CDRs of the antibody that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody without those alterations. In one embodiment, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and/or framework residues is described in: Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

A "blocking antibody" or "antagonist antibody" refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking antibodies or antagonist antibodies partially or completely inhibit the biological activity of the antigen.

"Small molecule" or "small organic molecule" is defined herein as an organic molecule having a molecular weight of less than about 500 Daltons.

"PRO binding oligopeptide" or "PRO binding oligopeptide" refers to an oligopeptide that is capable of binding to PRO with sufficient affinity to make the oligopeptide useful as a diagnostic and/or therapeutic agent targeting PRO. In certain embodiments, the extent of binding of the PRO binding oligopeptide to an unrelated, non-PRO protein is less than about 10% of the binding of the PRO binding oligopeptide to PRO, as measured by, for example, a surface plasmon resonance assay. In certain embodiments, the PRO binding oligopeptide has a dissociation constant (Kd) of less than or equal to 1 μM, less than or equal to 100 nM, less than or equal to 10 nM, less than or equal to 1 nM, or less than or equal to 0.1 nM.

"PRO binding organic molecule" or "PRO binding organic molecule" refers to an organic molecule that is different from an oligopeptide or antibody as defined herein and that is capable of binding to PRO with sufficient affinity to make the organic molecule useful as a diagnostic and/or therapeutic agent targeting PRO. In certain embodiments, the extent of binding of the PRO binding organic molecule to an unrelated, non-PRO protein is less than about 10% of the binding of the PRO binding organic molecule to PRO, as measured by, for example, surface plasmon resonance assays. In certain embodiments, the PRO binding organic molecule has a dissociation constant (Kd) of less than or equal to 1 μM, less than or equal to 100 nM, less than or equal to 10 nM, less than or equal to 1 nM, or less than or equal to 0.1 nM.

The dissociation constant (Kd) of any molecule binding to a target polypeptide can be conveniently measured using surface plasmon resonance assays. This assay can be performed on a BIAcore ^™ -2000 or BIAcore ^™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized target polypeptide CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The target polypeptide was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/min until approximately 10 response units (RU) of the coupled protein were obtained. Following injection of the target polypeptide, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of the binding molecules (0.78 nM to 500 nM) in PBS containing 0.05% Tween-20 (PBST) were injected at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k _on ) and dissociation rates (k _off ) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio k _off /k _on . See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293: 865-881. If the on-rate of the antibody exceeds 10 ⁶ M ^-1 s ^-1 according to the surface plasmon resonance assay above, the on-rate can be determined using a fluorescence quenching technique by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

"Liposome" refers to a small vesicle composed of various types of lipids, phospholipids and/or surfactants that can be used to deliver agents (such as drugs) to mammals. Similar to the lipid arrangement of biological membranes, the components of liposomes are usually arranged in a bilayer form.

The word "label" as used herein refers to a detectable compound or composition. The label can be detectable by itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, can catalyze the chemical alteration of a substrate compound or composition that produces a detectable product. Radionuclides that can serve as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109.

An "isolated" biological molecule such as a nucleic acid, polypeptide, or antibody is one that has been identified and separated and/or recovered from a component of its natural environment.

Reference herein to "about" a value or parameter includes (and describes) embodiments involving that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

It is understood that the aspects and embodiments of the invention described herein include "consisting of" and/or "consisting essentially of" the aspects and embodiments.

II. General Techniques for Practicing the Compositions and Methods of the Invention

Nucleotide variants associated with lupus are provided herein. These variants provide biomarkers for lupus and/or predispose to or contribute to the development, persistence, and/or progression of lupus. Thus, the invention disclosed herein is useful in a variety of contexts, for example, in methods and compositions related to lupus diagnosis and treatment.

Detection of genetic variations

The nucleic acid according to any of the above methods can be genomic DNA; RNA transcribed from genomic DNA; or cDNA generated from RNA. The nucleic acid can be derived from a vertebrate, such as a mammal. A nucleic acid is said to be "derived from" a particular source if it is obtained directly from the source, or if it is a copy of a nucleic acid found in the source.

Nucleic acids include copies of nucleic acids, such as copies derived from amplification. Amplification may be desirable in some cases, for example, to obtain a desired amount of material to detect variations. For example, PRO-associated polynucleotides or portions thereof can be amplified from nucleic acid material. The amplicon can then be subjected to a variation detection method (such as those described below) to determine whether a variation is present in the amplicon.

Variations can be detected by certain methods known to those skilled in the art. Such methods include, but are not limited to, DNA sequencing; primer extension assays, including allele-specific nucleotide incorporation assays and allele-specific primer extension assays (e.g., allele-specific PCR, allele-specific ligation chain reaction (LCR), and gap-LCR); allele-specific oligonucleotide hybridization assays (e.g., oligonucleotide ligation assays); cleavage protection assays, in which protection from the action of cleavage agents is used to detect mismatched bases in nucleic acid duplexes; analysis of MutS protein binding; electrophoretic analysis comparing the mobility of variant and wild-type nucleic acid molecules; denaturing gradient gel electrophoresis (DGGE, as in, e.g., Myers et al. (1985) Nature 313:495); analysis of RNase cleavage at mismatched base pairs; analysis of chemical or enzymatic cleavage of heteroduplex DNA; mass spectrometry (e.g., MALDI-TOF); genetic bit analysis (GBA); 5' nuclease assays (e.g.,); and assays using molecular beacons. Some of these methods are discussed in more detail below.

Detection of variations in target nucleic acids can be achieved by molecular cloning and sequencing of target nucleic acids using techniques known in the art. Alternatively, amplification techniques such as polymerase chain reaction (PCR) can be used to amplify target nucleic acid sequences directly from genomic DNA preparations from tumor tissue. The nucleic acid sequence of the amplified sequence can then be determined, and variations can be identified therefrom. Amplification techniques are well known in the art, for example, polymerase chain reaction is described in Saiki et al., Science 239:487, 1988; U.S. Patent Nos. 4,683,203 and 4,683,195.

The target nucleic acid sequence can also be amplified using a ligase chain reaction (which is known in the art). See, for example, Wu et al., Genomics 4:560-569 (1989). In addition, a technique called allele-specific PCR can also be used to detect variations (e.g., substitutions). See, for example, Ruano and Kidd (1989) Nucleic Acids Research 17:8392; McClay et al. (2002) Analytical Biochem. 301:200-206. In certain embodiments of this technology, allele-specific primers are used, wherein the 3' end nucleotides of the primers are complementary to the specific variation in the target nucleic acid (i.e., capable of specific base pairing with the specific variation in the target nucleic acid). If the specific variation is not present, no amplified product is observed. The Amplification Refractory Mutation System (ARMS) can also be used to detect variations (e.g., substitutions). ARMS is described in, for example, European Patent Application Publication No. 0332435, and Newton et al., Nucleic Acids Research, 17:7, 1989.

Other methods useful for detecting variations (e.g., substitutions) include, but are not limited to, (1) allele-specific nucleotide incorporation assays, such as single base extension assays (see, e.g., Chen et al. (2000) Genome Res. 10:549-557; Fan et al. (2000) Genome Res. 10:853-860; Pastinen et al. (1997) Genome Res. 7:606-614; and Ye et al. (2001) Hum. Mut. 17:305-316); (2) allele-specific primer extension assays (see, e.g., Ye et al. (2001) Hum. Mut. 17:305-316; and Shen et al. Genetic Engineering News, Vol. 23, March 15, 2003), including allele-specific PCR; (3) 5′ nuclease assays (see, e.g., De La Roche-Posay, et al., Genetic Engineering News, Vol. 23, March 15, 2003), including allele-specific PCR; Vega et al. (2002) BioTechniques 32:S48-S54 (describing assays); Ranade et al. (2001) Genome Res. 11:1262-1268; and Shi (2001) Clin. Chem. 47:164-172); (4) assays using molecular beacons (see, e.g., Tyagi et al. (1998) Nature Biotech. 16:49-53; and Mhlanga et al. (2001) Methods 25:463-71); and (5) oligonucleotide ligation assays (see, e.g., Grossman et al. (1994) Nuc. Acids Res. 22:4527-4534; Patent Application Publication No. US 2003/0119004 A1; PCT International Publication No. WO 01/92579 A2; and U.S. Patent No. 6,027,889).

Variations can also be detected by mismatch detection methods. Mismatch refers to a hybridized nucleic acid duplex that is not 100% complementary. Lack of complete complementarity may be due to deletion, insertion, inversion, or substitution. An example of a mismatch detection method is the mismatch repair detection (MRD) assay, which is described in, for example, Faham et al., Proc. Natl Acad. Sci. USA 102: 14717-14722 (2005) and Faham et al., Hum. Mol. Genet. 10: 1657-1664 (2001). Another example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al., Proc. Natl. Acad. Sci. USA, 82: 7575, 1985, and Myers et al., Science 230: 1242, 1985. For example, the method of the present invention may involve the use of a labeled ribonucleic acid probe complementary to a human wild-type target nucleic acid. The riboprobe is annealed (hybridized) together with the target nucleic acid derived from the tissue sample and then digested with the enzyme RNase A that can detect some mispairings in the double-stranded RNA structure. If RNase A detects a mispairing, it cuts at the mispairing site. Thus, when the annealed RNA preparation is separated on an electrophoresis gel matrix, if RNase A has detected and cuts the mispairing, an RNA product smaller than the full-length double-stranded RNA of the riboprobe and mRNA or DNA will be seen. The riboprobe need not be the full length of the target nucleic acid, but can be a part of the target nucleic acid, provided that it covers the position suspected of having a variation.

In a similar manner, DNA probes can be used to detect mismatches, for example, via enzymatic or chemical cleavage. See, for example, Cotton et al., Proc. Natl. Acad. Sci. USA, 85:4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, 72:989, 1975. Alternatively, mismatches can be detected by a shift in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, for example, Cariello, Human Genetics, 42:726, 1988. Using riboprobes or DNA probes, a target nucleic acid suspected of containing a variation can be amplified prior to hybridization. Southern hybridization can also be used to detect changes in target nucleic acids, particularly if the changes are gross rearrangements, such as deletions and insertions.

Restriction fragment length polymorphism (RFLP) probes for target nucleic acid or surrounding marker genes can be used to detect variations, such as insertions or deletions. Insertions and deletions can also be detected by cloning, sequencing, and amplification of target nucleic acids. Single stranded conformation polymorphism (SSCP) analysis can also be used to detect allelic base change variants. See, for example, Orita et al., Proc. Natl. Acad. Sci. USA 86: 2766-2770, 1989, and Genomics, 5: 874-879, 1989.

Compositions of the present invention

The present invention provides isolated polynucleotide compositions comprising a polynucleotide comprising a SNP or a fragment thereof. In one embodiment, the polynucleotide is a PRO-associated polynucleotide.

Specifically, the present invention provides compositions comprising unique SNP sets and/or combinations, which can be used as genetic profiles or labels indicating a subject at risk of developing lupus, or indicating a disease or its symptoms or conditions. The polymorphisms disclosed herein are useful as biomarkers for assessing the risk of developing lupus, and are useful as targets for designing diagnostic reagents. In some embodiments, SNPs are not related to genes. In other embodiments, SNPs are related to genes and can be located in regions between or within genes, and more specifically, can be located in coding regions or non-coding regions. Genes associated with the SNPs of the present invention can be associated with unknown genes, or can be associated with known genes (e.g., ITGAM or BLK).

The SNPs identified herein provide targets for the development of therapeutic agents for the diagnosis and treatment of genetically identified lupus patients, including the diagnosis and targeted treatment of a subpopulation of lupus patients that exhibit a unique genetic signature comprising one or more of the SNPs of the present invention. For example, in one embodiment, genes containing the genetic variations identified herein, nucleic acids associated with these genes (e.g., DNA or RNA), and proteins encoded by these genes can be used as targets for the development of therapeutic agents (e.g., small molecule compounds, antibodies, antisense/RNAi agents, etc.) or directly as therapeutic agents for the treatment of lupus (e.g., therapeutic proteins, etc.).

Thus, in one aspect, the present invention provides a collection of one or more SNPs that form a unique genetic signature for assessing the risk of developing lupus. In one aspect, the unique genetic signature comprises about 1-10, 10-20, 20-30, 30-40, or 40-50 SNPs selected from any of the SNPs listed in Figures 1-17 and Tables 1-10.

On the one hand, the unique genetic signature comprises one or more SNPs, two or more SNPs, three or more SNPs, four or more SNPs, five or more SNPs, six or more SNPs, seven or more SNPs, eight or more SNPs, nine or more SNPs, ten or more SNPs, eleven or more SNPs, twelve or more SNPs, thirteen or more SNPs, fourteen or more SNPs, fifteen or more SNPs, sixteen or more SNPs, seventeen or more SNPs, eighteen or more SNPs, nineteen or more SNPs or twenty or more SNPs selected from any SNP listed in Figures 1-17 and Tables 1-10. On the one hand, the SNPs of the genetic signature are selected from Table 6. In another aspect, the SNP is selected from rs9888739, rs13277113, rs7574865, rs2269368, rs6889239, rs2391592 and rs21177770. In another aspect, the SNP is selected from rs2187668, rs10488631, rs7574865, rs9888739, rs13277113, rs2431697, rs6568431, rs10489265, rs2476601, rs2269368, rs1801274, rs4963128, rs5754217, rs6445975, rs3129860, rs10516487, rs6889239, rs2391592, and rs2177770.

In another embodiment, the invention provides a kind of polynucleotide (such as DNA or RNA) of separation or its fragment, its length is at least about 10 Nucleotide, wherein said polynucleotide or its fragment comprise: a) with the genetic variation of the nucleotide position corresponding to the position of the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10, or (b) the complement of (a).In one embodiment, the polynucleotide of described separation is genomic DNA, and it comprises the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10.In another embodiment, the polynucleotide of described separation is RNA, and it comprises the SNP (SNP) selected from any SNP listed in Figure 1-17 and Table 1-10.

In one embodiment of the invention, genetic variation in the region upstream of the B lymphoid tyrosine kinase (BLK) and C8orf13 transcription start sites (chromosome 8p23.1) is associated with disease risk in both US and Swedish case/control series (rs13277113, OR = 1.39, meta P = 1 x ^10-10 ), and is also associated with altered mRNA levels in B cell lines. In another embodiment, variation in the integrin αM (ITGAM) and integrin αX (ITGAX) regions (chromosome 16p11.2) is associated with SLE in combined samples (rs11574637, OR = 1.33, meta P = 3 x ^10-11 ). In a comprehensive genome-wide association scan in SLE, the inventors have identified, and then confirmed through replication, two novel genetic loci: a) a promoter region allele associated with reduced BLK expression and increased C8orf13 expression and b) a SNP (or variant) in the ITGAM/ITGAX region that is in strong linkage disequilibrium with two common non-synonymous alleles of ITGAM.

In one embodiment, the polynucleotide or fragment thereof is at least about 10 nucleotides long, or at least about 15 nucleotides long, or at least about 20 nucleotides long, or at least about 30 nucleotides long, or at least about 40 nucleotides long, or at least about 50 nucleotides long, or at least about 60 nucleotides long, or at least about 70 nucleotides long, or at least about 80 nucleotides long, or at least about 90 nucleotides long, or at least about 100 nucleotides long, or at least about 110 nucleotides long, or at least about 120 nucleotides long, or at least about 130 nucleotides long, or at least about 140 nucleotides long, or at least about 150 nucleotides long, or at least about 160 nucleotides long. In some embodiments, the fragment or full-length polynucleotide can also include the part or whole natural occurrence flanking region of SNP.In this context, term " about " refers to that described nucleotide sequence length adds or subtracts 10% of described length.

In another embodiment, the sequence of the polynucleotide comprises a genetic variation within a linkage disequilibrium region (e.g., as listed in any of Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene. In another embodiment, the complement of any of the above polynucleotides is provided. In another embodiment, a PRO encoded by any of the above polynucleotides is provided.

In one embodiment, the polynucleotide of separation provided herein is for example detected with radioisotope, fluorescent agent or developer.In another embodiment, the polynucleotide of separation is a primer.In another embodiment, the polynucleotide of separation is an oligonucleotide, for example an allele-specific oligonucleotide.In another embodiment, the oligonucleotide can be for example long 7-60 nucleotide, long 9-45 nucleotide, long 15-30 nucleotide or long 18-25 nucleotide.In another embodiment, the oligonucleotide can be for example PNA, morpholino-phosphoramidate (morpholino-phosphoramidate), LNA or 2 '-alkoxy alkoxy (alkoxyalkoxy).As provided herein, the oligonucleotide is useful as for example hybridization probe for detecting genetic variation.

In one embodiment, the present invention provides a composition comprising a plurality of polynucleotides capable of specifically hybridizing to at least one, two, three, four, or five PRO-associated polynucleotides, or complements of such PRO-associated polynucleotides, each PRO-associated polynucleotide comprising a genetic variation at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the polynucleotides are provided in the form of an array, a gene chip, or a gene collection (e.g., a collection of genes or fragments thereof, provided separately or as a mixture). In another embodiment, allele-specific oligonucleotides are provided that hybridize to a region of a PRO-associated polynucleotide comprising a genetic variation (e.g., a substitution). In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. The allele-specific oligonucleotide comprises a nucleotide that base pairs with the genetic variation when hybridized to the region of the PRO-associated polynucleotide. In another embodiment, the complement of the allele-specific oligonucleotide is provided. In another embodiment, a microarray comprising an allele-specific oligonucleotide or its complement is provided. In another embodiment, the allele-specific oligonucleotide or its complement is an allele-specific primer. In one embodiment, the allele-specific oligonucleotide comprises a genetic variation in a PRO-related polynucleotide sequence, wherein the PRO-related polynucleotide encoding is a PRO encoded by a sequence in a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in the non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene. In another embodiment, the complement of any of the above polynucleotides is provided.

Allele-specific oligonucleotides can be used with control oligonucleotides that are identical to the allele-specific oligonucleotides except that the nucleotide that specifically base pairs with the genetic variation is replaced with a nucleotide that specifically base pairs with the corresponding nucleotide present in the wild-type PRO-associated polynucleotide. Such oligonucleotides can be used in competitive binding assays under hybridization conditions that allow the oligonucleotide to distinguish between PRO-associated polynucleotides comprising the genetic variation and PRO-associated polynucleotides comprising the corresponding wild-type nucleotide.

Using routine methods based on, for example, the length and base composition of the oligonucleotides, one skilled in the art can derive suitable hybridization conditions under which (a) the allele-specific oligonucleotide preferentially binds to a PRO-associated polynucleotide comprising a genetic variation relative to a wild-type PRO-associated polynucleotide, and (b) the control oligonucleotide preferentially binds to a wild-type PRO-associated polynucleotide relative to a PRO-associated polynucleotide comprising a genetic variation. Exemplary conditions include high stringency conditions, such as hybridization conditions of 5x standard saline phosphate EDTA (SSPE) and 0.5% _NaDodSO4 (SDS) at 55°C, followed by a wash with 2x SSPE and 0.1% SDS at 55°C or room temperature. In another embodiment, a binding agent is provided that preferentially binds to a PRO comprising an amino acid variation (relative to a wild-type PRO). In one embodiment, the amino acid variation is any genetic variation derived from a nucleotide position corresponding to a SNP listed in any of Figures 1-17 and Tables 1-10 (including, for example, any specific SNP in any of these Figures or Tables). In another embodiment, the binding agent is an antibody.

How to use

The present invention also provides various compositions suitable for use in practicing the methods of the present invention. In one embodiment, the present invention comprises at least one nucleic acid molecule useful for detecting one or more genetic variations as disclosed in Figures 1-17 and Tables 1-10. Such nucleic acid molecules can be used in the methods of the present invention, for example, to detect, measure, and treat lupus. In some embodiments, the nucleic acid molecule is attached to a solid substrate as described herein.

In another embodiment, the invention provides an array that can be used in the method of the present invention. In one embodiment, the array of the present invention comprises an individual or set of nucleic acid molecules useful for detecting one or more genetic variations. For example, the array of the present invention can comprise a series of discretely placed allele-specific oligonucleotide individuals or allele-specific oligonucleotide sets. Several technologies for attaching nucleic acids to solid substrates such as glass slides are well known in the art. One method is to incorporate modified bases or analogs containing reactive modules (such as amine groups, derivatives of amine groups, or another group with a positive charge) that can be attached to a solid substrate into the synthesized nucleic acid molecules. The synthesized product is then contacted with a solid substrate (such as a glass slide) coated with an aldehyde or other reactive group. Aldehydes or other reactive groups will form covalent bonds with the reactive modules on the amplified product, and the amplified product will become covalently attached to the glass slide. Other methods (such as those using aminopropylsilane (amino propryl silican) surface chemistry) are also known in the art.

Can use certain methods known to those skilled in the art to obtain biological samples according to any of the above methods. Biological samples can be obtained from vertebrates (particularly mammals). Tissue biopsy is usually used to obtain representative fragments of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or fluid known or believed to contain tumor cells of interest. For example, samples of lung cancer damage can be obtained by resection, bronchoscopy, fine needle aspiration (fine needle aspiration), bronchial brushing or from sputum, pleural fluid or blood. The variation in the target nucleic acid (or encoded polypeptide) can be detected from the tumor sample or from other body samples (such as urine, sputum or serum). (Cancer cells fall off from the tumor and appear in such body samples.) By screening such body samples, simple early diagnosis can be achieved for diseases such as cancer. In addition, the variation in the target nucleic acid (or encoded polypeptide) can be more easily monitored by testing such body samples for the variation in the target nucleic acid (or encoded polypeptide). In addition, methods for enriching tumor cells in tissue preparations are known in the art. For example, the tissue can be separated from paraffin or cryostat sections. Cancer cells can also be separated from normal cells by flow cytometry or laser capture microdissection.

After determining that a subject, or a tissue or cell sample, contains a genetic variation disclosed herein, an effective amount of a suitable lupus therapeutic agent can be administered to the subject to treat the lupus condition in the subject. Diagnosis of the various pathological conditions described herein in mammals can be performed by a skilled practitioner. Diagnostic techniques are available in the art that allow, for example, diagnosis or detection of lupus in mammals.

Lupus therapeutic agents can be administered according to known methods, such as intravenous administration (such as bolus injection or continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Optionally, administration can be carried out via mini-pump infusion using a variety of commercially available devices.

The effective dosage and schedule for administering a lupus therapeutic agent can be determined empirically, and making such determinations is within the skill of the art. Single or multiple doses may be employed. For example, an effective dosage or effective amount of an interferon inhibitor used alone may range from about 1 mg/kg to about 100 mg/kg of body weight or more per day. Interspecies calibration of dosages may be performed in a manner known in the art, such as disclosed in Mordenti et al., Pharmaceut. Res., 8:1351 (1991).

When lupus therapeutics are administered in vivo, normal dosage amounts can vary from about 10 ng/kg per day up to 100 mg/kg of mammal body weight or more, preferably about 1 μg/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature; see, for example, U.S. Patent Nos. 4,657,760; 5,206,344; or 5,225,212. Different formulations are expected to be effective for different therapeutic compounds and different conditions, and, for example, administration targeted to one organ or tissue may necessitate a different mode of delivery than administration targeted to another organ or tissue.

It is contemplated that additional therapies may also be employed in the methods. The one or more additional therapies may include, but are not limited to, administration of steroids and other standard of care regimens for the condition in question. It is contemplated that such additional therapies may be employed in a separate pharmaceutical formulation from, for example, the targeted lupus therapeutic agent.

The present invention also provides a method for detecting the presence of lupus by detecting a variation in a PRO or PRO-related polynucleotide derived from a biological sample. In one embodiment, the biological sample is obtained from a mammal suspected of having lupus.

The present invention also provides a method for determining the genotype of a biological sample by detecting the presence of a genetic variation in a PRO-associated polynucleotide derived from the biological sample. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In another embodiment, the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene. In another embodiment, the biological sample is known to contain or is suspected of containing a PRO or PRO-associated polynucleotide comprising the variation. In another embodiment, the biological sample is a cell line, such as a primary or immortalized cell line. In one such embodiment, genotyping provides a basis for classifying or subclassifying a disease.

The present invention also provides a method for identifying cells known to contain or suspected of containing a PRO or PRO-related polynucleotide comprising a variation in a biological sample from a mammal, by detecting a variation in a PRO or PRO-related polynucleotide derived from a cell of the biological sample. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In another embodiment, the PRO-related polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The present invention also provides a method for diagnosing lupus in a mammal, which is achieved by detecting the presence of a variation in a PRO or PRO-related polynucleotide derived from a biological sample obtained from the mammal, wherein the biological sample is known to contain or suspected of containing a PRO or PRO-related polynucleotide comprising the variation. The present invention also provides a method for aiding the diagnosis of lupus in a mammal, which is achieved by detecting the presence of a variation in a PRO or PRO-related polynucleotide derived from a biological sample obtained from the mammal, wherein the biological sample is known to contain or suspected of containing a PRO or PRO-related polynucleotide comprising the variation. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In another embodiment, the PRO-related polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene.

Multiple algorithms known in the art and described herein can be used to assess the risk of lupus and the response to therapy. Variants related to phenotype can interact in an additive, allele dose-dependent manner. In some embodiments of the present invention, an algorithm based on a stratification scheme can be used to assess the risk of lupus, the severity of the disease, and the response to therapy. Based on the number of risk alleles carried, lupus cases can be stratified into groups. In one embodiment, the risk allele is defined as the allele enriched in lupus cases relative to the control from the locus. For example, in one embodiment, if a total of 19 alleles from 18 loci are listed, the maximum possible number of risk alleles is equal to 38. As described herein, the lupus cases stratified by the number of risk alleles and the tertiles of the resulting distribution can be measured. The tertiles of the lupus cases can then be examined for the difference in disease severity, risk, and the response to therapy. In another embodiment, a method for predicting whether a subject with lupus will respond to a therapeutic agent targeting a PRO or a PRO-related polynucleotide is provided, which is achieved by determining whether the subject comprises a variation in a PRO or a PRO-related polynucleotide, wherein the presence of a variation in a PRO or a PRO-related polynucleotide indicates that the subject will respond to the therapeutic agent. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In another embodiment, the PRO-related polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The present invention also encompasses methods for detecting the presence of a genetic variation at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10 in a subject or a sample obtained therefrom, which is accomplished by (a) contacting a nucleic acid in the subject or sample with any of the polynucleotides described above under conditions suitable for formation of a hybridization complex between the nucleic acid and the polynucleotide; and (b) detecting whether the polynucleotide undergoes specific base pairing with the nucleic acid at the nucleotide position. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene.

The present invention also provides methods for detecting the presence of a genetic variation in a nucleic acid associated with a PRO, which is accomplished by (a) contacting the nucleic acid with an allele-specific oligonucleotide specific for the genetic variation under conditions suitable for hybridization of the allele-specific oligonucleotide to the nucleic acid; and (b) detecting the presence of allele-specific hybridization. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene. In another embodiment, the allele-specific oligonucleotide is an allele-specific primer.

The present invention also provides a method for assessing a subject's predisposition to lupus, which is achieved by detecting the presence of a variation in a PRO or a PRO-related polynucleotide in the subject, wherein the presence of a variation in a PRO or a PRO-related polynucleotide indicates that the subject has a predisposition to lupus. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In another embodiment, the PRO-related polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The present invention also provides a method for subclassifying lupus in a mammal, the method comprising detecting in a biological sample derived from the mammal the presence of a variation at a nucleotide position corresponding to a position of a single nucleotide polymorphism (SNP) as listed in any of Figures 1-17 and Tables 1-10, wherein the biological sample is known to contain or is suspected of containing a PRO or PRO-associated polynucleotide comprising the variation. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation comprises a SNP as listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-associated polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP as listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene. In one embodiment, the subcategories are characterized by tissue/organ involvement (eg, lupus nephritis), sex, and/or ethnicity.

In one embodiment of the detection method of the present invention, the detection comprises performing a method selected from the group consisting of: a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; a 5' nuclease assay; an assay using a molecular beacon; and an oligonucleotide ligation assay.

The present invention also provides a method for identifying a therapeutic agent for effectively treating lupus in a patient subpopulation, the method comprising associating the efficacy of the agent with the presence of a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) in the patient subpopulation, wherein the SNP is one of the SNPs listed in Figures 1-17 and Tables 1-10, thereby identifying the agent as effectively treating lupus in the patient subpopulation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of the SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises the SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-associated polynucleotide encoding is a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises the SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The methods of the present invention provide information useful for determining appropriate clinical intervention steps, if and as appropriate. Thus, in one embodiment of the methods of the present invention, the methods further comprise a clinical intervention step based on the results of the evaluation of the presence or absence of a variation as disclosed herein in a PRO or PRO-related polynucleotide. For example, appropriate intervention may involve preventive and therapeutic steps, or adjustments to any then-current preventive or therapeutic steps, based on the genetic information obtained by the methods of the present invention.

As will be apparent to one skilled in the art, in any of the methods of the invention, while detecting the presence of a variant can positively indicate a characteristic of a disease (e.g., the presence or subtype of a disease), not detecting a variant can also be instructive by providing a contrary characterization of the disease.

The present invention also provides a method for amplifying a nucleic acid comprising a PRO-associated polynucleotide or fragment thereof, wherein the PRO-associated polynucleotide or fragment thereof comprises a genetic variation. In one embodiment, the method comprises (a) contacting the nucleic acid with a primer that hybridizes to a sequence 5' or 3' to the variation, and (b) extending the primer to generate an amplification product comprising the genetic variation. In one embodiment, the method further comprises contacting the amplification product with a second primer that hybridizes to a sequence 5' or 3' to the genetic variation, and extending the second primer to generate a second amplification product. In one such embodiment, the method further comprises amplifying the amplification product and the second amplification product by, for example, a polymerase chain reaction.

In some embodiments, the genetic variation is at a nucleotide position corresponding to the position of the SNP of the present invention. In one such embodiment, the genetic variation comprises the SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-associated polynucleotide encoding is a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises the SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in the non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

Further methods of the present invention include methods of treating lupus in a mammal, comprising the steps of obtaining a tissue or cell sample from the mammal, examining the tissue or cell for the presence of a mutation as disclosed herein, and, after determining the presence of the mutation in the tissue or cell sample, administering to the mammal an effective amount of a suitable therapeutic agent. Optionally, the method comprises administering to the mammal an effective amount of a targeted lupus therapeutic agent and, optionally, a second therapeutic agent (e.g., a steroid, etc.).

In one embodiment, a method for treating lupus is provided, comprising administering to the subject an effective amount of a PRO antagonist or agonist. In one embodiment, the subject exhibits a variation in a PRO or a PRO-related polynucleotide. In one embodiment, the variation is a genetic variation. In one embodiment, the genetic variation is at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one such embodiment, the genetic variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the PRO-related polynucleotide encodes a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The present invention also provides a method for treating a lupus condition in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent effective to treat the condition. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene.

The present invention also provides a method for treating a subject with a lupus condition, the method comprising administering to the subject a therapeutic agent known to be effective in treating the condition in a subject having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene.

The present invention also provides a method for treating a subject with a lupus condition, the method comprising administering to the subject a therapeutic agent previously shown in at least one clinical study to be effective in treating the condition, wherein the agent is administered to at least five human subjects in the clinical study, each of the human subjects having a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene. In one embodiment, the at least 5 subjects have a total of 2 or more different SNPs for the group of at least 5 subjects. In one embodiment, the at least 5 subjects have the same SNP for the entire group of at least 5 subjects.

The present invention also provides a method for treating lupus subjects in a specific lupus patient subpopulation, comprising administering to the subject an effective amount of a therapeutic agent approved as a therapeutic agent for the subpopulation, wherein the subpopulation is characterized at least in part by an association with a genetic variation at a nucleotide position corresponding to a SNP listed in Figures 1-17 and Tables 1-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene. In one embodiment, the subpopulation is of European descent. In one embodiment, the present invention provides a method comprising manufacturing a lupus therapeutic agent, packaging the agent and instructions for administering the agent to a subject who suffers from or is believed to suffer from lupus, and the subject has a genetic variation at a position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene.

The present invention also provides a method for specifying a therapeutic agent used in a subpopulation of lupus patients, the method comprising providing instructions for administering the therapeutic agent to the subpopulation of patients, wherein the subpopulation of patients is characterized by a genetic variation at a position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 5-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in the genomic DNA of a coding gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in a coding region of the gene. In one embodiment, the subpopulation is of European descent.

The present invention also provides a method for marketing a therapeutic agent for use in a subpopulation of lupus patients, the method comprising informing a target audience about the use of the therapeutic agent for treating the subpopulation of patients, the subpopulation of patients being characterized by the presence of a genetic variation in the subpopulation at a position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 5-10. In one embodiment, the variation comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the variation is a SNP in a PRO-associated polynucleotide encoding a PRO encoded by a sequence within a linkage disequilibrium region (e.g., as listed in Figures 1-17 and Tables 1-10). In one embodiment, the genetic variation is in genomic DNA encoding a gene (or its regulatory region), wherein the gene (or its regulatory region) comprises a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the SNP is in a non-coding region of the gene. In one embodiment, the SNP is in the coding region of the gene. In one embodiment of any of the above methods comprising the use of a therapeutic agent, such agent comprises a lupus therapeutic agent as disclosed herein.

The present invention also provides a method for modulating signaling through a B cell receptor in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively modulates signaling through a B cell receptor.

The present invention also provides a method for regulating Th17 cell differentiation in a subject known to have a genetic variation at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) listed in Figures 1-17 and Tables 1-10, the method comprising administering to the subject a therapeutic agent that effectively regulates Th17 cell differentiation.

Reagent test kit

In one embodiment of the present invention, a kit is provided. In one embodiment, the kit comprises any polynucleotide described herein, optionally, and an enzyme. In one embodiment, the enzyme is at least one enzyme selected from the group consisting of a nuclease, a ligase, and a polymerase.

In one embodiment, the present invention provides a kit comprising a composition of the present invention and instructions for use for detecting lupus by determining whether the subject's genome comprises a genetic variation as disclosed herein. In one embodiment, the composition of the present invention comprises a plurality of polynucleotides capable of specifically hybridizing to at least one, two, three, four, or five PRO-associated polynucleotides or complements of such PRO-associated polynucleotides, each PRO-associated polynucleotide comprising a genetic variation at a nucleotide position corresponding to the position of a SNP listed in any of Figures 1-17 and Tables 1-10. In one embodiment, the composition of the present invention comprises a polynucleotide encoding at least a portion of a PRO. In one embodiment, the composition of the present invention comprises a nucleic acid primer capable of binding to at least a portion of a PRO-associated polynucleotide and causing polymerization (e.g., amplification) of at least a portion of the PRO-associated polynucleotide. In one embodiment, the composition of the present invention comprises a binding agent (e.g., a primer, a probe) that specifically detects a PRO-associated polynucleotide (or its complement) (or a corresponding gene product). In one embodiment, the composition of the present invention comprises a binding agent that specifically binds to at least a portion of a PRO. In one embodiment, the invention provides an article of manufacture comprising a therapeutic agent and instructions for using the agent to treat a lupus patient having a variation in a PRO-associated polynucleotide as disclosed herein.

For use in the applications described or proposed above, the present invention also provides a kit or article. Such a kit may comprise a carrier means that is compartmentalized to hold one or more container means, such as vials, tubes, etc., in a tightly constrained manner, each container means containing one of the separate components to be used in the method. For example, one of the container means may be equipped with a detectable label or a detectably labeled probe. Such a probe may be a polynucleotide specific for a PRO-associated polynucleotide. If the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit may also include a container containing nucleotides for amplifying the target nucleic acid sequence and/or a container containing a reporter means (such as a biotin-binding protein, such as avidin or streptavidin) bound to a reporter molecule (such as an enzyme label, a fluorescent label, or a radioisotope label).

Typically, the kits of the invention will include the container described above and one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is for a specific therapeutic or non-therapeutic application and may also indicate directions for in vivo or in vitro use, such as those described above.

The kits of the present invention have many embodiments. A typical embodiment is a kit comprising a container, a label on the container, and a composition contained in the container; wherein the composition comprises a detection agent for PRO or PRO-related polynucleotides, the label on the container indicates that the composition can be used to assess the presence of PRO or PRO-related polynucleotides in at least one type of mammalian cell, and instructions for using the detection agent to assess the presence of PRO or PRO-related polynucleotides in at least one type of mammalian cell. The kit may further comprise a set of instructions and materials for preparing a tissue sample and applying antibodies and probes to the same section of the tissue sample. For example, a kit may comprise a container, a label on the container, and a composition contained in the container; wherein the composition comprises a polynucleotide that can hybridize under stringent conditions with the complement of a PRO-related polynucleotide, the label on the container indicates that the composition can be used to assess the presence of PRO-related polynucleotides in at least one type of mammalian cell, and instructions for using the polynucleotides to assess the presence of PRO-related RNA or DNA in at least one type of mammalian cell.

Other optional components in the kit include one or more buffers (e.g., blocking buffer, washing buffer, substrate buffer, etc.), other reagents such as substrates that can be chemically altered by the enzyme label (e.g., chromogen), epitope retrieval solution, control samples (positive and/or negative controls), control slides, etc.

Sales Methods

The invention herein also encompasses a method for marketing a lupus therapeutic agent or a pharmaceutically acceptable composition thereof, comprising advertising, teaching, and/or detailing to a target audience the use of the agent or pharmaceutical composition thereof for treating a patient or patient population suffering from lupus whose sample obtained from the patient or patient population suffering from lupus shows the presence of a genetic variation as disclosed herein.

Marketing refers to communications, usually paid, made via non-personal media in which the originator is identified and the message is controlled. Marketing for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. The term also includes commercial information announcements appearing in any printed communication media designed to arouse the interest of the general public in order to persuade, inform, publicize, motivate, or otherwise change behavior in a manner that leads to the purchase, support, or endorsement of the inventions herein.

Marketing of the diagnostic methods described herein may be accomplished by any means. Examples of marketing media for delivering such information include television, radio, movies, magazines, newspapers, the Internet, and billboards, including commercials, i.e., information that appears in broadcast media.

The type of marketing used will depend on many factors, such as the nature of the target audience to be communicated, such as hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and relevant jurisdictional laws and regulations governing the marketing of drugs and diagnostics. Marketing can be personalized or customized based on user profiles defined by service interactions and/or other data, such as user demographics and geographic location.

The following are examples of methods and compositions of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example

Bibliographic information regarding references cited (and represented by numbers) in Examples 1-3 is provided at the end of Example 3. Bibliographic information regarding references cited (and represented by numbers) in Examples 4-6 is provided at the end of Example 6.

Example 1: Materials and methods for genome-wide association scans in systemic lupus erythematosus

This example describes the materials and methods used to perform a genome-wide scan for SLE in a large sample of 1311 SLE cases and 3340 controls. Of the 500,000 variants (which capture an estimated 85% of the common variation across the human genome), 24 were genotyped and tested for association with SLE.

Subjects

Genotypes were determined for SLE case samples from the following collections: a) 338 subjects from the Autoimmune Biomarkers Collaborative Network (ABCoN), a repository funded by the NIH/NIAMS. ²⁵ b) 141 subjects from the Multiple Autoimmune Disease Genetics Consortium (MADGC). ²⁶ c) 613 subjects from the Lupus Genetics Project at the University of California San Francisco (UCSF). ^{10, 27} and d) 335 subjects from the University of Pittsburgh Medical Center (UPMC) plus ⁸ samples collected at the Feinstein Institute for Medical Research. All SLE cases self-reported Caucasian ancestry. The diagnosis of SLE (meeting four or more of the American College of Rheumatology (ACR)-defined criteria) was confirmed in all cases by medical record review (94%) or by standard written documentation from the treating rheumatologist ( ⁶ %). Clinical data were reviewed at each institution and tabulated. Figure 4 shows the counts and percentages ^for each of the 11 ACR classification criteria for SLE.

A total of 3583 control samples were examined in the association analysis. As part of this project, 1861 control samples were selected from the New York Cancer Project (NYCP) collection ³⁰ based on self-reported race, sex, and age and then genotyped. In addition, genotype data from 1722 self-reported Caucasian control samples were obtained from the publicly available iControlDB database <www.illumina.com/pages.ilmn?ID=231>.

For replication, genotypes were determined from DNA samples collected independently from 793 Swedish SLE patients (all meeting four or more of the SLE classification criteria as defined by the ACR) and 857 healthy Swedish control individuals. The patients were recruited from the Rheumatology Clinic and University Hospital in Lund, Uppsala, Karolinska ( ^Solna ). The Institutional Review Boards of all collaborating institutions approved these studies, and all participants gave informed consent.

Genotyping

At the Feinstein Institute, control samples (N = 1861) from the NYCP were genotyped on the Illumina HumanHap550 genotyping bead chip (BeadChip) ^31. 1465 samples (464 cases, 1001 controls) were genotyped on the HumanHap550v1 chip, and 1875 samples (1015 cases, 860 controls) were genotyped on the HumanHap550v3 chip. Genotype data from 1452 of these control samples were submitted to iControlDB and made publicly available before publication. Another independent collection of 1722 Caucasian samples genotyped using the HumanHap550 bead chip was obtained from studies 66 and 67 of iControlDB <www.illumina.com/pages.ilmn? ID=231>. Case samples were genotyped in serial phases at the Feinstein Institutes; Series 1 consisted of 479 cases from ABCoN and MADGC, Series 2 included 613 cases from UCSF, and Series 3 consisted of 387 cases from UPMC and the Feinstein Institutes. 545,080 single nucleotide polymorphisms (SNPs) present on both HumanHap550 versions were included in the analysis. Case and control samples with an average call rate of less than 80% across the chip were genotyped again.

In the Swedish replicate collection, SNPs rs11574637 and rs13277113 were genotyped using a homogeneous single base primer extension assay with fluorescence polarization detection performed at the SNP Technology Platform in Uppsala (www.genotyping.se) and reagents from Perkin-Elmer. The genotype call rate in the sample was ⁹⁶ %, and reproducibility was 100%, based on duplicate determinations of 4.6% of genotypes. A three-generation CEPH pedigree with 20 members was genotyped in parallel with the study samples, and no departures from Mendelian inheritance were observed for any of the SNPs.

Data quality filters

Samples with an average call rate of 95% or less (N = 42) or with reported individual sex discordant with observed sex (N = 21) were excluded from the analysis. Each sample was assessed for identity by state (IBS) across the genome and examined for cryptic relatedness. One sample from each pair estimated to be a duplicate or one- to three-degree-of-kin was removed (Pi_hat > 0.10 and Z1 ≥ 0.15, N = 161). Three of these pairs consisted of one case and one control; controls were removed. SNPs with a frequency of less than 1% in cases (N = 21,644) or a HWEP of less than or equal to 1 x ^10-6 in controls (N = 2819) were excluded from the analysis. SNPs with a missingness greater than 5% were removed (N = 6074). SNPs were tested for the probability of a significant difference in missingness between cases and controls; SNPs with a P value of less than or equal to 1 x ^10-5 were removed (N = 7646). SNPs were also tested for batch effects: for example, between the ABCoN sample and all other cases; SNPs with P less than 1×10 ⁻⁹ were removed (N=13).

Population outliers were detected using EIGENSTRAT ^33. Samples that deviated from the mean by more than 6 standard deviations in any of the first 10 principal components were excluded from the analysis (N=141). Data from the 3340 remaining control samples were randomly assigned proportionally to each SLE case series, resulting in a control:case ratio of approximately 2.5 (Table 1).

Series 1 consisted of 411 cases and 1047 controls, Series 2 consisted of 595 cases and 1516 controls, and Series 3 consisted of 305 cases and 777 controls. Overall, 93% of the cases were female, and 62% of the controls were female. No significant differences in allele frequency were noted between males and females.

SNPs with greater than 2% missing data in at least one series and unequal distribution of missing data between cases and controls (differential missingness, P < 1 x ^10-3 ) were removed (N = 3323). SNPs in the pseudoautosomal region of chromosome X (N = 13) did not show a significant association and were excluded from further analysis. Sample and marker filtering was performed using the analysis module within the software program PLINK ^34. For each series, a total of 502,033 SNPs were entered into downstream analysis.

Data Analysis

The association of all SNPs with SLE susceptibility was calculated using a 2x2 contingency table. A genomic control inflation factor ( _λgc ) was then calculated for each sample series. The genomic control inflation factor is a measure based on the median chi-square that reflects whether the bulk of the distribution conforms to the null hypothesis ( _λgc = 1.0). A _λgc value greater than ¹ indicates that the mean chi-square association statistic is elevated due to systematic technical artifacts or the presence of population stratification. After removing low-quality data to minimize technical artifacts, evidence of inflation was recorded for each series: 1.14, 1.18, and 1.11 for series 1, 2, and 3, respectively. To correct for the presence of population stratification, the principal components for each series were calculated in EIGENSTRAT using a subset of SNPs. Remove (5011) SNPs with case MAF less than 2%, (1792) SNPs with control HWE P less than or equal to ^1x10-4 , or (50414) SNPs with missing data greater than 1%, and SNPs in abnormal LD pattern regions due to structural variations on chromosomes 6 (24-36Mb), 8 (8-12Mb), 11 (42-58Mb), and 17 (40-43Mb). Use the remaining 440,202 SNPs to calculate principal components. In each series, the first 4 principal components are used to adjust association statistics for all 502,033 SNPs. After the population stratification adjustment, the λ _gc of each series is close to 1.0 (see Table 1). The modified association statistics (Figures 12 A-12D) of each series are combined by weighted merging of the λ _gc mixed with Z scores. Top 50 loci are shown in Figure 5. Additionally, statistics for all SNPs from each series that passed the QC filter and combined association statistics are summarized in the table (not shown).

To test for heterogeneity between the three case-control studies for the most relevant variants, the Breslow-Day test implemented in PLINK was run for the SNPs with the best association in each of these regions: HLA DRB, STAT4, IRF5, BLK, and ITGAM/ITGAX. No significant heterogeneity was detected (each P greater than 0.2).

The association between each SNP and the subphenotype was calculated for the combined dataset using the combined odds ratio and Mantel-Haenszel heterogeneity test implemented in Stata 9.2 (www.stata.com/) ( FIG9 ). The calculated P values were not adjusted for multiple testing because the ACR criteria are known to be associated and a simple Bonferroni correction of α=0.05/11=0.0045 may be overly conservative.

Gene expression analysis

Gene expression measurements from Epstein-Barr virus-transformed B cell lines from 210 unrelated, healthy HapMap individuals, derived from a publicly available dataset (GENEVAR project, www.sanger.ac.uk/humgen/genevar/), were examined for association with variants significantly associated with SLE ³⁶ . Specifically, the median fluorescence intensity from four measurements of probes for BLK (GI_33469981-S), C8orf13 (GI_32698772-S), ITGAM (GI_6006013-S), ITGAX (GI_34452172-S), ACTB (β-actin, GI_5016088-S), and GAPDH (GI_7669491-S) were examined for 60 U.S. residents of Northern and Western European ancestry (CEU), 60 Yoruba (YRI), 45 Han Chinese individuals from Beijing (CHB), and 45 Japanese individuals from Tokyo (JPT). The expression data for BLK, C8orf13, GAPDH, and ACTB were stratified by rs13277113 genotype (obtained from HapMap (www.hapmap.org)) and the significance of differential expression was measured by a 2-tailed t-test with equal variance. Similarly, the expression data for ITGAM, ITGAX, GAPDH, and ACTB were stratified by genotype at rs11574637 and tested for significance using a t-test. Expression data normalized to a log scale across HapMap populations as described by the GENEVAR project yielded similar results as median fluorescence intensity.

By examining and data mining recently published studies (www.sph.umich.edu/csg/liang/asthma/), associations of BLK and C8orf13 expression with cis-genetic variation were obtained in an independent cohort of 400 EBV-transformed B cells. ³⁷ Specifically, the proxy of rs13277113 (rs4840568) was measured for association with expression levels of BLK (probe 206255_at) and C8orf13 (probe 226614_s_at), as described by Dixon et al. ³⁷ .

Example 2: Identification of C8orf13/BLK and ITGAM/ITGAX as novel susceptibility loci

Genome-wide association analysis

A total of 502,033 polymorphic SNPs were passed through quality control filters on the Illumina chip, and three case-control series were used to test association with SLE in a phased manner (Table 1). Combined association statistics were calculated by adding the Z score converted from the EIGENSTRAT modified chi-square test statistic, weighted for series size, and adjusted for the residual λ _gc of each series (see methods).

Figure 1 shows a comparison of observed meta-analysis P values relative to the null distribution. Significant deviations from the null distribution were observed in the tail of the distribution (Figure 1, panel A, black diamonds), which may indicate the presence of a true positive association. Strong associations with SLE were documented for three established risk loci. In the HLA class II region, rs2187668 was ^a near-perfect predictor of the DRB1*0301 allele and was the variant most strongly associated with SLE in the combined analysis (P= ^3x10-21 ). An additional 157 HLA region SNPs, many of which were associated with the DRB1*0301 allele, had observed P values less than ^5x10-7 (Figure 1, panel B). Strong associations were observed for variants linked to the well-validated risk haplotype of interferon regulatory factor 5 (IRF5) (e.g., rs10488631, P=2x10-11) ^. In addition, an association with STAT4 was observed (rs7574865, P = 9 x ^10-14 ). STAT4 has recently been reported to be associated with both SLE and rheumatoid arthritis. ¹⁰ The SLE dataset presented here overlaps with the ^earlier report and includes an additional 341 cases and 2905 controls not included in the previous analysis. In addition, the P values for the top STAT4 SNPs reported here have been adjusted for population stratification.

Removing variants in HLA, IRF5, and STAT4 from the chi-squared expected versus observed analysis did not eliminate the p-value deviation from the null distribution (Figure 1 Panel A, circles), suggesting the existence of additional SLE loci. As shown in Figure 1 Panel B, multiple SNPs near the B-lymphoid tyrosine kinase (BLK) gene and in the region containing the integrin alpha M (ITGAM) and integrin alpha X (ITGAX) genes were highly associated with SLE in the combined analysis. These genes or regions have not been previously linked to SLE susceptibility.

BLK/C8orf13

Several variants on the short arm of chromosome 8 (8p23.1) are associated with SLE (Fig. 2, Table 2, Fig. 8). Relative to controls, the "A" allele of rs13277113 is highly enriched in U.S. SLE cases (P = ^8x10-8 , combined OR = 1.39, 95% CI = 1.26-1.54). To confirm this initial observation, the types of independent collections of 793 SLE cases and 857 matched controls from Sweden were determined in terms of rs13277113, and a convincing association of the minor "A" allele with SLE was also observed (P = ^3.6x10-4 , OR = 1.33, 95% CI = 1.13-1.55; Table 2). Meta-analysis of rs13277113 using both American and Swedish samples showed P = 1.4×10 ⁻¹⁰ , which exceeded the stringent genome-wide significant association threshold of P < 5×10 ^{−8 39} .

rs13277113 is located between two genes transcribed in opposite directions: BLK (an Src family tyrosine kinase that signals downstream of the B-cell receptor) and C8orf13 (a ubiquitously expressed gene of unknown function) (Figure 2). No known BLK or C8orf13 coding region variants are in linkage disequilibrium (LD) with rs13277113.

Common genetic variation has been shown to be associated with cis-gene expression levels. ^8,36,37,40 To determine whether relevant promoter SNPs could influence BLK and/or C8orf13 mRNA expression, a gene expression dataset generated from Epstein-Barr virus-transformed B lymphocyte cell lines from 210 unrelated HapMap samples was interrogated. ³⁶ Strikingly, the risk "A" allele of rs13277113 was associated with lower BLK mRNA expression levels (Figure 2, Panel B). Homozygotes for the A allele exhibited expression levels approximately 50% lower than those for the G allele, while A/G heterozygotes had intermediate levels. Interestingly, expression of the C8orf13 gene was also associated with the risk haplotype, but in the opposite direction. The A allele of rs13277113 was associated with higher C8orf13 expression in the transformed lines, while the G allele was significantly associated with lower expression (Figure 2, Panel C). A/G heterozygotes again exhibited intermediate expression levels. Based on the genotype at rs13277113, expression of many control mRNAs (e.g., β-actin, GAPDH) was unchanged in the cell lines ( FIG6 ), and consistent allelic differences in BLK expression were observed across all HapMap populations ( FIG7 ). These results were confirmed by analyzing an independent dataset of gene expression and genome-wide SNPs in 400 non-HapMap transformed B cell lines. ³⁷ In this dataset, a marker associated with rs13277113 (rs4840568, r ² =0.77) was associated with both decreased expression of BLK (P=8.9× ^{10 −27} , probe 206255_at) and increased expression of C8orf13 (P=4.6×10 ⁻³⁵ , probe 226614_s_at).

Multiple conserved transcription factor binding sites, including motifs for IRF1, PPARG, and the interferon-stimulated response element, are located in the 5' region of BLK and C8orf13. However, neither rs13277113 nor related variants ( ^r² >0.5) alter known transcription factor binding sites or other known functional nucleic acid motifs. We hypothesize that rs13277113, or variants strongly associated with rs13277113, alter BLK and C8orf13 mRNA expression levels.

ITGAM/ITGAX

Variants within the integrin α-chain gene cluster on chromosome 16 were also significantly associated with SLE (Figure 3, Table 2). A reproducible association of the "C" allele of rs11574637 was observed across three SLE series (P = 5 × ^10-7 , OR = 1.30, 95% CI = 1.17-1.45). Importantly, the "C" allele of rs11574637 showed similar strong enrichment in the Swedish replication series (P = 4 × ^10-7 , OR = 1.59, 95% CI = 1.33-1.91; Table 2), and a meta-analysis showed a combined P = 3 × ^10-11 . We conclude that variants linked to rs11574637 mark a confirmed SLE risk allele and that the ITGAM/ITGAX locus contributes to SLE pathogenesis.

Rs11574637 is part of a large, approximately 150 kb block of associated SNPs that encodes several genes, including the 5' portion of ITGAM and ITGAX (Fig. 3, panel A). Both ITGAM and ITGAX are expressed at detectable levels in EBV-transformed B cells, but rs11574637 is not significantly associated with mRNA expression levels of either gene (data not shown). Of potential interest, SNP rs11574637 is associated with two ITGAM non-synonymous variants. In the control population, the Pro1146Ser variant (rs1143678, P = 2.5 x ^10-5 ) was associated with the disease-associated rs11574637 variant with an ^r2 of 0.85. The "C" allele of rs1143678 and the 1146Ser allele formed haplotypes on 18.2% of control chromosomes; the "C" allele was also present on an additional 2% of haplotypes that lacked the 1146Ser allele. A second nonsynonymous allele (rs1143683, Ala858Val) was not directly genotyped in this study but was highly associated with Pro1146Ser ( ^r² = 0.85 in HapMap CEU). Further studies will be needed to determine whether ITGAM nonsynonymous variants or other alleles underlie the association within the ITGAM/ITGAX region.

Association with clinical features of SLE

Finally, the association between the two top SNPs, rs11574637 (BLK) and rs13277113 (ITGAM), and the presence of each ACR criterion was examined using combined case series 1-3 ( FIG. 9 , and see methods). The strongest association was an inverse correlation between the rs11574637 minor allele and the presence of arthritis, OR=0.73 (95% CI=0.59-0.91, P=0.0045). Both variants were modestly associated with hematologic criteria: rs11574637, OR=1.21 (95% CI=1.00-1.47, P=0.04) and rs13277113, OR=1.23 (95% CI=1.03-1.46, P=0.02). No other significant associations were observed.

discuss

This study describes the results of a comprehensive genome-wide association study conducted in SLE. By studying a large number of SLE cases (1311) and an even larger group of controls (3340), we identified the major alleles that contribute to SLE risk. Strong signals observed in the HLA region, specifically IRF5 and STAT4, served as positive controls for this experiment and confirmed that these loci are among the most important genetic factors in this disease.

The Src family tyrosine kinase BLK is an interesting new candidate gene for SLE. BLK expression is highly restricted to the B lymphocyte lineage. ⁴¹ In mice, BLK expression is first observed in circulating late pro-B cells, continues throughout B cell development, and is subsequently downregulated in plasma B cells. ⁴² BLK knockout mice have no gross phenotype, ⁴³ and functional studies in human B cells have not yet been performed. Without being bound by theory, BLK is one of the tyrosine kinases that transduce signals downstream of the B cell receptor and may have redundant roles in mice, given the lack of a phenotype in knockout animals. There is precedent for significant species differences in the roles of B cell receptor-related kinases. For example, Bruton's tyrosine kinase (BTK) deficiency in humans leads to X-linked agammaglobulinemia and a complete deficiency of B cells. ⁴⁴ However, Btk deficiency in mice is associated with a much milder phenotype, with functionally impaired mature B cell generation. ⁴⁵

Signaling through the B cell receptor is important for establishing the B cell repertoire during B cell development through induction of anergy, deletion, and receptor editing. ^{46, 47} As shown here, the risk allele at BLK is associated with reduced expression of BLK mRNA in transformed B cell lines. Without being limited to theory, altered BLK protein levels may affect tolerance mechanisms in B cells, predisposing individuals to systemic autoimmunity. A similar mechanism has recently been shown for Ly108, one of the major genetic loci in the NZM2410 mouse model of lupus. ⁴⁸ Thus, in one embodiment of the present invention, one skilled in the art can use the information provided herein to assess the effect of risk haplotypes on the expression of the ubiquitously expressed gene C8orf13.

The second locus identified in this scan was ITGAM/ITGAX. Although ITGAX was not excluded based on the strong LD extending into the 5' portion of ITGAX in this region, data suggest that ITGAM may be a relevant gene in this region. ITGAM (also known as CD11b, Mac-1, and complement receptor type 3) is a well-characterized integrin α-chain molecule expressed by various myeloid cell types, including dendritic cells, macrophages, monocytes, and neutrophils. ^49-51 ITGAM forms a heterodimer with ITGB2 (CD18) and mediates adhesion between various cell types in the immune system and between myeloid cells and the endothelium. ⁵² ITGAM-deficient mice show enhanced disease progression and inflammation in several models of autoimmunity, including lupus. ^53-55 Recent data suggest that ITGAM may function normally to suppress Th17 differentiation, ⁵⁶ a pathway that has been linked to the induction of autoimmunity. Interestingly, CD11b expression has been reported to be elevated on neutrophils from patients with active SLE. ⁵⁷ The risk allele of ITGAM and its two highly related nonsynonymous alleles may predispose to altered protein function and/or expression regulation, thereby contributing to systemic autoimmunity.

In summary, our data identify two new susceptibility loci for SLE: BLK/C8orf13 on chromosome 8 and ITGAM/ITGAX on chromosome 16. The most likely candidate genes within these two loci are BLK and ITGAM. The identification of these genes provides important new insights into the genetic basis of SLE and also suggests potential new targets for treatment.

Example 3: Genome-wide association scan in systemic lupus erythematosus (SLE) and identification of new loci associated with SLE

In this embodiment, initial data set is made up of the case and control group from the whole genome association study described in embodiment 1 and embodiment 2 above, and wherein genotype is from IlluminaHumanHap550v1 chip and IlluminaHumanHap550v3 chip.Data set from IlluminaHumanHap550v1 chip is made up of 555352 kinds of SNP in each of 464 cases and 1962 controls.Data set from IlluminaHumanHap550v3 chip is made up of 561466 kinds of SNP in each of 971 cases and 1621 controls.For each data set, quality control filter is applied similarly to the mode described in embodiment 1 and embodiment 2 above.Gained data set from HumanHap550v1 chip is made up of 534523 kinds of SNP in each of 422 cases and 1881 controls. The resulting dataset from the HumanHap550v3 chip consisted of 549,273 SNPs in each of 929 cases and 1,558 controls.

The above-mentioned data set from IlluminaHumanHap550v1 chip is merged with the above-mentioned data set from IlluminaHumanHap550v3 chip.The data set of gained is made up of 564307 kinds of SNP in each of 1351 cases and 3439 parts of controls.This data set is merged with the genotype (553820 kinds of SNP in each of 4527 parts of samples used as a control) from CGEMS breast cancer and prostate cancer research.The data set of gained is made up of 570099 kinds of SNP in each of 1351 cases and 7966 parts of controls.Similarly apply quality control filter with the mode described above in embodiment 1 and embodiment 2.The data set of gained is made up of 446856 kinds of SNP in each of 1351 cases and 7966 parts of controls.

The above dataset was used to impute the genotype probability for each polymorphic CEU SNP in the Phase II HapMap using the program IMPUTE (www.stats.ox.ac.uk/~marchini/software/gwas/impute.html). The recommended effective population size (-Ne 11418) was used.

The association between SLE status and each imputed SNP was calculated using the program SNPTEST (www.stats.ox.ac.uk/~marchini/software/gwas/snptest.html). Population outliers were excluded; they were determined using the program EIGENSTRAT in a manner similar to that described above in Examples 1 and 2. Both additive and general frequentist models were tested.

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Example 4: Genome-wide association scan in 1310 SLE cases and 7859 controls

Methods: Sample information and genotyping of SLE cases and controls

The selection and genotyping of SLE case samples were described previously (1). Briefly, DNA samples from a) 338 subjects from the Autoimmune Biomarker Collaborative Network (ABCoN), a repository funded by the NIH/NIAMS (2), b) 141 subjects from the Multiple Autoimmune Disease Genetics Consortium (MADGC) (3), (ABCON + MADGC = case series 1), c) 613 subjects from the University of California, San Francisco (UCSF) Lupus Genetics Project (4, 5) (case series 2), and d) 335 subjects from the University of Pittsburgh Medical Center (UPMC) (6) plus 8 samples collected at the Feinstein Institutes for Medical Research (case series 3) were genotyped using the Illumina 550K array. All SLE cases were North Americans of European ancestry, as determined by self-report. The diagnosis of SLE (fulfilling four or more American College of Rheumatology (ACR)-defined criteria (7)) was confirmed in all cases by medical record review (94%) or by standard written documentation by the treating rheumatologist (6%). Clinical data for these case series are presented elsewhere (4, 3, 2, 6, 5).

A total of 8147 control samples genotyped using the Illumina 550K array were examined in the association analysis. Three sources (all North Americans of European ancestry) were used for the controls: 1861 samples from the New York Health Project (NYHP) collection (8); 1722 samples from the publicly available iControlDB database (www.illumina.com/pages.ilmn?ID=231); and 4564 samples from the publicly available Cancer Genetics Markers of Susceptibility (CGEMS) project (http://cgems.cancer.gov/). Genotyping of the NYHP samples was previously described (1).

Genotype data quality filters

Sample and SNP filtering was performed using the analysis modules within the software programs PLINK (9) and EIGENSTRAT (10), as described below.

a) SLE cases, NYCP samples, and iControlDB samples

The Illumina 550K SNP array, version 1 (HH550v1), was used to determine the genotypes of 464 cases and 1962 controls, and the Illumina 550K SNP array, version 3 (HH550v3), was used to determine the genotypes of 971 cases and 1621 controls, as described in (1). Samples with reported sex that did not match the observed sex (HH550v1: 10, HH550v3: 11) and samples with >5% missing genotypes (HH550v1: 25, HH550v3: 21) were excluded from the analysis. Cryptic kinship between SLE cases and controls was determined by evaluating the identity of status (IBS) across the entire genome for all possible paired sample combinations. One of each pair of samples that was estimated to be a duplicate or one to three generations of relatives was excluded (Pi_hat≥0.10 and Z1≥0.15; HH550v1:88, HH550v3:73). SNPs with an HWE P of less than or equal to ^1x10-6 in controls (HH550v1:3176, HH550v3:2240) and SNPs with greater than 5% missing data (HH550v1:12605, HH550v3:7137) were removed. SNPs were tested for significant differences in the frequency of missing data between cases and controls, and SNPs with a P of less than or equal to ^1x10-5 in the differential missingness test performed in PLINK were removed (HH550v1:5027, HH550v3:2804). SNPs were also tested for significant allele frequency differences between sexes; all SNPs had a p greater than or equal to ^1x10-9 in the control. The data were checked for the presence of batch effects (e.g., between the ABCoN sample and all other cases), and SNPs with allele frequency differences of less than ^1x10-9 p were excluded (HH550v1:18, HH550v3:10). Variants with heterozygous haploid genotypes were set to deletions (HH550v1:2305, HH550v3:875). In addition, variants with minor allele frequencies less than 0.0001 were removed (HH550v1:97, HH550v3:57).

b) CGEMS samples

The 2287 parts of mammary cancer samples that are separated and 2277 parts of prostate cancer samples are used to analyze the heterozygous haploid genotype.For 2277 parts of prostate cancer samples and 2287 parts of mammary cancer samples that are separated, heterozygous haploid genotype is arranged into disappearance (prostate: 2717, mammary gland: 0).Exclude the sample (prostate: 0, mammary gland: 2) that the sex of report and observed sex do not match and missing data are greater than 5% sample (prostate: 15, mammary gland: 1).To the secret kinship of sample test, as described above, and be that every pair of sample of replica or one to three generations of relatives removes a part (Pi_hat ≥ 0.10 and Z1 ≥ 0.15 from estimation; Prostate: 12, mammary gland: 7).Remove the SNP (prostate: 3254, mammary gland: 2166) that MAF is less than 0.0001.

c) All samples

Additional data quality filters were applied to the combined dataset consisting of all SLE cases and controls. SNPs with greater than 5% missing data (N = 65,421) and samples with greater than 5% missing data (N = 0) were removed. Replicate samples were tested using 957 independent SNPs with a MAF greater than or equal to 0.45, and no replicates were found. SNPs with a HWE P less than or equal to ^1x10-6 in controls (N = 2174) and SNPs with greater than 2% missing data (N = 5522) were removed. SNPs were tested for significant differences in the proportion of missing data between cases and controls and SNPs with excess missing data differences were removed (P ≤ ^1x10-5 , N = 16080). SNPs were tested for significant differences between sexes, and all SNPs had a P greater than or equal to ^1x10-9 in controls. SNPs were also examined for the presence of batch effects; specifically, between CGEMS breast cancer samples and all other controls, and between CGEMS prostate cancer samples and all other controls, and SNPs with P less than ^1x10-9 were removed (N=73). After applying the above quality filters, 480,831 SNPs remained.

EIGENSTRAT was used to test the presence of population outliers for cases and controls. For the purpose of determining the principal components of variation (EIGENSTRAT) to detect population outliers, SNPs with a MAF of less than 2% in cases (N = 16,068), a HWEP of less than or equal to ^1x10-4 in controls (N = 977), or missing data greater than 1% (N = 17,029) were excluded; SNPs in regions with abnormal LD patterns due to structural variation on chromosomes 6 (24-36 Mb), 8 (8-12 Mb), 11 (42-58 Mb), and 17 (40-43 Mb); and SNPs in the pseudo-autosomal region of chromosome X (N = 12). Samples with a deviation from the mean of more than 6 standard deviations in any of the first 10 principal components were removed (N = 148).

The final dataset had 1310 cases, 7859 controls, and 480,831 SNPs, and the genomic control inflation factor (λ _gc ) (11) was 1.06 after applying the above-mentioned data quality filters.

Imputing unobserved genotypes

The extensive linkage disequilibrium present in the human genome allows in some cases the inference of untyped variants with high confidence. IMPUTE (a program for imputing unobserved genotypes in whole-genome case-control studies based on a set of known haplotypes (HapMap Phase II haplotypes, www.hapmap.org)) was used in the analysis (www.stats.ox.ac.uk/~marchini/software/gwas/impute.html).

Imputed GNE cases and controls from NYCP, iDB, and CGEMS

After quality control filters, there were 1310 GNE cases, 3344 NYCP and iDB controls, 4515 CGEMS controls, and 446,856 SNPs. The program IMPUTE (v0.3.1) was run, including CEU haplotypes, legends, and a map file aligned to NCBI Build 35. The effective population size was set to the recommended value of 11418. No strand file was used; strand alignment checking was enabled in IMPUTE. Cases, NYCP and iDB controls, and CGEMS controls were imputed separately, and each chromosome was imputed separately. 2,562,708 SNPs were imputed.

Use SNPTEST (v1.1.3) to carry out association test to both actual and inferred genotypes.For the SNP of determined genotype, use actual genotype. Association test is the Cochran-Armitage test for additive genetic effect, wherein uses "-appropriate" ("-proper") option to fully consider the uncertainty of genotype. Only retain the SNP (2,481,907 kinds of SNP [97%]) with information score more than 0.50 (i.e. frequentist_add_proper_info>0.50).

Results. A non-redundant list of SLE loci associated with SLE in an analysis of 1310 cases and 7859 controls (P < 1 x ^10-5 ) is presented in Table 1. A ranked list was generated by presenting the single variant with the lowest P value in the +/- 100 kb interval from the analysis of 2.3 x ¹⁰⁶ SNPs, as described above.

Table 1: Loci associated with SLE in the analysis of 1310 cases and 7859 controls ( ^P≤1x10-5 ). A rank-ordered list was generated as follows, i.e., showing the single variant with the lowest P value in the +/-100 kb interval, from an analysis of ^2.3x106 SNPs, as described. Shown are SNPs (dbSNP id), chromosomes, positions (base pair positions in human genome Build35), minor allele frequencies in SLE cases and controls, P values from SNPTEST (under an additive model, corrected for imputed accuracy), imputed information scores (assessment of imputed accuracy), and odds ratios (with 95% confidence intervals).

Example 5: Meta-analysis of SLE risk loci reported in GNE association scans

method

Review of SLE literature and criteria for confirmed SLE loci

A total of 16 alleles met one of the criteria described below for confirmed SLE risk loci (Table 2).

1) SLE risk loci with at least 2 independent reports of ^P≤1x10-5 .

The literature was searched for loci with two independent reports of association with P ≤ 1 x 10 ^-5 in non-overlapping SLE cohorts. The literature search represented publications prior to April 2008. Identical variants (or alternatives with r ² > 0.3) showing association with SLE in the same direction of effect were required. A total of seven alleles met this requirement, including HLA-DRB1*0301 (HLA-DR3, (18, 19)), HLA-DRB1*1501 (HLA-DR2, (18, 19)), protein tyrosine phosphatase non-receptor type 22 (PTPN22, (20, 21)), interferon regulatory factor 5 (IRF5, (22, 23)), signal transducer and activator of transcription 4 (STAT4, (5, 21)), B lymphoid tyrosine kinase (BLK, (21, 1)), and integrin alpha M (ITGAM, (1, 24)). The same alleles or the best surrogate (r ² >0.85) from the genome-wide association scan of 1310 SLE cases and 7859 controls described here were imputed into the analysis (Table 2).

2) There is one reported SLE risk locus with ^P≤1x10-5 .

A literature search was performed for SLE risk loci with a reported P≤1×10 ⁻⁵ in one publication up to April 2008, and a total of 18 loci were identified.

At 13 of these loci, the same variant or near-perfect replacement (r ² >0.9) was genotyped in the 1310 SLE cases and 7859 controls genomic scans described above (Table 4). Meta-analysis using the methods described below was performed on these 13 loci, and 8 of these loci achieved P ≤ 5 x 10 ⁻⁸ . Loci that achieved genome-wide significance (marked by single genes within the locus) included: pituitary tumor transforming protein 1 (PTTG1), APG5 autophagy 5-like (ATG5), CTD-binding SR-like protein rA9 (KIAA1542), ubiquitin-conjugating enzyme E2L3 (UBE2L3), PX domain-containing serine/threonine kinase (PXK), Fc fragment low affinity IIa receptor for IgG (FCGR2A), tumor necrosis factor (ligand) superfamily 4 (TNFSF4), and B-cell scaffold protein with ankyrin repeats 1 (BANK1). Variants that achieved genome-wide significance in the meta-analysis were carried forward into the analysis (Table 5, Table 2). For the remaining five loci, no reported variants or near-perfect surrogates ( ^r2 >0.9) were detected in the genome-wide association scan of 1310 SLE cases and 7859 controls (Table 2). However, one variant in interleukin-1 receptor-associated kinase 1 (IRAK1) had an observed P≤1×10 ⁻⁴ and was pushed into the analysis ( Table 1 ).

meta-analysis

Corrected association statistics for each series were combined by summing the Z scores weighted for group size.

Example 6: Summary

Summary of SLE risk loci

Two main approaches were used to identify SLE risk loci: a) an analysis of 1310 SLE cases and 7859 controls, and b) a meta-analysis using previously reported SLE risk loci.

A non-redundant list of variants with strong association with SLE risk (P<1×10 ⁻⁶ ) is provided in Table 6 .

Algorithms for assessing SLE risk and response to therapy

It is known that variants related to phenotype interact in an additive, allele dose-dependent manner (38,39). In an exemplary embodiment, the following algorithm can be used to assess lupus risk, disease severity, and response to therapy. Lupus cases can be stratified into groups based on the number of risk alleles carried. In this exemplary embodiment, the risk allele is defined as the allele from the locus that is enriched in lupus cases relative to the control. For example, in Table 6, there are a total of 19 alleles from 18 loci, making the maximum possible number of risk alleles equal to 38. Lupus cases stratified by the number of risk alleles and the resulting distribution tertiles can be determined. The tertiles of lupus cases can then be examined in terms of the differences in disease severity, risk, and response to therapy.

Table 6: Lupus risk loci

*Chromosome position of the variant in human genome NCBI Build 35 (Hg17, May 2004) (http://www.ncbi.nlm.nih.gov/genome/guide/human/release_notes.html#b35), in base pairs

References

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10. A. L. Price et al., Nat Genet 38, 904 (2006).

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12. R. R. Graham et al., Arthritis research 3, 299 (2001).

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16. S. Purcell et al., American Journal of Human Genetics 81,559 (2007).

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18.K.Hartung,Baur,M.P.,Coldewey,R.,Fricke,M.,Kalden,J.R.,Lakomek,H.J.,Peter,H.H.,Schende l, D., Schneider, P.M., Seuchter, S.A., Stangel, W., Deicher, H.R.G., J. Clin. Invest. 90, 1346 (1992).

19. Z. Yao et al., Eur J Immunogenet 20, 259 (1993).

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21. J.B. Harley et al., Nat Genet 40, 204 (2008).

22. S. Sigurdsson et al., Am J Hum Genet 76,528 (2005).

23. R.R. Graham et al., Nat Genet 38,550 (2006).

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25. C. Kyogoku et al., Am J Hum Genet 75,504 (2004).

26. J.B. Harley, K.L. Moser, P.M. Gaffney, T.W. Behrens, Curr Opin Immunol 10, 690 (1998).

27. M.M. Fernando et al., PLoS Genet 3, e192 (2007).

28. R.R. Graham et al., Proc Natl Acad Sci U S A 104, 6758 (2007).

29. D.S. Cunninghame Graham et al., Nature Genetics 40, 83 (2008).

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32. C. O. Jacob et al., Arthritis Rheum 56, 4164 (2007).

33. J.C. Edberg et al., Hum Mol Genet 17, 1147 (2008).

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36. K. A. Frazer et al., Nature 449,851 (2007).

37. P.I. de Bakker et al., Nat Genet 37, 1217 (2005).

38. J. Maller et al., Nat Genet 38, 1055 (2006).

39. G. Lettre et al., Nat Genet 40, 584 (2008).

Example 7: Sequencing Summary

method

Genomic DNA from 192 SLE patients and 96 healthy controls was whole-genome amplified before resequencing. All exons and selected noncoding regions (2.5 kb promoter region upstream of exon 1) of B lymphoid kinase (BLK), integrin alpha M (ITGAM), and integrin alpha X (ITGAX) were resequenced.

Initial allele calling was performed by software provided by “Polymorphic.” All coding polymorphisms as well as common non-coding alleles were manually inspected to confirm allele calls and to create genotyping files for association and haplotype analysis.

Variants of ITGAM/ITGAX are provided in Tables 7 and 9 and Tables 8 and 10. The variants in Tables 7 and 9 are not present in the database dbSNP build 129. The variants in Tables 8 and 10 were discovered by sequencing ITGAM/ITGAX and BLK.

Table 7: Variants in ITGAM and ITGAX exon and promoter regions

Table 8: Variants in ITGAM and ITGAX exon and promoter regions

Table 9: Variants in the BLK exon and promoter regions

Table 10: Variants in the BLK exon and promoter regions

Example 8

Subjects and study design

A genome-wide association study for SLE was conducted. The Illumina HumanHap550 genotyping bead chip (555,352 SNPs) was used to determine the genotypes of 1,079 SLE cases and 1,411 controls. The SLE cases were from three different groups. Control samples were selected based on available HLA typing, race, gender, and age. The majority of controls (approximately 277) were selected so that the frequencies of the HLA DR2 and DR3 haplotypes would match those found in SLE.

Three versions of Illumina HumanHap550 are available. The number of SNPs shared between versions 1 and 3 is 545,080; only these SNPs were analyzed. Version 1 was used for all cohort 1 and cohort 2 samples and 1001 control samples. Version 3 was used for all cohort 3 samples and 410 control samples.

Rework chips with an average call rate < 80%. After all reworks are complete, remove samples with a call rate < 90%.

Initially, samples were divided into two groups for analysis. The first group (Group 1) consisted of all group 1 and group 2 samples (466 cases) and 724 control samples. The second group (Group 2) consisted of all group 3 samples (613 cases) and remaining 687 control samples.

Filtering in Group 1

The samples were checked for agreement between genotype-determined sex and clinical records; discrepancies were found in 10 samples (3 cases, 7 controls) and these 10 samples were removed from further analysis.

The samples were then tested for intercontinental admixture using the program STRUCTURE (an online link can be accessed by typing "pritch.bsd.uchicago.edu/structure" and ".html" as the suffix). (Essentially as described in Pritchard et al., Genetics (2000), 155:945-959; Falush et al., Genetics (2003), 164:1567-1587; Falush et al., Molecular Ecology Notes (2007), doi:10.1111/j.1471-8286.2007.01758.x). HumanHap550 includes a "DNA Test Panel" of 276 SNPs that are ideal for determining percent ancestry for the CEU, YRI, and CHB+JPT populations of the HapMap project. (CHB and JPT cannot be distinguished using these SNPs.) Genotypes for 274 of the 276 SNPs in the DNA test panel were determined across all HapMap populations; STRUCTURE was run with the genotypes of these 274 SNPs in a collection consisting of the remaining Group 1 samples (463 cases, 717 controls) plus one sample from each lineage in the HapMap project (i.e., 20 CEPH samples from Utah (CEU), 30 Yoruba samples (YRI), 45 Han Chinese samples (CHB), and 44 Japanese samples (JPT)). The HapMap samples were included as positive controls and used to aid the clustering algorithm. STRUCTURE was run three times independently with the same parameters: using a correlated allele frequency model without prior population information and an admixture ancestry model, with three populations, 30,000 burn-in steps, followed by 100,000 Markov-Chain Monte Carlo steps. The three runs had very similar ancestry coefficients for each sample, and each HapMap sample had greater than 93.0% ancestry to its geographic origin; each CEU sample had greater than 97.0% CEU ancestry. Samples with less than 90.0% CEU ancestry in any of the three runs (28 cases, 24 controls) were removed from further analysis.

For the remaining samples (435 cases, 693 controls), SNPs with a call rate less than 95% were removed from further analysis (23,275 SNPs (4%)). Then, SNPs with a Hardy-Weinberg probability of 0.001 or less in controls were removed from further analysis (15,622 SNPs (3%)).

Filtering in Group 2

The samples examined did not clearly show agreement between genotype-determined sex and clinical records.

SNPs with call rates less than 95% were removed from further analysis (34,998 SNPs (6%)).

The samples were then tested for intercontinental admixture using STRUCTURE, as described above. Samples with less than 90.0% CEU ancestry in any of the three runs (21 cases, 24 controls) were removed from further analysis.

For the remaining samples (592 cases, 663 controls), SNPs with a Hardy-Weinberg probability of 0.001 or less in controls were removed from further analysis (22,202 SNPs (4%)).

Combine Group 1 and Group 2

Groups 1 and 2 were combined for the final analysis.

The remaining samples in group 1 (435 cases, 693 controls) were combined with the remaining samples in group 2 (592 cases, 663 controls) to generate the final group (1027 cases, 1356 controls). Only the remaining SNPs in both group 1 and group 2 (496,458 SNPs) were further analyzed.

All samples (1076 cases, 1404 controls) without gender discrepancies were checked to see if they might be duplicates or related. First, all sample pairs were compared between 800 SNPs scattered throughout the genome. Duplication candidates and related candidates were then checked between 540,000+ SNPs. Three groups of outliers were detected. The first group (20 pairs) had greater than 95% identity between each pair and were considered to be duplicates. The second group (17 pairs) had 67-77% identity between each pair and were considered to be related. The third group (5 pairs) had 58-63% identity between each pair and were considered to be related. (The average identity between samples was 51-55%.) Overall, 39 samples (29 cases, 10 controls) were removed from the final group.

SNPs in mitochondrial DNA (19 SNPs) were removed from further analysis.

The resulting primary panel (998 cases, 1346 controls, 496,439 SNPs) was used in the following analyses.

The same analysis was also performed on specific subsets of the main groups:

Subset 1: females only (907 cases, 967 controls) and Subset 2: cases with lupus nephritis, and all controls (286 cases, 1346 controls).

Analysis and Results

All SNPs in the primary panel were analyzed using EIGENSTRAT, a program essentially as described in Price et al., Nature Genetics (2006), 38:904-909 (an online link can be accessed by typing "genepath.med.harvard.edu/~reich/EIGENSTRAT" followed by ".htm" as the suffix), which also corrects for population stratification. Outliers were removed using the first 10 principal components for 5 rounds before correcting for stratification. The EIGENSTRAT chi-square statistic was then calculated, and the one-tailed probability of the chi-square distribution was calculated using the CHIDIST function of Microsoft Excel with 1 degree of freedom.

To determine the top candidate regions, we first reduced the number of candidate SNPs by using a P value threshold: for subset 1 (female) and subset 2 (nephritis), SNPs with a P greater than ^2.0x10-5 were removed from further analysis, while for the main group (998 cases, 1346 controls), SNPs with a P greater than ^7.0x10-5 were removed from further analysis. In the female subset, 19 SNPs were retained. In the nephritis subset, 35 SNPs were retained. In the main group, 47 SNPs were retained. Then, linkage disequilibrium (LD) regions containing each SNP were determined by examining the LD plots, which were implemented using the HelixTree program (available by typing www.goldenhelix.com/pharmhelixtreefeatures and ".html" as a suffix to access the online link) (Golden Helix, Montana, USA). D' and ^r were calculated using the EM algorithm, which was implemented using only the genotypes of the cases and controls. Regions were delineated by eye using D' ≥ approximately 0.9 as the boundary.

Once each region is depicted, the genes in each region are viewed using a genome browser established in the art (e.g., the UCSC genome browser, which is essentially documented in Kuhn et al., Nucleic Acids Res. (2007), 35 (database release): D668-73; online links can be accessed by typing "genome.ucsc" and ".edu" as a suffix, compiled in March 2006). Immune-specific gene expression was examined as determined in the IRIS study (Abbas et al., Genes and Immunity (2005), 6: 319-331, including its online supplementary information). Top candidate regions were identified by, for example, the presence of immune-specific genes in the region. In the nephritis subset, 11 regions containing 20 candidate SNPs were selected, which may contain at least one SLE risk allele (Figures 12A-12D). In the female subset, 6 individual regions containing 9 candidate SNPs were selected (Figures 13A-13B). In the main group, 6 individual regions containing 8 candidate SNPs were selected (Figures 14A-14B). A total of 23 regions containing 37 candidate SNPs were selected. It should be noted that the SNPs are listed under the study group with the strongest SNP results, so that repeated hits between study groups are not shown. In addition, hits in the MHC region are not included. The LD regions drawn based on the data in Figures 12A-14B were determined and summarized in Figures 15-17 respectively.

Claims (23)

1. Use of one or more polynucleotides in the preparation of a detection reagent or article for use in the following methods, Each of the one or more polynucleotides is capable of allelically hybridizing to the allele of a single nucleotide polymorphism (SNP) contained in a genetic marker that indicates lupus or causes lupus. The method diagnoses lupus in subjects or assesses their risk of developing lupus by detecting the presence of the genetic tag in biological samples obtained from the subjects. The genetic tag mentioned therein contains a single nucleotide polymorphism (SNP) of rs7574865. 2. The use of claim 1, wherein the genetic tag further comprises at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs10489265 and rs13277113. 3. The use of claim 2, wherein the genetic tag comprises single nucleotide polymorphisms (SNPs) of rs7574865, rs10488631, rs10489265, and rs13277113. 4. The use of claim 1, wherein the genetic tag further comprises at least one single nucleotide polymorphism (SNP) selected from the group consisting of: rs2269368, rs5754217, rs1801274, rs2476601, and rs10516487. 5. The use of claim 4, wherein the genetic tag comprises single nucleotide polymorphisms (SNPs) of rs7574865, rs4963128, rs2269368, rs5754217, rs1801274, rs6568431, rs2476601, and rs10516487. 6. The use of claim 1, wherein the genetic tag further comprises at least one single nucleotide polymorphism (SNP) selected from the group consisting of: rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs3129860, rs2187668, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, and rs6445975. 7. The use of claim 6, wherein the genetic tag comprises single nucleotide polymorphisms (SNPs) of rs7574865, rs10488631, rs4963128, rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs6568431, rs3129860, rs2187668, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, rs6445975, and rs2431697. 8. The use of claim 1, wherein the genetic tag further comprises at least one single nucleotide polymorphism (SNP) selected from the group consisting of: rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs3129860, rs2187668, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, and rs6445975. 9. The use of claim 8, wherein the genetic tag comprises single nucleotide polymorphisms (SNPs) of rs7574865, rs10488631, rs4963128, rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs6568431, rs3129860, rs2187668, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, rs6445975, and rs2431697. 10. The use of any one of claims 1-9, wherein the method is used to diagnose lupus in a subject. 11. The use of any one of claims 1-9, wherein the method is used to assess whether a subject is at risk of developing lupus. 12. The use of any one of claims 1-9, wherein the detection comprises performing a method selected from the group consisting of: allele-specific primer extension assay; allele-specific nucleotide incorporation assay; allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assay using molecular beacons; and oligonucleotide ligation assay. 13. The use of claim 10, wherein the detection comprises performing a method selected from the group consisting of: allele-specific primer extension assay; allele-specific nucleotide incorporation assay; allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assay using molecular beacons; and oligonucleotide ligation assay. 14. The use of claim 11, wherein the detection comprises performing a method selected from the group consisting of: allele-specific primer extension assay; allele-specific nucleotide incorporation assay; allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assay using molecular beacons; and oligonucleotide ligation assay. 15. The test reagent or article according to any one of claims 1-14. 16. A kit comprising at least one enzyme and one or more polynucleotides, wherein each of the one or more polynucleotides is capable of allele-specific hybridization to the allele of a single nucleotide polymorphism (SNP) of rs7574865. 17. The kit of claim 16, further comprising one or more polynucleotides, each of said polynucleotides being capable of allelically hybridizing to at least one additional single nucleotide polymorphism (SNP) allele selected from the group consisting of: rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs3129860, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, and rs6445975. 18. The kit of claim 16 or 17, wherein the enzyme is a polymerase. 19. The kit of claim 16 or 17, wherein the enzyme is a ligase. 20. The kit of claim 16 or 17, wherein each of the one or more polynucleotides is bound to a marker. 21. The kit of claim 17, wherein the at least one additional single nucleotide polymorphism (SNP) comprises rs10488631, rs10489265, and rs13277113. 22. The kit of claim 17, wherein the at least one additional single nucleotide polymorphism (SNP) comprises rs4963128, rs2269368, rs5754217, rs1801274, rs6568431, rs2476601, and rs10516487. 23. The kit of claim 17, wherein the at least one additional single nucleotide polymorphism (SNP) comprises rs10488631, rs4963128, rs2269368, rs6889239, rs5754217, rs1801274, rs1143679, rs6568431, rs3129860, rs2187668, rs13277113, rs2476601, rs10516487, rs10489265, rs2391592, rs6445975, and rs2431697.