CN102803509B - For the method treating, diagnose and monitoring lupus - Google Patents

Abstract

Provide discriminating, the method diagnosing and predicting lupus, including the sub-phenotype (subpehnotype) of some lupus, and the method providing treatment lupus, including some subgroups of patient.The method provided is based on one group of allele relevant to systemic lupus erythematosus (sle) (SLE) risk genes seat, described risk genes seat includes that BLK, TNIP1, PRDM1, JAZF1, UHRF1BP1, IL1O, IFIH1, CFB, CEC16A, IL12B and SH2B3, described risk genes seat have effect to SLE risk.Additionally provide the method differentiating effective lupus therapeutic agent, and the method that prediction is to the response of lupus therapeutic agent.

Description

Methods for treating, diagnosing and monitoring lupus

Cross References to Related Applications

This application claims priority to US Provisional Application No. 61/278,510, filed October 7, 2009, which is hereby incorporated by reference in its entirety.

sequence listing

This application contains a Sequence Listing submitted in ASCII format via EFS-Web, which is hereby incorporated by reference in its entirety. The ASCII copy, created on September 20, 2010, is named P4325R1W.txt and is 57,896 bytes in size.

field

Methods of identifying, diagnosing, predicting and assessing the risk of developing lupus, and methods of treating lupus are provided. Also provided are methods for identifying effective lupus therapeutics and predicting responsiveness to lupus therapeutics.

background

Lupus is an autoimmune disease that is estimated to affect nearly one million Americans, primarily women between the ages of 20-40. Lupus involves antibodies that attack connective tissue. The major form of lupus is systemic lupus (systemic lupus erythematosus; SLE). SLE is a chronic autoimmune disease with strong genetic and environmental components (see eg 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; D'Cruz et al., Lancet (2007), 369:587-596). Various other forms of lupus are known, including but not limited to cutaneous lupus erythematosus (CLE), lupus nephritis, and neonatal lupus.

Untreated lupus can be fatal as it progresses from attacking the skin and joints to attacking internal organs, including the lungs, heart, and kidneys (with a major focus on kidney disease), making early and accurate lupus diagnosis and/or assessment of lupus risk especially The essential. Lupus manifests primarily as a series of flare-ups, with intervals of little or no disease symptoms. Renal injury, measured by the amount of proteinuria in the urine, is one of the most acute areas of injury associated with pathogenicity in SLE and accounts for at least 50% of the mortality and morbidity of the disease.

Clinically, SLE is a heterogeneous disorder characterized by high affinity autoantibodies (autoAbs). Autoantibodies play an important role in the pathogenesis of SLE, and the diverse clinical picture of the disease is caused by deposition of antibody-containing immune complexes in blood vessels leading to inflammation in the kidney, brain, and skin. Autoantibodies also have a direct pathogenic role in promoting hemolytic anemia and thrombocytopenia. SLE is associated with antinuclear antibodies, production of circulating immune complexes, and activation of the complement system. SLE has an incidence of about 1 in 700 women between the ages of 20 and 60. SLE can affect any organ system and can cause severe tissue damage. A large number of autoantibodies with different specificities exist in SLE. Patients with SLE often develop clinical features with anti-DNA, anti-Ro, and antiplatelet specificity and are able to initiate the disease, such as glomerulonephritis, arthritis, serositis, neonatal complete heart block, and hematologic Abnormal autoantibodies. These autoantibodies may also be associated with central nervous system disorders. Arbuckle et al. describe the appearance of autoantibodies before the clinical onset of SLE (Arbuckle et al. N. Engl. J. Med. 349(16): 1526-1533 (2003)). Lupus, including SLE, is not easy to diagnose, leading physicians to adopt a multifactorial sign and symptom-based classification approach (Gill et al., American Family Physician 68(11):2179-2186 (2003)).

One of the greatest challenges in the clinical management of complex autoimmune diseases such as lupus is the accurate early identification of the disease in patients. In recent years, many strain and candidate gene studies have been performed, identifying genetic factors that contribute to SLE susceptibility. Haplotypes carrying the class II HLA alleles DRB1*0301 and DRB1*1501 were significantly associated with disease and the presence of antibodies to nuclear autoantigens. See, eg, Goldberg MA et al., Arthritis Rheum. 19(2):129-32 (1976); Graham RR et al., Am J Hum Genet. 71(3):543-53 (2002); and Graham RR et al. , Eur J Hum Genet. 15(8):823-30 (2007). Recently, variants of interferon regulatory factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT) were discovered as significant risk factors for SLE. See eg, Sigurdsson S et al., Am J Hum Genet. 76(3):528-37 (2005); Graham RR et al., Nat Genet. 38(5):550-55 (2006); Graham RR et al., Proc Natl Acad Sci USA 104(16):6758-63 (2007); and Remmers EF et al., NEngl J Med. 357(10):977-86 (2007). The identification of IRF5 and STAT4 as SLE risk genes provides support for the notion that, under certain circumstances, the type I interferon (IFN) pathway plays an important role in the pathogenicity of SLE disease. Type I IFN is present in the serum of SLE cases, and IFN production correlates with the presence of immune complexes comprising Ab and nucleic acid (reviewed in Ronnblom et al., J Exp Med 194:F59 (2001); see also Baechler EC et al. , Curr Opin Immunol.16(6):801-07(2004); Banchereau J et al., Immunity25(3):383-92(2006); Miyagi et al., J Exp Med 204(10):2383-96(2007 )). Most cases of SLE show a prominent type I IFN gene expression "signature" in blood cells (Baechler et al., Proc Natl Acad Sci USA 100:2610 (2003); Bennett et al., J Exp Med 197:711 (2003 )), and have elevated serum levels of IFN-inducible cytokines and chemokines (Bauer et al., PLoSMed 3:e491 (2006)). Immune complexes containing native DNA and RNA stimulate toll-like receptors (TLRs) 7 and 9 expressed by dendritic cells and B cells to produce type I interferons, which further stimulate immune complex formation (reviewed in Marshak-Rothstein et al., Annu Rev Immunol 25, 419 (2007)).

In addition, several studies have been performed to identify reliable biomarkers for diagnostic and prognostic purposes. However, no clinically validated diagnostic markers (eg, biomarkers) have been identified that would allow physicians or others to accurately define the pathophysiological aspects of SLE, clinical activity, response to therapy, or risk of developing disease, despite the Multiple candidate genes and alleles (variants) thought to contribute to SLE susceptibility were identified. For example, at least 13 common alleles have been reported to contribute to SLE risk in individuals of European ancestry (Kyogoku et al., Am J Hum Genet 75(3):504-7 (2004); Sigurdsson et al., Am J Hum Genet 76(3):528-37 (2005); Graham et al., Nat Genet 38(5):550-55 (2006); Graham et al., Proc Natl Acad Sci USA 104(16):6758-63 (2007); Remmers et al., N Engl J Med357(10): 977-86 (2007); Cunninghame Graham et al., Nat Genet 40(1): 83-89 (2008); Harley et al., Nat Genet 40(2): 204- 10(2008); Hom et al., N Engl JMed 358(9):900-9(2008); Kozyrev et al., Nat Genet 40(2):211-6 (2008); Nath et al., Nat Genet 40(2) : 152-4 (2008); Sawalha et al., PLoS ONE 3(3): e1727 (2008)). Alleles inferred to be causal are known to be HLA-DR3, HLA-DR2, FCGR2A, PTPN22, ITGAM, and BANK1 (Kyogoku et al., Am J Hum Genet 75(3):504-7 (2004); Kozyrev et al., Nat Genet 40(2):211-6(2008); Nath et al., Nat Genet40(2):152-4(2008)), while the risk haplotypes of IRF5, TNFSF4 and BLK may affect the expression of mRNA and protein levels in SLE (Sigurdsson et al., Am J Hum Genet 76(3):528-37 (2005); Graham et al., Nat Genet 38(5):550-55 (2006); Graham et al., Proc Natl Acad Sci US A 104(16):6758-63 (2007); Cunninghame Graham et al., Nat Genet 40(1):83-89 (2008); Hom et al., N EnglJ Med 358(9):900-9 (2008)). Causal alleles for STAT4, KIAA1542, IRAK1, PXK, and other genes such as BLK have not been identified (Remmers et al., N Engl J Med 357(10):977-86 (2007); Harley et al., Nat Genet 40(2 ): 204-10 (2008); Hom et al, N Engl J Med 358(9): 900-9 (2008); Sawalha et al, PLoS ONE 3(3): e1727 (2008)). These and other lupus-associated genetic variations are also described in International Patent Application No. PCT/US2008/064430 (International Publication No. WO 2008/144761). Although these genetic variants contribute significantly to SLE risk and to various aspects of the disease described so far, more information remains to be determined regarding the contribution of genetic variants to, for example, the significant clinical heterogeneity of SLE.

Therefore, it would be highly advantageous to have additional molecular-based diagnostic methods that can be used to objectively identify the presence and/or classify disease in patients, define pathophysiological aspects of lupus, clinical activity, response to therapy, prognosis , and/or risk of developing lupus. Furthermore, it would be advantageous to have molecular-based diagnostic markers associated with various clinical and/or pathophysiological and/or other biological indicators of disease. Thus, there is a continuing need to identify novel risk loci and polymorphisms associated with lupus as well as other autoimmune diseases. Such correlations are of great benefit in identifying the presence of lupus in patients or determining susceptibility to developing the disease. Such correlations also facilitate the identification of lupus pathophysiological aspects, clinical activity, response to treatment, or prognosis. Furthermore, statistically and biologically significant and reproducible information on such correlations can be used as an integral part of efforts to identify specific subgroups of patients expected to benefit significantly from a particular therapeutic agent Therapeutic agents that have shown therapeutic benefit in such specific lupus patient subpopulations or have shown therapeutic benefit in clinical studies.

The invention described herein fulfills the above needs and provides other benefits.

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

overview

The methods provided herein are based, at least in part, on the discovery of a novel set of loci associated with SLE and contributing to disease risk (SLE risk loci). In addition, a panel of alleles associated with these SLE risk loci is provided. Causal alleles within the BLK locus associated with biological effects that increase SLE risk were also included. In addition, risk loci associated with other autoimmune diseases and increased SLE risk are provided.

In one aspect, there is provided a method of identifying lupus in a subject, the method comprising detecting the presence of a variation in a SLE risk locus in a biological sample derived from the subject, wherein the SLE risk locus is BLK, wherein the variation in the BLK locus Occurs at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP), wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8 , wherein the subject is suspected of having lupus. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods in Nuclease Assays; Assays Using Molecular Beacons; and Oligonucleotide Ligation Assays.

In another aspect, there is provided a method of identifying lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 4 in a biological sample derived from the subject, wherein at least one of the loci The variation occurs at a nucleotide position corresponding to the position of the SNP of at least one locus described in Table 4, and wherein the subject is suspected of having lupus. In certain embodiments, at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or at least 13 loci, or Variants were detected in 26 loci. In one embodiment, at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. In one embodiment, the variation of at least one locus comprises the SNPs described in Table 4. In certain embodiments, the presence of a variation in at least one SLE risk locus described in Table 4 is detected (wherein the variation in at least one locus occurs at a SNP associated with at least one locus described in Table 4 at the nucleotide position corresponding to the position) and the presence of variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs at the nucleotide position corresponding to the position of the SNP, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is a thymine) combination at chromosome 11389322 of human chromosome 8. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods in Nuclease Assays; Assays Using Molecular Beacons; and Oligonucleotide Ligation Assays.

In yet another embodiment, there is provided a method of identifying lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 6 in a biological sample derived from the subject, wherein at least one The variation in the locus occurs at a nucleotide position corresponding to the position of the SNP of at least one locus described in Table 6, and wherein the subject is suspected of having lupus. In certain embodiments, variations are detected at at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, at least one locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In one embodiment, the variation in at least one locus comprises the SNPs described in Table 6. In certain embodiments, the presence of variation in at least one SLE risk locus described in Table 6 is detected (wherein the variation in at least one locus occurs at a SNP position corresponding to at least one locus described in Table 6 at the corresponding nucleotide position) and the presence of variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs at the nucleotide position corresponding to the position of the SNP, wherein the SNP is rs922483 (SEQ ID NO: 13 ), where the variation is a thymine) combination at chromosome 11389322 of human chromosome 8. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods in Nuclease Assays; Assays Using Molecular Beacons; and Oligonucleotide Ligation Assays.

In yet another aspect, there is provided a method of identifying lupus in a subject, the method comprising detecting the presence of a variation in at least one of the SLE risk loci described in Table 4 and at least one of the SLE risk loci described in Table 6 in a biological sample derived from the subject. The presence of a variation in an SLE risk locus, wherein the variation in each locus occurs at a nucleotide position corresponding to the SNP position of each locus described in Tables 4 and 6, and wherein the subject suspects have lupus. In certain embodiments, the variation is detected in at least three loci, or at least four loci, or at least five loci, or at least 7 loci, or at least 10 loci. In one embodiment, at least one locus described in Table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL10, and at least one locus described in Table 6 is selected from IFIH1, CFB, CLEC16A, IL12B, and SH2B3. In one embodiment, the variation in at least one locus described in Table 4 and the variation in at least one locus described in Table 6 comprises the SNPs described in Table 4 and Table 6, respectively. In certain embodiments, the presence of variation in at least one SLE risk locus described in Table 4 is detected (wherein the variation in at least one locus occurs at a SNP position corresponding to at least one locus described in Table 4 nucleotide position), and the presence of variation in at least one SLE risk locus described in Table 6 (wherein the variation in at least one locus occurs at a SNP position corresponding to at least one locus described in Table 6 ), in combination with the presence of a variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs at a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8). In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods in Nuclease Assays; Assays Using Molecular Beacons; and Oligonucleotide Ligation Assays.

In one aspect, a method of predicting responsiveness to a lupus therapeutic agent in a subject with lupus is provided, the method comprising determining whether the subject comprises a variation in a SLE risk locus, wherein the SLE risk locus is BLK, wherein the variation in the BLK locus Occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 82, wherein the variation occurs in the BLK SLE risk locus Presence indicates responsiveness of a subject to a therapeutic agent.

In another aspect, there is provided a method of predicting the responsiveness of a subject with lupus to a lupus therapeutic, the method comprising determining whether the subject includes a variation in at least one SLE risk locus described in Table 4, wherein at least one of the loci The variation occurs at the nucleotide position corresponding to the SNP position of at least one locus described in Table 4, wherein the presence of the variation in at least one locus indicates responsiveness of the subject to the therapeutic agent. In certain embodiments, the subject is at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or at least 13 loci, or 26 loci including variants. In one embodiment, at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. In one embodiment, the variation in at least one locus comprises the SNPs described in Table 4. In certain embodiments, the method includes determining whether the subject includes a variation in at least one SLE risk locus described in Table 4 (wherein the variation in at least one locus occurs in at least one locus associated with Table 4 on the nucleotide position corresponding to the SNP position of BLK), and the variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs on the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO :13), wherein the variation is a thymine) combination at chromosome 11389322 of human chromosome 8, wherein the presence of variation in at least one of the loci described in Table 4 and the presence of variation in the BLK locus indicate that the subject is responsive to the therapeutic agent responsiveness.

In yet another aspect, there is provided a method of predicting the responsiveness of a subject with lupus to a lupus therapeutic, the method comprising determining whether the subject includes a variation in at least one SLE risk locus described in Table 6, wherein at least one of the loci The variation in occurs at the nucleotide position corresponding to the SNP position of at least one locus described in Table 6, wherein the presence of the variation in the at least one locus indicates responsiveness of the subject to the therapeutic agent. In certain embodiments, the subject comprises variation in at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, at least one locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In one embodiment, the variation in at least one locus comprises the SNPs described in Table 6. In some embodiments, the method includes determining whether the subject includes a variation in at least one SLE risk locus described in Table 6 (wherein the variation in at least one locus occurs in at least one locus associated with Table 6 on the nucleotide position corresponding to the SNP position of BLK), and the variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs on the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO :13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8) combination, wherein the presence of variation in at least one of the loci described in Table 6 and the presence of variation in the BLK locus indicate that the subject is responsive to the therapeutic agent responsiveness.

In other aspects, there is provided a method of predicting the responsiveness of a subject suffering from lupus to a lupus therapeutic agent, the method comprising determining whether the subject includes a variation in at least one SLE risk locus described in Table 4 (wherein at least one of the loci The variation occurs at the nucleotide position corresponding to the SNP position of at least one locus described in Table 4), and the variation is included in at least one SLE risk locus described in Table 6 (wherein the SNP in at least one locus The variation occurs at the nucleotide position corresponding to the SNP position of at least one locus described in Table 6), wherein the presence of the variation in at least one locus described in Table 4 and at least one gene described in Table 6 The presence of variation in a locus indicates responsiveness of a subject to a therapeutic agent. In certain embodiments, the subject comprises variation in at least three loci, or at least four loci, or at least five loci, or at least seven loci, or at least ten loci. In one embodiment, at least one locus described in Table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL10, and at least one locus described in Table 6 is selected from IFIH1, CFB, CLEC16A, IL12B, and SH2B3. In one embodiment, the variation in at least one locus described in Table 4 and the variation in at least one locus described in Table 6 comprises the SNPs described in Table 4 and Table 6, respectively. In certain embodiments, the method includes determining whether the subject includes a variation in at least one SLE risk locus described in Table 4 (wherein the variation in at least one locus occurs in at least one locus associated with Table 4 The corresponding nucleotide position of the SNP position), and the variation in at least one SLE risk locus described in Table 6 (wherein the variation in at least one locus occurs in at least one locus described in Table 6 at the nucleotide position corresponding to the SNP position of ), and the variation in the BLK SLE risk locus (wherein the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8) in combination, wherein the presence of variation in at least one locus described in Table 4 and the presence of variation in at least one locus described in Table 6 And the presence of a variation in the BLK locus indicates responsiveness of the subject to the therapeutic agent.

In still another aspect, there is provided a method of diagnosing or predicting lupus in a subject, the method comprising detecting the presence of a variation in a SLE risk locus in a biological sample derived from the subject, wherein the SLE risk locus is BLK, wherein the BLK gene The variation in the locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8, and the BLK locus The presence of a variant in is diagnostic or predictive of lupus in the subject.

In yet another aspect, there is provided a method of diagnosing or predicting lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 4 in a biological sample derived from the subject, wherein: The chemical sample is known or suspected to include nucleic acid comprising a variation in at least one SLE risk locus described in Table 4; the variation in at least one locus includes the SNP described in Table 4 or is located at the SNP corresponding to the SNP described in Table 4 at nucleotide positions; and the presence of a variation in at least one locus is diagnostic or predictive of lupus in the subject. In certain embodiments, at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or at least 13 loci, or Variants were detected in 26 loci. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. In certain embodiments, the method includes detecting the presence of a variation in at least one of the SLE risk loci described in Table 4, and in combination with the presence of a variation in the BLKSLE risk locus, wherein: the biological sample is known or suspected to include Nucleic acid comprising a variation in at least one SLE risk locus described in Table 4 and a variation in the BLK locus, the variation in at least one locus described in Table 4 includes the SNP described in Table 4 or is located in the SNP described in Table 4 The nucleotide position corresponding to the above-mentioned SNP, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is in human No. 8 Thymine at chromosome 11389322 of the chromosome and the presence of a variation in at least one of the loci described in Table 4 and the presence of a variation in the BLK locus are diagnostic or predictive of lupus in the subject.

In still another aspect, there is provided a method of diagnosing or predicting lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 6 in a biological sample derived from the subject, wherein: The chemical sample is known or suspected to include nucleic acid comprising a variation in at least one SLE risk locus described in Table 6; the variation in at least one locus includes the SNP described in Table 6 or is located at the SNP corresponding to the SNP described in Table 6 at nucleotide positions; and the presence of a variation in at least one locus is diagnostic or predictive of lupus in the subject. In certain embodiments, variations are detected at at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In certain embodiments, the method includes detecting the presence of a variation in at least one of the SLE risk loci described in Table 6, and in combination with the presence of a variation in the BLK SLE risk locus, wherein: the biological sample is known or suspected to include The nucleic acid comprising the variation in at least one SLE risk locus described in Table 6 and the variation in the BLK locus, the variation in at least one locus described in Table 6 includes the SNP described in Table 6 or is located in the same range as Table 6 At the nucleotide position corresponding to the SNP, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is in human 8 Thymine at chromosome 11389322 of chromosome number, and the presence of a variation in at least one of the loci described in Table 6 and the presence of a variation in the BLK locus is a diagnosis or prognosis of lupus in the subject.

In yet another aspect, there is provided a method of diagnosing or predicting lupus in a subject, the method comprising detecting the presence of a variation in at least one of the SLE risk loci described in Table 4 and in Table 6 in a biological sample derived from the subject The presence of variation in at least one SLE risk locus, wherein: the biological sample is known or suspected to include: a variation in at least one SLE risk locus described in Table 4 and at least one SLE described in Table 6 The nucleic acid of the variation in the risk locus; The variation in at least one locus includes the SNP described in Table 4 and Table 6 respectively or is located on the nucleotide position corresponding to the SNP described in Table 4 and Table 6; and in Table 4 The presence of a variation in said at least one locus and the presence of a variation in at least one of the loci described in Table 6 is diagnostic or predictive of lupus in the subject. In certain embodiments, variations are detected at at least three loci, or at least four loci, or at least five loci, or at least seven loci, or at least ten loci. In one embodiment, at least one SLE risk locus described in Table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10, and at least one SLE risk locus described in Table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In certain embodiments, the method comprises detecting the presence of a variation in at least one of the SLE risk loci described in Table 4, and the presence of a variation in at least one of the SLE risk loci described in Table 6, and the association with the BLKSLE risk gene The presence combination of the variation in locus, wherein: biological sample is known or suspected to comprise: comprise the variation in at least one SLE risk locus described in Table 4 and the variation in at least one SLE risk locus described in Table 6 and The nucleic acid of the variation in the BLK locus, the variation in at least one locus described in Table 4 includes the SNP described in Table 4 or is located at a nucleotide position corresponding to the SNP described in Table 4, and is described in Table 6 The variation in at least one locus includes the SNP described in Table 6 or is located at the nucleotide position corresponding to the SNP described in Table 6, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position , wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8, and the presence of the variation in at least one of the loci described in Table 4 and the presence of the variation in Table 6 The presence of a variation in at least one of the loci described above, and the presence of a variation in the BLK locus are diagnostic or predictive of lupus in the subject.

In another aspect, there is provided a method of aiding in the diagnosis or prediction of lupus in a subject, the method comprising detecting the presence of a variation in a SLE risk locus in a biological sample derived from the subject, wherein the SLE risk locus is BLK, wherein the BLK gene The variation in the locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8, and the BLK locus The presence of a variant in is diagnostic or predictive of lupus in the subject.

In yet another aspect, there is provided a method of aiding in the diagnosis or prediction of lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 4 in a biological sample derived from the subject, wherein: The biological sample is known or suspected to include a nucleic acid comprising a variation in at least one SLE risk locus described in Table 4; the variation in at least one locus includes the SNP described in Table 4 or is located at the SNP corresponding to the SNP described in Table 4 and the presence of a variation in at least one locus is diagnostic or predictive of lupus in the subject. In certain embodiments, at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or at least 13 loci, or Variants were detected in 26 loci. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. In certain embodiments, the method comprises detecting the presence of a variation in at least one of the SLE risk loci described in Table 4, and in combination with the presence of a variation in the BLK SLE risk locus, wherein: the biological sample is known or suspected to include The nucleic acid comprising the variation in at least one SLE risk locus described in Table 4 and the variation in the BLK locus, the variation in at least one locus described in Table 4 includes the SNP described in Table 4 or is located in the same range as Table 4 At the nucleotide position corresponding to the SNP, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is in human 8 Thymine at chromosome 11389322 of chromosome number, and the presence of a variation in at least one of the loci described in Table 4 and the presence of a variation in the BLK locus is a diagnosis or prognosis of lupus in the subject.

In yet another aspect, there is provided a method of aiding in the diagnosis or prediction of lupus in a subject, the method comprising detecting the presence of a variation in at least one SLE risk locus described in Table 6 in a biological sample derived from the subject, wherein: The biological sample is known or suspected to include a nucleic acid comprising a variation in at least one SLE risk locus described in Table 6; the variation in at least one locus includes the SNP described in Table 6 or is located at the SNP corresponding to the SNP described in Table 6 and the presence of a variation in at least one locus is diagnostic or predictive of lupus in the subject. In certain embodiments, variations are detected at at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In certain embodiments, the method includes detecting the presence of a variation in at least one of the SLE risk loci described in Table 6, and in combination with the presence of a variation in the BLK SLE risk locus, wherein: the biological sample is known or suspected to include The nucleic acid comprising the variation in at least one SLE risk locus described in Table 6 and the variation in the BLK locus, the variation in at least one locus described in Table 6 includes the SNP described in Table 6 or is located in the same range as Table 6 At the nucleotide position corresponding to the SNP, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position, wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is in human 8 Thymine at chromosome 11389322 of chromosome number, and the presence of a variation in at least one of the loci described in Table 6 and the presence of a variation in the BLK locus is a diagnosis or prognosis of lupus in the subject.

In yet another aspect, there is provided a method of aiding in the diagnosis or prediction of lupus in a subject, the method comprising detecting the presence of a variation in at least one of the SLE risk loci described in Table 4 in a biological sample derived from the subject and in Table 4 The presence of variation in at least one SLE risk locus described in 6, wherein: the biological sample is known or suspected to include: comprising the variation in at least one SLE risk locus described in Table 4 and at least one of the SLE risk loci described in Table 6 The nucleic acid of the variation in the SLE risk locus; The variation in at least one locus includes the SNP described in Table 4 and Table 6 respectively or is located on the nucleotide position corresponding to the SNP described in Table 4 and Table 6; The presence of a variation in at least one of the loci described in 4 and the presence of a variation in at least one of the loci described in Table 6 is a diagnosis or prognosis of lupus in the subject. In certain embodiments, variations are detected at at least three loci, or at least four loci, or at least five loci, or at least seven loci, or at least ten loci. In one embodiment, at least one SLE risk locus described in Table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10, and at least one SLE risk locus described in Table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In certain embodiments, the method comprises detecting the presence of a variation in at least one of the SLE risk loci described in Table 4, and the presence of a variation in at least one of the SLE risk loci described in Table 6, and the association with the BLKSLE risk gene The presence combination of the variation in locus, wherein: biological sample is known or suspected to comprise: comprise the variation in at least one SLE risk locus described in Table 4 and the variation in at least one SLE risk locus described in Table 6 and The nucleic acid of the variation in the BLK locus, the variation in at least one locus described in Table 4 includes the SNP described in Table 4 or is located at a nucleotide position corresponding to the SNP described in Table 4, and is described in Table 6 The variation in at least one locus includes the SNP described in Table 6 or is located at the nucleotide position corresponding to the SNP described in Table 6, and the variation in the BLK locus occurs at the nucleotide position corresponding to the SNP position , wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is thymine at chromosome 11389322 of human chromosome 8, and the presence of the variation in at least one of the loci described in Table 4 and the presence of the variation in Table 6 The presence of a variation in at least one of the loci described above, and the presence of a variation in the BLK locus are diagnostic or predictive of lupus in the subject.

In one aspect, there is provided a method of treating a lupus disorder in a subject, wherein the genetic variation is known to exist at a nucleotide position corresponding to a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), Where the SLE risk locus is BLK, wherein the variant is thymine at chromosome 11389322 of human chromosome 8, the method comprises administering to the subject a therapeutic agent effective to treat the disorder.

In another aspect, there is provided a method of treating a lupus disorder in a subject whose genetic variation is known to be at a nucleotide position corresponding to the SNP described in Table 4 in at least one SLE risk locus described in Table 4 above, methods comprising administering to a subject a therapeutic agent effective in treating a condition. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10.

In another aspect, there is provided a method of treating a lupus disorder in a subject whose genetic variation is known to be at a nucleotide position corresponding to the SNP described in Table 6 in at least one SLE risk locus described in Table 6 above, methods comprising administering to a subject a therapeutic agent effective in treating a condition. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3.

In another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective in treating the disorder in the subject at a nucleotide position corresponding to a SNP in the SLE risk locus There is a genetic variation where the SNP is rs922483 (SEQ ID NO: 13) and the SLE risk locus is BLK, where the variation is thymine at chromosome 11389322 of human chromosome 8.

In another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective in treating the disorder in the subject, the subject being at least one of the SLE risk loci described in Table 4 associated with Table 4 4 There is a genetic variation at the nucleotide position corresponding to the SNP. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10.

In another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective in treating the disorder in the subject, said subject being at least one of the SLE risk loci described in Table 6 associated with Table 6 6. There is a genetic variation at the nucleotide position corresponding to the SNP. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3.

In yet another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent that has been shown to be effective in treating the disorder in at least one clinical study, wherein the therapeutic agent is administered in the study to at least 5 human subjects each having a genetic variation at a nucleotide position corresponding to a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13) and the SLE risk locus is BLK , where the variation is thymine at chromosome 11389322 on human chromosome 8.

In yet another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent that has been shown to be effective in treating the disorder in at least one clinical study, wherein the therapeutic agent is administered in the study Giving at least 5 human subjects each having a genetic variation at a nucleotide position corresponding to a SNP described in Table 4 in at least one SLE risk locus described in Table 4. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10.

In yet another aspect, there is provided a method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent that has been shown to be effective in treating the disorder in at least one clinical study, wherein the therapeutic agent is administered in the study Giving at least 5 human subjects each having a genetic variation at a nucleotide position corresponding to a SNP described in Table 6 in at least one SLE risk locus described in Table 6. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3.

In another aspect, there is provided a method comprising preparing a lupus therapeutic, comprising packaging the therapeutic and administering the therapeutic to a subject having, or believed to have, lupus at a SNP in a SLE risk locus The corresponding position has a genetic variation, where the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, where the variation is thymine at chromosome 11389322 of human chromosome 8.

In yet another aspect, there is provided a method comprising preparing a lupus therapeutic, comprising packaging the therapeutic and administering the therapeutic to a subject having or believed to have lupus and at least as described in Table 4 The positions corresponding to the SNPs described in Table 4 in one SLE risk locus have genetic variation.

In yet another aspect, there is provided a method comprising preparing a lupus therapeutic, comprising packaging the therapeutic and administering the therapeutic to a subject having or believed to have lupus, and at least as described in Table 6 The positions corresponding to the SNPs described in Table 6 in one SLE risk locus have genetic variation.

In one aspect, there is provided a method of selecting a patient with lupus for treatment with a lupus therapeutic, the method comprising detecting the presence of a genetic variation at a nucleotide position corresponding to a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, wherein the variation is thymine at chromosome 11389322 of human chromosome 8. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays.

In other aspects, there is provided a method of selecting a patient with lupus for treatment with a lupus therapeutic, the method comprising detecting a nucleoside corresponding to a SNP described in Table 4 in at least one SLE risk locus described in Table 4 The presence of genetic variation at the acid position. In certain embodiments, at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or at least 13 loci, or Variants were detected in 26 loci. In one embodiment, at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. In one embodiment, the variation at at least one locus comprises the SNPs described in Table 4. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays.

In other aspects, there is provided a method of selecting a patient with lupus for treatment with a lupus therapeutic, the method comprising detecting a nucleoside corresponding to a SNP described in Table 6 in at least one SLE risk locus described in Table 6 The presence of genetic variation at the acid position. In certain embodiments, variations are detected at at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B3. In one embodiment, the variation at at least one locus comprises the SNPs described in Table 6. In one embodiment, detecting comprises performing a primer extension assay; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays.

In another aspect, there is provided a method of assessing whether a subject is at risk of developing lupus, the method comprising detecting the presence of a genetic signature indicative of a risk of developing lupus in a biological sample obtained from the subject, wherein the genetic The signature includes a set of at least three SNPs, each of which occurs in the SLE risk loci described in Table 4 and/or Table 6. In certain embodiments, the genetic signature comprises a set of at least 4 SNPs, or at least 5 SNPs, or at least 7 SNPs, or at least 10 SNPs. In some embodiments, the SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B, and SH2B3. In certain embodiments, the genetic signature further comprises a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, wherein the variation is on chromosome 8 of human chromosome Thymine at position 11389322.

In another aspect, there is provided a method of diagnosing lupus in a subject, the method comprising detecting the presence of a genetic signature indicative of lupus in a biological sample obtained from said subject, wherein said genetic signature comprises a set of at least three SNPs, Each SNP occurs in the SLE risk loci described in Table 4 and/or Table 6. In certain embodiments, the genetic signature comprises a set of at least 4 SNPs, or at least 5 SNPs, or at least 7 SNPs, or at least 10 SNPs, or at least 15 SNPs, or at least 20 SNPs, or at least 30 SNPs. In one embodiment, the SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B and SH2B3. In certain embodiments, the genetic signature further comprises a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, wherein the variation is on chromosome 8 of human chromosome Thymine at position 11389322.

Brief introduction to the drawings

Figure 1 shows an overview of the experimental design of a targeted replication study of certain SNPs to identify additional SLE risk loci as described in Example 1.

Figure 2 shows novel genome-wide significant associations in SLE, and identification of novel novel associations within TNIP1 (A), PRDM1 (B), JAZF1 (C), UHRF1BP1 (D) and IL10 (E) described in Example 1 risk loci. (F) Histogram of P-values for independent SNPs for the conditions described in Example 1 and control replicates; the dashed line indicates the expected outcome density under the null distribution.

Figure 3 shows the time to candidate (P<1x10 ^-5 ) and validation (P<5x10 ^-8 ) status in the meta-analysis stratified by P value in the original GWAS described in Example 1. Variation percentage.

Figure 4 shows the linkage disequilibrium block (shown as ^r2 ) in the BLK promoter region described in Example 2. Figure 4 discloses 'C>T-rs922483' as SEQ ID NO:13.

Figure 5 shows the results of a luciferase reporter gene expression assay for the BLK promoter region with various haplotypes as described in Example 2. (A) In BJAB cells, SNP rs922483C>T (SEQ ID NO: 13); (B) in Daudi cells, SNP rs922483C>T (SEQ ID NO: 13); (C) in BJAB cells, SNP rs1382568A >C/G>C; (D) in Daudi cells, SNP rs1382568A>C/G>C; (E) in BJAB cells, SNP rs4840568G>A; (F) in Daudi cells, SNP rs4840568G>A; Data shown represent means of triplicate determinations +/- standard error of the mean; dotted bars show results for haplotypes marked on left side of graph; hatched bars: risk haplotype 22-ACT; empty bars: No risk haplotype 22-GAC; *p<0.05, **p<0.01, ***p<0.001, ns = not significant (t-test). Figure 5A-F discloses the '22 (GT) repeat' as SEQ ID NO:15. Figures 5C-F also disclose that 'rs922483C>T' is SEQ ID NO:13.

Figure 6 shows the SNP rs1382568A>C/G>C in Daudi cells with 18 (GT) repeats (SEQ ID NO: 14) or 22 (GT) repeats (SEQ ID NO: 15) as described in Example 2 Results of the luciferase reporter gene expression assay of the BLK promoter region. Data shown represent mean of duplicate determinations +/- standard error of the mean; ns = not significant (t-test). Figure 6 discloses that '18 (GT) repeat' is SEQ ID NO:14, '22 (GT) repeat' is SEQ ID NO:15, and 'rs922483C>T' is SEQ ID NO:13.

Figure 7 shows the sequence of the SNP, rs922483 (SEQ ID NO: 13), and the position in the SNP of the causal allele of the BLK locus as described in Example 2. Positions of causal alleles are shown in bold brackets; C/T variants are shown in bold.

Detailed description of the invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, within the skill of the art. Such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Grait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987 and regularly updated); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994) fully explains such techniques . In addition, primers, oligonucleotides and polynucleotides for use in the present invention can be generated using standard techniques known in the art.

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

definition

For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural and vice versa. As used in this specification and the appended claims, the singular forms ("a", "an", and "the") include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "protein" includes a plurality of proteins; reference to "cell" includes a mixture of cells, and so on. In the event of a conflict between a definition set forth below and a definition in any document incorporated herein by reference, the definition set forth below applies.

As used herein, "lupus" or "lupus disorder" is an autoimmune disease or disorder that generally involves antibodies that attack connective tissue. The major forms of lupus are systemic lupus, systemic lupus erythematosus (SLE), which includes cutaneous SLE and subacute cutaneous SLE, and other types of lupus (including nephritic, extrarenal, encephalitic, pediatric, nonrenal, discoid, and hair removal).

The term "polynucleotide" or "nucleic acid" used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, can be made before or after assembly of the polymer. A sequence of nucleotides may be interrupted by non-nucleotide elements. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps"; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as having uncharged linkages (e.g., methylphosphonate, phosphotriester , phosphoramide, carbamate (cabamate), etc.) and those with charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), containing overhanging moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptide, poly-L-lysine, etc.), those with intercalating agents (such as acridine, psoralen, etc.), those containing chelating agents (such as metals, radioactive metals, boron, metal oxides, etc.) , those containing an alkylator, those having modified linkages (eg, alpha-anomeric nucleic acids, etc.), and polynucleotides in unmodified form. Furthermore, any hydroxyl groups normally present in carbohydrates can be substituted, for example by phosphate groups, phosphate groups, protected by standard protecting groups, or activated to make other linkages to other nucleotides, or can be bonded to a solid support conjugate. The 5' and 3' OH can be phosphorylated or partially substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. The polynucleotide may also comprise ribose or deoxyribose sugars in the form of analogs generally known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro- or 2'-Azido-ribose, carbocyclic sugar analogues, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranoses, furanoses, sedum Heptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleosides. One or more phosphodiester linkages may be replaced by other linking groups. These other linking groups include, but are not limited to, through P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 (" Amidate"), P(O)P, P(O)OR', CO, or CH2 ("formacetal") substituted phosphoric acid embodiments wherein R or R' are independently H or optionally comprise an ether (-- O--) substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. It is not necessary for all bonds in a polynucleotide to be the same. The foregoing applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" refers to short single-stranded polynucleotides that are at least about 7 nucleotides in length and less than about 250 nucleotides in length. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides applies equally and fully to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and permits polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.

The term "genetic variation" or "nucleotide variation" refers to an alteration (e.g., one or more nuclear Nucleotide insertions, deletions, inversions, or substitutions, such as single nucleotide polymorphisms (SNPs). Unless otherwise indicated, the term also includes 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 DNA at which different alleles or other nucleotides exist in a population. A SNP position is often preceded and followed by a highly conserved allelic sequence (eg, a sequence that differs in less than 1/100 or 1/1000 of the members of the population). Individuals can be homozygous or heterozygous for alleles at each SNP position.

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

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

The terms "genetic variation at a nucleotide position corresponding to a SNP position", "nucleotide variation at a nucleotide position corresponding to a SNP position" and grammatical variants thereof refer to relatively corresponding DNA positions in a polynucleotide sequence The genetic variation on the position in the genome occupied by the SNP. Unless otherwise indicated, the term also includes corresponding variations in the complement of the nucleotide sequence.

The term "array" or "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes (eg, 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 a method of producing one or more copies of a reference nucleic acid sequence or its complement. Amplification can be linear or exponential (eg PCR). "Copy" does not imply perfect sequence complementarity or identity relative to the template sequence. For example, copies may contain nucleotide analogs such as deoxyinosine, deliberate sequence changes (such as those introduced by primers containing sequences that hybridize to the template but are not fully complementary), and/or Sequence error.

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

The term "allele-specific primer" refers to an allele-specific oligonucleotide, which is a primer.

The term "primer extension assay" refers to an assay in which nucleotides are added to a nucleic acid, resulting in a longer nucleic acid or "extension product" that is detected directly or indirectly. Nucleotides may 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 passed through The polymerase extends, 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 is hybridized 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 hybridization probe, resulting in 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 produces a detection signal level that is higher than that emitted by a free oligonucleotide.

The term "oligonucleotide ligation assay" refers to an assay in which an allele-specific oligonucleotide and a second oligonucleotide are hybridized and ligated together (directly or via intervening nucleotides indirectly), and directly or indirectly detect the ligated product.

The term "target sequence", "target nucleic acid" or "target nucleic acid sequence" generally refers to a polynucleotide sequence of interest suspected or known to contain nucleotide variations therein, including copies of such target nucleic acid produced by amplification.

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

The terms "SLE risk loci" and "confirmed SLE risk loci" refer to any one of the loci identified in Table 4 and Table 6 and the BLK locus.

The terms "SLE risk allele" and "confirmed SLE risk allele" refer to variations present in the SLE risk locus. Such variations include, but are not limited to, single nucleotide polymorphisms, insertions and deletions. Some exemplary SLE risk alleles are shown in Tables 4 and 6.

As used herein, a subject "at risk" of developing lupus may or may not have detectable disease or symptoms of disease, and may or may not have exhibited detectable disease or symptoms of disease prior to the methods of treatment described herein. "At risk" means that a subject has one or more risk factors, which are measurable parameters associated with having lupus, as described herein and known in the art. Subjects with one or more of these risk factors have a higher probability of developing lupus than subjects without one or more of these risk factors.

The term "diagnosis" is used herein to refer to the identification and classification of a molecular or pathological state, disease or disorder. For example, "diagnosing" can refer to a particular type of lupus condition, such as the identification of SLE. "Diagnosis" can also refer to the classification of specific subtypes of lupus, eg by tissues/organs involved (lupus nephritis), by molecular characteristics (eg subgroups of patients characterized by genetic variations in specific genes or nucleic acid regions).

The term "aiding diagnosis" is used herein to refer to methods of assisting in making a clinical determination regarding the presence or nature of a particular type of symptom or condition in lupus. For example, a method to aid in the diagnosis of lupus can include measuring the absence of one or more SLE risk loci or the presence of an SLE risk allele in a biological sample from an individual.

The term "prognosis" is used herein to refer to disease symptoms attributable to an autoimmune disorder, including, for example, the prediction of the likelihood of relapse, flare, and drug resistance of an autoimmune disease such as lupus. The term "prediction" is used herein to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or group of drugs.

As used herein, "treatment" refers to clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be performed prior to or during the course of clinical pathology. Desirable therapeutic effects include preventing the occurrence or recurrence of the disease or a disorder or symptom thereof, alleviating a disorder or symptom of the disease, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or palliation of the disease state, and achieving remission or improved forecasts. In some embodiments, the methods and compositions of the invention are used in an attempt to delay the onset of a disease or disorder.

"Effective amount" refers to an amount effective (dose and time required) to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective (dose and time required) to achieve the desired prophylactic result. Since prophylactic doses are used in subjects prior to or at an early stage of disease, the prophylactically effective amount will usually, but not necessarily, be less than the therapeutically effective amount.

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 (eg, mice and rats). In certain embodiments, the mammal is a human.

As used herein, "patient subgroup" and grammatical variants thereof refer to a subgroup of patients characterized by one or more unique measurable and/or identifiable characteristics that separate the subgroup of patients from the from other patients in the broader disease category. Such characteristics include disease subtype (eg, SLE, lupus nephritis), gender, lifestyle, health history, organs/tissues involved, treatment history, and the like.

A "control subject" refers to a healthy subject who has not been diagnosed with lupus or a lupus disorder and does not suffer from any signs or symptoms associated with lupus or a lupus disorder.

The term "sample" as used herein refers to a composition obtained or derived from a subject of interest comprising cells and/or other molecular entities to be characterized and/or identified, for example in terms of physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "biological sample" or "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized.

"Tissue or cell sample" means a collection of similar cells obtained from a subject or patient's tissue. The source of the tissue or cell sample may be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluid, such as cerebrospinal fluid , amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time during pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines. Alternatively, the tissue or cell sample is obtained from a diseased tissue/organ. Tissue samples may contain compounds that are not naturally mixed 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 obtained or not considered to be suffering from the method or composition of the present invention. The cell or tissue of origin of the identified disease or condition. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy portion of the body of the same subject or patient in which a disease or disorder 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 in whom a disease or condition is not being identified using a composition or method of the invention subject or patient.

For purposes herein, a "section" of a tissue sample means a single portion or monolithic tissue sample, eg, a thin slice of tissue or cells excised from the tissue sample. Given that the present invention is understood to include methods of analyzing the same section of a tissue sample at both the morphological and molecular levels, or for both proteins and nucleic acids, it is understood that multiple sections of the tissue sample can be taken and analyzed in accordance with the present invention .

"Correlating" or "correlating" means comparing the performance and/or results of a first analysis or procedure with the performance and/or results of a second analysis or procedure in any manner. For example, the results of a first analysis or procedure can be used to perform a second procedure, and/or the results of a first analysis or procedure can be used to determine whether a second analysis or procedure should be performed. With regard to embodiments of the gene expression analysis or procedure, the results of the gene expression analysis or procedure can be used to determine whether a particular treatment regimen should be pursued.

As used herein, the word "label" refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a nucleic acid probe or antibody, and facilitates the detection of the agent to which it is conjugated or fused. The label may itself be detectable (eg, radioisotope or fluorescent label), or in the case of an enzymatic label, it may catalyze the chemical alteration of a detectable substrate compound or composition.

A "medicament" is an active drug that treats 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 means a decreased response to standard doses of a drug or to a standard regimen of treatment.

As used in accordance with the present invention, the term "decreased sensitivity" to a particular therapeutic agent or treatment option means a reduced response to standard doses of the therapeutic agent or to a standard regimen of treatment, which can be achieved by increasing the dose of the therapeutic agent or the intensity of the treatment to compensate (at least in part) for the reduced response.

A subject's "predicted response" and variations thereof may be assessed with any endpoint that demonstrates benefit to the patient, including, but not limited to, (1) some degree of inhibition of disease progression, including slowing and complete cessation; (2) Reduction in number of disease episodes and/or symptoms; (3) Reduction in lesion size; (4) Inhibition (i.e., reduction, slowing, or complete cessation) of disease cell infiltration into adjacent surrounding organs and/or tissues (5) Inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; (6) Reduction of autoimmune response, which may, but does not necessarily lead to regression or detachment of disease focus; (7) A disease associated with the disorder (8) an increase in the length of time exhibited without disease after treatment; and/or (9) a decreased mortality at a given time point after treatment.

As used herein, "lupus therapeutic agent", "therapeutic agent effective in the treatment of lupus" and grammatical variations thereof mean, when provided in an effective amount, known, clinically shown, or expected by a clinician to provide treatment in a subject with lupus Agent of benefit. In one embodiment, the phrase includes any active agent marketed by the manufacturer as a clinically accepted active agent, or otherwise administered by a licensed clinician, which, when provided in an effective amount, is expected to treat lupus Provides a healing effect on objects of . In one embodiment, lupus therapeutic agents include non-steroidal anti-inflammatory drugs (NSAIDs), which include acetylsalicylic acid (eg, 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 therapeutics include acetaminophen (eg, Tylenol), corticosteroids, or antimalarials3 (eg, chloroquine, hydroxychloroquine). In one embodiment, lupus therapeutics include immunomodulatory drugs (eg, 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- receptors (such as anakinra), anti-interleukin 10, anti-interleukin 6 receptor, anti-interferon alpha, anti-B lymphocyte stimulator), co-stimulatory inhibitors (such as anti-CD154, CTLA4-Ig (eg abatacept), B cell energy regulators (eg LJP 394 (eg abetimus)). In one embodiment, lupus therapeutics include hormone therapy (eg, DHEA) and antihormonal therapy (eg, the antiprolactin agent bromocriptine). In one embodiment, the lupus therapeutic is an agent that provides immunoadsorption, is an anti-complement factor (e.g., anti-C5a), T cell vaccination, transfection of cells with the T cell receptor zeta chain, or peptide therapy (e.g., targeting anti-DNA idiotype edratide).

As used herein, a therapeutic agent having "approval for sale" or "approved as a therapeutic agent" or grammatical variations of these phrases means approved, licensed, registered by a relevant governmental entity (e.g., federal, state, or local regulatory agency, department, office) or authorized for sale by and/or through and/or on behalf of commercial entities (e.g., for-profit entities) for the treatment of specific disorders (e.g., lupus) or subgroups of patients (e.g., patients with lupus nephritis, specific ethnicity, gender, lifestyle, disease risk spectrum patients, etc.) active agent (eg pharmaceutical preparation, form of medicament). Relevant government entities include, for example, the US Food and Drug Administration (FDA), the European Medicines Agency (EMEA), and their equivalents.

"Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins with similar structural characteristics. Antibodies display binding specificity for a particular antigen, while immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. Polypeptides of the latter type are produced, for example, at low levels by the lymphatic system and at elevated levels by myeloma.

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, polyclonal antibodies Specific antibodies (eg, 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 antibodies, human antibodies, humanized antibodies and/or affinity matured antibodies.

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 refers especially to antibodies having heavy chains comprising an Fc region.

"Antibody fragment" comprises a portion of an intact antibody, especially comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules;

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

"Fv" is the smallest antibody fragment that contains the entire antigen binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. Together, the six CDRs of the Fv confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment comprises the variable domain of the heavy chain and the variable domain of the light chain, and also comprises the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the C-terminus of the CH1 domain of the heavy chain that contain one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for a Fab' in which the cysteine residues of the constant domains bear a free sulfhydryl group. F(ab') ₂ was originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a population comprising a single antibody, except for possible mutations that may be present in minor amounts, such as naturally occurring mutations. same. Thus, the modifier "monoclonal" indicates the property that the antibody is not a mixture of isolated antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a target-binding polypeptide sequence obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method may be to select a unique clone from a pool of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the sequence of the selected binding target may be further altered, e.g., to improve affinity for the target, to humanize the sequence of the binding target, to improve its production in cell culture, to reduce its in vivo Antibodies that are immunogenic, to produce multispecific antibodies, etc., and that contain the altered target-binding sequence are also monoclonal antibodies of the present invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the property that the antibody was obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used according to the present invention can be produced by various techniques, such as 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. 1998); Hammerling et al., in Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 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.Natl.Acad.Sci.USA101(34):12467-12472(2004); and Lee et al. , J.Immunol.Methods 284(1-2):119-132(2004)) and for producing human or human immunoglobulin loci or genes encoding human immunoglobulin sequences in animals WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 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, et al.); Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-859 (1996); Neuberger, Nature Biotechnol. 14: 826 ( 1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

The monoclonal antibodies herein specifically include "chimeric" antibodies, in which part of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and The remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and 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)).

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which the antibody is derived from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate with Residues from the hypervariable region of the receptor are substituted for residues from the hypervariable region of the desired specificity, affinity and/or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are substituted by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications can be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all Or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For further details see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1998); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

A "human antibody" is an antibody comprising an amino acid sequence corresponding to that of an antibody produced by a human and/or that has been produced using any of the techniques disclosed herein for the production of 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)); use of human myeloma and mouse-human heteromyeloma (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, pages 55-93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)); and in the absence of endogenous immunoglobulin production Monoclonal antibodies are produced in transgenic animals (such as mice) that produce the repertoire of human antibodies (see, for example, 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 human antibody specifically excludes humanized antibodies comprising antigen-binding residues from non-human animals.

An "affinity matured" antibody is one that has one or more alterations in one or more of its CDRs that result in improved affinity of the antibody for an antigen compared to a parent antibody that does not possess these alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by methods known in the art. Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. 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. Random mutagenesis of HVR and/or framework residues is described in Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

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

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

As used herein, the word "label" refers to a detectable compound or composition. The label may itself be detectable (eg, radioisotope or fluorescent label), or in the case of an enzymatic label, it may catalyze a chemical alteration of a substrate compound or composition resulting in a detectable product. Radionuclides that may 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" biomolecule, such as a nucleic acid, polypeptide or antibody, is one that has been identified and separated and/or recovered from at least one component of its natural environment.

Reference herein to "about" a value or parameter includes (described) embodiments that refer to that value or parameter per se. For example, a description referring to "about X" includes description of "X."

general technology

This article provides nucleotide variations associated with lupus. These variations provide biomarkers for lupus and/or predisposing to or contributing to the onset, persistence and/or progression of lupus. Accordingly, the invention disclosed herein finds use in a variety of settings, such as in methods and compositions related to the diagnosis and treatment of lupus.

In certain embodiments, the methods involve prediction, ie, the prediction of disease symptoms attributable to an autoimmune disorder, including, for example, the likelihood of relapse, flaring, and drug resistance of an autoimmune disease such as lupus. 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, eg, treatment with a particular therapeutic agent, and/or its probability of being disease-free for a certain period of time. Treatment decisions can be made clinically using the predictive methods of the present invention by selecting the treatment modality most appropriate for any particular patient. The predictive method of the present invention is to predict whether a patient is likely to respond favorably to a treatment regimen (such as a given treatment regimen, including, for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.), or whether the patient is likely to be Valuable tool for long-term survival following treatment regimens. The diagnosis of SLE can be based on the current American College of Rheumatology (ACR) criteria. Active disease can be defined by one of the British Isles Lupus Activity Group's (BILAG) "A" criteria or by two of the BILAG "B" criteria. Modified from Tan et al. "The Revised Criteria for the Classification of SLE" Arth Rheum 25 (1982) Some signs, symptoms or other indicators for the diagnosis of SLE may be cheek eruptions such as cheek eruptions, discoid eruptions, or red raised plaques photosensitivity, such as a reaction to sunlight, leading to the development or increase of a rash; oral ulcers, such as those in the nose or mouth, that are usually painless; arthritis, such as non-erosive arthritis involving two or more peripheral joints ( Arthritis with undamaged bone around the joint); serositis, pleurisy, or pericarditis; renal dysfunction such as excess protein in urine (greater than 0.5 gm/day or 3+ on test stick) and/or cellular Casts (abnormal composition derived from urine and/or white blood cells and/or renal tubular cells); neurological signs, symptoms, or other indicators, seizures (convulsions), and/or psychosis in the absence of medication, or known Metabolic disturbances causing these effects; and hematologic signs, symptoms, or other indicators, such as hemolytic anemia or leukopenia (white blood cell count less than 4,000 cells/mm ³ ) or lymphopenia (less than 1,500 lymphocytes/mm ³ ) or thrombocytopenia (less than 100,000 platelets/mm ³ ). Leukopenia and lymphopenia generally must be detected at two or more times. Thrombocytopenia generally must be detected in the absence of drugs known to induce thrombocytopenia. The present invention is not limited to these signs, symptoms or other indicators of lupus.

Detection of Genetic Variations

The nucleic acid of any of the above methods may be genomic DNA, RNA transcribed from genomic DNA, or cDNA produced from RNA. Nucleic acids may be derived from vertebrates, such as mammals. A nucleic acid is said to be "derived from" a particular source if it was obtained directly from that source or if it is a copy of a nucleic acid found in that source.

Nucleic acid includes copies of the nucleic acid, eg, copies resulting from amplification. Amplification may be desirable in certain circumstances, for example in order to obtain a desired amount of material for detection of a variant. The amplicon can then be subjected to a variation detection method, such as the method 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. These 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 linker strand reaction (LCR) and gap LCR); allele-specific oligonucleotide hybridization assays (e.g., oligonucleotide ligation assays); cleavage protection assays in which errors in nucleic acid duplexes are detected with substances that protect from cleavage Base matching; MutS protein binding assay; electrophoretic analysis comparing the mobilities of variant and wild-type nucleic acid molecules; denaturing gradient gel electrophoresis (DGGE, such as, for example, Myers et al. (1985) Nature 313:495); RNase at mismatched bases Analysis of cleavage; analysis of chemical or enzymatic cleavage of heteroduplex DNA; mass spectrometry (eg MALDI-TOF); genetic bit analysis (GBA); 5' nuclease assay (eg ); and assays utilizing molecular beacons. Some of these methods are discussed in further detail below.

Detection of variations in a target nucleic acid can be accomplished by molecular cloning and sequencing of the target nucleic acid using techniques well 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 identified therefrom. Amplification techniques are well known in the art, eg polymerase chain reaction as described in Saiki et al., Science 239:487, 1988; US Patent Nos. 4,683,203 and 4,683,195.

The target nucleic acid sequence can also be amplified using the ligase chain reaction known in the art. See, eg, Wu et al., Genomics 4:560-569 (1989). In addition, variants (such as substitutions) can also be detected using a technique known as allele-specific PCR. See eg Ruano and Kidd (1989) Nucleic Acids Research 17:8392; McClay et al. (2002) Analytical Biochem. 301:200-206. In certain embodiments of this technique, allele-specific primers are used, wherein the 3' terminal nucleotide of the primer is complementary to (i.e. capable of specific base pairing with) a specific variation in the target nucleic acid. If the specific variation is absent, no amplification product is observed. The Amplification RefractoryMutation System (ARMS) can also be used to detect mutations (eg, substitutions). ARMS are described, for example, in European Patent Application Publication No. 0332435 and in Newton et al., Nucleic Acids Research, 17:7,1989.

Other methods 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 assay (see eg Ye et al. (3) 5' nuclease assay (see for example De La Vega et al. (2002) BioTechniques 32: S48-S54 (description (2001) GenomeRes.11:1262-1268; and Shi (2001) Clin.Chem.47:164-172); (4) assays utilizing molecular beacons (see, e.g., Tyagi et al. (1998) NatureBiotech. 16:49-53; and Mhlanga et al. (2001) Methods 25:463-71); and (5) oligonucleotide ligation assays (see, eg, Grossman et al. (1994) Nuc. Acids Res. 22:4527-4534; patent Application Publication No. US 2003/0119004A1; PCT International Publication No. WO 01/92579A2; and U.S. Patent No. 6,027,889).

Variants can also be detected by mismatch detection. Mismatches are hybridizing nucleic acid duplexes that are not 100% complementary. The lack of perfect complementarity can be caused by deletions, insertions, inversions or substitutions. An example of a mismatch detection method is the mismatch described in, e.g., Faham et al., Proc. Natl Acad. Sci USA 102: 14717-14722 (2005) and Faham et al., Hum. Repair detection (MRD) assay. Another example of a mismatch cleavage technique is the RNase protection method described in Winter et al., Proc. For example, the methods of the invention may involve the use of labeled riboprobes that are complementary to a human wild-type target nucleic acid. The riboprobes are annealed (hybridized) together with target nucleic acids derived from tissue samples, followed by digestion with the enzyme RNase A, which is capable of detecting some mismatches in duplex RNA structures. If RNase A detects a mismatch, it cleaves at the mismatch site. Thus, when renatured RNA preparations are separated on an electrophoretic gel matrix, if RNase A has detected and cleaved the mismatches, smaller than full-length duplex RNAs with riboprobes and mRNA or DNA will be seen. of RNA products. The riboprobe need not be the full length of the target nucleic acid, but may be a portion of the target nucleic acid as long as it contains the position suspected of having a variation.

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

Variations, such as insertions or deletions, can be detected with restriction fragment length polymorphism (RFLP) probes for the target nucleic acid or surrounding marker genes. Insertions and deletions can also be detected by cloning, sequencing and amplification of target nucleic acids. Single-strand conformation polymorphism (SSCP) analysis can also be used to detect base-change variants of alleles. See, eg, Orita et al., Proc. Natl.

Microarrays are multiplexed technologies, typically using an array of thousands of nucleic acid probes that hybridize to, for example, cDNA or cRNA samples under high stringency conditions. Probe-target hybridization is typically detected and quantified by detecting fluorophore-, silver- or chemiluminescence-labeled targets to determine the relative abundance of nucleic acid sequences in the target. In a typical microarray, probes are attached to a solid surface by covalent bonds (via epoxysilane, aminosilane, lysine, polyacrylamide, or others) to a chemical matrix. Solid surfaces are eg glass, silicon wafers or microscopic beads. Various microarrays are commercially available, including those produced by, for example, Affymetrix, Inc. and Illumina, Inc.

Biological samples can be obtained by certain methods known to those skilled in the art. Biological samples can be obtained from vertebrates, and especially mammals. A representative tumor tissue block is usually obtained by tissue biopsy. Alternatively, tumor cells can be obtained directly in the form of tissue or fluid known or believed to contain the tumor cells of interest. For example, samples of lung cancer lesions can be obtained by excision, bronchoscopy, fine needle aspiration, bronchial brushing, or from saliva, pleural fluid, or blood. Variations in the target nucleic acid (or encoded polypeptide) can be detected from tumor samples or from other bodily samples such as urine, saliva or serum in which tumor cells are shed from the tumor and are present. By screening such body samples, simple early diagnosis of diseases such as cancer can be achieved. Furthermore, the progress of therapy can be more easily monitored by testing such body samples for variations in the target nucleic acid (or encoded polypeptide). Furthermore, methods are known in the art for enriching tissue preparations for tumor cells. For example, tissue can be isolated from paraffin or cryostat sections. Cancer cells can also be isolated from normal cells by flow cytometry or laser capture microdissection.

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

The lupus therapeutic agent may be administered according to known methods, such as intravenous administration as a bolus injection or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerobrospinally, subcutaneously, intraarticularly, intrasynovially, intrathecally, Oral, topical or inhalation routes. Optionally, administration can be by micropump infusion using a variety of commercially available devices.

Effective dosages and schedules for administering lupus therapeutics can be determined empirically and are within the skill in the art to make such determinations. Single or multiple doses may be utilized. For example, an effective dosage or amount of an interferon inhibitor used alone may range from about 1 mg/kg body weight to about 100 mg/kg body weight per day. Interspecies increases and decreases in dosage may be performed in a manner known in the art, eg, as disclosed in Mordenti et al., Pharmaceut. Res., 8:1351 (1991).

When utilizing in vivo administration of a lupus therapeutic agent, normal doses may vary from about 10 ng/kg to up to 100 mg/kg of mammalian body weight per day or more, preferably from about 1 μg/kg/day to 10 mg/kg/day, depending on the route of administration . Guidance on specific dosages and methods of delivery is provided in the literature; see, eg, US Pat. Nos. 4,657,760, 5,206,344, or 5,225,212. It is expected that different formulations will be effective for different therapeutic compounds and different disorders, and targeting administration to one organ or tissue may, for example, necessitate delivery in a different manner than another organ or tissue.

It is contemplated that other therapies may also be utilized in this method. The one or more other therapies may include, but are not limited to, administration of steroids and other standard of care regimens for the disorder in question. It is contemplated that such other therapies may be used as separate active agents from, for example, targeted lupus therapeutics.

Reagent test kit

For use in the applications described or suggested above, kits or preparations are also provided. Such kits may comprise carrier means divided so as to receive in a tight seal one or more container means, eg vials, tubes etc., each container means comprising a separate element for use in the method. For example, a container means can include probes that are or can be detectably labeled. Such probes may be polynucleotides specific for polynucleotides comprising SLE risk loci. Wherein, the kit uses nucleic acid hybridization to detect the target nucleic acid, the kit may also have a container containing one or more nucleotides for amplifying the target nucleic acid sequence, and/or a container containing a reporter tool, such as a biological A protein-binding protein such as avidin or streptavidin conjugated to a reporter molecule such as an enzyme-labeled, fluorescently-labeled or radioisotope-labeled reporter.

Kits generally include the above container and one or more other containers including materials required from a market and user standpoint, including buffers, diluents, filters, needles, syringes, and instructions for use. A label may be placed on the container to indicate that the composition is intended for a particular therapeutic or non-therapeutic use, and may also indicate instructions for in vivo or in vitro use, such as those described above.

Other optional components in the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents such as substrates that are chemically altered by enzyme labels (e.g., chromosomal Original), epitope retrieval solution, control samples (positive and/or negative controls), control slides, etc. Other components are enzymes, for example including but not limited to nucleases, ligases or polymerases.

sales method

Also provided is a method for marketing a lupus therapeutic, or a pharmaceutically acceptable composition thereof, comprising advertising, informing and/or illustrating to a target audience that the therapeutic, or pharmaceutical composition thereof, has been treated in the presence of the present invention in a sample obtained therefrom. Use of a disclosed genetic variation in a lupus patient or subgroup of patients.

Sales are generally paid communications through impersonal media, where the advertiser is identified and the message is controlled. For the purposes of this article, sales include publicity, PR campaigns, product placement, sponsorship, underwriting and promotion. This term also includes sponsored informational announcements appearing in any print communication medium designed to appeal to a large number of readers in order to persuade, inform, promote, stimulate, or otherwise change the preference for purchasing, supporting, or approving the invention mode of behavior.

Marketing of diagnostic methods may be accomplished by any means. Examples of sales media for delivering such information include television, radio, movies, magazines, newspapers, the Internet, and billboards, including advertisements, which are information that appear in broadcast media.

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

Example

In all examples, references to certain publications are noted by number with full bibliographic information at the end of the Examples section.

Example 1

Identification of novel risk loci for SLE

methods and objects

object

SLE cases, samples used for genome-wide association scans (GWAS), and controls from the NewYork Health Project (NYHP) collection have been described previously (Mitchell et al., J Urban Health 81(2):301-10 (2004)) Selection and genotyping (Hom et al., N Engl J Med 358(9):900-9 (2008)). As detailed below, this SLE case consisted of 3 case series: a) 338 from the NIH/NIAMS-funded repository Autoimmune Biomarkers Collaborative Network (ABCoN) (Bauer et al., PLoS medicine 3(12):e491 (2006)). cases and 141 cases from Multiple Autoimmune Disease Genetics Consortium (MADGC) (Criswell et al., Am J Hum Genet 76(4):561-71 (2005)); b) from University of California San Francisco (UCSF) Lupus Genetics 613 cases from the Project (Seligman et al., Arthritis Rheum 44(3):618-25 (2001); Remmers et al., N Engl JMed 357(10):977-86 (2007)); and c) from the University of Pittsburgh Medical 335 cases from the Center (UPMC) (Demirci et al., Ann Hum Genet 71 (Pt 3): 308-11 (2007)) and 8 cases from The Feinstein Institute for Medical Research. Controls were 1861 samples from the NYHP specimen collection, 1722 samples from the publicly available iControlDB database (available at Illumina Inc.) and from the publicly available National Cancer Institute Cancer Genetic Markers of Susceptibility (CGEMS) project (available at 4564 samples obtained under URL: cgems(dot)cancer(dot)gov).

Genome-wide dataset of 1310 SLE cases and 7859 controls

We previously described the selection and genotyping of SLE case samples (Hom et al., N Engl J Med 358(9):900-9 (2008)). All SLE cases were North Americans of European ancestry, as determined by self-report and confirmed by genotyping. The diagnosis of SLE was confirmed in all cases (meeting four or more criteria defined by the American College of Rheumatology [ACR] [Hochberg et al., Arthritis Rheum 40(9):1725 [1997]]). Clinical data for these case series are presented elsewhere (Seligman et al, Arthritis Rheum 44(3):618-25 (2001); Criswell et al, Am JHum Genet 76(4):561-71 (2005); Bauer et al, PLoS medicine 3(12):e419(2006); Demirci et al., Ann Hum Genet 71(Pt 3):308-11(2007); Remmers et al., N Engl J Med 357(10):977-86(2007) . Genotyping and selection of NYHP samples was previously described (Hom et al., N Engl J Med 358(9):900-9 (2008)). Table 1 describes the number of effective samples organized by site.

Sample and SNP filtering was performed with the analysis module within the software programs PLINK and EIGENSTRAT described below (see also Purcell et al., Am J Hum Genet 81(3):559-75 (2007); Price et al., Nat Genet 38(8) : 904-09 (2006)). Genome-wide SNP data were used in this study to facilitate close matching of cases and controls, providing genotypes at confirmed and suspected SLE loci.

Table 1. Number of samples analyzed in genome-wide and locus-organized replication studies

^a depicts a sample of a genome-wide association scan (Hom, G. et al., N Engl J Med 358:900-9 (2008)). ^bIndependent SLE cases from US cohorts from the PROFILE SLE Consortium, University of California San Francisco (UCSF) (Thorburn, CM et al., Genes Immun8:279-87 (2007)), University of Pittsburgh Medical Center (UPMC), Minnesota University (UMN), and Johns Hopkins University (JHU). US controls were from the New York Health Project (Gregersen et al.) and Alzheimer's cases and controls were from the University of Pittsburgh and NCRAD. ^c SLE cases and controls from Stockholm, Karolinska, Solna, Uppsala, Lund and Sweden. ^d 823 controls from Stockholm were genotyped using the Illumina 317K SNP array. SNPs in these samples were assigned and analyzed as described in Methods.

Custom SNP Arrays

A custom array was designed with 10,848 SNPs that passed the quality control measurements described below. The complete array has 12,864 SNPs, but 2016 SNPs did not meet quality control measures, leaving 10,848 SNPs for analysis. The custom array consisted of 3,188 SNPs selected based on nominal P<0.05 in a genome-wide association scan for SLE, 505 SNPs from 25 previously reported SLE risk loci, 42 SNPs were selected from other autoimmune diseases following a literature search for confirmed risk alleles, and 7113 SNPs were used to identify and control for population substructure. The latter group includes SNPs that have been used to define differences in continental populations (Kosoy, R. et al., Hum. Mutat. 30:69-78 (2009)) and SNPs enriched in the substructure of European populations (Tian, C. et al., PLoS Genet 4, e4(2008)). Custom arrays were produced by Illumina, Inc. using its iSelect Custom BeadChip and our provided rs identification numbers for SNPs that passed the quality control filter described below.

Quality control and imputation

For the US data, a total of 1,464 US cases and 3,078 US controls were genotyped on the custom Illumina chip, also referred to herein as the custom 12K chip. Use stringent quality control (QC) standards to ensure high-quality data are included in the final analysis. Specifically, a) 116 individuals with >5% missing data were excluded, and b) based on implicit kinship (cryptic relatedness) and based on Identical by State (IBS) status (PIHat>0.15) The sample was replicated and 279 individuals were excluded. Only SNPs with a) < 5% missing data, b) Hardy-Weinberg Equilibrium (HWE) p-value > 1x10 ^-6 , c) Minor Allele Frequency (MAF) > 0.01% are included and d) SNPs with a p-value > 1 x 10 ⁻⁵ in the test for the loss-of-difference deletion of cases and controls. Batch effects for SNPs were also examined. After applying the above filters, the final set of 1144 cases and 3003 controls and 11024 SNPs was available for analysis. All QC tests were performed using PLINK (Purcell et al., Am J Hum Genet 81(3):559-75 (2007)).

For the Swedish data, a panel of 888 cases and 527 controls genotyped on a custom 12K chip was available for analysis. A combination of separate 1115 Swedish controls genotyped on the Illumina, Inc. 317K Human HapMap SNP Bead Array (also referred to herein as the 317K array) was also incorporated into the analysis. Follow the steps below to combine the two datasets. First, an overlapping dataset of 6,789 SNPs between 12K and 317K data was generated. Use this dataset to test for implied kinship and replicate samples for the Swedish replicate group. Results 313 samples were excluded (PI Hat > 0.15). Following quality control checks, 863 cases and 523 controls shipped genotyped on a custom 12K chip and 831 controls genotyped on a 317KI Illumina chip were analyzed. Second, 831 Swedish controls genotyped with 317K array imputation (see below), resulting in a larger set of overlapping SNPs. Among the remaining SNPs, 4605 SNPs were captured by imputation. A final set of 11394 overlapping SNPs was finally analyzed. SNPs in this dataset were filtered using the same threshold as above. The remaining 1250 SNPs not captured by imputation were analyzed only in the initial set of Swedish samples genotyped on the 12K chip.

Using the Phase II HapMap CEU sample as a reference, 831 haplotypes genotyped with the 317K array were imputed using MACH (Markov Chain-based haplotyping software program, available at URL sph.umich.edu/csg/abecasis/MACH). Swedes control. Phase II HapMap CEU refers to samples from the human haplotype project known to be Utah residents with Northern and Western European ancestry (CEU) released from "Phase II" data (see also Li et al., AmJHum GenetS79 at 2290(2006)). Stringent quality control checks were applied to 317K SNPs prior to imputation. A subset of ^293,242 markers passing the following criteria (1) MAF > 1%, (2) deletion rate < 5% and (3) HWE p-value > 1×10 −6 were included in the imputation. After imputation, SNPs with low imputation quality, ie, MACH reported R squared_Hat(RSQR_HAT)<0.40, were discarded. An overlapping set of 11394 markers was available for analysis. To account for uncertainties in imputation, probability scores were used in the analysis rather than genotype calls.

To impute genome-wide association study samples, genotype data for meta-analysis were obtained from 1310 SLE cases genotyped with the Illumina 550K genome-wide SNP platform (see Hom, G. et al., NEngl J Med 358:900-9 (2008)). The selection and genotyping of SLE case samples was described previously (Hom, G. et al., N Engl J Med 358:900-9 (2008)). In addition to the above 3583 controls (Hom, G. et al., N Engl J Med 358:900-9 (2008)), 4564 cancer genetic susceptibility markers (Cancer Genetics Control samples for the Markers of Susceptibility (CGEMS) project (available at URL: cgems.cancer.gov). The entire 7859 control samples were tested using the data quality control filter described above (Hom, G. et al., N Engl J Med 358:900-9 (2008)). Genotyping using the HapMapPhase II CEU sample as reference was then inferred using IMPUTE version 1 (available at URL www.stats.ox.ac.uk/~marchini/software/gwas/impute.html) (Marchini, J. et al., Nat . Genet. 39:906-913 (2007)). Association statistics were generated using SNPTEST (available at URL www.stats.ox.ac.uk/~marchini/software/gwas/snptest_v1.1.4.html) (Marchini, J. et al., Nat. Genet. 39:906-913( 2007)). Specifically, association statistics were generated using an additive model (Frequentist 1 option in SNPTEST), adjusting for uncertainty in imputed genotypes (-proper option in SNPTEST). A sorted list of correlation statistics is used to select the replicated regions described.

Population Stratification of Replicated Samples

For each replication cohort, ancestry informative markers were used to correct for possible population stratification. Using a subset of 5486 irrelevant ancestry-informative markers that passed stringent quality control criteria, the EIGENSTRAT software was used to infer the top ten major genetic variation components (Price et al., Nat Genet 38(8): 904-09 (2006)). Outliers (defined as σ>6) were removed from each sample set. Specifically, 27 genetic outliers were removed from the US group and 45 from the Swedish group, respectively. Some degree of population stratification along the first two eigenvectors was observed in both the US and Swedish replication sets. To correct for case-control stratification, one of the following strategies was used: (1) the correction for the Cochran-Armitage test statistic incorporated in EIGENSTRAT was applied to the US replicate dataset and the Swedish replicate dataset for which genotype data were available; ( 2) In analyzing the imputed Swedish data, use principal components as covariates in the logistic regression model.

Correlation analysis

For the US data, some inflation of the test statistic was observed after implementing an allelic association test with an uncorrected degree of freedom of 1 (PLINK (Purcell, S. et al., Am J Hum Genet 81:559 -75(2007))). To correct for population stratification in the US sample, principal component analysis (EIGENSTRAT) was performed using 5486 irrelevant ancestry-informative markers. First, genetic outliers (defined as σ > 6) were removed. Second, the Cochran-Armitage trend chi-square test statistic for each genotyped SNP in 1129 cases and 2991 controls was calculated, and then the first four eigenvectors were used to adjust the test statistic for each SNP in EIGENSTRAT. A two-tailed p-value based on the test statistic for each SNP was calculated. After correcting for population stratification, the λ _gc for the US sample was 1.05.

For the Swedish data, hidden population stratification of the Swedish group was examined using 5486 ancestry informative markers genotyped in 12K samples plus an additional Illumina 317K control. After removal of genetic outliers, 834 cases and 515 controls were genotyped on a custom 12K chip, and 823 controls were genotyped on an Illumina 317K chip. The test statistic correction performed in EIGENSTRAT was used for an overlapping set of 6789 SNPs between the two Illumina arrays. To correct for the stratification of the 4605 SNP groups genotyped in 12K samples and imputed in Illumina 317K samples, the first four eigenvectors determined above were used as covariation in the logistic regression model performed in SNPTEST, since EIGENSTRAT Genotype data not intended for imputation. A small set of 1250 markers not captured by Illumina 317K SNP imputation was analyzed in only 834 cases and 515 controls genotyped on a custom 12K array. After correcting for population stratification, the lambda _gc for the Swedish sample was 1.10.

Meta-analysis

Meta-analysis was performed using the weighted z-score method. To combine results from different groups, alleles were mapped on the forward strand of the National Center for Biotechnology Information (NCBI) Human Genome Reference Sequence No. 36 to avoid ambiguities associated with C/G and A/T SNPs. NCBI's Human Genome Reference Sequence is available at URL: www.ncbi.nlm.nih.gov . See also Pruitt et al., Nucl. Acids Res. 35 (database issue): D61-D65 (2007). P-values for each group were converted to z-scores considering the direction of the effect relative to an arbitrary reference allele. A weighted sum of z-scores was calculated by taking the square root of the sample size for each group and then dividing their sum by the square root of the total sample size, weighting each z-score. The combined z-scores for the Swedish and American replication groups were converted to one-tailed p-values. The z-scores from the meta-analysis were converted to two-tailed p-values and the evidence for association was calculated. SNPs crossing a threshold of ^5x10-8 were considered to be overwhelmingly associated with SLE. Loci that failed genome-wide significance with combined p-values of less than 1×10 ⁻⁵ were considered strong candidates. The meta-analysis method was performed using the freely available METAL software package (available at URL: www.sph.umich.edu/csg/abecasis/Metal ). To calculate pooled odds ratios, the Cochran-Mantel-Haenszel (CMH) method implemented by METAL software was used. Odds ratios were calculated relative to the risk allele for each SNP. In addition, the weighted average allele frequency in controls was calculated relative to the risk allele for each SNP.

Percent Variance Explained

The percentage of variance explained was calculated for SNPs previously associated with SLE and for SNPs with meta p-values less than ^1x10 in our replication study. A liability threshold model was used where SLE was assumed to have underlying liability scores normally distributed with mean 0 and variance 1 . Assume that the prevalence of SLE in the general population is 0.1%. To calculate thresholds for each genotype, the allele frequencies in the controls and the effect sizes corresponding to the odds ratios (ORs) in our analysis were used.

interaction analysis

To look for epistasis effects between top signals, lists of all SNPs were compiled in Tables 2, 4 and 6 and interaction analyzes were performed for each replicate group using the epistasis option performed in PLINK. For greater statistical power, a case-only analysis was performed. After correcting for the number of tests, no SNP-SNP interactions were found at a significance level of p<0.05.

conditional analysis

In each genomic region exhibiting a strong association with SLE, the SNP exhibiting the strongest signal was selected. Use PLINK as a condition for this SNP and look for other SNPs that show a strong association with SLE.

Large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL10 in systemic lupus erythematosus novel risk loci

Existing genome-wide association (GWA) and candidate gene studies have identified at least 15 common risk alleles that reached genome-wide significance (P<5x 10 ^-8 ). These include genes important for adaptive immunity and autoantibody production (HLA class II alleles, BLK, PTPN22, and BANK1), and genes with roles in innate immunity and interferon signaling (ITGAM, TNFAIP3, STAT4, and IRF5) (Cunninghame Graham, DS et al., Nat. Genet. 40:83-89 (2008); Graham, RR et al., Nat. Genet. 40(9): 1059-61 (2008); Graham, RR et al. , J Intern Med 265: 680-88 (2009); Harley, JB et al., Nat. Genet. 40: 204-10 (2008); Hom, G. et al., N Engl J Med 358: 900-9 (2008 ); Kozyrev, SV et al., Nat. Genet. 40: 211-6 (2008); Sawalha, AH et al., PLoSONE 3: e1727 (2008); Sigurdsson, S. et al., Am J Hum Genet 76: 528- 37 (2005)). To identify additional risk loci, targeted replication studies were performed on SNPs of 2446 loci exhibiting nominal p-values <0.05 in the existing GWAS7 scan of 1310 cases and 7859 controls. SNPs from 25 previously reported SLE risk loci, 42 SNPs from 35 loci suggestive of other autoimmune diseases, and more than 7000 ancestry-informative markers were also genotyped. An overview of the experimental design is shown in Figure 1. The above SNPs were incorporated into an Illumina custom-made SNP array. Array Genotyping Independent Cases and Controls from the US and Sweden. 823 Swedish controls were genotyped using the Illumina 310K SNP array, and variants were analyzed as described in Methods, above.

Specifically, as described above, custom SNP arrays consisting of >12,000 variants were designed and genotyped into 2 independent SLE case and control populations from the United States (1129 SLE cases and 2991 controls) and Sweden (834 SLE cases and 1338 controls). The U.S. controls included 2215 Alzheimer's disease case/control samples, which were considered acceptable controls because the genetic basis of SLE and Alzheimer's disease were expected to be independent of each other. Afterwards, data quality filtering was applied to remove low performing samples and SNPs, population outliers and duplicate/related individuals (see 'Methods' above). After these quality control measures, a final set of 10848 SNPs, as shown in Figure 1, was tested. Association statistics were calculated for 3735 variants and corrected for population stratification using 7113 ancestry-informative markers (see 'Methods' above).

We first examined 25 variants (from 23 loci) previously reported to be associated with SLE (see Table 2). We also found additional evidence of association for 21 variants (P<0.05), including 9 loci that reached genome-wide significance (P<5×10 ⁻⁸ ) in the existing combined dataset. Among the genome-wide significant results were HLA class II DR3 (DRB1*0301), IRF5, TNFAIP3, BLK, STAT4, ITGAM, PTPN22, PHRF1 (KIAA 1542) and TNFSF4 (OX40L). This analysis provided additional evidence for variants at nine loci with significance reported at the genome-wide level in a previous study: HLA*DR2, TNFAIP3 (rs6920220), BANK1, ATG5, PTTG1, PXK, FCGR2A , UBE2L3 and IRAK1/MECP2.

An earlier candidate gene study identified MECP2 as a potential risk allele for SLE (Sawalha, A.H. et al., PLoS ONE 3: e1727 (2008)). However, in the available dataset, SNPs near IRAK1, a key gene for toll-like receptor 7 and 9 signaling, within the identified linkage disequilibrium region around MECP2 showed the strongest evidence for association . Similar findings have been reported recently (Jacob, C.O. et al., Proc. Natl. Acad. Sci. USA (2009)), and additional work is needed to determine the causal alleles in the IRAK1/MECP2 locus. We found additional evidence of association for 3 loci—TYK2, ICA1, and NMNAT2—that previously showed significance but had no genome-wide evidence of association (Harley, J.B. et al. Nat. Genet. 40:204-10 (2008); Sigurdsson, S. et al., Am J Hum Genet 76:528-37 (2005)). For the 4 previously suggested variants—LYN, SCUBE1, TLR5, and LY9—no evidence of any association was observed in the combined dataset values.

To identify novel SLE risk loci, we examined a total of 3188 SNPs from 2446 different loci that showed evidence of association with SLE in our genome-wide dataset (Hom, G. et al., N EnglJ Med 358, 900-9 (2008)), the data set included 502033 SNPs genotyped in 1310 SLE cases and an expanded set of 7859 controls. Using this dataset, >2.1M variants were imputed using the Phase II HapMap CEU sample as a reference (see 'Methods' above), and an ordered list of association statistics was generated. Variants with P<0.05 were selected for possible inclusion on custom replicate arrays. For efficient genotyping, groups of correlated variants ( ^r2 >0.2) were identified and at least 2 SNPs were selected from each group with the lowest p-value<0.001. For the remaining groups, the SNP with the lowest p-value in the group was included. Among replicate samples, association statistics were calculated (see Methods) and significant enrichment was observed for replicate outcomes relative to the expected null distribution. Excluding previously reported SLE risk alleles, there were 134 loci with P<0.05 (expected 64, P=2×10 ⁻¹⁵ ) and 12 loci with P<0.001 (expected 1, P=1×10 ⁻⁹ ), Indicates the presence of true positives.

Figures 2A-2 each show that within a 500kb region around loci defined by TNIP1 (Figure 2A), PRDM1 (Figure 2B), JAZF1 (Figure 2C), UHRF1BP1 (Figure 2D) and IL-10 (Figure 2E), relative Genomic position on the x-axis, association results from genome-wide association scans plotted on the y-axis. In each of Figures 2A-2E, solid squares indicate the meta-analysis P values for the most relevant markers. For each of Figures 2A-2E, P-values from genome scans are marked to represent the LD of variants associated with the genome: dotted circles indicate ^r2 >0.8, dashed circles, ^r2 >0.5; streaked circles, ^r2 >0.2; open circle r ² <0.2. Recombination rates in the CEU HapMap (solid black line) and known human genes are shown below each graph along the bottom of each graph in Figures 2A-2E. In Figure 2B (PRDM1), a previously reported and independent SLE risk locus (rs2245214) near the ATG5 gene is marked by a solid black circle. Figure 2F shows a histogram of P values for 1256 independent SNPs ( ^r2 <0.1 with any other SNP in the array) among 1963 case and 4329 control replicate samples. Under the null distribution, the expected resulting density is marked with a dashed line in Figure 2F. As shown in Figure 2F, a significant enrichment of results less than P<0.05 was observed.

Thus, the replication study identified 5 novel SLE risk loci with combined P-values (P<5×10 ⁻⁸ ) above the genome-wide significance threshold: TNIP1 , PRDM1 , JAZF1 , UHRF1BP1 , and IL10. Detailed statistical associations for these and other loci are shown in Table 4 below.

The variant rs7708392 on 5q33.1 was significantly associated with SLE in all 3 groups with a combined P = 3.8x10 ^-13 (Fig. 2A), which is located on interacting protein 1 of TNF-α-inducible protein 3 (TNFAIP3) (TNIP1) intron. Variants close to TNIP1 were recently found to have an effect on psoriasis risk (Nair, RP et al., Nat Genet 41:199-204 (2009)), however the SLE and psoriasis variants are separated by 21 kb and appear to be distinct genetic signals ( r ² =0.001). TNIP1 and TNFAIP3 are interacting proteins (Heyninck, K. et al., FEBS Lett 536:135-40 (2003)), however, the precise role of TNIP1 in regulating TNFAIP3 is unknown. Several different variants near TNFAIP3 are associated with SLE (Graham, RR et al., Nat. Genet. 40(9): 1059-61 (2008), Musone, SL et al., Nat. Genet. 40(9): 1062- 64 (2008)), rheumatoid arthritis (Plenge, RM et al., Nat Genet 39:1477-82 (2007)), psoriasis (Nair, RP et al., Nat Genet 41:199-204 (2009)) and The association of type I diabetes (Fung, EY et al., Genes Immun 10: 188-91 (2009)) suggests that this pathway has an important role in the regulation of autoimmune diseases.

The second identified risk variant (rs6568431, P=7.12x10 ^-10 ) is between PR domain-containing 1 (PRDM1, also known as BLIMP1) with ZNF domains and APG5 autophagy 5-like (ATG5). identified within the intergenic region. The signal of rs6568431 appears to be different from the previously reported SLE risk allele rs2245214 in ATG5 (Harley, JB et al., Nat Genet 40:204-10 (2008)) (see Table 4) because rs6568431 shares r ² < 0.1, while rs2245214 was still significantly associated with SLE after the conditional logistic regression incorporating rs6568431 (P<1×10 ⁻⁵ ) ( FIG. 2B ).

The promoter region of 1 (JAZF1) juxtaposed with another zinc finger gene was the third newly identified SLE locus (rs849142, P=1.54×10 ⁻⁹ ) ( FIG. 2C ). Interestingly, the same variant was previously associated with risk of type 2 diabetes (Zeggini, E. et al., Nat Genet 40:638-45 (2008)) and difference in height (Johansson, A. et al., Hum Mol Genet 18 : 373-80 (2009)) associated. The isolated prostate cancer allele rs10486567 close to JAZF1 (Thomas, G. et al., Nat Genet 40:310-5 (2008)) did not show any evidence of association in this study.

A non-synonymous allele (R454Q) of ICBP90-binding protein 1 (UHRFBP1, rs11755393, P= ^2.22x10-8 ) defines a 4th novel risk locus in SLE. This allele is a non-conserved amino acid change in the putative binding partner of UHRF1, a transcription and methylation factor associated with multiple pathways (Arita, K. et al., Nature 455:818-21 (2008 )). The UHRFBP1 risk allele lies within an extended linkage disequilibrium region encompassing multiple genes, including small nuclear ribonucleoprotein polypeptide C (SNPRC), part of the RNA processing complex frequently targeted by SLE autoantibodies.

The fifth novel SLE locus identified was interleukin-10 (IL10; rs3024505, P = 3.95x10 ^-8 ) (Fig. 2E). IL-10 is an important immunoregulatory cytokine that functions by downregulating the immune response (Diveu, C. et al., CurrOpin Immunol 20:663-8 (2008)), and the association of variants in IL-10 with SLE has been inconsistently reported (Nath, SK et al., Hum Genet 118:225-34 (2005)). Variants associated with SLE are the same as those currently identified for the treatment of ulcerative colitis (Franke, A. et al., Nat Genet 40:1319-23 (2008)) and type 1 diabetes (Barrett, JC et al., Nature Genetics 41:703- 707 (2009)) contributed to the risk of the same SNP, suggesting that there may be a shared pathophysiology of the IL10 pathway between these disorders.

Using a significance threshold of P<1×10 ⁻⁵ in the combined replicate samples, 21 additional SLE candidate risk loci were identified (Table 4). Under the null distribution condition of the meta-analysis, less than 1 (0.01 ) locus was expected to have P<1×10 ⁻⁵ (P=8×10 ⁻⁷⁷ ), suggesting that several of these loci may be true positive loci. Interesting candidate genes in this list include: a) Interferon Regulatory Factor 8 (IRF8), which was suggested in a previous GWAS (Graham, RR et al., Nat. Genet. 40(9): 1059- 61 (2008)), and its family members IRF5 and IRF7, both fall into confirmed SLE risk loci; b) TAO kinase 3 (TAOK3), which is a missense allele of a kinase expressed in lymphocytes (rs428073 , N47S); c) lysosomal trafficking regulator (LYST), mutations of which cause Chediak-Higashi syndrome in humans, a complex disorder characterized by lymphoproliferative disorders; and d) interleukin 12 receptor beta 2 (IL12RB2 ), a locus that includes IL23R and SERPBP1 but appears to be distinct from the IL23R variants reported in the autoimmune diseases inflammatory bowel disease, psoriasis, and ankylosing spondylitis (Duerr, RH et al., Science 314:1461-3( 2006)).

A notable feature of current GWA studies is the large number of overlapping loci shared between different complex diseases (Zhernakova, A. et al., Nat Rev Genet 10:43-55 (2009)). We tested 42 variants (Tables 6 and 7) from 35 loci previously reported as autoimmune disease risk alleles associated with SLE. However, no single locus had an unadjusted P-value < 5x10 ^-8 , but enrichment of associated alleles was found. From the 35 tested loci (total of 42 variants), 5 alleles had an unadjusted P<0.0004 (less than one outcome expected by the probability of passing, P=4.4x10 ⁻¹² ), and in the Thirty-five previously described loci had P<0.05 after Bonferroni correction. For each of the five variants, the SLE-associated alleles matched previously reported alleles and had the same direction of effect (Table 6). A very significant association was observed for the missense allele of IFIH1 (rs1990760, p= ^3.3x10-7 ), which was previously associated with type 1 diabetes and Grave's disease (Smyth, DJ et al., Nat Genet 38:617- 9 (2006); Sutherland, A. et al., J Clin Endocrinol Metab 92:3338-41 (2007)). An association was also observed for the missense allele (R32Q) of complement factor B (CFB, rs641153), which is located in a region of HLA class III and is a validated risk allele for age-related macular degeneration (Gold , B. et al., Nat Genet 38:458-62 (2006)). SLE risk alleles were not in significant linkage disequilibrium (LD) with other SLE-associated HLA region variants (DR2/DR3), which remained significant after a conditional logistic regression analysis integrating DR2 and DR3. HLA is a complex genetic region, but surprisingly the allele of SNP rs641153 has nearly the same allele as the reported AMD risk allele (Gold, B. et al., Nat Genet 38:458-62 (2006)) protective effect. Additional studies of 5 candidate disease alleles are indicated.

In addition, Table 7 provides detailed summary statistics for the 42 variants identified in other autoimmune diseases. Interestingly, variants of CTLA4, IL23R, NOD2, and CD40, prominent risk factors in other autoimmune diseases, do not appear to exhibit any evidence of an association with SLE.

Using the 26 SLE risk alleles (21 loci previously reported in Table 2, and 5 novel SLE loci described above), some additional analyzes were performed. Pairwise interaction analysis with identified loci, compared previously with SLE (Harley, J.B. et al., Nat Genet 40:204-10 (2008)) and other complex diseases (Barrett, J.C. et al., Nat Genet 40:955 -62 (2008)), no evidence of any non-additive interactions was observed. Using conditional logistic regression analysis, we did not find any evidence that multiple independent alleles contribute to the risk of any single risk locus. The percentage of variance explained by each identified SLE risk allele was then estimated using the method described by Barrett et al. (Barrett, J.C. et al., Nat Genet 40:955-62 (2008)). HLA-DR3, IRF5, and STAT4 were each estimated to account for >1% of the genetic variance, while the remaining loci each accounted for less than 1% of the variance. Altogether, these 26 SLE risk loci explained about 8% of the total genetic susceptibility to SLE.

Targeted replication of GWAS results is an effective study design to validate additional risk loci (Hirschhorn, J.N. et al., Nat Rev Genet 6:95-108 (2005)). However, very few data are available on the probability of replication results lacking acceptable P-value criteria for genome-wide significance. In this study, replication included all variants with P<0.05 from the original GWAS study. As shown in Figure 3, the lower the P-value in a GWAS study, the higher the probability of achieving candidate or validation status in the replication meta-analysis. Interestingly, no candidate or validation results were obtained in existing studies of variant groups with GWAS P between 0.05 and 0.01, although said group accounted for ~1% of all variants tested in replication. 50%. These results can be used to guide the design of additional targeted studies, although it is true that the size of the original GWAS population, replication sample size, disease structure, and effect size of candidate variants need to be carefully considered in planning replication efforts.

These data provide further evidence that common variants in genes important for the secondary and innate defense functions of the immune system are important for establishing the risk of developing SLE. Although each identified allele is responsible for only a fraction of the overall genetic risk, these and other ongoing studies provide new insights into lupus pathogenesis, suggesting new targets and pathways for drug discovery and development.

Example 2

Resequencing and identification of causal alleles of BLK

As noted above, BLK was identified as a SLE-associated risk locus that reached genome-wide significance (P<5×10 ⁻⁸ ). To further characterize the genetic basis of this association and identify causal alleles, we performed desequencing studies of the BLK locus and reporter gene expression assays described below.

For the resequencing study, all 13 exons and the 2.5 kb upstream promoter sequence of the BLK locus were resequenced in DNA isolated from the NIH/NIAMS-funded Repository-Autoimmune Disease Biomarkers Collaboration Network (ABCoN) of 192 patients (Bauer et al., PLoS medicine 3(12):e491 (2006)), and the New York Cancer Program (NYCP) of 96 control individuals (Mitchell et al., J. Urban Health 81:301 -10(2004)). Genomic DNA was whole genome amplified according to the manufacturer's protocol (Qiagen, Valencia, CA., Cat. No. 150045) prior to sequencing.

The results of re-sequencing showed that 17 mutations (10 non-synonymous and 7 synonymous) were found in the coding region of the BLK gene (Table 8). None of these mutations exhibited a significantly higher frequency in cases than in controls. The overall frequency of non-synonymous mutations was not significantly higher in cases (14/191) than in controls (7/96).

In addition, multiple common mutations were identified in the non-coding region of BLK (shown in Table 9). Three SNPs (rs4840568, rs1382568 [triallelic SNP (A/C/G); the C allele was previously identified as a risk allele] and rs922483 (SEQ ID NO: 13)) showed similar The identified loci (Hom et al., N Engl J Med 358:900-09 (2008)) had an association of r ² >0.5 (rs13277113, odds ratio, 1.39, P=1×10 ⁻¹⁰ ). Figure 4 shows the HAP in the promoter region of BLK generated using Haploview (software freely available at URL www.broadinstitute.org/haploview/haploview; see Barrett JC et al., Bioinformatics 21:263-65 (2005)). Linkage disequilibrium (LD) blocks (shown as ^r2 ). The upper part of the figure shows a schematic representation of the BLK promoter region with the relative positions of the identified SNPs indicated. The ^r2 values between the enumerated SNPs are shown in boxes. The strength of LD between two SNPs is represented by the ^r2 value, provided in each box. The locus identified from GWAS (rs13277113) and the three SNPs identified by resequencing (rs4840568, rs1382568 and rs922483 (SEQ ID NO: 13)) are marked in black bold in the upper part of the figure.

This resequencing study did not reveal any common variants in the BLK coding region. However, three common variants in the promoter region (rs4840568, rs1382568, and rs922483 (SEQ ID NO: 13)) were identified as one or more potential causal alleles for the biological effects of BLK associated with increased SLE risk . Each of these variations was used in the luciferase reporter assay detailed below to further characterize the association.

Table 8. Mutations in the BLK coding region

Table 9. Common variations in BLK non-coding regions ('rs922483' disclosed as SEQ ID NO: 13)

A luciferase reporter gene assay was performed to study the effect of three SNPs - rs4840568, rs1382568 and rs922483 (SEQ ID NO: 13) - on BLK-mediated gene expression. The upstream sequence of BLK (-2256 to +55 bp) was amplified using genomic DNA from individuals carrying the at-risk or non-risk haplotype. Each PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA; Cat. No. K4500-01) and then subcloned into the pGL4 luciferase reporter gene vector (Promega, Madison, WI; Cat. No. E6651). Various haplotypes were generated using the construct carrying the risk-free haplotype as template for mutagenesis (Stratagene, La Jolla, CA; Cat. No. 10519-5).

The primers used for PCR amplification were as follows: Forward: CCACCTCTCTTCCGCCTTTCTCAT (SEQ ID NO.: 1); Reverse: TTTCATGGCTTGTGGCTTTCTGCC (SEQ ID NO.: 2). Primers used for mutagenesis are listed in Table 10 below.

Table 10. List of mutagenesis primers

Normalization was performed using the Renilla luciferase control reporter vector pRL-TK (Promega, Madison, WI; Cat. No. E2241). The cell line BJAB (persistent lymphoid cell line with B-cell characteristics (of bone marrow origin), lacking the Epstein-Barr virus genome, was derived from three human lymphomas; Klein et al., Proc.Natl.Acad.Sci.USA71:3283 -86 (1974)) or the Daudi cell line (American Type Culture Collection (ATCC) Cat. No. CCL-213) was used for transfection. For each transfection, use device (Lonza, Walkersville Inc., Walkersville, MD (Lonza Group Ltd., Switzerland); Cat. No. AAD-1001 ), 5 x ^{10 6} cells were transfected with 5 μg of each vector DNA. Daudi cells using cell lines Kit L (Lonza, Cat. No. VCA-1005) and Installation Procedure A-030. Cell lines used for BJAB cells Kit V (Lonza, Cat. No. VCA-1005) and Device program T-020. All transfections were performed in duplicate or triplicate. After transfection, cells were incubated at 37°C for 16 hours. After incubation, cells were harvested and used according to the manufacturer's instructions The reporter assay system (Promega, Madison, WI; Cat. No. E1960) measures luciferase activity.

The effect of each of the SNPs: rs4840568, rs1382568 and rs922483 (SEQ ID NO: 13) measured in the luciferase reporter assay described above, on BLK-mediated gene expression is shown in FIG. 5 . The different haplotypes produced by mutagenesis were compared with the risk-free (wild-type) haplotype 22-GAC (open bars in each of Figure 5A-F ) and the risky haplotype 22-ACT (shaded bars in each of Figure 5A-F )Compare.

Figures 5A and 5B show that SNP rs922483 (C>T) (SEQ ID NO: 13) leads to a significant effect on BLK-mediated gene expression in both BJAB (Figure 5A) and Daudi cells (Figure 5B). Haplotype 22-ACT exhibited an almost 50% reduction in transcriptional activity in both cell lines compared to the risk-free haplotype 22-GAC (open bars). Haplotypes containing the T allele consistently exhibited lower activity than haplotypes containing the C allele. Five independent experiments were performed in BJAB cells and six independent experiments were performed in Daudi cells. Data shown represent mean +/- standard error of the mean (s.e.m.) in triplicate determinations; *p<0.05, **p<0.01, ***p<0.001 (t-test).

Figures 5C and 5D show that SNP rs1382568 (A>C/G>C) did not lead to any significant effect on BLK-mediated expression in each cell line. Both haplotypes 22-GCC and 22-GGC (dotted bars) exhibit similar levels of luciferase activity compared to the risk-free haplotype 22-GAC (open bars). Five independent experiments were performed in BJAB cells and six independent experiments were performed in Daudi cells. Data shown represent means +/- s.e.m. of triplicate assays; *p<0.05, **p<0.01, ***p<0.001, ns=not significant (t-test).

Figures 5E and 5F show that SNP rs4840568 (G>A) did not lead to a significant effect on BLK-mediated expression in either BJAB cells or Daudi cells. The difference between the haplotype 22-AAC (dotted bars) and the risk-free haplotype 22-GAC (open bars) was not statistically significant in BJAB cells (Fig. 5E), but was statistically significant in Daudi cells Significantly (Fig. 5F). Considering that the haplotype 22-ACC (dotted bars) does not exhibit any defect in luciferase activity compared to the risk-free haplotype 22-GAC (open bars), the A allele is highly likely to be the causal allele large decrease (Fig. 5F). Data shown represent means +/- s.e.m. of triplicate assays; *p<0.05, **p<0.01, ***p<0.001, ns=not significant (t-test).

It was previously shown that (GT) repeats in the upstream region of the BLK promoter can function as enhancers of BLK gene expression (Lin et al., J Biol Chem 270:25968 (1995)). Therefore, we also tested whether the length of the (GT) repeat could affect the transcriptional activity of the BLK promoter. To carry out these experiments, using the strategies described above, genomic DNA samples from individuals carrying both 18(GT) repeats (SEQ ID NO: 14) or 22(GT) repeats (SEQ ID NO: 15) were selected for cloning. Five vectors were sequenced to verify that they contained (GT) repeats of the correct length. As shown in Figure 6, the haplotype containing 18 (GT) repeats (SEQ ID NO: 14) exhibited a similar expression to the haplotype containing 22 (GT) repeats (SEQ ID NO: 15) in the luciferase reporter assay. similar levels of transcriptional activity. Data shown represent means +/- s.e.m. in duplicate assays; ns = not significant (t-test).

Taken together, these results of the BLK resequencing work and the results of the luciferase reporter gene assay indicate that SNPrs922483 (C>T) (SEQ ID NO: 13) is the causal allele leading to reduced BLK transcription of Biological effects are associated with increased SLE risk. Furthermore, the results showed that the T allele of rs922483 (SEQ ID NO: 13) reduced BLK-mediated gene expression levels by 50%.

It is interesting to note that rs922483 (SEQ ID NO: 13) is located in an evolutionarily conserved region of the first exon of BLK, in a possible human transcription initiation site. The consensus sequence of the human Inr motif is identified as YYANWYY (IUPAC nucleotide code). Juven-Gershon et al., Dev. Biol. 339:225-229 (2010). In SNP rs922483 (SEQ ID NO: 13), the second base in the Inr region was altered relative to the common motif. Thus, the SLE risk haplotype Inr sequence is CTACCTC, whereas the "wild-type" haplotype Inr sequence is CCACCTC. It is suggested that modification of the second base in the conserved Inr motif may alter the affinity of the TFIID transcription complex, leading to the observed differences in transcription described above.

Claims (40)

1. A tool for detecting the presence of variations in the TNIP1 locus and optionally the presence of variations in at least one SLE risk locus described in Table 4 in a biological sample derived from an object is prepared for identifying lupus in an object The purposes in the test kit or preparation of wherein said TNIP1 locus is the cytosine of single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence as shown in SEQ ID NO:16 Alleles, and wherein the variation in at least one SLE risk locus described in Table 4 occurs at the nucleotide corresponding to the position of the single nucleotide polymorphism (SNP) of at least one locus described in Table 4 location, and wherein the subject is suspected of having lupus. 2. The use of claim 1, wherein the tool is used at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or Variants were detected in at least 13 loci, or 26 loci. 3. Use according to claim 1, wherein said tool is also used to detect the presence of a variation in the BLK locus, wherein the variation in BLK is at rs922483 in the nucleotide sequence as shown in SEQ ID NO: 13 Thymine alleles of single nucleotide polymorphisms (SNPs). 4. The use of any one of claims 1-3, wherein the variation in the at least one locus further comprises variation in one or more of PRDM1, JAZF1, UHRF1BP1 and IL10, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of the SNP at rs11755393 in the nucleotide sequence as set forth in SEQ ID NO: 19; and The variation in IL10 is the adenine allele of the SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20. 5. The use of any one of claims 1-3, wherein the variation in the at least one locus further comprises variation in one or more of IFIH1, CFB, CLEC16A, IL12B, and SH2B3, and wherein The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 6. The use of any one of claims 1-3, wherein the variation in the at least one locus also includes variation in one or more of PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B and SH2B3 , and where The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of SNP at rs11755393 in the nucleotide sequence as shown in SEQ ID NO: 19; The variation in IL10 is an adenine allele at SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20; The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 7. The use of any one of claims 1-3, wherein the variation in at least one SLE risk locus described in Table 4 comprises the SNPs described in Table 4. 8. The purposes of any one of claims 1-3, said tool is also used to detect other variations in at least one SLE risk locus described in Table 6, wherein other variations in at least one locus occur in the same On the nucleotide position corresponding to the SNP position of at least one locus described in Table 6. 9. The use of any one of claims 1-3, wherein detecting comprises performing a primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays. 10. The use of claim 9, wherein the primer extension assay is an allele-specific primer extension assay. 11. A tool for determining whether a subject is comprised of a variation in the TNIP1 locus and optionally a variation in at least one SLE risk locus described in Table 4 is used in preparation for predicting the responsiveness of a subject with lupus to a lupus therapeutic agent The purposes in the test kit or preparation of wherein said TNIP1 locus is the cytosine of single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence as shown in SEQ ID NO:16 Alleles, and wherein the variation in at least one SLE risk locus described in Table 4 occurs at a nucleotide position corresponding to a single nucleotide polymorphism (SNP) position of at least one locus described in Table 4 above, wherein the presence of a variation in the TNIP1 locus and optionally at least one of the loci described in Table 4 indicates responsiveness of the subject to a therapeutic agent. 12. The use of claim 11 , wherein the tool is used at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or Variants were detected in at least 13 loci, or 26 loci. 13. The purposes of claim 11, wherein said tool is also used to detect the presence of a variation in the BLK locus, wherein the variation in BLK is a single at rs922483 in the nucleotide sequence as shown in SEQ ID NO:13 Thymine alleles of nucleotide polymorphisms (SNPs). 14. The use of any one of claims 11-13, wherein the variation in at least one locus further comprises variation in one or more of PRDM1, JAZF1, UHRF1BP1, and IL10, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18 The variation in UHRF1BP1 is the guanine allele of the SNP at rs11755393 in the nucleotide sequence as set forth in SEQ ID NO: 19; and The variation in IL10 is the adenine allele of the SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20. 15. The use of any one of claims 11-13, wherein the variation in the at least one locus further comprises variation in one or more of IFIH1, CFB, CLEC16A, IL12B, and SH2B3, and wherein The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 16. The use of any one of claims 11-13, wherein the variation in the at least one locus also includes one or more of PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B and SH2B3 variation, and among them The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of SNP at rs11755393 in the nucleotide sequence as shown in SEQ ID NO: 19; The variation in IL10 is an adenine allele at SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20; The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 17. The use of any one of claims 11-13, wherein the variation in at least one SLE risk locus described in Table 4 comprises the SNPs described in Table 4. 18. The purposes of any one of claims 11-13, wherein said tool is also used to determine whether the subject comprises other variations in at least one SLE risk locus described in Table 6, wherein in at least one locus Other variations occur at nucleotide positions corresponding to the SNP positions of at least one of the loci described in Table 6. 19. A tool for detecting variation in the TNIP1 locus and optionally the presence of variation in at least one SLE risk locus described in Table 4 in a biological sample derived from an object is prepared for diagnosing or Use in a kit or preparation for predicting lupus, wherein the diagnosis or prediction comprises: (a) the biological sample is known to include or is suspected to include a nucleic acid comprising a variation in the TNIP1 locus and optionally at least one SLE risk locus described in Table 4; (b) the variation in the TNIP1 locus is a cytosine allele of a single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence as shown in SEQ ID NO: 16; (c) the variation in at least one SLE risk locus described in Table 4 includes the SNP described in Table 4 or is located on the nucleotide position corresponding to the SNP described in Table 4; and (d) the presence of a variation in the TNIP1 locus and optionally at least one of the loci described in Table 4 is diagnostic or predictive of lupus in the subject. 20. The use of claim 19, wherein the tool is used at least two loci, or at least three loci, or at least four loci, or at least five loci, or at least ten loci, or Variants were detected in at least 13 loci, or 26 loci. 21. The purposes of claim 19, wherein the variation in the at least one locus further comprises variation in one or more of PRDM1, JAZF1, UHRF1BP1 and IL10, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18 The variation in UHRF1BP1 is the guanine allele of the SNP at rs11755393 in the nucleotide sequence as set forth in SEQ ID NO: 19; and The variation in IL10 is the adenine allele of the SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20. 22. The use of claim 19, wherein the variation in at least one risk locus described in Table 4 comprises the SNPs described in Table 4. 23. The use of claim 19, wherein: (a) the biological sample is known to include or is suspected to also include nucleic acids comprising other variants in the BLK locus; (b) other variations in the BLK locus include a single nucleotide polymorphism (SNP), or are located at a nucleotide position corresponding to the position of a single nucleotide polymorphism (SNP), wherein the SNP is rs922483 (SEQ ID NO: 13), wherein the variation is a thymine at chromosome position 11389322 on human chromosome 8; and (c) The presence of other variants in the BLK locus is diagnostic or predictive of lupus in the subject. 24. The use of claim 19 or 23, wherein: (a) the biological sample is known to include or is suspected to also include nucleic acids comprising other variations in at least one SLE risk locus described in Table 6; (b) the other variation in at least one locus comprises or is located at a nucleotide position corresponding to a SNP described in Table 6; and (c) the presence of the other variant in at least one locus is diagnostic or predictive of lupus in the subject. 25. The use of any one of claims 19-23, wherein detecting comprises performing a primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays. 26. The use of claim 25, wherein the primer extension assay is an allele-specific primer extension assay. 27. A tool for detecting the presence of a genetic variation in the TNIP1 locus and optionally at least one SLE risk locus described in Table 4 is used in the preparation of a lupus patient selected for treatment with a lupus therapeutic agent Use in a kit or preparation for a patient, wherein the genetic variation in the TNIP1 locus is a single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence shown in SEQ ID NO: 16 Cytosine alleles. 28. The use of claim 27, wherein the variation in the at least one SLE risk locus further comprises variation in one or more of PRDM1, JAZF1, UHRF1BP1, and IL10, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of the SNP at rs11755393 in the nucleotide sequence as set forth in SEQ ID NO: 19; and The variation in IL10 is the adenine allele of the SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20. 29. The use of claim 27 or 28, wherein detecting comprises performing a primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; a 5' nuclease assay; Assays using molecular beacons; and methods for oligonucleotide ligation assays. 30. The use of claim 29, wherein the primer extension assay is an allele-specific primer extension assay. 31. Use of a tool for detecting the presence of a genetic marker indicative of a risk of developing lupus in a biological sample obtained from a subject in the manufacture of a kit or preparation for assessing whether a subject is at risk of developing lupus, wherein the The genetic signature includes a set of at least three single nucleotide polymorphisms (SNP), wherein one SNP occurs at the TNIP1 locus and each of the other SNPs occurs at the SLE risk loci described in Table 4 and Table 6, wherein The SNP in the TNIP1 locus is a single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence shown in SEQ ID NO:16. 32. The use of claim 31, wherein the genetic signature comprises a set of at least 4 SNPs, or at least 5 SNPs, or at least 7 SNPs, or at least 10 SNPs, or at least 15 SNPs, or at least 20 SNPs, or At least 30 SNPs. 33. The purposes of claim 31, wherein the SNPs of other SLE risk loci except TNIP1 are selected from SNPs in PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B and SH2B3, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of SNP at rs11755393 in the nucleotide sequence as shown in SEQ ID NO: 19; The variation in IL10 is an adenine allele at SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20; The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 34. The use of claim 31, wherein the genetic signature further comprises a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, wherein the variation is on human chromosome 8 thymine at chromosome 11389322. 35. Use of a means for detecting the presence of a genetic signature indicative of lupus in a biological sample obtained from a subject, wherein the genetic signature comprises a set of at least Three single nucleotide polymorphisms (SNPs), one of which occurs in the TNIP1 locus and each of the other SNPs occurs in the SLE risk loci described in Tables 4 and 6, wherein the SNP in the TNIP1 locus is a single nucleotide polymorphism (SNP) at rs7708392 in the nucleotide sequence shown in SEQ ID NO:16. 36. The use of claim 35, wherein the genetic signature comprises a set of at least 4 SNPs, or at least 5 SNPs, or at least 7 SNPs, or at least 10 SNPs, or at least 15 SNPs, or at least 20 SNPs, or At least 30 SNPs. 37. The purposes of claim 35, wherein the SNP in each SLE risk locus except TNIP1 locus is selected from the SNPs of PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B and SH2B3, and wherein The variation in PRDM1 is an adenine allele at SNP at rs6568431 in the nucleotide sequence as shown in SEQ ID NO: 17; The variation in JAZF1 is a thymine allele at SNP at rs849142 in the nucleotide sequence as shown in SEQ ID NO: 18; The variation in UHRF1BP1 is the guanine allele of SNP at rs11755393 in the nucleotide sequence as shown in SEQ ID NO: 19; The variation in IL10 is an adenine allele at SNP at rs3024505 in the nucleotide sequence as shown in SEQ ID NO:20; The variation in IFIH1 is a thymine allele at SNP at rs1990760 in the nucleotide sequence as shown in SEQ ID NO:21; The variation in CFB is the guanine allele of SNP at rs641153 in the nucleotide sequence as shown in SEQ ID NO: 22; The variation in CLEC16A is an adenine allele at SNP at rs12708716 in the nucleotide sequence as shown in SEQ ID NO: 23; The variation in IL12B is a guanine allele at SNP at rs6887695 in the nucleotide sequence as set forth in SEQ ID NO: 24; and The variation in SH2B3 is the thymine allele of the SNP at rs17696736 in the nucleotide sequence as shown in SEQ ID NO:25. 38. The use of claim 35, wherein the genetic signature further comprises a SNP in the SLE risk locus, wherein the SNP is rs922483 (SEQ ID NO: 13), and the SLE risk locus is BLK, and wherein the variation is 8 in people Thymine at position 11389322 of the chromosome. 39. The use of any one of claims 35-38, wherein detecting comprises performing a primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; Methods for nuclease assays; assays employing molecular beacons; and oligonucleotide ligation assays. 40. The use of claim 39, wherein the primer extension assay is an allele-specific primer extension assay.