HK1237003A1 - Methods for treating, diagnosing, and monitoring lupus - Google Patents

Description

Methods for treating, diagnosing and monitoring lupus

The present application is a divisional application of PCT application PCT/US2010/051589 entitled "method for treating, diagnosing and monitoring lupus" filed on 6/10/2010, with a date of entry into the national phase of china of 2012 6/2012 and with application number 201080055282.5.

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 61/278,510, filed on 7/10/2009, which is incorporated herein by reference in its entirety.

Sequence listing

This application contains a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy was created at 20/9/2010 under the name p4325r1w.txt, size 57,896 bytes.

FIELD

Methods of identifying, diagnosing, prognosing and assessing risk of developing lupus, and methods of treating lupus are provided. Methods for identifying effective lupus therapeutic agents and predicting responsiveness to lupus therapeutic agents are also provided.

Background

Lupus is an autoimmune disease that is estimated to affect almost one million americans, mainly women between the ages of 20-40. Lupus involves antibodies that attack connective tissue. The main form of lupus is systemic lupus (systemic lupus erythematosus; SLE). SLE is a chronic autoimmune disease with a strong genetic and environmental component (see, e.g., Hochberg MC, Dubois' Lupus Erythematosus, 5 th edition, Wallace DJ, Hahn BH, eds Baltimore: Williams and Wilkins (1997); Wakeland EK et al, Immunity 2001; 15 (3): 397-. Various other forms of lupus are known, including but not limited to Cutaneous Lupus Erythematosus (CLE), lupus nephritis, and neonatal lupus.

As it progresses from attacking the skin and joints to attacking the viscera, including the lungs, heart and kidneys (primary concern with kidney disease), untreated lupus can be fatal, making early and accurate diagnosis of lupus and/or assessment of risk of developing lupus particularly critical. Lupus is primarily characterized by a series of episodes (flare-up) with intervals of little or no disease. Renal injury, measured by the amount of proteinuria in urine, is one of the sharpest injury areas 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 manifestations of the disease are caused by the deposition of antibody-containing immune complexes in blood vessels leading to inflammation in the kidney, brain and skin. Autoantibodies also have a direct pathogenic effect in promoting hemolytic anemia and thrombocytopenia. SLE is associated with antinuclear antibodies, the generation of circulating immune complexes and the activation of the complement system. SLE has an incidence of about 1/700 in women between the ages of 20 and 60. SLE can affect any organ system and can cause severe tissue damage. There are a large number of autoantibodies with different specificities in SLE. SLE patients typically develop autoantibodies with anti-DNA, anti-Ro and anti-platelet specificity and that can initiate clinical features of the disease, such as glomerulonephritis, arthritis, serositis, neonatal complete cardiac block and hematological abnormalities. These autoantibodies may also be associated with central nervous system disorders. Arbuckle et al describe the emergence of autoantibodies prior to the clinical onset of SLE (Arbuckle et al N.Engl. J.Med.349 (16): 1526-1533 (2003)). Lupus (including SLE) is not easily diagnosed, resulting in physicians taking multi-factor signs and symptom-based classification approaches (Gill et al, American family physicians 68 (11): 2179-.

One of the biggest challenges in the clinical management of complex autoimmune diseases (e.g. lupus) is the accurate early identification of the disease in the patient. In recent years, many lines and candidate gene studies have been conducted to identify genetic factors that contribute to SLE susceptibility. Haplotypes carrying HLA class II alleles DRB1 ANG 0301 and DRB1 ANG 1501 are clearly associated with disease and the presence of antibodies to nuclear autoantigens. See, e.g., 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 JHum genet.15 (8): 823-30(2007). Recently, variants of interferon regulatory factor 5(IRF5) and the signal transducer and activator of transcription 4(STAT) have been discovered as significant risk factors for SLE. See, e.g., Sigurdsson S et al, Am JHum 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 concept that, in some cases, the type I Interferon (IFN) pathway plays an important role in the pathogenicity of SLE disease. Type I IFN is present in serum in SLE cases, and IFN production is associated with the presence of an immune complex comprising an Ab and nucleic acids (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, Immunity 25 (3): 383-92 (2006); Miyagi et al, J Exp Med 204 (10): 2383-96 (2007)). Most cases of SLE show a significant "signature" for type I IFN gene expression 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, PLoS Med 3: e491 (2006)). Immune complexes containing native DNA and RNA stimulate toll-like receptors (TLRs) 7 and 9 expressed by dendritic and B cells, producing 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 conducted to identify reliable biomarkers for diagnostic and prognostic purposes. However, no clinically confirmed diagnostic markers (e.g., biomarkers) have been identified that enable physicians or others to accurately define pathophysiological aspects of SLE, clinical activity, response to therapy, or risk of developing disease, despite the identification of multiple candidate genes and alleles (variants) thought to contribute to SLE susceptibility. For example, at least 13 common alleles have been reported to have an effect on 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 Genet38 (5): 550-55 (2006); Graham et al, Proc Natl Acad Sci U S104 (16): 6758-63 (2007); Remmers et al, N Engl J Med 357 (10): 977-86 (2007); Cunningham Graham et al, NatGenet 40 (1): Med 83-89 (2008); Harley et al, Nat Genet 40(2) (204-10); Hom et al, NEm et al, 358; Natl J2008-9 Nature 2008: 9, et al; 2008-2008: 2008 (900); Nature 2008-900), nat Genet 40 (2): 152-4 (2008); sawalha et al, PLoS ONE 3 (3): e1727 (2008)). Known alleles for which causal relationships have been inferred are 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 Genet 40 (2): 152-4(2008)), while the risk haplotypes of IRF5, TNFSF4 and BLK may act on SLE by affecting the expression levels of mRNA and protein (Sigurdsson et al, Am J Hum Genet 76 (3): 528-37 (2005); Graham et al, Nat Genet38 (5): 550-55 (2006); Graham et al, Proc Natl Acad Sci U A104 (16): 6758-63 (2007); Cunningham Graham et al, Nat Genet 40 (1): 83-89 (2008); Hom et al, N Engl J Med358 (9): 900-9 (2008)). Causal alleles of STAT4, KIAA1542, IRAK1, PXK, and other genes (e.g., 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 Med358 (9): 900-9 (2008); Sawalha et al, PLoS ONE 3 (3): e1727 (2008)). These and other genetic variations associated with lupus are also described in international patent application No. PCT/US2008/064430 (international publication No. WO 2008/144761). While these genetic variations contribute significantly to the risk of SLE and various aspects of the disease described to date, there remains a need to determine more information about the effect of genetic variations on significant clinical heterogeneity, such as SLE.

It would therefore be highly advantageous to have other molecular-based diagnostic methods that can be used to objectively identify the presence of a disease in a patient and/or classify the disease, define pathophysiological aspects of lupus, clinical activity, response to treatment, prognosis, and/or risk of developing lupus. Furthermore, it would be advantageous to have molecular-based diagnostic markers that correlate 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 and other autoimmune diseases. Such correlations are greatly advantageous for identifying the presence of lupus in a patient or determining a predisposition to develop disease. Such correlations are also useful for identifying pathophysiological aspects of lupus, clinical activity, response to treatment, or prognosis. Furthermore, statistical and biological significance and reproducible information associated with such correlations can be used as part of the effort to identify specific patient subpopulations that are expected to benefit significantly from treatment with a particular therapeutic agent, e.g., a therapeutic agent that benefits therapeutically in such specific lupus patient subpopulations or a therapeutic agent that exhibits therapeutic benefit in clinical studies.

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

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

SUMMARY

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

In one aspect, a method of identifying lupus in a subject is provided, the method comprising detecting in a biological sample derived from the subject the presence of a variation in a SLE risk locus, 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 a 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 selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods in oligonucleotide ligation assays.

In another aspect, there is provided a method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to the position of a SNP of at least one locus as set forth in table 4, and wherein the subject is suspected of having lupus. In certain embodiments, a variation is detected in 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. In one embodiment, the at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. In one embodiment, the variation of at least one locus comprises a SNP as set forth in table 4. In certain embodiments, the presence of a variation in at least one SLE risk locus as set forth in Table 4 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of at least one locus as set forth in Table 4) is detected in combination with the presence of a variation in a BLK SLE risk locus (wherein the variation in the BLK locus occurs at a nucleotide position corresponding to the position of the SNP, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is thymine at chromosome 11389322 of chromosome 8 of a human). In one embodiment, detecting comprises performing a primer extension assay selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods in oligonucleotide ligation assays.

In yet another embodiment, a method of identifying lupus in a subject is provided, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in at least one locus occurs at a nucleotide position corresponding to the position of a SNP of at least one locus as set forth in table 6, and wherein the subject is suspected of having lupus. In certain embodiments, the variation is detected 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 SH2B 3. In one embodiment, the variation in at least one locus comprises a SNP as set forth in table 6. In certain embodiments, the presence of a variation in at least one SLE risk locus as set forth in Table 6 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of at least one locus as set forth in Table 6) is detected in combination with the presence of a variation in a BLK SLE risk locus (wherein the variation in the BLK locus occurs at a nucleotide position corresponding to the position of the SNP, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 8 of a human). In one embodiment, detecting comprises performing a primer extension assay selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods in oligonucleotide ligation assays.

In yet another aspect, a method of identifying lupus in a subject is provided, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4 and the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in each locus occurs at a nucleotide position corresponding to a SNP position for each locus as set forth in table 4 and table 6, respectively, and wherein the subject is suspected of having lupus. In certain embodiments, a 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, the at least one locus described in table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10, and the at least one locus described in table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In one embodiment, the variation in at least one locus as set forth in table 4 and the variation in at least one locus as set forth in table 6 comprise a SNP as set forth in tables 4 and 6, respectively. In certain embodiments, the presence of a variation in at least one SLE risk locus as set forth in table 4 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of at least one locus as set forth in table 4), and the presence of a variation in at least one SLE risk locus as set forth in table 6 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of at least one locus as set forth in table 6) is detected in combination with the presence of a variation in a 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 chromosome 8 of human. In one embodiment, detecting comprises performing a primer extension assay selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods in oligonucleotide ligation assays.

In one aspect, a method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent 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 a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 82 of a human, wherein the presence of the variation in the BLK SLE risk locus is indicative of responsiveness of the subject to the therapeutic agent.

In another aspect, a method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent is provided, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a SNP position of the at least one locus as set forth in table 4, wherein the presence of a variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent. In certain embodiments, the subject comprises a variation in 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. In one embodiment, the at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. In one embodiment, the variation in at least one locus comprises a SNP as set forth in table 4. In certain embodiments, the method comprises determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 4 (wherein the variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 4), in combination with a variation in a 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 a thymine at chromosome 11389322 of chromosome 8 of the human), wherein the presence of the variation in the at least one locus as set forth in table 4 and the presence of the variation in the BLK locus are indicative of the subject's responsiveness to the therapeutic agent.

In yet another aspect, a method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent is provided, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a SNP position of the at least one locus as set forth in table 6, wherein presence of the variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent. In certain embodiments, the subject comprises a 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 SH2B 3. In one embodiment, the variation in at least one locus comprises a SNP as set forth in table 6. In certain embodiments, the method comprises determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 6 (wherein the variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6), in combination with a variation in a 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 a thymine at chromosome 11389322 of chromosome 8 of the human), wherein the presence of the variation in the at least one locus as set forth in table 6 and the presence of the variation in the BLK locus are indicative of the subject's responsiveness to the therapeutic agent.

In other aspects, methods of predicting responsiveness of a subject with lupus to a lupus therapeutic agent are provided, the methods comprising determining whether the subject includes a variation in at least one SLE risk locus as set forth in table 4 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 4), and includes a variation in at least one SLE risk locus as set forth in table 6 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6), wherein the presence of the variation in the at least one locus as set forth in table 4 and the presence of the variation in the at least one locus as set forth in table 6 are indicative of responsiveness of the subject to the therapeutic agent. In certain embodiments, the subject comprises a variation in at least three loci, or at least four loci, or at least five loci, or at least 7 loci, or at least ten loci. In one embodiment, the at least one locus described in table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 and the at least one locus described in table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In one embodiment, the variation in at least one locus as set forth in table 4 and the variation in at least one locus as set forth in table 6 comprise a SNP as set forth in tables 4 and 6, respectively. In certain embodiments, the method comprises determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 4 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 4), and a variation in at least one SLE risk locus as set forth in table 6 (wherein the variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6), and a variation in a BLK SLE risk locus (wherein the variation in a 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 a thymine at chromosome 89322 of chromosome No. 8 of human 1134), wherein the presence of the variation in at least one locus as set forth in table 4 and the presence of the variation in at least one locus as set forth in table 6 and the presence of the variation in the BLK locus Indicating the responsiveness of the subject to the therapeutic agent.

In yet another aspect, a method of diagnosing or prognosing lupus in a subject is provided, the method comprising detecting in a biological sample derived from the subject the presence of a variation in a SLE risk locus, wherein the SLE risk locus is BLK, 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 chromosome 8 in a human, and the presence of the 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 prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein: a biological sample is known to or suspected of comprising nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4; a variation in at least one locus comprises a SNP as set forth in table 4 or is located at a nucleotide position corresponding to a SNP as set forth in table 4; and the presence of a variation in at least one locus is a diagnosis or prognosis of lupus in the subject. In certain embodiments, a variation is detected in 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. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. In certain embodiments, the method comprises detecting the presence of a variation in at least one SLE risk locus as set forth in table 4, in combination with the presence of a variation in a BLK SLE risk locus, wherein: the biological sample is known or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4 and a variation in a BLK locus, the variation in the at least one locus as set forth in table 4 comprising or located at a nucleotide position corresponding to a SNP as set forth in table 4, and the variation in the BLK locus occurring at a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 8 of a human, and the presence of the variation in the at least one locus as set forth in table 4 and the presence of the variation in the BLK locus are diagnostic or predictive of lupus in a subject.

In yet another aspect, there is provided a method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein: a biological sample is known to or suspected of comprising nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6; a variation in at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and the presence of a variation in at least one locus is a diagnosis or prognosis of lupus in the subject. In certain embodiments, the variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In certain embodiments, the method comprises detecting the presence of a variation in at least one SLE risk locus as set forth in table 6, in combination with the presence of a variation in a BLK SLE risk locus, wherein: the biological sample is known or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6 and a variation in a BLK locus, the variation in the at least one locus as set forth in table 6 comprising or located at a nucleotide position corresponding to a SNP as set forth in table 6, and the variation in the BLK locus occurring at a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 8 of a human, and the presence of the variation in the at least one locus as set forth in table 6 and the presence of the variation in the BLK locus is diagnostic or predictive of lupus in a subject.

In yet another aspect, there is provided a method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4 and the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein: biological samples are known or suspected to include: a nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4 and a variation in at least one SLE risk locus as set forth in table 6; a variation in at least one locus comprises a SNP as set forth in tables 4 and 6 or is located at a nucleotide position corresponding to a SNP as set forth in tables 4 and 6, respectively; and the presence of a variation in at least one locus as set forth in table 4 and the presence of a variation in at least one locus as set forth in table 6 is a diagnosis or prognosis of lupus in the subject. In certain embodiments, a variation is detected 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, the at least one SLE risk locus described in table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 and the at least one SLE risk locus described in table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In certain embodiments, the methods comprise detecting the presence of a variation in at least one SLE risk locus as set forth in table 4, and the presence of a variation in at least one SLE risk locus as set forth in table 6, in combination with the presence of a variation in a BLK SLE risk locus, wherein: biological samples are known or suspected to include: nucleic acid comprising a variation in at least one SLE risk locus as set forth in Table 4 and a variation in at least one SLE risk locus and a variation in a BLK locus as set forth in Table 6, a variation in at least one locus as set forth in Table 4 comprising a SNP as set forth in Table 4 or at a nucleotide position corresponding to a SNP as set forth in Table 4, and the variation in at least one locus as set forth in Table 6 comprises a SNP as set forth in Table 6 or is located at a nucleotide position corresponding to a SNP as set forth in Table 6, and a 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 chromosome 8 in humans, and the presence of a variation in at least one locus as set forth in table 4 and the presence of a variation in at least one locus as set forth in table 6, and the presence of a variation in a BLK locus is a diagnosis or prognosis of lupus in the subject.

In another aspect, a method is provided that aids in diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in a SLE risk locus, wherein the SLE risk locus is BLK, 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 chromosome 8 in a human, and the presence of the 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 prognosis of lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein: a biological sample is known to or suspected of comprising nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4; a variation in at least one locus comprises a SNP as set forth in table 4 or is located at a nucleotide position corresponding to a SNP as set forth in table 4; and the presence of a variation in at least one locus is a diagnosis or prognosis of lupus in the subject. In certain embodiments, a variation is detected in 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. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. In certain embodiments, the method comprises detecting the presence of a variation in at least one SLE risk locus as set forth in table 4, in combination with the presence of a variation in a BLK SLE risk locus, wherein: the biological sample is known or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4 and a variation in a BLK locus, the variation in the at least one locus as set forth in table 4 comprising or located at a nucleotide position corresponding to a SNP as set forth in table 4, and the variation in the BLK locus occurring at a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 8 of a human, and the presence of the variation in the at least one locus as set forth in table 4 and the presence of the variation in the BLK locus are diagnostic or predictive of lupus in a subject.

In yet another aspect, there is provided a method of aiding in the diagnosis or prognosis of lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein: a biological sample is known to or suspected of comprising nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6; a variation in at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and the presence of a variation in at least one locus is a diagnosis or prognosis of lupus in the subject. In certain embodiments, the variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In certain embodiments, the method comprises detecting the presence of a variation in at least one SLE risk locus as set forth in table 6, in combination with the presence of a variation in a BLK SLE risk locus, wherein: the biological sample is known or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6 and a variation in a BLK locus, the variation in the at least one locus as set forth in table 6 comprising or located at a nucleotide position corresponding to a SNP as set forth in table 6, and the variation in the BLK locus occurring at a nucleotide position corresponding to the SNP position, wherein the SNP is rs922483(SEQ ID NO:13), wherein the variation is a thymine at chromosome 11389322 of chromosome 8 of a human, and the presence of the variation in the at least one locus as set forth in table 6 and the presence of the variation in the BLK locus is diagnostic or predictive of lupus in a subject.

In yet another aspect, there is provided a method of aiding in the diagnosis or prognosis of lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4 and the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein: biological samples are known or suspected to include: a nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4 and a variation in at least one SLE risk locus as set forth in table 6; a variation in at least one locus comprises a SNP as set forth in tables 4 and 6 or is located at a nucleotide position corresponding to a SNP as set forth in tables 4 and 6, respectively; and the presence of a variation in at least one locus as set forth in table 4 and the presence of a variation in at least one locus as set forth in table 6 is a diagnosis or prognosis of lupus in the subject. In certain embodiments, a variation is detected 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, the at least one SLE risk locus described in table 4 is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 and the at least one SLE risk locus described in table 6 is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In certain embodiments, the methods comprise detecting the presence of a variation in at least one SLE risk locus as set forth in table 4, and the presence of a variation in at least one SLE risk locus as set forth in table 6, in combination with the presence of a variation in a BLK SLE risk locus, wherein: biological samples are known or suspected to include: nucleic acid comprising a variation in at least one SLE risk locus as set forth in Table 4 and a variation in at least one SLE risk locus and a variation in a BLK locus as set forth in Table 6, a variation in at least one locus as set forth in Table 4 comprising a SNP as set forth in Table 4 or at a nucleotide position corresponding to a SNP as set forth in Table 4, and the variation in at least one locus as set forth in Table 6 comprises a SNP as set forth in Table 6 or is located at a nucleotide position corresponding to a SNP as set forth in Table 6, and a 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 chromosome 8 in humans, and the presence of a variation in at least one locus as set forth in table 4 and the presence of a variation in at least one locus as set forth in table 6, and the presence of a variation in a BLK locus is a diagnosis or prognosis of lupus in the subject.

In one aspect, a method of treating a lupus disorder in a subject is provided, wherein a genetic variation is known to exist at a nucleotide position corresponding to a SNP in a 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 chromosome 8 in a human, the method comprising 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 in which a genetic variation is known to be present at a nucleotide position in at least one SLE risk locus described in table 4 that corresponds to a SNP described in table 4, the method comprising administering to the subject a therapeutic agent effective to treat the disorder. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10.

In another aspect, there is provided a method of treating a lupus disorder in a subject in which a genetic variation is known to be present at a nucleotide position in at least one SLE risk locus described in table 6 that corresponds to a SNP described in table 6, the method comprising administering to the subject a therapeutic agent effective to treat the disorder. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

In another aspect, a method of treating a subject having a lupus disorder is provided, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in a subject having a genetic variation at a nucleotide position corresponding to a SNP in a 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 another aspect, there is provided a method of treating a subject having a lupus disorder, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in a subject having a genetic variation at a nucleotide position in at least one SLE risk locus described in table 4 that corresponds to a SNP described in table 4. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10.

In another aspect, there is provided a method of treating a subject having a lupus disorder, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in a subject having a genetic variation at a nucleotide position in at least one SLE risk locus described in table 6 that corresponds to a SNP described in table 6. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

In yet another aspect, a method is provided for treating a subject having 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 in which the therapeutic agent is administered to at least 5 human subjects each having a genetic variation at a nucleotide position corresponding to a SNP in a SLE risk locus, wherein the SNP is rs922483(SEQ ID NO:13) and the SLE risk locus is BLK, wherein the variation is a thymine at chromosome 11389322 of chromosome 8 in humans.

In yet another aspect, a method of treating a subject having a lupus disorder is provided, the method comprising administering to the subject a therapeutic agent that has been shown to be effective in at least one clinical study to treat the disorder, wherein the therapeutic agent is administered to at least 5 human subjects in the study, each of the subjects having a genetic variation at a nucleotide position corresponding to a SNP as set forth in table 4 in at least one SLE risk locus as set forth in table 4. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10.

In yet another aspect, a method of treating a subject having a lupus disorder is provided, the method comprising administering to the subject a therapeutic agent that has been shown to be effective in at least one clinical study to treat the disorder, wherein the therapeutic agent is administered to at least 5 human subjects in the study, each of the subjects having a genetic variation at a nucleotide position corresponding to a SNP as set forth in table 6 in at least one SLE risk locus as set forth in table 6. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

In another aspect, a method is provided that includes preparing a lupus therapeutic agent, comprising packaging the therapeutic agent with instructions for administering the therapeutic agent to a subject that has or is believed to have lupus and has a genetic variation at a position corresponding to a SNP in a 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 chromosome 8 in humans.

In yet another aspect, a method is provided that includes preparing a lupus therapeutic agent, comprising packaging the therapeutic agent with instructions for administering the therapeutic agent to a subject that has or is believed to have lupus and has a genetic variation at a position corresponding to a SNP as set forth in table 4 in at least one SLE risk locus as set forth in table 4.

In yet another aspect, a method is provided that includes preparing a lupus therapeutic agent, comprising packaging the therapeutic agent with instructions for administering the therapeutic agent to a subject that has or is believed to have lupus and has a genetic variation at a position corresponding to a SNP described in table 6 in at least one SLE risk locus described in table 6.

In one aspect, a method of selecting a patient with lupus for treatment with a lupus therapeutic agent is provided, the method comprising detecting the presence of a genetic variation at a nucleotide position corresponding to a SNP in a 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 selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

In other aspects, methods of selecting a patient with lupus for treatment with a lupus therapeutic agent are provided, the methods comprising detecting the presence of 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 certain embodiments, a variation is detected in 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. In one embodiment, the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. In one embodiment, the variation at the at least one locus comprises a SNP as set forth in table 4. In one embodiment, detecting comprises performing a primer extension assay selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

In other aspects, methods of selecting a patient with lupus for treatment with a lupus therapeutic agent are provided, the methods comprising detecting the presence of a genetic variation at a nucleotide position corresponding to a SNP set forth in table 6 in at least one SLE risk locus set forth in table 6. In certain embodiments, the variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci. In one embodiment, the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3. In one embodiment, the variation at the at least one locus comprises a SNP as set forth in table 6. In one embodiment, detecting comprises performing a primer extension assay selected from; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

In another aspect, there is provided a method of assessing whether a subject is at risk for developing lupus, the method comprising detecting in a biological sample obtained from the subject the presence of a genetic signature indicative of a risk for developing lupus, wherein the genetic signature comprises a set of at least three SNPs, each SNP occurring in a SLE risk locus as set forth in table 4 and/or table 6. In certain embodiments, the genetic tag 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 SH2B 3. 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 thymine at chromosome 11389322 of chromosome 8 in humans.

In another aspect, a method of diagnosing lupus in a subject is provided, the method comprising detecting in a biological sample obtained from the subject the presence of a genetic signature indicative of lupus, wherein the genetic signature comprises a set of at least three SNPs, each SNP occurring in a SLE risk locus as set forth in table 4 and/or table 6. In certain embodiments, the genetic tag 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, IFIHl, CFB, CLEC164, IL12B, and SH2B 3. 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 thymine at chromosome 11389322 of chromosome 8 in humans.

Brief description of the drawings

Figure 1 shows an experimental design overview of targeted replication studies of certain SNPs to identify other SLE risk loci as described in example 1.

Figure 2 shows significant associations of new whole genomes in SLE and identification of new risk loci within TNIP1(a), PRDM1(B), JAZF1(C), UHRF1BP1(D) and IL10(E) as described in example 1. (F) Histograms of P-values for individual SNPs for the case and control replicate samples described in example 1; the dashed line represents the expected resulting density at zero distribution.

FIG. 3 shows that in the original GWAS described in example 1, candidates were achieved in meta-analysis (meta-analysis) by P-value stratification (P < 1X 10)^-5) And verification (P < 5x 10)^-8) Percent variation of state.

FIG. 4 shows a linkage disequilibrium block (shown as r) in the BLK promoter region described in example 2²). FIG. 4 discloses that 'C > T-rs 922483' is SEQ ID NO: 13.

FIG. 5 shows the results of luciferase reporter expression assays with BLK promoter regions of 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 rs 1382568A > C/G > C; (D) in Daudi cells, SNP rs 1382568A > C/G > C; (E) in BJAB cells, SNP rs 4840568G > A; (F) in Daudi cells, SNP rs 4840568G > A; data shown represent the mean of triplicate determinations +/-standard error of the mean; the dotted bar shows the results for the haplotype indicated on the left side of the graph; hatched bars: the at risk haplotype 22-ACT; blank strip: the risk free haplotype 22-GAC; p < 0.05, p < 0.01, p < 0.001, ns is not significant (t test). Fig. 5A-F disclose that the '22 (GT) repeat' is SEQ ID NO: 15. fig. 5C-F also discloses that 'rs 922483C > T' is SEQ ID NO: 13.

FIG. 6 shows the results of luciferase reporter expression assays with 18(GT) repeats (SEQ ID NO: 14) or 22(GT) repeats (SEQ ID NO: 15) and the BLK promoter region of SNP rs 1382568A > C/G > C in Daudi cells as described in example 2. Data shown represent the mean of two replicate determinations +/-standard error of the mean; ns-no significance (t-test). Figure 6 discloses that the '18 (GT) repeat' is SEQ ID NO: 14. the '22 (GT) repeat' is SEQ id no: 15, and "rs 922483C > T" is SEQ ID NO: 13.

FIG. 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. The position of the causal allele is shown in bold brackets; C/T variation is shown in bold.

Detailed Description

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, which are within the skill of the art. Such as "Molecular Cloning: ALaborory Manual ", second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (edited by m.j. grait, 1984); "Animal Cell Culture" (edited by r.i. freshney, 1987); "Methods in enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al, 1987 and periodic updates); "PCR: such techniques are well explained in The literature of The Polymerase Chain Reaction ", (edited by Mullis et al, 1994). In addition, primers, oligonucleotides and polynucleotides useful 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, second edition, J.Wiley & Sons (New York, N.Y.1994) and March, advanced Chemistry Reactions, mechanics and Structure fourth edition, John Wiley & Sons (New York, N.Y.1992) provide the skilled artisan with a general guidance for many of the terms used in this application.

Definition of

For the purpose of interpreting the 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 "a protein" includes a plurality of proteins; the term "cell" includes mixtures of cells, and the like. In the event that the definitions set forth below conflict with definitions in any document incorporated by reference herein, the definitions set forth below apply.

As used herein, "lupus" or "lupus disorders" are autoimmune diseases or disorders that generally involve 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 nephritis, extrarenal, encephalitis, pediatric, non-renal, discoid, and alopecia).

The terms "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification of the nucleotide structure may be performed before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling element. Other types of modifications include, for example, "caps"; replacing one or more naturally occurring nucleotides with an analog; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramides, carbamates (cabamates), etc.) and with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those comprising a pendant moiety such as a protein (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators (alkylators), those with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. Furthermore, any hydroxyl group typically present in sugars can be substituted, for example, by phosphate groups, protected by standard protecting groups, or activated to make other bonds to other nucleotides, or can be conjugated to a solid support. The 5 'and 3' OH groups may 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, typically in the form of analogs known in the art, including, for example, 2 '-O-methyl-2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, α -anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, 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, embodiments in which the phosphoric acid is substituted by p (O) S ("thioester"), p (S) S ("dithioester"), O (NR 2 ("amidate"), p (O) P, P (O) OR ', CO, OR CH2 ("formacetal"), where R OR R' are independently H OR a substituted OR unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, OR araldyl optionally containing an ether (- -O- -) linkage. It is not necessary that all linkages in the polynucleotide be identical. The foregoing applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" refers to a short single-stranded polynucleotide of at least about 7 nucleotides in length and less than about 250 nucleotides in length. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of 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 generally allows polymerization of a complementary nucleic acid by providing free 3' -OH groups.

The term "genetic variation" or "nucleotide variation" refers to an alteration (e.g., an insertion, deletion, inversion or substitution of one or more nucleotides, such as a Single Nucleotide Polymorphism (SNP)) in a nucleotide sequence relative to a reference sequence (e.g., a sequence of a common and/or wild-type sequence and/or a major allele). 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.

A "single nucleotide polymorphism" or "SNP" refers to a single base position in DNA at which different alleles or other nucleotides are present in a population. SNP positions typically have highly conserved allelic sequences before and after (e.g., sequences that differ among members of a population less than 1/100 or 1/1000). Individuals may be homozygous or heterozygous for the allele at each SNP position.

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

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 variations thereof, refer to a genetic variation at a relatively corresponding DNA position in a polynucleotide sequence that is occupied by the SNP in a genome. 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 (e.g., oligonucleotides), on a substrate. The substrate may 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. The amplification may be linear amplification or exponential amplification (e.g., PCR). "copy" does not mean perfect sequence complementarity or identity with respect to the template sequence. For example, the copies may contain nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced by primers containing sequences that are hybridizable but not fully complementary to the template), and/or sequence errors that occur during amplification.

The term "allele-specific oligonucleotide" refers to an oligonucleotide that hybridizes to a region of a target nucleic acid containing a nucleotide variation (typically a substitution). "allele-specific hybridization" refers to the specific base pairing of a nucleotide in an allele-specific oligonucleotide with a nucleotide variation when the allele-specific oligonucleotide is hybridized to its target nucleic acid. An allele-specific oligonucleotide that is capable of allele-specific hybridization to 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 is (a) hybridized to a target nucleic acid at a region 3 'or 5' to a nucleotide variation, and (b) extended by a polymerase, thereby incorporating a nucleotide complementary to the nucleotide variation into an extension product.

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

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) hybridization is detected, either 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 nucleic acid degradation cleavage (nucleolytic cleavage) of the hybridized probe, resulting in a detectable signal.

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

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

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

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

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

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

As used herein, a subject that is "at risk" for developing lupus may or may not have a detectable disease or disease symptom, and may or may not have displayed a detectable disease or disease symptom prior to the treatment methods described herein. By "at risk" is meant that the subject has one or more risk factors, which, as described herein and known in the art, are measurable parameters associated with the development of lupus. 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, "diagnosis" can refer to the identification of a particular type of lupus disorder, e.g., SLE. "diagnosis" may also refer to the classification of a particular subtype of lupus, for example by the tissue/organ involved (lupus nephritis), by molecular characteristics (e.g. a subset of patients characterised by genetic variation in a particular gene or nucleic acid region).

The term "aiding diagnosis" is used herein to refer to a method of aiding in the clinical determination of the presence or nature of a particular type of symptom or condition associated with lupus. For example, a method of aiding in the diagnosis of lupus can comprise measuring the absence of one or more SLE risk loci or SLE risk alleles in a biological sample from an individual.

The term "prognosis" is used herein to refer to the prediction of the likelihood of disease symptoms attributable to an autoimmune disorder, including, for example, recurrence, flaring (flaring) 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 adversely to a drug or group of drugs.

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

An "effective amount" refers to an amount (dosage and time required) effective to achieve the desired therapeutic or prophylactic result. The "therapeutically effective amount" of a therapeutic agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount wherein a therapeutically beneficial effect outweighs any toxic or detrimental effect of the therapeutic agent. A "prophylactically effective amount" refers to an amount (in dosages and for periods of time required) effective to achieve the desired prophylactic result. Because prophylactic doses are used in subjects prior to or at an early stage of the disease, the prophylactically effective amount is typically, but not necessarily, lower 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 human and non-human primates) and rodents (e.g., mice and rats). In certain embodiments, the mammal is a human.

As used herein, "patient subpopulation" and grammatical variations thereof refers to a patient subpopulation characterized by one or more unique measurable and/or identifiable characteristics that distinguish the patient subpopulation from other patients within a broader disease category to which it belongs. Such features include disease subclasses (e.g., SLE, lupus nephritis), gender, lifestyle, health history, organs/tissues involved, history of treatment, and the like.

A "control subject" refers to a healthy subject that has not been diagnosed as having lupus or a lupus disorder, and does not have 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, which comprises cells and/or other molecular entities to be characterized and/or identified, for example, according to physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "biological sample" or "disease sample" and variations thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized.

By "tissue or cell sample" is meant a collection of similar cells obtained from a subject or patient 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 (asparate); blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from the subject at any time during pregnancy or development. The tissue sample may 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, and the like. As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" refers to a cell or tissue obtained from a source known or believed to not have a disease or condition being identified using the methods or compositions 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 same subject or patient in which the disease or disorder is being identified using the compositions or methods 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 an individual that is not the subject or patient in which the composition or method of the invention is being used to identify a disease or disorder.

For purposes herein, a "section" of a tissue sample means a single portion or piece of the tissue sample, such as a slice of tissue or cells cut from the tissue sample. If it is understood that the present invention encompasses methods of analyzing the same section of a tissue sample at both the morphological and molecular levels, or for both protein and nucleic acids, it is understood that multiple sections of a tissue sample may be taken and analyzed in accordance with the present invention.

"associated" or "related" means that the performance and/or results of a first analysis or procedure are compared in any way with the performance and/or results of a second analysis or procedure. For example, the results of the first analysis or procedure may be used to perform a second procedure, and/or the results of the first analysis or procedure may be used to determine whether a second analysis or procedure should be performed. For embodiments of gene expression analysis or protocols, the results of the gene expression analysis or protocol can be used to determine whether a particular treatment protocol should be performed.

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 detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

An "agent" 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.

The term "increased resistance" to a particular therapeutic agent or treatment selection, as used in accordance with the present invention, means a decreased response to a standard dose of drug or to a standard treatment regimen.

The term "reduced sensitivity" to a particular therapeutic agent or therapeutic selection, as used in accordance with the present invention, means a reduced response to a standard dose of the therapeutic agent or to a standard therapeutic regimen, wherein the reduced response can be compensated for (at least in part) by increasing the therapeutic agent dose or therapeutic intensity.

The subject's "predicted response" and variants thereof can be assessed with any endpoint that shows benefit to the patient, including, without limitation, (1) some degree of inhibition of disease progression, including slowing and complete cessation; (2) reduction in the number of episodes and/or symptoms of the disease; (3) reduction of lesion size; (4) inhibition (i.e., reduction, slowing, or complete cessation) of infiltration of disease cells into adjacent peripheral organs and/or tissues; (5) inhibition of disease spread (i.e., reduction, slowing, or complete cessation); (6) a reduction in an autoimmune response, which may, but need not, result in regression or detachment of a disease lesion; (7) a reduction in one or more symptoms associated with the disorder to some extent; (8) an increase in disease-free length after treatment; and/or (9) reduced mortality at a given time point after treatment.

As used herein, "lupus therapeutic agent," "therapeutic agent effective for treating lupus," and grammatical variations thereof, refers to an agent that, when provided in an effective amount, is known, clinically displayed, or expected by a clinician to provide a therapeutic benefit in a subject having lupus. In one embodiment, the phrase includes any agent sold by the manufacturer or otherwise used by a licensed clinician as a clinically acceptable agent that is expected to provide a therapeutic effect in a subject with lupus when provided in an effective amount. In one embodiment, lupus therapeutic agents include non-steroidal anti-inflammatory drugs (NSAIDs) including acetylsalicylic acid (e.g., aspirin), ibuprofen (Motrin), naproxen (Naprosyn), indomethacin (Indocin), nabumetone (Relafen), tolmetin (Tolectin), and any other embodiment including therapeutically equivalent active ingredients and formulations thereof. In one embodiment, the lupus therapeutic agent includes acetaminophen (e.g., Tylenol), a corticosteroid, or an antimalarial 3 (e.g., chloroquine, hydroxychloroquine). In one embodiment, the lupus therapeutic agent comprises an immunomodulatory drug (e.g., azathioprine, cyclophosphamide, methotrexate, cyclosporine). In one embodiment, the lupus therapeutic agent is an anti-B cell agent (e.g., anti-CD 20 (e.g., rituximab), anti-CD 22), an anti-cytokine agent (e.g., anti-tumor necrosis factor alpha, anti-interleukin-1-receptor (e.g., anakinra), anti-interleukin 10, anti-interleukin 6 receptor, anti-interferon alpha, anti-B lymphocyte stimulator), a costimulatory inhibitor (e.g., anti-CD 154, CTLA4-Ig (e.g., abatacept)), a B cell energy modulator (e.g., LJP 394 (e.g., abelimus))). In one embodiment, lupus therapeutic agents include hormonal therapy (e.g., DHEA) and anti-hormonal therapy (e.g., the anti-prolactin agent bromocriptine). In one embodiment, the lupus therapeutic agent is an agent that provides immunoadsorption, is an anti-complement factor (e.g., anti-C5 a), T cell vaccination, transfection of cells with a T cell receptor zeta chain, or peptide therapy (e.g., edratide targeting an anti-DNA idiotype).

As used herein, a therapeutic agent having "sales approval" or "approved as a therapeutic agent" or grammatical variations of these phrases refers to an active agent (e.g., pharmaceutical preparation, form of medicament) approved, licensed, registered, or authorized by and/or sold by and/or on behalf of a commercial entity (e.g., a profit entity) for use in treating a particular disorder (e.g., lupus) or patient subpopulation (e.g., patients with lupus nephritis, patients of a particular race, gender, lifestyle, disease risk profile, etc.). Relevant government entities include, for example, the U.S. Food and Drug Administration (FDA), the european medicines agency (EMEA), and equivalents thereof.

"antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins having similar structural features. Antibodies exhibit binding specificity to a particular antigen, while immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. The latter type of polypeptides is 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, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in greater detail herein). The antibody can be a chimeric antibody, a human antibody, a humanized antibody, and/or an affinity matured antibody.

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 especially refers to antibodies having a heavy chain comprising an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, in particular the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments, each with a single antigen-binding site) and a remaining "Fc" fragment (the name of which reflects its ability to crystallize readily). Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking the antigen₂And (3) fragment.

"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. The 6 CDRs of the Fv together confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with less affinity than the entire binding site.

The Fab fragment comprises a heavy chain variable domain and a light chain variable domain, and further comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the C-terminus of the heavy chain CH1 domain containing one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domains have a free thiol group. Initially as a pair of Fab 'fragments with hinge cysteines between them, F (ab')₂. Other chemical couplings of antibody fragments are also known.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a population comprising a single antibody is identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of isolated antibodies. In certain embodiments, such monoclonal antibodies generally include an antibody comprising a polypeptide sequence that binds a target, wherein the polypeptide sequence that binds the target is obtained by a method comprising selecting a single polypeptide sequence that binds the target 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 selected target-binding sequence may be further altered, for example to improve affinity for the target, to humanise the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and that antibodies comprising the altered target-binding sequence are also monoclonal antibodies of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be produced by a variety of 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, second edition 1998); hammerling et al inMonoclonal Antibodies and T-Cell Hybridomas563-681(Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al, Nature, 352: 624-628(1991) (ii) a 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.usa 101 (34): 12467-12472 (2004); and Lee et al, j.immunol.methods 284 (1-2): 119 (2004)) and techniques for producing human or human-like antibodies in animals having part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; 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. Pat. nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016; marks et al, bio. technology 10: 779 783 (1992); lonberg et al, Nature 368: 856-859 (1994); morrison, Nature 368: 812-813 (1994); fishwild et al, Nature Biotechnol.14: 845-859 (1996); neuberger, Nature Biotechnol.14: 826 (1996); and Lonberg and huskzar, lntern. rev. immunol.13: 65-93(1995)).

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

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody comprising minimal sequences derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity. In some cases, Framework Region (FR) residues of the human immunoglobulin are substituted by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or donor antibody. These modifications can be made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least 1, and typically 2, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally 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-; hurle and Gross, curr.op.biotech.5: 428-433(1994).

A "human antibody" is an antibody that comprises an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or that has been produced using any of the techniques disclosed herein for producing human antibodies. Such techniques include screening of human-derived combinatorial libraries, such as phage display libraries (see, e.g., Marks et al, J.mol.biol., 222: 581. Res., 19: 4133. 4137(1991)) and Hoogenboom et al; human Monoclonal antibodies are generated using human myeloma and mouse-human myeloma (heteromyeloma) cell lines (see, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.55-93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol., 147: 86 (1991)); and monoclonal antibodies in transgenic animals (e.g., mice) capable of producing all components of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al, Nature, 362: 255 (1993); Bruggemann et al, Yeast 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 CDRs that result in improved affinity of the antibody for an antigen, as compared to a parent antibody that does not have these alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies were generated by methods known in the art. Marks et al, Bio/Technology 10: 779-783(1992) describes 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-; yelton et al J.Immunol.155: 1994-2004 (1995); jackson et al, j.immunol.154 (7): 3310-9 (1995); and Hawkins et al, j.mol.biol.226: 889-896(1992) describes random mutagenesis of HVR and/or framework residues.

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 antagonist 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 be detectable by itself (e.g., radioisotope labels or fluorescent labels), or in the case of an enzymatic label, it may catalyze chemical alteration of a substrate compound or composition that produces a detectable product. Radionuclides that can be used as detection 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 (describes) embodiments that relate to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

General techniques

Provided herein are nucleotide variations associated with lupus. These variations provide biomarkers for lupus and/or predisposition to or contribute to the appearance, persistence and/or progression of lupus. Thus, the invention disclosed herein is useful in a variety of contexts, such as in methods and compositions relating to lupus diagnosis and treatment.

In certain embodiments, the methods relate to predicting, i.e., predicting the likelihood of disease symptoms attributable to an autoimmune disorder, including, for example, recurrence, flare (flare) and drug resistance of an autoimmune disease such as lupus. In one embodiment, the prediction relates to the extent of those reactions. In one embodiment, the prognosis relates to whether a patient will survive or improve after treatment, e.g., treatment with a particular therapeutic agent, and have no disease recurrence and/or probability thereof over a period of time. The predictive methods of the invention can be used clinically to make treatment decisions by selecting the treatment mode best suited for any particular patient. The predictive methods of the invention are valuable tools for predicting 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 therapy, etc.), or whether the patient is likely to survive long term following a treatment regimen. SLE can be diagnosed according to current American College of Rheumatology (ACR) criteria. Active disease may be defined by one British Isles Lupus Activity Group's (BILAG) "A" criteria or two BILAG "B" criteria. Some signs, symptoms or other indicators for diagnosing SLE modified from Tan et al, "The Revised criterion for The Classification of SLE" Arth Rheum 25(1982) may be cheek rash, such as cheek rash, discoid rash or red raised plaque; light sensitivity, such as response to sunlight, leading to the development or increase of rashes; canker sores, such as ulcers in the nose or mouth, are generally painless; arthritis, e.g. involving twoOr non-erosive arthritis of a plurality of peripheral joints (arthritis in which the bone around the joint is not damaged); serositis, pleuritis, or pericarditis; renal dysfunction, such as excess protein in urine (greater than 0.5 gm/day or 3+ on test bars) and/or cell casts (abnormal components derived from urine and/or leukocytes and/or renal tubular cells); neurological signs, symptoms or other indicators, seizures (convulsions), and/or psychosis without drugs, or metabolic disorders known to cause these effects; and hematological signs, symptoms or other indicators, such as hemolytic anemia or leukopenia (white blood cell count below 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 must generally 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 variation

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

Nucleic acids include copies of the nucleic acid, e.g., generated from amplified copies. Amplification may be desirable in some cases, for example, to obtain a desired amount of material for detection of a variation. The amplicon can then be subjected to a variation detection method, such as the methods 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 primer extension assaysSpecific PCR, allele-specific Ligation Chain Reaction (LCR) and gapped LCR); allele-specific oligonucleotide hybridization assays (e.g., oligonucleotide ligation assays); a cleavage protection assay in which mismatched bases in a nucleic acid duplex are detected with a protective agent from cleavage; MutS protein binding assay; electrophoretic analysis comparing the molecular mobility of the variant and wild-type nucleic acids; denaturing gradient gel electrophoresis (DGGE, as in, for example, Myers et al (1985) Nature 313: 495); analysis of RNase cleavage at mismatched base pairs; analysis of chemical or enzymatic cleavage of heteroduplex DNA; mass spectrometry (e.g., MALDI-TOF); genetic Bit Analysis (GBA); 5' nuclease assays (e.g.And assays using molecular beacons. Some of these methods are discussed in further detail below.

Detection of a variation in a target nucleic acid can be achieved 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 a target nucleic acid sequence directly from a genomic DNA preparation 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, for example, the polymerase chain reaction is described in Saiki et al, Science 239: 487, 1988; in U.S. Pat. nos. 4,683,203 and 4,683,195.

The target nucleic acid sequence may also be amplified using ligase chain reactions known in the art. See, e.g., Wu et al, Genomics 4: 560-569(1989). In addition, variations (e.g., substitutions) can also be detected using a technique known as allele-specific PCR. See, for example, Ruano and Kidd (1989) Nucleic Acids Research 17: 8392, adding a solvent to the mixture; McClay et al (2002) analytical biochem.301: 200-206. In certain embodiments of this technique, an allele-specific primer is used, wherein the 3' nucleotide of the primer is complementary to (i.e., capable of specific base-pairing with) a particular variation in the target nucleic acid. If the specific variation is not present, no amplification product is observed. The Amplification Refractory Mutation System (ARMS) can also be used to detect mutations (e.g., substitutions). ARMS are described, for example, in european patent application publication No. 0332435 and Newton et al, Nucleic Acids Research, 17: 7,1989, respectively.

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 assays (see, e.g., Ye et al (2001) hum. mut.17: 305-316; and Shen et al Genetic Engineering News, Vol.23, 15/3/2003), including allele-specific PCR; (3) 5' nuclease assay (see, e.g., De La Vega et al (2002) Bio technologies 32: S48-S54 (described)Measurement); ranade et al (2001) Genome Res.11: 1262-1268; and Shi (2001) clin. chem.47: 164-172); (4) assays using molecular beacons (see, e.g., Tyagi et al (1998) NatureBiotech.16: 49-53; and Mhlanga et al (2001) Methods 25: 463-71); and (5) oligonucleotide ligation assays (see, e.g., Grossman et al (1994) Nuc. acids Res.22: 4527-4534; patent application publication No. US 2003/0119004A 1; PCT International publication No. WO 01/92579A 2; and U.S. Pat. No. 6,027,889).

Variations can also be detected by mismatch detection methods. Mismatches are hybridization nucleic acid duplexes that are not 100% complementary. The lack of complete complementarity may be caused by a deletion, insertion, inversion or substitution. An example of a mismatch detection method is described, for example, in Faham et al, proc.natl acad.sci USA 102: 14717-14722(2005) and Faham et al, hum.mol.genet.10: 1657 Mismatch Repair Detection (MRD) assay in 1664 (2001). Another example of a mismatch cleavage technique is RNase protection, described in Winter et al, proc.natl.acad.sci.usa, 82: 7575, 1985 and Myers et al, Science 230: 1242, 1985. For example, the methods of the invention can involve the use of labeled ribonucleic acid probes that are complementary to a human wild-type target nucleic acid. The ribonucleic acid probe is renatured (hybridized) with a target nucleic acid derived from a tissue sample and subsequently digested with the enzyme RNase a, which is capable of detecting some mismatches in the duplex RNA structure. If RNase A detects a mismatch, it cleaves at the site of the mismatch. Thus, when isolating renatured RNA preparations on an electrophoretic gel matrix, if RNase A has detected and cleaved mismatches, a smaller RNA product will be seen than in the full-length duplex RNA of the ribonucleic acid probe with mRNA or DNA. The riboprobe need not be the full length of the target nucleic acid, but can be part of the target nucleic acid, so long as it contains a location suspected of having a variation.

Mismatches can be detected in a similar manner with DNA probes, for example by enzymatic or chemical cleavage. See, e.g., Cotton et al, proc.natl.acad.sci.usa, 85: 4397, 1988; and Shenk et al, proc.natl.acad.sci.usa, 72: 989, 1975. Alternatively, mismatches may be detected by electrophoretic mobility shift of the mismatched duplex relative to the paired duplex. See, e.g., Cariello, Human Genetics, 42: 726, 1988. The target nucleic acid suspected of containing the variation can be amplified with a ribonucleic acid probe or a DNA probe prior to hybridization. Southern hybridization can also be used to detect changes in a target nucleic acid, particularly if the change is a gross rearrangement, such as a deletion and an insertion.

Restriction Fragment Length Polymorphism (RFLP) probes for the target nucleic acid or surrounding marker genes can be used to detect variations, such as insertions or deletions. Insertions and deletions can also be detected by cloning, sequencing and amplification of the target nucleic acid. Single Strand Conformation Polymorphism (SSCP) analysis can also be used to detect base-altered variants of alleles. See, e.g., Orita et al, proc.natl.acad.sci.usa 86: 2766. 2770, 1989 and Genomics, 5: 874-879, 1989.

Microarrays are multiplex technologies, typically using an array of thousands of nucleic acid probes arranged to hybridize, for example, to a cDNA or cRNA sample under highly stringent conditions. The relative abundance of nucleic acid sequences in the target is typically determined by detecting fluorophore-, silver-, or chemiluminescent-labeled targets to detect and quantify probe-target hybridization. In a typical microarray, probes are attached to a solid surface by covalent bonds (via epoxy silane, amino silane, lysine, polyacrylamide, or others) to a chemical substrate. The solid surface is, for example, glass, a silicon wafer or a microscopic bead. Various microarrays are commercially available, including those produced by, for example, Affymetrix, inc.

Biological samples can be obtained by certain methods known to those skilled in the art. Biological samples can be obtained from vertebrates, and in particular mammals. A representative tumor tissue mass is typically obtained by tissue biopsy. Alternatively, the tumor cells may be obtained directly in the form of a tissue or fluid that is known or believed to contain the tumor cells of interest. For example, samples of lung cancer lesions may be obtained by resection, 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 a tumor sample or from other body samples such as urine, saliva, or serum (tumor cells shed from the tumor and appear in such body samples). By screening such body samples, a simple early diagnosis of diseases such as cancer can be achieved. Furthermore, by testing such body samples for variations in the target nucleic acid (or encoded polypeptide), the progress of the treatment can be more easily monitored. Furthermore, methods for enriching tissue preparations for tumor cells are known in the art. For example, tissue may be isolated from paraffin or cryostat sections. Cancer cells can also be isolated from normal cells by flow cytometry or laser capture microdissection.

Upon determining 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 can be administered to the subject to treat the lupus disorder in the subject. Diagnosis of the various pathological conditions described herein can be made in mammals by skilled practitioners. Diagnostic techniques are available in the art that allow, for example, the diagnosis or detection of lupus in mammals.

The lupus therapeutic agent may be administered according to known methods, such as intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracorobrosplanal, subcutaneous, intra-articular, intra-synovial, intrathecal, oral, topical or inhalation routes. Optionally, administration can be by micro-pump infusion using a variety of commercially available devices.

Effective dosages and schedules for administering lupus therapeutic agents can be determined empirically, and making such determinations is within the skill of the art. Single or multiple doses may be utilized. For example, an effective dose or amount of an interferon inhibitor used alone may range from about 1mg/kg body weight to about 100mg/kg body weight per day. The solvent may be removed in a manner known in the art, for example, as described by Mordenti et al, pharmaceut.res, 8: 1351(1991) inter-species increase or decrease of the dose is disclosed.

In vivo administration with lupus therapeutic agents, normal doses may vary from about 10ng/kg to up to 100mg/kg of mammalian body weight per day or more, preferably from about 1 μ g/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature; see, for example, U.S. patent nos. 4,657,760, 5,206,344, or 5,225,212. It is expected that different formulations will be effective for different therapeutic compounds and different disorders, administration targeted 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 the method. The one or more other therapies may include, but are not limited to, administration of steroids to the disorder in question and other criteria for a care regimen. It is contemplated that such other therapies may be used as active agents separate from, for example, lupus-directing therapeutic agents.

Reagent kit

Kits or preparations are also provided for use in the applications described or suggested above. Such kits may include carrier means divided to receive in a tight seal one or more container means, such as vials, tubes, etc., each container means including separate elements for use in a method. For example, one container means may comprise a probe that is or may be detectably labeled. Such probes can be polynucleotides specific for polynucleotides comprising SLE risk loci. Where the kit utilizes nucleic acid hybridization to detect a 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 means, such as a biotin-binding protein, e.g., avidin or streptavidin, bound to a reporter molecule, such as an enzyme label, a fluorescent label, or a radioisotope label.

The kit typically comprises the above-described container and one or more other containers comprising materials required from a market and user standpoint, including buffers, diluents, filters, needles, syringes, and instructions for use. The label may be located on the container to indicate that the composition is for a particular therapeutic or non-therapeutic application, and may also indicate for in vivo or in vitro use, such as those described above.

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

Marketing method

Also provided are methods for marketing lupus therapeutic agents or pharmaceutically acceptable compositions thereof, comprising promoting, informing and/or instructing a target audience of the use of the therapeutic agent or pharmaceutical composition thereof in treating lupus patients or subpopulations of patients from whom a sample obtained shows the presence of a genetic variation as disclosed herein.

Sales are typically paid travel through non-human media, where advertisers are identified and information is controlled. For purposes herein, sales include promotions, customs campaigns, product placement, sponsorships, underwriting, and promotions. This term also includes sponsored informational announcements appearing in any printed media of dissemination designed to appeal to a large audience to persuade, inform, promote, stimulate, or otherwise alter the behavior of the mode of purchasing, supporting, or approving preferences for the present invention.

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

In particular, the present invention relates to the following embodiments:

1. a method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in a SLE risk locus, 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 a thymine at chromosome 11389322 of chromosome 8 in a human, and wherein the subject is suspected of having lupus.

2. A method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to the position of a Single Nucleotide Polymorphism (SNP) of the at least one locus as set forth in table 4, and wherein the subject is suspected of having lupus.

3. The method of embodiment 2, wherein a variation is detected in 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.

4. The method of embodiment 2, wherein the at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL 10.

5. The method of embodiment 2, wherein the variation in at least one locus comprises a SNP as set forth in table 4.

6. A method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in at least one locus occurs at a nucleotide position corresponding to the position of a Single Nucleotide Polymorphism (SNP) of the at least one locus as set forth in table 6, and wherein the subject is suspected of having lupus.

7. The method of embodiment 6, wherein a variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci.

8. The method of embodiment 6, wherein at least one locus is selected from the group consisting of IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

9. The method of embodiment 6, wherein the variation in at least one locus comprises a SNP as set forth in table 6.

10. The method of embodiment 1, comprising detecting an additional variation in at least one SLE risk locus as set forth in table 4, wherein the additional variation in at least one locus occurs at a nucleotide position corresponding to the SNP position of at least one locus as set forth in table 4.

11. The method of embodiment 1, comprising detecting an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in at least one locus occurs at a nucleotide position corresponding to a SNP position of at least one locus as set forth in table 6.

12. The method of embodiment 10, comprising detecting an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in at least one locus occurs at a nucleotide position corresponding to a SNP position of at least one locus as set forth in table 6.

13. The method of embodiment 2, comprising detecting an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in at least one locus occurs at a nucleotide position corresponding to a SNP position of at least one locus as set forth in table 6.

14. The method of any one of embodiments 1-13, wherein detecting comprises performing a primer extension assay selected from the group consisting of; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

15. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, 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 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 11389322 of chromosome 8 of a human, wherein the presence of the variation in the BLK locus is indicative of responsiveness of the subject to the therapeutic agent.

16. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) position of the at least one locus as set forth in table 4, wherein presence of the variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent.

17. The method of embodiment 16, wherein a variation is detected in 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.

18. The method of embodiment 16, wherein the at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL 10.

19. The method of embodiment 16, wherein the variation in at least one locus comprises a SNP as set forth in table 4.

20. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) position of the at least one locus as set forth in table 6, wherein presence of the variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent.

21. The method of embodiment 20, wherein a variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci.

22. The method of embodiment 20, wherein at least one locus is selected from the group consisting of IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

23. The method of embodiment 20, wherein the variation in at least one locus comprises a SNP as set forth in table 6.

24. The method of embodiment 15, comprising determining whether the subject comprises an additional variation in at least one SLE risk locus as set forth in table 4, wherein the additional variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 4.

25. The method of embodiment 15, comprising determining whether the subject comprises an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6.

26. The method of embodiment 24, comprising determining whether the subject comprises an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6.

27. The method of embodiment 16, comprising determining whether the subject comprises an additional variation in at least one SLE risk locus as set forth in table 6, wherein the additional variation in the at least one locus occurs at a nucleotide position corresponding to the SNP position of the at least one locus as set forth in table 6.

28. A method of diagnosing or prognosing 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:

(a) the biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in the BLK locus;

(b) a variation 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 chromosome 8 of human; and

(c) the presence of a variation in the BLK locus is a diagnosis or prognosis of lupus in the subject.

29. A method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4;

(b) a variation in at least one locus comprises a SNP as set forth in table 4 or is located at a nucleotide position corresponding to a SNP as set forth in table 4; and

(c) the presence of the variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

30. The method of embodiment 29, wherein a variation is detected in 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.

31. The method of embodiment 29, wherein at least one locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL 10.

32. The method of embodiment 29, wherein the variation in at least one locus comprises a SNP as set forth in table 4.

33. A method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6;

(b) a variation in at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and

(c) the presence of the variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

34. The method of embodiment 33, wherein a variation is detected in at least two loci, or at least three loci, or at least four loci, or five loci.

35. The method of embodiment 33, wherein at least one locus is selected from the group consisting of IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

36. The method of embodiment 33, wherein the variation in at least one locus comprises a SNP as set forth in table 6.

37. The method of embodiment 28 wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising an additional variation in at least one SLE risk locus as set forth in table 4;

(b) the other variation in the at least one locus comprises a SNP as set forth in table 4 or is located at a nucleotide position corresponding to a SNP as set forth in table 4; and

(c) the presence of the other variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

38. The method of embodiment 28 wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising an additional variation in at least one SLE risk locus as set forth in table 6;

(b) the other variation in the at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and

(c) the presence of the other variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

39. The method of embodiment 37, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising an additional variation in at least one SLE risk locus as set forth in table 6;

(b) the other variation in the at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and

(c) the presence of the other variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

40. The method of embodiment 29, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising an additional variation in at least one SLE risk locus as set forth in table 6;

(b) the other variation in the at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and

(c) the presence of the other variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

41. The method of any one of embodiments 28-40, wherein detecting comprises performing a primer extension assay selected from the group consisting of; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

42. A method of treating a lupus disorder in a subject in which a genetic variation is known to exist at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) in a SLE risk locus, wherein the SNP is rs922483(SEQ ID NO:13) and the SLE risk locus is BLK, and wherein the variation is thymine at chromosome 11389322 of chromosome 8 in a human, the method comprising administering to the subject a therapeutic agent effective to treat the disorder.

43. A method of treating a lupus disorder in a subject in which a genetic variation is known to exist at a nucleotide position in at least one SLE risk locus as set forth in table 4 that corresponds to a Single Nucleotide Polymorphism (SNP) as set forth in table 4, the method comprising administering to the subject a therapeutic agent effective to treat the disorder.

44. A method of treating a lupus disorder in a subject in which a genetic variation is known to exist at a nucleotide position in at least one SLE risk locus as set forth in table 6 that corresponds to a Single Nucleotide Polymorphism (SNP) as set forth in table 6, the method comprising administering to the subject a therapeutic agent effective to treat the disorder.

45. A method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in the subject, the subject having a genetic variation in a SLE risk locus, wherein the SLE risk locus is BLK, wherein the variation in the BLK locus occurs at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) position, wherein the SNP is rs922483(SEQ ID NO:13), and wherein the variation is thymine at chromosome 11389322 of chromosome 8 of a human.

46. A method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in the subject, the subject having a genetic variation at a nucleotide position in the at least one SLE risk locus described in table 4 that corresponds to a Single Nucleotide Polymorphism (SNP) described in table 4.

47. The method of embodiment 46, wherein the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, and IL 10.

48. A method of treating a subject with a lupus disorder, the method comprising administering to the subject a therapeutic agent effective to treat the disorder in the subject, the subject having a genetic variation at a nucleotide position in the at least one SLE risk locus described in table 6 that corresponds to a Single Nucleotide Polymorphism (SNP) described in table 6.

49. The method of embodiment 48, wherein the at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

50. A method comprising preparing a lupus therapeutic agent and packaging the therapeutic agent with instructions for administering the therapeutic agent to a subject who has or is believed to have lupus and has a genetic variation at a position corresponding to a Single Nucleotide Polymorphism (SNP) in an SLE risk locus, wherein the SNP is rs922483(SEQ ID NO:13), the SLE risk locus is BLK, and wherein the variation is thymine at chromosome 11389322 of human chromosome 8.

51. A method comprising preparing a lupus therapeutic agent and instructions for packaging the therapeutic agent and administering the therapeutic agent to a subject who has or is believed to have lupus and has a genetic variation at a position in the at least one SLE risk locus of table 4 that corresponds to a Single Nucleotide Polymorphism (SNP) as set forth in table 4.

52. A method comprising preparing a lupus therapeutic agent and instructions for packaging the therapeutic agent and administering the therapeutic agent to a subject who has or is believed to have lupus and has a genetic variation at a position in the at least one SLE risk locus of table 6 that corresponds to a Single Nucleotide Polymorphism (SNP) as set forth in table 6.

53. A method of selecting a patient with lupus for treatment with a lupus therapeutic agent, comprising detecting the presence of a genetic variation at a position corresponding to a Single Nucleotide Polymorphism (SNP) in an SLE risk locus, wherein the SNP is rs922483(SEQ ID NO:13), the SLE risk locus is BLK, and wherein the variation is thymine at chromosome 11389322 of chromosome 8 in humans.

54. A method of selecting a patient with lupus for treatment with a lupus therapeutic agent, comprising detecting the presence of a genetic variation at a nucleotide position in at least one SLE risk locus described in table 4 that corresponds to a Single Nucleotide Polymorphism (SNP) described in table 4.

55. The method of embodiment 54, wherein the at least one SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10.

56. A method of selecting a patient with lupus for treatment with a lupus therapeutic agent, comprising detecting the presence of a genetic variation at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) as set forth in table 6 in at least one SLE risk locus as set forth in table 6.

57. The method of embodiment 56, wherein at least one SLE risk locus is selected from IFIH1, CFB, CLEC16A, IL12B and SH2B 3.

58. The method of any one of embodiments 53-57, wherein detecting comprises performing a primer extension assay selected from the group consisting of; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

59. A method of assessing whether a subject is at risk for developing lupus, the method comprising detecting in a biological sample obtained from the subject the presence of a genetic signature indicative of risk for developing lupus, wherein the genetic signature comprises a set of at least three Single Nucleotide Polymorphisms (SNPs), each SNP occurring in a SLE risk locus as set forth in tables 4 and 6.

60. The method of embodiment 59, 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.

61. The method of embodiment 59, wherein each SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B, and SH2B 3.

62. The method of embodiment 58, 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 thymine at chromosome 11389322 of chromosome 8 in humans.

63. A method of diagnosing lupus in a subject, the method comprising detecting in a biological sample obtained from the subject the presence of a genetic signature indicative of lupus, wherein the genetic signature comprises a set of at least three Single Nucleotide Polymorphisms (SNPs), each SNP occurring in a SLE risk locus as set forth in tables 4 and 6.

64. The method of embodiment 63, 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.

65. The method of embodiment 63, wherein each SLE risk locus is selected from TNIP1, PRDM1, JAZF1, UHRF1BP1, IL10, IFIH1, CFB, CLEC16A, IL12B, and SH2B 3.

66. The method of embodiment 63, 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 thymine at chromosome 11389322 of chromosome 8 in humans.

67. The method of any one of embodiments 59-66, wherein detecting comprises performing a primer extension assay selected from the group consisting of; an allele-specific primer extension assay; an allele-specific nucleotide incorporation assay; an allele-specific oligonucleotide hybridization assay; 5' nuclease assay; assays employing molecular beacons; and methods of oligonucleotide ligation assays.

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided above.

Examples

In all examples, references to certain publications are indicated by numbers, which have full bibliographic information at the end of the examples section.

Example 1

Identifying novel risk loci for SLE

Method and object

Object

The selection and genotyping of SLE cases, samples for Whole genome relevance scans (GWAS), and controls from the New York Health Project (NYHP) Collection (Mitchell et al, J Urban Health 81 (2): 301-10(2004)) (Hom et al, N Engl J Med358 (9): 900-9(2008)) have been described previously. As detailed below, the SLE cases consist of 3 case series: a) from an AutoimmuneBiomarkers tensile Network (ABCON) from NIH/NIAMS-sponsored depository (Bauer et al, PLoS media 3 (12)): e491(2006)) and 338 cases from Multiple Autoimmune Disease Genetics Consortium (MADGC) (Criswell et al, Am J Hum Genet 76 (4): 561-71 (2005)); b) from the University of California San Francisco (UCSF) Lupus Genetics Project (Seligman et al, Arthritis Rheum 44 (3)): 618-25 (2001); remmers et al, N Engl J Med 357 (10): 613 cases of 977-86 (2007); and c) from the University of Pittsburgh Medical Center (UPMC) (Demirci et al, an 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 4564 samples from the publicly available National Cancer Institute Cancer Genetic Markers of Suscientific (CGEMS) project (available at URL: CGEMS (dot) Cancer (dot) gov).

Whole genome data set of 1310 SLE cases and 7859 controls

We previously described the selection and genotyping of SLE case samples (Hom et al, N Engl J Med358 (9): 900-9 (2008)). All SLE cases were northern americans of european descent as determined by self-report and confirmed by genotyping. The diagnosis of SLE was confirmed in all cases by medical record review (94%) or by a standard file written by the attending rheumatologist (6%) [ Hochberg et al, Arthritis Rheum 40 (9): 1725[1997 ]) or 4 or more criteria defined by American College of Rheumatology [ ACR ]. Clinical data for these case series are given 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 Med358 (9): 900-9 (2008)). Table 1 describes the number of valid samples organized by site.

Sample and SNP filtration was performed using the analysis modules 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 Genet38 (8): 904-09 (2006)). Genome-wide SNP data was 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 site-directed tissue replication studies

^aSamples of whole genome association scans are described (Hom, G. et al, N Engl J Med 358: 900-9 (2008)).^bIndependent SLE cases from the american cohort are from the PROFILE SLE union, university of california at three colleges (UCSF) (Thorburn, c.m. et al, Genes Immun 8: 279-87(2007)), University of Pittsburgh Medical Center (UPMC), University of Minnesota (UMN), and university of John Hopkins (JHU). U.S. controls were from the New York health program (Gregersen et al) and Alzheimer's cases and controls were from the university of Pittsburgh and NCRAD.^cSLE cases and controls were from Stockholm, Karolinska, Solna, Uppsala, Lund andSweden。^d823 control from Stockholm were genotyped using Illumina 317K SNP array. SNPs in these samples were attributed and analyzed as described in "methods".

Customized SNP arrays

Custom arrays were designed with 10,848 SNPs measured by quality control as described below. The complete array had 12,864 SNPs, but 2016 SNPs failed quality control measurements, leaving 10,848 SNPs to enter the analysis. The custom array consisted of 3,188 SNPs selected based on a nominal P < 0.05 in association scans of the SLE genome-wide, 505 SNPs from 25 previously reported SLE risk loci, 42 SNPs selected from other autoimmune diseases after literature search for confirmed risk alleles, and 7113 SNPs to determine and control 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 that are substructural-rich in european populations (Tian, c. et al, PLoS Genet 4, e4 (2008)). A Custom array was produced by Illumina, inc. using its iSelect Custom beacon chip and the rs identification number we provided for the SNP that passed the quality control filtering described below.

Quality control and interpolation (imputation)

With respect to the us data, a total of 1,464 u.s. cases and 3,078 us control genotypes were typed on the custom Illumina chip, also referred to herein as the custom 12K chip. Strict Quality Control (QC) standards are used to ensure that the final analysis includes high quality data. Specifically, a) 116 individuals with > 5% missing data were excluded, and b) the data was analyzed based on implicit affinity (cryptic relatedness) and on state homology (identity byState; replicate samples of IBS) status (PI Hat > 0.15), excluding 279 individuals. Including only SNPs with a) < 5% missing data, b) Hadi-Winberg equilibrium (HWE) p-value > 1x10^-6C) Small allele frequency (MAF) > 0.01% and d) a p-value > 1x10 in tests with differential loss of deletion in case and control^-5The SNP of (1). The batch effect of SNPs was also examined. After applying the above-described filtering, 1144 cases and a final set of 3003 controls and 11024 SNPs were available for analysis. All QC tests were performed using PLINK (Purcell et al, Am J Hum Genet 81 (3): 559-75 (2007)).

With respect to 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 Human controls genotyped on an Illumina, inc.317k Human HapMap SNP bead array (also referred to herein as a 317K array) were also incorporated into the assay. The two sets of data sets were combined as follows. First, an overlapping data set of 6,789 SNPs between the 12K and 317K data was generated. This data set was used to examine implicit relationships and replicate samples of the Swedish copy group. As a result, 313 samples (PI Hat > 0.15) were excluded. After quality control checks, 863 cases and 523 controls shipped to be genotyped on a custom 12K chip and 831 controls genotyped on a 317KIllumina chip were analyzed. Second, the 831 Swedish human controls, genotyped with 317K array interpolation (see below), yielded a larger set of overlapping SNPs. Of the remaining SNPs, 4605 SNPs were captured by interpolation. The final set of 11394 overlapping SNPs was analyzed. SNPs in this dataset were filtered using the same threshold as above. The remaining 1250 SNPs not captured by interpolation (imputation) were analyzed only in the initial set of swedish human samples genotyped on a 12K chip.

831 Swedish controls genotyped with the 317K array were interpolated using MACH (a Markov Chain-based haplotype software program, available from URL sph. umich. edu/csg/abecasis/MACH) using the phase II HapMap CEU sample as a reference. Phase II HapMap CEU refers to samples from human haplotype items known as human haplotype items of utah inhabitants with northern and western european ancestors (CEUs) released from the "phase II" data (see also Li et al, am jhum gene S79 at 2290 (2006)). Before interpolation, a stringent quality control check was applied to the 317K SNP. The interpolation included 293,242 passes the following criteria (1) MAF > 1%, (2) deletion rate < 5%, and (3) HWE p value > 1x10^-6A subset of markers of (1). After interpolation, SNPs with low interpolation quality were discarded, i.e., R squared _ Hat (RSQR _ HAT) < 0.40 as reported by MACH. An overlapping 11394 marker set was available for analysis. To account for uncertainty in the interpolation, probability scores were used in the analysis instead of reading genotypes (genotype calls).

To interpolate the whole genome association study samples, genotype data for meta-analysis (meta-analysis) was from 1310 SLE cases genotyped with Illumina 550K whole genome SNP platform (see Hom, g. et al, NEngl J Med 358: 900-9 (2008)). Selection and genotyping of SLE case samples was previously described (Hom, g. et al, N Engl J Med 358: 900-9 (2008)). In addition to the 3583 controls described above (Hom, G. et al, N Engl J Med 358: 900-9(2008)), 4564 control samples (obtained from URL: CGEMS. Cancer. gov) from the publicly available Cancer genetic Susceptibility Markers of Suscapability (CGEMS)) program were included after approval. A total of 7859 control samples were tested using the above-described data quality control filtration (Hom, G. et al, N Engl J Med 358: 900-9 (2008)). Thereafter, IMPUTE version 1 (obtained from URLwww.stats.ox.ac.uk/. about marcini/software/gwas/impute.html) was used to infer genotyping using the HapMap PhaseII CEU sample as a reference (marcini, J.et al, nat. Genet.39: 906-913 (2007)). Association statistics were generated using SNPTEST (obtained from URL www.stats.ox.ac.uk/. about Marchini/software/gwas/SNPTEST _ v1.1.4.html) (Marchini, J. et al, nat. Genet. 39: 906-913 (2007)). Specifically, relevance statistics are generated using an additive model (Frequentist 1 option in SNPTEST) to adjust for uncertainty in the interpolated genotype (pro option in SNPTEST). The ordered list of association statistics is used to select the replication sections.

Population hierarchy of replica samples (Population hierarchy)

For each copy set (reproduction cohort), the possible population hierarchies are corrected using the ancestry information markers. The major genetic variation component in the top ten was inferred with EIGENSTRAT software using a subset of 5486 unrelated ancestral information markers that passed strict quality control criteria (Price et al, Nat Genet38 (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 outliers were removed from the swedish group, respectively. A degree of population stratification along the first two feature vectors was observed in both the us and swedish replication sets. To correct case-control stratification, one of the following strategies was used: (1) applying a correction of the Cochran-Armitage test statistic incorporated in EIGENSTRAT to the us and swedish replicate datasets for which genotype data is available; (2) in analyzing interpolated swedish human data, the principal components are used as covariates in the logistic regression model.

Relevance analysis

For the American data, some expansion (inflation) of the test statistics was observed after performing the uncorrected degree of freedom 1 allelic association test (PLINK (Purcell, S. et al, Am J Hum Genet 81: 559-75 (2007))). To correct for in American human samplesUsing 5486 unrelated ancestral information markers for major component analysis (EIGENSTRAT). First, genetic outliers (defined as σ > 6) were removed. Second, the Cochran-armintage trend card test statistics for each genotyped SNP in 1129 cases and 2991 controls were calculated, and the test statistics for each SNP in EIGENSTRAT were adjusted using the first four eigenvectors. The two-tailed p-value based on the test statistics for each SNP was calculated. Lambda of american human samples after correction of population stratification_gcIs 1.05.

For swedish human data, the swedish group was examined for hidden population stratification using 5486 ancestral informative markers genotyped in 12K samples and an additional Illumina 317K control. After removing genetic outliers, 834 cases and 515 controls were genotyped on a custom 12K chip, and 823 controls on an Illumina 317K chip. The test statistics performed in EIGENSTRAT were corrected for an overlapping set of 6789 SNPs between two Illumina arrays. To correct stratification of the 4605 SNP groups genotyped in the 12K sample and interpolated in the Illumina 317K sample, the first four feature vectors determined above were used as covariates of the logistic regression model performed in SNPTEST, since EIGENSTRAT was not intended for interpolated genotype data. A panel of 1250 markers not captured by Illumina 317K SNP interpolation were analyzed only in 834 cases and 515 controls genotyped on a custom 12K chip. Lambda of Swedish human samples after correction of population stratification_gcIs 1.10.

Meta-analysis

The meta-analysis was performed using a weighted z-score method. To combine the results of the different groups, alleles were mapped on the forward strand of the reference sequence No. 36 of the human genome at the National Center for Biotechnology Information (NCBI) to avoid ambiguity associated with C/G and a/T SNPs. The human genomic reference sequence for NCBI can be obtained from URL:www.ncbi.nlm.nih.gov. See also, Pruitt et al, nucleic acids res.35(database issue): D61-D65 (2007). Considering the direction of effect relative to an arbitrary reference allele, will eachThe P values of the groups were converted to z scores. The weighted sum of z-scores is calculated by taking the square root of the sample size of each group and then dividing its sum by the square root of the total sample size, weighting each z-score. The combined z-score of the swedish and american replica groups was converted to a single tail p-value. The z-score of the meta-analysis was converted to two-tailed p-values and evidence of relevance was calculated. Consider crossing threshold 5x10^-8The SNP of (a) is associated with the overwhelming SLE. With less than 1x10 without significance by whole genome^-5The locus of the combined p-value of (a) is considered a strong candidate. Using the freely available METAL software package (available from URL:www.sph.umich.edu/csg/abecasis/Metal) Meta-analytical method was performed. To calculate the summarized odds ratio, the Cochran-Mantel-Haenszel (CMH) method performed using METAL software was used. Odds ratios were calculated relative to the risk allele of each SNP. In addition, a weighted average allele frequency in the control was calculated relative to the risk allele for each SNP.

Percentage Variance interpretation (Percent Variance expained)

For the previous SLE related SNPs and in our replication studies with less than 1x10^-5SNP of meta p value, calculate percentage of variance interpretation. A liability threshold model (liability threshold model) was used, where SLE was assumed to have a potential liability score (liability) that is generally distributed with a mean of 0 and a variance of 1. The prevalence of SLE in the general population was assumed to be 0.1%. To calculate the threshold for each genotype, the allele frequencies in the control and the magnitude of the effect corresponding to the Odds Ratio (OR) in our analysis were used.

Interaction analysis

To look for superordinate effects between top signals, a list of all SNPs was compiled in tables 2, 4 and 6 and interaction analysis was performed for each replicate group using the superordinate option performed in PLINK. To obtain greater statistical efficacy, pure case analysis (case-only analysis) was performed. After correcting the test numbers, no SNP-SNP interaction was found at the significance level of p < 0.05.

Analysis of conditions

At each genomic region that showed a strong association with SLE, the SNP that showed the strongest signal was selected. PLINK was used as a condition for this SNP and other SNPs were sought that showed strong association with SLE.

Large-scale replication research identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as systemic lupus erythematosus Novel risk loci of

Existing genome-wide association (GWA) and candidate gene studies have identified at least 15 approaches to genome-wide significance (P < 5x 10)^-8) A common risk allele of (a). These include genes important for acquired immunity and autoantibody production (HLA class II alleles, BLK, PTPN22 and BANK1), and genes that play a role in innate immunity and interferon signaling (ITGAM, TNFAIP3, STAT4 and IRF5) (Cunninghame Graham, D.S. et al, Nat.Genet.40: 83-89 (2008); Graham, R.R. et al, Nat.Genet.40 (9): 1059-61 (2008); Graham, R.R. et al, InterJ Med 265: 680-88 (2009); Harley, J.B. et al, Nat.Genet.40: 204-10 (2008); Hom, G. et al, N Engl J J.900-9 (2008); Kozyr; 680, S.V.et al, Nat SaGenet.40: 204-10(2008), Hom, G. et al, N Engl J.J.2008: 900-9 (2008; Kozyr.680; Sigzyr.528, S.V.et al, Nat.40: 1726: plain J.17276, 2008) and S. 2008; PLUS) (2008; 2008) J.7: 2008) et al). To identify additional risk loci, targeted replication studies were performed on SNPs for 2446 loci that exhibited a nominal p-value < 0.05 in the existing GWAS7 scan for 1310 cases and 7859 controls. SNPs from 25 previously reported SLE risk loci, 42 SNPs from 35 loci suggested by other autoimmune diseases, and over 7000 ancestral informative markers were also genotyped. An overview of the experimental design is shown in figure 1. The above SNPs were incorporated into Illumina-customized SNP arrays. Arrays were genotyped for independent cases and controls in the us and sweden. 823 Swedish human control were genotyped using the Illumina 310K SNP array and the variants were analyzed as described in "methods" above.

Specifically, as described above, custom SNP arrays consisting of > 12,000 variants were designed, genotyped into 2 independent SLE case and control populations, from us (1129 SLE cases and 2991 controls) and sweden (834 SLE cases and 1338 controls), respectively. The american control included 2215 cases/control samples of alzheimer's disease, which was considered acceptable as a control because the genetic basis for SLE and alzheimer's disease was expected to be independent of each other. Data quality filtering is then applied to remove samples with poor performance and SNPs, population outliers and duplicate/related individuals (see "methods" above). After these quality control measurements, a final set of 10848 SNPs was examined, as shown in figure 1. Statistics of association were calculated for 3735 variants and 7113 ancestral informative markers were used to correct population stratification (see "methods" above).

First tested 25 variants (from 23 loci) previously reported to be associated with SLE (see table 2). We also found additional evidence for the association of 21 variants (P < 0.05), including reaching genome-wide significance (P < 5x 10) in the existing combined dataset^-8) Among the significant results for the whole genome were HLA class II DR3(DRB1 ＊ 0301), IRF5, TNFAIP3, BLK, STAT4, ITGAM, PTPN22, PHRFl (KIAA1542), and TNFSF4(OX 40L). this analysis provided additional evidence for variants at 9 loci, and significant whole genome levels were reported in a previous study, HLA ＊ DR2, TNFAIP3(rs6920220), BANK1, ATG5, PT 1, PXK, FCGR2A, UBE GR 2L3, and IRAK1/MECP 2.

Earlier candidate gene studies identified MECP2 as a potential risk allele for SLE (sawaha, a.h. et al, PLoS ONE 3: e1727 (2008)). However, in the existing data set, SNPs near IRAK1 showed the strongest evidence of association, with IRAK1 being the key gene for toll-like receptor 7 and 9 signaling, located within the identified linkage disequilibrium region around MECP 2. Similar findings have also been recently reported (Jacob, c.o. et al, proc.natl.acad.sci.usa (2009)), with additional work being required to determine causal alleles in the IRAK1/MECP2 locus. We found additional evidence for the association of 3 loci-TYK 2, ICA1, and NMNAT 2-these 3 loci previously showed significant but no genome-wide level association evidence (Harley, j.b. et al, nat. gene.40: 204-10 (2008); Sigurdsson, s. et al, Am J Hum gene 76: 528-37 (2005)). No evidence of any association was observed for the 4 previously suggested variants-LYN, SCUBE1, TLR5, and LY 9-in the combined dataset values.

To identify new SLE risk loci, we examined a total of 3188 SNPs from 2446 different loci (Hom, g. et al, N EnglJ Med358, 900-9(2008)) that showed evidence of association with SLE in our full genome dataset, which included 502033 SNPs that were genotyped in 1310 SLE cases and an expanded 7859 control set. Using this data set, > 2.1M variants were interpolated using the Phase II HapMap CEU sample as a reference (see "method" above) and an ordered list of correlation statistics was generated. Variants with P < 0.05 were selected for possible inclusion on the custom replication array. For efficient genotyping, related variants (r) were identified²> 0.2), and at least 2 SNPs are 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. In replicate samples, correlation statistics were calculated (see "method"), and significant enrichment of replication results relative to the expected zero distribution was observed. Excluding the previously reported SLE risk alleles, 134 loci harbor P < 0.05 (expected 64, P ═ 2x 10^-15) And 12 loci with P < 0.001 (expected 1, P ═ 1x10^-9) This indicates the presence of a true positive.

FIGS. 2A-2 each show the correlation results of the full-genome correlation scans plotted on the y-axis relative to the genomic position on the x-axis within a 500kb region around the locus defined by TNIP1 (FIG. 2A), PRDM1 (FIG. 2B), JAZF1 (FIG. 2C), UHRF1BP1 (FIG. 2D) and IL-10 (FIG. 2E). In each of FIGS. 2A-2E, the filled squares represent meta-assay P values for the most relevant markers. For each of FIGS. 2A-2E, P values from the genome scan are labeled to represent LD of the genome-wide related variants: with a dotted circle representing r²> 0.8, dotted circle, r²Is more than 0.5; with speckled circle, r²Is more than 0.2; hollow circle r²Is less than 0.2. Along the bottom of each of FIGS. 2A-2E, the recombination rates in CEU HapMap (solid black line) and known human genes are shown below each graph. In fig. 2B (PRDM1), the previously reported and independent SLE risk locus (rs2245214) near ATG5 gene is marked by a solid black circle. FIG. 2F shows 1256 independent SNPs (r versus any other SNP in the array) in 1963 cases and 4329 control replicate samples²< 0.1) P value histogram. The expected resulting density is indicated in fig. 2F with a dashed line at zero distribution. As shown in fig. 2F, significant enrichment of results less than P < 0.05 was observed.

Therefore, replication studies identified 5 new SLE risk loci with combined P values above the genome-wide significance threshold (P < 5x 10)^-8): TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL 10. Detailed statistical associations for these loci 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 the combination P ═ 3.8x 10^-13(FIG. 2A), which variant is located in the intron of the interacting protein 1(TNIP1) of TNF- α inducible protein 3(TNFAIP3) recently variants close to TNIP1 were found to have an effect on the risk of psoriasis (Nair, R.P. et al, Nat Genet 41: 199-204(2009)), whereas SLE and psoriasis variants are separated by 21kb and appear to be distinct genetic signals (r²0.001). TNIP1 and TNFAIP3 are interacting proteins (Heyninck, k. et al, FEBS Lett 536: 135-40(2003)), however, the precise role of TNIP1 in the regulation of TNFAIP3 is not known. Several different variants near TNFAIP3 are found in SLE (Graham, R.R. et al, Nat. Genet.40 (9): 1059-61(2008), Musone, S.L. et al, Nat. Genet.40 (9): 1062-64(2008)), rheumatoid arthritis (Plenge, R.M. et al, Nat Genet 39: 1477-82(2007)), psoriasis (Nair, R.P. et al, Nat Genet 41: 199-2)04(2009)) and type I diabetes (Fung, e.y. et al, genes immun 10: the relevance of 188-91(2009)) suggests that this pathway has an important role in the regulation of autoimmune diseases.

The second identified risk variant (rs6568431, P ═ 7.12x 10)^-10) Is identified in the intergenic region between PR domain containing 1(PRDM1, also known as BLIMP1) and APG5 autophagy 5-like (ATG5) with ZNF domain. The signal for rs6568431 appears to be different from the SLE risk allele rs2245214 in ATG5 previously reported (Harley, J.B. et al, Nat Genet 40: 204-10(2008)) (see Table 4) since rs6568431 and rs2245214 have r²< 0.1, while rs2245214 was still significantly associated with SLE after incorporation of conditional logistic regression of rs6568431 (P < 1x 10)^-5) (FIG. 2B).

The promoter region of 1(JAZF1) juxtaposed to another zinc finger gene is the 3 rd newly identified SLE locus (rs849142, P ═ 1.54x 10^-9) (FIG. 2C). Interestingly, the same variant was previously associated with the risk of type 2 diabetes (Zeggini, e. et al, Nat Genet 40: 638-45(2008)) and height differences (Johansson, a. et al, Hum MolGenet 18: 373-80 (2009)). Isolated prostate cancer allele rs10486567(Thomas, g. et al, Nat Genet 40: 310-5(2008)) near JAZF1 did not show any evidence of association in this study.

ICBP90 binding protein 1(UHRFBP1, rs11755393, P ═ 2.22x 10^-8) The non-synonymous allele of (R454Q) defines the 4 th new risk locus in SLE. This allele is a non-conservative amino acid change in a putative binding partner of UHRF1, and UHRF1 is a transcription and methylation factor associated with multiple pathways (Arita, k. et al, Nature 455: 818-21 (2008)). The UHRFBP1 risk allele is located in an extended linkage disequilibrium region encompassing multiple genes, including the small ribonucleoprotein polypeptide c (SNPRC), which is part of the RNA processing complex that SLE autoantibodies are often directed against.

The 5 th novel SLE locus identified was interleukin-10 (IL 10; rs)3024505，P＝3.95x 10^-8) (FIG. 2E). IL-10 is an important immunomodulatory cytokine that functions by down-regulating the immune response (Diveu, C. et al, CurrOpin Immunol 20: 663-8(2008)), and the association of variation in IL-10 with SLE has been reported to be inconsistent (Nath, S.K. et al, Hum Genet 118: 225-34 (2005)). SLE-associated variants are identical to SNPs currently identified as contributing risk to ulcerative colitis (Franke, a. et al, Nat Genet 40: 1319-23(2008)) and type I diabetes (Barrett, j.c. et al, Nature Genetics 41: 703-707(2009)), suggesting that there may be a shared pathophysiology of the IL10 pathway between these disorders.

Use of P < 1X10 in Combined replicate samples^-5Of SLE, 21 additional SLE candidate risk loci were identified (table 4). Under zero-distribution conditions of meta-analysis, less than 1 (0.01) locus is expected to have P < 1x10^-5(P＝8x10^-77) Candidate genes of interest in this list include a) interferon regulatory factor 8(IRF8), which was previously suggested in GWAS (Graham, R.R. et al, nat. Genet.40 (9): 1059-61(2008)), and its family members IRF5 and IRF7 both fall into the established SLE risk locus, b) TAO kinase 3(TAOK3), which is a missense allele of the kinase expressed in lymphocytes (rs428073, N47S), c) lysosomal trafficking regulator (LYST), whose mutation leads to Chediak-Higashi syndrome in humans, which is a complex disease characterized by lymphoproliferative disorders, and d) interleukin 12 receptor β 2(IL12RB2), which includes IL23R and SERPBP1, but differs from the inflammatory autoimmune disease and inflammatory autoimmune disease, inflammatory disease, ankylosing disease, IL 2006, 23R, and inflammatory disease, and so on human, IL12RB2, which appear to be reported in humans (Du 3, IRF 3523, 1463, R).

A significant feature of current GWA studies is the large number of overlapping loci that are shared between different complex diseases (zhakakova, a. et al, Nat Rev Genet 10: 43-55 (2009)). We tested 42 variants from 35 loci (tables 6 and 7) previously reported as autoimmune disease risk alleles associated with SLE. However, the device is not suitable for use in a kitchenWhereas, no single locus had an unregulated P value < 5X10^-8However, an enrichment of the relevant allele was found. From the 35 loci tested (42 variants in total), 5 alleles had unregulated P < 0.0004 (less than one result expected by chance, P ═ 4.4x 10)^-12) And has P < 0.05 after Bonferroni correction for the 35 previously described loci. For each of these 5 variants, SLE-associated alleles matched previously reported alleles and had the same direction of effect (table 6). A very significant association of missense alleles of IFIH1 was observed (rs1990760, P ═ 3.3x 10)^-7) This allele has previously been associated with type I diabetes and Grave's disease (Smyth, d.j. et al, Nat Genet 38: 617-9 (2006); sutherland, a. et al, J Clin Endocrinol Metab 92: 3338-41(2007)). The association of a missense allele of complement factor B (CFB, rs641153) (R32Q) located in the region of HLA class III, a validated risk allele of age-related macular degeneration, was also observed (Gold, B. et al, Nat Genet 38: 458-62 (2006)). SLE risk alleles have no significant Linkage Disequilibrium (LD) with other SLE-associated HLA region variants (DR2/DR3), and remain significant after integration of conditional logistic regression analysis of DR2 and DR 3. HLA is a complex genetic region, but surprisingly the allele of SNP rs641153 has almost the same protective effect as the reported AMD risk allele (Gold, b. et al, Nat Genet 38: 458-62 (2006)). Additional studies of 5 candidate disease alleles are indicated.

In addition, table 7 provides detailed summary statistics of the 42 variants identified in other autoimmune diseases. Interestingly, variants of CTLA4, IL23R, NOD2 and CD40, which are significant risk factors in other autoimmune diseases, did not appear to show any evidence of association with SLE.

Using 26 SLE risk alleles (21 loci previously reported in table 2, and 5 novel SLE loci as described above), some additional analyses were performed. Pairwise interaction analysis with the confirmed loci, consistent with previous literature for 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, no evidence was found that multiple independent alleles contribute to the risk of any single risk locus. Thereafter, the percentage of variance interpretation was estimated from each identified SLE risk allele 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 evaluated for > 1% genetic variance, while the remaining loci each accounted for less than 1% variance. In summary, these 26 SLE risk loci account for about 8% of the total genetic susceptibility to SLE.

Targeted replication GWAS results in validation of effective study design of other risk loci (Hirschhorn, J.N. et al, Nat Rev Genet 6: 95-108 (2005)). However, there is very little data available for the probability of duplicate results lacking acceptable P-value criteria for acceptable significance for the whole genome. In this study, replication encompasses all variants with P < 0.05 from the original GWAS study. As shown in figure 3, the lower the P-value in the GWAS study, the higher the probability of obtaining a candidate or validation state in the replicate meta-analysis. Interestingly, in the current study of the group of variants with GWAS P between 0.05 and 0.01, no candidate or validated results were obtained, although the group comprised-50% of all variants tested in replication. These results can be used to guide other targeted study design, although it is indeed necessary to carefully consider the size of the original GWAS population, the size of the replication sample, the disease structure and the amount of effect of candidate variants in planning replication work.

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

Example 2

Resequencing and identification of causal alleles of BLK

As described above, BLK was identified as achieving genome-wide significance (P < 5x 10)^-8) Is associated with SLE. To further characterize the genetic basis of this association and identify causal alleles, we performed re-sequencing studies on the BLK locus and reporter gene expression assays described below.

For the resequencing study, all 13 exons and the 2.5kb upstream promoter sequence of the BLK locus in DNA isolated from 192 patients (Bauer et al, PLoS media 3 (12): e491(2006)) of the NIH/NIAMS funded resource pool, the autoimmune disease biomarker cooperation network (ABCON), and 96 control individuals of the New York Cancer Program (NYCP) (Mitchell et al, J.Urban Health 81: 301-10 (2004)). Genomic DNA is a whole genome that was amplified prior to sequencing according to the manufacturer's protocol (Qiagen, Valencia, CA., cat # 150045).

The results of re-sequencing showed that 17 mutations (10 non-synonymous, 7 synonymous) were found in the coding region of the BLK gene (table 8). None of these mutations showed significantly higher frequency in the case than the control. The overall frequency of non-synonymous mutations was not significantly higher in case (14/191) than control (7/96).

In addition, multiple common mutations were identified in the non-coding region of BLK (shown in table 9). Three SNPs (rs4840568, rs1382568[ three allelic SNPs (A/C/G); C allele was previously identified as risk allele)]And rs922483(SEQ ID NO: 13)) showed a homology to the locus previously identified in GWAS (Hom et al, N Engl J Med 358: 900-09(2008))²Correlation > 0.5 (rs13277113, odds ratio, 1.39, P ═ 1x10^-10). FIG. 4 shows a Linkage Disequilibrium (LD) block (in r) in the promoter region of BLK generated using Haploview (software freely available from URL www.broadinstitute.org/Haploview/Haploview; see Barrett J.C. et al, biolnformatics 21: 263-65(2005)) (in r)²Display). The upper part of the figure shows a schematic representation of the BLK promoter region, indicating the relative positions of the identified SNPs. What is needed isR between the enumerated SNPs²The values are displayed in a box. LD Strength between two SNPs using r²A value representation is 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 bold black in the upper part of the figure.

This resequencing study did not reveal any common variations in the BLK coding region. However, three common variants of the promoter region (rs4840568, rs1382568 and rs922483(SEQ ID NO: 13)) were identified as one or more potential causal alleles of the biological effects of BLK associated with increased risk of SLE. Each such variation 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 the non-coding region of BLK ('rs 922483' disclosed as SEQ ID NO:13)

Luciferase reporter gene assays were performed to study the effect of three SNPs-rs 4840568, rs1382568 and rs922483(SEQ ID NO:13) on BLK-mediated gene expression. The upstream sequence of BLK (-2256 to +55bp) was amplified using genomic DNA from individuals carrying a haplotype at risk or not. Each PCR product was cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA; cat # K4500-01) and then subcloned into pGL4 luciferase reporter vector (Promega, Madison, Wis.; cat # E6651). Constructs carrying risk-free haplotypes were used as templates for mutagenesis (Stratagene, La Jolla, Calif.; Cat. No. 10519-5) to generate the various haplotypes.

The primers used for PCR amplification were as follows: forward direction: CCACCTCTCTTCCGCCTTTCTCAT (SEQ ID NO: 1); and (3) reversing: TTTCATGGCTTGTGGCTTTCTGCC (SEQ ID NO: 2). Primers used for mutagenesis are listed in table 10 below.

TABLE 10 mutagenesis primer List

Normalization was performed using the Renilla luciferase control reporter vector pRL-TK (Promega, Madison, Wis.; cat # E2241). Cell lines BJAB (a continuous lymphoid cell line with B cell characteristics (bone marrow derived), lacking Epstein-Barr virus genome, from three-person lymphomas; Klein et al, Proc. Natl. Acad. Sci. USA 71: 3283-86(1974)) or Daudi cell lines (American type culture Collection (ATCC) Cat. No. CCL-213) were used for transfection. For each transfection, use was made ofDevice (Lonza, Walkersville Inc., Walkersville, MD (Lonza Group Ltd., Switzerland); cat # AAD-1001) transfected 5X10 with 5. mu.g of each vector DNA⁶A cell. Cell line for Daudi cellsKit L (Lonza, cat # VCA-1005) anddevice procedure a-030. Cell line for BJAB cellsKit V (Lonza, cat # VCA-1005) anddevice program T-020. All transfections were performed in duplicate or triplicate. After transfection, cells were incubated at 37 ℃ for 16 hours. After incubation, the cells are harvested according toManufacturer's instruction useThe luciferase activity was measured by a reporter assay system (Promega, Madison, Wis.; cat. E1960).

Each SNP measured in the luciferase reporter assay system described above: the effect of rs4840568, rs1382568 and rs922483(SEQ ID NO:13) on BLK-mediated gene expression is shown in FIG. 5. The different haplotypes generated by mutagenesis were compared to the non-risky (wild type) haplotype 22-GAC (open bars in each of FIGS. 5A-F) and the risky haplotype 22-ACT (shaded bars in each of FIGS. 5A-F).

FIGS. 5A and 5B show that SNP rs922483(C > T) (SEQ ID NO:13) results in a significant effect on BLK-mediated gene expression in both BJAB (FIG. 5A) and Daudi cells (FIG. 5B). Haplotype 22-ACT showed 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 continue to exhibit lower activity than haplotypes containing the C allele. 5 independent experiments were performed in BJAB cells and 6 independent experiments were performed in Daudi cells. Data shown represent mean +/-standard error of mean (s.e.m.) in assays repeated three times; tp < 0.05, Tp < 0.01, Tp < 0.001(t test).

FIGS. 5C and 5D show that SNP rs1382568(A > C/G > C) did not result in any significant effect on BLK-mediated expression in each cell line. Both haplotypes 22-GCC and 22-GGC (dotted bars) exhibited similar levels of luciferase activity compared to the risk-free haplotype 22-GAC (open bars). 5 independent experiments were performed in BJAB cells and 6 independent experiments were performed in Daudi cells. Data shown represent mean values in assays repeated three times +/-s.e.m.; p < 0.05, p < 0.01, p < 0.001, ns is not significant (t test).

FIGS. 5E and 5F show that SNP rs4840568(G > A) did not result in significant effects on BLK-mediated expression in either BJAB cells or Daudi cells. The difference between haplotype 22-AAC (dotted bars) and risk-free haplotype 22-GAC (open bars) was not statistically significant in BJAB cells (FIG. 5E), but was statistically significant in Daudi cells (FIG. 5F). Considering that haplotype 22-ACC (dotted bar) did not show any defect in luciferase activity compared to the risk-free haplotype 22-GAC (open bar), the probability of the A allele becoming the causal allele was greatly reduced (FIG. 5F). Data shown represent mean values in assays repeated three times +/-s.e.m.; p < 0.05, p < 0.01, p < 0.001, ns is not significant (t test).

It was previously shown that (GT) repeats in the region upstream of the BLK promoter may 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 can affect the transcriptional activity of the BLK promoter. To perform these experiments, genomic DNA samples from individuals carrying both the 18(GT) repeat (SEQ ID NO: 14) or the 22(GT) repeat (SEQ ID NO: 15) were selected for cloning using the strategy described above. Five vectors were sequenced and verified to contain the correct length (GT) repeats. As shown in FIG. 6, the haplotype containing the 18(GT) repeat (SEQ ID NO: 14) exhibited a similar level of transcriptional activity in the luciferase reporter assay as the haplotype containing the 22(GT) repeat (SEQ ID NO: 15). Data shown represent the mean +/-s.e.m. in duplicate assays; ns is not significant (t-test).

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

Interestingly, it was noted that rs922483(SEQ ID NO:13) is located in an evolutionarily conserved region of the first exon of BLK, in the likely human transcription start site. The common sequence of the human Inr motif is identified as yyannwyy (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 is altered relative to the common motif. Thus, the SLE risk haplotype Inr sequence is CTACCTC, while the "wild-type" haplotype Inr sequence is CCACCTC. Suggesting 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 as described above.

Sequence listing

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Claims (10)

1. A method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in a SLE risk locus, 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 a thymine at chromosome 11389322 of chromosome 8 in a human, and wherein the subject is suspected of having lupus.

2. A method of identifying lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to the position of a Single Nucleotide Polymorphism (SNP) of the at least one locus as set forth in table 4, and wherein the subject is suspected of having lupus.

3. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, 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 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 11389322 of chromosome 8 of a human, wherein the presence of the variation in the BLK locus is indicative of responsiveness of the subject to the therapeutic agent.

4. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 4, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) position of the at least one locus as set forth in table 4, wherein presence of the variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent.

5. A method of predicting responsiveness of a subject with lupus to a lupus therapeutic agent, the method comprising determining whether the subject comprises a variation in at least one SLE risk locus as set forth in table 6, wherein the variation in at least one locus occurs at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) position of the at least one locus as set forth in table 6, wherein presence of the variation in at least one locus is indicative of responsiveness of the subject to the therapeutic agent.

6. A method of diagnosing or prognosing 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:

(a) the biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in the BLK locus;

(b) a variation 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 chromosome 8 of human; and

(c) the presence of a variation in the BLK locus is a diagnosis or prognosis of lupus in the subject.

7. A method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 4, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 4;

(b) a variation in at least one locus comprises a SNP as set forth in table 4 or is located at a nucleotide position corresponding to a SNP as set forth in table 4; and

(c) the presence of the variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

8. A method of diagnosing or prognosing lupus in a subject, the method comprising detecting in a biological sample derived from the subject the presence of a variation in at least one SLE risk locus as set forth in table 6, wherein:

(a) a biological sample is known to comprise or suspected to comprise nucleic acid comprising a variation in at least one SLE risk locus as set forth in table 6;

(b) a variation in at least one locus comprises a SNP as set forth in table 6 or is located at a nucleotide position corresponding to a SNP as set forth in table 6; and

(c) the presence of the variation in the at least one locus is a diagnosis or prognosis of lupus in the subject.

9. A method of treating a lupus disorder in a subject in which a genetic variation is known to exist at a nucleotide position corresponding to a Single Nucleotide Polymorphism (SNP) in a SLE risk locus, wherein the SNP is rs922483(SEQ ID NO:13) and the SLE risk locus is BLK, and wherein the variation is thymine at chromosome 11389322 of chromosome 8 in a human, the method comprising administering to the subject a therapeutic agent effective to treat the disorder.

10. A method of treating a lupus disorder in a subject in which a genetic variation is known to exist at a nucleotide position in at least one SLE risk locus as set forth in table 4 that corresponds to a Single Nucleotide Polymorphism (SNP) as set forth in table 4, the method comprising administering to the subject a therapeutic agent effective to treat the disorder.