MAIMUUDATUNTUU US009809854B2 TI ON MAN MINI (12 ) United States Patent ( 10 ) Patent No. : US 9 ,809 ,854 B2 Crow et al. (45 ) Date of Patent : Nov . 7 , 2017

Whitehead et al. (2005 ) Variation in tissue - specific expression (54 ) BIOMARKERS FOR DISEASE ACTIVITY among natural populations. Genome Biology, 6 :R13 . * AND CLINICAL MANIFESTATIONS Villanueva et al. (2011 ) Netting Neutrophils Induce Endothelial SYSTEMIC LUPUS ERYTHEMATOSUS Damage , Infiltrate Tissues, and Expose Immunostimulatory Mol ecules in Systemic Lupus Erythematosus . The Journal of Immunol @(71 ) Applicant: NEW YORK SOCIETY FOR THE ogy , 187 : 538 - 552 .* RUPTURED AND CRIPPLED Bijl et al. (2001 ) Fas expression on peripheral blood lymphocytes in MAINTAINING THE HOSPITAL , systemic lupus erythematosus ( SLE ): relation to lymphocyte acti vation and disease activity . Lupus, 10 :866 - 872 . * New York , NY (US ) Crow et al . (2003 )Microarray analysis of in lupus. Arthritis Research and Therapy , 5 :279 - 287 . * @(72 ) Inventors : Mary K . Crow , New York , NY (US ) ; Baechler et al . ( 2003 ) Interferon - inducible gene expression signa Mikhail Olferiev , Mount Kisco , NY ture in peripheral blood cells of patients with severe lupus . PNAS , (US ) 100 (5 ) : 2610 - 2615. * GeneCards database entry for IFIT3 ( obtained from < http : / /www . ( 73 ) Assignee : NEW YORK SOCIETY FOR THE . org /cgi - bin / carddisp .pl ? gene = IFIT3 > on May 26 , 2016 , RUPTURED AND CRIPPLED 15 pages ) . * Navarra et al. (2011 ) Efficacy and safety of belimumab in patients MAINTAINING THE HOSPITAL with active systemic lupus erythematosus : a randomised , placebo FOR SPECIAL SURGERY , New controlled , phase 3 trial . The Lancet , 377 :721 - 731. * York , NY (US ) Abramson et al . ( 1983 ) Arthritis Rheum . 26 :630 - 6 . American College of Rheumatology Ad Hoc Committee on Sys @( * ) Notice: Subject to any disclaimer , the term of this temic Lupus Erythematosus Response Criteria (2004 ) Arthritis patent is extended or adjusted under 35 Rheum . 50 : 3418 - 26 . Baechler et al . (2003 ) Proc Natl Acad Sci USA 100 : 2610 - 5 . U .S .C . 154 (b ) by 345 days . Barrat et al. (2005 ) J . Exp . Med . 202 : 1131 - 9 . Bauer et al . ( 2009 ) Arthritis Rheum . 60 : 3098 - 107 . (21 ) Appl. No. : 13/ 998 ,598 Bennett et al . (2003 ) J. Exp . Med . 197 :711 -23 . Brinkmann et al. ( 2004 ) Science 303 : 1532 - 5 . (22 ) Filed : Nov . 15 , 2013 Chaussabel et al. ( 2008 ) Immunity 29 : 150 -64 . Crow and Kirou ( 2008 ) Arthritis Res . Ther. 10 : 126 . (65 ) Prior Publication Data Crow (2007 ) Curr . Top . Microbiol. Immunol. 316 :359 - 86 . Crow and Wohlgemuth (2003 ) Arthritis Res. Ther . 5 :279 - 87 . US 2014 /0135225 A1 May 15 , 2014 Crow et al . (2003 ) Autoimmunity 36 :481 - 90 . Denny et al. (2010 ) J. Immunol. 6 :3284 -97 . Related U . S. Application Data De Waard et al . ( 1999 ) Gene 226 : 1 - 8 . Fan et al. ( 2004 ) Genome Res . 14 : 878 - 85 . (60 ) Provisional application No. 61/ 726 , 902, filed on Nov. Feng et al. (2006 ) Arthritis Rheum . 54 : 2951- 62 . 15 , 2012 Forsman and Dahlgren (2010 ) BMC Cell Biol . 11: 52 . Fukuda et al. (2009 ) Clin . Rheumatol. 28 : 301 - 4 . (51 ) Int. Ci. Garcia - Carrasco et al. (2002 ) J. Rheumatol. 29 :726 -30 . Garcia - Romo et al . ( 2011) Sci . Trans . Med . 3 :73ra20 . C12Q 1/ 68 ( 2006 .01 ) Gray et al. (2010 ) J. Immunol. 184 :6359 -66 . (52 ) U . S . CI. Hakkim et al. ( 2010 ) Proc Natl Acad Sci USA 107 : 9813 - 8 . CPC . . . C12Q 1 /6883 (2013 . 01 ); C12Q 2600 /158 Han et al . ( 2003 ) Immun . 4 : 177 - 86 . (2013 .01 ) Hargraves et al. ( 1948 ) Mayo Clin . Proc . 23 :25 - 8 . (58 ) Field of Classification Search Hargraves ( 1969 ) Mayo Clin . Proc . 44 :579 - 9 . None (Continued ) See application file for complete search history . Primary Examiner — Neil P Hammell (56 ) References Cited (74 ) Attorney , Agent, or Firm — Leason Ellis LLP U . S . PATENT DOCUMENTS (57 ) ABSTRACT 7 ,571 , 055 B2 * 8/ 2009 Behrens ...... C12Q 1/ 6883 This invention related to methods and assays for screening 435 /6 . 11 for, identifying , and predicting the severity and clinical 2010 /0113293 Al * 5 /2010 Pascual ...... C12Q 1 /6883 manifestations of systemic lupus erythematosus ( SLE ) . Spe 506 /8 cifically, this invention provides various biomarkers for the 2010 /0261172 A1 * 10 /2010 Yao ...... C12Q 1/ 6883 prediction of flares of the disease both in number and 435 / 6 . 12 severity , as well as clinical manifestations of the disease , and methods of using these biomarkers to correctly subclassify OTHER PUBLICATIONS patients with this disease , and prescribe appropriate treat Manzi et al . “ Systemic Lupus Erythematosus: Treatment and ment. The invention also provides for biomarkers of lupus Assessment” in Klippel et al. , Primer on the Rheumatic Diseases , disease activity , i. e ., flares, as well as biomarkers for the 13th ed . ( New York , Springer, 2008 ) , pp . 327 - 338 .* prediction of future flares, and methods of using these Affymetrix HG -U133A Annotation File ( filtered excerpt , obtained biomarkers . The invention also provides , in these biomark from < http :/ / www .affymetrix . com / Auth /analysis / downloads / na35 / ers , targets and methods for drug development and basic ivt/ HG -U133A .na35 .annot . csv .zip > on Apr. 29 , 2016 , 1 page ). * research for SLE . Mannucci et al. ( 2003) Von Willebrand factor cleaving protease ( ADAMTS - 13 ) in 123 patients with connective tissue diseases 6 Claims, 17 Drawing Sheets Journal of Hematology , 88 ( 8 ) :914 - 918 . * ( 5 of 17 Drawing Sheet( s ) Filed in Color ) US 9 ,809 , 854 B2 Page 2

( 56 ) References Cited OTHER PUBLICATIONS Irizarry et al . ( 2003 ) Biostatistics 4 : 249 -64 . Karlovich et al . ( 2009 ) BMC Med . Genomics . 2 : 33 . Kirou et al. ( 2004 ) Arthritis Rheum . 50 :3958 -67 . Kurien and Scofield (2006 ) Scand . J . Immunol. 64 : 227 - 35 . Kurien et al . ( 2000 ) Clin . Exp . Immunol. 120 : 209 - 17 . Lande et at . (2011 ) Sci. Transl . Med .3 : 73ra 19 . Lovgren et al. (2004 ) Arthritis Rheum . 50 : 1861 -72 . Mantovani et al. ( 1998 ) Ann . N Y Acad . Sci. 840 : 338 -51 . Milner and Day ( 2003 ) J. Cell . Sci . 116 : 1863 - 73 . Nathan ( 2006 ) Nat. Rev . Immunol. 6 : 173 - 82 . Samarajiwa et al. (2009 ) Nucleic Acids Research (Database Issue ) :D852 - 7 . Tan et at . ( 1982 ) Arthritis Rheum . 25 : 1271 - 7 . Theilgaard -Mönch et al . ( 2005 ) Blood 105: 1785 - 96 . Velculescu et at . ( 1995 ) Science 270 ;484 -487 . Velculescu et al. ( 1997) Cell 88 . Villanueva et al. ( 2011) J . Immunol. 187 : 538 - 52 . Yee et al. ( 2009 ) Rheumatology 48 :691 - 5 . Zhao et al. (2009 ) Drug Metab . Dispos .37 : 282 -91 . * cited by examiner U. S . Patentatent NovNov . 7 , 2017 Sheet 1 of 17 US 9 ,809 , 854 B2

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BIOMARKERS FOR DISEASE ACTIVITY 9 . Hematalogic disorder such as hemolytic anemia (low AND CLINICAL MANIFESTATIONS red blood cell count) or leukopenia (white blood cell SYSTEMIC LUPUS ERYTHEMATOSUS count< 4000 /ul ) ; 10 . Immunologic disorder including positive anti - smith , CROSS REFERENCE TO RELATED 5 anti- ds DNA , anti- phospholipid antibody, and/ or false APPLICATION positive serological test for syphilis ; and 11. Positive anti- nuclear antibody (ANA ) test. The present application claims priority to U . S . Patent Currently , a number of tests are performed to aid in Application Ser. No . 61/ 726 ,902 filed Nov . 15 , 2012 , which establishing a diagnosis of SLE in the context of the char 10 acteristic symptoms and signs of SLE . These include: anti is hereby incorporated by reference in its entirety . nuclear antibody ( ANA ) blood test; anti - double stranded FIELD OF THE INVENTION DNA test ; anti- Smith antibody test ; VDRL , a syphilis test ; complete blood count (CBC ) ; blood chemistry levels ; inflammatory markers — the erythrocyte sedimentation rate This invention relates to the field of screening for, iden 15 ( also called the ESR ) and C -reactive ; x - rays of tifying , and predicting the severity and clinical manifesta joints, and a biopsy from the skin or kidneys . tions of systemic lupus erythematosus (SLE ). Specifically , At this time there is no definitive diagnostic test for SLE , this invention provides various biomarkers for the prediction or any tests that predict the course and severity of the disease of flares of the disease both in number and severity , as well or which organs the disease is most likely to affect . More as clinical manifestations of the disease , and methods of 20 overove , genetic testing is not routinely performed in order to using these biomarkers to correctly subclassify patients with diagnose SLE . Thus , there is a real need in the art for this disease , and prescribe appropriate treatment. The inven definitive tests to determine the severity of a patient' s SLE , tion also provides for biomarkers of lupus disease activity , including the number and severity of flares of the disease . i . e . , flares , as well as biomarkers for the prediction of future Moreover , with the vast number of etiologies of the disease , flares , and methods of using these biomarkers . 25 as well as the number of therapies used for treatment , a test The invention also provides targets for drug development that provides information on which organs and organ sys and basic research for SLE . tems the disease will most affect would be useful. Better knowledge as to targets to concentrate upon when testing for BACKGROUND OF THE INVENTION drugs, as well as when performing basic research on SLE 30 would be of great value as well . Systemic lupus erythematosus (SLE ) , also known as The biomarkers described herein provide not only a novel lupus , is an autoimmune illness . and unique way to definitively screen , identify , and predict Although there are several different forms of lupus , the the severity , activity , and clinical manifestations of SLE , but classical lupus patient is usually a young woman with a provide a number ofmarkers for use in drug screening and combination of symptoms, such as fever, swollen lymph 35 research , and basic research on SLE . glands , rashes (particularly butterfly - shaped rashes on the face) , arthritis , fatigue, hair loss , chest and / or abdominal SUMMARY OF THE INVENTION pain , oral ulcers , and neuropsychiatric problems, such as headache , memory loss, mood disorders , and /or confusion . This invention is based upon the surprising discovery that While the cause of lupus is unknown , theories on its 40 changes in expression in three sets of signature genes origin include genetics , environment, infections, and the correlate to the severity of disease activity and clinical defective failure to process the products of an immune manifestations in subjects with SLE . Using the differences in response . Although it is a lifelong condition , symptoms tend expression of genes in three sets of signature genes, inter to cycle in alternate periods of flares and remission . Those feron inducible (“ IFIG ” ) , neutrophil granule , and neutro with lupus are at great risk of contracting kidney disease as 45 phil- related , SLE patients can be categorized into five well . groups. These five groups differ by the severity of the Treatment options include corticosteroids , anti -malarial disease including the number of, and severity of flares , as drugs, immunosuppressive drugs such as mycophenolate well as the clinical manifestations . Additionally , certain mofetil, cytotoxic agents such as cyclophosphamide , non protein markers can confirm these classifications . These steroidal anti - inflammatory drugs (NSAIDs ) , and certain 50 categories are useful for the health care provider in deter biologics, such as belimumab , rituximab , and others. mining treatment options, and the amount and type of The American College of Rheumatology has established monitoring a patient diagnosed with SLE may need . Genetic criteria that is used for studies, but can also aid in the testing in combination with testing for protein markers is a diagnosis . If four or more of the eleven criteria occur, a novel method for determining the severity and etiology of a patient may have SLE . These criteria are : 55 subject' s SLE , and can be used to determine the course of 1 . Malar rash ( rash on cheeks ); treatment and amount of monitoring of the subject, often 2 . Discoid rash ( red , scaly patches on skin that cause prior to any identifiable symptoms. This early identification scarring ) ; of disease activity and clinical manifestations is invaluable 3 . Photosensitivity ; in providing proper patient care especially given the unpre 4 . Oral ulcers ; 60 dictability of SLE . 5 . Nonerosive arthritis of two or more peripheral joints , Thus, one embodiment of the present invention is a with tenderness , swelling , or effusion ; method and /or assay for screening , identifying and /or pre 6 . Pleuritis or pericarditis ; dicting the severity and clinical manifestation of systemic 7 . Renal disorder as evidenced by more than 0 . 5 g per day lupus erythematosus in a subject , comprising obtaining protein in urine or cellular casts seen in urine under a 65 biological tissue and/ or fluid from the subject , purifying microscope ; and / or isolating nucleic acid , including , but not limited to , 8 . Neurologic disorder such as seizures or psychosis ; mRNA and cDNA , from the biological tissue and /or fluid , US 9 ,809 , 854 B2 detecting the expression of one or more genes in the IFIG and detecting the expression of one or more genes in the signature , listed in Table 2 , and comparing the expression of IFIG signature , listed in Table 2 , and one or more genes in the gene or genes with the expression of the gene or genes the neutrophil - related signature , listed in Table 4 , and com in a healthy control, wherein an increase in expression of paring the expression of the gene or genes with the expres one or more genes from the IFIG signature could indicate an 5 sion of the gene or genes in a healthy control, purifying and increase in flares, an increase in severe flares, and an isolating protein from the biological tissue and / or fluid , and increase in mucocutaneous manifestation of the disease . detecting the presence of TNFa , IL - 8 , and IL - 18 , wherein an A more preferred embodiment of the present invention is a method and / or assay for screening , identifying and / or increase in expression of one or more genes from the IFIG predicting the severity and clinical manifestation of systemic 10 signature and the neutrophil -related signature , and the pres lupus erythematosus in the subject, comprising obtaining ence of TNFO , IL - 8 , and IL - 18 , would indicate an increase biological tissue and/ or fluid from a subject, purifying in the number of flares , and an increase in mucocutaneous and / or isolating nucleic acid , including, but not limited to , manifestation of the disease . mRNA and cDNA , from the biological tissue and /or fluid , Another embodiment of the present invention is a method and detecting the expression of one or more genes in the 15 of01 treating a subject with SLE comprising obtaining bio IFIG signature , listed in Table 2 , and one or more genes in logical tissue and / or fluid from the subject, purifying and / or the neutrophil granule signature, listed in Table 3 . and isolating nucleic acid , including , but not limited to , mRNA comparing the expression of the gene or genes with the and cDNA , from the biological tissue and /or fluid , and expression of the gene or genes in a healthy control, wherein detecting the expression of one or more genes in the IFIG an increase in expression of one or more genes from the 20 signature , listed in Table 2 , and one or more genes in the IFIG signature and the neutrophil granule signature would neutrophil granule signature , listed in Table 3 , and compar indicate an increase in the number of flares , an increase in ing the expression of the gene or genes with the expression the number of severe flares , and an increase in vascular and of the gene or genes in a healthy control, wherein an increase renal manifestations of the disease . in expression of one or more genes from the IFIG signature Another preferred embodiment of the present invention is 25 and the neutrophil granule signature would indicate treating a method and / or assay for screening , identifying and / or the subject with agents for the treatment of vascular and predicting the severity and clinicalmanifestation of systemic renal manifestations of SLE , including, but not limited to , lupus erythematosus in the subject , comprising obtaining corticosteroids, such as prednisone in a medium to high dose biological tissue and / or fluid from a subject , purifying (7 . 5 to over 30 mg/ day ), or intravenous methylprednisolone and / or isolating nucleic acid , including, but not limited to , 30 in a high doses (known as pulse therapy, using greater than mRNA and cDNA, from the biological tissue and / or fluid , 50 mg/ day for 1 - 3 days ) ; cytotoxic drugs, such as cyclo and detecting the expression of one or more genes in the phosphamide ; immunosuppressive drugs , such as mycophe IFIG signature , listed in Table 2 , and one or more genes in nolate mofetil and azathioprine ; biologic agents , such as the neutrophil - related signature , listed in Table 4 , and com rituximab ; and anti -malarial drugs , such as hydroxychloro paring the expression of the gene or genes with the expres - 35 quine , as well as agents targeting the vascular system , sion of the gene or genes in a healthy control, wherein an including but not limited to anti -hypertensive drugs , statins , increase in expression of one or more genes from the IFIG and anti - coagulants , such as aspirin and coumarin , and signature and the neutrophil- related signature would indi monitoring the subject often , e. g ., at least monthly , possibly cate an increase in the number of flares, and an increase in weekly . mucocutaneous manifestation of the disease . 40 Another embodiment of the present invention is a method A further embodiment of the invention is a method and / or of treating a subject with SLE comprising obtaining bio assay for screening , identifying and/ or predicting the sever - logical tissue and / or fluid from the subject, purifying and / or ity and clinical manifestation of systemic lupus erythema isolating nucleic acid , including , but not limited to , mRNA tosus in a subject, comprising obtaining biological tissue and cDNA , from the biological tissue and / or fluid , and and / or fluid from the subject , purifying and/ or isolating 45 detecting the expression of one or more genes in the IFIG nucleic acid , including , but not limited to , mRNA and signature , listed in Table 2 , and one or more genes in the cDNA, from the biological tissue and / or fluid , and detecting neutrophil granule signature , listed in Table 3 , and compar the expression of one or more genes in the IFIG signature , ing the expression of the gene or genes with the expression listed in Table 2 , and one or more genes in the neutrophil of the gene or genes in a healthy control, purifying and granule signature , listed in Table 3 , and comparing the 50 isolating protein from the biological tissue and / or fluid , and expression of the gene or genes with the expression of the detecting the presence of anti - SSA /Ro autoantibodies , gene or genes in a healthy control, purifying and isolating wherein an increase in expression of one or more genes from protein from the biological tissue and /or fluid , and detecting the IFIG signature and the neutrophil granule signature , the presence of anti - SSA /Ro autoantibodies, wherein an wherein an increase in expression of one ormore genes from increase in expression of one or more genes from the IFIG 55 the IFIG signature and the neutrophil granule signature, and signature and the neutrophil granule signature , and the the presence of the anti - SSA /Ro autoantibodies would indi presence of the anti - SSA /Ro autoantibodies would indicate cate treating the subject with agents for the treatment of an increase in the number of flares , an increase in the number vascular and renalmanifestations of SLE , including , but not of severe flares , and an increase in vascular and renal limited to , corticosteroids such as prednisone in a medium to manifestation of the disease . 60 high dose ( 7 . 5 to over 30 mg/ day ) , or intravenous methyl A more preferred embodiment of the present invention is prednisolone in a high doses ( known as pulse therapy, using a method and /or assay for screening , identifying and /or greater than 50 mg /day for 1 - 3 days) ; cytotoxic drugs , such predicting the severity and clinicalmanifestation of systemic as cyclophosphamide ; immunosuppressive drugs , such as lupus erythematosus in a subject , comprising obtaining mycophenolate mofetil and azathioprine ; biologic agents , biological tissue and / or fluid from the subject , purifying 65 such as rituximab ; and anti -malarial drugs , such as hydroxy and / or isolating nucleic acid , including , but not limited to chloroquine , as well as agents targeting the vascular system , mRNA and cDNA , from the biological tissue and / or fluid , including but not limited to anti- hypertensive drugs , statins , US 9 , 809 , 854 B2 and anti- coagulants , such as aspirin and coumarin , and related signature listed in Table 4 prior to the treatment, monitoring the subject often , e .g ., at leastmonthly , possibly administering a therapeutic agent , or a test agent , or other weekly . treatment, detecting the expression of the same gene or Another preferred embodiment of the present invention is genes after treatment, and comparing the gene expression a method for treating a subject with SLE comprising obtain - 5 prior to treatment to the gene expression after treatment, ing biological tissue and / or fluid from a subject, purifying wherein a decrease in gene expression after treatment as and / or isolating nucleic acid , including , but not limited to , compared to the gene expression prior to treatment , indi mRNA and cDNA , from the biological tissue and /or fluid , and detecting the expression of one or more genes in the cates that the therapeutic agent or test agent or other treat IFIG signature , listed in Table 2 , and one or more genes in 10 mentSIC is effectively treating or ameliorating the subject' s the neutrophil - related signature , listed in Table 4 and com SLE paring the expression of the gene or genes with the expres A further embodiment would include purifying and iso sion of the gene or genes in a healthy control, wherein an lating protein from the biological tissue and / or fluid , and increase in expression of one or more genes from the IFIG detecting the presence of TNFa , IL - 8 , and / or IL - 18 , and / or signature and the neutrophil- related signatureature would indiindi. - 15 anuanti - SSA /Ro autoantibodies , prior to and after treatment, cate treating the subject with agents for the treatment of wherein a decrease in the level or amount of protein after mucocutaneous manifestation of the disease , including but treatment indicates that the therapeutic agent or test agent or not limited to , corticosteroids, such as prednisone in a low other treatment is effectively treating or ameliorating the to medium dose ( less than 20 mg /day ) ; anti -malarial medi- subject' s SLE . cations , such as hydroxychloroquine , and immunosuppres - 20 In all of the preceding methods and assays , a preferred sive agents , such as azathioprine, dapsone, and thalidomide, embodiment is that expression from more than one gene as well as topical agents that target mucocutaneous disor from a signature would be detected and / or measured . For the ders , including but not limited to , topical corticosteroids . IFIG signature , 1 to 83 genes can be detected and / or These subjects would typically be monitored less than measured , with IFIT1 and IFIT3 , being preferred . For the monthly , e . g ., quarterly , and would be especially counseled 25 neutrophil granule signature , 1 to 24 genes can be detected to avoid sunlight. and /or measured with OLFM4, CRISP3 , IF127 , DEFA4 , Another preferred embodiment of the present invention is MMP8 , ANXA3 , ARG1, MPO , DEFA1, LTF , and a method for treating a subject with SLE comprising obtain - CEACAM6 being preferred . For the neutrophil - related sig ing biological tissue and / or fluid from a subject, purifying nature , 1 to 23 genes , can be detected and / or measured , with and / or isolating nucleic acid , including but not limited to 30 TNFAIP6 , FPR1, FPR2 , LY96 , CYP4F3 , ILIR2 , PRRG4, mRNA and cDNA from the biological tissue and / or fluid , and DOCK4 , being preferred . and detecting the expression of one or more genes in the most preferred embodiment of the present invention is IFIG signature , listed in Table 2 , and one or more genes in a method and /or assay that comprise all five components . the neutrophil - related signature listed in Table 4 and com - It will also be understood that and polypeptides paring the expression of the gene or genes with the expres - 35 encoded by any of the genes listed in Tables 2 , 3 , and 4 can sion of the gene or genes in a healthy control, purifying and be detected and /or measured in all of the preceding methods isolating protein from the biological tissue and / or fluid , and and /or assays as a way of detecting and / or measuring gene detecting the presence of TNFa , IL - 8 , and / or IL - 18 , wherein expression . an increase in expression of one or more genes from the Additionally , the invention also relates to the surprising IFIG signature and the neutrophil - related signature and the 40 discovery that differential expression of genes in at least presence of TNFa , IL - 8 , and / or IL - 18 would indicate treat three signatures in an SLE patient along with increased von ing the subject with agents for the treatment of mucocuta Willebrand factor ( VWF ) and IL - 10 mark the active disease neous manifestation of the disease , including but not limited state , i . e . , flares . to , corticosteroids, such as prednisone in a low to medium Thus, a further embodiment of the present invention is a dose ( less than 20 mg/ day ) ; anti- malarial medications , such 45 method and / or assay for monitoring subjects with SLE for as hydroxychloroquine, and immunosuppressive agents, their responses to treatment, in both the regular care of the such as azathioprine , dapsone , and thalidomide, as well as subject, e .g ., administration of a known therapeutic agent topical agents that target mucocutaneous disorders , includ - and lifestyle changes , as well as in clinical trials of test ing but not limited to , topical corticosteroids. These subjects agents . This method and /or assay comprises obtaining bio would typically be monitored less than monthly , e . g ., quar - 50 logical tissue and / or fluid from a subject prior to treatment , terly , and would be especially counseled to avoid sunlight. purifying and /or isolating nucleic acid , including , but not It should be noted that the major benefit from these limited to , mRNA and cDNA , from the biological tissue methods of treatment is that they could be started prior to and / or fluid , and detecting the expression of one or more any overt symptoms in the subject. genes in the IFIG signature , listed in Table 2 , and /or detect Another embodiment of the invention is method of and /or 55 ing the expression of one or more genes in the neutrophil assay for monitoring subjects with SLE for their responses granule signature , listed in Table 3 , and / or one or more genes to treatment, in both the regular care of the subject, e . g ., in the plasma cell signature listed in Table 5 prior to the administration of a known therapeutic agent and lifestyle treatment, administering a therapeutic agent, or a test agent changes , as well as in clinical trials of test agents . This or other treatment, detecting the expression of the same gene method and/ or assay comprises obtaining biological tissue 60 or genes, and comparing the gene expression prior to treat and / or fluid from a subject prior to treatment, purifying ment to the gene expression after treatment, wherein a and / or isolating nucleic acid , including, but not limited to , decrease in gene expression after treatment as compared to mRNA and cDNA, from the biological tissue and / or fluid , the gene expression prior to treatment, indicates that the and detecting the expression of one or more genes in the therapeutic agent or test agent or other treatment is effec IFIG signature , listed in Table 2 , and /or detecting the expres- 65 tively treating or ameliorating the subject' s SLE . Preferably , sion of one or more genes in the neutrophil granule signature plasma proteins, von Willebrand factor (vWF ) and IL - 10 are listed in Table 3 , and/ or one or more genes in the neutrophil - also monitored . US 9 , 809 , 854 B2 In all of the preceding methods and assays , a preferred CEACAM6 being preferred . For the plasma cell signature , embodiment is that expression from more than one gene 1 to 48 genes, can be detected and /or measured , with CD38 , from a signature would be detected and /or measured . For the being preferred . For the T -cell /iNKT gene signature , 1 to 20 IFIG signature , 1 to 83 genes can be detected and/ or can be detected and/ or measured with KLRB 1 being measured , with IFIT1 and IFIT3 , being preferred . For the 5 preferred . neutrophil granule signature , 1 to 24 genes can be detected A preferred embodiment detects the expression of IFIT3 , and / or measured with OLFM4, CRISP3 , IF127 , DEFA4 , KLRB1, CD38 , and MMPS . MMP8 , ANXA3, ARG1, MPO , DEFA1, LTF , and Determining the expression of any of the genes in any of CEACAM6 being preferred . For the plasma cell signature , the signatures can be done by any method known in the art , 1 to 45 genes, can be detected and / or measured , with CD38 , 10 including, but not limited to , microarrays ; Southern blots ; being preferred. Northern blots ; dot blots ; primer extension ; nuclease pro Additionally , the invention also relates to the surprising tection ; subtractive hybridization and isolation of non -du discovery that differential expression of genes in four sig - plexed molecules using , for example , hydroxyapatite ; solu natures in an SLE patient when not flaring , indicates tion hybridization ; filter hybridization ; amplification increased number of future flares . 15 techniques such as RT- PCR and other PCR -related tech Thus, a further embodiment of the present invention is a niques such as PCR with melting curve analysis, and PCR method and /or assay for predicting future flares in a subject with mass spectrometry ; fingerprinting , such as with restric with SLE , who is not flaring, comprising obtaining biologi - tion endonucleases; and the use of structure specific endo cal tissue and / or fluid from the subject, purifying and/ or nucleases . mRNA expression can also be analyzed using isolating nucleic acid , including but not limited to mRNA 20 mass spectrometry techniques ( e . g ., MALDI or SELDI) , and cDNA from the biological tissue and /or fluid , detecting liquid chromatography , and capillary gel electrophoresis . the expression of the expression of one or more genes in the Any additional method known in the art can be used to IFIG signature , listed in Table 2 , and/ or detecting the expres - detect the presence or absence of the transcripts . sion of one or more genes in the neutrophil granule signature The expression of the genes from the subject with SLE listed in Table 3 , and /or one or more genes in the plasma cell 25 can be compared to a reference value of the expression of the signature listed in Table 5 , and / or one or more genes in the same genes in a healthy control. The levels of expressed T - cell/ iNKT signature listed in Table 6 , and comparing the genes may be measured as absolute or relative . Absolute expression of the genes with the expression of the genes in quantitation measure concentrations of specific RNA and a healthy control, wherein an increase in expression the requires a calibration curve . Relative quantification mea genes in the first three signatures and a decrease in the 30 sures fold change differences of specific RNA in comparison expression of the gene in the last signature , indicates an to housekeeping genes . Relative quantification is usually increase in future flares . adequate to investigate physiological changes in gene Yet another embodiment is a method of treating a subject expression levels. One reference value that can be used in with SLE comprising obtaining biological tissue and /or fluid the methods and assays of the invention is the fold change from the subject, purifying and/ or isolating nucleic acid , 35 between patients with SLE and healthy donors of expression including but not limited to mRNA and cDNA from the of each differentially expressed gene found in Table 9 . biological tissue and /or fluid , detecting the expression of Detection of the levels of anti -SSA /Ro autoantibodies, detecting the expression of the expression of one or more TNFa , IL - 8 , and IL - 18 , and vWF and IL - 10 , can be accom genes in the IFIG signature , listed in Table 2 , and /or detect - plished by any method known in the art, including methods ing the expression of one or more genes in the neutrophil 40 which result in qualitative results , such as ones where the granule signature listed in Table 3 , and / or one or more genes existence of the protein can be visualized , either by the in the plasma cell signature listed in Table 5 , and / or one or naked eye or by other means , and / or quantitative results . more genes in the T - cell/ iNKT signature listed in Table 6 , Such methods would include , but are not limited to , quan and comparing the expression of the genes with the expres - titative Western blots , immunoblots , quantitative mass spec sion of the genes in a healthy control, wherein an increase 45 trometry , enzyme- linked immunosorbent assays ( ELISAs) , in expression the genes in the first three signatures and a radioimmunoassays (RIA ), immunoradiometric assays decrease in the expression of the gene in the last signature , (IRMA ), and immunoenzymatic assays (IEMA ) and sand indicates consideration of treating the patient with agents wich assays using monoclonal and polyclonal antibodies . including, but not limited to , corticosteroids , such as pred - Reference values of fold change of proteins between patients nisone in a medium to high dose ( 7 . 5 to over 30 mg/ day ) , or 50 with SLE and healthy donors is found in Table 10 . intravenous methylprednisolone in a high doses (known as The present invention also provides for methods and tools pulse therapy, using greater than 50 mg/ day for 1 - 3 days) ; for drug design , testing of agents, and tools for basic cytotoxic drugs , such as cyclophosphamide ; immunosup - research into the causes and etiology of systemic lupus pressive drugs, such as mycophenolate mofetil and azathio erythematosus . prine ; biologic agents , such as rituximab ; and anti -malarial 55 One embodiment is a method and /or assay for screening drugs, such as hydroxychloroquine , as well as increasing the and /or identifying a test agent for the prevention and /or dose of any therapeutic agents currently be used by the treatment of SLE , comprising contacting or incubating the subject, and monitoring the subject more frequently, e . g ., test agent with a polypeptide encoded by one of the genes monthly or more frequently . listed in Tables 2 , 3 , 4 , 5 or 6 , and detecting the presence of In all of the preceding methods and assays , a preferred 60 a complex between the test agent, wherein if a complex embodiment is that expression from more than one gene between the test agent and the polypeptide is detected , the from a signature would be detected and /or measured . For the test agent is identified as a prevention and /or treatment for IFIG signature , 1 to 83 genes can be detected and /or SLE . measured, with IFIT1 and IFIT3 , being preferred . For the further embodiment is a method and / or assay for neutrophil granule signature , 1 to 24 genes can be detected 65 screening and / or identifying a test agent for the prevention and /or measured with OLFM4, CRISP3 , IF127, DEFA4 , and /or treatment of SLE , comprising contacting or incubat MMP8, ANXA3 , ARG1 , MPO , DEFA1, LTF , and ing the test agent with a polypeptide encoded by one of the US 9 , 809 , 854 B2 genes listed in Tables 2 , 3 , 4 , 5 , or 6 and a known ligand of gene signatures found in Tables 2 -6 would also be potential the polypeptide, and detecting the presence of a complex therapeutic targets drug development or research . between the test agent and the ligand , wherein if a complex The present invention also includes kits . between the test agent and the ligand is detected , the test agent is identified as a prevention and / or treatment for SLE . 5 BRIEF DESCRIPTION OF THE FIGURES Another embodiment of the present invention is a method For the purpose of illustrating the invention , there are and / or assay for screening and /or identifying a test agent for depicted in drawings certain embodiments of the invention . the prevention and/ or treatment of SLE , comprising con However , the invention is not limited to the precise arrange tacting or incubating the test agent with a polypeptide m ents and instrumentalities of the embodiments depicted in encoded by one of the genes listed in Tables 2 , 3 , 4 , 5 , or 6 " the drawings. and a known antibody of the polypeptide , and detecting the The patent or application file contains at least one drawing presence and quantity of unbound antibody, wherein the executed in color . Copies of this patent or patent application presence of the unbound antibody indicates that the test publication with color drawing ( s ) will be provided by the presence of the unbound antibody indicates that the test Office upon request and payment of the necessary fee . agent is binding to the polypeptide , and the test agent is 15 FIG . 1 is a heatmap and dendrogram showing the unsu identified as a prevention and /or treatment for SLE . pervised hierarchical clustering of donors ( shown in the These methods and assays can be done using polypeptides columns) and differentially expressed mRNA transcripts TNFA , IL - 8 , IL - 18, vWF, and IL - 10 . They can be performed ( shown in the rows) . Heatmap codes mean levels of expres with the polypeptides and test agents , and ligands and sion for each probe- set, calculated over all visits for each antibodies, if applicable , free in solution , or affixed to a solid 20 study subject before clustering, Unsupervised hierarchical support. The polypeptides and antibodies may be labeled by clustering is shown based on 433 mRNA transcripts differ any method known in the art . entially expressed in SLE and HD PBMC ( FC > 1 . 5 , p < 0 .05 , 5 % FDR ) . The first three groups of transcripts from the top High throughput screening can also be used to screen the of dendrogram were identified as follows: IFIG – interferon test agents . Small peptides or molecules can be synthesized 25 inducible genes , NG = neutrophil granules related , and and bound to a surface and contacted with the polypeptides 25 NR neutrophil related . SLE patients ( identified as IF # ) encoded by the gene signature transcripts , and washed . The were classified based on the dendrogram as follows: ( A ) bound peptide is visualized and detected by methods known linked with healthy donors ( identified as HD # ) shown in the art . mostly in blue ; ( B ) showing increase in IFN and NR A further embodiment of the present invention is a genes — blue turning to yellow and orange ; and ( C ) showing method and / or assay for screening and /or identifying a test su increase in IFN and NG genes - mostly yellow with orange . agent for the prevention and /or treatmentof SLE comprising FIG . 2 depicts the assignment of transcripts, in which is contacting or incubating a test agent to a nucleotide expression is different in PBMC of SLE patients , to func tional groups of genes . FIG . 2A is the graphical results of comprising any one of the genes listed in Tables 2 , 3, 4 , 5 , analyzing the top cluster in the dendrogram of FIG . 1 using or 6 , and determining if the test agent binds to the gene, 35 INTERFEROME. FIG . 2B shows the graphical results of wherein if the test agent binds to the nucleotide , the test analysis done on the second from top cluster in FIG . 1 , agent is identified as a therapeutic or preventative agent for labeled “ NG ” based upon previously published microarray SLE . analysis of bone marrow and peripheral blood neutrophils by A further embodiment of the present invention is a Theilgaard -Mönch et al. 2005 . FIG . 2C shows the results of method and /or assay for screening and /or identifying a test 40 analysis of the third cluster from the top of the dendrogram agent for the prevention and / or treatment ofSLE comprising in FIG . 1 labeled “ NR ” using a Gene Enrichment Profiler contacting or incubating a test agent with a nucleotide from Harvard University containing data from human nor comprising any one of the genes listed in Tables 2 , 3 , 4 , 5 , mal primary tissues ( 126 tissues represented by 557 microar or 6 which expresses a measurable phenotype, and measur- rays ) . Expression profiles were processed to identify tissue ing the phenotype before and after contact or incubation 45 specificity which is measured by an enrichment score . with the test agent, wherein if the expression of the mea - FIG . 3 depicts graphs of the average plasma levels of surable phenotype is decreased after the contact or incuba - autoantibodies and pro - inflammatory proteins among three tion with the test agent, the test agent is identified as a major groups of SLE patients during the study . FIG . 3A therapeutic or preventative agent for SLE . shows relative units of anti - SS - A /Ro autoantibody . FIG . 3B Themeasurable phenotype can be one that is native to the 50 shows concentrations of TNFa ; FIG . 3C shows concentra gene or one that is artificially linked , such as a reporter gene . tions of IL - 8 ; and FIG . 3D show concentrations of IL - 18 , for A further embodiment of the present invention is a lupus patients in groups A , B and C , defined based on method and /or assay for screening and /or identifying a test clustering analysis of gene expression data . The vertical line agent for the prevention and /or treatment of SLE , compris indicates a significant difference (p < 0 .05 ) compared to other ing transforming a host cell with a gene construct compris - 55 groups based on linear mixed model. Average levels from all ing any gene listed in Tables 2 , 3 , 4 , 5 or 6 , detecting the visits were calculated for each patient in the study for data expression of the gene in the host cell , contacting the test visualization and shown as black dots . agent with the host cell , and detecting the expression of the FIG . 4 is a heatmap and dendrogram showing the hier gene from the host cell after contact with the test agent or archical clustering of SLE patients based on transcripts , compound , wherein if the expression of the gene is reduced 60 selected as related to IFIG , neutrophil granule and neutro or decreased after contact with the test agent or compound , phil related signatures . The dendrogram depicts subjects the test agent is identified as a therapeutic or preventative (shown in columns) classified based on 195 mRNA tran agent for SLE . scripts , identified as interferon inducible genes , neutrophil The present invention also provides a method for deter - granules related and neutrophil - related genes ( shown in the mining target genes or proteins for drug development and 65 rows) . The attached color code on top of heatmap illustrates basic research regarding SLE . The invention also contem - how often individual subject target the same group when plates that the protein products of any of the genes in the clustering was performed 500 times , each time selecting US 9 , 809 , 854 B2 12 unique combination of visits . Deeper red color indicates are provided . A recital of one or more synonyms does not stable position in selected group . exclude the use of the other synonyms. The use of examples FIG . 5 is a heatmap and dendrograms that illustrate the anywhere in the specification , including examples of any classification of a validation set of 59 SLE patients . Level of terms discussed herein , is illustrative only , and in no way mRNA transcripts was measured by quantitative real time 5 limits the scope and meaning of the invention or any PCR . Seven genes ( in the rows) , representing interferon exemplified term . Likewise , the invention is not limited to inducible signature genes, neutrophil granule signature its preferred embodiments . genes and neutrophil - related signature genes were chosen to The term “ subject as used in this application means an classify SLE patients ( in the columns ) . Three major groups of SLE patients A , B , and C , and five subgroups ( A , B1, B2 , 10 malanimal with an immune system such as avians and mam C1, and C2 ) were observed . mals . Mammals include canines , felines , rodents , bovine, FIG . 6 shows graphs of disease activities in groups of SLE equines , porcines, ovines , and primates . Avians include , but patients based on hierarchical clustering and frequency of are not limited to , fowls , songbirds, and raptors . Thus , the visits per year . FIG . 6A shows frequency of severe flares . invention can be used in veterinary medicine , e . g . , to treat FIG . 6B shows frequency of visits with active mucocutaneus 1615 companion animals , farm animals , laboratory animals in involvement; FIG . 6C shows frequency of visits with active zoological parks, and animals in the wild . The invention is renal involvement; and FIG . 6D shows frequency of visits particularly desirable for human medical applications. with vascular involvement, based on BILAG score . The term “ patient ” as used in this application means a FIG . 7 is a graph depicting the amount of anti- SS - A human subject. In some embodiments of the present inven (Ro60 ) antibody in plasma samples of the various groups of 20 tion , the “ patient” is one suffering with systemic lupus SLE patients as measured by ELISA . The concentration erythematosus. higher than upper detection limit was considered as 200 The terms “ systemic lupus erythematosus ” and “ SLE” R .U ./ mL . p < 0 .01 . and “ lupus” will be used interchangeably herein and is FIG . 8 is graph depicting results of the 4 - gene microarray defined as a systemic autoimmune disease that can affect any score for nine patients ( denoted with IF # ) at a mean of 100 25 part of the body . As occurs in other autoimmune diseases , days before flare ( p = < 0 .02 ), at flare visit and a mean of 100 the immune system attacks the body ' s cells and tissue , days after flare visit (p = < 0 .02 ) . resulting in inflammation and tissue damage. SLE most FIG . 9 are graphs of the comparison of the 4 - gene often harms the heart , joints , skin , lungs, blood vessels , liver, microarray score and vWF plasma level as compared to kidneys , and nervous system . The course of the disease is flares in three representative SLE patients ( IF06 , IF09 , and 30 unpredictable. with periods of illness (called flares ) alter IF12 ) . FIGS. 9A , 9B , and 9C ( F06 , IF09 , and IF12 respec tively ) are the results of measurement of vWF in ug/ ml nating with remissions. versus days are shown in solid black lines. FIGS . 9D , 9E , The terms “ SLE disease activity index ” and “ SLEDAI” and 9F ( IF06 , IF09 , and IF12 respectively ) are the 4 - gene and “ SELENA SLEDAI” are used interchangeably in this microarray scores versus days as shown in solid black lines . 3525. application and means the weighted cumulative index of In all six figures, the SLEDAI 2K score is shown in open lupus disease activity ( American College of Rheumatology circles connected by black lines, black solid squares denote Ad Hoc Committee on Systemic Lupus Erythematosus severe flares, black solid diamonds denote pulse glucocor Response Criteria 2004 ) . The total score falls between 0 and ticoids, and open diamonds denote Cytoxan . 105 , with higher scores representing increased disease activ FIG . 10 is a graph depicting the best fit panel of biomark - 40 ity . The SLEDAI has been shown to be a valid and reliable ers versus SLEDAI score , using generalized linear mixed disease activity measure in multiple patient groups, and has model analysis. also has been shown to be sensitive to changes in disease FIG . 11 is a graph showing the percentage free from flares activity in children . versus days of patients with a low 4 - gene microarray score The terms “ British Isles Lupus Assessment Group Index at non - flaring visits and patients with a high 4 - gene microar - 45 2000 ” and “ BILAG ” are used interchangeably in this appli ray score at non - flaring visits . cation . BILAG is a score which accounts for disease activity FIG . 12 is a heatmap of gene expression analysis of high in eight systems. and low - flaring patients at non - flaring visit . Low - flaring The terms “ screen ” and “ screening ” and the like as used patients are denoted as “ L ” , and high - flaring patients are herein means to test a subject or patient to determine if they denoted as “ H ” . 50 have a particular illness or disease , or a particular manifes tation of an illness or disease . The term also means to test an DETAILED DESCRIPTION OF THE agent to determine if it has a particular action or efficacy . INVENTION The terms " identification " , " identify ” , “ identifying ” and the like as used herein means to recognize a disease state or Definitions 55 a clinical manifestation or severity of a disease state in a The terms used in this specification generally have their subject or patient. The term also is used in relation to test ordinary meanings in the art , within the context of this agents and their ability to have a particular action or efficacy . invention and the specific context where each term is used . The terms “ prediction ” , “ predict ” , “ predicting” and the Certain terms are discussed below , or elsewhere in the like as used herein means to tell in advance based upon specification , to provide additional guidance to the practi - 60 special knowledge . tioner in describing the methods of the invention and how to The term “ reference value ” as used herein means an use them . Moreover , it will be appreciated that the same amount or a quantity of a particular protein or nucleic acid thing can be said in more than one way . Consequently , in a sample from a healthy control or healthy donor . alternative language and synonyms may be used for any one The terms “ healthy control ” , “ healthy donor” and “HD ” or more of the terms discussed herein , nor is any special 65 are used interchangeably in this application and are a human significance to be placed upon whether or not a term is subject who is not suffering from systemic lupus erythema elaborated or discussed herein . Synonyms for certain terms tosus or any other rheumatic or autoimmune disease . US 9 ,809 , 854 B2 13 14 The terms “ treat ” , “ treatment” , and the like refer to a based quantitative real time PCR ( TaqMan® PCR ) . In the means to slow down , relieve, ameliorate or alleviate at least Examples section provided below , nucleic acid expression one of the symptoms of the disease , or reverse the disease profiles were obtained using Affymetrix GeneChip® oligo after its onset . nucleotide microarrays . The expression profiling at the pro The terms " prevent" , " prevention " , and the like refer to 5 tein level can be accomplished using any available technol acting prior to overt disease onset, to prevent the disease ogy to measure protein levels , e . g ., using peptide -specific from developing or minimize the extent of the disease or capture agent arrays . slow its course of development. The terms " gene signature” and “ signature genes” will be The term “ agent” as used herein means a substance that used interchangeably herein and mean the particular tran produces or is capable of producing an effect and would 10 scripts that have been found to be differentially expressed in include , but is not limited to , chemicals , pharmaceuticals , some SLE patients . biologics , small organic molecules , antibodies, nucleic The terms “ gene ” , “ gene transcript ” , and “ transcript" are acids, peptides , and proteins. used somewhat interchangeable in the application . The term The phrase " therapeutically effective amount” is used “ gene” , also called a " structural gene” means a DNA herein to mean an amount sufficient to cause an improve - 15 sequence that codes for or corresponds to a particular ment in a clinically significant condition in the subject, or sequence of amino acids which comprise all or part of one delays or minimizes or mitigates one or more symptoms or more proteins or enzymes, and may or may not include associated with the disease , or results in a desired beneficial regulatory DNA sequences , such as promoter sequences , change of physiology in the subject. which determine for example the conditions under which the As used herein , the term “ isolated ” and the like means that 20 gene is expressed . Some genes , which are not structural the referenced material is free of components found in the genes , may be transcribed from DNA to RNA , but are not natural environment in which the material is normally translated into an amino acid sequence . Other genes may found . In particular, isolated biological material is free of function as regulators of structural genes or as regulators of cellular components . In the case of nucleic acid molecules , DNA transcription . “ Transcript” or “ gene transcript” is a an isolated nucleic acid includes a PCR product, an isolated 25 sequence of RNA produced by transcription of a particular mRNA , a cDNA , an isolated genomic DNA , or a restriction gene . Thus , the expression of the gene can be measured via fragment. In another embodiment, an isolated nucleic acid is the transcript. preferably excised from the in which itmay be The term “ antisense DNA ” is the non -coding strand found . Isolated nucleic acid molecules can be inserted into complementary to the coding strand in double - stranded plasmids, cosmids, artificial , and the like . 30 DNA . Thus , in a specific embodiment , a recombinant nucleic acid The term " genomic DNA ” as used herein means all DNA is an isolated nucleic acid . An isolated protein may be from a subject including coding and non - coding DNA , and associated with other proteins or nucleic acids , or both , with DNA contained in introns and . which it associates in the cell , or with cellular membranes if The term “ nucleic acid hybridization ” refers to anti it is a membrane -associated protein . An isolated material 35 parallel hydrogen bonding between two single - stranded may be, but need not be , purified nucleic acids, in which A pairs with T ( or U if an RNA The term “ purified ” and the like as used herein refers to nucleic acid ) and C pairs with G . Nucleic acid molecules are material that has been isolated under conditions that reduce “ hybridizable ” to each other when at least one strand of one or eliminate unrelated materials , i . e ., contaminants . For nucleic acid molecule can form hydrogen bonds with the example , a purified protein is preferably substantially free of 40 complementary bases of another nucleic acid molecule other proteins or nucleic acids with which it is associated in under defined stringency conditions . Stringency of hybrid a cell; a purified nucleic acid molecule is preferably sub - ization is determined , e . g . , by ( i) the temperature at which stantially free of proteins or other unrelated nucleic acid hybridization and /or washing is performed , and ( ii ) the ionic molecules with which it can be found within a cell . As used strength and ( iii ) concentration of denaturants such as for herein , the term " substantially free ” is used operationally , in 45 mamide of the hybridization and washing solutions , as well the context of analytical testing of the material. Preferably, as other parameters . Hybridization requires that the two purified material substantially free of contaminants is at least strands contain substantially complementary sequences. 50 % pure ; more preferably , at least 90 % pure , and more Depending on the stringency of hybridization , however, preferably still at least 99 % pure . Purity can be evaluated by some degree of mismatches may be tolerated . Under “ low chromatography , gel electrophoresis , immunoassay , compo - 50 stringency ” conditions , a greater percentage of mismatches sition analysis , biological assay, and other methods known are tolerable (i . e . , will not prevent formation of an anti in the art . parallel hybrid ). The terms " expression profile ” or “ gene expression pro - The terms " vector ", " cloning vector" and " expression file ” refers to any description ormeasurement ofone ormore vector” mean the vehicle by which a DNA or RNA sequence of the genes that are expressed by a cell , tissue , or organism 55 ( e . g . a foreign gene ) can be introduced into a host cell, so as under or in response to a particular condition . Expression to transform the host and promote expression ( e .g . transcrip profiles can identify genes that are up - regulated , down - tion and translation ) of the introduced sequence . Vectors regulated , or unaffected under particular conditions . Gene include , but are not limited to , plasmids , phages, and viruses . expression can be detected at the nucleic acid level or at the Vectors typically comprise the DNA of a transmissible protein level. The expression profiling at the nucleic acid 60 agent, into which foreign DNA is inserted . A common way level can be accomplished using any available technology to to insert one segment of DNA into another segment of DNA measure gene transcript levels . For example , the method involves the use of enzymes called restriction enzymes that could employ in situ hybridization , Northern hybridization cleave DNA at specific sites ( specific groups of nucleotides) or hybridization to a nucleic acid microarray, such as an called restriction sites. A “ cassette ” refers to a DNA coding oligonucleotide microarray , or a cDNA microarray . Alterna - 65 sequence or segment of DNA which codes for an expression tively , the method could employ reverse transcriptase -poly - product that can be inserted into a vector at defined restric merase chain reaction (RT - PCR ) such as fluorescent dye - tion sites. The cassette restriction sites are designed to US 9 , 809 , 854 B2 15 16 ensure insertion of the cassette in the proper reading frame. ring, which have similar binding properties as the reference Generally , foreign DNA is inserted at one or more restriction nucleic acid , and which are metabolized in a manner similar sites of the vector DNA , and then is carried by the vector to the reference nucleotides. The nucleic acids may also be into a host cell along with the transmissible vector DNA . A modified by many means known in the art . Non - limiting segment or sequence of DNA having inserted or added 5 examples of such modifications include methylation , " caps” , DNA , such as an expression vector, can also be called a substitution of one or more of the naturally occurring “ DNA construct " or " gene construct .” A common type of nucleotides with an analog , and internucleotide modifica vector is a " plasmid” , which generally is a self -contained tions such as, for example , those with uncharged linkages molecule of double - stranded DNA , usually of bacterial (e . g ., methyl phosphonates , phosphotriesters , phosphoro origin , that can readily accept additional (foreign ) DNA and 10 amidates , and carbamates ) and with charged linkages ( e . g . , which can readily introduced into a suitable host cell . A phosphorothioates, and phosphorodithioates ) . Polynucle plasmid vector often contains coding DNA and promoter otides may contain one or more additional covalently linked DNA and has one or more restriction sites suitable for moieties , such as , for example , proteins ( e . g ., nucleases , inserting foreign DNA . Coding DNA is a DNA sequence toxins, antibodies, signal peptides, and poly - L - lysine ) , inter that encodes a particular amino acid sequence for a particu - 15 calators ( e . g . , acridine, and psoralen ) , chelators ( e . g . , metals , lar protein or enzyme. Promoter DNA is a DNA sequence radioactive metals , iron , and oxidative metals ), and alky which initiates, regulates, or otherwise mediates or controls lators . The polynucleotides may be derivatized by formation the expression of the coding DNA. Promoter DNA and of a methyl or ethyl phosphotriester or an alkyl phospho coding DNA may be from the same gene or from different ramidate linkage . Modifications of the ribose - phosphate genes , and may be from the same or different organisms. A 20 backbone may be done to facilitate the addition of labels, or large number of vectors , including plasmid and fungal to increase the stability and half - life of such molecules in vectors , have been described for replication and/ or expres - physiological environments . Nucleic acid analogs can find sion in a variety of eukaryotic and prokaryotic hosts . Non - use in the methods of the invention as well as mixtures of limiting examples include PKK plasmids (Clonetech ) , PUC naturally occurring nucleic acids and analogs . Furthermore , plasmids, PET plasmids (Novagen , Inc . , Madison , Wis . ) , 25 the polynucleotides herein may also be modified with a label PRSET or PREP plasmids ( Invitrogen , San Diego , Calif. ) , or capable of providing a detectable signal, either directly or PMAL plasmids (New England Biolabs , Beverly , Mass. ) , indirectly . Exemplary labels include radioisotopes, fluores and many appropriate host cells , using methods disclosed or cent molecules, and biotin . cited herein or otherwise known to those skilled in the The term “ polypeptide” as used herein means a compound relevant art. Recombinant cloning vectors will often include 30 of two or more amino acids linked by a peptide bond . one or more replication systems for cloning or expression , “ Polypeptide” is used herein interchangeably with the term one or more markers for selection in the host, e . g . antibiotic " protein . " resistance , and one or more expression cassettes . The term “ about” or “ approximately ” means within an The term " host cell ” means any cell of any organism that acceptable error range for the particular value as determined is selected , modified , transformed , grown , used or manipu - 35 by one of ordinary skill in the art, which will depend in part lated in any way, for the production of a substance by the on how the value is measured or determined , i. e ., the cell, for example , the expression by the cell of a gene , a limitations of the measurement system , i . e . , the degree of DNA or RNA sequence , a protein or an enzyme. Host cells precision required for a particular purpose , such as a phar can further be used for screening or other assays , as maceutical formulation . For example, " about” can mean described herein . 40 within 1 or more than 1 standard deviations, per the practice A “ polynucleotide ” or “ nucleotide sequence” is a series of in the art . Alternatively , “ about" can mean a range of up to nucleotide bases ( also called “ nucleotides ” ) in a nucleic 20 % , preferably up to 10 % , more preferably up to 5 % , and acid , such as DNA and RNA , and means any chain of two more preferably still up to 1 % of a given value. Alterna or more nucleotides. A nucleotide sequence typically carries tively , particularly with respect to biological systems or genetic information , including the information used by 45 processes , the term can mean within an order of magnitude , cellular machinery to make proteins and enzymes. These preferably within 5 - fold , and more preferably within 2 -fold , terms include double or single stranded genomic and cDNA , of a value . Where particular values are described in the RNA , any synthetic and genetically manipulated polynucle - application and claims, unless otherwise stated , the term otide , and both sense and anti -sense polynucleotide . This " about” meaning within an acceptable error range for the includes single - and double - stranded molecules , i . e . , DNA - 50 particular value should be assumed . DNA , DNA -RNA and RNA -RNA hybrids, as well as “ pro - Genes and Proteins Correlated to Clinical Outcomes of tein nucleic acids” (PNA ) formed by conjugating bases to an Systemic Lupus Erythematosus amino acid backbone . This also includes nucleic acids Patients with systemic lupus erythematosus (SLE ) have containing modified bases, for example thio -uracil , thio - variable manifestations and outcomes . This comprehensive guanine and fluoro -uracil . 55 molecular analysis of patients with SLE and healthy donors “ Nucleic acid ” refers to deoxyribonucleotides or ribo - (HD ) has identified subgroups of patients , primarily defined nucleotides and polymers thereof in either single - or double based on presence or absence of mRNA transcript signa stranded form . The nucleic acids herein may be flanked by tures , and has described significant relationships between natural regulatory (expression control) sequences , or may be those patient groups, and clinical and serologic features of associated with heterologous sequences , including promot - 60 lupus . The results set forth herein for the first time provide ers, internal ribosome entry sites ( IRES ) and other ribosome biochemical and genetic biomarkers for clinical outcomes of binding site sequences , enhancers , response elements , sup - SLE that will allow prediction of disease severity , which in pressors , signal sequences , polyadenylation sequences, turn allows for correct treatment choice at a much earlier introns, 5 ' - and 3 -non - coding regions , and the like . The term stage in the disease , even before symptoms occur. Further encompasses nucleic acids containing known nucleotide 65 more , these biochemical and genetic biomarkers will allow analogs or modified backbone residues or linkages, which a complete understanding of the mechanism of SLE , and are synthetic , naturally occurring, and non - naturally occur provide targets for testing new drugs and therapies for SLE . US 9 ,809 ,854 B2 17 18 Additionally , using the same data , biomarkers were also traps (NETS ) (Brinkmann et al . 2004 ) has suggested poten identified which correlate to disease activity , i . e ., flare tial relevance to lupus. This process may be important in the activity , and future flare activity . formation of anti -nuclear autoantibodies, endothelial dam As shown in the Examples 1 - 5 , peripheral blood mono age and autoimmune inflammation . Immune complexes that nuclear cells (PBMC ) and plasma samples from visits were 5 include anti -RNP antibodies can induce NET formation and collected longitudinally (up to 3 years ) from numerous SLE those NETs contribute to activation of endosomal TLRs and patients and 5 healthy donors . PBMC mRNA profiles for production of IFN - I by plasmacytoid dendritic cells (PDC ) each visit were established using Affymetrix GeneChips® . (Lande et al . 2011 ; Garcia - Romo et al . 2011) . Plasma levels of 44 autoantibodies and 41 pro - inflammatory Group B2 SLE patients showed differential expression of mediators were determined using Multi - Analyte Profiling 10 a less well -defined cluster of genes that includes TNFAIP6 , technology . A linear mixed model was used to identify FPR1, FPR2, LY96 , CYP4F3 , IL1R2, PRRG4, and DOCK4 . differentially expressed genes and clustering analysis was This group of genes is denoted as neutrophil- related or used for patient classification and transcript classification . neutrophil activation signature genes . Increased expression The gene signatures in SLE blood , type I interferon of representative transcripts in this cluster by group B2 inducible genes (IFIG ) and neutrophil granule genes, along 15 patients was confirmed by real- time - PCR . Based on tissue with neutrophil - related genes, classified SLE patients into 5 expression analysis and literature review , a common theme distinct groups. Patients with neither signature had only mild among those transcripts is increased expression by neutro disease , and no particular clinical manifestation . The other phils in peripheral blood ( FIG . 2C ) , and thus, they represent groups had more frequent flares, but were distinguishable a second neutrophil- related signature in SLE patients . based on gene expression , clinical manifestations , and 20 TNFAIP6 ( Tumor necrosis factor - inducible gene 6 or cytokine profile. TSG6 ) , the gene transcript in this cluster that showed the Each group with its identifying signature and clinical greatest fold increase , is a member of the hyaluronan manifestations is found in Table 1 . binding protein family and is associated with inflammation TABLE 1 Summary of Five Groups of SLE Patients Neutrophil Neutrophil- Increased Increased Increased granule gene related gene TNFa , IL - anti - SSA / Ro Increased severe Mucocutaneous Vascular IFIG signature signature 8 , IL - 18 autoantibodies Flares flares Involvement Involvement A B1 B2 XX

X

The IFIG signature is a feature of groups B1, B2, and C2. in arthritis models (Milner and Day 2003 ) . Formyl peptide Transcripts associated with the neutrophil granule gene receptor 1 and 2 (FPR1 and FPR2 ) are G -protein -coupled signature represented the most striking differentiating sig - an receptors highly expressed on neutrophils that recognize nature associated with groups C 1 and C2 , suggesting an d amage- related mitochondrial peptides and DNA , as well as important pathogenic role for the products of neutrophil microbial peptides and acute phase protein serum A granules in those patient groups, and were also associated (SAA ) (Forsman and Dahlgren 2010 ) . Engagement of FPR1 with flare . Neutrophil - specific transcripts were represented or FPR2 by their ligands activates neutrophil granulation . by genes encoding proteins present in primary azurophilic 45 Ly96 , also known as MD -2 , participates with TLR4 in granules and expressed at highest level in the bone marrow binding of lipopolysaccharide (LPS ) (Gray et al. 2010 ) . during myelopoiesis (Nathan 2006 ) . Induction of CYP4F3, a cytochrome P450 enzyme, regulates A role for neutrophils in the pathogenesis of SLE has a leukotriene B (4 ) metabolism in neutrophils (Zhao et al . long history , beginning with observations of so - called 2009 ) . ILIR2 receptor, induced by oxygen radicals and other “ Lupus Erythematosus (LE )” cells present in the bone 50 stimuli (Mantovani et al. 1998 ), is activated by inflammatory marrow of most SLE patients (up to 80 % ) (Hargraves et al. cytokines , binds IL - 1 and plays an anti - inflammatory role . In 1948 ) . The formation of LE cells is initiated when anti - addition to preferential expression of these neutrophil and nuclear autoantibodies penetrate neutrophils and interact inflammation - related transcripts , the plasma samples from with nuclear material, resulting in loss of nuclear structure . patients in group C demonstrated strikingly elevated levels The degraded nuclei are engulfed by nearby polymorpho - 55 of TNFa . TNFa is known to induce expression of TNFAIP6 nuclear leukocytes (Hargraves 1969) . Later , Abramson and and is itself a product of activated neutrophils. At the same colleagues pointed to the capacity of lupus sera to induce time interferon are capable to activate expression ofmany of neutrophil aggregation , particularly in patients with active those genes . This highlights a relationship between neutro central nervous system disease ( Abramson et al. 1983 ) . phils physiology and interferon signaling in SLE patients . The importance of neutrophils in SLE has re - emerged 60 More significantly , the SLE patient groups defined by with the description of the presence of low - density granu - gene expression analysis showed important differences in locytes in blood preparations from many lupus patients and clinical and serological parameters . Patients who demon the recognition that those cells produce IFN and are asso strated both IFIG and neutrophil granule gene signatures ciated with endothelial cell damage (Denny et al. 2010 ) . The (group C2) experienced higher rates of vascular involve discovery of NETosis , the process in which neutrophils 65 ment. Many of the patients in group C2 had a history of extrude intracellular material containing chromatin that Raynaud ' s phenomenon and some had serious vascular forms web - like structures called neutrophil extracellular manifestations. In addition , most group C2 patients had US 9 , 809 , 854 B2 20 elevated levels of anti- SSA /Ro autoantibodies. Anti -SSA anti - SSA / Ro antibodies in group C2 suggest that induction Ro autoantibodies are common in patients with Sjogren ' s of NETosis and its products might be relevant mechanisms syndrome and are associated with Raynaud ' s phenomena in the vasculopathy seen in many patients with SLE . Addi (Garcia - Carrasco et al. 2002 ) . Anti - SSA / Ro autoantibodies tionally , this analysis provides novel insights into a distinct appear in 30 - 50 % of SLE patients and have been associated 5 role for neutrophils and TNFa in the mucocutaneous disease with cutaneous vasculitis ( Fukuda et al. 2009 ) and neutro - experienced by some patients . penia (Kurien and Scofield 2006 ; Kurien et al. 2000 ) . Additionally , as shown in Example 6 , using K -mean Anti- SSA /Ro autoantibodies cross -react with the 64 kDa clustering from the microarray data described in Example 1 , autoantigen D1 protein on the neutrophil surface and may functionally related clusters including previously described induce neutrophil activation , or alternatively cell death and 10 IFIG , and neutrophil granule , along with plasma cell genes , neutropenia (Kurien et al. 2000 ) . This data suggests that and plasma factors von Willebrand factor and IL - 10 , were anti - SSA /Ro antibodies might promote NET formation , correlated to lupus disease activity . A four gene panel, with resulting in induction of the IFN - I pathway , along with genes representative of IFIG , neutrophil granule , and plasma damage to endothelial cells that results in vasculopathy cell , along with T cells /iNKT genes , were also indicative of (Hakkim et al. 2010 ; Villanueva et al. 2011 ). 15 current flare activity as well as future flare activity ( Example Lupus patients whose gene expression data cluster with 7 ) . group B2 show bright IFIG and a signature that appears to Signature Genes be neutrophil- related but lacks high level expression of This data show for the first time biomarkers , in the form neutrophil granule genes . Clinically , group B2 patients had of signature genes , which correlate to the severity of SLE more mucocutaneous flares than patients in group A , and 20 and its clinical manifestation . The expression of these genes their plasma samples show increased levels of pro - inflam - can be specifically used to screen for , identify and / or predict matory cytokines, particularly TNFO , IL - 8 , and IL - 18 . The the clinical manifestation and disease severity of subjects molecular pattern described for group B2 patients suggests who have been diagnosed with SLE . These biomarkers that mucocutaneous manifestations of SLE may be less include : dependent on contributions from low -density granulocytes 25 type I interferon inducible genes ( IFIG ) ; and their products than are vascular manifestations . neutrophil granule genes ; and This study provides new insights into the molecular neutrophil- related genes, associated with increased mechanisms that account for the heterogeneity of clinical expression by neutrophils in peripheral blood . disease in lupus. By incorporating analysis of longitudinal Genes in the type I interferon inducible gene signature biologic samples from SLE patients and HDs, along with 30 include those in Table 2 . consideration of points in time at which disease is either Genes in the neutrophil granule gene signature include stable or flaring, five distinct patient groups with distinct those in Table 3 . clinical and serologic features have been identified . The high Genes in the neutrophil - related gene signature include level neutrophil -granule signature and high prevalence of those in Table 4 . TABLE 2 Type I Interferon Inducible ( IFIG ) genes Gene Symbol Gene Title ANKRD22 ankyrin repeat domain 22 BST2 bone marrow stromal cell antigen 2 CCR1 chemokine ( C — C motif ) receptor 1 CMPK2 cytidine monophosphate (UMP - CMP ) kinase 2 , mitochondrial CXCL11 chemokine ( C — X — C motif ) ligand 11 DDX58 DEAD ( Asp -Glu - Ala - Asp ) box polypeptide 58 DDX60 DEAD ( Asp -Glu - Ala - Asp ) box polypeptide 60 DDX60L DEAD ( Asp -Glu - Ala - Asp ) box polypeptide 60 - like DTX3L deltex 3 - like (Drosophila ) EIF2AK2 eukaryotic translation initiation factor 2 -alpha kinase 2 EPSTI1 epithelial stromal interaction 1 (breast ) ETV7 ets variant 7 FBX06 F -box protein 6 FLJ42418 FLJ42418 protein GBP1 guanylate binding protein 1, interferon - inducible , 67 kDa GPR84 G protein - coupled receptor 84 H1FO H1 family , member 0 HERC5 hect domain and RLD 5 HERC6 hect domain and RLD 6 IFI27 interferon , alpha - inducible protein 27 IF135 interferon - induced protein 35 IF144 Interferon - induced protein 44 IFIH1 Interferon induced with helicase C domain 1 IFIT1 interferon - induced protein with tetratricopeptide repeats 1 IFIT2 interferon - induced protein with tetratricopeptide repeats 2 IFIT3 interferon - induced protein with tetratricopeptide repeats 3 IFITS interferon - induced protein with tetratricopeptide repeats 5 IFITM1 interferon induced transmembrane protein 1 ( 9 - 27 ) IFITM3 interferon induced transmembrane protein 3 ( 1 - 8U ) ILIRN interleukin 1 receptor antagonist IRF7 interferon regulatory factor 7 ISG15 ISG15 ubiquitin - like modifier JUP junction plakoglobin US 9 ,809 ,854 B2 21 TABLE 2 - continued Type I Interferon Inducible ( IFIG ) genes Gene Symbol Gene Title KLHDC7B kelch domain containing 7B KMO kynurenine 3 -monooxygenase (kynurenine 3 -hydroxylase ) LAMP3 lysosomal- associated membrane protein 3 LAP3 leucine aminopeptidase 3 LGALS3BP / / / lectin , galactoside -binding , soluble , 3 binding protein // / similar to LOC100133842 lectin , galactoside -binding , soluble , 3 binding protein LIPA lipase A , lysosomal acid , cholesterol esterase LOC100128718 Hypothetical protein LOC100128718 LOC147645 hypothetical protein LOC147645 LOC203274 Hypothetical protein LOC203274 LOC26010 viral DNA polymerase - transactivated protein 6 LYCE lymphocyte antigen 6 complex , E MS4A4A membrane - spanning 4 -domains , subfamily A , member 4 MT2A metallothionein 2A MX1 myxovirus ( influenza virus ) resistance 1 , interferon - inducible protein p78 (mouse ) MX2 myxovirus ( influenza virus ) resistance 2 (mouse ) NEXN nexilin ( F actin binding protein ) OAS1 2 ', 5 '- oligoadenylate synthetase 1 , 40 /46 kDa OAS2 2 - 5 '- oligoadenylate synthetase 2 , 69 /71 kDa OAS3 2 - 5 '- oligoadenylate synthetase 3 , 100 kDa OASL 2 - 5 '- oligoadenylate synthetase - like ODF3B outer dense fiber of sperm tails 3B PARP12 poly ( ADP - ribose ) polymerase family , member 12 PARP9 poly (ADP - ribose ) polymerase family , member 9 PLSCR1 phospholipid scramblase 1 RGL1 ral guanine nucleotide dissociation stimulator- like 1 RSAD2 radical S -adenosyl methionine domain containing 2 RTP4 receptor (chemosensory ) transporter protein 4 SAMDA sterile alpha motif domain containing 4A SAMD9 sterile alpha motif domain containing 9 SAMD9L sterile alpha motif domain containing 9 - like SCO2 SCO cytochrome oxidase deficient homolog 2 ( yeast ) SIGLEC1 sialic acid binding Ig- like lectin 1 , sialoadhesin SP100 SP100 nuclear antigen SP110 SP110 nuclear body protein SPTLC2 Serine palmitoyltransferase , long chain base subunit 2 STAT1 signal transducer and activator of transcription 1 , 91 kDa STAT2 signal transducer and activator of transcription 2 , 113 kDa TAPI transporter 1 , ATP -binding cassette , sub - family B (MDR / TAP) TLR7 toll - like receptor 7 TNFSF10 tumor necrosis factor ( ligand ) superfamily , member 10 TNFSF13B tumor necrosis factor ( ligand ) superfamily , member 13b TRIM22 tripartite motif - containing 22 TRIM5 tripartite motif - containing 5 TRIP6 thyroid hormone receptor interactor 6 TYMP thymidine phosphorylase UBE2L6 ubiquitin - conjugating enzyme E2L 6 USP18 ubiquitin specific peptidase 18 XAF1 XIAP associated factor 1 ZBP1 Z -DNA binding protein 1 ZCCHC2 zinc finger , CCHC domain containing 2

TABLE 3 50 TABLE 3 - continued Neutrophil Granule Genes Neutrophil Granule Genes Gene Symbol Gene Title Gene Symbol Gene Title ANXA3 annexin A3 55 DEFA4 defensin , alpha 4 , corticostatin ARG1 arginase , liver ERG v -ets erythroblastosis virus E26 oncogene homolog BPI bactericidal/ permeability -increasing protein (avian ) CAMP cathelicidin antimicrobial peptide HP / / / HPR haptoglobin // / haptoglobin -related protein CD24 CD24 molecule LCN2 lipocalin 2 CEACAM1 carcinoembryonic antigen - related cell adhesion LTF lactotransferrin molecule 1 (biliary glycoprotein ) 60 MMP8 matrix metallopeptidase 8 (neutrophil collagenase ) CEACAM6 carcinoembryonic antigen - related cell adhesion MMP9 matrix metallopeptidase 9 (gelatinase B , 92 kDa molecule 6 (non - specific cross reacting antigen ) gelatinase , 92 kDa type IV collagenase ) CEACAM8 carcinoembryonic antigen -related cell adhesion MPO myeloperoxidase molecule 8 OLFM4 olfactomedin 4 ????L1 chitinase 3 - like 1 ( cartilage glycoprotein -39 ) OLR1 oxidized low density lipoprotein ( lectin - like ) receptor 1 CRISP3 cysteine - rich secretory protein 3 65 RNASE3 ribonuclease , RNase A family , 3 ( eosinophil cationic DEFA1 defensin , alpha 1 protein ) US 9 , 809 , 854 B2 23 24 TABLE 3 - continued In another embodiment, a test for neutrophil- related genes could be done and a positive result could indicate at least Neutrophil Granule Genes that the subject is at an increased risk for flares , including Gene Symbol Gene Title severe flare, and increased mucocutaneous manifestation . 5 This would indicate to a clinician or health care provider SLC2A5 solute carrier family 2 ( facilitated glucose/ fructose transporter) , member 5 increased monitoring was needed for this subject, and per TCN1 transcobalamin I (vitamin B12 binding protein , R haps more aggressive therapy focused on mucocutaneous binder family ) indications. Tests for any combination of the three clusters of genes can also be performed . For example , in a further embodi TABLE 4 ment, the subject would be tested for both IFIG and neu trophil granule signature genes. A positive result of both Neutrophil- Related Genes signatures would indicate an increased risk for flares, includ Gene Symbol Gene Title ing severe flares, as well as increased vascular involvement . Subjects who have both these signatures also have the most ABCA1 ATP - binding cassette , sub - family A ( ABC1) , member 1 ADM adrenomedullin renal involvements and have the most active diseases . SLE AQP9 aquaporin 9 subjects who tested positive for both these signatures would CYP4F3 cytochrome P450 , family 4 , subfamily F , polypeptide 3 greatly benefit from early therapeutic intervention , as well as DDXOOL DEAD ( Asp -Glu - Ala - Asp ) box polypeptide 60 - like 20 increased monitoring . Moreover, the therapeutic interven DHRS9 dehydrogenase /reductase (SDR family ) member 9 DOCK4 Dedicator of cytokinesis 4 tion would be more focused on vascular and renal etiologies. FFAR2 free fatty acid receptor 2 Additionally , a subject who tested positive for the neutrophil FPR1 formyl peptide receptor 1 granule genes and negative for IFIG would also be at an FPR2 formyl peptide receptor 2 HECW2 HECT, C2 and WW domain containing E3 ubiquitin increased risk of flares, including severe flares , as well as protein ligase 2 25 increased vascular involvement , although the disease is not IL1R2 interleukin 1 receptor , type II as active in these patients who test positive for only the KCNJ15 potassium inwardly - rectifying channel, subfamily J, neutrophil - granule -related genes. member 15 LIMK2 LIM domain kinase 2 In yet a further embodiment, a subject would be tested for LRG1 leucine- rich alpha- 2 - glycoprotein 1 IFIG and neutrophil- related signature genes . A positive Ly96 Lymphocyte antigen 96 30 result ofboth of these signatures would indicate an increased MANSC1 MANSC domain containing 1 risk of flares and mucocutaneous involvement, with an PRRG4 Proline rich GLA 4 SLC22A4 solute carrier family 22 ( organic cation / ergothioneine active disease . This would indicate to a clinician that transporter ) , member 4 increased monitoring and therapy focused on mucocutane TNFAIP6 tumor necrosis factor , alpha -induced protein 6 ous manifestations of SLE should be started early in the TNFRSF10C tumor necrosis factor receptor superfamily , member 35 disease . A negative result on the neutrophil- related signature 10c, decoy without an intracellular domain TRPM6 transient receptor potential cation channel, subfamily M , genes but positive on the IFIG may indicate a less active member 6 disease . VNN3 vanin 3 A positive result for a particular gene signature would be considered an increase in gene expression of at least one 40 gene in the signature as compared to the expression of the By using these biomarkers , alone or preferably in con same gene in a healthy donor, preferably an increase in gene junction , important predictions and determinations can be expression of at least two genes in the signature as compared made regarding the course of an SLE patient' s progression . to the expression of the same gene in a healthy donor, While tests for these biomarkers can be performed at any preferably an increase in gene expression of at least five time after a diagnosis of SLE , preferably such tests would be 45 genes in the signature as compared to the expression of the performed as soon as possible after a positive diagnosis of same gene in a healthy donor, preferably an increase in gene SLE is made by a clinician . In that manner, the valuable expression in at least ten genes in the signature as compared insight into the clinical manifestations of the disease can be to the expression of the same gene in a healthy donor , and utilized in both choice of therapy as well as the determina - most preferably an increase in gene expression in at least tion for the amount and timing needed for monitoring by a 50 twenty genes in the signature as compared to the expression health care provider, often time prior to any noticeable of the same gene in a healthy donor. symptoms occur. A negative result for a particular gene signature would be In the one embodiment of the present invention , a test for considered either a decrease in or the same gene expression type I interferon inducible genes ( IFIG ) could be done and of at least one gene in the signature as compared to the a positive result could indicate at least that the subject is at 55 expression of the same gene in a healthy donor, preferably an increased risk for flares . This would indicate to a clinician either a decrease in or the same gene expression of at least or health care provider at least increased monitoring was two genes in the signature as compared to the expression of needed for this subject, and perhaps more aggressive the same gene in a healthy donor, preferably either a therapy . decrease in or the same gene expression of at least five genes In another embodiment, a test for neutrophil granule 60 in the signature as compared to the expression of the same genes could be done and a positive result could indicate at g ene in a healthy donor, preferably either a decrease in or the least that the subject is at an increased risk for flares , same gene expression of at least ten genes in the signature including severe flares, and increased vascular manifesta - as compared to the expression of the same gene in a healthy tion . This would indicate to a clinician or health care donor, and most preferably either a decrease in or the same provider increased monitoring was needed for this subject, 65 gene expression of at least twenty genes in the signature as and perhaps more aggressive therapy focused on vascular compared to the expression of the same gene in a healthy indications. donor. US 9 ,809 , 854 B2 25 26 A preferred embodiment is testing a subject for all three expressed signature neutrophil related genes can be used in gene signatures as soon as possible after a positive diagnosis the methods and assays of the present invention . Preferred of SLE so that predictions can be made regarding the activity neutrophil -related signature genes for use in the methods and severity of the disease as well as the areas that therapies and assays of the invention are TNFAIP6 , FPR1 , FPR2 , should be focused . Any single gene or any combination of 5 LY96 . CYP4F3 . ILIR2. PRRG4. and DOCK4. with genes from each signature can be used in the method and assays of the invention . A summary of the clinical indica TNFAIP6 , CYP4F3 and FPR1 being more preferred and tions of a positive result for each gene signature is found in TNFAIP6 being most preferred Table 1 . A preferred embodiment is found in Example 4 where Any one of the preferred or additional IFIG genes or any 10 seven genes were chosen from the three gene signatures , and combination of more than one up to all 83 differentially 10 with those seven genes, the five groups with varying clinical expressed signature IFIG genes can be used in the methods manifestations and disease activity were classified . These and assays of the present invention . Preferred IFIG signature genes were IFIT1 and IFIT3 which represented the IFIG genes to be use in the method of the invention are IF127 , signature , CEACAM6 and LTF which represented the neu IFIT1 and IFIT3 . 15 trophil granules gene signature and TNFAIP6 , CYP4F3 and Any one neutrophil granule signature gene or any com - FPR1 which represented the neutrophil - related gene signa bination of more than one up to all 24 differentially ture . expressed signature neutrophil granule genes can be used in Additional gene biomarkers have also been identified that the methods and assays of the present invention . Preferred correlate to disease activity, i . e . , flare , and predict and neutrophil granule signature genes for use in the methods 20 identify those SLE subjects who are at risk formore frequent and assays of the invention are OLFM4 , CRISP3 , IF127 , flares while they are not flaring . DEFA4 , MMPS , ANXA3 , ARG1, MPO , DEFA1, LTF , and These biomarkers include : CEACAM6 , with the two most preferred neutrophil granule type I interferon inducible genes ( IFIG ) found in Table 2 ; signature genes being CEACAM6 and LTF . neutrophil granule genes found in Table 3 ; Likewise , any one neutrophil- related signature gene or Immunoglobulin /plasma cell genes found in Table 5 ; and any combination of more than one up to all 23 differentially T -cells / iNKT genes found in Table 6 . TABLE 5 Immunoglobulin /plasma cell genes Gene Symbol Gene Title BHLHE41 basic helix - loop -helix family , member e41 CAV1 caveolin 1 , caveolae protein , 22 kDa CD38 CD38 molecule DTNB dystrobrevin , beta ELL2 elongation factor, RNA polymerase II , 2 FAM20B Family with sequence similarity 20 , member B FAM460 family with sequence similarity 46 , member C GLDC glycine dehydrogenase (decarboxylating ) IGF1 insulin - like growth factor 1 ( somatomedin C ) IGH @ // / IGHA1 // / immunoglobulin heavy locus // / immunoglobulin heavy constant IGHA2 // / IGHD III alpha 1 / // immunoglobulin heavy constant alpha 2 ( A2m IGHG1 / // IGHG3 // / marker ) / // immunoglobulin heavy constant delta / // IGHG4 / // IGHM III immunoglobulin heavy constant gamma 1 (Glm marker ) // / IGHV4 - 31 / / / immunoglobulin heavy constant gamma 3 (G3m marker ) // / LOC100126583 // / immunoglobulin heavy constant gamma 4 (G4m marker ) / // LOC642131 / / / immunoglobulin heavy constant mu // / immunoglobulin heavy LOC652128 / / / variable 4 - 31 / / / hypothetical LOC100126583 / // similar to VSIGO hCG1812074 // / similar to Ig heavy chain V - II region ARH -77 precursor // / V - set and immunoglobulin domain containing 6 IGH @ / / / IGHA1[ A1 / / / immunoglobulin heavy locus / // immunoglobulin heavy constant IGHA2 / // IGHG1 // / alpha 1 / / / immunoglobulin heavy constant alpha 2 ( A2m IGHG2 // / IGHG3 // / marker ) // / immunoglobulin heavy constant gamma 1 (Glm IGHM / / / IGHV4 - 31 marker ) // / immunoglobulin heavy constant gamma 2 (G2m / // LOC100126583 / // marker ) / // immunoglobulin heavy constant gamma 3 (G3m LOC652494 marker ) / // immunoglobulin heavy constant mu / / / immunoglobulin heavy variable 4 -31 // / hypothetical LOC100126583 // / similar to Ig heavy chain V - III region VH26 precursor IGH @ // / IGHA1 // / immunoglobulin heavy locus // / immunoglobulin heavy constant IGHA2 / / / alpha 1 / / / immunoglobulin heavy constant alpha 2 ( A2m LOC100126583 marker) / // hypothetical LOC100126583 IGH @ // / IGHA1 / / immunoglobulin heavy locus // / immunoglobulin heavy constant IGHG1 // / IGHG3 // / alpha 1 // / immunoglobulin heavy constant gamma 1 (Glm IGHM / // IGHV3 -23 marker ) / // immunoglobulin heavy constant gamma 3 (G3m / / / IGHV4- 31 marker ) // / immunoglobulin heavy constant mu / // immunoglobulin heavy variable 3 -23 // / immunoglobulin heavy variable 4 - 31 IGH @ // / IGHG1 III immunoglobulin heavy locus // / immunoglobulin heavy constant IGHG2 / / / IGHM / / / gamma 1 (Glm marker ) / / / immunoglobulin heavy constant IGHV4- 31 gamma 2 (G2m marker ) / // immunoglobulin heavy constant mu // / immunoglobulin heavy variable 4 - 31 IGHA1 / / / IGHD III immunoglobulin heavy constant alpha 1 / // immunoglobulin IGHG1 / // IGHG3 // / heavy constant delta / // immunoglobulin heavy constant gamma US 9 ,809 ,854 B2 27 28 TABLE 5 -continued Immunoglobulin /plasma cell genes Gene Symbol Gene Title IGHM // / IGHV3- 23 1 (Glm marker) / // immunoglobulin heavy constant gamma 3 / / / IGHV4- 31 / / / (G3m marker ) / // immunoglobulin heavy constant mu // / LOC100126583 immunoglobulin heavy variable 3 - 23 / / / immunoglobulin heavy variable 4 - 31 // / hypothetical LOC100126583 IGHAL / / / IGHD / / immunoglobulinimmunoglobulin heavy constant alpha 1 /// // / immunogloimmunoglobulin IGHG1 / / / IGHM III heavy constant delta // / immunoglobulin heavy constant gamma IGHV3- 23 / / / 1 (Glm marker) // / immunoglobulin heavy constantmu / // IGHV4 - 31 immunoglobulin heavy variable 3 - 23 // / immunoglobulin heavy variable 4 - 31 IGHD immunoglobulin heavy constant delta IGHG1 Immunoglobulin heavy constant gamma 1 (Glm marker ) IGHM immunoglobulin heavy constant mu IGJ immunoglobulin J polypeptide , linker protein for immunoglobulin alpha and mu polypeptides IGK @ // / IGKC immunoglobulin kappa locus // / immunoglobulin kappa constant IGK @ // / IGKC / // immunoglobulin kappa locus / // immunoglobulin kappa constant IGKV3- 20 / / / / // immunoglobulin kappa variable 3 - 20 / / / immunoglobulin IGKV3D - 11 // / kappa variable 3D - 11 / // immunoglobulin kappa variable 3D -15 IGKV3D - 15 // / ( gene/ pseudogene ) / // similar to hCG2043206 LOC440871 IGK @ // / IGKC / / / immunoglobulin kappa locus // / immunoglobulin kappa constant LOC647506 / / / // / similar to Ig kappa chain V - I region HK101 precursor / // LOC650405 / / / similar to Ig kappa chain V - I region HK102 precursor / // similar LOC652493 to Ig kappa chain V - I region HK102 precursor IGKC Immunoglobulin kappa constant IGKC immunoglobulin kappa constant IGKC / // IGKV1- 5 // / immunoglobulin kappa constant // / immunoglobulin kappa LOC100130100 / / / variable 1 - 5 // / similar to hCG26659 // / similar to Ig kappa chain LOC647506 / / / V - I region HK101 precursor // / similar to Ig kappa chain V - I LOC650405 / / / region HK102 precursor / // similar to Ig kappa chain V -I region LOC652493 // / HK102 precursor // / similar to Ig kappa chain V - I region HK102 LOC652694 precursor IGKC // / IGKV1- 5 // / immunoglobulin kappa constant // / immunoglobulin kappa LOC647506 / / / variable 1 - 5 / // similar to Ig kappa chain V - I region HK101 LOC652694 precursor // / similar to Ig kappa chain V - I region HK102 precursor IGKV1OR15 - 118 immunoglobulin kappa variable 1 /OR15 -118 pseudogene IGKV4 - 1 immunoglobulin kappa variable 4 - 1 IGL @ Immunoglobulin lambda locus IGL @ // / IGLC1 // / immunoglobulin lambda locus // / immunoglobulin lambda IGLV2 - 11 / // IGLV2- constant 1 (Mcg marker ) // / immunoglobulin lambda variable 2 18 // / IGLV2 - 23 11 / // immunoglobulin lambda variable 2 - 18 / // immunoglobulin lambda variable 2 - 23 IGL @ // / IGLV1 - 36 immunoglobulin lambda locus // / immunoglobulin lambda / / / IGLV1 -44 variable 1 - 36 / / / immunoglobulin lambda variable 1 -44 IGL @ // / LOC96610 immunoglobulin lambda locus // / BMSi homolog , ribosome assembly protein ( yeast) pseudogene IGLJ3 immunoglobulin lambda joining 3 IGLL3 immunoglobulin lambda -like polypeptide 3 IGLV2 - 11 // / IGLV2 immunoglobulin lambda variable 2 - 11 / // immunoglobulin 18 / / / IGLV2 - 23 lambda variable 2 - 18 // / immunoglobulin lambda variable 2 - 23 IGLV3 - 19 immunoglobulin lambda variable 3 - 19 LOC100130100 similar to hCG26659 LOC652493 similar to Ig kappa chain V - I region HK102 precursor LOC91316 glucuronidase , beta / immunoglobulin lambda - like polypeptide 1 pseudogene MGC29506 hypothetical protein MGC29506 RGS13 regulator of G -protein signaling 13 SSPN sarcospan (Kras oncogene - associated gene ) TNFRSF17 tumor necrosis factor receptor superfamily , member 17 TXNDC5 thioredoxin domain containing 5 (endoplasmic reticulum )

TABLE 6 TABLE 6 -continued T- cells /iNKT genes T -cells /iNKT genes 60 Gene Symbol Gene Title Gene Symbol Gene Title BEX5 brain expressed , X - linked 5 GATA2 GATA binding protein 2 CCR3 chemokine ( C — C motif ) receptor 3 IL4 interleukin 4 CDIC CDic molecule ILSRA interleukin 5 receptor, alpha CLIC5 chloride intracellular channel 5 65 ISM1 isthmin 1 homolog (zebrafish ) EPHA4 EPH receptor A4 KLRB1 killer cell lectin - like receptor subfamily B , member 1 US 9 , 809 , 854 B2 29 30 TABLE 6 - continued If required , a nucleic acid sample having the signature gene sequence ( s ) are prepared using known techniques. For T- cells / iNKT genes example , the sample can be treated to lyse the cells , using Gene Symbol Gene Title known lysis buffers , sonication , electroporation , with puri 5 fication and amplification occurring as needed , as will be MAF V -maf musculoaponeurotic fibrosarcoma oncogene homolog (avian ) understood by those in the skilled in the art . In addition , the NAP1L2 nucleosome assembly protein 1 - like 2 reactions can be accomplished in a variety of ways . Com NAP1L3 nucleosome assembly protein 1 - like 3 ponents of the reaction may be added simultaneously, or RORC RAR - related orphan receptor C STOGALNAC1 ST6 (alpha - N - acetyl- neuraminyl - 2 , 3 -beta - galactosyl- 1 , 3 ) - 10 sequentially , in any order. In addition , the reaction can N - acetylgalactosaminide alpha - 2 , 6 - sialyltransferase 1 include a variety of other reagents which can be useful in the STSSIA1 ST8 alpha - N - acetyl- neuraminide alpha - 2 , 8 methods and assays and would include but is not limited to sialyltransferase 1 TMEM176B transmembrane protein 176B salts , buffers , neutral proteins, such albumin , and detergents , TNFRSF10D tumor necrosis factor receptor superfamily , member 10d , which may be used to facilitate optimal hybridization and decoy with truncated death domain detection , and /or reduce non - specific or background inter TRPC1 transient receptor potential cation channel , subfamily C , actions . Also reagents that otherwise improve the efficiency member 1 USP53 ubiquitin specific peptidase 53 of the assay, such as protease inhibitors , nuclease inhibitors , and anti -microbial agents , can be used , depending on the sample preparation methods and purity . By using these biomarkers , in conjunction , important 20 Once prepared ,mRNA or other nucleic acids are analyzed predictions and determinations can be made regarding the by methods known to those of skill in the art . The nucleic course of an SLE patient' s progression . While tests for these acid sequence corresponding to a signature gene can be any biomarkers can be performed at any time after a diagnosisnosis ofo length , with the understanding that longer sequences are SLE , preferably such tests would be performed as soon as more specific . Preferably a nucleic acid corresponding to a possible after a positive diagnosis of SLE is made by a 25 signature gene is at least 20 nucleotides in length . Preferred clinician . In that manner , the valuable insight into the ranges are from 20 to 100 nucleotides in length , with from clinical manifestations of the disease can be utilized in both 30 toto 6060 nucleonucleotides being more preferred , and from 40 to choice of therapy as well as the determination for the amount 50 being most preferred . and timing needed for monitoring by a health care provider, In addition , when nucleic acids are to be detected pre ur. ferred methods utilize cutting or shearing techniques to cut In the one embodiment of the present invention , a test for the nucleic acid sample containing the target sequence into one or more genes from these four signatures would be done a size that will facilitate handling and hybridization to the and a positive result would indicate that a subject is at an target. This can be accomplished by shearing the nucleic increased risk for flares. This would indicate to a clinician acid through mechanical forces, such as sonication , or by increased monitoring of the patient as well as more aggres- 3 cleaving the nucleic acid using restriction endonucleases, or sive earlier therapy . A positive result is an increase in the any other methods known in the art . However , in most cases, IFIG gene , an increase in the neutrophil granule gene , an the natural degradation that occurs during archiving results increase in the immunoglobulinbulin // plasma cellcell gene but aa in " short ” oligonucleotides. In general, the methods and decrease in the T - cells / iNKT genes . Preferred genes toto be 40 assays of the invention can be done on oligonucleotides as used in the panel are : IFIT3, MMP8 , CD38 , and KLRB1. * short as 20 - 100 base pairs, with from 20 to 50 being All of these genes in all five signatures found in Tables 2 , preferred , and between 40 and 50 , including 44 , 45 , 46 , 47 , 3 , 4 , 5 , and 6 , can also be used as targets for developing 48 and 49 being the most preferred . therapies and research tools . A preferred method of the invention is performing gene Assays and Methods to Detect and Measure Signature Genes 45 expression profiling of the sample . Gene expression profil In order to detect any of these transcripts or genes , a ing refers to examining expression of one or more RNAs in sample of biological tissue or fluid from a subject who has a cell, preferably mRNA . Often at least or up to 10 , 100 , 100 , been positively diagnosed with SLE is obtained and pre - 10 , 000 or more different mRNAs are examined in a single pared and analyzed for the presence of the IFIG , neutrophil experiment. granule , neutrophil- related , immunoglobulin /plasma cell, 50 In a preferred method and assay of the invention , the gene and / or T - cells /iKNT signature genes , This can be achieved expression of themRNA or other nucleic acid obtained from in numerous ways, by a diagnostic laboratory , and /or a the subject with SLE is compared to the gene expression of health care provider. a healthy donor. Typically expression is compared to expres Most methods start with obtaining a sample of biological sion of a consistently expressed housekeeping gene tran tissue or fluid from the subject with SLE and extracting , 55 script, the relative expression determined , and then the isolating and / or purifying the nucleic acid ( e . g . , genomic expression of the subject is compared to the expression of DNA , cDNA , RNA ) from the tissue or fluid . the healthy control. The reference fold changes of each gene The nucleic acid can be obtained from any biological listed in Tables 2 -6 as compared to healthy controls is listed tissue . Preferred biological tissues include, but are not in Table 9 . limited to , epidermal, whole blood , and plasma . 60 In a preferred method to determine which group a subject The nucleic acid can be obtained from any biological with SLE would be classified as, the expression of 1 to 83 fluid . Preferred fluids include , but are not limited to , plasma, of IFIG signature genes is determined . In another preferred saliva , and urine . method , 1 to 24 of the neutrophil granule genes is deter In a preferred method , the nucleic acid is obtained from mined , and in another preferred method , 1 to 23 of the peripheral blood mononuclear cells . 65 neutrophil -related genes is determined . In a preferred The nucleic acid is extracted , isolated and purified from embodiment, the expression of at least gene from each the cells of the tissue or fluid by methods known in the art. signature is determined , and in a most preferred embodi US 9 , 809 , 854 B2 31 32 mentm , the expression of the genes , IFIT1, IFIT3 , target. It will be appreciated that when cDNA complemen CEACAM6 , LTF , TNFAIP6 , CYP4F3 and FPR1 are deter tary to the RNA of a cell is made and hybridized to a mined . microarray under suitable conditions, the level or degree of In a method to determine future flares in a subject with hybridization to the site in the array corresponding to any SLE , the expression of 1 to 83 of IFIG signature genes is 5 particular gene will reflect the prevalence in the cell of determined , 1 to 24 of the neutrophil granule genes is mRNA transcribed from that gene. For example, when determined , 1 to 45 of the immunoglobulin / plasma cell detectably labeled ( e . g ., with a fluorophore ) cDNA comple genes is determined , and 1 to 20 of the T - cell/ iKNT genes is mentary to the total cellular mRNA is hybridized to a determined . In a preferred embodiment, the expression of microarray , the site on the array corresponding to a gene IFIT3 , KLRB1, CD38 , and MMP8 are determined . 10 ( i .e . , capable of specifically binding a nucleic acid product Methods for examining gene expression , are often hybrid of the gene ) that is not transcribed in the cell will have little ization based , and include, Southern blots; Northern blots ; or no signal, while a gene for which mRNA is highly dot blots ; primer extension ; nuclease protection ; subtractive prevalent will have a relatively strong signal. hybridization and isolation of non -duplexed molecules By way of example , GeneChip® (Affymetrix , Santa using , for example , hydroxyapatite ; solution hybridization ; 15 Clara , Calif . ) , generates data for the assessment of gene filter hybridization ; amplification techniques such as RT - expression profiles and other biological assays . Oligonucle PCR and other PCR - related techniques such as PCR with otide expression arrays simultaneously and quantitatively melting curve analysis , and PCR with mass spectrometry ; “ interrogate ” thousands of mRNA transcripts . Each tran fingerprinting , such as with restriction endonucleases ; and script can be represented on a probe array by multiple probe the use of structure specific endonucleases. mRNA expres - 20 pairs to differentiate among closely related members of gene sion can also be analyzed using mass spectrometry tech - families. Each probe contains millions of copies of a specific niques ( e . g . , MALDI or SELDI) , liquid chromatography, oligonucleotide probe , permitting the accurate and sensitive and capillary gel electrophoresis . Any additional method detection of even low - intensity mRNA hybridization pat known in the art can be used to detect the presence or terns. After hybridization data is captured , using a scanner or absence of the transcripts . 25 optical detection systems, software can be used to automati Alternatively , the level of protein product of the genes can cally calculate the intensity values for each probe cell. Probe be measured from a protein sample from the biological cell intensities can be used to calculate an average intensity tissue or fluid using methods described below . for each gene , which correlates with mRNA abundance For a general description of these techniques, see also levels . Expression data can be quickly sorted based on any Sambrook et al . 1989; Kriegler 1990 ; and Ausebel et al. 30 analysis parameter and displayed in a variety of graphical 1990 . formats for any selected subset of genes . The preferred method for the detection of the transcripts Further examples of microarrays that can be used in the is the use of arrays or microarrays . These terms are used assays and methods of the invention are microarrays syn interchangeably and refer to any ordered arrangement on a thesized in accordance with techniques sometimes referred surface or substrate of different molecules, referred to herein 35 to as VLSIPSTM (Very Large Scale Immobilized Polymer as " probes. " Each different probe of any array is capable of Synthesis ) technologies as described , for example , in U . S . specifically recognizing and / or binding to a particular mol- Pat . Nos . 5 , 324 ,633 ; 5 , 744 ,305 ; 5 , 451, 683 ; 5 , 482 , 867 ; ecule , which is referred to herein as its “ target” in the context 5 ,491 , 074 ; 5 ,624 , 711; 5 , 795 ,716 ; 5 ,831 ,070 ; 5 , 856 , 101; of arrays. Examples of typical target molecules that can be 5 ,858 ,659 ; 5 ,874 ,219 ; 5 , 968 ,740 ; 5 , 974 , 164 ; 5 , 981, 185 ; detected using microarrays include mRNA transcripts , 40 5 , 981, 956 ; 6 , 025 ,601 ; 6 ,033 ,860 ; 6 ,090 ,555 ; 6 , 136 ,269 ; CRNA molecules, cDNA , PCR products , and proteins. 6 , 022 , 963 ; 6 , 083 ,697 ; 6 ,291 ,183 ; 6 ,309 ,831 ; 6 ,416 , 949 ; Microarrays are useful for simultaneously detecting the 6 ,428 ,752 and 6 , 482 , 591 . presence , absence and quantity of a plurality of different Other exemplary arrays that are useful for use in the target molecules in a sample. The presence and quantity , or invention include, but are not limited to , Sentrix® Array or absence , of the probe ' s target molecule in a sample may be 45 Sentrix® BeadChip Array available from Illumina® , Inc . readily determined by analyzing whether and how much of (San Diego , Calif . ) or others including beads in wells such a target has bound to a probe at a particular location on the as those described in U . S . Pat. Nos . 6 ,266 ,459 ; 6 , 355 ,431 ; surface or substrate . 6 ,770 ,441 ; and 6 ,859 , 570 . Arrays that have particle on the In a preferred embodiment , arrays used in the present surface can also be used and include those described in U . S . invention are “ addressable arrays " where each different 50 Pat . Nos . 6 ,489 ,606 ; 7 , 106 ,513 ; 7 , 126 , 755 ; and 7 , 164 ,533 . probe is associated with a particular “ address .” An array of beads in a fluid format , such as a fluid stream The arrays used in the present invention are preferable of a flow cytometer or similar device , can also be used in nucleic acid arrays that comprise a plurality of nucleic acid methods for the invention . Exemplary formats that can be probes immobilized on a surface or substrate . The different used in the invention to distinguish beads in a fluid sample nucleic acid probes are complementary to , and therefore can 55 using microfluidic devices are described , for example, in hybridize to , different target nucleic acid molecules in a U . S . Pat. No . 6 ,524 , 793 . Commercially available fluid for sample . Thus, each probe can be used to simultaneously mats for distinguishing beads include , for example , those detect the presence and quantity of a plurality of different used in XMAPTM technologies from Luminex or MPSSTM genes , e . g . , the presence and abundance of different mRNA methods from Lynx Therapeutics . molecules , or of nucleic acid molecules derived therefrom 60 A spotted microarray can also be used in a method of the ( for example , cDNA or CRNA ). invention . An exemplary spotted microarray is a The arrays are preferably reproducible, allowing multiple CodeLinkTM Array available from Amersham Biosciences . copies of a given array to be produced and the results from Another microarray that is useful in the invention is one each easily compared to one another. Preferably microarrays that is manufactured using inkjet printing methods such as are small , and made from materials that are stable under 65 SurePrintTM Technology available from Agilent Technolo binding conditions . A given binding site or unique set of gies . Other microarrays that can be used in the invention binding sites in the microarray will specifically bind to the include, without limitation , those described in U . S . Pat. Nos . US 9 , 809 , 854 B2 33 34 5 ,429 , 807 ; 5 ,436 , 327 ; 5 , 561 ,071 ; 5 , 583 , 211 ; 5 , 658, 734 ; juxtapositioned probes ( one featuring a fluorophore and the 5 , 837 , 858 ; 5 , 919 , 523 ; 6 , 287, 768 ; 6 , 287 ,776 ; 6 , 288 , 220 ; other , a suitable quencher ) can be used to determine the 6 ,297 ,006 ; 6 , 291, 193 ; and 6 ,514 ,751 . complementarity of the probe to the target sequence . This DASL can be used for quantitative measurements of RNA technique is sensitive enough to detect single -nucleotide target sequences as well as for DNA target sequences . DASL 5 polymorphisms ( SNP ) and can distinguish between various is described , for example , in Fan et al. 2004 . alleles by virtue of the dissociation patterns produced . Additional techniques for rapid gene sequencing and PCR with mass spectrometry uses mass spectrometry to analysis of gene expression include , SAGE (serial analysis detect the end product. Primer pairs are used and tagged with of gene expression ) . For SAGE, a short sequence tag ( typi- molecules of known masses , known as MassCodes . If DNA cally about 10 - 14 bp ) contains sufficient information to 10 from any of the agent of primer panel is present, it will be uniquely identify a transcript. These sequence tags can be amplified . Each amplified product will carry its specific linked together to form long serial molecules that can be Masscodes . The PCR product is then purified to remove cloned and sequenced . Quantitation of the number of times unbound primers , dNTPs , enzyme and other impurities . a particular tag is observed proves the expression level of the Finally , the purified PCR products are subject of ultraviolet corresponding transcript ( see , e . g ., Velculescu et al. 1995 ; 15 as the chemical bond with nucleic acid and primers are Velculescu et al. 1997 ; and de Waard et al . 1999 ). photolabile . As the Masscodes are liberated from PCR Screening and diagnostic method of the current invention products they are detected with a mass spectrometer . may involve the amplification of the target loci . A preferred When a probe is to be used to detect the presence of IFIG , method for target amplification of nucleic acid sequences is neutrophil granule, neutrophil -related , immunoglobulin / using polymerases, in particular polymerase chain reaction 20 plasma cell and / or T -cell / iNKT nucleic acids, the biological ( PCR ) . PCR or other polymerase - driven amplification meth - sample that is to be analyzed must be treated to extract the ods obtain millions of copies of the relevant nucleic acid nucleic acids. The nucleic acids to be targeted usually need sequences which then can be used as substrates for probes or to be at least partially single- stranded in order to form a sequenced or used in other assays . hybrid with the probe sequence. It the nucleic acid is single Amplification using polymerase chain reaction is particu - 25 stranded , no denaturation is required . However , if the larly useful in the embodiments of the current invention . nucleic acid to be probed is double stranded , denaturation PCR is a rapid and versatile in vitro method for amplifying must be performed by any method known in the art . defined target DNA sequences present within a source of The nucleic acid to be analyzed and the probe are incu DNA . Usually , the method is designed to permit selective bated under conditions which promote stable hybrid forma amplification of a specific target DNA sequence ( s ) within a 30 tion of the target sequence in the probe and the target heterogeneous collection of DNA sequences ( e . g . total sequence in the nucleic acid . The desired stringency of the genomic DNA or a complex cDNA population ). To permit hybridization will depend on factors such as the uniqueness such selective amplification , some prior DNA sequence of the probe in the part of the genome being targeted , and information from the target sequences is required . This can be altered by washing procedure , temperature , probe information is used to design two oligonucleotide primers 35 length and other conditions known in the art , as set forth in (amplimers ) which are specific for the target sequence and Sambrook et al. 1989 . which are often about 15- 25 nucleotides long . Labeled probes are , used to detect the hybrid , or alterna Mutation detection using the 5 ' 3 ' exonuclease activity tively , the probe is bound to a ligand which labeled either of Taq DNA polymerase ( TaqManTM assay ) can also be used directly or indirectly . Suitable labels and methods for label as a screening and diagnostic method of the current inven - 40 ing are known in the art , and include biotin , fluorescence , tion . Such an assay involves hybridization of three primers , chemiluminescence , enzymes , and radioactivity . the third primer being intended to bind just downstream of Assays using such probes include Southern blot analysis . one of the conventional primers which should be allele - In such an assay , a patient sample is obtained , the DNA specific . The additional primer carries a blocking group at processed , denatured , separated on an agarose gel , and the 3 ' terminal nucleotide so that it cannot prime new DNA 45 transferred to a membrane for hybridization with a probe . synthesis and at its 5 ' end carries a labeled group . In modern Following procedures known in the art ( e . g . , Sambrook et al . versions of the assay , the label is a fluorogenic group and the 1989 ), the blots are hybridized with a labeled probe and a third primer also carries a quencher group . If the upstream positive band indicates the presence of the target sequence . primer which is bound to the same strand is able to prime S outhern blot hybridization can also be used to screen for the successfully , Taq DNA polymerase will extend a new DNA 50 polymorphisms. In this method , the target DNA is digested strand until it encounters the third primer in which case its with one or more restriction endonucleases , size - fraction 5 ' > 3' exonuclease will degrade the primer causing release ated by agarose gel electrophoresis , denatured and trans of separate nucleotides containing the dye and the quencher, ferred to a nitrocellulose or nylon membrane for hybridiza and an observable increase in fluorescence . tion . Following electrophoresis , the test DNA fragments are PCR with melting curve analysis can also be used . PCR 55 denatured in strong alkali. As agarose gels are fragile , and with melting curve analysis is an extension of PCR where the DNA in them can diffuse within the gel, it is usual to the fluorescence is monitored over time as the temperature transfer the denatured DNA fragments by blotting on to a changes . Duplexes melt as the temperature increases and the durable nitrocellulose or nylon membrane , to which single hybridization of both PCR products and probes can be stranded DNA binds readily . The individual DNA fragments monitored . The temperature - dependent dissociation 60 become immobilized on the membrane at positions which between two DNA - strands can be measured using a DNA - are a faithful record of the size separation achieved by intercalating fluorophore such as SYBR green , EvaGreen or agarose gel electrophoresis . Subsequently , the immobilized fluorophore - labelled DNA probes. In the case of SYBR single - stranded target DNA sequences are allowed to asso green (which fluoresces 1000 - fold more intensely while ciate with labeled single - stranded probe DNA . The probe intercalated in theminor groove of two strands of DNA ) , the 65 will bind only to related DNA sequences in the target DNA , dissociation of the DNA during heating is measurable by the and their position on the membrane can be related back to large reduction in fluorescence that results . Alternatively , the original gel in order to estimate their size . US 9 , 809 , 854 B2 35 36 Northern blots , done in the same fashion , but utilizing target - specific sequence ; amplifying the probes forming the RNA , can also be used . hybridization complexes to produce amplicons ; and detect Dot -blot hybridization can also be used to screen for the ing the amplicons , wherein the detection of the amplicons IFIG , neutrophil granule , neutrophil- related , immunoglobu - indicates the presence of the nucleic acid sequences corre lin / plasma cell and/ or T - cell/ iNKT nucleic acids . Nucleic 5 sponding to the signature gene in the tissue sample ; and acid including genomic DNA , cDNA and RNA is obtained determining the expression level of the signature gene . from the subject with SLE , denatured and spotted onto a In the context of the present invention ,multiplexing refers nitrocellulose or nylon membrane and lowed to dry . The to the detection , analysis or amplification of a plurality of membrane is exposed to a solution of labeled single stranded nucleic acid sequences corresponding to the signature genes . probe sequences and after allowing sufficient time for probe - 10 In one embodimentmultiplex refers to the number of nucleic target heteroduplexes to form , the probe solution is removed acid sequences corresponding to a signature gene to be and the membrane washed , dried and exposed to an auto - analyzed in a single reaction , vessel or step . The multiplex radiographic film . A positive spot is an indication of the ing method is useful for detection of a single nucleic acid target sequence in the DNA of the subject and a no spot an sequence corresponding to a signature gene as well as a indication of the lack of the target sequence in the DNA of 15 plurality of nucleic acid sequences corresponding to a set of the subject. signature genes . Probes and Primers The expression level of nucleic acid sequences corre The expression patterns for signature genes are deter - sponding to a set of signature genes in a tissue sample can mined based on quantitative detection of nucleic acids or be determined by contacting nucleic acid molecules derived oligonucleotides corresponding to the signature genes , 20 from the tissue sample with a set of probes under conditions which means at least two nucleotides covalently linked where complementary probes form a hybridization complex together . Thus, the invention also provides a collection of with the signature gene - specific nucleic acid sequences, nucleic acids and oligonucleotides that correspond to a each of the probes including at least two universal priming signature gene or a set of signature genes, i. e ., IFIG signa - sites and a signature gene - specific nucleic acid sequence ; ture , neutrophil granule signature , neutrophil- related signa - 25 amplifying the probes forming the hybridization complexes ture , immunoglobulin /plasma cell signature and / or T - cell to produce amplicons ; detecting the amplicons , wherein the iNKT signature . A nucleic acid useful in the methods and detection of the amplicons indicates the presence of the assays of the invention is defined above . nucleic acid sequences corresponding to the set of signature The nucleic acids corresponding to signature genes can be genes in the tissue sample ; and determining the expression single stranded or double stranded , as specified , or contain 30 level of the target sequences , wherein the expression of at portions of both double stranded or single stranded least two , at least three , at least five signature gene - specific sequence. The nucleic acid can be DNA , both genomic and sequences is detected . cDNA , RNA or a hybrid , where the nucleic acid contains The presence of one, two or a plurality of nucleic acid any combination of deoxyribo - and ribo - nucleotides, and sequences corresponding to a set of signature genes can be any combination of bases, including , for example , uracil, 35 determined in a biological sample using single , double or adenine, thymine , cytosine , guanine , inosine , xanthine multiple probe configurations. In addition , mRNA signature hypoxanthine , isocytosine, isoguanine . A nucleic acid samples can initially be subjected to a " complexity reduc sequence corresponding to a signature gene can be a portion tion ” step , whereby the presence of a particular target is of the gene , a regulatory sequence, genomic DNA , cDNA confirmed by adding probes that are enzymatically modified RNA including mRNA and rRNA , or others . 40 in the presence of the signature gene -specific nucleic acid Anucleic acid sequence corresponding to a signature gene sequence . The modified probes are then amplified and can be derived from a biological sample , or from a second detected in a wide variety of ways . Preferred embodiments ary source such as a product of a reaction such as , for draw on multiplexing methods, which allow for the simul example , a detection sequence from an invasive cleavage taneous detection of a number of nucleic acid sequences , for reaction , a ligated probe from an OLA or DASL reaction , an 45 example , corresponding to a set of signature genes , as well extended probe from a PCR reaction , or PCR amplification as multiplexing amplification reactions, for example by product, (" amplicon ” ) . using universal priming sequences to do multiplex PCR A complementary nucleic acid sequence useful in the reactions. If desired , the initial step also can be both a methods of the invention can take many forms and probes complexity reduction and an amplification step . are made to hybridize to nucleic acid sequences to determine 50 Probes contemplated for use in the assays and methods of the presence or absence of the signature gene in a sample . In the present invention can be made by any method known in a preferred embodiment, a plurality of nucleic acid the art , including the procedures outlined below . sequences is detected . As used herein , " plurality ” or gram - In standard nucleic acid hybridization assays , probe must matical equivalents herein refers to at least 2 , 10 , 20 , 25 , 50 , be is labeled in some way, and must be single stranded . 100 or 200 different nucleic sequences , while at least 500 55 Oligonucleotide probes are short (typically 15 - 50 nucleo different nucleic sequences is preferred . More preferred is at tides ) single - stranded pieces of DNA made by chemical least 1000 , with more than 5000 or 10 , 000 particularly synthesis: mononucleotides are added , one at a time, to a preferred and more than 50 , 000 or 100 , 000 most preferred . starting mononucleotide, conventionally the 3' end nucleo Detection can be performed on a variety of platforms such tide, which is bound to a solid support . Generally, oligo as those set forth above . 60 nucleotide probes are designed with a specific sequence The expression level of a signature gene in a tissue sample chosen in response to prior information about the target can be determined by contacting nucleic acid molecules DNA . Oligonucleotide probes are often labeled by incorpo derived from the tissue sample with a set of probes under rating a 32P atom or other labeled group at the 5 ' end . conditions where perfectly complementary probes form a Conventional DNA probes are isolated by cell -based hybridization complex with the nucleic acid sequences cor - 65 DNA cloning or by PCR . In the former case, the starting responding to the signature genes, each of the probes includ - DNA may range in size from 0 . 1 kb to hundreds of kilobases ing at least two universal priming sites and a signature gene in length and is usually (but not always ) originally double US 9 , 809 , 854 B2 37 38 stranded . PCR - derived DNA probes have often been less the target site . In the presence of a suitably heat- stable DNA than 10 kb long and are usually , but not always, originally polymerase and DNA precursors ( the four deoxynucleoside double -stranded . triphosphates, dATP , DCTP , dGTP and DTTP ) , they initiate DNA probes are usually labeled by incorporating labeled the synthesis of new DNA strands which are complementary dNTPs during an in vitro DNA synthesis reaction by many 5 to the individual DNA strands of the target DNA segment, differentmethods including nick - translation , random primed and which will overlap each other labeling , PCR labeling or end - labeling . Proteins Correlated to Clinical Manifestations of SLE Labels can be radioisotopes such as 32P , 33P , 35S and PH , As stated above , and shown in Examples 3 , certain genes which can be detected specifically in solution or, more were associated with increased levels of certain anti - inflam commonly , within a solid specimen , such as autoradiogra - 10 matory proteins and with an increase in anti - SSA /Ro autoan phy . 32P has been used widely in Southern blot hybridiza - tibodies . Specifically , those subjects with SLE and the tion , and dot- blot hybridization . neutrophil -related gene signature also had an increase in Nonisotopic labeling systems which use nonradioactive TNF - a , IL - 8 , and IL - 18 , while those who had the neutrophil probes can also be used in the current invention . Two types granule signature had an increase in anti - SSA /Ro autoanti of non - radioactive labeling include direct nonisotopic label- 15 bodies . Thus , in order to confirm the clinicalmanifestations ing , such as one involving the incorporation of modified and disease activity associated with each of the gene signa nucleotides containing a fluorophore . The other type is tures, additional tests for these proteins can be done on the indirect nonisotopic labeling , usually featuring the chemical subject with SLE , in conjunction with the tests detecting the coupling of a modified reporter molecule to a nucleotide signature genes . precursor. After incorporation into DNA , the reporter groups 20 positive result of these biomarkers along with the gene can be specifically bound by an affinity molecule , a protein biomarkers can confirm the activity and severity of the or other ligand which has a very high affinity for the reporter disease as well as the clinicalmanifestations . More specifi group . Conjugated to the latter is a marker molecule or cally , a positive result of TNF -a , IL - 8 , and IL - 18 with a group which can be detected in a suitable assay. This type of positive result for IFIG and neutrophil -related gene would labeling would include biotin - streptavidin and digoxigenin . 25 indicate an increase in flares as well as mucocutaneous The invention also includes a collection of isolated probes involvement. A positive result of TNF - a . IL - 8 , and IL - 18 specific for the IFIG signature genes including any subset of along with an increase in IFIG only would indicate muco the 83 genes in Table 2 . cutaneous involvement. The invention also includes a collection of isolated probes Patients can also be tested for anti - SSA /Ro autoantibod specific for the neutrophil granule signature genes including 30 ies . A positive result for the antibodies along with a positive any subset of the 24 genes in Table 3 . result for IFIG and neutrophil granule signature genes , The invention also includes a collection of isolated probes would confirm a prediction of the most serious active specific for the neutrophil- related signature genes including diasdisease state of SLE with vascular involvement . A positive any subset of the 23 genes in Table 4 . result for the antibodies with a positive result of the neu The invention also includes a collection of isolated probes 35 trophil granule signature genes only would confirm an specific for the genes, IFIT1 , IFIT3 , CEACAM6 , LTF , increase in flares and activity of SLE along with vascular TNFAIP6 , CYP4F3 and FPR1. involvement . The invention also includes a collection of isolated probes As shown in Example 6 , IL - 10 and more significantly , specific for the immunoglobulin / plasma cell genes including vWF are increased during high disease activity , i .e . , flares. any subset of the 45 genes in Table 5 . 40 These protein biomarkers can be used in conjunction with The invention also includes a collection of isolated probes the four gene signature biomarkers , and more preferably , specific for the T - cell / iNKT signature genes including any with the IFIG , neutrophil granule , and plasma cell gene subset of the 20 genes in Table 6 . signatures , to monitor a patient ' s response to treatment, The invention also includes a collection of isolated probes either in regular care or in clinical trials . specific for the flare prediction signature genes comprising 45 These biomarkers , in addition to being useful for clini probes specific for IFIT3, KLRB1, CD38 , and MMP8 cians to predict disease activity , are also useful as targets for Primers for use in the various assays of the present developing therapies and research tools. invention are also an embodiment of the present invention . Assays and Methods to Detect Proteins The specificity of amplification depends on the extent to A sample of biological tissue or bodily fluid from a which the primers can recognize and bind to sequences other 50 subject with SLE , is obtained . than the intended target DNA sequences . For complex DNA The protein sample can be obtained from any biological sources , such as total genomic DNA from a mammalian cell, tissue . Preferred biological tissues include , but are not it is often sufficient to design two primers about 20 nucleo - limited to , epidermal, whole blood , and plasma. tides long . This is because the chance of an accidental The protein sample can be obtained from any biological perfectmatch elsewhere in the genome for either one of the 55 fluid . Preferred fluids include , but are not limited to , plasma, primers is extremely low , and for both sequences to occur by saliva , and urine. chance in close proximity in the specified direction is Protein is isolated and /or purified from the sample using normally exceedingly low . Although conditions are usually any method known in the art , including but not limited to chosen to ensure that only strongly matched primer- target immunoaffinity chromatography . duplexes are stable , spurious amplification products can 60 Any method known in the art can be used , but preferred nevertheless be observed . This can happen if one or both methods for detecting and measuring increase levels of the chosen primer sequences contain part of a repetitive DNA proteins in a protein sample include quantitative Western sequence, and primers are usually designed to avoid match - blot, immunoblot, quantitative mass spectrometry, enzyme ing to known repetitive DNA sequences , including large linked immunosorbent assays (ELISAs ), radioimmunoas runs of a single nucleotide 65 says (RIA ), immunoradiometric assays ( IRMA ) , and immu After the primers are added to denatured template DNA , noenzymatic assays ( IEMA ) and sandwich assays using they bind specifically to complementary DNA sequences at monoclonal and polyclonal antibodies . US 9 , 809 , 854 B2 39 40 Antibodies are a preferred method of detecting and mea organ involvement and disease severity . Due to toxicity , suring the inflammatory proteins in a sample . Such antibod cyclophosphamide is reserved for severe organ - threatening ies are available commercially or can be made by conven disease . At the other end of the spectrum , methotrexate or tional methods known in the art. Such antibodies can be azathioprine may be helpful for milder arthritis or skin monoclonal or polyclonal and fragments thereof, and immu - 5 disease . DMARDS can be used in patients whose condition nologic binding equivalents thereof. The term “ antibody has had an inadequate response to glucocorticoids . means both a homologous molecular entity as well as a Cyclophosphamide is used for immunosuppression in mixture, such as a serum product made up of several homologous molecular entities . cases of serious SLE organ involvement, especially severe In a preferred embodiment, such antibodies will immu - 10 CNS involvement, vasculitis , and lupus nephritis . noprecipitate the inflammatory proteins from a solution as Methotrexate is used for managing arthritis , serositis , well as react with inflammatory proteins on a Western blot , cutaneous , and constitutional symptoms. It blocks purine immunoblot, ELISA , and other assays listed above . synthesis and 5 - aminoimidazole - 4 - carboxamide ribonucle Antibodies for use in these assays can be labeled cova otide ( AICAR ) , thus increasing anti- inflammatory adenos lently or non - covalently with an agent that provides a 1515 Theine concentration at sites of inflammation . Methotrexate detectable signal . Any label and conjugation method known ameliorates symptoms of inflammation and is particularly in the art can be used . Labels, include but are not limited to , useful in arthritis treatment. enzymes , fluorescent agents , radiolabels , substrates, inhibi Azathioprine is an immunosuppressant and a less toxic tors , cofactors , magnetic particles , and chemiluminescent alternative to cyclophosphamide . It is used as a steroid agents . 20 sparing agent in nonrenal disease . Methods of Treatment and Monitoring and Targeting Treat - Mycophenolate is useful for maintenance in lupus nephri ment tis and other serious lupus cases . This agent inhibits inosine Current treatments for systemic lupus erythematosus are monophosphate dehydrogenase ( IMPDH ) and suppresses de guided by the individual patient' s manifestations , and novo purine synthesis by lymphocytes, thereby inhibiting include non - steroidal anti - inflammatory agents , anti -ma - 25 their proliferation . Mycophenolate also inhibits antibody larial agents , corticosteroid agents, and immunosuppres - production . sants . Fever, rash , musculoskeletal manifestations , and Intravenous immune globulin is used for immunosuppres serositis generally respond to treatment with hydroxychlo s ion in serious SLE flares . roquine , nonsteroidal anti - inflammatory drugs (NSAIDS ) , Belimumab inhibits the biologic activity of B -lymphocyte and steroids in low to moderate doses , as necessary , for acute 30 stimulator (BLYS ) , a naturally occurring protein required for flares . Medications such as methotrexate may be useful in survival and for development of B - lymphocyte cells into chronic lupus arthritis , and azathioprine and mycophenolate mature plasma B cells that produce antibodies. In autoim have been widely used in lupus of moderate severity . mune diseases , elevated BLYS levels are thought to contrib Nonsteroidal anti - inflammatory agents (NSAIDS ) pro ute to production of autoantibodies . This agent is indicated vide symptomatic relief for arthralgias , fever, headache , and 35 for active , autoantibody -positive SLE in patients in whom mild serositis . NSAIDS include: ibuprofen , naproxen , and standard therapy, including corticosteroids , antimalarials , diclofenac . immunosuppressives , and nonsteroidal anti - inflammatory Anti-malarial agents work with subtle immunomodula - drugs , is failing . tion without causing overt immunosuppression . These drugs Additionally , medications are often used by doctors to are useful in preventing and treating lupus skin rashes , 40 treat other conditions that commonly occur in patients with constitutional symptoms, arthralgias, and arthritis . Anti - lupus . Although these drugs do not specifically address the malarials also help to prevent lupus flares and have been underlying cause of lupus , they are used to treat other associated with reduced morbidity and mortality in SLE conditions that may be compounded or indirectly caused by patients followed in observational trials. Anti -malarial drugs lupus . include hydroxychloroquine. 45 Aspirin - Low doses of aspirin are often recommended Corticosteroid agents are used predominantly for anti - for lupus patients who have antiphospholipid antibodies and inflammatory activity and as immunosuppressants. Prepara - may reduce the risk of heart attack and stroke. tions include oral, intravenous, topical, and intra - articular Antiplatelet Medications (Platelet Antagonists ) — Some injections . Corticosteroids include methylprednisolone , lupus patients are at an increased risk for blood clots due to which is used for acute organ - threatening exacerbations. 50 the prevalence of a condition known as antiphospholipid Prednisone is the most common immunosuppressant for antibody syndrome (APS ) . Platelet antagonists help prevent treatment of autoimmune disorders and is the steroid most these clots and in doing so , also help to prevent heart attack , commonly prescribed for lupus . Low - dose oral prednisone stroke , and other complications . can be used for milder SLE , but more severe involvement Osteoporosis Medications (Bisphosphonates ) necessitates high doses of oral or intravenous therapy 55 Anti -hypertensives — 25 - 30 % of people with lupus expe Prednisone is usually given as tablets that come in 1 , 5 , rience hypertension . Themost common causes of high blood 10 , or 20 milligram (mg ) doses . Pills may be taken as often pressure in people with lupus are kidney disease and long as 4 times a day or as infrequently as once every other day. term steroid use . Usually , a low dose of prednisone is less than 20 mg/ day, a Anticoagulants — Anticoagulants are important to prevent medium dose is between 7 . 5 and 30 mg per day , and a dose 60 and treat thromboembolisms, a condition associated with of more than 30 mg qualifies as a high dose . anti - phospholipid antibodies . Lupus flares can be treated with an intra -muscular ( IM ) Gastrointestinal Medications injection of a drug called Triamcinolone . Statins — Studies have shown that people with lupus are Disease -modifying anti - rheumatic drugs (DMARDS ) are more likely to have clogged arteries that can lead to heart immunomodulatory agents that act as immunosuppressives 65 attack and stroke at a younger age . and cytotoxic and anti - inflammatory medications . The spe - Thyroid Medications Autoimmune thyroid disease is cific agent selection is generally indicated by the patient' s common in lupus. It is believed that about 6 % of people with US 9 , 809 , 854 B2 42 lupus have hypothyroidism ( underactive thyroid ) and about dose of any therapeutic agents currently be used by the 2 % have hyperthyroidism (overactive thyroid ) . subject, and monitoring the subject more frequently than Fibromyalgia Medications normal, e . g . , monthly or more frequently . Restasis A clinician or health care provider treating a subject who Patients diagnosed with SLE are counseled to see their 5 determines that the subject is in a current flare, would likely clinician or health care provider at least quarterly for moni focus the treatment on targets including , genes from the toring and testing . IFIG signature , the neutrophil granule signature , the plasma The current invention provides methods for providing cell signature, the T -cell /iNKT signature , vWF and / or IL - 10 . treatment based upon valid predictions as to the severity of Therapeutic agents that target vWF include , but are not SLE as well as the clinical manifestations . In this manner , a 10 1 subject diagnosed with SLE can be treated more effectively limited to , ARC - 1779 aptamer (Archemix - Baxter , Deerfield , with agents that target the particular clinical manifestations Ill. ), ALX - 0081 Nanobody ( Ablynx , Belgium ), and rPGP and the severity of the disease . Using the biomarkers pro 290 (Aarvon Bioscience , Woburn , Mass .) . vided herein for the first time allows clinicians and health The current invention also provides methods for moni care providers to tailor treatment more specificallyecifically based 15 toring!OM subjects and their responses to treatment, e .g , admin upon the profile of the patient. istration of agents , both oral and topical, life style alterations By way of example , a clinician or health care provider such as diet and exercise , and non - traditional treatment such treating a subject, who after testing is categorized in group as acupuncture . This is useful in both patient care as well as A , the group most similar to the healthy donors, would most clinical trials . Such a method comprises obtaining the likely treat the subject less aggressively . Treatment options 20 expression of at least one gene in at least one gene signature may include low doses of prednisone , and NSAIDs, or in a subject prior to any treatment. After a course of optionally no treatment and normal monitoring, i . e . , quar - treatment at a particular time period that a person of skill in terly . the art can determine, the measurement of expression of the A clinician or health care provider treating a subject, who same gene or genes is measured , and a decrease in expres after testing is categorized in group C1 or C2 , would most 25 sion would indicate the agent is effectively treating or likely treat the subject very aggressively , with agents that ameliorating the subject ' s SLE . The expression of at least target vascular and renal manifestations , and severe flares, one gene in at least one gene signature would be measured even if the subject has not manifested overt symptoms. before and after treatment . In a preferred embodiment at These agents would include, but are not limited to , corti - least one gene from each gene signature would be measured . costeroids such as prednisone in a medium to high dose ( 7 .5 30 Genes in any of the five signatures can be used . In a more to over 30 mg/ day ) or intravenous methylprednisolone in a preferred embodiment, more than one gene from each sig high doses (known as pulse therapy , using greater than 50 nature would be measured . In treatments that are to target mg/ day for 1 - 3 days ) ; cytotoxic drugs, such as cyclophos - vascular manifestations of disease , genes from the IFIG and phamide ; immunosuppressive drugs, such as mycophenolate neutrophil granule signature would be used , along with mofetil or azathioprine ; biologic agents , such as rituximab ; 35 anti- SSA / Ro antibodies. For treatments that are to target and anti -malarial drugs , such as hydroxychloroquine , as well mucocutaneous manifestations of the disease , genes from as agents targeting the vascular system , including but not the IFIG and neutrophil - related signature , along with TNF limited to anti -hypertensive drugs , statins , and anti - coagu - a , IL - 8 , and IL - 18 would be used . lants such as aspirin and coumarin . Additionally , the subject In a preferred embodiment, one gene from each of the would be monitored more frequently than normal, e . g . , 40 IFIG , neutrophil granule , and the neutrophil - related signa monthly or weekly . These subjects would also be counseled ture would be measured , and in a more preferred embodi to maintain habits that promote good vascular health , such ment, IFIT1 , IFIT3 , ANXA3, ARG1, MPO , DEFA1, LTF , as regular exercise , a low - fat diet , and no smoking . CEACAM6 , TNFAIP6 , FPRI, FPR2, LY96 , CYP4F3 , A clinician or health care provider treating a subject, who IL1R2 , PRRG4 , and DOCK4, would be measured before after testing is categorized in group B1 or B2 would most 45 and after treatment. In another embodiment , TNF - 2 , IL - 8 , likely treat the subject aggressively , with agents that target IL - 18 , and anti -SSA /Ro antibodies are also measured before mucocutaneous manifestations of SLE , and increased flares . and after treatment. These agents would include, but are not limited to , corti - In another preferred embodiment, genes from each of the costeroids, such as prednisone in a low to medium dose ( less IFIG , neutrophil granule , and plasma cell signature would be than 20 mg/ day ) ; anti -malarial medications, such as 50 measured , and in a more preferred embodiment, IFIT3 , hydroxychloroquine , and immunosuppressive agents , such MMP8 , and CD38 are measured . In a preferred embodi as azathioprine , dapsone, and thalidomide, aswell as topical ment, vWF and / or IL - 10 are also measured before and after agents that target mucocutaneous disorders , including but treatment. These biomarkers would monitor the effective not limited to , topical corticosteroids . These subjects would ness of a treatment for active disease . typically be monitored less than monthly , e . g ., quarterly , and 55 The present invention also provides a method for deter would be especially counseled to avoid sunlight. mining target genes or proteins for drug development. For A clinician or health care provider treating a subject, who example , a clinical trial that has been determined to target after testing is determined to be likely to have future flares , vascular manifestations of SLE would target genes in the would most likely treat the subject aggressively , with agents IFIG signature , and the neutrophil signature , as well as including but not limited to , corticosteroids , such as pred - 60 anti- SSA / Ro antibodies . A clinical trial that has been deter nisone in a medium to high dose ( 7 . 5 to over 30 mg/ day ) , or mined to target mucocutaneous manifestations would target intravenous methylprednisolone in a high doses (known as genes in the IFIG signature and the neutrophil- related sig pulse therapy , using greater than 50 mg/ day for 1 - 3 days ) ; nature , along with TNF -a , IL -8 , and IL - 18 . A clinical trial cytotoxic drugs , such as cyclophosphamide; immunosup - that has been determined to target the active stages of the pressive drugs, such as mycophenolate , mofetil or azathio - 65 disease, i. e ., flares, would target genes from the IFIG , prine ; biologic agents , such as rituximab ; and anti -malarial neutrophil granule , plasma cell , and T -cell / iNKT signature drugs , such as hydroxychloroquine, as well as increasing the as well as vWF and IL - 10 . US 9 , 809 , 854 B2 43 44 The invention also contemplates that the protein products peptides, proteins or fragments , or if the agent being tested of any of the genes in the gene signatures found in Tables 2 - 6 interferes with the formation of a complex between the would also be potential therapeutic targets for either moni peptide or protein and a known ligand . toring or drug development. Thus , the present invention provides for methods and Kits 5 assays for screening agents for treatment of SLE , compris It is contemplated that all of the assays disclosed herein ing contacting or incubating the test agent with a polypep can be in kit form for use by a health care provider and /or a diagnostic laboratory . tide or protein encoded by a gene in one of the gene Assays for the detection and quantitation of one or more signatures listed in Tables 2, 3, 4 , 5 , or 6 , or vWF, IL - 10 , of the gene signatures can be incorporated into kits . Such 10 TNF- a , IL - 8 and IL - 18 , and detecting the presence of a kits would include probes for one or more of the genes from complex between the polypeptide and the agent or the one or more signatures , i .e ., IFIG , neutrophil granule , neu presence of a complex between the polypeptide and a ligand , trophil - related , immunoglobulin / plasma cell signature and by methods known in the art . In such competitive binding T - cell/ iNKT, reagents for isolating and purifying nucleic assays , the polypeptide or fragment is typically labeled . Free acids from biological tissue or bodily fluid , reagents for 15 polypeptide is separated form that in the complex , and the performing assays on the isolated and purified nucleic acid , amount of free or uncomplexed polypeptide is measured . instructions for use , and reference values or the means for This measurement indicates the amount of binding of the obtaining reference values in a control sample for the test agent to the polypeptide or its interference with the included genes . binding of the polypeptide to a ligand" gand . A preferred kit for patient classification with regard to 20 High throughput screening can also be used to screen for disease activity and clinical manifestations would include therapeutic agents . Small peptides or molecules can be probes for at least one gene from each of the three signa - synthesized and bound to a surface and contacted with the tures, IFIG , neutrophil granule and neutrophil - related. A polypeptides encoded by the gene signature transcripts , and more preferred embodiment would include probes for IFIT1 , washed . The bound peptide is visualized and detected by IFIT3 , CEACAM6 , TNFAIP6 , CYP4F3 and FPR1. 25 methods known in the art . In a further embodiment, the kit would include reagents Antibodies to the polypeptides can also be used in com for testing for TNF - a , IL - 8 , andind IL - 18 , and / or anti- - SSA / Ro petitive drug screening assays . The antibodies compete with autoantibodies. Such a kit could include antibodies that recognize the peptide of interest , reagents for isolating the agent being tested for binding to the polypeptides. The and /or purifying protein from a biological tissue or bodily 30 antibodiesdete can be used to find agents that have antigenic fluid , reagents for performing assays on the isolatedbounty and 30 determinants on the polypeptides , which in turn can be used purified protein , instructions for use, and reference values or to develop monoclonal antibodies that target the active sites the means for obtaining reference values for the quantity or of the polypeptides. level of peptides in a control sample . The invention also provides for polypeptides to be used A preferred kit for monitoring treatment to disease activ - 35 for rational drug design where structural analogs of biologi ity would include probes from at least oneone gene from eacheach cally active polypeptides can be designed . Such analogs of the three signatures, IFIG . neutrophil granule , and , immu - would interfere with the polypeptide in vivo , such as by noglobulin /plasma cell , and reagents for testing for vWF and non - productive binding to target. In this approach the three IL - 10 . Such a kit could include antibodies that recognize the dimensional structure of the protein is determined by any peptide of interest, reagents for isolating and /or purifying 40 method known in the art including but not limited to X - ray protein from a biological tissue or bodily fluid , reagents for crystallography, and computer modeling. Information can performing assays on the isolated and purified protein , also be obtained using the structure of homologous proteins instructions for use , and reference values or the means for or target - specific antibodies . obtaining reference values for the quantity or level of Using these techniques, agents can be designed which act peptides in a control sample . 45 as inhibitors or antagonists of the polypeptides , or act as A preferred kit for predicting future flares would include decoys , binding to target molecules non -productively and probes for at least one gene from each of the four signatures , blocking binding of the active polypeptide . IFIG , neutrophil granule , immunoglobulin /plasma cell sig - Polypeptides encoded by any of the differentially nature and T -cell / iNKT. A more preferred embodiment expressed transcripts of the gene signatures found in Tables would include probes for IFIT3 , KLRB1, CD38 , and 50 2 , 3 , 4 , 5 , and 6 can be used . Additionally , testing can be MMP8 . done as described above using the proteins or fragments of A preferred embodiment of these kits would have the the proteins vWF, IL - 10 , TNF - a , 11 - 8 , and IL - 18 , as well as probes attached to a solid state . A most preferred embodi- anti- SS /Ro antibodies . ment would have the probes in a microarray format wherein A further embodiment of the present invention is gene nucleic acid probes for one or more of the genes from one 55 constructs comprising any one of the differentially expressed or more of the gene signatures would be in an ordered transcripts and a vector. These gene construct can be used for arrangement on a surface or substrate . testing of therapeutic agents as well as basic research Drug Screening Assays and Research Tools regarding SLE . These gene constructs can also be used to All of the biomarkers disclosed herein can be used as the transform host cells can be transformed by methods known basis for drug screening assays and research tools . 60 in the art. In one embodiment , polypeptides and proteins encoded The resulting transformed cells can be used for testing for by the transcripts in the gene signatures , IFIG , neutrophil therapeutic agents as well as basic research regarding SLE . granule , neutrophil - related , plasma cell , and T -cells / iNKT, Specifically , cells can be transformed with any one of the as well as vWF, IL - 10 , TNF - 2 , IL - 8 and IL - 18 , can be used differentially expressed transcripts , and contacted with a test in drug screening assays, free in solution , or affixed to a solid 65 agent. The resulting expression of the transcript can be support . All of these forms can be used in binding assays to detected and compared to the expression of the transcript in determine if agents being tested form complexes with the the cell before contact with the agent. US 9 ,809 , 854 B2 45 46 The expression of the transcripts in host cells can be U133 Plus 2 .0 GeneChips® (Affymetrix ) at 45° C . over detected and measured by any method known in the art , night. Chips were scanned in a GeneChip® scanner 3000 including but not limited to , reporter gene assays. (Affymetrix ). These gene constructs as well as the host cells trans Proteomic Study of Plasma Proteins formed with these gene constructs can also be the basis for 5 Plasma levels of 44 autoantibodies (autoimmune serology transgenic animals for testing both as research tools and for panel) and 41 inflammatory biomarkers (human inflamma therapeutic agents . Such animals would include but are not tion panels ver1. 0 ) were evaluated using the multi -analyte limited to , nude mice . Phenotypes can be correlated to the profiling (MAP ) technology (Rules -Based Medicine, Austin , genes and looked at in order to determine the genes effect on Tex . ) . The protein analytes included in this assay are listed the animals as well as the change in phenotype after admin - 10 in Table 8 . istration or contact with a potential therapeutic agent. Microarray Data Analysis and Quality . The gene constructs and host cells transformed with the Data from the Affymetrix U133 Plus 2 . 0 gene arrays ( CEL gene constructs can also be administered to murine models files ) were uploaded to GeneSpring GX11 software ( Agilent of SLE , for analyzing test agents as well as basic research . Technologies , Santa Clara , Calif .) , processed using the Any of the differentially expressed transcripts of the gene 15 robust multiarray analysis algorithm ( Irizarry et al. 2003 ) signatures found in Tables 2 , 3 , 4 , 5 , and 6 can be used . and log - transformed . Each * . CEL file represents an indi vidual subject' s visit . Affymetrix Expression Console soft EXAMPLES ware ver 1 . 1 was used to generate a detection p - value , which indicates the reliability of detection of the transcripts above The present invention may be better understood by ref- 20 background on the array. Probe -sets with a detection p -value erence to the following non - limiting examples , which are greater than 0 . 05 in more than 30 % of HDs or SLE patients presented in order to more fully illustrate the preferred were excluded from the analysis . embodiments of the invention . They should in no way be Proteomics Data Analysis . construed to limit the broad scope of the invention . Analytes below the detectable level in more than 50 % of 25 the SLE samples were excluded from analysis . The remain Example 1 ing low data points were adjusted to the least detectable level . All data are presented as a mean - standard deviation . Patients and Methods Serology results were calculated as a ratio of the median fluorescence intensity (MFI ) of reactivity with a specific Study Subjects 30 antigen to a negative control. Data were normalized to the All patients fulfilled at least four of the American College mean of each parameter, log2 transformed for data visual of Rheumatology (ACR ) criteria for SLE ( Tan et al. 1982 ). ization as well as for statistical and clustering analysis . Two cohorts of SLE patients were studied : a training set of P -values less than 0 . 05 were considered significant after 23 SLE patients, and a validation set of 58 SLE patients . multiple samples correction ( 5 % FDR ) . Fold change differ Demographic and clinical data of the study patients are 35 ences were calculated between geometric means and there summarized in Table 7 . fore were lower compared to fold change differences Episodes of mild /moderate and severe flare were estab - between arithmetic means . Fold change of more than 1 . 5 lished based on the Safety of Estrogens in Lupus Erythe - was considered significant. matosus : National Assessment — SLE Disease Activity Statistical Analysis of Microarray and Proteomics Data Index [SELENA - SLEDAI (SLEDAI ) ] score ( American 40 A linear mixed effects model was used to analyze gene College of Rheumatology Ad Hoc Committee on Systemic expression and proteomics data that included repeated mea Lupus Erythematosus Response Criteria 2004 ) . Mild or surements over time in individual patients and HDs, per moderate flares were defined as an increase in SLEDAI mitting estimation of variability among patient groups and score of more than 3 points from the previous visit . Severe based on flare status. flare was defined as change in SLEDAI to greater than 12 . 45 In experiments with a repeated measurements design it is New organ flares or requirement for medical intervention possible to accurately estimate the variability for expression were also considered flares. Longitudinal clinical data , along of each gene between groups using a linear mixed model with PBMC and plasma samples, were obtained over an (Karlovich et al. 2009 ) . average of 9 visits (range 2 - 26 ) for SLE patients and 5 visits The simplest model would include two sources of varia for HD . The duration of study follow - up for individual 50 tion in measured gene intensities: patient to patient variabil patients , averaged 860 days and ranged from 63 to 1941 ity and within patient variability . Let Y ; be the base 2 days . algorithm of normalized gene intensities of a particular gene Biologic Sample Collection transcript, i the studied groups [HD or SLE ], j the donor Blood samples from HD and SLE patients were processed [ j = 1 , . . . , 28 ], and t refers to time points . A linear model for within one hour of phlebotomy. Peripheral blood mononu - 55 the type of analysis herein is : clear cells ( “ PBMC” ) were purified using Ficoll PaqueTMPlus (GE Healthcare Life Sciences, Piscataway, Y; Fu + Time; j + Group ,+ T ;+ P ;+ ?ji ( 1 ) N . J . ) gradient centrifugation and preserved in RNeasy lysis where u = the grand mean , Time: - average deviance from u buffer (QIAGEN , Inc ., Valencia , Calif . ) . Samples were due to effect of time, Group - average deviance from u due stored at - 70° C . until RNA extraction . 60 to effect of disease, Tjt and P = random effect of time and RNA Isolation , Amplification , and Hybridization donor correspondingly and € are the additive stochastic RNA was isolated from 169 PBMC samples using the errors . RNeasy kit ( QIAGEN ) and quality assessed using the Agi - The model could be extended with the addition of the lent 2100 Bioanalyzer. Fifty nanograms of total RNA was effect of SLE flares . In that case the additional fixed effect used to prepare targets by Two -Cycle Target labeling kit 65 of flares should be added . ( Affymetrix , Santa Clara , Calif . , USA ) following the manu facturer ' s instructions and hybridized onto Y ;c u + Time; : +Group ,+ Flarejt + T ;x + P ; + €jt US 9 , 809, 854 B2 47 48 Flarejt = the effect of flares (non - flaring or flaring ) . based on autoantibodies. The results of the hierarchical Such models are both examples of generalized linear clustering are represented using “ heatmap ” plots , with the models and could be written in terms of the equation : orange color indicating high expression and blue corre Y = XB + Zu + e sponding to low expression of transcripts . In some cases, Where Y is a vector of n observations, B is a vector of fixed 5 due to the repetitive nature of experiments , average levels of effect , u is a vector of random effect (equal to the number of all time points for each transcript/ analyte per patient was patients ) , e is a vector of residual errors , X and Z are calculated before clustering analysis . incidence matrices for the fixed and random effects . The model is fitted to the data employing R ( R Develop - 10 TABLE 7 ment Core Team , 2012 ) and package lme4 (Bates and Maechler , 2009 ) under the important assumption that the General subject demographic and clinical characteristics residuals and patient p terms are normally distributed . Quan titatively , the hypothesis that the residual terms are normally SLE SLE distributed may be tested with the Shapiro normality test 15 HD ( training ) (validation ) ( p < 0 . 1 ) . To assess the validity of the mixed effect analyses, like Total subjects 5 23 58 lihood ratio tests were performed comparing the model with Male 2 (40 % ) 4 ( 17 % ) 7 (12 % ) fixed effect to the null models with only random effect from Median Age, years (Range ) 38 (35 -44 ) 27 ( 14 -50 ) 28 ( 17 -57 ) donors . Results in which the model including fixed effects 20 Asian Americans ( % ) 1 ( 20 % ) 3 (13 % ) 4 ( 7 % ) did not differ from the null model were rejected . Black Americans ( % ) 0 ( 0 % ) 6 ( 26 % ) 24 (41 % ) For each gene , fold change difference between groups was Hispanic Americans ( % ) 2 (40 % ) 8 (35 % ) 19 (33 % ) calculated based on log -means level for each donor. For each European Americans ( % ) 1 ( 20 % ) 5 (22 % ) 10 ( 17 % ) gene a generalized F - test was performed based on the Other/ Mix ( % ) 1 ( 20 % ) 1 (4 % ) 1 ( 2 % ) described model and the corresponding p -values were 25 Medion dicoce obtained . Alternatively , because it is debated whether the 7 ( 0 -16 ) 3 (0 -24 ) degree of freedom is a meaningful concept for linearmixing duration , years ( range ) models , p - values based on log likelihood ratio testing were Median ACR 6 (3 - 9 ) 6 (2 - 9 ) obtained (shown in tables ). Whenever it was possible , the Score , ( range ) model followed by Markov chain sampling was used for 20 SLEDAI ( at study initiation ), 6 (0 -25 ) 4 (0 - 16 ) more exact p values. Because numerous F - tests were per 30 Median , (range ) formed , a multiple testing correction procedure to control SLEDAI ( at study endpoint) , 4 ( 0 - 18 ) 4 ( 0 - 18 ) erroneously identified genes was applied . Median , ( range ) Hierarchical Clustering BILAG ( at study initiation ) , 7 ( 1 - 34 ) 5 (0 -16 ) Clustering of patients based on transcripts or analytesanalytes was 25w Median , (range ) performed using R package function hclust. The programprogram '' s " BILAG (at study endpoint) , 1 ( 0 - 14 ) 2 ( 0 - 15 ) parameters used included Pearson ' s centered , ward for Median , ( range ) patient classification based on differentially expressed tran scripts , and Euclidian centered , centroid for classification TABLE 8 Analytes studied in proteomics assay Samples Least Low High below Samples Detectable Plasma Plasma LDD in below LDD % Low % Low Analytes Units Dose (LDD ) Range Range HD ( 25 ) in SLE (144 ) in HD in SLE 1 Alpha - 2 Macroglobulin mg/ mL 0 .017 0 . 13 0 % 0 % 2 Alpha - 1 Antitrypsin mg/mL < 0 . 0029 1 . 2 3 . 1 0 % 0 % 3 Beta - 2 Microglobulin ug/mL 0 . 24 1 . 2 6 . 2 0 % 2 % 4 Brain - Derived ng/mL 0 ..044 00 .32 32 1616 0 % 1 % Neurotrophic 5 Complement 3 mg/mL < 0 .0024 0 . 76 2 . 1 0 % 0 % 6 C Reactive Protein ug/ mL 0 .038 0 . 25 50 0 % 0 % 7 Eotaxin pg/ mL 118 177 0 % 1 % 8 Factor VII ng /mL 106 443 0 % 0 % 9 Ferritin ng/mL 3 . 3 5 552 0 % 0 % 10 Fibrinogen mg/mL 0 . 0052 2 . 2 0 % 1 % 11 Haptoglobin mg/mL 0 . 0061 0 .047 7 . 6 0 % 8 % 12 ICAM - 1 ng/ mL 2 . 7 42 213 0 % 0 % 13 IL - 10 pg/ mL 5 . 4 1 . 8 38 4 % 0 % 14 IL - 12p70 pg/ mL 50 165 72 % 44 % 15 IL - 15 ng/ mL 0 . 2 4 . 6 36 % 26 % 16 IL - 18 pg/mL 20 1020 OOOOOOOOOOINOONOONwoo 0 % 0 % 17 IL - 1ra pg/mL 38 622 0 % 0 % 18 IL - 23 ng/ mL 1 . 2 30 12 % 6 % 19 IL - 6 pg/ mL 5 . 9 25 52 % 3 % 20 IL - 8 pg/ mL 3 . 3 59 4 % 0 % 21 MCP - 1 pg/ mL 8 . 8 35 4014010 0 % 0 % 22 MIP - 1 alpha 11 OBOTwoOOoooooooooooo- 56 % pg/ mL 89 29 % 23 MIP - 1 beta pg/mL 21 25 595 0 % 0 % US 9 , 809 , 854 B2 49 TABLE 8 -continued Analytes studied in proteomics assay Samples Least Low High below Samples Detectable Plasma Plasma LDD in below LDD % Low % Low Analytes Units Dose ( LDD ) Range Range HD (25 ) in SLE (144 ) in HD in SLE 24 MMP - 3 ng /mL 0 . 21 1 . 8 0 % 0 % 25 RANTES ng /mL 0 . 28 2 . 6 83 0 % 0 % 26 Stem Cell Factor pg/ mL 74 281 0 % 0 % 27 TIMP - 1 ng /mL 59 192 0 % 0 % 28 TNF - alpha pg/ mL 3 . 6 5919 72 % 24 % 29 TNF RII ng/ mL 0 . 3 3 . 1 79 0 % 0 % 30 VCAM - 1 ng /mL 5 . 4 284 1310 0 % 0 % 31 VDBP ( Vitamin D ug /mL 4 . 9 Pending Pending 0 % 0 % Binding 32 VEGF pg/ mL 28 91 1790 0 % 0 % 33 von Willebrand Factor ug/ mL 0 .43 5 . 3 74 0 % 0 % 34 B -Lymphocyte pg/ ml 55 pending pending OOOOOOOOO 4 % 0 % Chemoattractant ( BLC ) 35 IP - 10 (Inducible Protein - 10 ) pg/ ml 30 pending pending 0 % 0 % 36 MCP - 2 pg /ml 1515 pending pending oooooooooooooo 4 % 0 % 37 Gamma- Interferon - induced pg/ ml 100 pending pending 0 % 0 % Monokine 8 Prolactin ng /ml 0 . 5 0 . 88 42 0 % 0 % 39 BAFF pg /ml 11 0 % 0 % 0 ITAC pg/ ml 7 . 5 0 % 0 % 41 MIP - 3b pg/ ml 14 0 % 0 % 42 GM - CSF pg /mL 19 152 143 100 % 99 % 43 IFN - gamma pg/mL 3 . 6 9 . 5 123 92 % 85 % 44 IL - 12p40 ng/ mL 0 .55 2 . 7 144 100 % 100 % 45 IL - 17 pg /mL 8 . 5 14 80 144 100 % 100 % 46 IL - 1 alpha ng/ mL 0 . 0062 0 . 35 103 100 % 72 % 7 IL - 1 beta pg/ mL 8 . 7 100 76 % 69 % 48 IL - 2 pg/ mL 14* 61 144 100 % 100 % 9 IL - 3 ng/ mL 0 . 032 1 . 2 144 100 % 100 % 50 IL - 4 pg/ mL 12 103 87 20 % 60 % 51 IL - 5 pg/ mL 6 . 9 62 uvuauunawa 118 72 % 82 % 2 IL - 7 pg /mL 16 3 . 7 125 144 100 % 100 % 53 MMP- 2 ng /mL 15 183 3070 NNNNNNNHANN 129 100 % 90 % 54 MMP- 9 ng /mL 8 . 3 1050 105 68 % 73 % 55 TNF - beta pg/ mL 8 . 9 120 144 100 % 100 % 56 MCP - 3 pg/ mL 1717 pending pending 25 111 100 % 77 %

Example 2 the mean expression value for each transcript over all visits 40 for each SLE patient and HD was used . PBMC mRNA Transcripts Differentially Expressed Top three clusters of genes , shown on the left side of Between SLE Patients and HDs Define Three dendrogram ( FIG . 1 ) , were suspected to be enriched with Patient Groups transcripts related to IFIG , neutrophil- granule genes and 45 neutrophil related genes respectively . The presence of low Based on the statistical method described in Example 1, density granulocytes in blood preparations of lupus patients 433 differentially expressed probe - sets between HDs and has been shown earlier (Denny et al. 2010 ) . SLE patients were identified ( fold change (FC )> 1. 5 , p < 0 .05 , FIG . 2 depicts the database and methods on how the 5 % false detection rate ( FDR ) ) , along with 9 transcripts due functionality for corresponding clusters was assigned . FIG . to the effect of flare status. See Table 9 . 50 2A shows that the presence of IFIG within the assigned As SLE patients are known to exhibit activation of type 1 cluster ( the top of FIG . 1 ) as determined using the INTER interferon genes in the PBMC , the present of interferon FEROME database . inducible genes (“ IFIG ” ) using the INTERFEROME data - FIG . 2B shows that genes with expression associated with base (Samarajiwa et al. 2009 ) was tested . The transcripts primary , secondary and tertiary neutrophil granules tran most highly expressed in SLE compared to HD samples 55 scripts were identified based on previously published were dominated by IFN - 1 - induced genes ( IFIG ) and neu - microarray analysis of bone marrow and peripheral blood trophil granule genes , consistent with previous reports . neutrophils by Theiland -Munch 2005. Some genes were Approximately one quarter of transcripts (total number 112 ) specific for promyelocytes and metamyelocytes and not were identified as interferon regulated genes , with the major - observed in peripheral blood . According to this analysis of ity being regulated by type I interferon ( 96 genes by Type I 60 second from the top cluster (NG as shown on FIG . 1) was versus 74 genes by Type II and 48 genes by Type III enriched with neutrophil granules genes . interferons) . FIG . 2C shows using the Gene Enrichment Profiler , Machine learning analysis is widely used for grouping containing data from human normal primary tissues (126 transcripts showing similar expression pattern . An unsuper - tissues represented by 557 microarrays ), it was determined vised hierarchical clustering was performed to classify both 65 the likely source of the transcripts deemed as neutrophil transcripts deregulated in SLE samples and HDs who par - related (NR , third cluster from the top on FIG . 1 ) . Expres ticipated in the study . For simplicity and gene visualization , sion profiles were processed to identify tissue specificity US 9 , 809 , 854 B2 51. 52 which was measured by an enrichment score . The resulting previously been detected in PBMC from pediatric lupus heat map demonstrates high enrichment of selected gene patients (Bennett et al. 2003 ) . In addition , this analysis transcripts among human peripheral blood neutrophils. It detected overexpression of neutrophil related transcripts should be noted that part of the neutrophil - granule genes and within group B lupus patients . neutrophil related genes, based on INTERFEROME data - 5 The elevated levels of IFIG and neutrophil granule tran base analysis , are also interferon inducible genes and there - scripts highlight their significance in distinguishing SLE fore assumed to be largely expressed on neutrophils com - patients from HDs. However, some of the differentially pared to other . expressed transcripts, particularly those derived from The list of differentially expressed transcripts , their samples collected at times of disease flare , may represent an median intensity across all visits in SLE patients and HDs, 10 acute -phase reaction rather than a characteristic feature of their fold difference in expression in SLE patients compared lupus. Linear mixed model analysis of gene expression data to HDs and their level of significance are presented in Table from all individual visits of all study subjects was used to identify those transcripts that reflect lupus disease as well as Based on the heatmap shown in FIG . 1 , HDs are clearly those that are associated with lupus flare . Using that distinct from SLE patients . Hierarchical clustering classified 15 approach , 131 probe - sets were identified with a significant SLE patients into three principal groups ( A , B and C ) . Group effect related to lupus disease and lupus flares ( p < 0 .05 , 5 % A consisted of six patients that were placed close to the HDs. FDR ) . Those probe - sets are highlighted in bold in Table 9 . Group A patients generally hold levels of IFIG and neutro Of interest , well known IFIGs do not appear among those phil - granule transcripts similar to HD . The most populated probe - sets associated with flare . Instead , a number of tran group , C , included 12 patients . The majority of patients in 20 scripts related to neutrophil granule genes as well as neu group C show elevated expression of both IFIG and neu t rophil related transcripts , including TNFAIP6 , were among trophil - granule genes. Group B demonstrated prominent the 131 probe -sets identified by this analytical approach . In IFIG but lacks the neutrophil granule signature . Instead , a addition , lupus flares change the level of 9 probe- sets , which set of neutrophil related transcripts was increased . were normally expressed when patient was during non In general this analysis confirmed the prominent overex - 25 flaring state ( identified by an asterisk in Table 9 . pression of IFIG observed previously in PBMC of SLE Taken together, these analyses, based on multiple longi patients (Crow et al . 2007 ; Crow et al . 2003 ; Lovgren et al. tudinal PBMC samples from SLE patients and HDs, indicate 2004 ; Barrat et al. 2005 ; Crow et al. 2003 ) and also that representative transcripts of the IFIG and neutrophil demonstrated expression of neutrophil -granule transcripts in granule signatures was sufficient to define at least three a distinct patient group C . Many of the latter genes have distinct patient groups. US 9 , 809 , 854 B2 54

p-values 0.9973 0.9594 0.7699 0.8528 0.7738 0.0034 0.8528 0.0176 0.7215 0.7956 0.9269 0.8948 0.0125 0.8972 0.7595 0.5221 0.0793 0.8610 0.8164 0.5166 0.0084 0.0084 0.0034 0.7778 0.0025 0.8913 0.0279 0.5637 0.7441 0.9973 0.7183 0.0034 0.9973 0.3115 0.0034 0.0025 0.0025 0.5410 0.7738 0.8637 0.4156 Effectofflares Estimate -0.028 -0.036 0.160 0.118 0.172 1.160 0.118 0.818 0.144 0.095 -0.054 0.060 1.028 0.048 0.098 0.240 -0.374 0.056 0.111 0.213 1.173 0.860 1.178 0.105 1.012 0.045 0.784 -0.195 0.099 -0.012 0.104 0.936 0.016 0.320 1.097 0.739 1.151 0.173 0.094 0.046 -0.195

p-values 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 Effectofdisease 4.493 3.708 3.603 3.556 3.249 3.469 2.990 3.134 2.806 2.651 2.565 2.624 2.991 2.536 2.567 2.516 2.110 2.381 2.443 2.387 2.820 2.664 2.653 2.170 2.530 2.079 2.380 1.967 2.054 2.008 2.032 .325 1.892 2.067 2.318 2.099 2.308 1.835 1.801 1.711 1.537

Estimate 2

1936.1 745.9 459.5 831.3 408.2 914.6 532.7 691.9 755.0 1335.1 645.5 1943.3 988.3 93.0 1297.0 1969.3 798.3 1023.4 369.6 999.3 675.6 SLE 1076.5 6548.2 5038.5 5337.9 4618.3 7952.2 4887.1 2470.2 6717.4 1292.1 4995.8 1550.3 3444.3 2209.3 3782.4 1375.4 760.5 8287.7 1155.4 2410.2

MeanValues 21.1 62.1 44.2 97.5 87.1 16.1 86.2 391.1 128.9 352.1 492.1 726.8 114.5 365.4 143.2 900.2 108.3 137.7 173.0 217.3 394.9 138.1 777.8 303.5 295.2 119.7 180.6 199.9 185.8 322.5 179.2 HD 678.9 1095.4 945. 1051.0 200.6 440.8 870.5 533.3 2374.3 689.7

FoldChange 22.00 12.94 11.35 11.21 8.87 7.33 6.96 6.59 6.54 6.02 85.5 5.78 5.75 5.56 5.47 5.16 5.11 4.99 4.78 4.65 4.62 4.61 4.24 4.21 4.13 4.07 993. 3.98 3.96 3.88 3.78 3.72 3.57 3.51 3.50 3.41 3.39 3.33 3.32 3.20 3.18 EntrezGene 3429 10964 91543 91543 3434 4317 6614 4057 3437 129607 11274 9636 10321 4599 2537 3437 27074 10561 6614 10561 10562 3934 383 4940 4680 94240 1669 400941 51191 100133842 3959on 4061 4680 4938 3433 306 634 4318 3433 4938 4939 2766 TABLE9 GeneTranscriptsDifferentiallyExpressedinSLEPatients

matrixmetallopeptidase9(gelatinaseB,92kDa) mitochondrialkinase2,cytidinemonophosphate(UMP-CMP) carcinoembryonicantigen-relatedcelladhesionmolecule6 lectingalactoside,-bindingsoluble3bindingprotein. matrixmetallopeptidase8(neutrophilcollagenase) myxovirus(influenzavirus)resistance1. interferon-inducedproteinwithtetratricopeptiderepeats2 interferon-inducedproteinwithtetratricopeptiderepeats1 interferon-inducedproteinwithtetratricopeptiderepeats3 interferon-inducedproteinwithtetratricopeptiderepeats3 epithelialstromalinteraction1(breast) complexlocusEantigen,lymphocyte6 carcinoembryonicantigen-relatedcelladhesionmolecule6(non-specific crossreactingantigen) interferon-inducedproteinwithtetratricopeptiderepeats2 carcinoembryonicantigen-relatedcelladhesion radicalS-adenosylmethioninedomaincontaining2 radicalS-adenosylmethioninedomaincontaining2 sialicacidbindingIg-likelectin1,sialoadhesin lysosomal-associatedmembraneprotein3 sialicacidbindingIg-likelectin1,sialoadhesin 2,5'-oligoadenylatesynthetase140/46kDa 2',5-oligoadenylatesynthetase140/46kDa 2-5oligoadenylatesynthetase,69/71kDa protein27alphainducible-interferon, interferon-inducedprotein44like cysteine-richsecretoryprotein3 proteininducible6interferonalpha-, kDasynthetase3,100oligoadenylate-25 molecule1(biliaryglycoprotein) ubiquitinspecificpeptidase18ISG15ubiquitin-likemodifier interferon-inducedprotein44 Interferon-inducedprotein44 defensin,alpha4corticostatin hectdomainandRLD5 guanosinemonophosphatereductase olfactomedin4 lipocalin2 arginase,liver FLJ42418protein GeneTitle lactotransferrin A3annexin GeneSymbol IFI27 IFI44L RSAD2 RSAD2 IFIT1 MMP8 SIGLEC1 LTF IFIT3 CMPK2 USP18 ISG15 luntatoilCRISP3 MX1 IFI6 IFIT3 LAMP3 IF144 SIGLEC1 IFI44 OLFM4 LCN2 ARG1 OAS3 CEACAM6 EPSTI1 DEFA4 FLJ42418 HERC5 LGALS3BP LOC100133842 LY6E CEACAM6 andOAS1 IFIT2 ANXA3 CEACAM1 gasMMP9 IFIT2 OAS1 OAS2 GMPR

219519_sat 202018_sat 205483s_at 214453_sat 206177_sat 203757sat 203936_sat 205552_sat Affymetrix ProbeSetID 202411_at 204439_at 213797at 242625_at 203153at 231688at 229450_at 226702_at 219211_at 207802at 202086at 204415at 204747at 205569_at 44673_at 214059_at 212768sat 212531_at 218400at 227609_at 207269_at 231455_at 219863at 200923at 202145at 211657_at 202869_at 217502at 209369_at 209498at 226757_at 204972at 204187_at US 9 , 809 , 854 B2 55 56

p-values 0.9536 0.7549 0.9017 0.0025 0.7915 0.5875 0.0106 0.9973 0.8584 0.9972 0.9027 0.2650 0.0543 0.0152 0.6516 0.4061 0.2419 0.1473 0.8553 0.9511 0.5244 0.5189 0.0034 0.8173 0.2033 0.4672 0.2945 0.7810 0.0335 0.8553 0.0034 0.0025 0.0442 0.3969 0.0140 0.0334 0.3832 0.9973 0.0584 0.5244 0.9575 0.8584 0.0301 0.0025 0.2650 Effectofflares

-0.026 0.127 0.004 0.781 0.069 0.144 0.754 0.001 0.034 -0.016 -0.063 0.246 0.570 0.978 0.082 -0.292 0.166 -0.335 0.040 0.012 -0.119 -0.141 0.546 0.056 -0.309 -0.131 0.145 0.061 0.466 -0.044 0.746 1.132 0.443 0.124 0.612 0.453 -0.218 -0.003 0.423 0.154 -0.012 0.047 0.473 0.654 0.236 p-valuesEstimate 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0012 0.0006 0.0002 0.0024 0.0002 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 00020. 0.0002 0.0002 0.0002 0.0002 0.0003 0.0009 0.0002 0.0002 0.0006 0.0002 0.0012 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 Effectofdisease Estimate 1.712 1.802 1.727 2.061 1.684 1.716 2.000 1.588 1.601 1.526 1.544 1.664 1.752 1.907 1.546 1.167 1.554 1.330 1.476 1.438 1.386 1.367 1.718 1.469 1.311 1.331 1.502 1.419 1.530 1.322 1.665 1.837 1.487 1.406 1.556 1.459 1.064 1.283 1.426 1.428 1.336 1.291 1.421 1.547 1.377 SLE 591.2 2429.7 692.4 2005.0 34.9 2554.3 583.0 986.4 571.0 103.6 3443.9 55.8 648.8 6147.4 191.8 1238.9 2842.2 977.1 522.0 2203.1 161.0 438.9 2150.0 177.1 1749.3 293.8 301.3 1673.6 894.9 309.0 407.5 469.9 95.1169 830.427980 56.52010 117.53880 498.417003 30.11084 110.23931 948.929476 384.011934 1842.255754 222.47337 198.46149 5368.513723

8.9 MeanValues 197.1 207.7 341.0 208.8 31.1 14.3 9 70.8 357.7 257.3 61.5 114.7 68.4 82.0 356.2 95.3 107.2 192.7 HD 852.6 157.4 728.6 912.4 1242.6 241.5 2283. 1076.3 187.5 673.7 672.5 731.3 116.6 694.3

FoldChange 3.17 3.12 3.11 3.09 3.06 3.06 2.99 2.97 2.87 2.87 2.87 2.79 2.77 2.75 2.74 2.72 2.71 2.71 2.70 2.69 2.68 2.67 2.65 2.642.62 2.61 2.56 2.53 2.52 2.482.48 2.46 2.45 2.43 2.41 2.41 2.41 2.39 2.39 2.38 2.38 2.34 2.30 2.30

EntrezGene 1667/ 1668/ 54739 118932 3728 55076 26010 51513 7130 94240 3665 54739 113730 8638 728358 4051 83666 7145 5359 6373 219285 5610 55008 91624 117854 4600 6373 91624 5359 4939 100133941 5610 671 1116 100133941 6037 100133941 51339 64135 100133941 1890 83666 100133941 2352 8638

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

eukaryotictranslationinitiationfactor2-alphakinase eukaryotictranslationinitiationfactor2-alphakinase ribonuclease,RNaseAfamily3(eosinophilcationicprotein) dapper,antagonistofbeta-cateninhomolog1(Xenopuslaevis) defensin,alpha1/3neutrophil-specific cytochromeP450,family4subfamilyFpolypeptide3 myxovirus(influenzavirus)resistance2mouse tumornecrosisfactor,alpha-inducedprotein6Epithelialstromalinteraction1(breast) sterilealphamotifdomaincontaining9-like chitinase3-like1(cartilageglycoprotein39) interferoninducedwithhelicaseCdomain1 viralDNApolymerase-transactivatedprotein6 poly(ADP-ribose)polymerasefamily,member9 chemokine(C—Xmotif)ligand11 chemokine(C—Xmotif)ligand11 2-5'oligoadenylatesynthetase,69/71kDa bactericidal/permeability-increasingprotein poly(ADP-ribose)polymerasefamily,member9 interferonregulatoryfactor7 nexilin(Factinbindingprotein) tripartitemotif-containing6 nexilin(Factinbindingprotein) folatereceptor3(gamma) XIAPassociatedfactor1 ankyrinrepeatdomain22 transmembraneprotein45A XIAPassociatedfactor1 kelchdomaincontaining7B 2-5oligoadenylatesynthetaselike phospholipidscramblase1 hectdomainandRLD6 phospholipidscramblase1 2-5'oligoadenylatesynthetaselike defensin,alpha1 thymidinephosphorylase GeneTitle junctionplakoglobin etsvariant7 tensin1 CD24molecule CD24molecule CD24molecule CD24molecule CD24molecule

GeneSymbol DEFA1/ XAF1 ANKRD22 JUP TMEM45A LOC26010 ETV7 TNFAIP6 EPSTI1 IRF7 XAF1 KLHDC7B OASL DEFA3 /LOC728358 CYP4F3 PARP9 TNS1 PLSCR1 CXCL11 SAMD9L EIF2AK2 HERC6 NEXN TRIM6 MX2 CXCL11 NEXN PLSCR1 OAS2 CD24 EIF2AK2 BPI ????L1 CD24 RNASE3 CD24 DACT1 IFIH1 CD24 TYMP PARPO CD24 FOLR3 OASL

at_S 206133_at 238439_at 201015_sat 222154_sat 224225_sat 206025_sat 235276_at 208436sat 228617_at 236285_at 205033_sat 206515_at 223220s_at 221748_sat 202430_sat 210163_at 230036_at 204211_xat 223599_at 204994_at 211122_sat 1552309aat 202446_sat 206553_at 213294_at 216379_xat 209771_xat 219179_at 208650_sat 204858s_at 227807_at 208651_xat 210797_sat Affymetrix ProbeSetID 219410at 205660at 219352at 226103at 266_sat 205557at 209395at 243754at 206851at 219209at 239979at 206371at US 9 , 809 , 854 B2 57 58

p-values 0.7353 0.8264 0.8972 0.9973 0.8553 0.7183 0.9536 0.1192 0.8185 0.7448 0.0465 0.0948 0.0034 0.8972 0.9973 0.5238 0.0025 0.9941 0.9972 0.3020 0.3060 0.8584 0.8972 0.1908 0.1181 0.8948 0.5490 0.7781 0.9369 0.9042 0.3271 0.0930 0.8903 0.0063 0.0063 0.0176 0.1357 0.2878 0.4035 0.7998 0.0422 0.0248 Effectofflares Estimate 0.093 -0.062 0.055 0.050 -0.043 -0.068 -0.021 0.394 0.100 -0.079 0.642 0.542 1.164 0.047 -0.001 0.102 0.716 0.009 0.027 0.169 0.214 0.123 -0.016 0.308 0.333 0.040 -0.135 0.180 0.036 0.097 0.163 0.322 0.058 0.662 0.477 0.468 0.642 0.406 0.113 0.147 0.450 0.495

p-values 0.0002 0.0002 0.0002 0.0326 0.0002 0.0002 0.0439 0.0002 0.0002 0.0002 0.0048 0.0003 0.0006 0.0006 0.0002 0.0002 0.0003 0.0002 0.0006 0.0002 0.0002 0.0374 0.0002 0.0002 0.0002 0.0002 0.0003 0.0186 0.0002 0.0188 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0175 0.0062 0.0002 0.0019 0.0021 0.0006 Effectofdisease Estimate 1.278 1.149 1.336 1.007 1.168 1.180 0.958 1.372 1.232 1.096 1.433 1.449 1.728 1.205 1.201 1.172 1.438 1.141 1.086 1.202 1.226 0.937 1.104 1.267 1.218 1.071 0.986 0.863 1.075 0.986 1.080 1.165 1.042 1.294 1.232 1.199 1.232 1.064 1.068 0.991 1.142 1.199 SLE 1495.7 2173.1 327.8 438.5 868.8 2874.8 408.3 125.4 694.1 619.6 637.6 181.7 323.7 795.2 1360.4 1161.4 1236.8 3453.4 66.2 1794.7 1704.5 479.8 5113.5 2512.4 169.2 5883.6 507.4 793.0 2506.5 411.7 474.0 599.0 762.0 31.2 2336. 273.5 129.6 137.5 8024.2 1263.6 244.9 1319.8

MeanValues 906.9 132.4 118.0 378.8 69.8 52.2 298.5 276.7 187.5 64.2 83.0 347.6 587.1 532.4 473.3 24.7 798.8 897.7 114.8 73.7 229.9 355.4 156.0 235.7 313.0 393.3 11.5 81.0 128.3 49.3 45.4 578.8 93.4 614.2 HD 642.5 1250.4 1585.7 2372.6 1171.9 3062.3 1331.4 3964.6

FoldChange 2.30 2.29 2.28 2.26 2.25 2.25 2.25 2.24 2.23 2.22 2.22 2.22 2.20 2.19 2.18 2.16 2.16 2.15 2.15 2.13 2.13 2.12 2.10 2.09 2.07 2.022.02 2.01 2.00 2.00 1.99 1.99 1.99 1.99 1.98 1.98 1.97 1.97 1.96 1.92 1.92 1.92

EntrezGene 2812/ 23586 55601 219285 7504 6772 6772 51327 9732 51338 6772 4353 2867 7850 3430 24138 6947 219285 24138 23643 3042 51056 3557 91351 8743 23034 8743 5413 3005 4778 23586 55859 10170 4311 51312 10410 8349 6518 8876

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

transcobalaminI(vitaminB12bindingprotein,Rbinderfamily) signaltransducerandactivatoroftranscription1,91kDa interferoninducedtransmembraneprotein3(1-8U) signaltransducerandactivatoroftranscription1, membersubfamily4domainsA,membranespanning- signaltransducerandactivatoroftranscription1, interferon-inducedproteinwithtetratricopeptiderepeats5 member10superfamily,necrosisfactor(ligand)tumor membersuperfamily10,necrosisfactor(ligand)tumor glycoprotein1b(platelet),betapolypeptide/septin5 dehydrogenase/reductase(SDRfamily)member9 solutecarrierfamily2(glucose/fructose),member5 sterilealphamotifdomaincontaining9-like interferon-inducedproteinwithtetratricopeptiderepeats5 sterilealphamotifdomaincontaining9-like DEAD(Asp-GluAla)boxpolypeptide60like derivedkDa2),45erythroid-nuclearfactor( DEAD(Asp-GluAla)boxpolypeptide58DEAD(Asp-GluAla)boxpolypeptide60 X-linkedKxbloodgroup(McLeodsyndrome) interleukinreceptor1,typeII sterilealphamotifdomaincontaining4A DEAD(Asp-GluAla)boxpolypeptide58 solutecarrierfamily25,member37 interferon-inducedprotein35 H1histonefamily,member0 linked1X-expressed,brain erythroidassociatedfactor dedicatorofcytokinesis4 freefattyacidreceptor2 antigen96lymphocyte hemoglobin,mu leucineaminopeptidase3interleukin1receptorantagonist endopeptidasemetallo-membrane histonecluster2,H2be GeneTitle 91kDa 91kDa myeloperoxidase vanin1

GeneSymbol GP1BB/ DDX58 DDX60 SAMD9L XK STAT1 STAT1 ERAF DOCK4 MS4A4A STAT1 MPO FFAR ILIR2 IF135 IFIT5 TCN1 SAMD9L IFITS LY96 HBM LAP3 ILIRN DDX60L TNFSF10 SAMD4A TNFSF10 SEPT5 H1F0 NFE2 DDX58 BEX1 DHRS9 MME SLC25A37 IFITM3 HIST2H2BE SLC2A5 VNN1 M97935_MAat M97935_MBat 218943_sat HUMISGF3A/ 209969_sat 219607sat HUMISGF3A/ 211372_sat 209417_sat 203596_sat 203595sat 217933_sat 212657_sat 202687_sat 206655_sat 209930sat 224009_xat 222528_sat 212203_xat 202708_sat Affymetrix ProbeSetID 218986sat 235643at 206698_at AFFX 219672at 205003_at AFFX 203949_at 221345_at 243271_at 205513_at 226603at 237597at 206584_at 240336_at 1559585at 202688_at 212845_at 231078_at 208886_at 222793_at 233591at 218332at 203434sat 204430sat 205844at US 9 , 809 , 854 B2 59 09

p-values 0.8584 0.8285 0.0125 0.4164 0.9994 0.1359 0.9269 0.7988 0.0359 0.5994 0.8551 0.5494 0.1644 0.0125 0.9972 0.0638 0.3536 0.9426 0.0886 0.3773 0.3249 0.6456 0.0152 0.2309 0.6772 0.9575 0.0084 0.8972 0.0415 0.1978 0.0272 0.1036 0.8960 0.9284 0.3726 0.0034 0.0025 0.0235 0.4902 0.4840 0.0084 0.0207 0.0309 0.1703 Effectofflares 0.040 0.080 0.448 0.109 -0.004 0.357 0.023 0.046 -0.324 0.117 0.121 0.124 0.270 0.428 -0.001 0.24618979 0.120 0.017 0.232 -0.086 0.207 -0.144 0.537 0.143 0.073 0.016 0.530 0.038 0.451 0.245 0.526 -0.282 0.025 0.067 -0.176 0.828 0.590 0.339 -0.137 -0.187 0.798 0.428 0.463 -0.258 p-valuesEstimate 0.0002 0.0002 0.0002 0.0002 0.0002 0.0006 0.0002 0.0002 0.0006 0.0002 0.0275 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 0.0009 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0012 0.0006 0.0002 0.0372 0.0024 0.0021 0.0003 0.0003 0.0002 0.0006 0.0009 0.0002 0.0012 0.0014 Effectofdisease Estimate 0.949 1.056 1.145 1.042 1.017 1.043 0.941 1.013 0.730 1.043 0.871 1.033 1.113 1.104 0.900 0.992 0.982 0.924 1.047 0.835 1.085 0.815 1.181 0.971 0.931 0.914 1.109 0.913 1.103 1.052 1.130 0.747 0.903 0.782 0.857 1.271 1.140 1.008 0.806 0.760 1.150 1.054 1.093 0.724 SLE 319.5 3641.8 285.2 729.7 175.6 329.8 7925.0 25.0 30.2 271.9 1103.4 121.2 633.6 942.2 207.7 517.9 12301 698.2 1032.6 7262.2 480.5 1162.5 156.6 3970.6 564.5 2817.4 72.0 723.3 536.2 148.1 64.2 720.4 3402.4 652.8 104.2 548.8 332.0 136.5 533.7 174.6 296.0 1401.5 43.3 298.5

MeanValues 161.1 137.3 82.1 146.4 12.1 14.4 58.4 109.2 380.2 539.9 71.6 35.7 385.7 69.5 20.7 51.5 259.8 151.1 56.1 289.7 84.2 153.7 15.8 157.5 HD 1823.5 365.9 4149.9 146.5 460.9 291.7 480.0 264.5 644.6 3958.9 250.6 660.9 2160.1 302.9 1716.5 289.9 378.7 1856.7 283.4 758.2

FoldChange 1.92 1.91 1.91 1.91 1.91 1.90 1.89 1.89 1.88 1.88 87.1 1.87 1.86 1.86 1.86 1.85 1.84 1.84 1.84 1.83 1.83 1.83 1.83 1.83 1.82 1.82 1.82 1.82 1.81 1.81 1.81 1.81 1.80 1.80 1.80 1.79 1.78 1.78 1.77 77.1 1.77 1.77 1.77 1.77

EntrezGene 3013/ 54809 9997 10170 26270 113730 253827 8519 26010 440836 1191 118932 7298 10170 4602 54877 64108 54877 6772 1890 1154 53831 91351 3431 8743 8351 4939 2358 51514 26577 26228 9246 1191 2359 3772 6590 158471 8564 55084 54682 83716 54443 26228

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatientsGeneTranscriptsDifferentiallyExpressedinSLEPatients

interferoninducedtransmembraneprotein1(9-27) potassiuminwardly-rectifyingchannel,subfamilyJmember15 SCOcytochromeoxidasedeficienthomolog2(yeast) dehydrogenase/reductase(SDRfamily)member9 dehydrogenase/reductase(SDRfamily)member9 kynurenine3-monooxygenase(hydroxylase) V-mybmyeloblastosisviraloncogenehomolog(avian) signaltransducerandactivatoroftranscription1, tumornecrosisfactor(ligand)superfamily,member10 histonecluster1,H2ad/H3d sineoculisbindingproteinhomolog(Drosophila) cysteine-richsecretoryproteinLCCLdomaincontaining2 viralDNApolymerase-transactivatedprotein6 receptor(chemosensory)transporterprotein4 cytokineinducibleSH2-containingprotein DEAD(Asp-GluAla)boxpolypeptide60like procollagenC-endopeptidaseenhancer2 signaltransducingadaptorfamilymember1 signaltransducingadaptorfamilymember1 sterilealphamotifdomaincontaining9 methioninesulfoxidereductaseB3 outerdensefiberofspermtails3B zincfinger,CCHCdomaincontaining2 zincfinger,CCHCdomaincontaining2 2'-5oligoadenylatesynthetase,69/71kDa Drosophila)homolog(denticleless ubiquitin-conjugatingenzymeE2L6 secretoryleukocytepeptidaseinhibitorDrosophila)(homolog2prune MANSCdomaincontaining1 kelchdomaincontaining7B domain22repeatankyrin thymidylatesynthetase thymidinephosphorylase Gprotein-coupledreceptor84 SP110nuclearbodyprotein formylpeptidereceptor2 formylpeptidereceptor3 anillin,actinbindingprotein F-boxprotein6 GeneTitle clusterin 91kDa clusterin GeneSymbol HIST1H2ADIII - SAMD9 SCO2 DHRS9 FBX06 KLHDC7B MSRB3 IFITM1 LOC26010 ODF3B CLU ANKRD22 TYMS DHRS9 MYB ZCCHC2 RTP4 ZCCHC2 STAT1 TYMP CISH GPR84 DDX60L SP110 TNFSF10 HIST1H3D OAS2 FPR2 DTL PCOLCE2 STAP1 UBE2L6 CLU FPR3 KCNJ15 SLPI PRUNE2 KMO SOBP MANSC1 CRISPLD2 ANLN STAP1

223952_xat 201601_xat 208792_sat 219799_sat 219062_sat 222816_sat HUMISGF3A/ M97935_3at 223377xat 228152_sat 214329_xat 218585_sat 219295sat 205306x_at 220945_xat 222608_sat 1554343_aat Affymetrix ProbeSetID 219691_at 205241_at 231769at 1552639at 225782at 215617_at 231192at 238327at 239196at 202589_at 239988_at 204798at 219684_at AFFX 217497at 223767_at 209761sat 214472_at 228607_at 210772_at 220059_at 201649at 208791at 230422_at 210119_at 203021_at 212805_at 218974_at 221541_at US 9 , 809 , 854 B2 62

p-values1 0.8972 0.0025 0.5579 0.7013 0.2309 28860. 0.3832 0.0084 0.0025 0.2620 0.7810 0.0422 0.0248 0.4389 0.1584 0.0025 0.7810 0.0025 0.8480 0.9973 0.1277 0.9302 0.5702 0.0793 0.6341 0.7595 0.0279 0.7810 0.9375 0.4391 0.0025 0.0829 0.0176 0.0034 0.0192 0.0192 0.0106 Effectofflares 260.0 Estimate -0.020 0.472 -0.123 -0.119 0.274 -0.071 0.608 0.573 0.181 -0.053 0.401 0.609 0.219 0.287 0.519 0.087 0.594 -0.042 -0.003 0.179 -0.012 0.094 0.345 -0.097 0.049 0.346 0.045 0.008 0.137 0.499 0.284 0.439 0.344 0.542 0.570 0.730

p-values 0.0002 0.0002 0.0002 0.0003 0.0002 0.0003 0.0002 0.0014 0.0002 0.0002 0.0002 0.0014 0.0002 0.0002 0.0006 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0052 00020. 0.0012 0.0006 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 0.0021 0.00380.0077 Effectofdisease Estimate 0.856 1.029 0.806 0.757 0.978 0.928 0.764 1.121 1.071 0.863 0.792 0.937 1.135 0.994 0.989 1.073 0.860 1.055 0.791 0.785 0.886 0.694 0.865 0.984 0.765 0.752 0.955 0.748 0.787 0.822 0.979 0.965 0.941 0.960 1.010 0.987 1.092 SLE 1525.6 82.1 599.3 1086.6 220.2 2151.7 9329.8 388.6 401.1 173.7 3471.1 68.4 200.1 651.8 79.1 303.3 195.8 86.8 3050.5 2518.3 204.6 318.1 1169.2 163.3 988.9 398.2 394.0 2149.1 110.2 4199.0 491.3 86.9 847.7 13005.9 1896.3 35.2 346.2

MeanValues 853.3 42.5 329.3 647.0 98.4 223.1 213.3 96.9 33.1 105.4 326.7 41.3 167.4 99.4 44.5 116.9 179.0 674.3 84.1 609.2 228.4 65.4 273.9 45.0 522.3 13.9 169.7 HD 1007.9 5386.4 1982.5 1736.3 1486.0 240.9 1272.0 2637.7 7775.8 1216.3

FoldChange 1.76 1.76 1.75 1.75 1.74 1.74 1.73 1.73 1.73 1.73 1.73 1.72 1.71 1.71 1.71 71.1 1.70 1.70 1.70 1.70 1.70 1.69 1.69 1.68 1.68 1.68 1.68 1.67 1.67 1.67 1.67 1.67 1.67 1.66 1.65 1.65 1.65 EntrezGene 684 634 8564 1154 2289 2289 6772 5806 55711 3017 3669 56670 2357 388 259266 57568 8651 634 64761 151636 10643 54625 7153 1026 6672 10326 3431 51513 10673 84418 29128 79056 6280 133 4973 55350

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

moleculeadhesion1cellcarcinoembryonicantigen-related kynureninemonooxygenase3-(kynureninehydroxylase) oxidizedlowdensitylipoprotein(lectin-like)receptor1 signaltransducerandactivatoroftranscription1, rapidlyinducedbyIL-1pentraxinrelatedgene, interferonstimulatedexonucleasegene20kDa associatedlike2proliferation1signalinduced- carcinoembryonicantigen-relatedcelladhesion cyclindependent-kinaseinhibitor1A(p21,Cipl) tumornecrosisfactor(ligand)superfamily,member13b poly(ADP-ribose)polymerasefamily,member12 insulin-likegrowthfactor2mRNAbindingprotein3poly(ADP-ribose)polymerasefamily,member14 kDaDNAalpha170topoisomerase()II chromosome5openreadingframe32ubiquitin-likewithPHDandringfingerdomains1 bonemarrowstromalcellantigen2 cytokineinducibleSH2-containingprotein familymemberB,homologgeneras asp(abnormalspindle)homolog,microcephaly suppressorofcytokinesignaling1 molecule1(biliaryglycoprotein) signal-regulatoryproteinbeta1 FK506bindingprotein5 FK506bindingprotein5 reductase2CoAfattyacyl formylpeptidereceptor1 associated(Drosophila) ProlinerichGla(G-carboxyglutamic $100calciumbindingproteinA9 (biliaryglycoprotein) histonecluster1,H2bd succinatereceptor1 deltex3-like(Drosophila) SP100nuclearantigen SP110nuclearbodyprotein acid)4(transmembrane) GeneTitle etsvariant7 vanin3 91kDa beta adrenomedullin GeneSymbol BST2 CEACAM1 KMO CISH FKBP5 FKBP5 STAT1 PTX3 FAR2 HIST1H2BD ISG20 SUCNR1 FPR1 RHOB ASPM SIPAIL2 SOCS1 CEACAM1 PARP12 DTX3L IGF2BP3 PARP14 TOP2A CDKNIA SP100 SIRPB1 SP110 ETV7 TNFSF13B C5orf32 UHRF1 PRRG4 S100A9 ADM OLR1 VNN3

211889_xat 211138_sat 221223_xat 200887_sat 220615_sat 222067_xat 219918s_at 210001_sat 211883_xat 218543_sat 203820_sat 201291_sat 202284_sat 210218_sat 1554624_aat 209762_xat 223502s_at Affymetrix ProbeSetID 201641_at 204560at 224856_at 206157_at 33304_at 223939_at 205118_at 212099_at 225056_at 225415_at 239033_at 224701_at 221680sat 224707at 225655_at 238513_at 203535_at 202912_at 210004at 220528at US 9 , 809 , 854 B2 63 64

p-values 0.0235 0.0025 0.8553 0.8135 0.1978 0.0025 0.3366 0.1646 0.0025 0.8913 0.9973 0.2152 0.0106 0.8972 0.0791 0.2544 0.0025 0.1223 0.0778 0.0084 0.9575 0.5991 0.1161 0.2807 0.8675 0.8913 0.0025 0.2338 0.7810 0.0034 0.0449 0.8584 0.8135 0.0376 0.0925 0.1036 0.4360 Effectofflares 0.24200755 Estimate 0.433 0.448 0.108 0.088 0.413 0.415 -0.167 0.164 0.668 0.058 0.003 0.215 0.549 0.020 0.305 0.141 0.720 0.181 0.308 0.561 0.002 0.114 0.188 0.163 0.074 -0.045 0.443 0.127 0.045 0.405 0.344 0.077 0.038 0.318 0.299 0.297 0.117

p-values 0.0002 0.0014 0.0003 0.0303 0.0014 0.0237 0.0002 0.0084 0.0002 0.0002 0.0009 0.0002 0.0002 0.0009 0.0002 0.0033 0.0002 0.0012 0.0002 0.0002 0.0014 0.0002 0.0002 0.0002 0.0002 0.0002 0.0026 0.0002 0.0002 0.0002 0.0002 0.0002 0.0029 0.0002 0.0002 0.0002 0.0002 0.0002 Effectofdisease Estimate 0.887 0.931 0.940 0.678 0.671 0.794 0.900 0.561 0.816 0731. 0.695 0.683 0.848 0.951 0.706 0.851 0.780 1.020 0.817 0.863 0.927 0.687 0.768 0.825 0.804 0.775 0.685 0.956 0.746 0.670 0.870 0.891 0.633 0.695 0.867 0.837 0.800 0.720 1524.6 237.6 792.3 198.2 274.3 2790.2 561.3 65.7 1159.2 265.2 81.6 3615.6 3772.3 2204.5 9686.7 42.9 728.8 283.9 7605.6 7350.1 317.1 649.7 651.0 7620.2 337.7 660.0 685.1 56.2 12113.0 2587.3 290.3 346.7 1756.4 1111.6 800.5 132.9 1986.5 206.7 SLE

937.3 105.1 167.8 330.4 36.9 151.5 48.7 21.1 156.6 189.4 408.5 197.3 434.5 32.9 168.7 220.1 78.4 128.1 MeanValues 124.5 434.9 1449.4 681.2 2238.0 2537.8 1315.7 6058.5 9443. 4741.7 4865.9 399.8 4732.8 409.1 7660.0 1621.6 941.1 703.9 487.2 1408.3 HD

FoldChange 1.64 1.64 1.63 1.63 1.63 1.631.63 1.62 1.61 1.61 1.61 1.61 1.60 1.60 1.60 1.60 1.60 1.60 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.58 1.58 1.58 1.58 1.57 1.57 1.57 1.57 1.57 1.57 EntrezGene 648998 653361 654816n/ 654817 8876 5872 145567 257019 55711 23362 9517 9586 64135 54809 1230 366 8519 79733 9517 116844 2896 2357 6583 85363 6398 2896 9517 10855 51284 10581 3431 8778 390035 8531 3431 6556 79746 53829 81622 TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

interferoninducedtransmembraneprotein1(9-27) interferoninducedtransmembraneprotein2(1-8D) similartoNeutrophilcytosolfactor1(NCF-), serinepalmitoyltransferase,longchainbasesubunit2 Serinepalmitoyltransferase,longchainbasesubunit2 serinepalmitoyltransferase,longchainbasesubunit2 CAMPresponsiveelementbindingprotein5InterferoninducedwithhelicaseCdomain1 cation/ergothioneinetransporter),member4 subfamilyK,family52olfactoryreceptor solutecarrierfamily11(proton-coupleddivalent enoylCoenzymeAhydratasedomaincontaining3 purinergicreceptorP2Y,G-proteincoupled13 tetratricopeptiderepeatdomain7B pleckstrinandSec7domaincontaining3 chemokine(C—motif)receptor1 sialicacidbindingIg-likelectin5 metaliontransporters),member1 RAB13,memberRASoncogenefamily sterilealphamotifdomaincontaining9 leucine-richalpha2glycoprotein1 unc-93homologB1(C.elegans) FERMdomaincontaining3 FattyacylCoAreductase2 E2Ftranscriptionfactor8 formylpeptidereceptor1solutecarrierfamily22(organic tripartitemotif-containing5 secretedandtransmembrane1 SP110nuclearbodyprotein coldshockdomainproteinA SP110nuclearbodyprotein aquaporin9 toll-likereceptor7 member3pseudogene GeneTitle pseudogene vanin1 granulin granulin heparanase

GeneSymbol /NCF1 LOC648998 NCF1B /NCF1C VNN1 RAB13 TTC7B FRMD3 FAR2 PSD3 SPTLC2 CREB5 IFIH1 SAMD9 CCR1 AQP9 IFITM1 E2F8 SPTLC2 LRG1 GRN FPR1 SLC22A4 TRIM5 SECTM1 GRN SPTLC2 HPSE TLR7 IFITM2 SP110 SIGLEC5 OR52K3P CSDA SP110 SLC11A1 ECHDC3 P2RY13 UNC93B1

204961_sat 1558549sat 231274s_at 203355_sat 203127_sat 205931_sat 214022_sat 200678_xat 205119_sat 210705_sat 213716_sat 216041_xat 216202_sat 219403_sat 201161_sat 208392_xat Affymetrix ProbeSetID 202252at 226152_at 229893at 239108_at 216020_at 228531_at 205098at 205568_at 219990_at 225095_at 228648at 205896_at 220146at 1558011at 201315xat 208012xat 220000_at 232829_at 210423sat 219298_at 220005at 225869sat US 9 , 809 , 854 B2 65 99

0.7898 0.9973 0.0623 0.8972 0.0034 0.9511 0.0272 0.8972 0.0580 0.3121 0.0063 0.7998 0.1158 0.3143 0.2425 0.9495 0.0063 0.0925 0.7738 0.0384 0.0260 0.1584 0.0835 0.9511 0.5826 0.5733 0.7998 0.0925 0.0025 0.0335 0.2425 0.0152 0.1476 0.997239 0.9511 Effectofflares p-valuesEstimate 0.107 0.031 0.310 0.011 0.329 0.017 0.341 0.040 0.221 0.179 0.459 -0.030 0.256 0.249 -0.240 0.042 0.420 0.312 0.087 0.290 0.366 0.232 0.282 0.003 0.097 0.105 0.116 0.305 0.483 -0.263 -0.144 0.264 0.254 0.021 -0.033

0.0012 0.0002 0.0002 0.0002 0.0003 0.0002 0.0003 0.0002 0.0241 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0006 0.0002 0.0006 0.0006 0.0057 0.0116 0.0006 0.0002 0.0322 0.0029 0.0002 0.0014 0.0029 0.0112 Effectofdisease 0.58900017 0.8410002 0.6600002 0.8370002 0.6750002 0.9410002 Estimate 0.679 0.697 0.753 0.714 0.870 0.619 0.819 0.843 0.536 0.656 0.820 0.785 0.770 0.738 0.890 0.785 0.797 0.663 0.720 0.693 0.719 0.821 0.831 0.396 0.473 0.740 0.797 0.561 0.584

194.9 212.3 241.7 127.7 567.6 251.9 92.9 468.3 461.9 58.8 139.5 373.8 934.1 557.7 266.7 42.4 224.4 950.1 117.4 62.9 103.5 572.5 313.2 2112.4 4247.6 1169.4 1423.5 3515.9 1262.7 8195.6 342.0 1241.6 1175.6 1096.1 670.5 SLE

115.2 76.6 55.5 35.4 85.4 23.0 79.4 40.4 58.0 MeanValues 1249.9 2764.3 135.9 752.2 890.6 141.3 2318.6 839.8 4783.7 362.2 160.4 232.3 759.1 308.8 762.3 281.5 236.4 611.5 372.0 168.5 156.2 659.6 717.2 449.9 366.5 206.3 HD

E FoldChange 1.57 1.56 1.56 1.56u 1.56 1.55 55.1 1.55 1.55 1.55 1.55 1.55 1.55 1.55 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.53 1.53 1.53 1.53 1.53 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.51

EntrezGene 1378/ 11039/ les1379 m 4354 7097 7205 79930 aintain4502 11042 653188 55577 84674 2289 57520 100128718lis 284759 6546 id4811 645 19 120892 440836 160364lolitatud 7077 1164 81030 144455 22797 1230 25797 2814 23362 64218 51514 84333 5800 TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

biliverdinreductaseB(flavinreductaseNADPH) ATP-bindingcassette,subfamilyA(ABC1)member1 complementcomponent(3b/4b)receptor1 55kDaproteinpalmitoylated1,membrane caspaserecruitmentdomainfamily,member6 solutecarrierfamily8(sodium/calciumexchanger), domain(Ig),immunoglobulindomainsema transmembranedomain(TM)andshort proteintyrosinephosphatase,receptortypeO glucuronidase,betapseudogene/ glucuronidase,betapseudogene/ CDC28proteinkinaseregulatorysubunit2 pleckstrinandSec7domaincontaining3 semaphorin)4Adomaincytoplasmic,( (Knopsbloodgroup)/complementcomponent thyroidhormonereceptorinteractor6 HECT,C2andWWdomaincontainingE3 Outerdensefiberofspermtails3BC-typelectindomain family12,memberATIMPmetallopeptidaseinhibitor2 chemokine(C—motif)receptor1 platelet)glycoproteinV( denticlelesshomolog(Drosophila) polycombgroupringfinger5 glucuronidase,betapseudogene FK506bindingprotein5 ubiquitinproteinligase2 signal-regulatoryproteinbeta2 leucine-richrepeatkinase2 E2Ftranscriptionfactor7 dockingprotein3 HypotheticalproteinLOC100128718 Z-DNAbindingprotein1 factorECtranscription glutaminyl-peptidecyclotransferase (3b/4b)receptor1-like toll-likereceptor2 metallothionein2A N-acetylglucosaminekinase GeneTitle member1 nidogen1

GeneSymbol CR1/CRIL /SMA4 i MPP1saith TLR2 TRIP6 DOK3 MT2A LOC653188 SMA5 NAGK CARD6 FKBP5 HECW2 LOC100128718 SIRPB2 SLC8A1 NID1 BLVRB ABCA1 LRRK2 ODF3B CLEC12A TIMP2tersebut CKS2 ZBP1 E2F7 TFEC CCR1 ???? GP5 PSD3 SEMALA DTL PCGF5 PTPRO 1561615_sat Affymetrix ProbeSetID 239205_sat 202974_at 204924at 209129at 223553_sat 212185_xat 215043_sat 218231_at 224414_sat 224840at 232080_at 1556643_at 1559034_at 202007_at 202201_at 203505at 229584_at 232375_at 235681_at 243934at 1569401at 203167at 204170_sat 208087_sat 228033_at 232383at 205099_sat 205174sat 207926at 218613_at 219259_at 222680_sat 227935s_at 208121s_at US 9 , 809 , 854 B2 67 89

p-values 0.8164 0.0517 0.5293 0.0517 0.2472 0.7595 0.1788 0.2180 0.1117 0.0709 0.1006 0.6238 0.1822 0.9972 0.4840 0.9973 0.8336 0.8264 0.3383 0.4453 0.8972 0.9511 0.7233 0.8528 0.3941 0.3878 0.1392 0.3020 0.0772 0.0152 0.1543 0.0025 0.1978 0.4840 0.9828 0.5579 0.0456 0.8057 0.5994 0.9973 Effectofflares Estimate 0.027 0.244 -0.096 0.348 -0.114 -0.029 -0.188 -0.122 -0.188 -0.241 -0.269 -0.142 -0.157 0.006 -0.141 0.007 -0.094 -0.090 -0.097 -0.141 0.020 -0.018 0.108 0.070 -0.100 0.207 -0.183 -0.158 -0.315 -0.226 -0.222 -0.239 -0.090 -0.076 -0.010 -0.138 -0.259 -0.091 -0.100 0.001

p-values 0.0002 0.0002 0.0036 0.0009 0.0002 0.0002 0.0006 0.0002 0.0002 0.0009 0.0043 0.0052 0.0002 0.0181 0.0003 0.0024 0.0064 0.0101 0.0002 0.0002 0.0099 0.0002 0.0162 0.0118 0.0002 0.0173 0.0002 0.0006 0.0081 0.0002 0.0014 0.0002 0.0002 0.0002 0.0104 0.0068 0.0002 0.0017 0.0002 0.0003 Effectofdisease 0.522 Estimate 0.627 0.783 0.787 -0.662 -0.612 -0.726 -0.671 -0.710 -0.698 -0.701 -0.635 -0.631 -0.563 -0.693 -0.557 -0.600 -0.622 -0.718 -0.666 -0.567 -0.589 -0.498 -0.512 -0.633 -0.475 -0.777 -0.726 -0.856 -0.794 -0.807 -0.812 -0.676 -0.677 -0.633 -0.710 -0.780 -0.685 -0.682 -0.563 SLE 8277.9 2025.4 30.5 507.0 158.3 474.6 34.1 21.1 2966.3 662.2 36.6 202.8 20.4 1017.5 172.9 1512.1 214.1 235.0 208.6 219.6 27.3 65.1 128.8 231.1 72.6 143.4 362.6 2955.9 159.6 328.0 417.9 242.6 186.6 1271.2 39.9 169.7 189.3 97.0 270.0 450.0

MeanValues 19.0 360.1 231.6 701.5 49.2 30.4 904.2 49.7 293.7 30.2 250.4 312.0 358.9 302.3 334.6 42.6 102.7 182.3 346.4 114.1 213.4 540.9 196.8 503.0 594.2 365.9 274.0 59.1 230.2 304.4 143.1 437.2 668.6 HD 5473.7 1337.4 4148.1 1417.7 2225.0 4311.8 1938.9

FoldChange 1.51 1.51 1.51 1.51 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 EntrezGene 10346 648998 5747 58484 5797 115 8910 4291 925 911 2823 150759 64065 64783 4190 80205 7634 3800 1880 9590 10000 23178 56172 23176 11096 5342

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

ADAMmetallopeptidasewiththrombospondintype1motif,5 neutrophiloxidasefactor)(p47-phoxNCF47K similartoNeutrophilcytosolfactor1(NCF-) (NeutrophilNADPHoxidasefactor1)47kDa proteintyrosinephosphatase,receptortypeM ChromodomainhelicaseDNAbindingprotein9 Malatedehydrogenase1,NAD(soluble) PASdomaincontainingserine/threoninekinase ankylosis,progressivehomolog(mouse) NLRfamily,CARDdomaincontaining4 V-aktmurinethymomaviraloncogenehomolog tripartitemotif-containing22 diseaseprotein)(NOXO2 PTK2proteintyrosinekinase2 myeloidleukemiafactor1 RNAbindingmotifprotein15 anchorprotein12PRKA)kinase(A 3(proteinkinaseB,gamma) (47kDaautosomalchronicgranulomatous cyclase9adenylate sarcoglycan,epsilon M6Aglycoprotein hypotheticalproteinLOC150759PERP,TP53apoptosiseffector Zincfingerprotein80 kinesinfamilymember5C Gprotein-coupledreceptor183 plasminogen-likeB2 GeneTitle CD8amolecule CD1cmolecule septin8 GeneSymbol TRIM22 LOC648998 PTK2 NLRC4 PTPRM ADCY9 SGCE MLF1 CDSA CD1C GPM6A LOC150759 PERP RBM15 MDH1 CHD9 ZNF80 KIF5c GPR183 AKAP12 AKT3 PASK ANKH 41160 ADAMTS5 PLGLB2

1552553_aat Affymetrix ProbeSetID 213293_sat 214084_xat 241453at 203329_at 204497_at 204688_at 204783at 205758_at 205987_at 209469_at 213703_at 217744_sat 228030_at 228455_at 229434_at 230713at 232511_at 235203at 235374at 236999_at 239540_at 239654_at 241775at 1556467at 1565852_at 203130_sat 205419_at 210517_sat 212609_sat 213534sat 223093at 226627_at 227755_at 229357_at 230120_sat 230230_at 230961_at 235651_at 235716_at US 9 , 809 , 854 B2 69

p-values 0.7558 0.9973 0.8972 0.9495 0.7998 0.7586 0.7915 0.0223 0.1966 0.9575 0.9269 0.0906 0.4871 0.3024 0.7810 0.8553 0.1114 0.9269 0.6284 0.8553 0.3624 0.9973 0.8553 0.7190 0.9199 0.9779 S 0.7988060 Effectofflares 0.1177586 -0.1093969 0390.8994- 0.01209779 0.127 Estimate 0.001 0.047 -0.044 -0.071 0.068 -0.051 -0.273 -0.213 -0.032 -0.020 -0.262 -0.121 -0.122 0.056 -0.028 -0.197 -0.042 -0.100 -0.019 -0.124 -0.012 0.052 -0.113 0.036 0.017

p-values 0.0421 0.0002 0.0017 0.0075 0.0019 0.0002 0.0247 0.0002 0.0012 0.0012 0.0003 0.0002 0.0002 0.0003 0.0415 0.0003 0.0009 0.0002 0.0002 0.0003 0.0002 0.0002 0.0024 0.0019 0.0002 0.0002 0.0002 0.0019 0.0014 0.0177 0.0038 Effectofdisease Estimate -0.448 -0.581 -0.565 -0.629 -0.675 -0.556 -0.530 -0.677 -0.606 -0.679 -0.653 -0.660 -0.788 -0.733 -0.559 -0.648 -0.842 -0.738 -0.653 -0.650 -0.697 -0.768 -0.752 -0.725 -0.664 -0.757 -0.678 -0.559 -0.681 -0.562 -0.557

68.6 82.3 395.7 82.2 85.6 60.1 478.3 147.8 35.0 69.5 487.3 93.9 436.9 37.6 93.5 155.6 485.7 117.1 88.8 36.1 38.3 17.6 206.9 61.4 264.3 127.3 231.8 217.3 170.3 227.7 274.5 SLE MeanValues 106.7 123.7 597.3 122.5 128.3 90.4 727.9 237.0 56.7 110.9 739.5 155.9 661.6 53.4 138.6 244.9 700.0 179.1 141.9 55.9 61.4 26.6 345.1 93.1 405.0 189.4 343.8 328.9 262.5 332.0 411.8 HD

FoldChange 0.65 0.65 0.65 0.65 0.65 0.65 0.65 650. 0.65 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 EntrezGene 100130070 III 100130775 100131787 / 100131905 III 100132291 100132488 Ill 6232 6281 8481 54165 324822875 23705 9975 23261 10142 51077 23178 339290 6167 54796 57562 5169 55553 59084 256356 10432 90338

TABLE9-continued PatientsTranscriptsDifferentiallyExpressedinSLEGeneGeneTranscriptsDifferentiallyExpressedinSLEPatients

hydroxyprostaglandindehydrogenase15-(NAD) nuclearreceptorsubfamily1,groupDmember2 ribosomalproteinS27/ IllribosomalproteinS27pseudogene6/ ectonucleotidepyrophosphatase/phosphodiesterase3 /ribosomalproteinS27pseudogene21 S27pseudogene23ribosomal/proteinS27 DCN1defective,incullinneddylation1domain ectonucleotidepyrophosphatase/phosphodiesterase calmodulinbindingtranscriptionactivator1Akinase(PRKA)anchorproteinyotiao9 FCF1smallsubunit(SSU)processomecomponent PASdomaincontainingserine/threoninekinase SRY(sexdeterminingregionY)-box6 ectonucleotidepyrophosphatase/phosphodiesterase S100calciumbindingproteinA10 containing1(S.cerevisiae) glycerolkinase5(putative) RNAbindingmotifprotein14 oral-facialdigitalsyndrome1 celladhesionmolecule1 homolog(S.cerevisiae) RibosomalproteinL37 zincfingerprotein160 pseudogene13 ribosomalprotein pseudogene29 ribosomalproteinS27pseudogene7 4(putativefunction) HypotheticalLOC339290 basonuclin2 5(putativefunction) GeneTitle KIAA1377

RPS27P13/ RPS27P21/ RPS27P23/ RPS27P29/ RPS27P6/ GeneSymbol RPS27/ RPS27P7 l S100A10 OFD1 DCUN1D1 e HPGD ENPP4 CADM1 NR1D2 CAMTA1 AKAP9 FCF1 PASK LOC339290 RPL37 BNC2 KIAA1377 ENPP3 SOX6 ENPP5 GK5 RBM14 ZNF160

1556239_aat Affymetrix ProbeSetID 236621at 238568_sat 238909at 241751_at 241845at 242428_at 242920at 1569482_at 203913sat 204160s_at 209030_sat 209750_at 213268_at 215483at 215567_at 216945_xat 217506_at 224766at 230722at 232166at 232737sat 235526at 237054_at 238121_at 238623at 239064at 239635_at 239954_at 242673at 242693at US 9 , 809 , 854 B2

0.6418 0.8675 0.7998 0.4315 0.7810 0.5502 0.0990 0.0334 0.2411 0.5826 0.1788 0.7158 0.2472 0.9284 0.8948 0.6500 0.9754 0.9972 0.8960 0.3726 0.3271 0.2435 0.0176 0.7998 0.9511 0.5994 0.9694 0.1584 89130. 0.8960 0.8528 0.2945 0.1279 0.0025 0.4360 0.1511 0.3020 0.1220 0.9973 Effectofflares Estimatep-values o -0.129 0.053 -0.078 0.099 -0.074 -0.107 -0.265 -0.193 -0.138 0.077 -0.160 -0.056 0.113 -0.042 0.055 -0.129 -0.009 0.012 -0.037 -0.122 -0.248 -0.118 -0.309 -0.076 -0.022 0.095 0.016 0.301 0780.- -0.064 0.056 0.259 -0.182 -0.317 -0.101 -0.177 -0.169 -0.187 -0.024

p-values 0.0003 0.0014 0.0077 0.0002 0.0116 0.0014 0.0012 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0019 0.0066 0.0014 0.0003 0.0026 0.0045 0.0002 0.0050 0.0002 0.0002 0.0003 0.0003 0.0002 0.0031 0.0144 0.0033 0.0003 0.0003 0.0300 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0075 Effectofdisease Estimate -0.672 -0.595 -0.636 -0.641 -0.677-0.707 -0.851 -0.799 -0.740 -0.629 -0.740 -0.671 -0.669 -0.655 -0.597 -0.666??? -0.616 -0.585 -0.576??? -0.810 -0.845??? -0.758 -0.923 -0.632 -0.707 -0.652 -0.616 -0.484 -0.741 -0.694 -0.614 -0.573 -0.807???? -0.918 ??? -0.828 -0.786??? ???-0.861 -0.805??? -0.674??? SLE 163.3 279.9 144.4 1338.0 124.0 63.9 183.3 1729.4 826.7 980.9 3406.1 567.2 102.9 210.0 322.6 285.3 378.7 104.0 50.1 65.9 25.8 246.7 84.3 130.3 156.7 41.5 16.6 103.8 237.2 245.6 52.1 142.0 335.2 131.7 128.2 1634.9 70.5 1798.5 342.8

MeanValues 246.4 437.3 232.6 202.4 120.3 260.4 895.8 166.1 320.3 494.9 433.6 588.8 177.6 78.1 104.1 46.1 399.2 130.1 202.3 251.6 67.7 27.4 178.7 376.3 386.7 80.8 211.3 523.0 225.4 205.5 108.9 539.2 HD 2010.2 2600.4 1306.9 1457.7 5181.8 2518.7 2812.0

FoldChange 0.64 0.64 0.64 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.61 0.61 0.61 0.61 0.61 0.61 0.61 0.61

EntrezGene 3075/ 64417 255394 5066 6431 5616 3823 1975 26053 5066 445347 /6967 57583 4756 9159 6670 960 7050 51449 8707 26056 27122 9440 79188 8028 9765 6601 4199 4145 51276 11043 445347 3078 445347 /6967

TABLE9-continued PatientsGeneTranscriptsDifferentiallyExpressedinSLEGeneTranscriptsDifferentiallyExpressedinSLEPatients

protein/TalternatereadingframegammaTCR TCRgammaalternatereadingframeprotein/T killercelllectin-likereceptorsubfamilyC,member3 subtilisintype7convertase/kexinproprotein RAB11familyinteractingprotein5(classI) dependentregulatorofchromatin,subfamilyc eukaryotictranslationinitiationfactor4B leukemia(trithoraxhomolog,Drosophila); malicenzyme1,NADP(+)-dependentcytosolic complementfactorH/ Chromosome5openreadingframe28 peptidylglycinealpha-amidatingmonooxygenasesplicingfactor,arginine/serine-rich6 peptidylglycinealpha-amidatingmonooxygenase CD44molecule(Indianbloodgroup) polypeptide2galactosyltransferase, laevisXenopus)homologdickkopf3( megakaryocyte-associatedtyrosinekinase TCRgammaalternatereadingframeprotein neogeninhomolog1(chicken) UDP-Gal:betaGlcNAcbeta1,3 Myeloid/lymphoidormixed-lineage containing16FYVEdomainfinger,zinc SWI/SNFrelated,matrixassociatedactin t-complex11(mouse)like2 autismsusceptibilitycandidate2 cellreceptorgammaconstant2 TGFB-inducedfactorhomeobox1 prenylcysteineoxidase1 mediatorcomplexsubunit17 Transmembraneprotein43 cellreceptorgammaconstant2 linkedkinase-protein,Y protein181transmembrane Sp3transcriptionfactor translocatedto,10 zincfingerprotein571 GeneTitle member2 midline2 related1

GeneSymbol TARP/ CFH/CFHR1 TARP/ C5orf28 TCP11L2 PAM SFRS6 PRKY KLRC3 EIF4B AUTS2 PAM TRGC2 TMEM181 NEO1 PCSKT SP3 CD44 TGIF1 PCYOX1 B3GALT2 RAB11FIP5 DKK3 MED17 TMEM43 MLLT10 ZFYVE16 SMARCC2 ME1 MATK ZNF571 MID2 TARP TRGC2

1557828_aat Affymetrix ProbeSetID 1559413_at 1566608_at 202336_sat 206108_sat 206279at 207723_sat 211937_at 212599_at 212958_xat 216920_sat 225127_at 225270_at 229692_at 232431_at 232521at 232529_at 1565868_at 1566901at 203803_at 210121_at 210879_sat 214247_sat 232483_st 233480_at 237542_at 238257at 240859at 244425at 1557300sat 1561973at 204059_sat 206267_sat 206648at 209733_at 209813_xat 215388_sat 215806_xat 232889_at US 9 , 809 , 854 B2 74

p-values 0.3510 0.9284 0.4156 0.4871 0.1300 0.8553 0.9511 0.1353 0.1502 0.2862 0.0034 0.0791 0.8948 0.0335 0.1074 0.5826 0.8972 0.9973 0.7998 0.0293 0.6456 0.1747 0.4453 0.6382 0.0176 0.8675 0.1464 0.6382 0.0896 0.3086 0.8892 0.7233 0.9199 0.3033 0.9973 0.1433 0.6003 0.1258 0.3318 0.0939 0.5309 0.8972 0.8553 Effectofflares Estimate -0.142 0.023 -0.161 -0.161 0.219 -0.035 0.015 -0.134 -0.188 -0.155 -0.456 -0.164 -0.035 -0.348 -0.149 0.128 -0.020 0.002 -0.076 -0.385 -0.097 -0.223 -0.161 -0.150 -0.212 -0.084 -0.150 -0.095 -0.126 -0.111 0.092 -0.058 0.054 0.226 0.004 -0.203 -0.101 -0.230 -0.144 -0.232 -0.065 -0.051 0.075

p-values 0.0002 0.0210 0.0002 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0021 0.0002 0.0002 0.0003 0.0012 0.0002 0.0002 0.0002 0.0019 0.0002 0.0093 0.0002 0.0009 0.0002 0.0003 0.0002 0.0002 0.0031 0.0077 0.0057 0.0002 0.0002 00020. 0.0003 0.0002 0.0002 0.0003 0.0002 Effectofdisease Estimate -0.852 -0.652 -0.802 -0.763 -0.627 -0.729 -0.798 -0.807 -0.811 -0.863 -1.002 -0.885 -0.779 -0.998 -0.844 -0.609 -0.781 -0.751 -0.757 -1.010 -0.812 -0.896 -0.821 -0.821 -0.914 -0.721 -0.882 -0.841 -0.847 -0.800 -0.817 -0.783 -0.769 -0.605 -0.678 -0.992 -0.816 -0.942 -0.901 -0.892 -0.844 -0.774 -0.795 SLE 127.7 190.6 239.3 321.2 76.2 23.0 233.8 289.0 1670.0 30.5 111.1 46.2 324.5 66.1 66.3 559.6 33.1 110.9 187.9 681.8 81.7 380.1 352.8 141.5 399.2 179.9 83.3 28.3 364.7 433.4 255.9 116.1 164.2 48.7 350.9 3359.6 123.2 409.6 2753.4 50.5 77.6 207.1 36.4

MeanValues 211.5 304.1 381.0 502.0 117.3 38.7 370.7 446.2 55.0 168.9 77.0 527.2 102.2 108.6 871.4 52.3 181.6 330.8 938.1 134.7 626.5 578.8 228.0 688.0 276.9 139.6 46.5 694.1 412.7 201.2 285.5 85.2 564.6 213.5 705.0 101.1 141.2 340.9 67.7 HD 2602.4 618.8 5524.3 4993.3

FoldChange 0.61 0.61 0.61 0.61 0.61 0.61 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.58 0.58 0.58 0.580.58 0.58 0.58 0.58 0.58 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 EntrezGene 5729 23177 284756 40161222875 10219 445347 /6967 1305 57125 56172 4090 4163 6574 54796 9528 2624 51668 130872 29883 6165 2254 1285 9265 55007 158248 27332 3820 149628 85379 11076 4094

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

TCRgammaalternatereadingframeprotein/T Solutecarrierfamily20(phosphatetransporter),member1 killercelllectin-likereceptorsubfamilyG,member1 heatshockproteinfamilyB(small),member11 fibroblastgrowthfactor9(glia-activating) familywithsequencesimilarity118,memberA killercelllectin-likereceptorsubfamilyB,member1 prostaglandinD2receptor(DP) chromosome20openreadingframe197mitochondrialcarriertriplerepeat6 ectonucleotidepyrophosphatase/phosphodiesterase ankylosis,progressivehomolog(mouse) AHAI,activatorofheatshock90kDaprotein CCR4-NOTtranscriptioncomplex,subunit7 collagen,typeIValpha3(Goodpastureantigen) repeatdomain16tetratricopeptide pyrinandHINdomainfamily,member1 tubulinpolymerizationpromotingprotein centrosomalprotein68kDa cellreceptorgammaconstant2 collagen,typeXIIIalpha1plexindomaincontaining1 SMADfamilymember5mutated incolorectalcancers transmembraneprotein59 GATAbindingprotein2 ATPasehomolog2(yeast) v-mafmusculoaponeuroticfibrosarcomaoncogene 4(putativefunction) ribosomalproteinL35a Zincfingerprotein638 homolog(avian) GeneTitle basonuclin2 cytohesin3 KIAA1671protein

CTA-22169.4 GeneSymbol TARP/ PTGDR CEP68 C20orf197 MCART6 ENPP4 KLRG1 TRGC2 COL13A1 PLXDC1 ANKH SMAD5 ??? SLC20A1 BNC2 TMEM59 GATA2 HSPB11 AHSA2 CNOT7 RPL35A FGF9 COL4A3 CYTH3 FAM118A TTC16 ZNF638 KLRB1 PYHIN1 TPPP MAF

1561578s_at 1566324_aat Affymetrix ProbeSetID 234165_at 236419_at 239442at 239808at 1553311at 204161_sat 210288_at 211144_xat 211343sat 219700at 220076_at 225219at 226225at 227273_at 230494_at 238478_at 241018_at 241036_at 209710at 214163at 226665_at 233019_at 233713_at 238026at 243915at 206404at 222073at 225147_at 226475_at 230543at 236833at 239243at 243578at 1563473at 214470_at 216748at 225525_at229026 _at 229147_at 230104_sat 233406_at US 9 , 809 , 854 B2 75

p-values 0.9880 0.0405 0.6131 0.3020 0.7998 0.1544 0.8960 0.0207 0.6234 0.6341 0.5410 0.8135 0.1112 0.8762 0.8528 0.0376 0.9426 0.0702??? 0.8528 0.3624 0.0025??? 0.1966??? 0.2347 0.6199??? 0.7549 0.8528??? 0.1788??? 0.4268 0.1444??? 0.1006?????? 0.0034 0.3099??????? 0.2710 0.9269 0.1223 0.9269 0.1155 0.3079 0.1223 Effectofflares Estimate 0.001 -0.211 -0.076 0.153 0.068 -0.244 0.068 -0.268gen -0.100 -0.094 0.170 -0.062 -0.222 -0.050 -0.082 -0.307 -0.003 -0.255 0.123 -0.093 -0.272 -0.114 -0.088 -0.038 0.057 -0.162 -0.129 -0.174 -0.200 -0.353 -0.171 -0.144 -0.029 -0.148 -0.030 1840.- -0.138 -0.160

p-values 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0003 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002-1063 0.0002-896 Effectofdisease -0.8700002 -0.9610002 -0.9350002 -0.7440006 -0.7780002 -0.9760006 -0.8600002 -1.00400002 -0.9750002 -0.9220002 -0.7300019 0.0002-881 0.0012-886 -1.17300002 -0.9220002 -1.12800002 -0.8120021 -1.00400002 -1.37900012 Estimate -1.103 -1.008 -1.017 -1.101 -1.028 -1.109 -1.076 -1.111 -1.156 -1.256 -1.224 -1.222 -1.153 -1.222 -1.290 -1.279 -1.221 -1.289

49.5 2815.3 42.4 403.9 31.9 306.3 439.8 78.6 202.7 1226.6 119.4 35.1 157.6 128.7 216.2 162.6 70.7 90.5 168.5 298.0 1227.5 189.5 512.5 294.9 345.5 129.4 2976.8 246.0 162.9 847.1 1774.2 458.1 1220.9 98.0 99.4 72.5 154.1 211.9 142.3 SLE

MeanValues 87.6 4900.1 73.0 690.7 55.2 479.7 731.1 141.6 328.9 2196.9 216.7 59.7 275.7 221.4 406.9 282.3 137.6 169.6 308.5 577.2 1769.7 304.8 915.6 547.2 646.1 281.6 5639.7 471.4 358.2 1858.6 3612.2 940.9 2468.6 236.9 240.5 161.5 291.2 451.0 283.3 HD

FoldChange 0.56 0.56 0.56 0.56 0.56 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.54 0.54 0.54 0.53 0.53 0.53 0.53 0.52 0.52 0.52 0.52 0.51 0.49 0.49 0.49 0.49 0.48 0.48 0.47 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 EntrezGene 22836 6095 221002 3067 23236 26049 6228 8320 79844 3486 79844 26051 4675 54206 94120 4092 2205 4747 339988 5521 23022 22822 7049 5797 59352 162966 4603 11098 7049 23705 23127 10842 54477 90102 284367

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatients

proteinphosphatase1,regulatory(inhibitor)subunit16B proteintyrosinephosphatase,receptortypeM. pleckstrinhomologydomaincontaining,familyA pleckstrinhomology-likedomain,familyBmember2 phospholipaseC,beta1(phosphoinositide-specific) familywithsequencesimilarity169,memberA laevisXenopus)homolog(eomesodermin nucleosomeassemblyprotein1-like3 proteinphosphatase2(formerly2A),regulatory pleckstrinhomology-likedomain,familyA transforminggrowthfactor,betareceptorIII transforminggrowthfactor,betareceptorIII glycosyltransferase25domaincontaining2 affinityI,highfragmentofIgEFc leucine-richrepeatcontainingGproteincoupled readingframe16chromosome7open sialicacidbindingIg-likelectinpseudogene,3 RasGEFdomainfamily,member1A zincfinger,DHHC-typecontaining11insulin-likegrowthfactorbindingprotein3 zincfinger,DHHC-typecontaining11 ERBBreceptorfeedbackinhibitor1 v-mybmyeloblastosisviraloncogenehomolog Rho-relatedBTBdomaincontaining3RARrelated-orphanreceptorA like3synaptotagmin- SMADfamilymember7 receptorfor;alphapolypeptidepolypeptideneurofilamentlight, proteinassociatedpalladincytoskeletal, ribosomalproteinS23 LOC339988proteinhypothetical subunitB,betaisoform zincfingerprotein600 protease,serine23 celladhesionmolecule1 GeneTitle histidinedecarboxylase member1 6receptor like1avian)-( member5

GeneSymbol n RHOBTB3 RORA RASGEF1A HDC PLCB1 FAM169A RPS23 EOMES ZDHHC11 IGFBP3 ZDHHC11 PPP1R16B NAP1L3 ERRFI1 SYTL3 SMAD7 FCERIA NEFL LOC339988 PPP2R2B PALLD PHLDA1 TGFBR3 PTPRM LGR6 ZNF600 MYBL1 PRSS23 TGFBR3 CADM1 GLT25D2 C7orf16 PLEKHA5 PHLDB2 SIGLECP3

1552283_sat 1562698x_at 1555579_sat Affymetrix ProbeSetID 202975_sat 226682_at 226777_at 230563_at 1557459at 207067s_at 213222at 213954_at 227722at 231776_at 244726_at 210095_sat 221646_sat 233813_at 204749at 224657at 232752_at 1562255at 204790_at 211734_sat 221916at 213849_sat 200897_sat 225842_at 226625_at 227819at 242463xat 213906_at 202458at 204731at 209031at 209883_at 220231_at 220952_sat 225688_sat 232686_at US 9 , 809 , 854 B2 77

p-values 0.1474 0.7312 0.8553 0.0935 0.8553 0.7586 0.3340 0.1984 0.3099 0.3383 0.7356 0.2419 0.5981 0.6274 Effectofflares Estimate -0.214 -0.100 -0.038 -0.246 -0.034 -0.186 -0.107 -0.138 -0.124 -0.168 -0.054 -0.173 -0.077 0.122

p-values 0.0002 0.0002 0.0006 0.0002 0.0002 0.0026 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 Effectofdisease Estimate -1.310 -1.194 -1.182 -1.417 -1.314 -1.273 -1.347 -1.390 -1.445 -1.632 -1.568 -1.726 -1.894 -1.756 SLE 325.9 226.4 225.4 124.5 29.2 74.4 225.5 108.8 434.4 311.1 14.0 27.9 86.1 73.5

MeanValues 267.2 78.3 129.2 47.8 88.0 HD 701.9 491.8 467.9 540.0 271.8 1077.2 790.7 263.0 182.8

FoldChange 0.44 0.44 0.43 0.43 0.41 0.41 0.40 0.40 0.39 0.35 0.35 0.32 0.29 0.28 EntrezGene 55605 440823 55007 407738 9891 83416 9254 79966 54843 11098 167681 4646 9638

TABLE9-continued GeneTranscriptsDifferentiallyExpressedinSLEPatientsGeneTranscriptsDifferentiallyExpressedinSLEPatients

familywithsequencesimilarity19(chemokineC—motif)-like, fasciculationandelongationproteinzeta1(zygin myocardialinfarctionassociatedtranscript(non familywithsequencesimilarity118,memberA calciumchannel,voltage-dependentalpha2/delta NUAKfamily,SNF1-likekinase1 OTE:BoldedareprobesetsshowingsignificanteffectfromFLAREbasedonp-valuesobtainedbyMarcovchainMonteCarlosampling kinesinfamilymember21A stearoyl-CoAdesaturase5like2synaptotagmin - proteincoding) Fcreceptor-like5 protease,serine23 protease,serine35 GeneTitle A1member subunit2 VImyosin

GeneSymbol KIF21A MIAT FAM118A FAM19A1 NUAK1 FCRL5 CACNA2D2 SCD5 SYTL2 PRSS23 PRSS35 MYO6 FEZ1 Affymetrix ProbeSetID 226003_at 228658at 219629at 230923_at 204589_at 231647_sat 204811_sat 224901_at 225496sat 226279_at 235874_at 203216_sat 203562_at 1560263_at US 9 ,809 ,854 B2 79 80 Example 3 The data herein demonstrated that three distinct groups of patients , defined by gene expression analysis of multiple Proteomic Patterns among Groups of SLE Patients patient peripheral blood and plasma samples collected over Autoantibodies in the form of immune complexes con - 5 time, were associated with distinct autoantibody and cytok tribute to lupus pathology , and distinct autoantibody speci ines profile in serum samples. Patients with the presence of ficities have been associated with particular clinical mani festations of disease. The titer of 40 autoantibodies for each IFIG and neutrophil- related signature were associated with SLE visit was determined , using multiplex analysis of higher plasma levels of TNFa , IL - 8 and IL - 18 . Patients with plasma samples. Five autoantibodies were significantly neutrophil granule signature were associated with a high deregulated (FC > 1. 5 ; p < 0 .05 , 5 % FDR ) between SLE level of anti -SSA /Ro autoantibodies. TABLE 10 Autoantibodies and plasma factors differentially expressed between SLE patients and healthy donors (minimum fold change 1 . 5 ; p < 0 .05 ) . HD vs SLE Flare Analyte Fold Change Estimates p - values Estimates p - values Autoantibodies Double Stranded DNA ( ds DNA ) 2 . 97 0 .0037 0 .86 0 .0016 Antibody # SSA Antibody 7. 3 2 . 90 0 .0166 - 0 .22 0 .2806 RNP ( c ) Antibody# 2 . 9 1 .43 0 . 1136 0 .65 0 .0016 Histone Antibody# 1 . 5 0 . 49 0 . 1939 0 . 83 0 . 0000 Histone H2b Antibody# 1. 5 0 .46 0 .0971 0 .61 0 . 0016 Plasma factors ( inflammatory biomarkers )

1 . 90 0 . 0004 - 0 .23 0 . 7343 Prolactin ?? B - Lymphocyte Chemoattractant (BLC ) :??? 1 . 71 0 .0117 0 . 36 0 . 1030 IL - 10 # ? 1 . 26 0 . 0003 0 . 99 0 .0002 IP - 10 ( Inducible Protein - 10 ) # ?? 1 . 46 0 . 0020 0 .54 0 . 0212 ? MIP - 3b ? 1 . 14 0 . 0050 0 . 15 0 . 5961

= TNF RII # ? 0 . 96 0 . 0084 0 . 85 0 . 0000

= MIP - 1 beta ? 0 .91 0 . 0248 0 . 12 0 . 8073 TNF -alpha # 2 .0 0 . 91 0 . 0248 0 . 78 0 .0209 IL - 8 # 1. 9 0 . 86 0 . 0272 0 . 93 0 . 0457 BAFF 1 . 8 0 . 81 0 . 0084 0 . 16 0 . 7343 C Reactive Protein # 1 .7 0 . 49 0 . 5629 3 . 77 0 .0000 Beta - 2 Microglobulin # 1 .7 0 . 68 0 . 0268 0 . 48 0 . 0000 IL - 18 # 1. 6 0 .69 0 .0125 0 . 72 0 . 0002 IL - 6 # 1 . 6 0 .66 0 . 0084 0 . 56 0 .0212 MCP - 2 1. 5 0 .85 0 .0188 0 . 23 0 . 3031 von Willebrand Factor# 1 . 5 0 . 43 0 . 1904 1 . 10 0 . 0000 IL - 23 # 1. 5 0 .47 0 . 2282 - 0 .67 0 . 0406 Eotaxin # 0 . 6 - 0 . 38 0 . 5629 - 0 . 77 0 . 0002 Ferritin # 0 . 4 - 1 . 44 0 . 0855 2 .41 0 .0000 The effect of disease and flare on the level of autoantibodies is illustrated by fold increase and 5 % FDR with corrected p - values. # identifies those autoantibodies and plasma factors that showed a significant effect from flares . patients and HDs based on linear mixed model analysis Example 4 ( Table 10 ) . Plasma anti - dsDNA , anti -histone , anti -histone H2b and anti -RNP ( c ) antibodies increased significantly dur Cross- Sectional Evaluation of PBMC Profiles in ing the flares . 50 There was also an observed significant and striking SLE Patients increase of anti - SSA /Ro autoantibodies in group C of SLE patients ( p < 0 .05 ) (FIG . 3A ) . A new unsupervised hierarchical clustering was per Analysis of 41 inflammatory biomarkers identified | 1019 formed based on transcripts assigned to the top 3 clusters : factors with significantly different plasma levels in SLE and 55 IFIG , neutrophil granules and neutrophil related signatures HD samples ( FC > 1 . 5 , p < 0 . 05 , 5 % FDR ) ( Table 10 ) . Among and obtained similar classification of SLE patients ( groups the inflammatory biomarkers identified , prolactin showed A , B and C ) as shown in the dendrogram (FIG . 4 ) . Based on the greatest increase in SLE patients compared with HDs. linear mixed model the majority of those transcripts remain Linear mixed model analysis demonstrated a significant upregulated even during non - flaring visits , meaning that it is effect of 14 analytes on lupus disease and an overlapping but 60 likely that cross -sectional analysis would also classify SLE not identical group of 13 plasma factors ( p < 0 .05 , 5 % FDR ) patient into similar group . To evaluate this hypothesis , on lupus flares , as indicated in Table 10 . hierarchical clustering was performed 500 times, each time Inflammatory biomarker data were compared in the selecting a random visit for each patient. The attached color patient groups defined by gene expression analysis . Patients code in FIG . 4 indicates how often particular subject in group B showed a higher level of tumor necrosis factor a 65 remained in the same group when the data from random ( TNF a ), IL - 8 and IL - 18 . ( p < 0 .05 for all ) (FIGS . 3B , C , and visits were used instead of taking an average . It was clear D ). that the majority of patients from category A and C remain US 9 , 809, 854 B2 82 in the same branches of dendrogram irrespective of visit . from group B2 and C 1 (non - significant ) . Group B2 also Patients from group B were less stable and more often were showed higher incidence of serositis disorders (OR 2 . 8 misclassified . However overall it remained possible to clas ( 1 . 0 - 10 ) p < 0 . 05 ). ACR criteria for renal disorders , which sify patients based on a single visit . represent one of the major challenges for lupus patients , Quantitative real time PCR (RT - PCR ) analysis was per - 5 were detected among patients from group C1 and C2 . formed for an additional set of 59 SLE patients . Seven genes The most illustrative parameter of disease activity is lupus were chosen from top three clusters ; IFIT1 and IFIT3 flare. In order to evaluate parameters , such as flares, which represented IFIG signature, CEACAM6 and LTF repre vary over the time, a generalized linear mixed model as sented neutrophil granules signature and TNFAIP6 , described in Example 1 was used . Based on SELENA CYP4F3 and FPR1 represented neutrophil related signature . 10 SLEDAI index , flares can be categorized to severe and Level of HPRT1 remained constant in peripheral blood of mild /moderate . The rate of total, severe and mild /moderate SLE patients based on microarray data and it was chosen as flares per year for each category was compared first . Patients housekeeping gene . See FIG . 5 . from group B1 and C2 developed an average of 1 . 9 ( 1 . 3 - 2 . 8 ) Based on results of RT- PCR , SLE patients were classified flares/ year , and 1 . 6 ( 1 . 2 - 2 . 4 ) flares /year , respectively . That to similar groups A , B and C , according to unsupervised 15 was significantly higher (p < 0 .03 and p < 0. 01 , respectively ) hierarchical clustering : group A lacked either signature ; in comparison to that of group A which had 1 . 1 ( 0 . 8 - 1 . 5 ) group B showed IFIG signature , but lacked neutrophil flares /year . With regard to severe flare occurrences, the granules signature ; and group C showed prominent neutro - highest rate of severe flares was for groups C1 ( 0 . 6 ( 0 . 2 - 1 . 6 ) phil granules signature . flares/ year) and C2 ( 0 . 6 ( 0 . 3 - 1 . 4 ) flares /year ). That was As more samples were studied further classifications 20 significantly higher rate ( p < 0 . 03 and p < 0 .01 , respectively ) in could be made .Group B could be subdivided in two groups, comparison with group A ( 0 .2 flares/ year ). B1 and B2 , based on absence or presence of neutrophil SLEDAI is a useful instrument to evaluate disease activity related signature . Group C could also be subdivided into two over time. In this study SLEDAI score tended to be higher groups , C1 and C2, based on the absence or presence of IFIG in groups B2 and C2 , without reaching significance . BILAG signature. Among the 59 SLE patients used for validation a 25 score is an organ specific index , based upon the physician ' s new visit of patient IF58 , which was from our initial set of intention to treat the patient, which scores SLE disease patients , were used to confirm the absence of IFIT1 and activity in eight organ -based systems ( American College of IFIT3 expression for that patient. Rheumatology Ad Hoc Committee on Systemic Lupus Ery In summary , classification of SLE patients based on the thematosus Response Criteria 2004 ). BILAG scores were IFIG , neutrophil - granules and neutrophil related signatures 30 significantly higher among SLE patients from group C2 ( 4 subdivides SLE patient in 5 groups . ( 4 . 5 - 7 . 6 ) , p < 0 .02 ) . Subsequently , the statistical analysis was focused on Example 5 components of the BILAG score . The rate of visits of patients with BILAG score A , B and C per year, were Clinical Characteristics of Defined SLE Patient ' s 35 calculated . The grades A , B and C of BILAG score indicate Groups current involvement of a system in pathological process . Based on components of BILAG score , it was observed that It was hypothesized that classification of SLE patients patients from group B2 , who showed prominent IFIG and based on hierarchical clustering of mRNA data might reflect neutrophil - related gene signatures, were more prone to have distinct clinical phenotypes . 40 mucocutaneous involvements ( estimate 2 . 3 ( 1 . 6 - 3 . 3 ) visits / Demographic data for five groups of SLE patients were year, p < 0 .04 ). In contrast , patients from group C1, lacking summarized in Table 11. In general , the prevalence of SLE IFIG signature , rarely display mucocutaneous involvement is far higher in females than in males . In our study 11 out of ( estimate 0 . 4 ( 0 . 2 - 0 . 8 ) visits / year, p < 0 . 01 ) . 81 patients were males and male /female ratio was close to The next prominent difference was that patients with the frequently reported ratio of 1 : 9 . Unexpectedly , the 45 neutrophil granule gene signature , groups C1 and C2 , were distribution of male patients among groups was biased with more prone to develop vascular involvements (estimate 1 . 3 significant prevalence of male patients in group C1 (OR 5 . 6 , (0 . 7 - 2 . 3 ) visits / year p < 0 . 05 and 1 . 7 ( 1 . 1 - 2 . 6 ) visits / year 95 % CI (1 . 5 -41 ) , p < 0 .03 ) . Groups A and B1 have minor p < 0 .01 , respectively ) . Patients showing both neutrophil proportion ofmale cases . Interestingly , patients from group granule and IFIG gene signatures, group C2 , were more B1 were significantly older at the first visit (median age 41 50 prone to manifest renal flares by BILAG ( estimate 1 . 5 years ( range 19 - 52 years p < 0 .04 ) , and preferentially Euro - ( 1 - 2 . 4 ) visits / year p < 0 .05 ) . Groups B2 and Ci, both dis pean Americans ( 4 out of 6 , OR 3 . 4 95 % CI ( 1 . 0 - 17 ) , playing IFIG signatures , showed increase rate of general p = 0 .05 ) . No other difference in patient' s demographic was involvement (estimate 3 .1 ( 2 .3 -4 . 2 ) p < 0 .01 and 2 .6 (2 - 3. 5 ) observed . p < 0 .01 visits / year, respectively ) . Major clinical characteristics for SLE patients, including 55 In summary , it was observed that patients from group C2 ACR criteria for SLE , flare status, components of SLEDAI were more active and more often developed renal disease , and BILAG scores , were collected longitudinally for each which is a significant clinical challenge for SLE patients. patient visits . ACR score and ACR criteria for the last follow The fact that patients from group C2 were more active visit for each patient was compared among groups ( Table was further supported by the observation that the overall 12 ) . There was no difference in ACR score between groups . 60 dose of prednisone over the last month was higher for this However , no patients from group C 1 developed photosen - group compared to others ( 12 . 3 ( 9 . 2 - 15 .6 ) mg/ day p < 0 .01 ) . sitivity , in contrast to other groups (OR 0 . 1 95 % CI ( 0 - 2 . 0 ). These data are summarized in Table 13 . FIG . 6 shows a p < 0 . 05 ) . There were patients from group B1 and C2 whom graphical representation of each group with regard to severe developed photosensitivity more often (non - significant ) . flares, and clinical manifestations , including mucocutane Patients from group C 1 were more prone to develop 65 ous, renal, and vascular. neurological disorders (non - significant) . At the same time, The results herein show that mRNA transcripts differen there were no cases of discoid disorders among patients tially expressed among patient groups A , B1, B2, C1 and C2 US 9 ,809 ,854 B2 83 84 were not only associated with different degrees of disease pathological mechanism among the groups was also differ activity and frequency of clinical flares, but might also ent, as groups C1 and C2 were more prone to vascular identify distinct pathogenic mechanisms associated for involvements , while groups B1 and B2 were more prone to instance with mucocutaneous versus vascular involvement. mucocutaneus disorders . See Table 13 . The results of laboratory blood tests collected longitudi- 5 nally for the majority of the patients were also compared . TABLE 11 Group C2 of SLE patients , in accordance with their clinical manifestations , showed non - significant increase in erythro Demographic characteristics obtained for groups cytes sedimentation rate (ESR ) ( 35 . 8 mm /h ( 31 . 1 -40 . 4 ) N / S ) of SLE patients ( A , B1, B2 , C1, C2) and C -reactive protein concentration (CRP ) ( 1 . 3 mg/ dL 10 - ( 1- 1. 5 ) N /S ). Compared to other groups , significant decrease in lymphocytes count ( (ALC ) for group C2 was observed A B1 B2 C1 C2 ( 1 . 12 / pt ( 0 . 98 - 1 . 25 ) p < 0 .02 ) . Level of complement C3 was Total 22 9 15 6 2u|29 also low in group A (84 mg/ dL (78 - 91 ) , N / S ) , whereas group Sex Male om B1 had significantly higher level of C3 compared to other 15 ( % ) ( 5 % ) of( 0 % ) ( 20 % ) (50 % ) * (14 % ) groups (96 mg/ dL (87 - 106 ) p < 0 .02 ) . SLE patients , from p - value ogmere 0 .03 group C1, where male prevalence was noted , had the lowest Age , years 25 .5 41 28 29 .5 28 erythrocytes sedimentation rate ( 13 . 4 mm / h (3 . 8 -23 . 4 ) ( range ) ( 14 - 57) ( 19 - 52 ) * ( 17- 50) ( 25 -43 ) ( 17- 49 ) p < 0 .01 ) , and at the same time showed increased neutrophil 0 . 036 counts ( ANC ) in peripheral blood (5 .67 /uL ( 4 .64 - 6 .69 ) 20 Disease duration , 4 .5 4 3 o6 3 p < 0 .02 ) . These results are summarized in Table 14 . years (0 -24 ) (0 -11 ) (0 -19 ) (0 - 16 ) nganari(0 -23 ) The classification of SLE patients based on the selected ( range ) mer- IFIG , neutrophil - granules and neutrophil related genes was Asian Americans 2 1 1 -1 2 capable of sub - dividing SLE patient in groups , with distinct (9 % ) ( 11 % ) (7 % ) ( 17 % ) (7 % ) clinical, laboratory and demographic association . In general, 25 BlackBlack AmericansAmericans 9 -1 4 2 14 SLE patients from group A and B1 received less doses of ( % ) (41 % ) ( 11 % ) (27 % ) ( 33 % ) ( 48 % ) prednisone in the course of their disease as compared to European Americans 5 4 2 2 2 other groups ( 5 .6 ( 3 - 8 ) mg/ day and 5 . 8 ( 1 . 6 - 10 . 4 ) mg/ day , ( % ) (23 % ) (44 % )* (13 % ) (33 % ) (7 % ) respectively ) , while SLE patients from group C2 received p -value 0 . 06 0 .07 significantly higher doses ( 12. 3 ( 9 . 2 - 15 . 6 ) mg/ day p < 0 .01 ) 30 Hispanic Americans 5 2 8 -1 1111 ( Table 13 ) . Moreover, patients from group C2 were more ( % ) ( 23 % ) (22 % ) (53 % ) ( 17 % ) ( 38 % ) prone to develop flares ( including severe flares ). Those , Other 1 1 0 0 0 along with BILAG score and rate of renal involvement, ( 5 % ) ( 11 % ) ( 0 % ) (0 % ) ( 0 % ) indicated that group C2 of SLE patients was associated with -| a more dangerous course of disease . That group was also 35 NoteNote : | associated with higher levels of anti -SS - A (Ro ) autoantibody Confidence intervals and p - values were obtained for categorical data using odds ratio test ( FIG . 7 ) . Although groups B2 and C1 of SLE patients were for small samples . Confidence intervals and p - values for continuous parameter such as age , was calculated using generalized linear model ( see details in methods) . Significantly less active than group C2 , those patients remain under the different parameters were bolded and labeled with asterisk mark . higher risk compared to groups Al and B1 . Interestingly the TABLE 12 Average ACR score and number of individuals correspond to ACR criteria for groups of SLE patients ( A , B1, B2 , C1 , C2 ) A B1 B2 C1 C2 ACR score , al6 5 6 ( range ) ( 3 - 9 ) ( 3 - 7 ) ( 2 - 9 ) ( 4 - 6 ) ( 2 - 9 ) Malar Rash , 13 4 16 ( % ) (59 % ) ( 44 % ) ( 60 % ) (33 % ) ( 55 % ) Discoid Rash , 3 2 6 ( % ) ( 14 % ) ( 22 % ) ( 0 % ) ( 0 % ) ( 21 % ) Photosensitivity , 15 ( 27 % ) ( 56 % ) (33 % ) (0 % ) (52 % ) p - value 0 .05 Oral Ulcers , 10 ( % ) ( 36 % ) (22 % ) ( 33 % ) (33 % ) ( 34 % ) Arthritis , 21 13 5 24 (95 % ) (89 % ) (87 % ) (83 % ) (83 % ) Serositis , 9 2 10 1 12 ( % ) (41 % ) ( 22 % ) (67 % ) ( 17 % ) (41 % ) 0 .04 Renal Disorder , 4 4 18 ( % ) (36 % ) (44 % ) (40 % ) (67 % ) (62 % ) Neurological disorder, 2 1 4 2 6 ( % ) ( 9 % ) (11 % ) (27 % ) ( 33 % ) ( 21 % ) Hematologic disorder , 18 6 10 3 21 ( 82 % ) ( 67 % ) (67 % (50 % ) (72 % ) US 9 , 809 , 854 B2 85 86 TABLE 12 -continued Average ACR score and number of individuals correspond to ACR criteria for groups of SLE patients ( A , B1 , B2 , C1 , C2 ) A B1 B2 ci C2 Immunologic disorder, 21 6 12 24 ( % ) ( 95 % ) (67 % ) ( 80 % ) (83 % ) ( 83 % ) ANA 22 151 ) 28 (100 % ) (100 % ) (100 % ) (100 % ) (97 % ) Note : p - values were obtained for categorical data using odds ratio test for small samples . ANA - indicates positive for antinuclear autoantibodies test.

TABLE 13 Estimated SLEDAI and BILAG scores and estimated rates of visits/ year when flares or systemic involvement occur Estimates (95 % CI) , p - value B1 B2 C1 C2 Flares/ 1. 1 (0 .8 -1 .5 ) 1 .1 (0 .6 -1 . 8 ) 1. 9 (1 .3 - 2 . 8 ) 1. 3 (0 . 8 - 2 .2 ) 1 . 7 ( 1 . 2 - 2 . 4 ) * year 0 .0261 Severe Flares/ 0 . 2 ( 0 . 1 - 0 . 4 ) 0 . 2 ( 0 . 1 - 0 .6 ) 0 . 2 ( 0 . 1 - 0 . 5 ) 0 . 6 (0 . 2 - 1 . 6 ) * 0 . 6 ( 0 . 3 - 1 . 4 ) * year 0 .0296 0 .0036 SLEDAI 3 . 2 ( 2 . 4 - 3 . 9 ) 3 . 4 ( 2 . 5 - 5 . 9 ) 4 . 1 ( 3 . 8 - 7 . 5 ) 2 . 9 ( 1 . 5 - 4 . 3 ) 3 . 7 ( 3 . 1 - 5 .8 ) BILAG 2 . 7 ( 2 . 1 - 3 . 4 ) 2 . 9 ( 2 . 1 - 4 . 8 ) 3 . 1 ( 2 . 6 - 5 . 2 ) 2 .6 ( 1 . 4 - 3 . 9 ) 4 ( 4 .5 - 7 . 6 ) * 0 . 0078 General/ 1 . 8 ( 1 . 4 - 2 . 2) 2 . 2 ( 1. 5 - 3 . 1 ) 3 . 1 ( 2 . 3 - 4 . 2 )* 1. 4 (0 . 9 - 2 . 3 ) 2 . 6 ( 2- 3 . 5 ) * year 0 . 0004 0 . 0046 Mucocutaneous / 1 . 3 ( 1 - 1 . 7 ) 1 . 9 ( 1 . 3 - 2 . 9 ) 2 . 3 ( 1 .6 - 3 . 3 ) * 0 . 4 ( 0 . 2 - 0 . 8 ) * 1 . 4 ( 1 - 2 ) year 0 . 0026 0 . 0047 Neurological 0 .2 (0 .1 - 0 . 3 ) 0 .6 (0 . 2- 1 . 7 ) 0 . 8 (0 . 4 - 2 . 2 ) 0 . 9 ( 0 . 4 - 2 . 6 ) 0 . 6 (0 . 3 - 1 . 6 ) year Muskoskeletal 2. 2 (1 . 8 - 2. 7 ) 1. 7 (1 . 1 -2 . 5 ) 2. 7 (2 -3 . 7 ) 1 . 2 ( 0 . 7 - 2 ) * 2 ( 1 . 5 - 2 . 6 ) year 0 .0244 Cardiac 0 . 1 ( 0 - 0 . 3 ) 0 . 4 ( 0 . 2 - 0 . 6 ) year 0 . 6 (0 . 4 - 0 . 9 ) 0 . 2 (0 . 1 - 0 . 5 ) 0 . 6 (0 . 4 - 1. 1 ) Vascular 0 . 7 (0 . 5 - 1 ) 0 . 9 (0 .5 - 1 . 6 ) 1 . 1 (0 . 6 - 1 . 7 ) 1 . 3 ( 0 . 7 - 2 . 3 ) * 1 . 7 ( 1 . 1 - 2 . 6 ) * year 0 . 0394 0 . 0001 Renal/ 0 .5 (0 .3 - 0 .8 ) 1 .3 (0 .7 - 2 . 3 ) 1. 2 (0 .7 - 2 . 1 ) 1 (0 . 5 - 1. 9 ) 1 . 5 ( 1 - 2 . 4 ) * year 0 .05 Hemotological/ 1. 7 (1 . 4 -2 . 2) 1. 1 (0 . 7- 1 .8 ) 1. 3 (0 . 9- 1 . 9) 1. 2 (0 . 7- 2 ) 2 . 1 ( 1 .6 - 2 .8 ) year Prednisone mg day 5. 6 (3 - 8 ) 5. 8 (1 . 6 -10 .4 ) 8. 9 (5 . 4 - 12. 8 ) 6 .6 (1 . 7 - 11. 5 ) 012 . 001 .3 ( 9. 2 - 15. 6 ) * Note : Flares and Severe flares were determined using SELENA / SLEDAI instruments . SLEDAI?SLE disease activity index 2000 ; BLAG - British Isles Lupus Assessment Group 2000 index ; BLAG score accounts disease activity in eight systems. Disease activity in particular system was considered positive for BLAG equal A , B and C . Estimate indicates average rate of visits per year . The corresponding p - value indicates level of significance in comparison to group A . 50

TABLE 14 Major laboratory findings for groups of SLE patients ( A , B1, B2 , C1, C2 ) . Estimate (95 % CI) , p -value A B1 B2 C1 C2 C3 mg/ dL 8484 9696 88 94 86 (78 -91 ) (87 - 100 )* (81 -95 ) (83 - 105) (81 - 91 ) 0 .02 C4 mg/ dL 18 . 9 18 . 1 16 . 1 16 . 8 18 . 4 ( 16 . 5 - 21. 4 ) ( 14 . 2 - 21 . 8 ) (13 . 4 -18 . 9 ) ( 12. 2 - 21. 3 ) ( 16 . 3 - 20 . 3 ) ESR mm / hr 22 27 . 6 20 . 9 13 . 4 35 . 8 ( 16 .4 -27 .7 ) (18 . 1- 37 . 2 ) (14 . 6 - 27 .4 ) ( 3 .8 - 23. 4 ) (31 . 1 - 40 . 4 ) 0 .0002 CRP mg/ dL 0 . 8 0 . 7 1 1 . 3 Thi ( 0 . 7 - 1 .3 ) (0 . 2 - 1 . 4 ) (0 . 5 - 1 . 1 ) ( 0 .5 - 1 .5 ) ( 1 - 1 . 5 ) US 9 , 809 , 854 B2 87 88 TABLE 14 -continued Major laboratory findings for groups of SLE patients (A , B1, B2, C1, C2) . Estimate (95 % CI) , p -value A B1 B2B2 ci C2 HB mg/ dL 12 12 . 3 12 . 1 12 . 5 11 . 8 ( 11 . 7 - 12 . 4 ) ( 11 . 8 - 12 . 8 ) (11 . 7 - 12 . 5 ) (11 . 9 - 13. 2 ) ( 11 . 6 - 12 . 1 ) WBC / L 6 . 2 6 . 5 5 . 7 7 . 6 6 . 1 ( 5 . 4 - 7 ) ( 5 . 2 - 7 . 7 ) ( 4 . 8 - 6 . 7 ) ( 6 . 1 - 9 ) (5 . 5 -6 . 8 ) ANC /uL 4 . 18 4 . 49 4 .22 5 .67 4 . 46 ( 3 .61 - 4 . 75) ( 3 .56 - 5 . 33 ) ( 3 .58 - 4 .88 ) ( 4 .64 - 6 .69 ) * ( 3 .99 - 4 .92 ) 0 .014 ALC / L 1 . 38 1 . 22 1 . 17 1 . 32 1 . 12 ( 1 . 21- 1 .54 ) ( 0 . 97- 1 .46 ) ( 0 . 98 - 1 .35 ) ( 1 .02 - 1 . 6 ) ( 0 .98 -1 .25 ) * 0 .0144 PLT /uL 0 ..27 0 .. 3 0 . 29 0 .24 0 . 27 (0 . 25 - 0 . 29 ) ( 0 .27 -0 .33 ) (0 .27 - 0 .31 ) ( 0 . 2 - 0 .28 ) ( 0 .25 -0 .29 ) Note : Estimates , confidence intervals and p - values were obtained based on linear mixed model followed by Markov chain Monte Carlo methods for detecting p values.

20 Example 6 were significantly different between SLE and healthy con trols . These were von Willebrand factor (vWF ) , Eotaxin , Biomarkers for Lupus Disease Activity IL - 10 , and anti - Ro antibody. Using Pearson correlation , vWF showed the highest level Using K -means clustering of the microarray data in 25 of significance when relating levels of all patient visits to Example 1, twenty - five of the most significant clusters with SLEDAI score ( R = 0 . 5 ). Moreover , paired t- test analysis the greatest level of variance over time were selected . showed a consistent increase in the level of vWF during Using the generalized linear mixed model analysis severe flare ( p < 0 .02 , mean fold change 1 .6x ) as compared to described in Example 1 , the best combination of transcript both the first non - flaring visit before and the first non - flaring signatures was identified as the interferon , plasma cell. 30 visit after . In generalized linear mixed model analysis for neutrophil related , and neutrophil granule ( Table 15 ) . correlation of plasma factors with SLEDAI score , vWF gave Representative gene transcripts from various clusters the strongest correlation with SLEDAI. Only vWF was were chosen to verify this correlation . Select genes from a significantly higher at the time of flare as compared to non - flare visits . cluster can reflect the entire cluster in relation to disease 35 The level of vWF in plasma was correlated with the activity . The criteria for the selection of the gene transcripts 4 - gene score . As shown by FIG . 9 , level of vWF in plasma was : was significantly correlated with 4 - gene score. FIG . 9 shows 1. Each component of score (probe set ) should be representative examples of fluctuation of vWF ( FIGS . 9A , expressed at a high level and represent functional B , C ) and 4 - gene microarray score (FIGS . 9D , E , F ) in three characteristics of the cluster . 40 selected SLE patients . 2 . The obtained score should reflect SLEDAI score vWF is produced by endothelial cells and is required for ( Spearman correlation ). normal hemostasis and vascular function . Levels of circu 3 . The obtained score should coincide with severe flare lating vWF are increased following endothelial cell damage ( p < 0 .01 ) and show decrease after severe flare . and during acute phase responses . The significantly Using these criteria and the results of generalized linear 45 increased level of vWF during lupus flare in the majority of mixed effects model between the clusters and SLEDAI set SLE patients highlights the role of endothelial injury as a forth in Table 15 , four genes from four different clusters major pathogenic mechanism in SLE and identifies vWF as were chosen for further microarray analysis : IFIT3 from an informative biomarker for patientmanagement and clini C08 , KLRB1 form C10 , CD38 from C27 , and MMP8 from cal studies. C49 . 50 When the transcripts and plasma analytes were combined , the five factors providing the best correlation with SLEDAI TABLE 15 were von Willebrand factor, IL - 10 , interferon transcripts , plasma cell transcripts , and neutrophil granule transcripts . Relationship between SLEDAI and Gene Clusters See FIG . 10 . Number of 5555 Cluster # Cluster Name genes t -value p - value Example 7 C17 Neutrophil -related genes 23 4 . 24 . . 0001 C10 T - cells /iNKT genes 20 - 3 . 42 0 . 0009 Biomarkers for Future Flare Activity C49 Neutrophil granule genes 3 . 37 0 . 0010 C08 IFIG 2 . 99 0 .0033 60 Using microarrays and statistical analysis as described in C27 Immunoglobulin /plasma 1 . 93 0 . 0561 Example 1 , and the 4 - gene microarray described in Example cell genes 6 , and the clinical characteristics described in Example 5 , a correlation between gene expression and future flares was As shown in FIG . 8 , the 4 -gene microarray score for ten sought. SLE patients increased during flare visits . 65 Participating SLE patients were divided into two groups Next using the generalized linear mixed model analysis of according to 4 - gene score level, with those defined as having the plasma analytes in Table 10 four were identified that a high score in the top 25th percentile and those defined as US 9 ,809 , 854 B2 89 90 having a low score in the bottom 25th percentile . The score TABLE 16 was determined at the first patient visit with a SLEDAI score < 4 and identified as time 0 . An increase of > 3 in the SLEDAI Disease Manifestations and SLEDAI > 4 score was considered as a flare event. of high and low flaring SLE patients Based on the Kaplan -Meier plot , SLE patients with a high 5 4 - gene score at time 0 were more likely to develop a flare High ( 11) Low (11 ) P value during the follow - up visits than those with a low 4 - gene Malar rash 7 (64 % ) 4 ( 36 % ) N / S SCscore (estimated level of significance p < 0 . 1 N / S ) . Discoid 3 (27 % ) 1 ( 9 % ) N / S The disease manifestations of these groups , as well as Photosensitivity 6 (55 % ) 2 ( 18 % ) 0 . 091 1 10 Ulceration 6 (55 % ) 3 (27 % ) N / S their number of SLEDAI2K > 4 visits / year, average pred Arthritis 10 (91 % ) 10 (91 % ) N / S nisone intake, and average C3 level is summarized in Table Serositis 7 (64 % ) 2 ( 18 % ) 0 .04 16 . Renal 5 ( 45 % ) 7 (64 % ) N / S The difference between mean level of gene transcript Neurological 2 (18 % ) 3 ( 27 % ) N / S expression between high and low flaring patients for all 15 Hematological 7 (64 % ) 6 (55 % ) N / S non - flaring visits greater than 100 days prior to or after a Immunological 10 (91 % ) 9 (82 % ) N / S severe flare was determined . ANA 10 (91 % ) 11 (100 % ) N / S As shown in FIG . 11 , patients with a low 4 - gene score of SLEDAI2K 24 visits /year 4 . 9 ( 1 . 8 ) 1 . 5 ( + 1 . 8 ) < 0 . 01 * genes comprising IFIT3 , KLRB1, CD38 , and MMP8 at Average oral prednisone 8 (73 % ) 3 (27 % ) 0 . 04 non - flaring visits were significantly less likely to develop 28 mg/ day flares (p < 0 .01 ) . Average C3 level, mg/ dl 72 ( + 11) 97 ( + 22) < 0 .01 * Additionally , as shown in FIG . 12 , unsupervised hierar chical clustering of SLE patients based on the level of 41 deregulated transcripts between high and low flaring Additional genes that could be useful as biomarkers for patient' s groups . The red color of tree correspond to low future flares are listed in Table 17 . These transcripts were flaring SLE patients (labeled as L ), the green tree color different between high and low flaring SLE patients at correspond to highly flaring SLE patients (labeled as H ). non - flare visits . TABLE 17 Gene Candidates for Biomarkers for Future Flares Hi vs p Probe Set ID Gene Symbol Gene Title Low values 206632 _ s _ at uAPOBEC3B apolipoprotein B mRNA editing 3 . 9 0 .0102 enzyme (3B ) 234989 _ at NCRNA00084 non -protein coding RNA 84 1 . 8 0 . 0005 202145 at LY6E lymphocyte antigen 6 complex , locus E 1 . 6 0 .0162 225954 s at MIDN midnolin 1 . 5 0 . 0098 220712 _ at Clorf60 chromosome 8 open reading frame 1 . 5 0 . 0092 60 228806 _ at RORC RAR - related orphan receptor C 0 .7 0 .0047 230708 _ at PRICKLE1 prickle homolog 1 ( Drosophila ) 0 . 7 0 . 0014 209905 at HOXAS homeobox A9 0. 7 0 .0064 201280 s at DAB2 disabled homolog 2 , mitogen 0 . 7 0 .0278 l responsive phosphoprotein 208022 _ s _ at CDC14B CDC14 cell division cycle 14 0 . 7 0 .0367 homolog B NNNNN 235121 at ZNF542 zinc finger protein 542 0 .6 0 . 0074 216905 _ s _ at ST14 suppression of tumorigenicity 14 0 . 6 0 .0005 ( colon carcinoma) 218711 _ s _ at SDPR serum deprivation response 0 . 6 0 . 0422 (phosphatidylserine b . protein ) 212298 _ at NRP1 neuropilin 1 0. 6 0 .0041 217216 _ x _ at MLH3 mutL homolog 3 ( E . coli) 0 . 6 0 .0006 203819 _ s _ at IGF2BP3 insulin -like growth factor 2 mRNA 0 . 6 0 .0236 binding protein 3 214651 sat HOXA9 homeobox A9 0 . 6 0 .0153 214469 _ at HIST1 H2AE histone cluster 1, H2ae 0 . 6 0 . 0348 214307 at HGD homogentisate 1 , 2 - dioxygenase 0 . 6 0 . 0449 (homogentisate oxidase ) 1557167 _ at HCG11 HLA complex group 11 0 . 6 - 0 .0001 1557169 x at HCG11 HLA complex group 11 0 .6 - 0 . 0001 243802 _ at DNAH12 dynein , axonemal, heavy chain 12 0 . 6 0 .04 223631 _ s _ at C19orf33 chromosome 19 open reading frame 0 .6 0 . 0097 33 230051_ at C10orf47 open reading frame 0 . 6 0 .0316 47 230520 _ at AIG1 androgen - induced 1 0 . 6 0 .012 1554918 a at ABCC4 ATP - binding cassette , sub - family C 0 .6 0 . 0151 member 4 208180 _ s _ at HIST1H4B Histone cluster 1 , H4b 0 . 5 0 .0459 209839 _ at DNM3 dynamin 3 0 . 5 0 . 0255 220496 at CLEC1B C - type lectin domain family 1 , 0 . 5 0 .0401 member B US 9 , 809 , 854 B2 91 TABLE 17 - continued Gene Candidates for Biomarkers for Future Flares Hi vs p Probe Set ID Gene Symbol Gene Title Low values 223777 _ at MGC13005 hypothetical LOC84771 0 . 4 0 .0071 203290 _ at HLA -DQA1 major histocompatibility complex , 0 . 2 0 .0118 class II, DQ alpha 1

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(2005 ) Blood 105 : 1785 - 96 immunoenzymatic assay ; Velculescu et al. ( 1995 ) Science 270 : 484 -487 65 d . comparing the level of vWF with a reference level of Velculescu et al. ( 1997 ) Cell 88 vWF in a sample of blood obtained from the subject Villanueva et al. (2011 ) J . Immunol. 187 :538 -52 prior to the treatment; and US 9 , 809 , 854 B2 93 94 e . determining the treatment as being effective for treating c . assaying for the level of vWF in a sample of blood from SLE when the levels of IFIT3 , MMP8 , CD38 , and vWF the subject, wherein assaying for the vWF is performed from the subject are decreased and the level of KLRB1 using a method comprising at least one of ELISA , from the subject is increased as compared to the western blot, immunoblot, quantitative mass spectrom reference levels . 4 . The method of claim 3 , wherein the subject is human . 5 etry , radioimmunoassay, immunoradiometric assay , or 5 . A method of detecting increased risk of future flares of immunoenzymatic assay ; systemic lupus erythematosus (SLE ) in a subject diagnosed d . comparing the level of vWF with a reference level of with SLE , consisting of: VWF in a sample of blood from the first or a second a . assaying gene expression levels of IFIT3 , MMP8 , CD38 , and KLRB1 in a sample of blood from the healthy control; subject, wherein assaying the gene expression levels is e . determining that the subject has an increased risk of performed using a method comprising at least one of : future flares of SLE when the levels of IFIT3 , MMP8 , PCR , RNA sequencing , or microarray ; CD38 , and vWF from the subject are increased and the comparing the gene expression levels of IFIT3 , MMP8 , level of KLRB1 from the subject is decreased as CD38 , and KLRB1 in the sample from the subject with 15 compared to the reference levels . reference gene expression levels of IFIT3 , MMP8 , CD38 , and KLRB1 in a sample of blood from a first 6 . The method of claim 5 , wherein the subject is human . healthy control; * * * * *