[0001] Spondylarthritis (SpA) is a family of seronegative arthritis. The various clinical forms include axial features, peripheral arthritis, enthesitis, and extra-articular features such as uveitis, psoriasis, and inflammatory bowel disease. The prototypic form of these disabling diseases is ankylosing spondylitis (AsS), a highly heritable arthropathy with 80-90% of susceptibility attributable to genetic factors. The main genetic risk factor is the MHC class I molecule HLA-B27, carried by 80-90% of patients. However, the presence of HLA-B27 explains only 20-40%) of the genetic risk of developing AS, suggesting an important role for additional genes in AS pathogenesis (1). To better understand the pathophysiology of SpA, several genome-wide association studies (GWAS) have been performed. These studies have uncovered genetic linkage to AS of non-HLA genes, in particular of key components of the IL-23 signaling pathway such as IL23R and IL12B, as well as genes associated with other pathways such as ERAP1 and ANTXR2 (2-5).

Nonetheless, how genetic variation at these loci affects immune function and contributes to pathology remains unknown.

[0002] For almost two decades, IFN-y-secreting Thl cells have been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases. Thl cell development from naive CD4 ⁺ precursor T cells is initiated by antigenic stimulation and strongly enhanced by the heterodimeric cytokine IL-12, which activates the transcription factor STAT4 in developing Thl cells. STAT4 induces expression of genes involved in the differentiation and effector functions of Thl cells, such as TBX21 (which encodes the Thl lineage transcription factor T-bet), the gene encoding the signaling subunit of the IL-12 receptor, (IL12RB2) and IFNG, which codes for the Thl signature cytokine IFN-γ (6-8).

[0003] The key role of Thl cells in autoimmunity has been challenged by the identification of IL-23, a member of the IL-12 family of heterodimeric cytokines, and of IL- 17-secreting Thl 7 cells as key players in mouse models of autoimmunity (9). IL-12 and IL- 23 share a common subunit, p40, encoded by the IL12B gene, which pairs with IL-23pl9 to form bioactive IL-23 and with IL-12p35 to form IL-12 (10). Mice with a deletion of the IL- 23pl9 subunit, but not mice with a deletion of the IL-12p35 subunit are protected from disease in several experimental models of autoimmunity, such as experimental autoimmune encephalomyelitis (EAE) (11, 12), collagen- induced arthritis (CIA) (13), and inflammatory bowel disease (IBD) (14, 15). It was shown that IL-23, but not IL-12, induced the secretion of IL-17 from activated memory T cells (16), and subsequent studies revealed that IL-23 is essential for the maintenance and function of a distinct subset of effector CD4 ⁺ T cells, termed Thl7 cells, which are characterized by the secretion of the pro -inflammatory cytokines IL-17A and IL-17F (12, 17). Together, these studies provided strong evidence that IL-23 and Thl7 cells play a critical role in chronic inflammation and autoimmunity in mouse models. A role for IL-17 and Thl7 cells in human chronic inflammatory disease has been supported by reports showing increased frequencies of IL-17-producing cells in the peripheral blood of SpA patients, compared with healthy donors (18-22). Thl7 cells, however, may not be the only T cell subset to promote inflammation. Several studies have demonstrated that both Thl7 and Thl cells induced disease in experimental models of autoimmune diseases (23, 24), suggesting that CD4 ⁺ T cell populations with distinct functional properties may contribute to the pathogenesis of chronic inflammatory diseases.

[0004] There is a need for methods of diagnosing an autoimmune disease or a likelihood of having or developing an autoimmune disease in a subject. There is also a need for methods of treating an autoimmune disease that match therapies to subjects in useful ways. This disclosure provided methods, reagents, and other aspects that meet partially or wholly one or more of these and/or other needs.

SUMMARY

[0005] This disclosure provides methods of diagnosing a spondylarthritis (SpA) or a likelihood of having or developing an SpA in a subject. In some embodiments the methods comprise a) providing a nucleic acid sample from the subject; b) performing a genotyping assay of (i) at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6; wherein the second gene is different than the first gene; c) determining whether the at least two genotyped SNP loci comprise at least one of a protective allele and a risk allele; and d) diagnosing the subject as having an SpA or having an increased risk of having or developing an SpA if the at least two genotyped SNP loci comprise genotypes selected from at least one risk allele and the absence of at least one protective allele. [0006] In some embodiments the SNP loci genotyped in b) are selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the risk alleles in c) comprise allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the protective alleles in c) comprise allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695. In some embodiments, if the at least two SNP loci comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the subject is diagnosed as having an SpA or a likelihood of having or developing an SpA.

[0007] In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rs 1343151, rsl0045431, rs744166, and rs6503695 in the nucleic acid sample from the subject. In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl004819, rsl 1209032, rs7574865, and rs3093024 in the nucleic acid sample from the subject. In some embodiments the methods comprise performing a genotyping assay of at least one SNP locus linked to IL23R, at least one SNP locus linked to IL12B, at least one SNP locus linked to STAT3, at least one SNP locus linked to STAT4, and at least one SNP locus linked to CCR6. In some embodiments, if the genotyped SNP loci do not comprise genotypes selected from at least one risk allele and the absence of at least one protective allele then the subject is diagnosed as not having an SpA or a likelihood of having or developing an SpA.

[0008] In some embodiments the genotyped SNP loci comprise rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments, if the SNP loci do not comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the subject is diagnosed as not having an SpA or a likelihood of having or developing an SpA.

[0009] In some embodiments the SpA is characterized by an increase in at least one type of CD4+ cell effector function selected from IL17A function, IL17F function, IL23R function, RORC function, IFNG function, TNFA function, IL12RB2 function, and TBX21 function. In some embodiments the CD4+ cell effector function comprises at least one of Thl cell function and Thl7 cell function.

[0010] In some embodiments the SpA is ankylosing spondylitis (AsS).

[0011] In some embodiments the methods further comprise comparing the genotypes of the SNP loci to a reference table comprising SpA disease risk values for the genotyped SNP loci to thereby assign a disease risk value to the subject.

[0012] This disclosure also provides methods of treating or preventing an SpA in a subject. In some embodiments the methods comprise a) providing a nucleic acid sample from the subject; b) performing a genotyping assay of (i) at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the first and second genes are different; c) determining whether the at least two genotyped SNP loci comprise at least one of a protective allele and a risk allele; and d) administering an SpA therapeutic agent to the subject if the at least two genotyped SNP loci comprise genotypes selected from at least one risk allele and the absence of at least one protective allele.

[0013] In some embodiments the SNP loci genotyped in b) are selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the risk alleles in c) comprise allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the protective alleles in c) comprise allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[0014] In some embodiments, if the at least two SNP loci comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP

rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rsl343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the SpA therapeutic agent is administered to the subject.

[0015] In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rs 1343151, rsl0045431, rs744166, and rs6503695 in the nucleic acid sample from the subject. In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024 in the nucleic acid sample from the subject.

[0016] In some embodiments the methods comprise performing a genotyping assay of at least one SNP locus linked to IL23R, at least one SNP locus linked to IL12B, at least one SNP locus linked to STAT3, at least one SNP locus linked to STAT4, and at least one SNP locus linked to CCR6.

[0017] In some embodiments if the genotyped SNP loci do not comprise genotypes selected from at least one risk allele and the absence of at least one protective allele then the SpA therapeutic agent is not administered to the subject.

[0018] In some embodiments the genotyped SNP loci comprise rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments if the genotyped SNP loci do not comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the SpA therapeutic agent is not administered to the subject.

[0019] In some embodiments the SpA is characterized by an increase in at least one type of CD4+ cell effector function selected from IL17A function, IL17F function, IL23R function, RORC function, IFNG function, TNFA function, IL12RB2 function, and TBX21 function in the subject. In some embodiments the CD4+ cell effector function comprises at least one of Thl cell function and Thl7 cell function.

[0020] In some embodiments the SpA is ankylosing spondylitis (AsS).

[0021] In some embodiments the methods further comprise comparing the genotypes of the SNP loci to a reference table comprising SpA disease risk values for the genotyped SNP loci, to thereby assign a disease risk value to the subject.

[0022] This disclosure also provides kits for diagnosing an SpA, a likelihood of having or developing an SpA, or a likelihood of response to an SpA therapeutic. In some embodiments the kits comprise at least one probe or primer for performing a genotyping assay of (i) at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the second gene is different than the first gene. In some embodiments the probe hybridizes specifically with a nucleic acid molecule comprising a risk allele at the SNP locus. In some embodiments the probe hybridizes specifically with a nucleic acid molecule comprising a protective allele at the SNP locus. In some embodiments the primer amplifies a nucleic acid fragment comprising the SNP locus. In some embodiments the primer specifically amplifies a nucleic acid fragment comprising a risk allele at the SNP locus. In some embodiments the primer specifically amplifies a nucleic acid fragment comprising a protective allele at the SNP locus.

[0023] In some embodiments the SNP loci are selected from rsl 1209026,

rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the at least one probe or primer are for detecting at least one allele selected from allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the at least one probe or primer are for detecting at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rs 10045431, allele G at SNP rs744166, and allele C at SNP rs6503695. In some embodiments the at least one probe or primer are for detecting at least one allele selected from at least one allele A at SNP rs 1004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the subject is diagnosed as having an SpA or a likelihood of having or developing an SpA.

[0024] In some embodiments the kits further comprise at least one of a positive control sample and a negative control sample, said positive and negative control samples comprising a nucleic acid comprising a SNP locus selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 or a protein encoded by a gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 shows that SpA patients carrying the protective IL23R rsl 1209026 allele express lower levels of genes associated with the differentiation and function of Thl7 and Thl cells. CD3 CD4 T cells were sorted from peripheral blood of SpA patients and stimulated for 48h with anti-CD3/anti-CD28 beads, without the addition of cytokines (-) or in the presence of IL-12 and anti-IL-4 (IL-12) or IL-23 and anti-IL-4 and anti-IFN-γ (IL-23), as indicated. The expression level of Thl 7 (upper panels) and Thl (lower panels) marker genes was determined by real-time PCR. Data were normalized to 18S RNA and represented as fold-change relative to anti-CD3/anti-CD28 stimulated CD4 ⁺ T cells from a reference sample. Eight patients carried the minor protective IL23R R381Q variant of rsl 1209026 (AG), 41 patients were homozygous for the major allele (GG).

[0026] Figs. 2A-2C show that genetic variation at loci linked to SpA affects the secretion of IL-17F, IFN-γ, and TNF-a. Genotyping was performed using TaqMan allelic discrimination assays as described in the Examples. SNPs at IL23R (Fig. 2 A), at genes associated with the IL-23/Thl7 axis such as CCR6, IL12B, STAT3, and STAT4 (Fig. 2B), and at genes linked to SpA but not associated with the IL-23 signaling pathway (Fig. 2C) were selected from the literature. (P) and (R) indicate protective and risk-associated minor alleles, respectively. Major and minor alleles are indicated below the X-axis. CD3 CD4 ⁺ T cells were sorted from peripheral blood of SpA patients and stimulated for 48h with anti-CD3/CD28- coated beads. The indicated cytokines in the supernatants of stimulated cells were measured using Luminex assays. Horizontal bars represent the median values of cytokine

concentrations, -values were calculated using a Mann- Whitney test.

[0027] Fig. 3 shows a linkage disequilibrium (r ² ) map of SNPs analyzed in the studies reported in the Examples. The map was generated with Hap!oview 4.2 using European HapMap CEPH (CEU) samples.

[0028] Fig. 4 shows SpA patients carrying a protective IL23R allele express lower levels of genes associated with the differentiation and function of Thl7 and Thl cells.

CD3 CD4 ⁺ T cells were sorted from peripheral blood of SpA patients and stimulated for 48h with anti-CD3/anti-CD28 beads, without the addition of cytokines (-) or in the presence of IL-12 and anti-IL-4 (IL-12) or IL-23 and anti-IL-4 and anti-IFN-γ (IL-23), as indicated. The expression level of Thl 7 (upper panels) and Thl (lower panels) marker genes was determined by real-time PCR. Data were normalized to 18S RNA and represented as fold- change relative to anti-CD3/anti-CD28 stimulated CD4 ⁺ T cells from a reference sample. 27 patients carried the minor protective rsl343151 allele (A) and 22 were homozygous for the common allele (G). Horizontal bars in the graph indicate the median. P- values were calculated using a Mann- Whitney test and are indicated above the graph; the horizontal bars above the graphs indicate the groups that were compared.

[0029] Figs. 5A-5C show an overview of the effect of genetic variation at loci linked to SpA on Thl 7 and Thl cell functions. Genotyping was performed using TaqMan allelic discrimination assays as described in the Examples. SNPs linked to SpA at IL23R (A), genes associated with the IL-23/Thl7 axis such as CCR6, IL12B, STAT3, and STAT4 (B), and at genes linked to SpA but not associated with the IL-23 signaling pathway (C) were selected from the literature. SNP rs7574865 at STAT4 and rs2476601 at PTPN22 were also included because they are strongly associated with increased disease risk in RA and SLE. Major and minor alleles are indicated below the X-axis. (P) and (R) indicate protective and risk- associated minor alleles, respectively. To provide a better overview, the expression of the 4 Thl7 marker genes (IL17A, IL17F, IL23R and ROR and 4 Thl markers (IFNG, TNFA, IL12RB2 and TBX21) were combined after normalization and represented as "mean expression level" and plotted on the Y-axis (indicated by Thl 7 and Thl below the X-axis). Mean expression levels were calculated by setting the highest fold-change value for each gene to 100 and scaling the other values accordingly, followed by calculating the mean value for Thl7 marker genes and Thl markers (indicated by "Thl7" and "Thl" below the X-axis).

[0030] Fig. 6 shows that genetic variation at the IL12B locus affects effector T cell functions. Patients carrying the protective rs 10045431 variant (A) at IL12B express lower levels of Thl 7 and Thl markers. The expression level of the indicated genes in stimulated CD4 ⁺ T cells was determined as in Figure 1.

[0031] Fig. 7 shows genetic variation at the CCR6 locus affects effector T cell functions. Patients carrying the risk-associated minor allele of CCR6 rs3093024 (A) express higher levels of Thl7 and Thl markers. The expression level of the indicated genes in stimulated CD4 ⁺ T cells was determined as in Figure 1.

[0032] Figs. 8A-8B shows the effect of genetic variants at the STA T3 and STA T4 loci on effector T cell functions. (A) Patients carrying the protective rs6503695 variant (A) at STAT3 express similar levels of Thl 7 and Thl markers as those carrying the major (T) allele. (B) The risk-associated minor allele of STAT4 rs7574865 (T) is associated with higher expression levels of the Thl markers IL12RB2 and TBX21. The expression level of the indicated genes in stimulated CD4 ⁺ T cells was determined as in Figure 1.

[0033] Figs. 9A-9B shows that genetic variation at ERAP1 and ANTXR2 has no impact on the expression of Thl 7 and Thl marker genes. CD4 ⁺ T cells from patients carrying the ERAPl rs27434 allele associated with increased disease risk (A) (Fig. 9A) or those carrying the protective ANTXR2 rs4333130 allele (C) (Fig. 9B) do not show altered expression of genes associated with Thl 7 and Thl cell functions. The expression level of the indicated genes in stimulated CD3 CD4 ⁺ T cells from SpA patients was determined and represented as in Figure 1.

[0034] Figs. 10A-10B shows that SpA patients carrying protective IL23R alleles express lower levels of genes associated with the differentiation and function of Thl 7 and Thl cells. The data set is from a replication cohort of 24 patients (See Table 2). CD3 CD4 ⁺ T cells were sorted from peripheral blood of SpA patients and stimulated for 48h with anti- CD3/anti-CD28 beads, without the addition of cytokines. The expression level of Thl 7 (upper panels) and Thl (lower panels) marker genes was determined by real-time PCR. Data were normalized to HPRT R A and represented as fold-change relative to anti-CD3/anti- CD28 stimulated CD4 ⁺ T cells from a reference sample. Horizontal bars in the graph indicate the median. P- values were calculated using a Mann- Whitney test and are indicated above the graph. (A) Patients carrying the protective minor (A) IL23R rsl 1209026 allele express lower levels of the Thl7 marker genes IL17A, IL17F, IL23R, RORC as well as Thl markers IFNG, TNF, IL12RB2 and TBX21, compared to patients carrying the common (G) allele. (B) The minor protective IL23R rs 1343151 (A) allele is associated with a significantly lower expression levels of Hi 7 A, ILl 7F, IL23R, RORC as well as IFNG, TNF and IL12RB2, compared to the common (G) allele in SpA patients.

[0035] Figs. 11A-11B shows that genetic variation at the IL12B and CCR6 loci affects effector T cell functions. The data set is from a replication cohort of 24 independent patients (See Table 2). (A) Patients carrying the protective rsl 0045431 variant (A) at IL12B express lower levels of the Thl 7 and Thl markers. (B) The risk-associated minor allele of CCR6 rs3093024 (A) is associated with significant higher expression levels of ILl 7 A, ILl 7F, IL23R, as well as IFNG and IL12RB2, compared with the common (G) allele. The expression level of the indicated genes in stimulated CD4 ⁺ T cells was determined by real-time PCR as in Figure 10.

[0036] Figs. 12A-12B show the effect of genetic variants at the STAT3 and STAT4 loci on effector T cell functions; data from cohort 2. (A) Patients carrying the protective rs6503695 variant (A) at STAT3 express similar levels of Thl 7 and Thl markers as those carrying the major (T) allele. (B) The risk-associated minor allele of STAT4 rs7574865 (T) is associated with higher expression levels of the Thl markers IL12RB2 and TBX21. The expression level of the indicated genes in stimulated CD4 ⁺ T cells was determined as in Figure 10.

[0037] Figs. 13A-13B shows The effect of genetic variation at ERAP1 and ANTXR2 on the expression of Thl 7 and Thl marker genes; data from cohort 2. CD4 ⁺ T cells from patients carrying the ERAPl rs27434 allele associated with increased disease risk (A) (A) or those carrying the protective ANTXR2 rs4333130 allele (C) (B) do not show altered expression of genes associated with Thl 7 and Thl cell functions. The expression level of the indicated genes in stimulated CD3 CD4 ⁺ T cells from SpA patients was determined and represented as in Figure 10. [0038] Fig. 14A shows the combinatorial effect on CD4 effector functions of multiple SNPs at genes associated with the IL-23/Thl7 pathway. For each patient, the presence of a susceptibility minor allele (IL23R rsl004819, CCR6 rs3093024) was scored as +1 and the presence of a protective minor allele (IL23R rsl 1209026, IL23R rs 1343151, IL12B

10045431) as -1. The resulting sum was plotted on the x-axis. Relative expression levels of IL17 A (left panel), IL17F (middle panel) and IFNG (right panel) were determined as described in Fig. 1 and plotted on the y-axis. Analyzed SNPs displayed no or weak linkage disequilibrium (see Supplementary Figure 2)

[0039] Fig. 14B shows that SNPs at loci not associated with the IL-23 pathway do not show a combinatorial effect on CD4 effector functions. The presence of a susceptibility minor allele (ERAP1 rs27434, 2pl5 rsl0865331) was scored as +1 and the presence of a protective minor allele (ANTXR2 rs4333130, lq32 rsl 1584383, 21q22 rs2242944) was scored as -1. The resulting sum was plotted on the x-axis as in (Fig. 14A). Relative expression levels of IL17 A (left panel), IL17F (middle panel) and IFNG (right panel) were plotted on the y-axis.

[0040] Fig. 15A shows the combinatorial effect on CD4 effector functions of multiple SNPs at genes associated with the IL-23/Thl7 pathway. The cumulative genetic risk of each patient for 5 alleles targeting the IL-23 pathway (IL23R rsl 1209026, rsl 004819 and rsl343151, IL12B rsl0045431, CCi?6 rs3093024) was estimated using a multiplicative model. Patients were ranked according to the cumulative risk and grouped according to high and low cumulative risk (CR), using the median as cut-off. The expression level of Thl7 (upper panels) and Thl (lower panels) marker genes was determined by real-time PCR as in Figure 1.

[0041] Fig. 15B shows the same analysis as in (Fig. 15A) was performed using 5 SNPs associated with SpA, but not in the IL-23 pathway (ERAP1 rs27434, ANTXR2 rs4333130, 2pl5 rsl0865331, lq32 rsl 1584383, 21q22 rs2242944).

[0042] Figs. 15C and 15D show that patients carrying the IL23R R381Q variant have reduced frequencies of memory and CCR6 ⁺ CD4 ⁺ T cells. PBMC were stained with anti-CD4 Pacific Blue, anti-CD45RO FITC, and anti-CCR6 PE (BD Bioscience). The frequencies of CD45RO ⁺ and CCR6 ⁺ T cells within the CD4 ⁺ T cell population were plotted for patients carrying the protective minor allele (AG) or those homozygous for the major allele of IL23R rsl 1209026 (GG). (C) Immunofluorescence staining for a representative patient of each genotype. (D) Frequencies of CD4 ⁺ CD45RO ⁺ (left panel) and CD4 ⁺ CCR6 ⁺ cells (right panel) in SpA patients. -values were calculated using a Mann- Whitney test.

[0043] Figs. 16A-C show that IL-23 and IL-12 differentially affect expression of molecules controlling differentiation and function of Thl and Thl7 cells. CD3 CD4 ⁺ T cells were sorted from peripheral blood of SpA patients and stimulated with anti-CD3/anti-CD28 beads without the addition of cytokines (none) or in the presence of IL-23 (A and B) or IL-12 (C) for 48h.

[0044] As shown in (A), IL-23 enhances secretion of IFN-γ and IL-17A from stimulated CD4 ⁺ T cells from SpA patients. The production of IFN-γ and IL-17A was determined using Luminex assays.

[0045] As shown in (B), IL-23 upregulates expression of Thl 7 and Thl marker genes. In addition to a strong induction of the Thl 7 markers, IL-23 treatment also increased expression of IFNG, IL12RB2, and TBX21. mRNA levels were determined by real-time PCR.

[0046] As shown in (C), IL-12 downregulates IL17 A and RORC expression, but upregulates expression of IL23R. The expression level of Thl7 markers IL17A, IL17F, IL23R and RORC and Thl marker genes IFNG, TNF, IL12RB2 and TBX21 was determined by realtime PCR (B and C). Data were normalized to 18S RNA and represented as fold-change relative to anti-CD3/anti-CD28 stimulated CD4 ⁺ T cells from a reference sample. Sample pairing (with or without addition of cytokine) is indicated by thin lines. -values were calculated using a two-tailed Wilcoxon matched-pairs test.

DETAILED DESCRIPTION

A. Introduction

[0047] Recent genome-wide association studies have revealed numerous genetic associations between specific single nucleotide polymorphisms (SNPs) and immune- mediated inflammatory diseases. A current challenge is to associate the genetic variants to specific disease states that allow diagnosis of subjects and identification of subjects likely to receive a benefit from administration of a therapeutic agent. A hypothesis of this disclosure is that identification of novel combinations of SNP genotypes having a contributory or synergistic effect on effector mechanisms implicated in pathogenesis of SpA will provide combinations of SNP genotypes that identify subjects with an SpA disease state and/or likely to respond to a particular treatment with a useful degree of accuracy. The examples report on investigation of the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4 ⁺ T lymphocyte subsets involved in chronic inflammatory disease.

[0048] Expression of Thl 7 and Thl cytokines and transcription factors in CD4 ⁺ T cells isolated from SpA patients was measured and correlated with the patient's genotype at loci genetically linked to spondyloarthritis. The results show that the effector functions of Thl 7 and Thl cells in SpA patients are under combinatorial control by multiple SNPs at genes associated with the IL-23/Thl7 pathway. SpA patients carrying risk-associated alleles of genes in this pathway expressed the highest levels of genes involved in the differentiation and function of Thl 7 and Thl cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Thl 7 and Thl genes, suggesting that these SNPs may contribute to SpA pathogenesis through distinct cellular mechanisms. The results show that genetic variation at genes associated with the IL- 23 signaling pathway affect the effector functions of Thl 7 and Thl cells in SpA patients and provide a framework to delineate the mechanisms by which genetic variants contribute to pathology in SpA specifically and autoimmune disease in general. Because of the central role of expression of Thl 7 and Thl cytokines and transcription factors in CD4 ⁺ T cells in SpA pathology, the results also provide methods and reagents for measuring an imporant aspect of disease state in subjects. The methods and reagents may be used, for example, to measure or diagnose disease state and to select patients for therapeutic intervention.

B. Definitions

[0049] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Certain references and other documents cited herein are expressly incorporated herein by reference. Additionally, all UniProt/SwissProt records cited herein are hereby incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0050] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0051] Before the present proteins, compositions, methods, and other embodiments are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The term "comprising" as used herein is synonymous with "including" or "containing", and is inclusive or open-ended and does not exclude additional, unrecited members, elements or method steps.

[0053] This disclosure refers to single nucleotide polymorphism (SNP) database entries by SNP identification number, as well as other information on the internet. The skilled artisan understands that information on the internet, SNP database entries, is updated from time to time and that, for example, the reference number used to refer to a particular sequence can change. Where reference is made to a public database of SNP information or other information on the internet, it is understood that such changes can occur and particular embodiments of information on the internet can come and go. Because the skilled artisan can find equivalent information by searching on the internet, a reference to an internet web page address or a sequence database entry evidences the availability and public dissemination of the information in question.

[0054] As used herein "SNP" refers to "single nucleotide polymorphism." A single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an individual. Most SNPs have only two alleles, and one is usually more common in the population. A SNP may be present in an exon or an intron of a gene, an upstream or downstream untranslated region of a gene, or in a purely genomic location (i.e., non-transcribed). When a SNP occurs in the coding region of a gene, the SNP may be silent (i.e., a synonymous polymorphism) due to the redundancy of the genetic code, or the SNP may result in a change in the sequence of the encoded polypeptide (i.e., a non-synonymous polymorphism). In the instant disclosure, SNPs are identified by their Single Nucleotide Polymorphism Database (dbSNP) rs number, e.g., rs4263839. The dbSNP is a free public archive for genetic variation within and across different species developed and hosted by the National Center for iotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI).

[0055] A polymorphic site, such as a SNP, is usually preceded by and followed by conserved sequences in the genome of the population of interest and thus the location of a polymorphic site can often be made in reference to a consensus nucleic acid sequence (e.g., of thirty to sixty nucleotides) that bracket the polymorphic site, which in the case of a SNP is commonly referred to as the "SNP context sequence." Context sequences for the SNPs disclosed herein may be found in the NCBI SNP database available at:

www.ncbi.nlm.nih.gov/snp as well as in other publicly available sources that skilled artisans regularly reference for such information. Alternatively, the location of the polymorphic site may be identified by its location in a reference sequence (e.g., GeneBank deposit) relative to the start of the gene, mRNA transcript, BAC clone or even relative to the initiation codon (ATG) for protein translation. The skilled artisan understands that the location of a particular polymorphic site may not occur at precisely the same position in a reference or context sequence in each individual in a population of interest due to the presence of one or more insertions or deletions in that individual as compared to the consensus or reference sequence. It is routine for the skilled artisan to design robust, specific and accurate assays for detecting the alternative alleles at a polymorphic site in any given individual, when the skilled artisan is provided with the identity of the alternative alleles at the polymorphic site to be detected and one or both of a reference sequence or context sequence in which the polymorphic site occurs. Thus, the skilled artisan will understand that specifying the location of any

polymorphic site described herein by reference to a particular position in a reference or context sequence (or with respect to an initiation codon in such a sequence) is merely for convenience and that any specifically enumerated nucleotide position literally includes whatever nucleotide position the same polymorphic site is actually located at in the same locus in any individual being tested for the presence or absence of a genetic marker of the invention using any of the genotyping methods described herein or other genotyping methods well-known in the art.

[0056] As used herein, a "risk allele" is an allele of a SNP that when present in the genome of a subject indicates that the subject has an increased risk of having or developing an SpA compared to a subject having a genome that does not comprise the risk allele. In some embodiments the subject is also likely to benefit from treatment with an SpA

therapeutic agent.

[0057] As used herein, a "protective allele" is an allele of a SNP that when present in the genome of a subject indicates that the subject has a decreased risk of having or developing an SpA compared to a subject having a genome that does not comprise the protective allele. Accordingly, if a subject's genome does not comprise at least one copy of a protective allele the subject has an increased rist of having or developing an SpA compared to a subject whose genome comprises at least one copy of the protective allele. In some embodiments a subject whose genome comprises at least one copy of a protective allele of a SNP is unlikely to benefit from treatment with an SpA therapeutic agent. In some

embodiments a subject whose genome does not comprise at least one copy of a protective allele of a SNP is likely to benefit from treatment with an SpA therapeutic agent.

[0058] A SNP locus is linked to a gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6 if the genotype of the SNP locus correlates with the activity of the protein encoded by the gene in a tissue of a subject. Such a linkage may be established using standard genetic techniques well known to skilled artisans such as those used in the examples of this application. [0059] As used herein, the term "isolated" refers to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%>, about 30%>, about 40%>, about 50%>, about 60%), about 70%), about 80%>, about 90%>, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%), about 97%), about 98%>, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0060] In some embodiments, polymeric molecules (e.g., a polypeptide sequence or nucleic acid sequence) are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%), at least 95%, or at least 99% identical. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar. The term "homologous" necessarily refers to a comparison between at least two sequences (nucleotides sequences or amino acid sequences). In some embodiments, two nucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, or at least about 90% identical for at least one stretch of at least about 20 amino acids. In some embodiments, homologous nucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Both the identity and the approximate spacing of these amino acids relative to one another must be considered for nucleotide sequences to be considered homologous. In some embodiments of nucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In some embodiments, two protein sequences are considered to be homologous if the proteins are at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, or at least about 90% identical for at least one stretch of at least about 20 amino acids. [0061] The term "polynucleotide", "nucleic acid molecule", "nucleic acid", or "nucleic acid sequence" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single- stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. The nucleic acid (also referred to as polynucleotides) may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.

[0062] A "synthetic" RNA, DNA or a mixed polymer is one created outside of a cell, for example one synthesized chemically.

[0063] The term "nucleic acid fragment" as used herein refers to a nucleic acid sequence that has a deletion, e.g., a 5 '-terminal or 3 '-terminal deletion compared to a full- length reference nucleotide sequence. In an embodiment, the nucleic acid fragment is a contiguous sequence in which the nucleotide sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. In some embodiments fragments are at least 10, 15, 20, or 25 nucleotides long, or at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides long. In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some

embodiments such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.

[0064] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term "degenerate oligonucleotide" or "degenerate primer" is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0065] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32, and even more typically at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266: 131-141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)). [0066] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%>, 85%, or at least about 90%>, or at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0067] Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. "Stringent hybridization conditions" and "stringent wash conditions" in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of

hybridization.

[0068] In general, "stringent hybridization" is performed at about 25°C below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions. "Stringent washing" is performed at temperatures about 5°C lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50%) of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51. For purposes herein, "stringent conditions" are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65°C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65°C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing. [0069] The term "spondylarthritis" of "SpA" is a group of diseases involving the axial skeleton and having a negative serostatus. Seronegative refers to the fact that these diseases are negative for rheumatoid factor, indicating a different pathophysiological mechanism of disease than what is commonly seen in rheumatoid arthritis. The term includes ankylosing spondilitis reactive arthritis, enteropathic spondylitis or spondylitis associated with inflammatory bowel disease, psoriatic arthritis, and undifferentiated

spondylo arthropathy.

[0070] The term "ankylosing spondilitis" or "AsS" is one form of SpA. In comparison to SpA conditions, however, AsS has the greatest potential to cause restricted movement in the spine. The rate of spinal limitation and the extent of damage to other joints varies greatly from patient to patient, but AsS has the capacity to introduce significant limitations and disabilities into the lives of young patients. AsS is typically a difficult condition to diagnose due to its slow onset of symptoms, lack of specific laboratory tests, and delay in development of x-ray changes. An experienced physician can detect subtle limitations in mobility of the spine and expansion of the chest wall using certain

measurements and can reproduce tenderness over the sacroiliac joints, both of which further support AsS as a possible diagnosis.

[0071] X-rays are important to document the presence of inflammation in the sacroiliac joints. While these changes may take several years to be noticeable, an experienced physician is able to detect subtle abnormalities, which can confirm a diagnosis of AsS. As the disease progresses, fusion of the vertebrae can also be visualized, the end result being the "bamboo spine" appearance. Ideally, the diagnosis of AS should be made before this complication is encountered. In patients early in the course of their disease, magnetic resonance imaging (MRI) can detect changes in the sacroiliac joint and other areas of the spine before they are apparent on x-ray. When encountering inflammation in other joints or tendons, plain x-rays may be unhelpful, but either MRI or ultrasound can demonstrate inflammation in the joints or soft tissues and give further evidence for the diagnosis.

[0072] While conventional laboratory tests may be abnormal in AsS, they are less helpful in making the diagnosis. Markers of inflammation may be elevated, but these findings are both nonspecific and not consistently observed even in active AsS. Among all of the spondyloarthropathies, AsS is mostly strongly associated with a genetic marker known as HLA-B27. This marker is seen in about 90% of Caucasian and 50% of Afro-American individuals with AsS, but is also found in about 8% of the general population. Only in cases where the suspicion for AsS is high does this test help with the diagnosis.

[0073] All of the above investigations can be helpful in distinguishing AsS from other more common causes of chronic lower back pain and designing a proper treatment plan. Still, some patients are difficult to diagnose initially and may be labelled as "undifferentiated spondyloarthropathy." Many of these patients will eventually "declare themselves" and demonstrate clear features of ankylosing spondylitis or other related conditions such as psoriatic arthritis. The remainder continues to show incomplete features and designing a treatment plan can be challenging in this group of individuals. The methods and reagents provided by this disclosure provide new approaches for diagnosing AsS and/or for measuring the increased CD4+ effector function and/or increased autoimmune inflammation that characterizes AsS and related conditions.

[0074] The phrase "increased likelihood" refers to an increase in the probability that an event will occur. For example, some methods herein allow prediction of whether a subject will display an increased likelihood of developing symptoms of an SpA or of responding to treatment with an SpA therapeutic agent in comparison to a subject whose genome does not comprise at least one SpA risk allele or lack at least one SpA protective allele.

[0075] The term "SpA therapeutic agent" means any agent that is administered to a subject to reduce the likelihood that the subject will develop at least one SpA and/or to reduce the severity of at least one symptom of the SpA. The at least one manifestation may, for example, be occurrence of autoimmune inflammation and/or occurrence of increased CD4+ effector function. A skilled artisan will appreciate that, in view of this disclosure, it is possible to practice the methods disclosed utilizing any SpA therapeutic, including any SpA therapeutic developed after the filing date of this application. Exemplary SpA therapeutics include, without limitation: TNF blockers (e.g., etanercept (Enbrel), infliximab (Remicade) or adalimumab (Humira)); IL-1 blockers (e.g., Anakinra); agents that block co-stimulation (e.g., CTLA4-Ig (Abatacept)); anti-IL-6R antibody (Tocilizumab); anti-IL-17A; anti-IL-23 (e.g., Ustekinumab (Stelara®)); and JAK3 inhibitors (e.g., Tofacitinib).

[0076] In some embodiments the SpA therapeutic agent includes any therapeutic agent that blocks, attenuates, or reduces the activity of one or more components of the IL-23 signaling pathway so as to reduce or arrest IL-23 signaling pathway transduction, the nuclear transcription activation, and/or the enhanced downstream cytokine production resulting therefrom. Specific examples include antagonists that prevent the interaction of IL-23 ligand with IL-23 receptor [e.g., anti-ligand, including components p40 (IL-12B) and pl9 (IL-23 A) antagonists, anti-receptor, including components IL-12RB1 and IL-23R antagonists], antagonists which prevent the interaction of the receptor binding components (e.g., TYK2, JAK2), and intracellular signaling molecules (e.g., STAT-1, STAT-3, STAT-4, STAT-5). The form of such IL-23 pathway antagonists can include antibodies, and antigen binding fragments thereof, small molecules, interfering R A ("R Ai", e.g.- siR A, shRNA, miRNA), oligopeptides, etc. In some embodiments the IL-23 pathway antagonist binds directly to IL-23R. In some embodiments the IL-23 pathway antagonist binds directly to an IL-23R ligand. Examples of IL-23 pathway antagonists include Ustekinumab (Stelara®).

[0077] AsS may be treated with any autoimmune therapeutic agent. It is most commonly treated with nonsteroidal anti- inflammatory drugs (NSAIDs), such as naproxen (Aleve®, Naprosyn®) and indomethacin (Indocin®) and tumor necrosis factor (TNF) blockers, such as Adalimumab (Humira®), Etanercept (Enbrel®), Infliximab (Remicade®), and Golimumab (Simponi®).

[0078] The term "I/L-23 signaling pathway" includes all components that provide and/or receive a signal resulting from the binding of the cytokine IL-23 with its receptor, including the resulting intracellular signal transduction and resulting in the nuclear transcription activation. For example, this includes the IL-23 ligand components p40 (IL- 12B) and pl9 (IL-23 A), and the receptor components IL-12RB1 and IL-23R, the receptor binding components TYK2 and JAK2, and the cytoplasmic signal transducer STAT-3. Additionally, cyokine production resulting from IL-23 signaling, "downstream IL-23 cytokines," includes IL-1, IL-6, IL-17A, IL-17F, IL-21 , IL-22, IL-26 and TNF-a and GM- CSF, chemokines (e.g., KC, MCP-1, MIP-2) and matrix metalloproteases.

[0079] The term "subject" refers to any animal that suffers from an SpA. For example, the term includes humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), and household pets (e.g., dogs, cats, rodents, etc.). The subject may be suffering from an SpA disease or may be at risk of developing an SpA disease. [0080] As used herein, the terms "treat," "treatment," "treating," and "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down and/or stop the progression or severity of a condition associated with a disease or disorder. The terms include reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with an increase in CD4+ cell effector function in a subject. Such diseases include SpA diseases such as AsS. Treatment is generally

"effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.

Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The terms "treat," "treatment," "treating," and "amelioration" in reference to a disease also include providing relief from the symptoms or side-effects of the disease (including palliative treatment).

C. Genotyping Assays to Identify SNP Alleles in Nucleic Acid Samples

[0081] In certain methods of this disclosure a nucleic acid sample from a subject is provided. As recognized by the skilled artisan, nucleic acid samples containing a particular SNP may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand. Similarly, reference to a particular genotype obtained for a SNP on both copies of one strand of a chromosome is equivalent to the complementary genotype obtained for the same SNP on both copies of the other strand.

[0082] The term "nucleic acid sample" refers to a sample from a subject suspected of having an SpA, known to have SpA, known to be in remission from an SpA, or simply at risk of developing an SpA. In some embodiments the subject is undergoing treatment with an SpA therapeutic agent. In some embodiments the subject is not undergoing treatment with an SpA therapeutic agent. The test sample may originate from various sources in the subject without limitation. If genomic DNA in the sample will be analyzed the tissue sample can come from any tissue source that comprises genomic DNA of the subject, including, without limitation, synovial fluid, blood, blood-derived product (such as buffy coat, serum, and plasma), lymph, urine, tear, saliva, hair bulb cells, cerebrospinal fluid, buccal swabs, feces, synovial fluid, synovial cells, sputum, or tissue samples. In addition, one of skill in the art would realize that some samples would be more readily analyzed following a fractionation or purification procedure, for example, isolation of DNA from whole blood. If mRNA in the sample will be analyzed the tissue sample will generally be taken from a tissue of the subject in which the gene associated with the SNP is known to be expressed, which may be a tissue that is affected by at least one symptom of the SpA disease or may be from a tissue that is not affected by at least one symptom of the SpA disease.

[0083] In some embodiments the test sample is collected from the subject and then tested with little or no sample processing. In some embodiments the sample is processed, such as for example and without limitation processing to isolate all or a portion of the nucleic acid in the sample, such as genomic DNA in the sample, total RNA in the sample, or mRNA in the sample.

[0084] In general, methods for detecting an allele at a SNP locus can be divided into two groups: (1) methods based on hybridization analysis of polynucleotides, and (2) other methods based on biochemical detection or sequencing of polynucleotides. The method used may be based on analysis of a starting nucleic acid that is total RNA or mRNA obtained from the subject. In some embodiments cDNA is made from the mRNA as part of the method. Alternatively, the method used may be based on analysis of a starting nucleic acid that is genomic DNA obtained from the subject.

[0085] Any method known in the art or later developed may be used, in view of the teachings of this disclosure, to detect an allele at a SNP locus present in a starting sample that is genomic DNA obtained from a subject. Exemplary methods include, by way of example only, large-scale SNP genotyping, exonuclease-resistant nucleotide detection, solution-based methods, genetic bit analyses, primer guided nucleotide incorporation, allele specific hybridization, and other techniques. Any method of detecting a marker may use a labeled oligonucleotide.

[0086] Numerous methods and devices are well known to the skilled artisan to identify the presence or absence of a SNP allele and to genotype an SNP locus. DNA

(genomic and cDNA) for SNP detection can be prepared from a biological sample by methods well known in the art, e.g., phenol/chloroform extraction, PURE GENE DNA® purification system from GentAS Systems (Qiagen, CA). Detection of a DNA sequence may include examining the nucleotide(s) located at either the sense or the anti-sense strand within that region. The presence or absence of a SNP allele in a patient may be detected from DNA (genomic or cDNA) obtained from PCR using sequence-specific probes, e.g., hydrolysis probes from Taqman, Beacons, Scorpions, or hybridization probes that detect a SNP allele. For the detection of the SNP, sequence specific probes may be designed such that they specifically hybridize to the genomic DNA for the alleles of interest or, in some cases, an RNA of interest. For example, primers and probes for SNPs may be designed based on context sequences found in the NCBI SNP database available at: www.ncbi .nlm.nih.gov/snp. These probes may be labeled for direct detection or contacted by a second, detectable molecule that specifically binds to the probe. The PCR products also can be detected by DNA-binding agents. Said PCR products can then be subsequently sequenced by any DNA sequencing method available in the art. Alternatively the presence or absence of an allele can be detected by sequencing using any sequencing methods such as, but not limited to, Sanger- based sequencing, pyrosequencing or next generation sequencing (Shendure J. and Ji, H., Nature Biotechnology (1998), Vol. 26, Nr 10, pages 1135-1145). Optimised allelic discrimination assays for SNPs may be purchased from Applied Biosystems (Foster City, California, USA).

[0087] Various well-known techniques can be applied to interrogate a particular SNP, including, e.g., hybridization-based methods, such as dynamic allele-specific hybridization (DASH) genotyping, SNP detection through molecular beacons (Abravaya K., et al. (2003) Clin Chem Lab Med. 41 :468-474), Luminex xMAP technology, Illumina Golden Gate technology and commercially available high-density oligonucleotide SNP arrays (e.g., the Affymetrix Human SNP 5.0 GeneChip performs a genome-wide assay that can genotype over 500,000 human SNPs), BeadChip kits from Illumina, e.g, Human660W-Quad and Human 1.2M-Duo); enzyme-based methods, such as restriction fragment length polymorphism (RFLP), PCR-based methods (e.g., Tetra-primer ARMS-PCR), Invader assays (Olivier M. (2005) Mutat Res. 573(1-2): 103-10), various primer extension assays (incorporated into detection formats, e.g., MALDI-TOF Mass spectrometry, electrophoresis, blotting, and ELISA-like methods), Taqman assays, and oligonucleotide ligase assays; and other post- amplification methods, e.g., analysis of single strand conformation polymorphism (Costabile et al. (2006) Hum. Mutat. 27(12): 1163-73), temperaure gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography, high-resolution melting analysis, DNA mismatch-binding protein assays (e.g., MutS protein from Thermus aquaticus binds different single nucleotide mismatches with different affinities and can be used in capillary

electrophoresis to differentiate all six sets of mismatches), SNPLex® (proprietary SNP detecting system available from Applied Biosystems), capillary electrophoresis, mass spectrometry, and various sequencing methods, e.g., pyrosequencing and next generation sequencing, etc. Commercial kits for SNP genotyping include, e.g., Fluidigm Dynamic Array® IFCs (Fluidigm), TaqMan® SNP Genotyping Assay (Applied Biosystems),

MassARRAY® iPLEX Gold (Sequenom), and Type-it Fast® SNP Probe PCR Kit (Quiagen).

[0088] In some embodiments, the presence or absence of an allele or SNP in a patient is detected using a hybridization assay. In a hybridization assay, the presence or absence of the genetic marker is determined based on the ability of the nucleic acid from the sample to hybridize to a complementary nucleic acid molecule, e.g., an oligonucleotide probe. A variety of hybridization assays are available. In some, hybridization of a probe to the sequence of interest is detected directly by visualizing a bound probe, e.g., a Northern or Southern assay. In these assays, DNA (Southern) or RNA (Northern) is isolated. The DNA or RNA is then cleaved with a series of restriction enzymes that cleave infrequently in the genome and not near any of the markers being assayed. The DNA or RNA is then separated, e.g., on an agarose gel, and transferred to a membrane. A labeled probe or probes, e.g., by incorporating a radionucleotide or binding agent (e.g., SYBR® Green), is allowed to contact the membrane under low-, medium- or high-stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe. In some embodiments, arrays, e.g., the MassARRAY system (Sequenom, San Diego, California, USA) may be used to genotype a subject.

[0089] Sequence-Specific Oligonucleotide (SSO) typing uses PCR target

amplification, hybridization of PCR products to a panel of immobilized sequence-specific oligonucleotides on beads, detection of probe-bound amplified product by color formation followed by data analysis. Those skilled in the art would understand that the described Sequence-Specific Oligonucleotide (SSO) hybridization may be performed using various commercially available kits, such as those provided by One Lambda, Inc. (Canoga Park, CA) coupled with Luminex® technology (Luminex, Corporation, TX). LABType® SSO is a reverse SSO (rSSO) DNA typing solution that uses sequence-specific oligonucleotide (SSO) probes and color-coded microspheres to identify alleles. The target DNA is amplified by polymerase chain reactions (PCR) and then hybridized with the bead probe array. The assay takes place in a single well of a 96-well PCR plate; thus, 96 samples can be processed at one time.

[0090] Sequence Specific Primers (SSP) typing is a PCR based technique which uses sequence specific primers for DNA based allele typing. The SSP method is based on the principle that only primers with completely matched sequences to the target sequences result in amplified products under controlled PCR conditions. Allele sequence-specific primer pairs are designed to selectively amplify target sequences which are specific to a single allele or group of alleles. PCR products can be visualized on an agarose gel. Control primer pairs that matches non-allelic sequences present in all samples act as an internal PCR control to verify the efficiency of the PCR amplification. Those skilled in the art would understand that low, medium and high resolution genotyping with the described sequence-specific primer typing may be performed using various commercially available kits, such as the Olerup SSP™ kits (Olerup, PA) or (Invitrogen) or Allset and™Gold DQA1 Low resolution SSP (Invitrogen).

[0091] Sequence Based Typing (SBT) is based on PCR target amplification, followed by sequencing of the PCR products and data analysis. In some cases, RNA, e.g., mature mRNA or pre-mRNA, can also be used to determine the presence or absence of SNP alleles. Analysis of the sequence of mRNA transcribed from a given gene can be performed using any known method in the art including, but not limited, to Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR ELISA, TaqMan-based quantitative RT-PCR (probe-based quantitative RT-PCR) and SYBR green-based quantitative RT-PCR. In one example, detection of mRNA levels involves contacting the isolated mRNA with an oligonucleotide that can hybridize to mRNA encoded by a coding sequence comprising a SNP allele. The nucleic acid probe can typically be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, or 100 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed. In one format, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. Amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between. In general, amplification primers are from about 10 to about 30 nucleotides in length and flank a region from about 50 to about 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. PCR products can be detected by any suitable method including, but not limited to, gel electrophoresis and staining with a DNA-specific stain or hybridization to a labeled probe.

[0092] In some embodiments, the presence of a SNP allele in a subject is determined by measuring RNA levels using, e.g., a PCR-based assay or reverse-transcriptase PCR (RT- PCR). In some embodiments, quantitative RT-PCR with standardized mixtures of

competitive templates can be utilized.

[0093] A SNP allele can also be identified by detecting an equivalent genetic marker thereof, which can be, e.g., a second SNP allele on the same haplotype as the first SNP allele. Two particular alleles at different loci on the same chromosome are said to be in linkage disequilibrium (LD) if the presence of one of the alleles at one locus tends to predict the presence of the other allele at the other locus. The second SNP may be an allele of a polymorphism that is currently known. Other second SNPs may be readily identified by the skilled artisan using any technique well-known in the art for discovering polymorphisms.

D. SNP Loci

[0094] The Examples demonstrate that SNP alleles linked to the IL23R, IL12B, STAT3, STAT4, and CCR6 genes are either associated with an increased or a reduced risk of developing an SpA disease, such as AsS. Specifically, alleles of the following SNP loci are either associated with an increased or a reduced risk of developing an SpA disease, such as AsS: rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[0095] The following SNP alleles are risk alleles: allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. Thus, the presence of at least one copy of one of these alleles in the genome of a subject indicates that the subject has an increased risk of having or developing an SpA disease. For that reason the presence of at least one copy of one of these alleles in the genome of a subject indicates that the subject is likely to receive a therapeutic benefit from administration of an SpA therapeutic agent.

[0096] The following SNP alleles are protective alleles: allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695. Thus, the presence of at least one copy of one of these alleles in the genome of a subject indicates that the subject has a decreased risk of having or developing an SpA disease. Accordingly, the absence of at least one copy of one of these alleles in the genome of a subject indicates that the subject has an increased risk of having or developing an SpA disease in comparison to a similar subject whose genome comprises at least one protective allele. For that reason the absence of at least one copy of one of these alleles in the genome of a subject indicates that the subject is likely to receive a therapeutic benefit from administration of an SpA therapeutic agent.

[0097] Without wishing to be bound by theory, it is believed that the data in the examples demonstrate that this set of SNP loci (rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024) as well as all possible subsets of it (e.g. two SNP loci chosen from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024; three SNP loci chosen from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024; etc.) are partucularly useful for identifying the likelihood that a subject has an SpA disease or has an increased likelihood of having or developing an SpA disease because these SNP loci are linked to genes that function in a common pathway of CD4+ effector function that is linked to a disease mechanism in SpA diseases such as AsS.

[0098] The examples show that the presence of at least one allele selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 in the genome of a subject correlates with the occurrence of an increase in the expression or activity of molecules that mediate CD4+ effector functions, including IL17A, IL17F, IL23R, RORC, IFNG, TFNA, IL12RB2, and TBX21 function, while the absence of at least one allele selected from allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 in the genome of a subject correlates with the abscence of an increase in the expression or activity of molecules that mediate CD4+ effector functions, including IL17A, IL17F, IL23R, RORC, IFNG, TFNA, IL12RB2, and TBX21 function. Accordingly, this disclosure provides methods of assaying CD4+ cell effector function in a subject by assaying for the presence of a variant mutation in at least one allele selected from allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 in the nucleic acid sample of the subject. The Examples also show that the presence of at least one allele selected from allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 in the genome of a subject correlates with an increased risk that the subject will develop or has developed an SpA disease such as AsS, compared to subjects whose genome does not comprise the at least one allele selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865. (See Table 3.) Accordingly, this disclosure also provides methods of diagnosing an SpA disease or an increased risk of having or developing an SpA disease in a subject, and methods of treating or preventing an SpA disease, the methods comprising determining whether the subjects genome comprises at least one allele selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, as discussed further below.

[0099] The examples show that the presence of at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695 in the genome of a subject correlates with the absence of an increase in the expression or activity of molecules that mediate CD4+ effector functions, including IL17A, IL17F, IL23R, RORC, IFNG, TFNA, IL12RB2, and TBX21 function, while the absence of at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695in the genome of a subject may correlate with an increase in the expression or activity of molecules that mediate CD4+ effector functions, including IL17A, IL17F, IL23R, RORC, IFNG, TFNA, IL12RB2, and TBX21 function. Accordingly, this disclosure provides methods of assaying CD4+ cell effector function in a subject by assaying for the presence of at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695 in the nucleic acid sample of the subject. The Examples also show that the presence of at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695 in the genome of a subject correlates with a reduced risk that the subject will develop or has developed an SpA disease such as AsS, compared to subjects whose genome does not comprise the at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695. (See Table 3.) Accordingly, this disclosure also provides method of diagnosing an SpA disease or a likelihood of having or developing an SpA disease in a subject, and methods of treating or preventing an SpA disease, the methods comprising determining whether the subjects genome comprises at least one allele selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, as discussed further below.

E. Methods of Diagnosing an SpA Disease

[00100] Methods of diagnosing a spondylarthritis (SpA) or a likelihood of having or developing an SpA in a subject are provided. The methods may comprise a) providing a nucleic acid sample from the subject; b) performing a genotyping assay of (i) at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the second gene is different than the first gene; c) determining whether the at least two genotyped SNP loci comprise at least one of a protective allele and a risk allele; and d) diagnosing the subject as having an SpA or having an increased risk of having or developing an SpA if the at least two genotyped SNP loci comprise genotypes selected from at least one risk allele and the absence of at least one protective allele.

[00101] In some embodiments of the methods, b) comprises performing a genotyping assay of at least one third SNP locus linked to a third gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the third gene is different than the first and second genes. In some embodiments the methods comprise performing a genotyping assay of at least one fourth SNP locus linked to a fourth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fourth gene is different than the first, second, and third genes. In some embodiments the methods comprise performing a genotyping assay of at least one fifth SNP locus linked to a fifth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fifth gene is different than the first, second, third, and fourth genes. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the first gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the second gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the third gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the fourth gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the fifth gene.

[00102] In some embodiments the SNP loci genotyped in b) are selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the risk alleles in c) comprise allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the protective alleles in c) comprise allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[00103] In some embodiments, if the at least two SNP loci comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP

rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rsl343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the subject is diagnosed as having an SpA or a likelihood of having or developing an SpA.

[00104] In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, and rs6503695 in the nucleic acid sample from the subject. In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl004819, rsl 1209032, rs7574865, and rs3093024 in the nucleic acid sample from the subject.

[00105] In some embodiments the methods comprise performing a genotyping assay of at least one SNP locus linked to IL23R, at least one SNP locus linked to IL12B, at least one SNP locus linked to STAT3, at least one SNP locus linked to STAT4, and at least one SNP locus linked to CCR6. In some embodiments, if the genotyped SNP loci do not comprise genotypes selected from at least one risk allele and the absence of at least one protective allele then the subject is diagnosed as not having an SpA or a likelihood of having or developing an SpA. In some embodiments the genotyped SNP loci comprise rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments, if the SNP loci do not comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the subject is diagnosed as not having an SpA or a likelihood of having or developing an SpA.

[00106] In some embodiments the SpA is characterized by an increase in at least one type of CD4+ cell effector function selected from IL17A function, IL17F function, IL23R function, RORC function, IFNG function, TNFA function, IL12RB2 function, and TBX21 function. In some embodiments the CD4+ cell effector function comprises at least one of Thl cell function and Thl7 cell function. In some embodiments the SpA is ankylosing spondylitis (AsS).

[00107] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all eleven of the SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[00108] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rs 11209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695.

[00109] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, or all four of the SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00110] In some embodiments of the methods the genotyping assay is performed on at least one, at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl 343151 , rsl 0045431, rs744166, rs6503695; and is also performed on at least one, at least two, at least three, or all four of the SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00111] In some embodiments the methods comprise determining whether at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, allele A at SNP rsl004819, allele A at SNP rsl 1209032 , allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00112] In some embodiments the methods comprise determining whether at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[00113] In some embodiments the methods comprise determining whether at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00114] In some embodiments the methods comprise determining whether at least one, at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and determining whether at least one, at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00115] Table 4 lists the SNP loci that are most predictive of modified differentiation and function of Thl7 and Thl cells. SNP loci linked to various genes associated with differentiation and function of Thl 7 and Thl cells were analyzed. For the purposes of this disclosure the function of the gene product of each of these genes is a CD4+ cell effector function. Expression of each of these genes and/or of the gene product in the experiments in the examples is a proxy for the function of the gene product.

[00116] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL17A function. In such embodiments the at least one SNP locus is selected from rsl343151, rsl0045431, and rsl 0489629. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rs 1343151, allele A at SNP rsl 0045431, and allele C at SNP rsl0489629.

[00117] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL17F function. In such embodiments the at least one SNP locus is selected from rs 1343151 and rsl 0045431. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rs 1343151 and allele A at SNP rsl 0045431.

[00118] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL23R function. In such embodiments the at least one SNP locus is rsl0489629. In some embodiments the method comprises determining whether the SNP locus comprises allele C at SNP rsl 0489629.

[00119] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with RORC function. In such embodiments the at least one SNP locus is rs 1343151. In some embodiments the method comprises determining whether the SNP locus comprises allele A at SNP rs 1343151. [00120] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IFNG function. In such embodiments the at least one SNP locus is rs3093024. In some embodiments the method comprises determining whether the SNP locus comprises allele allele A at SNP rs3093024.

[00121] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with TFNA function. In such embodiments the at least one SNP locus is rs7574865. In some embodiments the method comprises determining whether the SNP locus comprises allele T at SNP rs7574865.

[00122] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL12RB2 function. In such embodiments the at least one SNP locus is rs 1343151. In some embodiments the method comprises determining whether the SNP locus comprises allele A at SNP rs 1343151.

[00123] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with TBX21 function. In such embodiments the at least one SNP locus is selected from rsl0045431, rs7574865, and rsl343151. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rsl0045431, allele T at SNP rs7574865, and allele A at SNP rsl343151.

[00124] In some embodiments the method further comprises comparing the genotype of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 to a reference table comprising SpA disease risk values for the at least two SNP loci to thereby assign a disease risk value to the subject. The " SpA disease risk" value is a value that represents the likelihood that a subject has an SpA disease and/or that the subject will develop an SpA disease. In some embodiments the value is an odds ratio. In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven of the SNP loci comprising alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl 343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, allele A at SNP rsl004819, allele A at SNP rsl 1209032 , allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

[00125] In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695 to SpA disease risk.

[00126] In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

[00127] In some embodiments the table comprises data correlating a genotype comprising at least one, at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and comprising at least one, at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

F. Methods of Treating or Preventing an SpA Disease.

[00128] Methods of treating or preventing an SpA in a subject are also provided. The methods may comprise a) providing a nucleic acid sample from the subject; b) performing a genotyping assay of (i) at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the second gene and the first gene are different; c) determining whether the at least two genotyped SNP loci comprise at least one of a protective allele and a risk allele; and d) administering an SpA therapeutic agent to the subject if the at least two genotyped SNP loci comprise genotypes selected from at least one risk allele and the absence of at least one protective allele. [00129] In some embodiments of the methods, b) comprises performing a genotyping assay of at least one third SNP locus linked to a third gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the third gene is different than the first and second genes. In some embodiments the methods comprise performing a genotyping assay of at least fourth first SNP locus linked to a fourth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fourth gene is different than the first, second, and third genes. In some embodiments the methods comprise performing a genotyping assay of at least one fifth SNP locus linked to a fifth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fifth gene is different than the first, second, third, and fourth genes. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the first gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the second gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the third gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the fourth gene. In some embodiments the genotyping assay is performed on at least two, at least three, at least four, or more SNP loci linked to the fifth gene.

[00130] In some embodiments the SNP loci genotyped in b) are selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the risk alleles in c) comprise allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the protective alleles in c) comprise allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695. In some embodiments, if the at least two SNP loci comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the SpA therapeutic agent is administered to the subject. [00131] In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rs 1343151, rsl0045431, rs744166, and rs6503695 in the nucleic acid sample from the subject. In some embodiments the methods comprise performing a genotyping assay of at least two SNP loci selected from rsl004819, rsl 1209032, rs7574865, and rs3093024 in the nucleic acid sample from the subject.

[00132] In some embodiments the methods comprise performing a genotyping assay of at least one SNP locus linked to IL23R, at least one SNP locus linked to IL12B, at least one SNP locus linked to STAT3, at least one SNP locus linked to STAT4, and at least one SNP locus linked to CCR6. In some embodiments, if the genotyped SNP loci do not comprise genotypes selected from at least one risk allele and the absence of at least one protective allele then the SpA therapeutic agent is not administered to the subject. In some

embodiments the genotyped SNP loci comprise rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments, if the genotyped SNP loci do not comprise genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP

rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rsl343151, the absence of allele A at SNP rsl 0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695, the SpA therapeutic agent is not administered to the subject.

[00133] In some embodiments the SpA is characterized by an increase in at least one type of CD4+ cell effector function selected from IL17A function, IL17F function, IL23R function, RORC function, IFNG function, TNFA function, IL12RB2 function, and TBX21 function in the subject. In some embodiments the CD4+ cell effector function comprises at least one of Thl cell function and Thl7 cell function. In some embodiments the SpA is ankylosing spondylitis (AsS).

[00134] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all eleven of the SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[00135] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl 0045431, rs744166, rs6503695.

[00136] In some embodiments of the methods the genotyping assay is performed on at least two, at least three, or all four of the SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00137] In some embodiments of the methods the genotyping assay is performed on at least one, at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl 343151 , rsl 0045431, rs744166, rs6503695; and is also performed on at least one, at least two, at least three, or all four of the SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00138] In some embodiments the methods comprise determining whether at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, allele A at SNP rsl004819, allele A at SNP rsl 1209032 , allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00139] In some embodiments the methods comprise determining whether at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[00140] In some embodiments the methods comprise determining whether at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. [00141] In some embodiments the methods comprise determining whether at least one, at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and determining whether at least one, at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00142] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL17A function. In such embodiments the at least one SNP locus is selected from rsl343151, rsl0045431, and rsl0489629. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rs 1343151, allele A at SNP rsl 0045431, and allele C at SNP rsl0489629.

[00143] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL17F function. In such embodiments the at least one SNP locus is selected from rsl 343151 and rsl 0045431. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rs 1343151 and allele A at SNP rsl0045431.

[00144] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL23R function. In such embodiments the at least one SNP locus is rsl0489629. In some embodiments the method comprises determining whether the SNP locus comprises allele C at SNP rsl 0489629.

[00145] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with RORC function. In such embodiments the at least one SNP locus is rs 1343151. In some embodiments the method comprises determining whether the SNP locus comprises allele A at SNP rsl343151.

[00146] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IFNG function. In such embodiments the at least one SNP locus is rs3093024. In some embodiments the method comprises determining whether the SNP locus comprises allele allele A at SNP rs3093024. [00147] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with TFNA function. In such embodiments the at least one SNP locus is rs7574865. In some embodiments the method comprises determining whether the SNP locus comprises allele T at SNP rs7574865.

[00148] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with IL12RB2 function. In such embodiments the at least one SNP locus is rs 1343151. In some embodiments the method comprises determining whether the SNP locus comprises allele A at SNP rs 1343151.

[00149] In some embodiments the methods comprise a genotyping assay performed on at least on SNP locus associated with TBX21 function. In such embodiments the at least one SNP locus is selected from rsl0045431, rs7574865, and rsl343151. In some embodiments the method comprises determining whether the at least one SNP locus comprises an allele selected from allele A at SNP rsl0045431, allele T at SNP rs7574865, and allele A at SNP rsl343151.

[00150] In some embodiments the method further comprises comparing the genotype of at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 to a reference table comprising SpA disease risk values for the at least two SNP loci to thereby assign a disease risk value to the subject. The " SpA disease risk" value is a value that represents the likelihood that a subject has an SpA disease and/or that the subject will develop an SpA disease. In some embodiments the value is an odds ratio. In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven of the SNP loci comprising alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl 343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, allele A at SNP rsl004819, allele A at SNP rsl 1209032 , allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

[00151] In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695 to SpA disease risk.

[00152] In some embodiments the table comprises data correlating a genotype comprising at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

[00153] In some embodiments the table comprises data correlating a genotype comprising at least one, at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and comprising at least one, at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 to SpA disease risk.

[00154] In some embodiments the performing a genotyping assay in at least one SNP locus is performed on a nucleic acid sample from a subject that has not been diagnosed with an SpA disease. In some embodiments the result of the assaying is identification that the subject is at risk of developing an SpA disease.

[00155] In some embodiments the performing a genotyping assay in at least one SNP locus is performed on a nucleic acid sample from a subject before initiation of treatment with an SpA therapeutic agent.

[00156] In some embodiments the performing a genotyping assay in at least one SNP locus is performed on a nucleic acid sample from a subject after initiation of treatment with a first SpA therapeutic agent. In some embodiments the first SpA therapeutic agent is not sufficient to adequatly manage the subject's symptoms. In some embodiments the presence of the variant mutation in the at least one SNP locus is detected in the subject and that result is used as a basis to initiate treatment with a second SpA therapeutic agent. In some embodiments treatment with the first SpA therapeutic agent is ceased.

[00157] In some embodiments the method of treating or preventing an SpA disease in a subject does not comprise performing a genotyping assay of at least one SNP locus selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 in a nucleic acid sample from the subject. For example, the genotype of the subject may be known because it was determined independently of the method. This may occur, for example, where a genotyping assay of at least one SNP locus selected from rsl 1209026, rsl 0489629, rsl 1465804, rs 1343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 was used to diagnose the subject as having an SpA disease or an increased likelihood of developing an SpA disease. Such a subject may then be identified and treated by

administering an SpA therapeutic agent.

[00158] Accordingly, this disclosure also provides methods of treating or preventing an SpA disease in a subject, comprising; identifying a subject comprising at least two SNP loci selected from rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl 0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 that comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024; and administering a therapeutically effective amount of an SpA therapeutic agent to a subject to thereby treat or prevent the SpA disease in the subject.

[00159] This disclosure also provides methods of treating or preventing an SpA disease in a subject, comprising: identifying a subject comprising at least two SNP loci selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 that do not comprise allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and administering a therapeutically effective amount of an SpA therapeutic agent to a subject to thereby treat or prevent the SpA disease in the subject.

[00160] This disclosure also provides methods of treating or preventing an SpA disease in a subject, comprising: identifying a subject comprising at least two SNP loci selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 that comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024 and do not comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and administering a therapeutically effective amount of an SpA therapeutic agent to a subject to thereby treat or prevent the SpA disease in the subject.

G. Kits for Use in Diagnosing and Treating an SpA

[00161] Kits for genotyping at least one SNP locus linked to a gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6 are also provided. In some embodiments the kits are for genotyping at least one SNP locus selected from rsl 1209026, rs 10489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024 are also provided. In some embodiments the kits are for genotyping at least one first SNP locus linked to a first gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, and (ii) at least one second SNP locus linked to a second gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the first and second genes are different.

[00162] In some embodiments the kits are also for genotyping at least one third SNP locus linked to a third gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the third gene is different than the first and second genes. In some embodiments the kits are also for genotyping at least one fourth first SNP locus linked to a fourth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fourth gene is different than the first, second, and third genes. In some embodiments the kits are also for genotyping at least one fifth SNP locus linked to a fifth gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6, wherein the fifth gene is different than the first, second, third, and fourth genes. In some embodiments the kits are for genotyping at least two, at least three, at least four, or more SNP loci linked to the first gene. In some embodiments the kits are for genotyping at least two, at least three, at least four, or more SNP loci linked to the second gene. In some embodiments the kits are for genotyping at least two, at least three, at least four, or more SNP loci linked to the third gene. In some embodiments the kits are for genotyping at least two, at least three, at least four, or more SNP loci linked to the fourth gene. In some embodiments the kits are for genotyping at least two, at least three, at least four, or more SNP loci linked to the fifth gene.

[00163] In some embodiments the SNP loci are selected from rsl 1209026,

rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. [00164] The kits typically comprise at least one probe and/or at least one primer that comprises a sequence designed to hybridize under stringent conditions with a complementary sequence in the subject's genome such that the probe or the primer is useful in conducting an assay to detect the presence of at least one allele of an SNP locus linked to a gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6. In some embodients the kits comprise at least one probe and/or at least one primer that comprises a sequence designed to hybridize under stringent conditions with a complementary sequence in the subject's genome such that the probe or the primer is useful in conducting an assay to detect the presence of at least one allele of an SNP locus selected from rs 11209026, rs 10489629, rs 11465804, rs 1343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[00165] A probe is a nucleic acid that hybridizes to its complementary sequence under stringent hybridization conditions. Typically a probe nucleic acid comprisese from 10 to 200 nucleotides, from 20 to 200 nucleotides, from 15 to 30 nucleotides, from 20 to 50

nucleotides, or from 25 to 100 nucleotides. In some embodiments the probe comprises about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides that are complementary to its target sequence. The target sequence is typically a genomic sequence comprising a SNP of this disclosure. In some embodiments the target sequence is a cDNA sequence comprising a SNP of this disclosure or an RNA sequence encoded by a genomic sequence comprising a SNP of this disclosure.

[00166] A primer is a nucleic acid that initiates DNA replication, transcription, or reverse transcription in an assay. By Primers are typically from about 7 to 100 nucleotides in length such as from 10 to 20, 15 to 30, 20 to 40, 20 to 50, or 40 to 100 nucleotides in length. Kits of this disclosure may include a pair of primers that together may be used to amplify a nucleic acid sequence in an assay, the amplified nucleic acid sequence comprising a SNP of this disclosure.

[00167] The kits of this disclosure may comprise a set of at least one probe and/or at least one primer designed to detect the presence of at least one SNP of this disclosure in a sample. In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of any set of SNPs identified in this disclosure, such as a set of SNPs assayed in any method disclosed herein. In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one SNP locus selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024. In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of allele A at SNP rsl 004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024. In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of allele A at SNP rsl 1209026, allele C at SNP rsl0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[00168] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least two SNP loci comprisng genotypes selected from at least one allele A at SNP rsl 004819, at least one allele A at SNP rsl 1209032, at least one allele T at SNP rs7574865, at least one allele A at SNP rs3093024, the absence of allele A at SNP rsl 1209026, the absence of allele C at SNP rsl 0489629, the absence of allele G at SNP rsl 1465804, the absence of allele A at SNP rs 1343151, the absence of allele A at SNP rsl0045431, the absence of allele G at SNP rs744166, and the absence of allele C at SNP rs6503695.

[00169] In some embodiments the kit comprises a set of probes and/or primers designed to detect at least one allele of at least two SNP loci selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, and rs6503695 in a nucleic acid sample from a subject. In some embodiments the kit comprises a set of probes and/or primers designed to detect at least one allele of at least two SNP loci selected from

rsl004819, rsl 1209032, rs7574865, and rs3093024 in a nucleic acid sample from a subject.

[00170] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of at least one SNP locus linked to IL23R, at least one SNP locus linked to IL12B, at least one SNP locus linked to STAT3, at least one SNP locus linked to STAT4, and at least one SNP locus linked to CCR6. In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of the SNP loci rsl 1209026, rsl 0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[00171] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all eleven ofthe SNP loci selected from rs 11209026, rsl0489629, rsl l465804, rsl343151, rsl0045431, rs744166, rs6503695, rsl004819, rsl 1209032, rs7574865, and rs3093024.

[00172] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695.

[00173] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of at least two, at least three, or all four ofthe SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00174] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least one allele of at least one, at least two, at least three, at least four, at least five, at least six, or all seven of the SNP loci selected from rsl 1209026, rsl0489629, rsl 1465804, rsl343151, rsl0045431, rs744166, rs6503695; and to detect the presence of at least one allele of at least one, at least two, at least three, or all four of the SNP loci selected from rsl 004819, rsl 1209032, rs7574865, and rs3093024.

[00175] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven SNP loci alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695, allele A at SNP rsl004819, allele A at SNP rsl 1209032 , allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00176] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven SNP loci alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rs 1343151, allele A at SNP rsl 0045431, allele G at SNP rs744166, and allele C at SNP rs6503695.

[00177] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or eleven SNP loci alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00178] In some embodiments the kit comprises a set of probes and/or primers designed to detect the presence of at least two, at least three, at least four, at least five, at least six, or seven of the SNP loci comprise alleles selected from allele A at SNP rsl 1209026, allele C at SNP rsl 0489629, allele G at SNP rsl 1465804, allele A at SNP rsl343151, allele A at SNP rsl0045431, allele G at SNP rs744166, and allele C at SNP rs6503695; and at least one, at least two, at least three, or four of the SNP loci comprise alleles selected from allele A at SNP rsl004819, allele A at SNP rsl 1209032, allele T at SNP rs7574865, and allele A at SNP rs3093024.

[00179] The kits may optionally comprise a positive control probe or primer that hybridizes with a genomic locus other than the SNP loci linked to a gene selected from IL23R, IL12B, STAT3, STAT4, and CCR6. The kits may optionally further comprise a negative control probe or primer.

[00180] In some embodiments at least one of the probes and/or primers in the kit are attached to a solid support such as an array or a bead.

[00181] The kits may optionally include a detectable marker. The marker may be chemically bound to a probe or primer in the kit. Suitable detectabel markers are well known in the art and include without limitation fluorescent lables and radioactive labels. Detectable marker systems typically used in the art with any of the assay methods disclosed herein may be included in the kit.

[00182] The kits may optionally include at least one buffering agent. The kits may also optionally include at least one nucleic acid polymerase.

EXAMPLES

[00183] The following examples serve to more fully describe the manner of using the invention. These examples are presented for illustrative purposes and should not serve to limit the true scope of the invention. A. Patients and Methods Used in the Examples

1. Patients

[00184] Peripheral blood samples were obtained from patients with spondyloarthritis in the Department of Rheumatology (Cochin Hospital, Paris, France). All the patients met either the modified New York criteria (25) for ankylosing spondylitis or the Amor criteria (26) for spondyloarthritis. Age, sex, disease duration, HLA-B27 positivity and the type of clinical presentation (axial involvement, peripheral arthritis, enthesis and non rheumato logic symptoms) were recorded. Current treatment was also noted at the time of sampling (Table 1). The patients were all adults and had never received biologic anti-rheumatic agents. The mean age of the patients was 38 years (19-81). Among the 49 patients, 18 were female. 79% of the patients were HLA-B27 positive. 31 patients had ankylosing spondylitis, 16 psoriatic arthritis, 2 IBD (Inflammatory Bowel Disease) related arthritis and one reactive arthritis associated with urethritis. To confirm the findings, a replication cohort of 24 SpA patients with similar demographics (Table 2) was recruited.

[00185] The study was approved by the Ethical Review Committee "Comite de Protection des Personnes He de France III" and written informed consent was given by all patients prior to inclusion in the study.

TABLE 1

40 M + Y Y Y N NSAID/MTX

51 F - Y N Y N NSAID

45 M + Y N N N NSAID

46 M + Y Y N N NSAID

46 M + Y N Y psoriasis NSAID

41 F + Y Y Y N NSAID

28 M + Y N N N NSAID

33 M - Y N Y N NSAID

21 M + Y N Y N 0

27 F + Y N Y N NSAID

30 M + Y Y N urethritis NSAID/MTX

48 F + Y N N Crohn's NSAID disease/

psoriasis

28 M + Y N N N NSAID

38 M - Y N Y psoriasis NSAID

36 F - Y Y N psoriasis NSAID

42 M + Y Y N N NSAID

32 F + Y N N N NSAID

32 M + Y Y Y psoriasis NSAID

39 M - Y N Y N NSAID

24 F + Y N Y N NSAID

28 M + Y Y Y psoriasis NSAID

19 M + Y N N N NSAID

30 M + Y N N N NSAID

44 M + Y Y N psoriasis MTX

32 F + Y N N N NSAID

34 M - Y N Y N NSAID

30 F + Y N Y psoriasis NSAID 34 35 M - Y N Y N NSAID/sulfasalazi ne

35 34 F + Y N N N 0

36 31 F + Y N N N 0

37 28 M + Y N N psoriasis NSAID

38 45 F - Y N Y psoriasis MTX

39 60 M + Y N Y uveitis 0

40 40 M + Y N N uveitis NSAID

41 41 M + N Y Y psoriasis NSAID

42 81 F + Y Y N uveitis Colchicine

43 44 F + N Y Y N NSAID

44 37 F + Y N Y uveitis NSAID

45 63 M + Y N Y psoriasis NSAID

46 56 M + Y Y Y psoriasis/uv 0

eitis

47 63 M - Y Y Y psoriasis NSAID/MTX

48 50 F + Y N Y uveitis NSAID

49 34 M + Y N N psoriasis 0

Patients recruited to this study. NSAID, non-steroidal anti-inflammatory drug; MTX, methotrexate.

TABLE 2

Patient Age Gender HLA- Type of injury Treatment

# B27

(years)

Axial Peripheral Enthesitis Non- rheumato- logic

symptoms

1 41 M + Y N N uveitis NSAID

2 35 M + Y N N psoriasis 0

3 35 F + Y N N N 0

4 46 F - Y N Y psoriasis MTX

5 36 M - Y N Y N NSAID/sulfasalazi ne

6 24 M + Y Y N psoriasis 0

7 27 F + Y N Y N 0

8 34 M + Y Y Y psoriasis NSAID

9 81 F + Y Y N uveitis MTX/colchicine

10 44 F + Y Y Y uveitis/psori NSAID/MTX asis

11 63 M - Y Y Y psoriasis MTX

12 46 M + Y Y N psoriasis MTX

13 35 M - Y N Y N 0

14 39 M - Y N Y psoriasis NSAID

15 31 F + Y N Y psoriasis NSAID

16 29 M + Y N N N 0

17 32 M + Y Y N urethritis 0

18 18 M + Y N N N NSAID

19 54 M + Y N N N 0

20 30 M + Y N N N NSAID

21 44 F + Y N Y uveitis 0

22 29 F - Y N N N NSAID

23 26 M + Y N N N NSAID

24 52 M + Y N Y N NSAID/MTX

Patients recruited to the replication cohort (24 patients). NSAID, non-steroidal anti-inflammatory drug; MTX, methotrexate.

2. Cell purification and stimulation

[00186] Peripheral Blood Mononuclear cells (PBMC) were isolated by density gradient centrifugation (Lymphoprep, Axis-Shield). Cells were stained with anti-CD3 Pacific Blue (BD Bioscience) and anti-CD4 APC (Miltenyi Biotec) antibodies and sorted using a FACS Aria II (BD Biosciences). Sorted CD3 CD4 T cells were cultured at a concentration of 10 ⁶ cells/ml in RPMI 1640 medium (Gibco) completed with 10% fetal calf serum

(Hyclone) and 1% penicillin and 1% streptomycin (Gibco). Cells were stimulated with Dynabeads coated with anti-CD3 and anti-CD28 (one bead/cell; Invitrogen) in the presence or in the absence of IL-12 (lOng/ml; Roche) and anti-IL-4 (^g/ml; BD Biosciences) or IL- 23 (lOOng/ml; Peprotech), anti-IL-4 (^g/ml) and anti-IFN-γ (^g/ml; BD Biosciences). Cells and supernatants were harvested after 48h of stimulation for analysis of mRNA expression and cytokines production, respectively.

3. Statistical analysis

[00187] A two-tailed Mann- Whitney test was used for comparison of gene or protein expression levels in CD4 ⁺ T cells carrying or not a minor allele in the different stimulation conditions. A two-tailed Wilcoxon matched-pairs test was used to analyze the induction of Thl7 and Thl marker genes by IL-23 or IL-12. Data were evaluated using Prism software (GraphPad).

[00188] Univariate analysis was performed by comparing the expression level of each gene according to the presence/absence of the minor allele for each SNP. If the mean differences were significant at a -value < 0.20, the SNP was selected for the multivariate analysis. Multivariate analysis by linear regression using a stepwise selection mode of variables was performed using the software SAS v9.3.

[00189] Genetic risk calculations were performed following a procedure published online (http://www.decodeme.com/health-watch-information/risk-calcu lation). Briefly, we first calculated the relative risk for each allele, taking into account the odds ratio and frequency of minor and major alleles. The cumulative risk was then computed using a multiplicative model, assuming that the factors behave independently, as is the case for SNPs that are not, or only in weak linkage disequilibrium. We then ranked the patients according to the cumulative risk for these 5 SNPs. The median was used to group the patients according to high and low cumulative risk.

4. Real-time Quantitative PCT

[00190] For cohort 1 (49 patients, Table 1), total RNA was extracted with the RNeasy Mini kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Archive kit (Applied Biosystems). RNA levels were assessed using custom designed Taqman Low Density Arrays on a 7900 Real-Time PCR system (Applied Biosystems), following the manufacturer's protocol. Reactions were run in triplicate and expression levels were normalized to 18S RNA. The following Taqman assays (Applied Biosystems) were used: IL17A (Hs00174383_ml), IL17F (Hs00369400_ml), IL23R (Hs00332759_ml), RORC (Hs01076112_ml), IFNG (Hs00174143_ml), TNF (Hs00174128_ml), IL12RB2 (Hs00155486_ml), TBX21 (Hs00203436_ml), and 18S (Hs99999901_sl).

[00191] For cohort 2, (24 patients, Table 2), total RNA was extracted with the RNeasy Micro kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Archive kit (Applied Biosystems). RNA levels were assessed using BioMark technology (Fluidigm), following the manufacturer's protocol (Advanced Development Protocol 17). Reactions were run in triplicate and expression levels were normalized to HPRT RNA. The following qPCR gene expression assays (Solaris Thermo Fisher Scientific) were used: HPRT (AX-008735-00- 0200), IFNG (AX-019379-00-0200), IL12RB2 (AX-007932-00-0200), IL17A (AX-007937- 00-0200), IL17F (AX-007942-00-0200), IL23R (AX-007976-00-0200), RORC (AX-003442- 00-0200), TBX21 (AX-005217-00-0200), and TNF (AX-010546-00-0200).

5. Cytokine measurements

[00192] Cytokine production was assessed in culture supernatants using Procarta® Immunoassays (Panomics) according to manufacturers' recommendations.

6. SNP Genotyping

[00193] SNPs associated with SpA were genotyped using pre-developed TaqMan allelic discrimination assays (Applied Biosystems, Table 3) following the manufacturer's protocol. Reactions were performed using an Applied Biosystems 7900HT Fast Real-Time PCR System with the SDS 2.3 software for allele discrimination (Applied Biosystems) or using a BioMark PCR System (Fluidigm) following the manufacturers' recommendations.

TABLE 3

1L12B rsl0045431 A Protective A=0.1763 0.83

STAT3 rs744166 G Protective G=0.4547 0.84

STAT3 rs6503695 C Protective C=0.3315 0.86

STAT4 rs7574865 T Risk T=0.25 1.27

CCR6 rs3093024 A Risk A=0.3938 1.13

ERAP1 rs27434 A Risk A=0.3512 1.19

PTPN22 rs2476601 A Risk A=0.0421 1.31

ANTX22 rs4333130 C Protective C=0.3471 0.82

21q22 rs2242944 A Protective A=0.4734 0.72

2pl5 rsl0865331 A Risk A=0.4199 1.27 chlq32 rsl 1584383 C Protective C=0.1694 0.74

Genotyping assays used in this study. MAF: minor allele frequency.

7. Linkage Disequilibrium map

[00194] The linkage disequilibrium (r2) map was generated with Hap lo view 4.2 (http://www.broad.mit.edu/mpg/haploview/) using scores from the The International HapMap Project.

B. Example 1: Genetic variation at the IL23R locus affects expression of genes associated with development and function of Thl 7 and Thl cells.

[00195] To assess if genetic variants at IL23R and other genes in the IL-23/IL-17 pathway affect CD4 ⁺ T cell functions, CD4 ⁺ T cells were isolated from the peripheral blood of 49 SpA patients (Table 1) and stimulated in the presence or absence of IL-12 or IL-23. The expression of genes associated with the development and function of Thl 7 and Thl cells was then analyzed. Patients were genotyped at loci that are genetically linked to SpA (Table 3) (2- 5). Patients were grouped according to the presence of the major (G) or minor (A) allele at rsl 1209026, a SNP at the IL23R locus. The minor allele of rsl 1209026 causes an R381Q amino acid exchange in the cytoplasmic portion of IL-23R and is less frequent in SpA patients than in healthy controls. It was found that patients carrying the protective minor allele of rsl 1209026 expressed substantially lower levels of IL-17A transcripts, compared to patients carrying the common allele (Figure l, P = 0.0361 for samples stimulated in the absence of cytokines, and P = 0.0239 for samples stimulated in the presence of IL-23).

Similarly, lower expression levels were observed for IL-17F and the Thl7-associated transcription factor RORC, (Figure 1). Presence of the R381Q variant also strongly affected Thl cell function, as substantially reduced IFNG, TNF, IL12RB2, and TBX21 transcript levels were observed in CD4 ⁺ T cells from patients carrying the minor allele, both in cells stimulated in the absence of cytokines, or cells stimulated in the presence of IL-12 or IL-23 (Figure 1). Consistently, lower levels of inflammatory cytokines (IFN-γ, IL-17F, and TNF-a) were detected in the supernatants of CD4 ⁺ T cells from IL23 R R381Q carriers. In contrast, levels of the homeostatic cytokine IL-2 were not altered in CD4 ⁺ T cells from patients carrying the protective R381Q IL23R variant (Figure 2 A, top row). Reduced expression of Thl 7 and Thl marker genes was not restricted to patients carrying the IL23R R381Q variant, but could also be observed when patients were grouped according to the presence or absence of the protective minor allele (A) of rs 1343151, another SNP in the IL23R locus, that is only in weak linkage disequilibrium with rsl 1209026 (r ² =0.074, Figure 3). In particular, patients carrying the protective allele had significantly lower transcript levels of IL17 A, IL17F and RORC, as well as TNF A, IL12RB2, and TBX21 (Figure 4). The analysis was extended to four additional SNPs at the IL23R locus that have been associated with SpA (2). Patients carrying a protective minor allele at rsl 0489629 or rsl 1465804 showed a similar decrease in the expression of Thl 7 and Thl marker genes. Consistently, an inverse pattern was observed for the minor alleles at rsl 004819 and rsl 1209032, which have been associated with increased disease risk (Figure 5A for transcript levels, protective minor alleles marked as "P", risk minor alleles marked as "R", and Figure 2A for cytokine protein levels) (2).

C. Example 2: Genetic variation in genes associated with the IL-23/Thl7 pathway alters Thl7 and Thl responses

[00196] These findings show that genetic variation at the IL23R locus affects both Thl 7 and Thl cell functions. To determine if this effect was restricted to IL23R or could be extended to additional molecules in the IL-23/Thl7 pathway, the impact on CD4 ⁺ T cell function of genetic variation at IL12B, the gene encoding the IL-12p40 subunit shared by IL- 12 and IL-23 was analyzed. GWAS have revealed an association of IL12B with psoriasis, Crohn's disease and SpA (27-29), providing further evidence for genetic overlap of these three diseases. It was found that patients carrying the protective rsl 0045431 allele (A) at IL12B showed a trend towards lower levels of Thl 7 and Thl marker gene expression (Figure 6), as well as decreased effector cytokine production (Figure 2B). [00197] GWAS have also uncovered a link between Crohn's disease or SpA and genetic variants at CCR6 and STAT 3 (4, 30).

[00198] CCR6 is a chemokine receptor highly expressed by Thl7 and a subset of Thl cells (31, 32). It was found that CD4 ⁺ T cells from SpA patients carrying the disease- associated rs3093024 allele (A) at CCR6 expressed significantly higher levels ofIL17A, IL17F, IFNG, and TNF (Figure 7).

[00199] STAT3 is a transcription factor activated by multiple cytokines, including those implicated in the differentiation and function of Thl 7 cells, such as IL-6, IL-21 and IL- 23 (33). However, no significant change was observed in the expression of Thl7 and Thl marker genes in SpA patients carrying the protective minor rs6503695 allele (C) (Figure 8 A) or the minor rs744166 allele (G), two SNPs at STAT3 (Figure 5B) (4). The effect of genetic variation at STAT4 was also analyzed. That gene encodes the transcription factor

downstream of IL-12 signaling, which has been associated with increased disease risk in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (34). In contrast to what was observed for the STAT3 variants, patients carrying the minor (T) allele rs7574865 at STAT4 expressed substantially higher levels of TBX21 and IL12RB2 (Figure 8B). Of note, the signaling subunit of the IL-12 receptor and the Thl -specific transcription factor T-bet are both direct transcriptional targets of STAT4 (6, 7), suggesting that genetic variation at STAT4 directly affects Thl cell differentiation. Together, these findings demonstrate that the effector functions of Thl 7 and Thl cells are controlled by multiple SNPs at genes associated with the IL-23/Th 17 pathway.

[00200] In addition to molecules associated with the IL-23 pathway, GWAS have revealed association of AS with variants at ERAP1, ANTXR2, lq32 and two intergenic regions at chr2pl5 and chr21q22 (2-5). Non-synonymous variants of ERAP1, an endoplasmic reticulum aminopeptidase, have reduced enzymatic activity (5). Moreover, association of ERAP1 with AS was only observed in HLA-B27-positive individuals, indicating that processing of antigenic peptides for MHC class I presentation may contribute to AS pathogenesis. Accordingly, a further experiment asked whether genetic variation at these loci, which are not directly linked to the IL-23 signaling pathway, affect the expression of Thl7 or Thl marker genes. No differences in the levels of inflammatory cytokine gene expression (Figure 9A) or cytokine production (Figure 2C) in patients carrying a susceptibility minor allele at ERAP1, rs27434 were detected. Similarly, no a link between CD4 ⁺ T cell function and the protective ANTXR2 rs4333130 allele (Figure 9B), or genetic variants at lq32 (rsl 1584383), chr 2pl5 (rsl0865331) and chr 21p22 (rs2242944) (Figure 5C) was detected.

These findings suggest that SNPs not associated with the IL-23/IL-17 axis do not specifically affect Thl7/Thl cell function, but act on SpA pathology through a different cellular pathway.

[00201] A multivariate analysis was performed to determine the independent

contribution of each SNP and their hierarchy on the expression of Thl and Thl7 marker genes. This analysis revealed that SNP IL23R rsl343151 has the highest independent

predictive value for the expression of IL17A, IL17F, RORC, TBX21 and IL12RB2, followed by HJ 25 rsl 0045431, predicting the expression of IL17A, IL17F and TBX21 (Table 4).

TABLE 4: Multivariate Analysis

Prediction of IL17A

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

IL23R rsl0489629, IL23R rsl343151, IL23R rsl 1209032, IL12B Coefficient estimate

-value rs 10045431 , STA T4 rs7574865, CCR 6 rs3093024, 21 q22 rs2242944 (±SD)

7£2 K rsl343151 -12.43 (±4.36) 0.007 IL12B rsl 0045431 -5.21 (±2.66) 0.056 IL23R rsl 0489629 7.90 (±4.31) 0.074

Prediction of IL17F

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

IL23R rsl0489629, IL23R rsl343151, IL23R rsl 1209032, IL12B Coefficient estimate

-value rsl 0045431, CC ½ rs3093024 (±SD)

7£2 K rsl343151 -14.52 (±6.80) 0.038 IL12B rsl 0045431 -12.77 (±6.80) 0.067

Prediction of IL23R

Variables included in the model: IL23R rsl 1209026, IL23R rsl 0489629,

Coefficient estimate

7£2 K rsl343151, IL12B rsl2131065, S7¾7 rs7574865, CCR6 rs3093024 -value

(±SD)

IL23R rsl 0489629 -20.46 (±10.60) 0.060

Prediction of RORC

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

Coefficient estimate

IL23R rsl0489629, IL23R rsl343151, ST AT 4 rs7574865 -value

(±SD)

7£2 K rsl343151 -4.01 (±1.39) 0.006 Prediction of IFNG

Variables included in the model: IL23R rsl 1209026, IL23R rs 1343151,

Coefficient estimate

IL23R rsl 1209032, ST AT 4 rs7574865, CCR6 rs3093024 -value

(±SD)

CCR6 rs3093024 53.96 (±33.79) 0.117

Prediction of TNFA

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

IL23R rsl343151, IL12B rsl 0045431, STAT4 rs7574865, CCR63093024, Coefficient estimate

-value

21q22 rs2242944, 2pl5 rsl0865331 (±SD)

S7¾7 rs7574865 1.97 (±1.22) 0.113

Prediction of IL12RB2

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

IL23R rsl0489629, IL23R rsl343151, IL23R rsl 1209032, IL12B Coefficient estimate

-value rsl0045431, S7¾7 rs7574865, CCR63093024 (±SD)

7L25R rsl343151 -4.65 (±2.14) 0.034

Prediction of TBX21

Variables included in the model: IL23R rsl 1209026, IL23R rsl 004819,

IL23R rsl0489629, IL23R rsl343151, IL12B rsl0045431, ST AT 4 Coefficient estimate

-value rs7574865, CCR6 rs3093024 (±SD)

IL12B rsl 0045431 -3.67 (±1.83) 0.051

S7¾7 rs7574865 3.44 (±1.93) 0.081

7L25R rsl343151 -3.28 (±1.88) 0.089

Table 4: Prediction of the effects of multiple SNPs on the expression of genes associated with the

differentiation and function of Thl 7 and Thl cells. Multivariate analysis was performed by linear regression using a stepwise selection mode of variables. SNPs were selected if the mean differences were significant at a P- value < 0.20 in the univariate analysis.

[00202] To replicate the observed effect of genetic variation at genes in the IL- 23/Thl7 pathway on Thl and Thl 7 cell functions, gene expression in CD4 ⁺ T cells isolated from peripheral blood from an independent cohort of 24 SpA patients (cohort 2, Table 2) was analyzed. Consistent with the results obtained with the first cohort (Figure 1 and Figure 4), patients in cohort 2 carrying protective minor alleles of rsl 1209026 or rs 1343151 at IL23R expressed substantially lower levels of the Thl7 marker genes IL17A, IL17F, IL23R and

RORC as well as Thl marker genes IFNG, TNF, IL12RB2 and TBX21, compared to patients carrying the common alleles (Figures 10A and 10B). Similarity, it was found that patients of cohort 2 carrying the protective rs 10045431 allele (A) at IL12B showed a trend towards lower levels of Thl7 and Thl marker gene expression (figure 11A), whereas patients carrying the risk-associated CCR6 rs3093024 allele expressed significantly higher levels of III 7 A, III 7F, IL23R, as well as IFNG and IL12RB2 (Figure 1 IB). It was noted that patients carrying the protective minor allele (C) of STAT3 rs6503695 show a modest reduction of the expression level of several Thl 7 and Thl markers; in particular IL17A and TNF (Figure 12A). A modest effect of the risk-associated (T) allele of STAT4 rs7574865 on the expression of the STAT4 target genes IFNG and IL12RB2 was also observed (Figure 12B), consistent with the results obtained with the first cohort (Figure 8B). Finally, SNPs associated with SpA but not associated with genes in the IL-23/Thl7 axis, such as ERAP1 rs27734 or ANTXR2 rs4333130 have little effect on the expression of Thl and Thl 7 marker genes (Figures 13A and 13B). Together, the data obtained from cohort 2 support the conclusion that genetic variation at genes associated with the IL-23/Thl7 pathway control the effector functions of Thl and Thl 7 cells.

D. Example 3: Combinatorial control of the effector functions of Thl7 and Thl cells

[00203] Since the data demonstrate that genetic variation at several loci affects CD4 ⁺ effector functions in SpA patients, another experiment asked whether it is possible to measure a combined effect of multiple SNPs in the IL-23 signaling pathway on Thl7/Thl inflammatory functions. A first analysis showed that SpA patients carrying predominantly susceptibility alleles expressed overall higher levels of IL17A, IL17F, and IFNG. In contrast, patients carrying the highest number of protective alleles expressed the lowest levels of these cytokine genes (Figure 14). To obtain a statistical evaluation of this effect, the expression levels of inflammatory cytokines were correlated with the genetic risk associated with 5 alleles targeting the IL-23 pathway, which displayed no or weak linkage disequilibrium (IL23R rsl 1209026, rsl004819 and rsl343151, ILI2B rsl0045431 and CCR6 rs3093024). The cumulative genetic risk of each patient for these SNPs was estimated using a

multiplicative model. The patients were then ranked according to the cumulative risk for these 5 SNPs. Finally, the patients were grouped according to high and low cumulative risk and analyzed to determine if the expression of Thl or Thl 7 markers differed in the two groups. It was found that patients having a higher cumulative risk express significantly higher levels of Thl and Thl 7 marker genes than patients with a lower cumulative risk (Figure 15 A). To define if this is restricted to SNPs at loci associated to the IL-23 signaling pathway, the same analysis was performed using 5 SNPs associated with SpA, but not targeting molecules in the IL-23 pathway. This analysis revealed no differences in the two groups (Figure 15B), suggesting that genetic risk associated with SNPs in the IL-23 pathway, but not in other signaling pathways, correlates with Thl and Thl7 effector functions.

[00204] A possible explanation for these findings is that the genotype of a patient affects the frequency, within the CD4 compartment, of memory CD4 ⁺ T cells or of CCR6 ⁺ T cells that can produce effector cytokines. It was found that patients carrying the R381Q IL- 23R variant had lower frequencies of CD45RO ⁺ (memory) and CCR6 ⁺ CD4 ⁺ T cells (figures 15C and 15D), although the frequency of activated (HLA-DR ) cells in the memory or in the CCR6 ⁺ compartment was similar in the two groups (not shown). These data indicate that genetic variation in the IL-23 pathway may have an impact on the homeostasis of CD4 effector cell populations and affect the frequency of inflammatory T cell subsets.

E. Discussion of Example Results

[00205] This investigation of the link between genetic variation at loci associated with SpA and CD4 ⁺ T cell function shows that polymorphisms at loci in the IL-23/Thl7 pathway affect the expression of genes involved in the differentiation and function of Thl 7 and Thl cells in SpA patients. In particular, the presence of the IL23R R381Q variant had a strong impact on the expression of genes implicated in the differentiation and function of both effector subsets. The effect of this variant on CD4 ⁺ T cell function is still incompletely understood. One study, conducted in healthy controls, showed that memory CD4 ⁺ and CD8 ⁺ T cells from individuals carrying the IL23R R381Q variant had decreased IL-23 -induced IL- 17A expression, and lower frequencies of circulating IL-17-producing CD4 ⁺ and CD8 ⁺ T cells (35). A similar study by Di Meglio et al. did not find differences in the frequencies of peripheral Thl 7 cells but revealed reduced IL-17 production in response to IL-23 stimulation of in vitro polarized Thl7 cells from healthy donors carrying the IL23R R381Q allele (36). Pidasheva et al. showed that the IL23R R381Q allele was associated with reduced IL-23 - induced STAT3 phosphorylation and decreased numbers of IL-23 -responsive T cells (37). The authors of these studies suggested that the IL23R R381Q variant represents a "loss-of- function" allele that leads to significant protection against autoimmunity (35-37). On the other hand, an additional study investigated signaling downstream of IL-23R using retroviral transduction of IL-23R variants of human T cell blasts. This study did not identify defects in IL-23 signaling (measured as STAT3 activation and IFN-γ production) in T cells transduced with the IL-23R R381Q variant (38).

[00206] In the studies reported herein, conducted in SpA patients, it was found that IL23R R381Q carriers expressed significantly lower levels of both Thl7 and Thl marker genes. However, presence of this genetic variation was not sufficient to protect from pathology, as 8 out 49 SpA patients recruited to this study carried this allele. This suggests that the combinatorial action of multiple SNPs, rather than a single genetic variant, determines the pathologic outcome.

[00207] Consistently, the results show that the effector functions of Thl7 and Thl cells in SpA patients are under combinatorial control by multiple genetic variants in IL12B, IL23R, and CCR6. Of note, SNPs that are associated with SpA, but are not at loci related to the IL- 23/Thl7 pathway, did not affect CD4 ⁺ T cell activity, indicating that they may impact on different pathophysiological mechanisms. The approach followed by this disclosure, which correlates for each patient the presence of multiple disease-associated or protective polymorphisms with defined cell functions provides a strategy to delineate the mechanisms by which genetic variants contribute to pathology. This concept is complementary to a recent study in which individual genetic variants predisposing to inflammatory disease were clustered into groups based on the pattern of sharing of these alleles across multiple diseases (39). Although no functional analyses were performed, that study suggested that the resulting clusters of SNPs represent different molecular pathways.

[00208] In addition to affecting the expression of Thl7 marker genes, it was found that genetic variation at genes associated with the IL-23/Thl7 pathway had a strong effect on genes controlling the differentiation and function of Thl cells, such as TBX21, IL12RB2 and IFNG. It was observed that IL-23 signaling enhanced production of IFN-γ (Figure 16A) and upregulated transcripts encoding TBX21, IL12RB2 and IFNG (Figure 16B) in anti-CD3/CD28 stimulated CD4 ⁺ T cells from SpA patients, possibly as a result of direct regulation of Thl marker genes by IL-23, as indicated by a previous report (40). It was also noted that expression of IL23R was strongly increased by IL-23 (Figure 16B), as well as IL-12 (Figure 16C). Previous work has demonstrated an important role for IL-12 in the regulation of the IL12RB2 gene (6), which is only 50 kb downstream of the IL23R gene in humans. These findings point to a previously not appreciated level of co-regulation of the IL23R and

IL12RB2 loci. Furthermore, the finding that IL-23, which stabilizes Thl7 cells and that IL-12, which induces Thl cells, can cross-regulate the expression of the signaling subunits of their respective receptors provides evidence that some of the functions of Thl 7 and Thl cells may overlap. Together, the data reported herein support the notion that genetic variants in the IL12B gene (encoding the IL-12p40 subunit shared by IL-12 and IL-23) and the IL23R gene act as important rheostats in tuning both Thl 7 and Thl responses. Furthermore, the genetic link of SNPs in IL12B and IL23R to SpA, psoriasis and IBD suggests a role for both Thl 7 and Thl cells in the pathogenesis of these human diseases. The findings therefore provide an important link between genotype, cell function and pathology.

[00209] In contrast to SNPs at IL23R, IL12B and CCR6, genetic variants at STAT3 had only a minor effect on the expression of Thl 7 and Thl markers. The STAT3 transcription factor is a central player in integrating signals from multiple cytokines in various tissues. Deletion of STAT3 in the germline leads to embryonic lethality in mice (41). Null-mutations have not been reported in humans but patients with Job's or hyper IgE syndrome (HIES) carry dominant-negative mutations of STAT3 and lack Thl 7 cells (42-44), demonstrating an important role of this transcription factor in the homeostasis of Thl7 cells in vivo. Although we currently cannot explain why genetic variation at STAT3 does not seem to alter the effector function of Thl 7 cells in the assay system used in the Examples, a possible hypothesis is that the key role of this factor in multiple organ systems may restrict genetic variation that strongly alters its expression or functional activity.

[00210] Recent reports have demonstrated that the IL-23 pathway operates also in other immune cell populations, such as CD8 ⁺ T cells (35), γδ T cells (22, 45), innate lymphoid cells (46) and mast cells (47, 48). Kenna et al. reported that γδ T cells expressing IL23R and secreting IL-17, but not CD4 ⁺ Thl 7 cells, are enriched in peripheral blood of patients with ankylosing spondylitis (AS) (22) and proposed that IL-23R-expressing γδ T cells may play a role in AS pathogenesis, providing a link between the observed genetic association of IL23R and AS. More recently, Sherlock et al. demonstrated in a mouse model that IL-23 acts on a previously not identified entheseal resident CD3 CD4 CD8 ^" T cell population expressing IL-23R and the transcription factor RORyt. Stimulation of these cells by IL-23 resulted in the production of inflammatory cytokines and chemokines such as IL-6, IL-17, IL-22 and CXCL1 and lead to the development of entheseal inflammation and bone remodeling, the hallmarks of spondylo arthritic disease (49). It will be interesting to determine how genetic variation at molecules in the IL-23 pathway affects these cell populations in the context of inflammatory diseases.

[00211] The introduction of biological therapies targeting IL-1, IL-6, IL-17 and IL-23 has increased the treatment options for inflammatory diseases (1); however, as is the case for TNF blockers, these reagents are not effective in all patients. The prior art has not provided a way to predict responsiveness of patients to these treatments. The data reported herein enable the design of specific treatments, tailored to the genetic architecture of each individual patient.

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[00261] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.