WO2008039445A2 - Polymorphisms in the human xbp-1 gene are associated with inflammatory bowel disease - Google Patents

Abstract

The instant invention is based, at least in part, on the finding that XBP-I plays a role in intestinal inflammation that resembles inflammatory bowel disease and that a single SNP in the 5' untranslated region of the human XBPl gene is significantly associated with IBD predicting a direct role for XBPl in the pathogenesis of this disease. Accordingly, the present invention provides methods for detecting at least one SNP in the human XBP-I gene as well as methods to determine the predisposition of a human subject to develop inflammatory bowel disease.

Description

POLYMORPHISMS IN THE HUMAN XBP-I GENE ARE ASSOCIATED WITH

INFLAMMATORY BOWEL DISEASE

Related Applications

This application claims priority to U.S. Provisional Application Serial No. 60/846,950, titled "Polymorphisms in the Human XBP-I Gene are Associated with Inflammatory Bowel Disease" filed September 25, 2006, the entire contents of which is incorporated herein by this reference.

Government Funding

Work described herein was supported, at least in part, by National Institutes of Health (NIH) under grants DK44319, P30 DK034854, AI32412, POl AI56296. The government may therefore have certain rights in this invention.

Background of the Invention

A single layer of intestinal epithelial cells (EEC) is the structure in immediate contact with the commensal microbiota and provides an immunologically functional barrier between these luminal microbes and the subepithelial hematopoietic system. IEC function is considered to play a role in inflammatory bowel disease (IBD)

(Podolsky,D.K. N. Engl. J. Med. 347, 417-429 (2002)), a disorder thought to result from immune activation by commensal microbiota that presents as Crohn's disease (CD) or ulcerative colitis (UC) (Podolsky,D.K. N. Engl. J. Med. 347, 417-429 (2002)). In the small intestine, the four epithelial cell lineages, absorptive epithelium, goblet, Paneth and entero endocrine cells, derive from a common, constantly renewing intestinal epithelial stem cell (Reya,T. & Clevers,H. Nature 434, 843- 850 (2005)). Paneth cells, located at the crypt base, contain several anti-bacterial peptides, α-defensins (cryptdins), and other antimicrobial proteins. A subset of CD is genetically linked to mutations in the intracellular pattern recognition receptor NOD2/CARD15 (Ogura, Y. et al. Nature 411, 603-606 (2001); Hugot,J.P. et al. Nature 411, 599-603 (2001)) in association with reduced expression of bactericidal Paneth cell cryptdins (Wehkamp,J. et al. Proc. Natl. Acad Sci. U. S. A 102, 18129-18134 (2005)). Nod2 deficient mice also exhibit decreased cryptdin expression and impaired clearance of oral Listeria monocytogenes infection (Kobayashi, K.S. et al. Science 307, 731-734 (2005)). However, factors apart from NOD2 must regulate Paneth cell function in IBD since CD patients without the CD-associated N0D2 polymorphisms have decreased Paneth cell α-defensins (Wehkamp,J. et al. Proc. Natl. Acad Sci. JJ. S. A 102, 18129-18134 (2005)). Nod2 deficient mice also do not develop spontaneous or induced intestinal inflammation (Kobayashi, K.S. etal. Science 307, 731-734 (2005)).

Single nucleotide polymorphisms (SNPs), resulting from variations, insertions, or deletions, result in base changes that contribute to phenotypic diversity. Polymorphisms in genes or regulatory regions of genes have been correlated with the development of, or susceptibility, to diseases or other conditions. The genetic risk factors associated with the development of inflammatory bowel disease (IBD) is very important. The identification of genetic polymorphisms that are tightly liked with IBD are desirable and will aid in the diagnosis or prognosis of the disease.

Summary of the Invention

The present invention is based, at least in part, on the discovery that X-box binding protein- 1 (XBPl), a key effector of the Unfolded Protein Response (UPR), is involved in both regulating Paneth cell function and the inflammatory state of the epithelium. As shown in the appended examples, selective disruption of XBPl in intestinal epithelium results in Paneth cell loss and spontaneous intestinal inflammation that resembles human IBD. Furthermore, it has been discovered that a single SNP in the 5' untranslated region of the human XBPl gene (rs6005893) is significantly associated (P - 0.00084) with IBD predicting a direct role for XBPl in the pathogenesis of this disease.

Accordingly, in one aspect the present invention provides methods to determine the predisposition of a human subject to develop inflammatory bowel disease, comprising detecting at least one single nucleotide polymorphism (SNP) in the human XBP-I gene, to thereby determine the predisposition of a human subject to develop inflammatory bowel disease. In one embodiment, a polymorphism in a NOD2-CARD15 gene is further detected.

In another aspect, the invention provides a method for detection of at least one SNP in the human XBP-I gene, which method comprises determining a nucleotide at position -3230 relative to the start ATG of the human XBP-I gene, and thereby detecting the absence or presence of at least one SNP. In one embodiment, the single nucleotide polymorphism at position -3230 is the presence of G and/or T.

In one embodiment, the method to detect the single nucleotide polymorphism is primer extension of at least one PCR product and MALDI-TOF analysis.

In one aspect, the invention provides an isolated and purified allele-specific oligonucleotide probe of about 5 to about 50 nucleotides which specifically detects a human XBP-I polymorphism at position -3230 relative to the start ATG of the human XBP-I gene. In another aspect, the invention provides a diagnostic kit comprising an oligonucleotide that specifically detects a human XBP-I polymorphism at position minus (-) 3230 relative to the start ATG of the human XBP-I gene.

Brief Description of the Figures

Figures Ia-Ih show activation of the UPR in human CD, spontaneous enteritis and Paneth cell loss in XBP I^'7" mice. a. Inflamed (CD-I) and non-inflamed (CD-NI) ileal biopsies from CD patients and healthy control (Ctrl) subjects were analyzed for grp78 and total XBPl mRNA expression (levels in Controls were arbitrarily set at 1, and CD-I and CD-NI levels expressed as ratio to Controls; lefty axis). XBPl mRNA splicing is expressed as ratio of XBPls/XBPu (right y axis), b. grp78 mRNA expression in Xbpl - deleted mouse intestinal epithelium (XBP I^'7"), normalized to β-actin). c. Spontaneous enteritis in XBP Y^1' mice (upper panels and lower left panel), and normal histology of XBP1^+/+ mice (lower right panel). Upper left, cryptitis with villous shortening, crypt regeneration and architectural distortion; upper right, neutrophilic crypt abscesses; lower left, duodenitis with surface ulceration and granulation tissue, d. Paneth cells with typical eosinophilic granules on H&E stained sections at the base of crypts in XBP1^+/+, but not XBPl^^'epithelium. Electron microscopy (EM) with only rudimentary electron-dense granules and a contracted ER in XBPl^7" basal crypt epithelial cells, normal configuration in XBP^+/+ mice. Immunohistochemistry (IHC) for the granule proteins lysozyme and pro-cryptdin in XBP1^+/+ and XBPl^"7' epithelia. e. Goblet cell staining by PAS in XBP1^+/+ and XBPl^"7" epithelia. EM exhibited smaller cytoplasmic mucin droplets and a contracted ER in XBP I^'7' goblet cells. No structural abnormalities were found in neighboring absorptive epithelia in XBP Y^1' mice. f. Enumeration of Paneth cells and goblet cells in small intestines, g. XBPl^^^Cre-ER¹² mice were administered 5 daily doses of 1 mg tamoxifen to induce deletion of the XBP l^floxneo gene in the intestinal epithelium. XBPl, cyrptdin-5 (Defcr5), and Chop mRNA (all expressed normalized to β-actin; left.y axis) expression in epithelium during and after tamoxifen treatment. Percentage of crypts with Paneth cells on H&E stainings (right y axis), h. TUNEL and H&E staining on small intestinal sections of tamoxifen-treated

_collected _{at the} indicated days.

Figures 2a-2h show XBPl^"7" deficiency in epithelium results in impaired antimicrobial function and increased susceptibility to DSS colitis, a. Small intestinal crypts isolated from XBPl⁺⁷⁺ and XBPl^'7' animals were stimulated lOμM carbamyl choline (CCh). Supernatants were precipitated, resolved on SDS-PAGE and detected by anti-lysozyme IgG. b. Intestinal epithelial cell-specific XBPl deleted mice (XBPl^'7'; n=9) and XBPl sufficient litteπnates (XBPl⁺⁷⁺; «=P; 5-10 weeks of age) were perorally infected with 3.6* 10⁸ Z,. monocytogenes. Faeces was aseptically collected 1Oh after infection and colony forming units (c.f.u.) of L. monocytogenes determined. Data presented as c.f.u. per mg dry weight of faeces, c. Oral infection with L. monocytogenes was performed as in (b), liver and spleen aseptically harvested 72h after infection, and c.f.u. of L. monocytogenes determined (XBP1^+/+ n=20; XBPl^"7' n=17). Data expressed as c.f.u. per organ. Two-tailed Mann- Whitney test for (b) and (c). d. 4.5% DSS was administered in drinking water for 5 days and then replaced by regular drinking water in XBP1^+/+ (n=9) and XBPl^7' (n=12) littermates (age 6-12 weeks). Wasting was monitored by daily weight and presented as % of initial weight. One-tailed Student's t test. e. Presence of rectal bleeding during DSS colitis was assessed daily and scored as absent (0), as traces of blood at anus or the base of the tail (1), or as clearly visible rectal blood (2). Mean ± s.e.m.; XBP1^+/+ (n=9), XBPl^7" (n=12). Two-tailed Mann-Whitney test. f. Individual signs of inflammation of colonic tissue harvested on day 8 of DSS colitis were scored blindly. Two-tailed Mann- Whitney test. g. Typical colonic histology on day 8 of DSS colitis. Arrows, borders of ulcers, h. mRNA expression (normalized to β-actin) of inflammatory mediators were quantified in colonic specimens on day 8 of DSS colitis were analyzed for key inflammatory mediators by qPCR. n=4 per group. Mean ± s.e.m. Two-tailed Mann- Whitney test.

Figures 3a-3g show that XBPl^7' epithelia exhibit an increased inflammatory state, a. Small intestinal formalin-fixed sections were stained with rabbit anti- phospho-JNK antibody, and revealed a patchy staining pattern in XBPl^"7", but not XBP1^+/+ sections. Control rabbit mAb did not exhibit any staining, b. Small intestinal MODE-K cells were transduced with a small interfering retrovirus (iXBP) or a control retrovirus (Control). XBPl mRNA knock-down in iXBP-transduced cells was 88% as determined by qPCR. MODE-K.iXBP and MODE-K. Control were stimulated for the indicated periods of time with Flagellin (1 μg/ml) and TNFα (50 ng/ml) and analyzed for P-JNK and total JNK by Western, c. MODE-K.iXBP (filled circles) and MODE-K. Ctrl (open circles) cells were stimulated for 4h with flagellin, and supernatants assayed by ELISA for CXCLl. d. Experiment as in (c), except for cells were stimulated with TNF-α. e. MODE-K.iXBP (circles) and MODE-K. Ctrl (diamonds) cells were stimulated with either lOμg/ml flagellin (filled symbols) or cultured in media alone (open symbols) for 4h, in the presence of the specific JNK inhibitor SP600125. Supernatants were assayed for CXCLl. f. In a similar experiment as in (e), MODE-K cells were stimulated with 50 ng/ml TNF-α (filled symbols) or cultured in media alone (open symbols), g. MODE-K.iXBP (filled circles) and MODE- K. Ctrl (open circles) were loaded with the glycolipid antigen α-galactosylceramide (αGC), fixed, and co-cultured with the CD Id-restricted NKT cell hybridoma DN32.D3. MODE-K antigen presentation was measured as IL-2 release from DN32.D3 into the supernatant. Figure 4 shows genetic variants and haplotype structure of the 81.1kb region containing the entire XBP-I gene. The approximate location of each SNP is shown in reference to the positions on the HapMap build 20 (Chr22: 27,504,552 - 27,585,713). The SNPs indicated by the underline (rs5762809, rs2269578, rs6005893, rs5762812) have not been previously validated in HapMap. The SNP that is associated with IBD (rs6005893; P = 0.00084) is boxed. We have constructed haplotypes using the genotype data from the current study of 1226 Italian cases and controls. The number in each box indicates the D¹ value between each SNP pair using the block definition of Gabriel et al. {Science 296, 2225-2229 (2002)) (D' values = 1 are not shown), indicative of high linkage disequilibrium (LD). Within this block of LD, there are 5 common haplotypes (frequency >1%). Frequencies of these haplotypes are very similar (0.64; 0.12; 0.07; 0.05; 0.030) to those using the data for the CEU population from the International HapMap project (www.hapmap.org). Despite this strong LD, no other SNP identified in the region is significantly correlated (r2 0.01 -0.18) with rs6005893.

Figure 5 shows the generation oϊXbpf°^x mice. a. Schematic representation of the gene targeting strategy. A floxedXbpl allele was generated by homologous recombination in W4/129 embryonic stem (ES) cells. The targeting vector contains a loxP site in intron 3 and a floxed neomycin resistance gene cassette (neo) in the intron 2 of theXbpl gene. A targeted ES cell clone identified by Southern blot was injected into C57BL/6 blastocysts to get the chimeras, which was subsequently bred to establish Xbpf^0*""⁰ strain. XbpP¹⁰*"^*0 mice were mated with EIIacre transgenic mice to induce a partial Cre-mediated recombination. Male mice with most deletion of the neo cassette only were mated with the wild type female mice to obtain Xbpl^flox strain, b. Breeding of Xbpl*¹⁰* mice with Cre transgenic mice results in the deletion of exon 2 of the Xbpl gene. To test the effect of the Cre-mediated exon 2 deletion, Xbpf^ox mice were crossed to MxI -ere transgenic mice which express Cre upon poly(I:C) administration. Total RNAs isolated from the liver were analyzed for the expression of XBP-I mRNA by a Northern blot. XBP 1 mRNA produced from Xbpl ^Jhx/Jhx:Aώcl -cre mice which received poly(I:C) (Xbpl^) was slightly smaller than the wild type XBPl mRNA. c. The mutant XBPl mRNA was characterized by RT-PCR followed by DNA sequencing. The mutant XBPl mRNA lacked the exon 2, which resulted in the change of the translational reading frame, introducing a premature translational termination codon. Lack of functional XBPIs protein inXbpl^m mice was also confirmed.

Figures 6a-6d show that epithelial XBPl deletion decreases mRNA expression of Paneth cell-specific genes, but does not affect enteroendocrine cells and intestinal epithelial permeability, a. Small intestinal mucosal scrapings (n=8 per group) were analyzed for cryptdin-1 (Defer 1), cryptdin-4 (Defcr4), cryptdin-5 (Defcr5), lysozyme (Lysz), mucin-2 (Muc2), cathelicidin (Campl), and XBPl (primers binding in the floxed region) mRNA expression, b. Livers and spleens of XBPl⁰⁰³^⁰³VQe (XBPl^7") and χBPl^floxΩox (XBPl⁺⁷+) mice were analyzed for XBP 1 mRNA levels (primers binding in the floxed region) quantified by qPCR. c. The marker for enteroendocrine cells, chromogranin, was detected by IHC in small intestines of XBP1^+/+ and XBPl^'7' mice, d. XBP1^+/+ and XBPl^7' mice were orally administered with FITC-dextran, and FITC- dextran serum levels assayed 4h later.

Figures 7a-7b show that XBPl deletion does not regulate genes involved in intestinal cell fate decisions, a. Expression of indicated genes was analyzed in small intestinal mucosal scrapings of XBP1^+/+ and XBP I^7' mice. n=3 per group (except for Tcf4, n=6). Two-tailed Student's t-test. No significant differences were observed, b. Small intestinal paraffin-embedded sections were stained by anti-β-catenin. Arrows point to nuclear β-catenin.

Figure 8 shows that XBPl deletion leads to the presence of apoptotic cells in the epithelium, a. Apoptotic nuclei were identified in XBP1^+/+ (XBPl^floχ/floxVCre) and XBPl^7"(XBPl^flox/flox) sections with anti-active (cleaved) caspase-3 and TUNEL.

Arrows point to apoptotic cells, b. Deletion of XBPl gene inXBPl^flo™^βoΛtelιn^CreER^T2 mice was induced by a 5 day administration of 1 mg tamoxifen i.p. daily, and apoptotic cells stained by an anti-active (cleaved) caspase-3 antibody. Time-points in the figure indicate the length of tamoxifen administration. Arrows point to apoptotic cells.

Figuer 9 shows that XBPl deletion results in a distorted villus: crypt ratio and hyperproliferation of intestinal epithelial cells, a. Jejunal sections of XBPl^flox/n°^x (XBP 1^+/+; n=7) and XBPl^flox/floxVCre (XBPl⁷-; n=8) mice were assessed for their villusxrypt ratio on H&E stainings (ratios of > 4:1 are considered normal for jejunum), b. XBPl*¹⁰³^⁰" (XBP1^+/+) and XBPl^floχ/floxVCre (XBPl^7') mice _were administered i.p. with bromodeoxyuridine (BrdU), and small intestinal sections harvested after 1 h and 24 h. The 1 h time-point labels the pool of proliferating IEC in the crypts (mostly transit amplifying IEC), whereas the 24 h time-point assesses the migration along the crypt-villus axis indicating the turn-over of the IEC compartment.

Figures 10a- 10b show that antibiotic treatment during 7% DSS colitis abrogates genotype-related differences in susceptibility to colitis, a. XBP1^+/+ (n=6) and XBPl ^A (n=3) littermates were treated with antibiotics (neomycin sulfate, 1.5g/l; metronidazole, 1.5g/l) in drinking water during the 5 days of high-dose DSS (7%; commensal flora- depleted mice are less susceptible to DSS colitis, which requires an increase in DSS dose to achieve colonic inflammation) administration as well as during the subsequent time on regular drinking water. Wasting was monitored by daily weight measurements, b. Colonic specimens harvested on day 8 of 7% DSS colitis in the presence of antibiotics were histologically assessed for inflammation. Two-tailed Mann-Whitney test.

Detailed Description The instant invention is based, at least in part, on the finding that XBP-I plays a role in intestinal inflammation that resembles inflammatory bowel disease and that a single SNP in the 5' untranslated region of the human XBPl gene (rs6005893) is significantly associated (P = 0.00084) with IBD predicting a direct role for XBPl in the pathogenesis of this disease. Accordingly, the present invention provides methods for detecting at least one SNP in the human XBP-I gene as well as methods to determine the predisposition of a human subject to develop inflammatory bowel disease.

/. Definitions

The term "polymorphism" refers to the coexistence of more than one form of a gene, or portion thereof, or a segment of DNA. A portion of a gene or segment of DNA of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region." A polymorphic locus can be a single nucleotide, the identity of which differs in the other alleles. A polymorphic locus can also be more than one nucleotide long. The allelic form occurring most frequently in a selected population is often referred to as the reference and/or wildtype form. Other allelic forms are typically designated or alternative or variant alleles. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic or biallelic polymorphism has two forms. A trialleleic polymorphism has three forms.

In one embodiment, a polymorphism is a single nucleotide polymorphism. The term "single nucleotide polymorphism" (SNP) refers to a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. A SNP usually arises due to substitution of one nucleotide for another at the polymorphic site. SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymorphic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" (thymidine) at the polymorphic site, the altered allele can contain a "C" (cytidine), "G" (guanine), or "A" (adenine) at the polymorphic site. SNP's may occur in protein-coding nucleic acid sequences, in which case they may give rise to a defective or otherwise variant protein, or genetic disease. Such a SNP may alter the coding sequence of the gene and therefore specify another amino acid (a "missense" SNP) or a SNP may introduce a stop codon (a "nonsense" SNP). When a SNP does not alter the amino acid sequence of a protein, the SNP is called "silent." SNP's may also occur in noncoding regions of the nucleotide sequence. This may result in defective protein expression, e.g., as a result of alternative spicing, or it may have no effect.

The term "linkage" describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci, or genetic markers. The term "linkage disequilibrium," also referred to herein as "LD," refers to a greater than random association between specific alleles at two marker loci within a particular population. In general, linkage disequilibrium decreases with an increase in physical distance. If linkage disequilibrium exists between two markers, or SNPs, then the genotypic information at one marker, or SNP, can be used to make probabilistic predictions about the genotype of the second marker.

As used herein, the term "detect" with respect to polymorphic elements includes various methods of analyzing for a polymorphism at a particular site in the genome. The term "detect" includes both "direct detection," such as sequencing, and "indirect detection," using methods such as amplification amd/or hybridization.

As used herein, the term "XBP-I " refers to a X-box binding human protein that is a DNA binding protein and has an amino acid sequence as described in, for example, Liou, H-C. et. al. (1990) Science 247:1581-1584 and Yoshimura, T. et al. (1990) EMBO J. 9:2537-2542, and other mammalian homologs thereof, such as described in Kishimoto T. et al., (1996) Biochem. Biophys. Res. Commun. 223:746-751 (rat homologue). Exemplary proteins intended to be encompassed by the term "XBP-I" include those having amino acid sequences disclosed in GenBank with accession numbers A36299 [gi: 105867], NP_005071 [gi:4827058], P17861 [gi:139787], CAA39149 [gi:287645], and BAA82600 [gi:5596360] or e.g., encoded by nucleic acid molecules such as those disclosed in GenBank with accession numbers AF027963 [gi: 13752783]; NM_013842 [gi: 13775155]; or M31627 [gi: 184485]. XBP-I is also referred to in the art as TREB5 or HTF (Yoshimura et al. 1990. EMBO Journal. 9:2537; Matsuzaki et al. 1995. J. Biochem. 117:303). As used herein, the term "XBP-I gene" refers to the coding sequence of XBP-I found in genomic DNA, as well as the intronic sequences and 5' and 3' untranslated/regulatory regions of the XBP-I gene. For example, in one embodiment, an XBP-I gene includes, for example, about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb of genomic DNA upstream of the XBP-I ATG initiation codon or downstream of the XBP-I termination codon.

As used herein, the term "NOD2-CARDJ5 " refers to the caspase recruitment domain family member 15. The nucleotide and amino acid sequence of N0D2- CARD15 can be found in, for example, GenBank Accession No.: gi:11545911, the contents of which are incorporated by reference. Several polymorphisms associated with susceptibility to inflammatory bowel disease have been identified and include, for example, a frameshift variant and two missense variants (Hugot, et al. Nature 411, 599- 603 (31 May 2001) and Ogura, et al. Nature 411, 603-606 (31 May 2001)), the contents of each of which are incorporated by reference. One of skill in the art can readily determine the presence or absence of these polymorphisms.

//. Isolation of Genetic Material

The subject SNPs are useful as markers, e.g., to make assessments regarding the propensity of an individual to develop inflammatory bowel disease or a related condition, and/or regarding the ability of an individual to respond to a certain course of treatment.

Genetic material suitable for use in such assays can be derived from a variety of sources. For example, nucleic acid molecules {e.g., mRNA or DNA, preferably genomic DNA) can be isolated from a cell from a living or deceased individual using standard methods. Cells can be obtained from biological samples, e.g., from tissue samples or from bodily fluid samples that contain cells, such as blood, urine, semen, or saliva. The term "biological sample" is intended to include tissues, cells and biological fluids containing cells which are isolated from a subject, as well as tissues, cells and fluids present within a subject.

Body samples may be obtained from a subject by a variety of techniques known in the art including, for example, by the use of a biopsy or by scraping or swabbing an area or by using a needle to aspirate. Methods for collecting various body samples are well known in the art. Tissue samples suitable for use in the methods of the invention may be fresh, frozen, or fixed according to methods known to one of skill in the art. In one embodiment, suitable tissue samples are sectioned and placed on a microscope slide for further analyses. In another embodiment, suitable solid samples, i.e., tissue samples, are solubilized and/or homogenized and subsequently analyzed as soluble extracts. The subject detection methods of the invention can be used to detect polymorphic elements in DNA in a biological sample in intact cells (e.g., using in situ hybridization) or in extracted DNA, e.g., using Southern blot hybridization. In one embodiment, immune cells are used to extract genetic material for use in the subject assays.

///. Uses of Polymorphic Elements Of The Invention The subject polymorphisms of the invention are useful as markers in a variety of different assays. The subject polymorphisms of the invention can be used, e.g., in diagnostic assays, prognostic assays, and in monitoring clinical trials for the purposes of predicting outcomes of possible or ongoing therapeutic approaches. The results of such assays can, e.g., be used to prescribe a prophylactic course of treatment for an individual, to prescribe a course of therapy after onset of inflammatory bowel disease (EBD), or to alter an ongoing therapeutic regimen.

Accordingly, one aspect of the present invention relates to diagnostic assays for detecting polymorphisms, e.g., SNPs, in a biological sample (e.g., cells, fluid, or tissue) to thereby determine whether an individual is afflicted with DBD, or is at risk of developing EBD. In one embodiment, the methods of the invention can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a specific allelic variant, e.g., SNP, of one or more polymorphic regions of an XBPl gene. The allelic differences can be: (i) a difference in the identity of at least one nucleotide or (ϋ) a difference in the number of nucleotides, which difference can be a single nucleotide at multiple sites or several nucleotides. The invention also provides methods for detecting differences in an XBPl gene such as chromosomal rearrangements, e.g., chromosomal dislocation.

The subject assays can also be used to determine whether an individual is at risk for passing on the propensity to develop a disease or disorder to an offspring. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing inflammatory bowel disease. The invention can also be used in prenatal diagnostics.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, determining one or more polymorphic elements in the sample and comparing the polymorphisms present in the control sample with those in a test sample.

The invention also encompasses kits for detecting the polymorphic elements in a biological sample. For example, the kit can comprise a primer capable of detecting one or more SNP sequences in a biological sample. The kit can further comprise instructions for using the kit to detect SNP sequences in the sample. IV. Detection of Polymorphisms

Practical applications of techniques for identifying and detecting polymorphisms relate to many fields including disease diagnosis.

DNA polymorphisms can occur, e.g., when one nucleotide sequence comprises at least one of 1) a deletion of one or more nucleotides from a polymorphic sequence; 2) an addition of one or more nucleotides to a polymorphic sequence; 3) a substitution of one or more nucleotides of a polymorphic sequence, or 4) a chromosomal rearrangement of a polymorphic sequence as compared with another sequence. As described herein, there are a large number of assay techniques known in the art which can be used for detecting alterations in a polymorphic sequence.

In one embodiment, analysis of polymorphisms is amenable to highly sensitive PCR approaches using specific primers flanking the sequence of interest. Oligonucleotide primers corresponding to XBP-I sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. In one embodiment, detection of the polymorphism involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077-1080; and Nakazawa et al. (1994) PNAS 91:360-364). In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.

This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, DNA) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically amplify a subject SNP under conditions such that hybridization and amplification of the sequence occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting polymorphisms described herein.

In one preferred embodiment, detection of single nucleotide polymorphisms ("SNP") and point mutations in nucleic acid molecule is based on primer extension of PCR products by DNA polymerase. This method is based on the fact that the nucleoside immediately 5¹ adjacent to any SNP/point mutation site is known, and the neighboring sequence immediately 3' adjacent to the site is also known. A primer complementary to the sequence directly adjacent to the SNP on the 3' side in a target polynucleotide is used for chain elongation. The polymerase reaction mixture contains one chain-terminating nucleotide having a base complementary to the nucleotide directly adjacent to the SNP on the 5' side in the target polynucleotide. An additional dNTP may be added to produce a primer with the maximum of a two-base extension. The resultant elongation/termination reaction products are analyzed for the length of chain extension of the primer, or for the amount of label incorporation from a labeled form of the terminator nucleotide. (See, e.g., U.S. Patent No. 6,972,174, the contents of which are incorporated by reference).

In one preferred embodiment, a polymorphism is detected by primer extension of PCR products, as described above, followed by chip-based laser deionization time-of- flight (MALDI-TOF) analysis, as described in, for example U.S. Patent No. 6,602,662, the contents of which are incorporated by reference.

Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In one embodiment, after extraction of genomic DNA, amplification is performed using standard PCR methods, followed by molecular size analysis of the amplified product (Tautz, 1993; Vogel, 1997). In one embodiment, DNA amplification products are labeled by the incorporation of radiolabeled nucleotides or phosphate end groups followed by fractionation on sequencing gels alongside standard dideoxy DNA sequencing ladders. By autoradiography, the size of the repeated sequence can be visualized and detected heterogeneity in alleles recorded. In another embodiment, the incorporation of fluorescently labeled nucleotides in PCR reactions is followed by automated sequencing. (Yanagawa, T., et al., (1995). / Clin Endocrinol Metab 80: 41-5 Huang, D., et al, (1998). J Neuroimmunol 88: 192-8.

In other embodiments, polymorphisms can be identified by hybridizing a sample and control nucleic acids to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, MJ. et al. (1996) Nature Medicine 2: 753-759). For example, polymorphisms can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. etal. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of polymorphisms. This step is followed by a second hybridization array that allows the characterization of specific polymorphisms by using smaller, specialized probe arrays complementary to all polymorphisms detected.

In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence XBPl, or a region surrounding XBPl and detect allelic variants, e.g., mutations, by comparing the sequence of the sample sequence with the corresponding reference (control) sequence. Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert {Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Patent No. 5,547,835 and international patent application Publication Number WO 94/16101, entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Patent No. 5,547,835 and international patent application Publication Number WO 94/21822 entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation" by H. Koster), and U.S Patent No.5,605,798 and International Patent Application No. PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et al. (1996) Adv Chromatogr 36: 127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident to one skilled in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleotide is detected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Patent No. 5,580,732 entitled "Method of DNA sequencing employing a mixed DNA-polymer chain probe" and U.S. Patent No. 5,571,676 entitled "Method for mismatch-directed in vitro DNA sequencing".

In some cases, the presence of a specific polymorphism of XBPl in DNA from a subject can be shown by restriction enzyme analysis. For example, a specific nucleotide polymorphism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA hetero duplexes (Myers, et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage" starts by providing hetero duplexes formed by hybridizing a control nucleic acid, which is optionally labeled, e.g., RNA or DNA, comprising a nucleotide sequence of an XBPl allelic variant with a sample nucleic acid, e.g., RNA or DNA, obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single- stranded regions of the duplex such as duplexes formed based on basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Sl nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, Cotton et al. (1988) Proc. NatlAcadSci USA 85:4397; Saleeba et al (1992) Methods Enzymol.

217:286-295. In a preferred embodiment, the control or sample nucleic acid is labeled for detection.

In another embodiment, an allelic variant can be identified by denaturing high- performance liquid chromatography (DHPLC) (Oefner and Underhill, (1995) Am. J. Human Gen. 57:Suppl. A266). DHPLC uses reverse-phase ion-pairing chromatography to detect the heteroduplexes that are generated during amplification of PCR fragments from individuals who are heterozygous at a particular nucleotide locus within that fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57:Suppl. A266). In general, PCR products are produced using PCR primers flanking the DNA of interest. DHPLC analysis is carried out and the resulting chromatograms are analyzed to identify base pair alterations or deletions based on specific chromatographic profiles (see O'Donovan et al. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility is used to identify the type of XBP-I polymorphism. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad Sci USA 86:2766; see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single- stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In another preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5). In yet another embodiment, the identity of an allelic variant of a polymorphic region is obtained by analyzing the movement of a nucleic acid comprising the polymorphic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313 :495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 1275).

Examples of techniques for detecting differences of at least one nucleotide between two nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes may be prepared in which the known polymorphic nucleotide is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allele specific oligonucleotide hybridization techniques may be used for the simultaneous detection of several nucleotide changes in different polylmorphic regions of XBP-I. For example, oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid. Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238; Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also termed "PROBE" for Probe Oligo Base Extension. In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) MoI. Cell Probes 6:1).

In another embodiment, identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Patent No. 4,998,617 and in Landegren, U. et al, (1988) Science 241 : 1077-1080. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g., biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al, (1990) Proc. Natl. Acad Sci. (U.S.A.) 87:8923-8927. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Several techniques based on this OLA method have been developed and can be used to detect specific allelic variants of a polymorphic region of an XBP-I gene. For example, U.S. Patent No. 5593826 discloses an OLA using an oligonucleotide having 3 -amino group and a 5'-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage. In another variation of OLA described in Tobe et al ((1996) Nucleic Acids Res 24: 3728), OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.

In another embodiment, the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Patent No. 4,656, 127). According to the method, a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is used for determining the identity of the nucleotide of a polymorphic site (Cohen, D. et al (French Patent 2,650,840; PCT Application No. WO91/02087). As in the Mundy method of U.S. Patent No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ is described by Goelet, P. et al (PCT Application No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. etal. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. S. et al, Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P, Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C, et al, Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al, Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. etal, Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al, GATA 9: 107-112 (1992); Nyren, P. et al, Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA™ in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A. -C, et al, Amer.J. Hum. Genet. 52:46-59 (1993)).

The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe/primer nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a polymorphic elements. In addition, a readily available commercial service can be used to analyze samples for the polymorphic elements of the invention.

V. Primers for Amplification of Polymorphic Elements

Given that the human XBP-I coding sequence and flanking sequences are publically available, primers can readily be designed to amplify the polymorphic sequences and/or detect XBP-I polymorphisms by one of ordinary skill in the art. For example, an XBP-I sequence comprising a polymorphism (e.g., SNP) of the invention can be identified in the NCBI Variation Database (dbSNP using the SNP IDs presented in Table 4) or by homology searching of another database containing human genomic sequences (e.g., using Blast or another program) and the location of the SNP sequence and/or flanking sequences can be determined and the appropriate primers identified and/or designed by one of skill in the art.

For example, the sequences flanking the XBP-I SNPs presented in Table 4 are as follows:

rs5762870:

TCCATTCCCC CAGGGCTAGG AGGATGGGGT CTCTACCTTC ACTATTTTCT TTAGAAAGAA GAAAGAAAGA AGAAAGATAG TGATGGCGGT AAGGACAACA GGGCCCTCGG GGGCACATGG CTATGCTGTG GCCTGGAGAG AGGCAGACTC TTCTCAGAGG AGGCTAAGGG CCTCTGGCTA GAGCATGGCT ACAAGCAAGG TGGCTAACAT GAAGAGACCA TGGTGCAGAG GCAGATGCCA GCAGGCTAAA GGACAATTCT GGAATGGGGC AGTGGGAGGG GTCCTGTGTC TTTGGGCTTC TTTGATCCCT GAGACACTTT GGACGGGACA GAGCCTTGGC CACCAACGGG GTCATGTCTG TTTAAACAAA ATTTAACACA GGAGGTAAAT CATTAACTCA Y [C/T] TGAAGAAACA AACAGGAGTG CAGGCCATAT CTCTAGCACC AGGATTCAAG AGCCACGGGG ATGTTAAGAA CTCCTGAATA TCAGAAGTGG CTGCCACTCA TGGCTTCTGT AAGTGAGTGA AAGCATTTGG TGAGCACCTT CTATAAGCAA GTCTTCGTGC CAGGTGCTGT GGGAACAGAT GCATGAAGAG GCTCAGAGTC TGACCTGAGC TTGGAGCTGG GGGAATACGA GCTCACAGCC ATTGTGAGCA TGCCCATGCC TGTCTCCAGG CCCACCACGG CCcatccatc atctcatgta attctcatgc ctctgaggta gggcaaggat tgcctgcatt tcatagacaa ggaaacccaa gctcagagag ggagagcaac ttgcctcggc cacacagctT

rsl0222249: tgagacagag tctcgcaccg tcccccgggc tggagggcag tggcccgatc tcggctcact gcaatctctg cctcccaggt tcaagtgatt ctcctgcctc agcctcccaa gcagctggga ttacaggtgc ccaccaccac gctcagctaa tttttttttt gtatttttag tagagacacg gtttcattat gttggccaag ctggtctcaa actcctgacc tcatgatctg cctgcctcag cctcccaaag tgctgggatt acagacgtga gccactgtgc ctggcctttt ttttttttta aatcatgacc atcctagtgg atgtgaaata atatctcatt gtgcatttcc ctgaaggcta ataatgcaaa gaatcattta atgtacttgt tggccattgg tacaactatt tttttggTAT CATCTCtttt ttaaaataaa aaaatttGAA ATACCAAAAG ATATATAGGA CAACACAACA AACATTTTTG TTCCCACCAC R [A/G]

TGACTTAAGA AATAggccag gcacagtggt ggctcacgcc tgtaatccca gcactttggg aggccgaggt gggcggatca cgaggtcagg agatggagac caccctggct aacacggtga aaccccgtct ctactaaaaa tacaaaaaat tagccgggca tggtggcacg ctcctgtagt accagctact cgggaggctg aggcaggaga atggcgtgaa cctgggaggc ggagcttgca gtgagccaag atggccccac tgcactgcag cctgggcgac agagcgagac tccaggcgac agagcaagac tccatctcaa aaaaaaaaaa aaaaagaaaa gaaaagaaAT AAAACATTgg ctgggtgcag tggctcacac ctgtaatccc agcactttgg gaggcctagg tgaatcactt gagctcacaa gtttgaaacc agcctgggca acatagtgaa accccgtctc tataaaaata caaaaattag gttgggtgtg

rs5762852:

AGCCAGCATG ACTTTGGAGC ATAGTCACTG AATGAATGTC CTTGGGTTGG GAAGCTGAAG CTCTGGGTCT GATATGTGCA CTGCTGCTAA CTCGCTGTAT GACCTCACAT GAGGCTCTTT

TCACATGTAC CTCTCACAGC TCTATAAGGA ACGACCCAGG CCAGAAACCT GCCAAATCCT GTCCAGTACT ATGATCTACA AAACGGATCT GCCTCAGGAA AGCCAGGGAA GGTGGGGCTG GATGGAGTGT GTTCCAGCTG GAACATCTGC TCTGGGCCAG GCACAGTGGG GAAAGCCAAG GTGGAT

Y [C/T] CACTATGGCC CTGATCTCAT GATTGGGGTG AGTCACATTT TTATGATTAA TAAACAATAG CACGTTCAAG TCCCCACTAA TTcaaatatt tactaggggt ctattctgtg ccaggcaaag gcatatggat acataagata aataagacac agtccttacc ctcaaggagt tcGGTGTGGA CAGGGATTGA TAAAACTGTG AAGTCAAGGA GACCTTTGTG AAATAGGGtg tagaaagtaa aagttcctct tcaaagtttc ctttcttgtt aaagaataaa tcacaactgt taaaaataat agtttctttt aaagattaac ttTATGCTAG ACATGCTCAC AGGCAGGTag tacattctat gtagtacttt aaccaaggta tctgtgctgg acgtgctcac aggcatgtcc cagctcgcag cctataccca ttccttattt aggaatatta ttacttttta aaaatccttt cataagcagc ttcctctttt cctttgtCTT

rs5762839: cagtggtgcc ttctcggctc actgcaacct ccacctcccg ggttcaagcg attctcgtgc ctcagcctcc cgagtagctg ggactacagg cgcgtaccac aacgcccggc tagtttttgt attttgagta gagacggggt ttcaccatgt tggccaacct ggtctcgaac tcctggcccc aagggatccg cccactttgg ccacccaaaa tgctgggatt acaggtgtga gccaccatgc ctggcctctc ttagtaattt gcaagtatat gatacaatgt tattaactat agtcaccata ttgtacaata AGACTGTACT TAGGCTTCAA AAGTTGAAAA GCAGGATGAA GGAAGAAAAC TGCCTCATGT CAACGTCTAT CCACACCTCC ACCCATCTAA

Y [C/T]

GAATACTTAC CAATCACCTA CTCCACCAGA TAGCACCAAA TAGCCAAAAC AAAAAGCCCA GCCTTTCAGT CAGGATGATT TTGTTTACCA GTGATGGAAC CCTACTTtgt gtgtgtgtat gtgtgtgtgt gtgagagaga gaaagagaga gagagagaCC CATTTTATGT GGACACTTAA

GGCCTGGGTG GAATGAGCTG GGTGGCAGGC AAGTAGAGGG TCGCAGGCCC ATGCCTGACT

CTGGGAGGGG ATGCCAGGCA GTGGGGACTG GCCCAGGCAG GCCAAGGGGA GTGCTGGAAG

GGTGGCCCAG GCAGCCAGAC CCATTGGGGG AAACTGAAGG CCAGCCCTTG GGAAGGTATC TAAGCCAGGG GGTGCGGGGC CAGAGCCTGG CCCAGGGAAG

ΓS5762814: tcaagcgatt ctcctgcctc agcctcccaa gtagctggaa ttacaggcat gtgccaccat gcccagctaa ttttgtattt ttagtagaga cggggtttct ccatgtgggt gaggctggtc ttgaactccc aacctcaggt gatccacccc cctcggcctc ccaaagtgct ggaattacag gcgtgagcca ccgctcctgg ccCTGAATGT ACTCATTTTA AAGCACTAAG GCAACAGTTC TAGTTACCTA CACGGCACCT ATTATACTTT TTCTTCCTTA TTAAAAAAAA CTATTTTGTT CAAGATGCCC ACTTGAAAAC ACTCAGCTTC CAGACTCCCT GGAGTTTATG GATAGTTACA TAATGTGGTT CAGGCCAATG TATACATACG TGGAAGTTCT R [A/G]

GGCACAGACG ATTCCAGGAA AGTTTGTTAA GTGGAGGAGA CTCAACTGGG AAGGCTTTCA CCTTTTGCCT GTTGCTCTTT TCCTTTTTTC CCCTCTTCCT TGCCTGGAAT GCTGATGAAA CTTCCAAAGA TTGAGAGGTT CAGCCGCCAT CTCACAAATA TGCGGATTGG AGTCACATGC TAAGGACGTC AGAGGGGAAA GACAGAAGGA GTCTGGGCTC CTGATGGCAC CATGGAGTGG CCATTTAGCT CTAGACTGCC CATTTCCAGA TGTCACATTA CATAAGAATT ATAATTCCTG TGGTAGCTGG GTTCTTGTTA CATGCCATTA AATACAGTCC TCGCTTTTAC TGACAAtttt tttttttttt ttgagaccga gtcttgctct gtcgcccagg

rs5762812: GGTTATATTA TCTTCTTAAT CATGCTGTTT TGTTTTATGT ATGTCTCTTT ATAAACAACA TATAGCTGGC TTTGGGGTTT CTCAGAGCTC TCTTTTAAGA AGAGaatata aatcatttgt atttattttt gattatttat ttatttGGAC TTATTTCTGC CATGTTATTT CACATTTTTT GTTTACTATA TTTCTcaagg caggaggatt gcttgaggcc aggagttcaa gaccaggctg ggaaacatat tgtgaccttg tctctacaaa aaatttaaaa aaattaacca ggcgtggtga cttgcacctg tagtcccagc tgctcggaag acggaggtgg taagatccct tgaacccagg agttcgaggc tgcagtaaac tatgagctat gattgaacca Y [C/T] tgtactccaa cctgggagac agaatgagct cctgcctcta aaaaacaaaa atttttttaa aaaaaaggaa aaTGACTTAC CAGCCATTTT TATGTTTATG GAAATGTTTA TGGAAAGAAA GCATATGTAg caatattaat atgagacaga atttatcata aaggcattaa aaggacaaag aagggtattt catgttgatt gaaggtacaa gttaccatat atacatcttt atgaacatAG CAACATAATT TTGAAATGTA TAggcctagc atgtcggctc acacctgtaa tctcagcact ttgggaggct gaggtcaggg gttcaagacc agcctggtca acatggcgaa accccgtctc tattaaaaat acaaaaatta actgggcatg gtggcacgtg

rs6005893:

Ggcgcctgta atctcagcta ctggggaggc tgaggcagga taatgtcttg aacctgggag gcagaggttg caattagcca agattgtgcc attgcactcc agcctggtca acagagtgag actttctcaa aaaaaaaaaa aaaaaaaaaa aaaGTcatta ttcagaaaga atcagtcagg aaagggtgga caaaacagga acagaaatAA ATAGGCATTC CAGAAAAGGG AAAACAGAAA AATACTGTAC ACTGGTaagc agaaaacaga aaataacatg gcaggaataa gtccaaatat ataaataata aaacataaat gtgaatgggt tatattatct tcttaaTCAT GCTGTTTTGT TTTATGTATG TCTCTTTATA AACAACATAT AGCTGGCTTT K [G/T]

GGGTTTCTCA GAGCTCTCTT TTAAGAAGAG aatataaatc atttgtattt atttttgatt atttatttat ttGGACTTAT TTCTGCCATG TTATTTCACA TTTTTTGTTT ACTATATTTC Tcaaggcagg aggattgctt gaggccagga gttcaagacc aggctgggaa acatattgtg accttgtctc tacaaaaaat ttaaaaaaat taaccaggcg tggtgacttg cacctgtagt cccagctgct cggaagacgg aggtggtaag atcccttgaa cccaggagtt cgaggctgca gtaaactatg agctatgatt gaaccattgt actccaacct gggagacaga atgagctcct gcctctaaaa aacaaaaatt tttttaaaaa aaaggaaaaT

rs2269578:

GATTTTCCCC CCTTAAGCGG ACTTATTTCC ATCCGGAGTG ACAGAATTTA ATTCCAAACC

S [C/G]

AGAGCTTTCC AGACTGACGA ATTTTTACCG GGACTAACAG AGAATTACCT CAGCCTGACA

rs5762809:

CCCAGCGTGG CGGATCCGGT CAGTCCGGGC TTCCTGCCCC GCCCCGCGCC ACGCTGGCAC CGACCCGCCA GGCCAAAGGC GACGGAGCCC TGCGGGGCGG CCAGTGCTGG GTccccgctC ccagcccctg cccctgcccc tgtccctagt cccggcttca gatctggccc cagaccccgg ccccTCGCCC ACCTCCTCAG CGCCTTCTCC TCGGGGCTCA GGTGCGTGAG GCGCTGTCGC TTGCGCGCCT GGGGCAGCCC CCCGCTCGCT GCCTCCGGGC TGGCCCCTCT CTGGGCTGGC ACCATGAGCG GCAGGGCCTG GCCGGCCGGG GCTCCGGCGG CGGAGGCGGG CTGCCCCGAC AGAAGCAGAA CTTTAGGGGT CCCGTCGGCC GGGTTCGGCG Y [C/T] GGCTGCCACC ACCACCATAG CTCCAGACTA CGCACCGCGC ACCGCGCGCC GCAGCCGCCC AGCGCCCAGC CTCGCCGCGC CCGGCCTTTC TACGGTCGTG GCCCTCCGCG ATTGGCCAGC GTCGCGTGAC GCACGGCCGA GCTCGGCGTC CATTGGTCCG GCCTGCCCGG CGGGGCGTTT CAGGACCGTG GCTATGGAGT CCGGCGTGGC AGCGGCAATC CCTGGCCAAA GGTACTTGGG GTCATTTTCC GCGGGGGGTT ACGTGCGGGA GCGTGTCCTC CACAAACGGA TTTTCCCCCC TTAAGCGGAC TTATTTCCAT CCGGAGTGAC AGAATTTAAT TCCAAACCGA GAGCTTTCCA GACTGACGAA TTTTTACCGG GACTAACAGA GAATTACCTC

rs2097461: GTCTCAGAGG GTATCTCTAA GACTAGGGGC TTGGTATATA TGTGGTCAAA ACGAATTAGT TCATTAATGG CTTCCAGCTT GGCTGATGAC GTCCCCACTG ACAGAGAAAG GGAGGCTGGT AAGGAACTGG GTCCTTCTGG GTAGACCTCT GGGAGCTCCT CCAGGCTGGC AGGCTCTGGG GAAGGGCATT TGAAGAACAT GACTGGGTCC AAGTTGTCCA GAATGCCCAA CAGGATATCA GACTGTAAGA GGCAAAAATT AAATGAAGTA CAACTGTCAG AATACAATGG AAAATCTAAC TGGAACACTT TGTACTGGGT TCCATAATGT AAATTAGTCA TTATGTGATA AGATGACCTC GGGACCCACC AGACCCATTT ATCTACACTT CACTCCATGT

Y [C/T]

CTATATTACC TGGAACTAGG AAGGTAGTTG ATGTTCACCT CCAACCCCAC CAAAAACTAA CTTCAACCCT CATCTGTCTA GTTAGGGATG TCAAGCATCA AACAGATGGA ATTAACTGGT TATATAGCTC TTTAATAAGT CAGAATGATC CCTACCTCTG AATCTGAAGA GTCAATACCG CCAGAATCCA TGGGGAGATG TTCTGGAGGG GTGACAACTG GGCCTGCACC TGCTGCAGAG GTGCACGTAG TCTGAGTGCT GCGGACTCAG CAGACCCGGC CACTGGCCTC ACTTCATTCC CCTGGGAGGA AAGACCAAAG TGAATAAACA GCTTCAAGTG CCCAAGGAAA TGCTTGCTAG ACAGCTGTGA TTCTCAACTT TAAAGAATTA CTTTTCAAAA

IS2267131:

AGagatacca ttatccaact ttacagatga ggaaataagt gattatatat aacccctggg ttacccagct taccagtggc agagttgaaa atagggagtt tcagtctaga aaatgctctt aattcccata ataAGGGCAA GAGAATCTCG ATATGTGATG GTGTGTCCTT GTTTAACATT TATAAGATGG TGTAGCAAAT AGTggttagg gttctagctc ttccacttac tagctgtgat attggtcagt taGGCTCAAG AATCTGTCTC AGAAAGACAG TAACTGCTCA AATTATAGCT TATATCACTT ATGGCAGTCC AGAATTAAAG CAGGCAGTAA TTAAGGTGGA AAAGCCTTCA GTCACTTTAA AAAGCGTTAG GCTCACTTCA GTTGGATTAA

Y [C/T] TCCTACCACA GCACACAGAT AAACATCTGG AGAGAAAACA AGACTTACGG CCCTAGAAGA AATCAAACAA GGATGCTGCA TTGTACCTTT TAATTGCATG GGTAGTTTTA AATAAATGGA GAAAGCACCT TTCAGAAGCT ACACTAGCAG GAAAAAATTC CATCAAGCAT TTACATAGTA AATTTCTATA ATTTCACAAA AGATTCTTGA TCTTACTTGA AGTATACATG AGGGAAAGAG CCCCCTCAGC AGGTGTTCCC GTTGCTTACA GAAGCAAACT AAAGGACCTA AAACTGGAGG CAAGCCAGGA TGCCAAAAAG GGGGAAGAGA AATGATAAAG AACCATTCAT AAATTCCATG TCTACTTCAA GACATTTGTC TAATGACCCT TACATAATAA

rs5762795: TTCTCTGGAA ACCCCTCCCT GAACTCCTTT CTGATAAGCC AAAGCCATCC TCTAATACTA AGAACTATAC AGGAGAGAGC CAAGCTAAGC ATGTAGCTGC TGGCACTGCC TTCCCTCAGA GAACTGAACT GTTTTCGGAA CCTCGGCCAA CAGGGATGTC TCTTTATAAT AGTTTGGAGA CAGCTACTAG CACAGAAGAA GAGATGGAAC TCTAGAAACC AATTTCTACA CTAAAGTTGT CAAATGTTAG AAGAATCCTG TGTTCAGTTA TGAGACTCTT TGCATAGTAT AGGGACTTGA AAGTTTTATG AGACGGGTGT AATAATATCT CCACCTGTGA TTTGGGGGTG GGACTCTTAT TTTGGGTAGC CATTTATTGA CTTCACCTTT TTGCCAAGGA M [A/C]

GTTTGTCTCA AGGGAAAAGC AGTTTTCTGT GGGGCTTATT AAAGGAATGT TGGTTTACAT TGTCTTCAAA GACAAGTATA GAAGCTGTAT GTGTAAGGGT GACTTAAATC ATATGTCACA TTGTCTAAAC TATTCAGACA CTTGGAGAAT ATTCTCCTTG AATTAAAAAA GATGATTAAG AAGGATGCTC CTACAACTGT ATCCTGACAG TTAAGTCACA GCTTAATGTG TAGATATGAG CTGTTTACAG TGGTGACTAT ATATAATTGG GGAGAAGAAG GGAAGAGAGC AGCAGTAGCT TAAGCCTGTT GCTAAGAAAT TTAATTTCTT AGCAACTTGT AATTTAGTTA TCAATTCAAT ATAGCTCTGT TGATTAAATA GCCGATAGTA TTGTGGCTCT

rs5752797:

TGATGAAGTT TTTACAAAGA AAATGATTGA GTCTATGTAA GTTTTGGTAC CTGATTTCTA

TTTAATTATG ATCTTGGAGG GAAGACCCAA TGTCTAGATT CTGCCCACCC TGTCCTCATT

AGCTCTTTTT ATCTCCTGAT CCCCAGGCAA TTGGAGATTG GCTTTTTCCT AGTTCATTAG GGGTGAGCAA AGTGCTTTAC AAGCAGGAGT TTGAAGTATG GGGAAGTGGC TGCCTTGCCT ACTTACTCTT TGGTTCCTTT GAAGACCTGA AATGGACTGC AGTCTGCTAT TTGGAAATGA GATGACTGGC AAGAGAGCCC ACTCTCAAGT TCCTGTGATA CAGGAACCAC ATTTTAAAGG CTCTTGTTTA CCTGGCTTTC TAAGTGACCC AGGAACAACA Y [C/T] TGCGGCCTAG GAAAAAAAAT AAAGTGGCCC AACAGTGAAA GCAGTTGCTG CAAAGACTGC AAAACAAAGC TCATATTCTT GGCTCATAGC ATAGTCCTTC CAGGCCTTTA GCAGAACTGT TTTGCTCTTA ATCTAAACAC AGTTTATGAG CTCAGTTATC TGGTTTCCTA GATCATCAGA GTAGAAGCAG AAACAGCAAA TGAGAGCAGA ACAAACAAAA CTCCTAACAG GGAGCTGTGA CTACACTGTG AGGACAAGAG AGGAAACTGG AAAGCAAACA CAATAACCTA AAAAGTAAAG TGGCCTGGGG CCCCTGAGTA ATGATTAGCC CAGCTCTAAT CCTGTAGTCC GTGAAGCCTC CGTGATCTCA AATTGTCATT ATTCACTATG TAAAATAGTA

Using the sequences provided above, one of skill in the art can readily design oligonocleoitdes to amplify and/or detect the polymorphism within these sequences. In one embodiment, a primer for amplification of a polymorphic elements is at least about 5-10 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 15-20 base pairs in length. In one embodiment, a primer for amplification of a polymorphic element is at least about 20-30 base pairs in length. In one embodiment, a primer for amplification of a polymorphic element is at least about 30-40 base pairs in length. In one embodiment, a primer for amplification of a polymorphic element is at least about 40-50 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 50-60 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 60-70 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 70-80 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 80-90 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 90-100 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 100-110 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 110- 120 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 120-130 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 130-140 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 140-150 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 150-160 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 160- 170 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 170-180 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 180-190 base pairs in length. In one embodiment, a primer for amplification of a polymorphic elements is at least about 190-200 base pairs in length.

In one embodiment, a primer for amplification of a polymorphic element of the invention is located at least about 200 base pairs away from (upstream or downstream of) the polymorphism to be amplified (i.e., leaving about 200 nucleotides from the end of the primer sequence to the polymorphism). In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 150 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 100 base pairs away from (upstream or downstream) of the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 75 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 50 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 25 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 10 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 5 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In another embodiment, a primer for amplification of a polymorphism of the invention is located at least about 2 base pairs away from (upstream or downstream of) the polymorphic sequence to be amplified. In yet another embodiment a primer for amplification of a polymorphism of the invention is adjacent to the polymorphic sequence to be amplified.

The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.

This invention is further illustrated by the following examples which should not be construed as limiting. EXAMPLES

The notation of references in the following sections refers to the list of publications following the Examples section.

Materials and Methods Human Biopsy Samples

Ileal biopsies were obtained from 3 randomly selected patients with clinically, endoscopically and histologically confirmed diagnosis of CD, as well as 4 healthy control patients. The diagnosis of CD was confirmed by established criteria of clinical, radiological and endoscopic analysis, and from histology reports. Informed consent was obtained and procedures performed according to the approval by the local ethics committee of the Medical University of Innsbruck. Biopsies were collected in RNAlater (Ambion), RNA isolated using RNAeasy columns (Qiagen), reverse transcribed, and used for quantitative PCR and XBPl splicing assays. Mice

One XBPl^fl0X/+ (129;B6) and one XBPl^floχ/" (129;B6;Balb/c) were initially mated with Villin (V)-Cre transgenic mice (Madison, B.B. et al. J. Biol. Chem. 277, 33275-33283 (2002)) (Jackson Laboratories) to eventually obtain XBP 1 ^flox/floxVCre transgenic mice. Colony maintenance involved mating XBPl^flox/floxVCre x χBPl^flox/flox as well as XBPl^fl0X/wtVCre x XBPl ^flox/flox .To exclude XBPl -unrelated phenotypes, we bred XBPl^flox/wtVCre x XBPl^flox/wt to obtain XBPl^VCre mice, which were confirmed to be histologically and clinically indistinguishable from XBP 1*** or XBPl^flox/wt mice. All experiments reported were performed with sex- and age-matched littermate "XBP I^'7"" (Le. XBPl^noχ/floxVCre), and "XHPl⁺*" (Le. XBPl^flox*^ox or XBPl^floχ/wt) mice obtained through the husbandry regimen reported above.

For experiments involving time-dependent Cre-mediated deletion of the floxed XBPl gene, we mated χBPl^floxneo/+ (129;B6) mice (see Fig. 5) with VCreER^ (129;B6) mice (el Marjou, F. et al. Genesis. 39, 186-193 (2004)). Cre recombinase was activated by administration of lmg tamoxifen (MP Biomedicals) intraperitoneally daily over 5 consecutive days. EIIaCre (Lakso, M. et al. Proc. Natl. Acad. Sci. U. S. A 93, 5860- 5865 (1996); Holzenberger, M. et al. Nucleic Acids Res. 28, E92 (2000)) andλώclCre (Kuhn, R., et al. Science 269, 1427-1429 (1995)) transgenic mice were obtained from Jackson Laboratories. All mice were PCR-genotyped of genomic DNA isolated by phenol extraction and isopropanol precipitation of proteinase K-digested tails. Reagents

Rabbit antibodies directed towards phospho-JNK, total- JNK and active (cleaved) caspase-3 were from Cell Signaling Technology. Rabbit anti-lysozyme antibody was from DakoCytomation, and anti-pro cryptdin antibody (Ayabe, T. et al. J. Biol. Chem. 277, 5219-5228 (2002)). Flagellin was obtained from Invivogen, and TNFα from Peprotech. The specific JNK-I, -2,-3 inhibitor SP600125 (Bennett, B.L. et al.. Proc.Natl. Acad. ScL U. S. A 98, 13681-13686 (2001)) (Sigma) was dissolved at 5OmM in DMSO. Carbamyl choline was from Sigma, and used at a final concentration of lOμM. Immunohistochemistry

Tissues were collected in 10% neutral buffered formalin and embedded in paraffin. Sections were deparaffinized in isopropanol and graded alcohols, followed by antigen retrieval with Retrievagen A solution according to manufacturer's protocol (Becton Dickinson), and endogenous peroxidase quenched by H₂O₂. Sections were then blocked for 30min with normal goat serum, and incubated overnight at 4⁰C with primary antibodies at dilutions recommended by the manufacturers. Secondary biotinylated anti-rabbit antibody (1:200) was added for 30 minutes followed by detection with streptavidin-HRP and development with DAB⁺ chromogen according to manufacturer's (DakoCytomation) recommendations. Slides were counterstained with Mayer's hematoxylin, dehydrated, and mounted with Eukitt. TUNEL staining

Apoptotic cells were detected on paraffin embedded small intestinal sections using TUNEL-POD kit from Roche Applied Sciences according to the manufacturer's protocol. Oral L. monocytogenes Infection

Sex and age matched groups of XBP1^+/+ and XBPl^7" littermates were infected intragastrically using gastric gavage at 3.6><10⁸ L. monocytogenes strain 10403s per mouse. AU procedures with infected animals were performed in BL-2 safety cabinets. For colony forming units (c.f.u.) assay, faecal pellets were aseptically collected 1Oh after oral infection, and mice euthanized 72 hours after infection, and liver and spleen aseptically harvested. Faecal pellets, liver and spleen were homogenized in PBS, and serial dilutions of the homogenates plated on LB plates containing 200μg/ml streptomycin, incubated at 37 ⁰C for 18 hours and c.f.u. were counted. L. monocytogenes burden in faeces was expressed as c.f.u. per mg dry weight, whereas liver and spleen data were expressed as c.f.u. per organ. Dextran Sodium Sulphate Colitis

Sex and age-matched littermate mice (8 to 12 weeks old) were given DSS (ICN Biomedicals Inc.) in the drinking water for 5 days as indicated and provided regular water thereafter. As indicated, mice were also treated with neomycin sulfate (1.5 g/L) and metronidazole (1.5 g/L) (Sigma) in the drinking water. 4.5% DSS was used for induction of colitis, except for those experiments where the commensal flora was depleted by antibiotic treatment, which is known to require higher concentrations (7%) of DSS to induce inflammatory changes (Maeda, S. et al. Science 307, 734-738 (2005); Rath, H.C. et al. Infect. Immun. 69, 2277-2285 (2001)). Weight was recorded daily at the same time, and rectal bleeding assessed according to an arbitrary score (0, absent; 1, traces of blood at anus or the base of the tail; 2, clearly visible rectal blood). For histological and mRNA expression studies, mice were killed on day 8 after initiation of DSS treatment. Histological scoring of colonic tissue fixed in neutral buffered formalin and embedded in paraffin was performed according to ten Hove et al. (Gut 50, 507-512 (2002)) RNA was isolated from colonic tissue using RNeasy kit (Qiagen) to study mRNA expression of mediators known to be involved in this type of experimental colitis. Electron Microscopy

Small intestinal tissue from sex-matched XBP1^+/+ and XBP l^'A littermates were fixed with 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% picric acid in 10OmM sodium cacodylate buffer. After washing with 10OmM sodium cacodylate buffer, tissues were treated for Ih with 1% osmium tetroxide and 1.5% potassium ferrocyanide, and then 30 min with 0.5% uranyl acetate in 5OmM maleate buffer, pH 5.15. After dehydration in ethanol, tissues were treated for 1 h in propylenoxide and then embedded in Epon/Araldite resin. Ultrathin sections were collected on EM grids and observed by using a JEOL 1200EX transmission electron microscope at an operating voltage of 60 kV. Crypt Isolation

Small intestinal crypts were isolated following published protocols (Ayabe, T. et al. Nat. Immunol. 1, 113-118 (2000)). In brief, the small intestinal lumen of adult mice was rinsed with ice-cold PBS and segments were everted and shaken in Ca++ and Mg++-free PBS buffer containing 3OmM EDTA to elute crypts. Villi and crypts eluted during 5min intervals were recovered by centrifugation at 70Og and crypt fractions identified by light microscopy. Crypt numbers were estimated by hemocytometry and 2,000 crypts resuspended in iPIPES buffer containing lOμM carbamyl choline (CCh; Sigma) and incubated for 30min at 37⁰C. Supernatants were harvested and proteins precipitated by trichloroacetic acid (TCA), and precipitates resuspended in Laemmli's buffer and resolved on 12% SDS-PAGE. Rabbit anti- lysozyme (DakoCytomation) was used for detection by Western blotting. In vivo Intestinal Permeability Experiments

Age-matched XBP1^+/+ and XBP Y^1' littermates were perorally administered with 0.6mg/g body weight of a 80mg/ml solution of FITC-dextran (Sigma), and peripheral blood collected 4h later. Dilutions of FITC-dextran in PBS were used as a standard curve, and absorption of 50μl serum or standard measured in a fluorometer at 488nm. Bromodeoxyuridine (BrdU) Incorporation

XBP1^+/+ and XBPl^"7" littermates were injected with lmg BrdU (Becton Dickinson) in 500μl PBS, and small intestinal tissue harvested after Ih or 24h in 10% neutral buffered formalin. Paraffin embedded tissue was sectioned and stained with anti-BrdU antibody according to manufacturer's protocol (Becton Dickinson).

Epithelial RNA Isolation and Quantification

Murine small intestines from age-matched XBP1^+/+ and XBPl^"7" littermates were opened longitudinally, rinsed with cold PBS, everted on a plain surface, RNAlater added onto the epithelial surface and the epithelium immediately scraped off using RNAse-free glass slides. Total RNA was isolated using RNAeasy columns (Qiagen), reverse transcribed and quantified by SYBR green PCR (Biorad). For microarray analysis, RNAs isolated from 3 specimens per genotype were pooled, and cDNA synthesis, hybridization, and laser scanning of the array carried out at the Biopolymers Core Facility (Harvard Medical School) with mouse genome 430 2.0 array (Affymetrix, Santa Clara, CA) as recommended by the manufacturer. The data analysis was performed by using Agilent GeneSpring GX and Affymetrix GCOS software under default parameter setting. Quantitative PCR cDNA was reverse transcribed from 1 μg of total RNA using oligo-dT primers and Qiagen's omniscript kit according to manufacturer's recommendations, lμl of cDNA was used per qPCR reaction, using SYBR green reaction mix (Biorad). Primers used had either been previously published (Kobayashi, K. S. et al. Science 307, 731- 734 (2005)), retrieved from publicly available databases (p ga. mgh. harvard . edu/primerbank/). or designed using VectorNTI software (Invitrogen), using recommended annealing temperature conditions or temperatures predicted by VectorNTI software. Product sizes ranged between 100 and 300bp. All primer sequences are available upon request. 40 cycles of PCR amplification were performed in an iCycler (Biorad), and quantities calculated based upon CT values and dilutions of arbitrary standard cDNAs. Specificity was confirmed by running products on 2% agarose gels. XBPl Splicing Assay

XBPl splicing was analyzed by specific primers flanking the unconventional splicing site yielding PCR product sizes of 164 and 138bp for XBPIu and XBPIs, respectively. Products were resolved on 2% agarose gels, and band intensity determined densitometrically (Optiquant Software, Perkin Elmer). XBPl Silencing in MODE-K Cells

The SV40 large T antigen-immortalized small intestinal epithelial cell line MODE-K (Vidal, K., et al. J. Immunol. Methods 166, 63-73 (1993)) was maintained in DMEM supplemented with 10% FCS, penicillin, streptomycin, glutamine, HEPES buffer, and non-essential amino acids. An XBPl -specific RNAi vector and a control vector were constructed exactly as reported by Lee et al. (Proc. Natl. Acad. Sci. U. S. A 100, 9946-9951 (2003)), except for that SFGΔU3hygro was used instead of SFGΔU3neo. Retroviral supernatant was prepared and used to transduce MODE-K cells as described (Iwakoshi, N.N. et al. Nat. Immunol. 4, 321-329 (2003)). Uninfected cells were removed by culturing cells in the presence of 750μg/ml hygromycin (Invitrogen) for 3 days. Suppression of XBPl mRNA expression by RNAi was confirmed by qPCR for XBPl. MODE-K.iXBP and MODE-K. Ctrl were seeded for CXCLl experiments (Song,F. et al. J. Immunol. 162, 2275-2280 (1999)) at IxIO⁵ cells per well in 96 well plates, allowed to adhere for 2-4 hours, then supernatant removed and stimulated with flagellin and TNFα for 4h. Similar results as reported in this manuscript were obtained when MODE-K cells were allowed to adhere for 24-48h to form confluent mono-layers. In experiments using the specific JNK inhibitor SP600125, MODE-K cells were pre-incubated for 30min with the indicated concentration of SP600125, cells supernatants removed and cells stimulated in fresh media with flagellin and TNFα in the presence or absence of SP600125.

For experiments assessing the CD Id-restricted antigen presenting function of MODE-K cells (van de WaI, Y. et al. Gastroenterology 124, 1420-1431 (2003)), IxIO⁵ MODE-K.iXBP and MODE-K. Ctrl cells were seeded in 96 well plates and allowed to adhere for 2-4h. The CD Id-binding model glycolipid α-galactosyl-ceramide (αGC) was then added at a concentration of lOOng/ml along with the indicated concentrations of SP600125. After 2h of incubation, MODE-K cells were washed and fixed with glutaraldehyde, followed by quenching with glycine, exactly as described (Kang, SJ. & CresswelLP. Nat. Immunol. 5, 175-181 (2004)). After 4 washes with media, the CD Id- restricted NKT cell hybridoma DN32.D3 (Bendelac, A. et al. Science 268, 863-865 (1995)), which is activated upon recognition of αGC presented by CD Id, was added to fixed MODE-K cells, and supernatants harvested after 18h assessed for IL-2 secretion by ELISA (BD Pharmingen). For experiments assessing JNK phosphorylation, MODE-K cells were seeded at 1x10⁶ per well in ImI in 6 well plates, allowed to form confluent mono-layers over 48- 72h, then washed with PBS, and stimulated with flagellin and TNFα for the indicated periods of time. Cells were then washed in ice-cold PBS and lysed in 500μl RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease (Complete®, Roche Applied Science) and Ser/Thr and Tyr phosphatase (Upstate) inhibitors. Western Blot

Protein content of lysates was determined by BCA assay, and equal amounts of lysates containing Laemmli buffer were boiled at 95⁰C for 5min, and resolved on 10% SDS-PAGE (for MODE-K cell lysates) or 12% SDS-PAGE (for TCA precipitates of purified crypts). Proteins were transferred to Protran membranes (Whatman), blocked with 5% milk in TBS-T, and incubated with the manufacturer-recommended concentrations of primary antibody in 3-5% BSA in TBS-T at 4⁰C overnight, washed, and incubated with a 1 :2,000 dilution of HRP-conjugated anti-rabbit secondary antibody in 3-5% milk in TBS-T for 45min at room temperature. Bands were visualized using Super Signal chemo luminescent substrate (Pierce). DNA Samples for SNP Genotyping

An IBD cohort of 1226 Italian cases and controls and 228 Canadian and Italian family trios (father-mother-affected child), for a total of 1910 DNA samples was examined. Specifically, 421 healthy unrelated controls, 746 patients with CD, 743 patients with UC, and 456 parents of IBD cases were genotyped. Canadian samples were collected from multiple hospitals in the province of Quebec: Chicoutimi (Complexe Hospitalier de Ia Sagamie), Montreal (McGiIl University Health Centre and Jewish General Hospital), Quebec City (Pavilion lΗόtel Dieu de Quebec), and Sherbrooke (Centre Universitaire de Sante de l'Estrie). Italian samples come from a population enrolled at S Giovanni Rotondo 'CSS' Hospital. In all populations considered, the diagnosis of IBD and classification as CD or UC was confirmed by established criteria of clinical, radiological and endoscopic analysis, and from histology reports. A review of the patient's chart, as well as an interview with the patient, was done to complete the phenotypic data. Written informed consent was obtained from all participants and ethics approval was granted in each of the participating institutions. SNPs Selection

We genotyped a region of 81.1kb (Chr22: 27,504,552 - 27,585,713; HapMap build #20) which includes the XBPl gene of 6kb (Chr22: 27,515,103 - 27,521,114), 64.6kb upstream of the XBPl gene, and 10.5kb downstream of the XBPl gene, which contains a segment of the hypothetical protein gene FLJ33814. First, we searched the HapMap database (www.hapmap.org; Build #20) for all SNPs in a 200 kb region surrounding the XBPl gene and downloaded the genotype data for the CEU population. Using Haploview version 3.31 we identified an ~81 kb block (blocks were defined according to the method of Gabriel et al) of strong LD containing the entire XBPl gene. Haplotype tagging SNPs (htSNPs) identified by the Haploview program as tagging all haplotypes greater than 1% were selected for genotyping. To incorporate some redundancy into SNP selection (in case some assays for the htSNPs failed design or genotyping), where possible, we chose 2 htSNPs per haplotype. Second, we supplemented these tagging SNPs with coding SNPs and SNPs located within the 5kb region upstream of the XBPl gene by searching the PubMed (www.ncbi.nhn.nih.gov/projects/SNP/), Ensembl (www.ensembl.org) and UCSC (genome.ucsc.edu) databases. The 21 SNPs for which we were able to design genotyping assays can be found in Table 4. Genotyping

Genotyping assays were designed for the Sequenom Mass Array iPLEX platform using the Sequenom Assay Design software version 3.0. The EBD samples (trios, cases, and controls) as well as the 90 CEU samples included in the International HapMap project were genotyped by primer extension of multiplex PCR products followed by a chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). A total of 13 SNPs were validated and we obtained a concordance >99.9% between our genotype data and the CEU data available on HapMap.

Genotyping call rates for the IBD samples ranged from 94.2 to 100% with an average of 98.3%. Genetic Analysis

Association in cases and controls was assessed by a standard Chi-Square test and association in trios was determined by transmission disequilibrium test (TDT), both performed in Haploview version 3.31. To combine data from cases-controls and trios populations, the number of risk alleles transmitted in trios or found in cases was reformatted as a mean or expected count (E_T and EC), observed count (OT and OC), and variance (varT and varC). A combined Z-score was then calculated by summation as Z=[(OT+OC)-(ET+EC)]/V(varT+varC). Although there was substantial correlation between the typed SNPs (see Figure 1), a conservative Bonferroni approach to correct for multiple testing (n—13) was used and determined a significance threshold of P < 0.004.

Example 1:

In eukaryotes, endoplasmic reticulum (ER) stress activates three distinct unfolded protein response (UPR) signalling pathways through ER transmembrane inositol-requiring enzyme- lα and β (IRElα and β), protein kinase- like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Wu, J. & Kaufman,R. J. Cell Death. Differ. 13, 374-384 (2006)). IREl excises an intron from transcription factor XBPl, its only known substrate as an endoribo nuclease, by an unconventional splicing event that generates XBPIs, a potent inducer of a subset of UPR target genes (Calfon, M. et al. Nature 415, 92-96 (2002)). XBPIs is required for ER expansion (Shaffer, A.L. et al. Immunity. 21, 81-93 (2004))and the development of highly secretory cells such as hepatocytes, plasma cells (Reimold, A.M. et al. Nature 412, 300-307 (2001)), and pancreatic and salivary gland epithelial cells ¹1. As IEC, and Paneth and goblet cells in particular, are highly secretory cells, it was hypothesized that XBPl might also regulate the function of these cells, and, consequently, mucosal defense and inflammation. The activation state of the UPR in the intestine was assessed by analyzing grp78 (BiP), an ER chaperone, and XBPl expression levels along with XBPl splicing status in ileal biopsies from healthy individuals and CD patients. Figure Ia shows increased grp78 levels in inflamed CD biopsies. Total XBPl levels were similar in controls and CD patients; however, the levels of active, spliced XBPl (XBPIs) were increased two-fold in both inflamed and non- inflamed CD biopsies. A two-fold increase in XBPIs levels is significant, given the tightly regulated turn-over of XBPIs (Yoshida,H., et al. J. CellBiol. 172, 565-575 (2006)), and along with increased grp78 levels indicates the presence of ER stress in these CD tissues. To investigate the function of XBPl in vivo, a mouse model with a conditional deletion of XBPl limited to the intestinal epithelium was generated. χBpi^Ωox/flox mice were generated and crossed onto Villin (V)-Cre transgenic mice (Fig. 5), where the Villin promotor directs Cre recombinase activity specifically in the small and large intestinal epithelium (el Marjou, F. et al. Genesis. 39, 186-193 (2004)). XBP 1 ^flox/floxVCre offspring were born at a Mendelian ratio and developed normally. XBPl exon 2 was efficiently deleted (99% in XBPl^flox/floxVCre (¹¹XBPl⁷-") small intestinal epithelia, 87% in colonic epithelia) (Fig. 6). XBPl mRNA levels in other organs (e.g. spleen and liver) were not affected (Fig. 6b). XBPT^7' small intestinal epithelia exhibited evidence of increased ER stress as grp78 was increased in the baseline state (Fig. Ib). Microarray analysis of epithelial cell scrapings confirmed this upregulation of BiP and an almost 9-fold upregulation of Chop (Ddit3) in XBP Y^1' mice indicative of ER stress (Table 2). In association with this increased ER stress, adult (8-32 wks) mice held under specific pathogen free (SPF) conditions exhibited small intestinal mucosal inflammation in 7/18 (39%) XBP1 ^A but not in any (0/20) XBPl^+/+mice (Chi square P<0.01; Fig. Ic). The inflammatory changes ranged in severity from lamina propria polymorphonuclear infiltrates, to crypt abscesses and frank ulcerations, and were often accompanied by a numeric increase in intraepithelial lymphocytes. No fistulae or granulomas were detected. XBPl^7" mice exhibited several changes in the cellular composition and architecture of the small intestine epithelial cell compartment. XBPl^"7" intestine was completely devoid of Paneth cells, in contrast to XBP1^+/+ mice (Fig. Id) or VCreXBPlwt/wt mice which had normal numbers of Paneth cells. Electron microscopy (EM) revealed that instead of the large electron-dense granules and expanded ER present in XBP1^+/+ Paneth cells, XBP I^7' Paneth cells exhibited only a few rudimentary electron- dense granules of minute size, and a compressed ER (Fig. Id). XBP1^+/+ Paneth cell granules store lysozyme and pro-forms of cryptdins, which were barely detectable in XBPl^7" crypts (Fig. Id). Messenger RNA expression of cryptdins- 1, -4, and -5 was reduced 34, 30, and 182-fold, respectively, and lysozyme expression reduced 7-fold (Fig. 6a) in XBPl^7" compared to XBP1^+/+ epithelium. In contrast, levels of cathelicidin, an antimicrobial peptide expressed by small intestinal and colonic absorptive epithelial cells were normal (Fig. 6a). Furthermore, XBP Y^1' small intestinal epithelia had reduced numbers and size of goblet cells by H&E and periodic acid Schiff (PAS) stains (Fig. Ie). Goblet cells were normal in the large intestine. EM of XBPl^7" jejunum showed a decrease in goblet cell granule size and differences in shape, along with a substantial reduction in ER size (Fig. Ie). mRNA for Muc2, a goblet cell-specific protein was reduced 4.5-fold in XBPl^'7' compared to XBP1^+/+ epithelium (Fig. 6a). Enteroendocrine cells were similarly sparsely distributed in XBP1^+/+ and XBPl^7' small intestinal epithelia (Fig. 6c). Absorptive epithelia did not exhibit any ultrastructural abnormalities in XBPl^{" "} mice (Fig. Ie), and assessment of the epithelial barrier function by oral gavage of fluoresceinated dextran was similar in XBPl^7" and XBP1^+/+ mice (Fig. 6c). Thus, XBPl^7" mice exhibited a major defect in Paneth cells and, to a lesser extent, goblet cells in the small intestine (Fig. If). Differentiation and proliferation of IECs is tightly regulated by several signal transduction pathways and transcription factors (Reya, T. & Clevers, H. Nature 434, 843- 850 (2005); Fre, S. et al. Nature 435, 964-968 (2005); van Es, J.H. et al. Nat. CellBiol. 7, 381-386(2005)). Quantitative PCR (β-catenin, Tcf4, Mathl, Hesl; Fig. 7a) and microarray analysis (Table 2) of XBPl^7" and XBP1^+/+ intestinal epithelial mRNA did not reveal alterations in known factors involved in intestinal cell fate decisions, and XBPl deletion did not alter nuclear localization of β-catenin (Fig. 7b). This rendered it unlikely that XBPl directs a transcriptional program that specifies Paneth and goblet cell lineage commitment. It was hypothesized that Paneth cells, due to their high secretory activity, might undergo programmed cell death from an inability to manage ER stress in the absence of an intact IREl/XBPl signalling pathway, similar to the relationship between zymogens and XBPl in pancreatic acinar cells (Lee, A.H., et al. EMBOJ. 24, 4368-4380 (2005)). Indeed, a few pyknotic, apoptotic cells were detected in some crypts in XBPl^7" but not in XBP1^+/+ mice (anti-active caspase-3⁺ and TUNEL⁺; Fig. 8a). In the steady state, Paneth cells are replenished from epithelial progenitors every ~30 days, while absorptive epithelia and goblet cells renew every 3-4 days (Reya, T. & Clevers, H. Nature 434, 843- 850 (2005)). To obtain temporal control of XBPl deletion and thereby circumvent the problems associated with a low-frequency event (apoptosis) in a replenishing cell population, XBPl^fl0xneo^^0xnfiOVillinCre-ER^T2 mice were generated (Fig. 6). Here, Cre recombinase is expressed as a fusion protein with a mutated estrogen receptor, requiring tamoxifen for its nuclear localization, while transcription is driven by the Villin promoter (el Marjou, F. et al. Genesis. 39, 186-193 (2004)). Along with efficient deletion of XBPl after initiation of tamoxifen treatment (Fig. Ig), Paneth cells were reduced by 98% on day 7, paralleled by a similar decrease in cryptdin-5 mRNA transcripts. Pyknotic epithelial nuclei staining positive by TUNEL (Fig. Ih) and active caspase-3 (Fig. 8b) were observed after 2.7 days, peaking at day 5, and declining on day 7. Apoptotic cells were present at the base of crypts, but also in villous epithelium, consistent with apoptosing Paneth and goblet cells, respectively (Fig. Ih). A gradual increase of Chop (Ddit3) mRNA was observed, similar to that observed in XBPl ^~'^~ mice (Table 2), after tamoxifen administration suggesting a link between the UPR and apoptotic pathways in XBPl -deficient mice as mediated by Chop (Fig. Ih) (Wu, J. & Kaufman,R.J. Cell Death. Differ. 13, 374-384 (2006)). The overall architecture of the small intestinal epithelium exhibited villus shortening with a reduction of the villus: crypt ratio (Fig. 9), indicative of a regenerative response in XBPl^7" mice. To assess proliferation in the stem cell compartment and turnover rate of IECs, bromodeoxyuridine (BrdU) pulse chase experiments were performed. A Ih pulse of BrdU labelled the proliferative pool of intestinal stem cells (predictably mostly transit-amplifying cells (Reya, T. & Clevers, H. Nature 434, 843- 850 (2005)), and was similar in XBP1^+/+ and XBPl^7" mice (Fig. 9). However, 24h after BrdU injection, labeled cells were found higher up in the crypt- villus axis in XBP Y^1' mice, indicating increased migration rate (Fig. 9). Thus XBPl affects IEC homeostasis both through controlling cell renewal and cell death. Upon stimulation with the muscarinic receptor agonist carbamyl choline (CCh),

Paneth cells release cryptdins, lysozyme and other constituents of their granules.¹⁶ As expected, supernatants from isolated CCh-stimulated XBP Y^1' crypts did not contain appreciable amounts of lysozyme, compared to XBP1^+/+ crypts (Fig. 2a). To assess the consequences of Paneth cell deficiency, we orally infected XBP1^+/+ and XBP Y^1' mice with Listeria monocytogenes, a gram positive intracellular pathogen. Ten hours after infection, 100-fold higher numbers of colony forming units (c.f.u.) of L. monocytogenes were recoverable from faeces of XBPl^7" compared to XBP1^+/+ mice (Fig. 2b). Translocation into liver and spleen analyzed after 72h revealed a 10-fold higher number of L. monocytogenes recovered from XBP Y^1' livers, but similar numbers from spleen (Fig. 2c). These data suggest that XBPl in the intestinal epithelium acts via Paneth cells to decrease the luminal burden of L. monocytogenes.

DSS is an agent that destroys mucosal epithelial cells and disrupts their barrier function allowing for increased bacterial invasion. As shown in Fig. 2d,e, XBP I^"7' mice administered 4.5% DSS in the drinking water exhibited more severe wasting and more rectal bleeding compared to XBP1^+/+ littermates. Histologically, XBPl^"7' colons displayed increased areas of mucosal erosions, edema, and cellular infiltration along with increased crypt loss as compared to XBP1^+/+ littermates (Fig. 2f,g). Addition of antibiotics abrogated the difference in severity of DSS colitis between XBPl⁺/* and XBP Y^1' mice (Fig. 10a,b) highlighting the importance of the commensal flora in the colitis observed here (Fig. 2d-h).

TNFα is a central mediator of inflammation in DSS colitis and human IBD (Targan, S.R. et al. N. Engl. J. Med. 337, 1029-1035 (1997)) and is regulated by microbial and non-microbial factors. TNFα mRNA expression was upregulated in XBP1^"Λ as compared to XBP1^+/+ colonic tissue from DSS treated mice (Fig. 2h). IREl has a crucial role in ER stress-induced JNK activation by recruiting the TNFR associated adaptor protein TRAF2 (Urano, F. et al. Science 287, 664-666 (2000)) and ER stress-induced JNK activation is dependent on TNFRl (Yang, Q. et al. EMBORep. 7, 622-627 (2006)). TNFRl acts downstream of IREl and both proteins are present in the same complex under ER stress conditions (Yang, Q. et al. EMBORep. 7, 622-627 (2006)). JNK phosphorylation status in XBP Y^1' mice was therefore analyzed. A phospho(P)-JNKl/2 antibody exhibited a patchy staining pattern in XBP 1^"Λ small intestinal epithelia, but not in XBP1^+/+ controls (Fig. 3 a). Levels of JNK1/2 phosphorylation are increased in the colonic mucosa of IBD patients (Waetzig, G.H., et al. J. Immunol. 168, 5342-5351 (2002)).

For functional study of JNK phosphorylation, we silenced XBPl expression in the mouse EEC line MODE-K using a small interfering RNA retrovirus (iXBP). MODE-K. iXBP compared to MODE-K. Ctrl cells showed increased JNK phosphorylation upon stimulation with TNFα and flagellin, a major bacterial antigen associated with the induction of colitis (Lodes, M.J. et al. J. Clin. Invest 113,1296- 1306(2004)) (Fig. 3b). As a surrogate marker to assess the inflammatory state of epithelial cells, the chemokine CXCLl was examined. MODE-K. iXBP cells showed increased release of CXCLl into the supernatant after stimulation with TNFα (Fig. 3c) and flagellin (Fig. 3d). The specific JNK inhibitor SP600125 is known to improve DSS colitis concomitant with reduced colonic TNFα levels and epithelial cell apoptosis, typically increased in DSS colitis . Addition of SP600125 resulted in a dose- dependent decrease of TNFα- and fiagellin-stimulated CXCLl secretion from silenced and non-silenced MODE-K cells (Fig. 3e,f). Non-specific effects of SP600125 were excluded by assessing the CD Id-restricted antigen presenting function of MODE-K cells (van de WaI₅Y. et al. Gastroenterology 124, 1420-1431 (2003)) (Fig. 3g). Overall, these data suggest that in the absence of XBPl, IEC exhibit a spontaneous increase in the tone of the JNK/SAPK signalling pathway, which integrates multiple pro-inflammatory signals. This is associated with increased TNFα secretion and epithelial responsiveness to cytokines, such as TNFα, and bacterial antigens, such as flagellin. Apart from that, increased JNK signalling might also contribute to induction of apoptosis of Paneth and goblet cells in XBPl^epithelia (Fig. Ih). Genetic deletion of XBPl hence results in induction of ER stress in intestinal epithelia at the baseline state, similar to what we have observed in human CD biopsy specimens (Fig. Ia). This in turn has two major consequences; increased JNK phosphorylation and hence a pro-inflammatory state, and depletion of Paneth cells with consequent effects on the regulation of the intestinal microbiota. These changes in epithelial cell function induced by loss of XBPl expression are revealed as spontaneous intestinal inflammation. XBPl is thus a unique component of the UPR in IBD pathogenesis since deletion of IREl β does not cause either loss of Paneth cells or spontaneous intestinal inflammation (Bertolotti, A. et al. J. Clin. Invest 107, 585-593 (2001)). Since XBPl regulates the two known inducers of IBD:¹ the inflammatory state of the mucosa (via JNK), and the commensal bacterial flora (via Paneth cells) it was a potential genetic risk factor for IBD. A recent genome scan with 358 autosomal microsatellites in 260 IBD patients identified nominal evidence for linkage with EBD of a locus on chromosome 22ql2.1, which is 0.3Mb apart from the XBPl gene . 1489 IBD patients were therefore tested (743 patients with UC and 746 with CD; Table 3) and matched controls (421 healthy unrelated controls and 456 parental controls) for 13 SNPs representing all haplotype tagging SNPs (htSNPs) as well as coding and promoter region variation in the genomic region containing XBPl (Fig. 4, Table 4). A single SNP (rs6005893) was found to be significantly associated with IBD (P = 0.00084, Table 1). This SNP was associated with both the UC and CD phenotypes (UC, P = 0.0021 and CD, P = 0.018), is located approximately 3.5 kb upstream of the transcription start site and is very common in the European derived populations under examination (Table 1). This variant confers an estimated relative risk of 1.6 (95% CI, 1.19-2.14). These results provide evidence that a genetic variant in the XBPl gene is a risk factor for IBD. It should be noted, however, that although none of the other known variants were found to be associated, we can not definitively implicate rs6005893 as the causative mutation given the extensive LD in the region such that other variants not yet identified could actually be causal. Nonetheless, taken together with the functional role of XBPl revealed in the current mouse studies, the identification of an association between IBD and rs6005893 strongly supports a key role for XBPl as a genetic locus for IBD that includes both UC and CD.

Therefore, the key elements of IBD pathogenesis - a genetically conferred inflammatory state of the host immune response to, and regulation of, the intestinal microbiota -can be accounted for and unified by the biology of a single gene product, XBPl, that is functionally active within the IEC. The selective deletion of this single gene, whose homologue confers risk to human IBD, in the intestinal epithelium, is sufficient to induce spontaneous enteritis that histologically resembles several features of human IBD. While inappropriate T helper cell activation has been a key paradigm of IBD pathophysiology and undoubtedly is important for its tissue- destructive consequences, the data and previously discovered genetic risk factors of CD converge now on epithelial cell dysfunction as key to IBD pathogenesis. Specifically, the observations that Nod2 (Ogura, Y. et al. Nature 411, 603-606 (2001); Hugot, J.P. et al. Nature 411, 599-603 (2001)) is expressed by Paneth cells (LaIa, S. et al. Gastroenterology 125, 47-57 (2003)) and Nod2-- mice exhibit increased L. monocytogenes translocation upon oral, but not parenteral, challenge (Kobayashi, K. S. et al. Science 307, 731-734 (2005)), implicate Paneth cells and their interactions with luminal microbes in the pathogenesis of IBD. Quite differently, N0D2 expression by myeloid cells has been linked to Toll-like receptor 2 signalling (Watanabe, T., et al. Nat. Immunol. 5, 800-808 (2004)) and increased sensitivity to development of DSS- associated colitis in an IL-I mediated pathway (Maeda, S . et al. Science 307, 734-738 (2005)), indicating unique contributions of specific cell types in IBD pathogenesis. DLG5 (Sto 11, M. et al. Nat. Genet. 36, 476-480 (2004)) is predicted to be important in the maintenance of epithelial cell integrity, while SLC22A4 and SLC22A5 encode organic cation transporters, which are expressed in colonic epithelium, macrophages and T cells (Peltekova,V.D. et al. Nat. Genet. 36, 471-475 (2004)). Taken together these observations suggest that human IBD may be initiated by and based upon a genetically encoded dysfunction of the epithelium. Finally, XBPl confers risk for both forms of IBD, CD and UC. IBD is undoubtedly a polygenic disease, with CD and UC often considered the final common pathways of a variety of inherited traits (and contributing environmental factors) that converge in one or the other form of presentation. CD and UC have many overlapping clinical, immune and genetic characteristics and several families with IBD have been reported with cases of both diseases within the same family (Halme, L. et al. World J. Gastroenterol. 12, 3668-3672 (2006)). XBPl is be a unifying risk factor that regulates pathogenic factors within the IEC common to both CD and UC. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

What is claimed is:

1. A method for determining the predisposition of a human subject to develop inflammatory bowel disease, said method comprising detecting at least one single nucleotide polymorphism (SNP) in the human XBP-I gene, to thereby determine the predisposition of a human subject to develop inflammatory bowel disease.

2. The method of claim 1, further comprising detecting a polymorphism in a NOD2-CARD15 gene.

3. A method for detection of at least one SNP in the human XBP-I gene, which method comprises determining a nucleotide at position -3230 relative to the start ATG of the human XBP-I gene, and thereby detecting absence or presence of at least one SNP.

4. The method of claim 3, wherein the single nucleotide polymorphism at position -3230 is the presence of G and/or T.

5. The method of any one of claims 1, 3, or 4 in which the single nucleotide polymorphism is determined by primer extension of at least one PCR product and MALDI-TOF analysis.

6. An isolated and purified allele-specifϊc oligonucleotide probe of about 5 to about 50 nucleotides which specifically detects a human XBP-I polymorphism at position -3230 relative to the start ATG of the human XBP-I gene.

7. A diagnostic kit comprising an oligonucleotide that specifically detects a human XBP-I polymorphism at position -3230 relative to the start ATG of the human XBP-I gene.