WO2008039445A2 - Polymorphismes du gène xbp-1 humain associés à une maladie intestinale inflammatoire - Google Patents

Polymorphismes du gène xbp-1 humain associés à une maladie intestinale inflammatoire Download PDF

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WO2008039445A2
WO2008039445A2 PCT/US2007/020658 US2007020658W WO2008039445A2 WO 2008039445 A2 WO2008039445 A2 WO 2008039445A2 US 2007020658 W US2007020658 W US 2007020658W WO 2008039445 A2 WO2008039445 A2 WO 2008039445A2
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xbpl
xbp
gene
cells
mice
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Richard S. Blumberg
Laurie H. Glimcher
John Rioux
Arthur Kaser
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President And Fellows Of Harvard College
The Brigham And Women's Hospital, Inc.
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • a single layer of intestinal epithelial cells 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)
  • Paneth cells located at the crypt base, contain several anti-bacterial peptides, ⁇ -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 ⁇ -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)).
  • SNPs Single nucleotide polymorphisms
  • 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.
  • 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.
  • XBPl X-box binding protein- 1
  • URR Unfolded Protein Response
  • selective disruption of XBPl in intestinal epithelium results in Paneth cell loss and spontaneous intestinal inflammation that resembles human IBD.
  • rs6005893 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.
  • 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.
  • SNP single nucleotide polymorphism
  • a polymorphism in a NOD2-CARD15 gene is further detected.
  • 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.
  • the single nucleotide polymorphism at position -3230 is the presence of G and/or T.
  • the method to detect the single nucleotide polymorphism is primer extension of at least one PCR product and MALDI-TOF analysis.
  • 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.
  • 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.
  • 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.
  • XBPl, cyrptdin-5 (Defcr5), and Chop mRNA (all expressed normalized to ⁇ -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 collecte d 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 lO ⁇ M carbamyl choline (CCh). Supernatants were precipitated, resolved on SDS-PAGE and detected by anti-lysozyme IgG.
  • CCh carbamyl choline
  • 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).
  • iXBP small interfering retrovirus
  • Control Control retrovirus
  • Control were stimulated for the indicated periods of time with Flagellin (1 ⁇ g/ml) and TNF ⁇ (50 ng/ml) and analyzed for P-JNK and total JNK by Western, c.
  • MODE-K.iXBP filled circles
  • MODE-K. Ctrl open circles
  • cells were stimulated for 4h with flagellin, and supernatants assayed by ELISA for CXCLl.
  • MODE-K.iXBP circles
  • MODE-K.iXBP (circles) and MODE-K.
  • Ctrl (diamonds) cells were stimulated with either lO ⁇ g/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.
  • MODE-K cells were stimulated with 50 ng/ml TNF- ⁇ (filled symbols) or cultured in media alone (open symbols), g. MODE-K.iXBP (filled circles) and MODE- K.
  • the SNPs indicated by the underline have not been previously validated in HapMap.
  • LD linkage disequilibrium
  • Figure 5 shows the generation o ⁇ Xbpf° 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.
  • neo floxed neomycin resistance gene cassette
  • XbpP 10 *" *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.
  • 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: A ⁇ cl -cre mice which received poly(I:C) (Xbpl ⁇ ) was slightly smaller than the wild type XBPl mRNA.
  • 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.
  • Livers and spleens of XBPl 003 ⁇ 03 VQe (XBPl 7" ) and ⁇ BPl flox ⁇ ox (XBPl +7 +) mice were analyzed for XBP 1 mRNA levels (primers binding in the floxed region) quantified by qPCR.
  • 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.
  • Figure 8 shows that XBPl deletion leads to the presence of apoptotic cells in the epithelium, a.
  • Apoptotic nuclei were identified in XBP1 +/+ (XBPl flo ⁇ /flox VCre) and XBPl 7" (XBPl flox/flox ) sections with anti-active (cleaved) caspase-3 and TUNEL.
  • Figuer 9 shows that XBPl deletion results in a distorted villus: crypt ratio and hyperproliferation of intestinal epithelial cells
  • XBPl* 103 ⁇ 0 " (XBP1 +/+ ) and XBPl flo ⁇ /flox VCre (XBPl 7' ) mice were e administered i.p.
  • 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.
  • Wasting was monitored by daily weight measurements, b.
  • polymorphism refers to the coexistence of more than one form of a gene, or portion thereof, or a segment of DNA.
  • 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.
  • a polymorphism is a single nucleotide polymorphism.
  • SNP single nucleotide polymorphism
  • 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.
  • the polymorphic site is occupied by a base other than the reference base.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • N0D2-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.
  • 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.
  • nucleic acid molecules ⁇ e.g., mRNA or DNA, preferably genomic DNA
  • 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.
  • 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.
  • immune cells are used to extract genetic material for use in the subject assays.
  • 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.
  • EBD inflammatory bowel disease
  • 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.
  • a biological sample e.g., cells, fluid, or tissue
  • 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.
  • allelic differences can be: (i) a difference in the identity of at least one nucleotide or ( ⁇ ) 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.
  • 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.
  • kits for detecting the polymorphic elements in a biological sample 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
  • 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.
  • assay techniques known in the art which can be used for detecting alterations in a polymorphic sequence.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • 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.
  • nucleic acid e.g., genomic, DNA
  • 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 5 1 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.
  • 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.
  • MALDI-TOF chip-based laser deionization time-of- flight
  • 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.
  • 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.
  • the incorporation of fluorescently labeled nucleotides in PCR reactions is followed by automated sequencing.
  • 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).
  • 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.
  • 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).
  • a specific polymorphism of XBPl in DNA from a subject can be shown by restriction enzyme analysis.
  • 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.
  • protection from cleavage agents can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA hetero duplexes (Myers, et al. (1985) Science 230:1242).
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • RNA/DNA hetero duplexes Myers, et al. (1985) Science 230:1242).
  • 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.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Sl nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.
  • control and sample nucleic acids 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.
  • control or sample nucleic acid is labeled for detection.
  • 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).
  • 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).
  • alterations in electrophoretic mobility is used to identify the type of XBP-I polymorphism.
  • SSCP single strand conformation polymorphism
  • 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.
  • 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).
  • 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).
  • DGGE denaturing gradient gel electrophoresis
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • OLA oligonucleotide ligation assay
  • 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.
  • 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.
  • 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.
  • OLA 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • Goelet, P. et al An alternative method, known as Genetic Bit Analysis or GBATM 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.
  • 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.
  • 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.
  • a readily available commercial service can be used to analyze samples for the polymorphic elements of the invention.
  • primers can readily be designed to amplify the polymorphic sequences and/or detect XBP-I polymorphisms by one of ordinary skill in the art.
  • 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.
  • sequences flanking the XBP-I SNPs presented in Table 4 are as follows:
  • GAATACTTAC CAATCACCTA CTCCACCAGA TAGCACCAAA TAGCCAAAAC AAAAAGCCCA GCCTTTCAGT CAGGATGATT TTGTTTACCA GTGATGGAAC CCTACTTtgt gtgtgtgtat gtgtgtgtgt gtgagagaga gaaagagaga gagagagaCC CATTTTATGT GGACACTTAA
  • rs5762812 GGTTATATTA TCTTCTTAAT CATGCTGTTT TGTTTTATGT ATGTCTCTTT ATAAACAACA TATAGCTGGC TTTGGGGTTT CTCAGAGCTC TCTTTTAAGA AGAGaatata aatcatttgt atttatttttt gattatttat ttatttGGAC TTATTTCTGC CATGTTATTT CACATTTTTT GTTTACTATA TTTCTcaagg caggaggatt gcttgaggcc aggagttcaa gaccaggctg ggaaacatat tgtgaccttg tctctacaaa aaatttaaaaaattaacca ggcgtggtga cttgcacctg tagtcccagc tgctcggaag acggaggtgg taagatccct tgaaccca
  • 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
  • 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
  • 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 TT TTGCCAAGGA M [A/C]
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Colony maintenance involved mating XBPl flox/flox VCre x ⁇ BPl flox/flox as well as XBPl fl0X /wtVCre x XBPl flox/flox .
  • XBPl flox/wt VCre x XBPl flox/wt we bred XBPl flox/wt VCre 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 no ⁇ /flox VCre), and "XHPl + *" (Le. XBPl flox * ox or XBPl flo ⁇ /wt ) mice obtained through the husbandry regimen reported above.
  • 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 TNF ⁇ 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 lO ⁇ M. 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 H 2 O 2 . Sections were then blocked for 30min with normal goat serum, and incubated overnight at 4 0 C 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> ⁇ 10 8 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.
  • mice 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.
  • 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 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.
  • 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 50 ⁇ l 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 500 ⁇ l 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).
  • 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).
  • 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 ⁇ g of total RNA using oligo-dT primers and Qiagen's omniscript kit according to manufacturer's recommendations, l ⁇ l of cDNA was used per qPCR reaction, using SYBR green reaction mix (Biorad).
  • 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 SFG ⁇ U3hygro was used instead of SFG ⁇ U3neo.
  • 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 750 ⁇ g/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.
  • 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)).
  • the CD Id- restricted NKT cell hybridoma DN32.D3 (Bendelac, A. et al. Science 268, 863-865 (1995)), which is activated upon recognition of ⁇ GC 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).
  • MODE-K cells were seeded at 1x10 6 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 TNF ⁇ for the indicated periods of time. Cells were then washed in ice-cold PBS and lysed in 500 ⁇ l 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.
  • RIPA buffer 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS
  • Protein content of lysates was determined by BCA assay, and equal amounts of lysates containing Laemmli buffer were boiled at 95 0 C 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 4 0 C 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
  • 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.
  • htSNPs Haplotype tagging SNPs
  • 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).
  • MALDI-TOF chip-based matrix-assisted laser desorption ionization time-of-flight
  • Genotyping call rates for the IBD samples ranged from 94.2 to 100% with an average of 98.3%. Genetic Analysis
  • ER stress activates three distinct unfolded protein response (UPR) signalling pathways through ER transmembrane inositol-requiring enzyme- l ⁇ and ⁇ (IREl ⁇ and ⁇ ), protein kinase- like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Wu, J. & Kaufman,R. J. Cell Death. Differ. 13, 374-384 (2006)).
  • URR unfolded protein response
  • IREl ⁇ and ⁇ ER transmembrane inositol-requiring enzyme- l ⁇ and ⁇
  • PERK protein kinase- like ER kinase
  • ATF6 activating transcription factor 6
  • 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 1 1.
  • 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.
  • XBPIs active, spliced XBPl
  • 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.
  • ⁇ Bpi ⁇ ox/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/flox VCre offspring were born at a Mendelian ratio and developed normally.
  • XBPl exon 2 was efficiently deleted (99% in XBPl flox/flox VCre ( 11 XBPl 7 -”) small intestinal epithelia, 87% in colonic epithelia) (Fig. 6).
  • XBPl mRNA levels in other organs e.g.
  • 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).
  • 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).
  • 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 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.
  • 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).
  • 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 ⁇ -catenin, Tcf4, Mathl, Hesl; Fig.
  • 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).
  • 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)).
  • XBPl fl0xneo ⁇ 0xnfiO VillinCre-ER T2 mice were generated (Fig. 6).
  • 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)).
  • Villin promoter el Marjou, F. et al. Genesis. 39, 186-193 (2004).
  • 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.
  • 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.
  • 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).
  • XBPl muscarinic receptor agonist carbamyl choline
  • 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.
  • DSS is an agent that destroys mucosal epithelial cells and disrupts their barrier function allowing for increased bacterial invasion.
  • XBP I 7' mice administered 4.5% DSS in the drinking water exhibited more severe wasting and more rectal bleeding compared to XBP1 +/+ littermates.
  • 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).
  • TNF ⁇ 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. TNF ⁇ mRNA expression was upregulated in XBP1 " ⁇ 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.
  • 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 " ⁇ 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)).
  • iXBP cells showed increased release of CXCLl into the supernatant after stimulation with TNF ⁇ (Fig. 3c) and flagellin (Fig. 3d).
  • the specific JNK inhibitor SP600125 is known to improve DSS colitis concomitant with reduced colonic TNF ⁇ levels and epithelial cell apoptosis, typically increased in DSS colitis . Addition of SP600125 resulted in a dose- dependent decrease of TNF ⁇ - 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 WaI 5 Y. et al. Gastroenterology 124, 1420-1431 (2003)) (Fig. 3g).
  • 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 TNF ⁇ secretion and epithelial responsiveness to cytokines, such as TNF ⁇ , and bacterial antigens, such as flagellin.
  • XBPl is thus a unique component of the UPR in IBD pathogenesis since deletion of IREl ⁇ 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.
  • 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.
  • XBPl a single gene product
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention repose, au moins en partie, sur la découverte selon laquelle le gène XBP joue un rôle dans l'inflammation intestinale qui s'apparente à une maladie intestinale inflammatoire et qu'un polymorphisme nucléotidique SNP dans la région non traduite en 5' du gène XBPI humain est associé de manière significative à la maladie intestinale inflammatoire, ce qui permet de pronostiquer le rôle direct de l'XBPI dans la pathogénèse de la maladie. De plus, la présente invention concerne des procédés qui permettent de détecter au moins un SNP dans le gène XBP-I humain, ainsi que des procédés qui permettent de déterminer la prédisposition d'un sujet humain à développer une maladie intestinale inflammatoire.
PCT/US2007/020658 2006-09-25 2007-09-25 Polymorphismes du gène xbp-1 humain associés à une maladie intestinale inflammatoire WO2008039445A2 (fr)

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WO2010014905A2 (fr) * 2008-07-31 2010-02-04 President And Fellows Of Harvard College Polymorphismes associés à une maladie intestinale inflammatoire
US9957506B2 (en) 2013-09-25 2018-05-01 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US9956236B2 (en) 2011-02-07 2018-05-01 Cornell University Methods for increasing immune responses using agents that directly bind to and activate IRE-1
US10655130B2 (en) 2012-03-09 2020-05-19 Cornell University Modulation of breast cancer growth by modulation of XBP1 activity

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US6632641B1 (en) * 1999-10-08 2003-10-14 Metrigen, Inc. Method and apparatus for performing large numbers of reactions using array assembly with releasable primers

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US6632641B1 (en) * 1999-10-08 2003-10-14 Metrigen, Inc. Method and apparatus for performing large numbers of reactions using array assembly with releasable primers

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KASER A. ET AL.: 'Transcription factor Xbp1 regulates paneth cell function and the inflammatory tone of the intestinal epithelium' GASTROENTEROLOGY - ABSTRACT FROM THE 108TH ANNUAL MEETING OF THE AMERICAN-GASTROENTEROLOGICAL-ASSOCIATION vol. 132, no. 4, SUPPL. 2, April 2007, pages A7 - A8 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010014905A2 (fr) * 2008-07-31 2010-02-04 President And Fellows Of Harvard College Polymorphismes associés à une maladie intestinale inflammatoire
WO2010014905A3 (fr) * 2008-07-31 2010-07-08 President And Fellows Of Harvard College Polymorphismes associés à une maladie intestinale inflammatoire
US9956236B2 (en) 2011-02-07 2018-05-01 Cornell University Methods for increasing immune responses using agents that directly bind to and activate IRE-1
US10655130B2 (en) 2012-03-09 2020-05-19 Cornell University Modulation of breast cancer growth by modulation of XBP1 activity
US9957506B2 (en) 2013-09-25 2018-05-01 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US10421965B2 (en) 2013-09-25 2019-09-24 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US10450566B2 (en) 2013-09-25 2019-10-22 Cornell University Compounds for inducing anti-tumor immunity and methods thereof

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