WO2008048902A2 - Procédés d'utilisation de polymorphismes nucléotidiques simples dans le gène il23r pour prévoir ou diagnostiquer une maladie intestinale inflammatoire - Google Patents

Procédés d'utilisation de polymorphismes nucléotidiques simples dans le gène il23r pour prévoir ou diagnostiquer une maladie intestinale inflammatoire Download PDF

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WO2008048902A2
WO2008048902A2 PCT/US2007/081269 US2007081269W WO2008048902A2 WO 2008048902 A2 WO2008048902 A2 WO 2008048902A2 US 2007081269 W US2007081269 W US 2007081269W WO 2008048902 A2 WO2008048902 A2 WO 2008048902A2
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haplotypes
jewish
block
il23r
risk
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WO2008048902A3 (fr
WO2008048902A8 (fr
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Jerome I. Rotter
Kent D. Taylor
Stephan Targan
Richard H. Duerr
Mark S. Silverberg
Steven R. Brant
John D. Rioux
Judy H. Cho
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Institut de Cardiologie de Montreal
Mt Sinai Hospital
BROAD INSTITUTE OF MIT
Yale University
Cedars Sinai Medical Center
Johns Hopkins University
University of Pittsburgh
Montreal Heart Inst
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Institut de Cardiologie de Montreal
Mt Sinai Hospital
BROAD INSTITUTE OF MIT
Yale University
Cedars Sinai Medical Center
Johns Hopkins University
University of Pittsburgh
Montreal Heart Inst
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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • 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

  • the invention relates generally to the fields of inflammation and autoimmunity and autoimmune disease and, more specifically, to genetic methods for diagnosing inflammatory bowel disease, Crohn's disease, and other autoimmune diseases.
  • CD Crohn's disease
  • UC ulcerative colitis
  • IBD idiopathic inflammatory bowel disease
  • CD and UC are thought to be related disorders that share some genetic susceptibility loci but differ at others.
  • the interleukin 23/interleukin 17 pathway in general, has been the focus of published research in inflammatory bowel disease and other chronic, immunologically- mediated disorders. Antibodies directed against the subunit of IL-23 that is shared with IL-12 (p40) have shown promise as a treatment for inflammatory bowel disease and other chronic, immune-mediated disorders.
  • IL-12 IL-12
  • the IL23/IL17 pathway has been generally linked with inflammation, there has been little if any evidence of a specific mechanism.
  • much of the attention regarding the involvement of IL-23 has been focused upon the interaction between the p40 subunit and the IL12Rbeta1 receptor subunit, due to a perceived higher affinity as compared to the interaction between the p35 subunit and the IL23R receptor subunit.
  • Various embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual, comprising determining the presence or absence of one or more risk haplotypes at the IL23R locus, where the presence of one or more risk haplotypes is diagnostic of susceptibility to Crohn's Disease.
  • the risk haplotypes can be selected from the group consisting of Block 2 H3 haplotype and Block 3 H8 haplotype, or any combination thereof, with the presence of two risk haplotypes presenting a greater susceptibility than the presence of one or none, and the presence of one risk haplotype can present a greater susceptibility than the presence of none, but less than the presence of two.
  • Risk haplotypes can also include one or more variant alleles selected from rs1569922, rs1004819, rs2201841, rs10889677, rs11209032 and rs1495965.
  • the presence of one or more risk haplotypes can also be diagnostic of susceptibility to Crohn's Disease in a non-Jewish individual, and can be indicative of a higher median anti-12 antibody level.
  • Protective haplotypes can also include the presence of an rs1569922 variant allele, one or more variant alleles selected from the group consisting of rs11209026, rs7517847, rs10489629, rs11465804 and rs1343151 , and an Arg381Gln variant. Finally, the presence of one or more protective haplotypes can be diagnostic of protection against Crohn's Disease in a non-Jewish individual.
  • Figure 1 depicts association signals in the IL23R gene region on chromosome 1 p31 in accordance with various embodiments of the present invention.
  • A Genomic locations of genes on chromosome 1p31 between 67,260,000-67,580,000 base pairs (Build 35).
  • B Pair-wise r 2 plot for HapMap SNPs.
  • the IL23R gene is contained within two linkage disequilibrium blocks, and the association signals are strongest in the centromeric block which contains exons 5-11 and extends into the intergenic region between IL23R and IL12RB2.
  • Figure 2 depicts three haplotype blocks in accordance with various embodiments of the present invention. Block 2 and Block 3 were observed to have associations with CD.
  • Figure 3 depicts IL23R Block 2 Haplotypes associated with CD in accordance with various embodiments of the present invention.
  • Figure 4 depicts IL23R Block 2 haplotypes association with CD risk in accordance with various embodiments of the present invention.
  • Figure 5 depicts IL23R Block 2 haplotypes associated with protection against CD in Non-Jewish individuals in accordance with an embodiment of the present invention.
  • Figure 6 depicts IL23R Block 3 haplotypes associated with CD in accordance with various embodiments of the present invention.
  • Figure 7 depicts IL23R Block 3 haplotypes associations with CD risk in accordance with various embodiments of the present invention.
  • Figure 8 depicts IL23R Block 3 haplotype associated with protection against CD in non-Jewish individuals in accordance with an embodiment of the present invention.
  • Figure 9 depicts IL23R haplotype combinations that are associated with CD in accordance with various embodiments of the present invention.
  • Figure 10 depicts IL23R haplotype combinations that are associated with CD in accordance with various embodiments of the present invention.
  • Figure 11 depicts IL23R and anti-12 antibodies association with Block 2 haplotypes in non-Jewish individuals in accordance with an embodiment of the present invention. Higher anti-12 levels are associated with Block 2 risk haplotypes in non- Jewish individuals.
  • Figure 12 depicts IL23R and anti-12 antibodies association with Block 3 haplotypes in Jewish individuals in accordance with an embodiment of the present invention.
  • Figure 13 depicts IL23R and anti-12 antibodies association with Block 3 haplotypes in non-Jewish individuals in accordance with various embodiments of the present invention. There are higher median anti-12 levels with Block 3 risk haplotype.
  • the inventors performed a genome-wide association study testing 308,332 autosomal single nucleotide polymorphisms (SNPs) on the lllumina HumanHap300 Genotyping BeadChip. Based on these studies, the inventors found single nucleotide polymorphisms (SNPs) and haplotypes that are associated with increased or decreased risk for inflammatory bowel disease, including but not limited to CD and UC. These SNPs and haplotypes are suitable for genetic testing to identify at risk individuals and those associated with such markers as the serum expression of anti-12 antibodies. Identification of genes that cause inflammatory bowel disease may support a new generation of therapies and genetic testing to identify at risk individuals, predict disease course and suggest the right therapy for individual patients.
  • SNPs single nucleotide polymorphisms
  • haplotypes are suitable for genetic testing to identify at risk individuals and those associated with such markers as the serum expression of anti-12 antibodies. Identification of genes that cause inflammatory bowel disease may support a new generation of therapies and genetic testing to identify at risk
  • IL23R as an inflammatory bowel disease gene. Additionally, the inventors have found both protective and risk allelic variants (hereinafter also referred to as a "SNP") and haplotypes for CD. The inventors have also found that in non-Jewish individuals, risk haplotypes were associated with a higher median anti-12 antibody levels. The detection of SNPs and/or haplotypes in IL23R may be used to identify at risk individuals, predict disease course and suggest the right therapy for individual patients. "Haplotype” as used herein refers to a set of single nucleotide polymorphisms (SNPs) on a gene or chromatid that are statistically associated.
  • SNP single nucleotide polymorphisms
  • Protective and “protection” as used herein refer to a decrease in susceptibility to IBD, including but not limited to CD and UC.
  • “Jewish” as used herein refers to an individual of Jewish descent. "Higher median anti-12 antibody levels” as used herein refers to levels determined relative to a standard consisting of pooled sera obtained from normal individuals. Using results expressed as ELISA units, sera with circulating anti-12 antibody levels exceeding the reference range value are termed higher median anti- antibody levels.
  • This gene encodes a subunit of the receptor for the pro-inflammatory cytokine, interleukin-23 (IL23), and was therefore an interesting functional candidate.
  • FBAT family-based association testing
  • the inventors also observed distortion of allele transmission to non-Jewish, UC-affected offspring, providing evidence for association of IL23R with non-Jewish UC. There was no evidence for association of Arg381Gln or other IL23R region markers in the Jewish UC families.
  • all ten IL23R markers in Table 2 showed significant association with IBD, even after Bonferroni correction for analyzing 308,332 SNPs in the original genome-wide association study (p-values ranged from 7 x 10 ⁇ 19 to 3.55 x 10 ⁇ 9 ; corrected p-values ranged from 2.16 x 10 ⁇ 13 to 1.09 x 10 "3 ).
  • the IL23R gene is contained within two large linkage disequilibrium blocks, and markers in the centromeric block containing exons 5-11 and part of the intergenic region between IL23R and IL12RB2 have the strongest association signals (Fig. 1). There is no significant association within IL12RB2 (Fig. 1).
  • the IL23R protein contains an extracellular domain (comprised of a signal sequence, an N-terminal immunoglobulin-like domain and two cytokine receptor domains), a single transmembrane domain, and a 252 amino acid cytoplasmic domain (C. Parham et al., J Immunol 168, 5699 (2002)).
  • Arg381 in the cytoplasmic domain, is the fifth amino acid internal to the transmembrane domain and is highly conserved between species.
  • the embodiments of the present invention provide for methods of diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease including but not limited to CD and/or UC.
  • the present invention provides for methods of diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease in non-Jewish individuals by determining the presence or absence of a risk variant.
  • the present invention provides methods of diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease in Jewish individuals by determining the presence or absence of a risk and/or protective variant.
  • the presence or absence of a risk and/or protective variant in IL-23R suggests an appropriate therapy for patients.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to CD in an individual by determining the presence or absence in the individual of a variant in the IL23R gene.
  • the present invention provides methods of diagnosing and/or determining susceptibility to CD in a non-Jewish individual by determining the presence or absence in the individual of a variant in the IL23R gene.
  • the present invention provides methods of diagnosing and/or determining protection against CD in a non-Jewish individual by determining the presence or absence in the individual of a variant in the IL23R gene.
  • the present invention provides methods of diagnosing and/or determining susceptibility to CD in a Jewish individual by determining the presence or absence in the individual of a variant in the IL23R gene.
  • the methods may include the steps of obtaining a biological sample containing nucleic acid including the IL23R gene from the individual and determining the presence or absence of a SNP an/or a haplotype in the biological sample.
  • the methods may further include correlating the presence or absence of the SNP and/or the haplotype to a genetic risk, a susceptibility for or protection against inflammatory bowel disease including but not limited to CD and/or UC, as described herein.
  • the methods may also further include recording whether a genetic risk, susceptibility for or protection against inflammatory bowel disease including but not limited to CD and/or UC exists in the individual.
  • biological sample means any biological matter from which nucleic acid molecules can be prepared.
  • material encompasses whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acid.
  • a method of the invention is practiced with whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA, for example, for enzymatic amplification or automated sequencing.
  • a method of the invention is practiced with tissue obtained from an individual such as tissue obtained during surgery or biopsy procedures.
  • One or more SNPs at the following bcations may be used to diagnose and/or predict a risk of susceptibility to or protection against CD in a Jewish or non-Jewish individual: rs1007819, rs7517847, rs10489629, rs2201841, rs11465804, rs11209026, rs1343151, rs10889677, rs11209032, rs1495965.
  • an rs11209026 variant (c1142G>A, p.Arg381 GIn) is diagnostic or predictive of protection against CD in an individual.
  • the presence of an rs1004819 variant is diagnostic or predictive of susceptibility to CD.
  • the presence of an rs7517847 variant is diagnostic or predictive of a protection against CD.
  • the presence of an rs10489629 variant is diagnostic or predictive of a protection against CD.
  • the presence of an rs2201841 variant is diagnostic or predictive of susceptibility to CD.
  • the presence of an rs11465804 variant is diagnostic or predictive of a protection against CD.
  • the presence of an rs1343151 variant is diagnostic or predictive of a protection against CD.
  • the presence of an rs10889677 variant is diagnostic or predictive of susceptibility to CD.
  • the presence of an rs11209032 variant is diagnostic or predictive of susceptibility to CD.
  • the presence of an rs1495965 variant is diagnostic or predictive of susceptibility to CD.
  • the inventors also observed three haplotype blocks in the IL23R gene. Blocks 2 and 3 were found to have associations with CD. Both protective and risk haplotypes for CD were identified in IL23R. In non-Jewish individuals, risk haplotypes were associated with higher median anti-12 levels. The results of the inventors' study implicate IL23R variation in the pathogenesis of CD.
  • Block 1 includes SNPs identified as rs11209008, rs12041056, rs1884444, and rs6671221.
  • Block 2 includes SNPs identified as rs1004819, rs790631 , and rs2863212.
  • Block 3 includes SNPs identified as rs7530511 , rs7528924, rs2201841 , rs11804284, rs1 1465804, rs10489628, and rs1343151. (See figure 2.)
  • IL23R Block 2 haplotypes are associated with CD. The association of Block 2 H2 was only found to be significant in non-Jewish individuals.
  • Block 2 H3 was found in both Jewish and non-Jewish individuals, but only found to be significant in non-Jewish individuals.
  • Figure 4 depicts IL23R Block 2 haplotypes and the associated CD risk.
  • Block 2 H3 is associated with a risk for CD and Block 2 H2 is associated with a protection against CD.
  • IL23R Block 2 protection is associated with CD in non-Jewish individuals.
  • the association of Block 2 H2 was only found to be significant in non-Jewish individuals.
  • the association of Block 2 H3 is in both Jewish and non-Jewish individuals, but only found to be significant in non-Jewish individuals.
  • IL23R Block 3 haplotypes are associated with CD.
  • Block 3 H1 is significant only in non-Jewish individuals.
  • Block 3 H8 is present in both Jewish and non-Jewish individuals, but only found to be significant in non-Jewish individuals.
  • Figure 7 depicts IL23R Block 3 haplotypes and the associated CD risk.
  • Block 3 H8 is associated with a risk for CD and Block 3 H1 is associated with a protection against CD.
  • Block 3 protection is associated with CD in non-Jewish individuals.
  • Certain IL23R Haplotype combinations are associated with CD, as depicted in figures 9 and 10.
  • Block 2 H3 and Block 3 H8 are associated with a risk for CD.
  • Block 2 H2 and Block 3 H1 are associated with a protection against CD.
  • Rs1569922, Block 2 H3, Block 3 H8 are associated with a risk for CD.
  • Rs1569922, Block 2 H2, Block 3 H1 are associated with protection against CD.
  • additbnal embodiments of the present invention provide for methods for diagnosing or predicting susceptibility to CD in an individual by determining the presence or absence of a haplotype in the individual.
  • the presence of Block 3 H1 haplotype may indicate a protection against CD. In one embodiment, the presence of Block 3 H1 haplotype may indicate a protection against CD in non-Jewish individuals.
  • the presence of Block 2 H2 haplotype may indicate a protection against CD.
  • the presence of Block 2 haplotype H2 may indicate protection against CD in non-Jewish individuals.
  • the presence of Block 2 H3 haplotype may indicate a susceptibility to CD.
  • the presence of Block 3 H8 haplotype may indicate a susceptibility to CD.
  • haplotype combinations may be associated with CD in a cumulative manner.
  • the presence of one or two haplotypes selected from the group consisting of Block 2 H3 and Block 3 H8 may confer an increased risk in susceptibility to CD.
  • the presence of two haplotypes selected from the group consisting Block 2 H3 and Block 3 H8 may confer a higher risk of susceptibility to CD as compared to the presence of one or none of these haplotypes. (See e.g., figure 9.)
  • the presence of one or two haplotypes selected from the group consisting of Block 2 H3 and Block 3 H8 may confer an increased risk of susceptibility to CD.
  • the presence of two haplotypes selected from the group consisting Block 2 H3 and Block 3 H8 may confer a higher risk of susceptibility to CD as compared to the presence of one or none of these haplotypes. (See e.g., figure 10.)
  • the presence of one of the following SNP rs1569922, haplotype Block 2 H3 or haplotype Block 3 H8 may indicate a risk of susceptibility to CD.
  • the presence of two of the following, in any combination, SNP rs1569922, haplotype Block 2 H3 and haplotype Block 3 H8 may indicate a higher risk of susceptibility to CD as compared to the presence of only one or none.
  • the presence of SNP rs1569922 and two haplotypes selected from the group consisting of Block 2 H3 and Block 3 H8, may indicate an even higher risk of susceptibility to CD as compared to the presence of only two, one or none.
  • the identities of the haplotypes and their nucleotide substitutions may be found in table 6.
  • the location of the SNPs may be found in table 5.
  • Additional embodiments of the present invention may also be used to diagnose or predict susceptibility to a particular subtype of Crohn's disease in a patient.
  • a subtype can be, for example, a clinical subtype of Crohn's disease with features of ulcerative colitis (U.S. Pat. No. 5,932,429) or clinical subtype of Crohn's disease having fibrostenosis.
  • a clinical subtype of Crohn's disease with features of ulcerative colitis U.S. Pat. No. 5,932,429
  • clinical subtype of Crohn's disease having fibrostenosis are examples of Crohn's disease having fibrostenosis.
  • these and other clinical subtypes of Crohn's disease also can be diagnosed by detecting the presence or absence of a SNP and/or a haplotype as described herein.
  • Figure 11 depicts IL23R and anti-12 antibodies association with Block 2 haplotypes in non-Jewish individuals.
  • Figure 12 depicts IL23R and anti-12 antibodies association with Block 3 haplotypes in Jewish individuals. This association was only observed in Jewish individuals.
  • Figure 13 depicts IL23R and anti-12 antibodies association with Block 3 haplotypes in non-Jewish individuals. Higher median I2 levels with Block 3 risk haplotype were observed.
  • the presence of a haplotype in Block 2 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies. In a particular embodiment, the presence of a haplotype in Block 2 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies in non- Jewish individuals. In one embodiment, the haplotype may be Block 2 H3. In another embodiment, the presence of SNP rs1004819 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies. In a particular embodiment, the presence of SNP rs1004819 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies in non-Jewish individuals. See figure 11.
  • the presence of a haplotype in Block 3 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies. In a particular embodiment, the presence of a haplotype in Block 3 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies in Jewish individuals. In one embodiment, the haplotype may be Block 3 H4. In another embodiment, the presence of SNP rs11465804 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies. In a particular embodiment, the presence of SNP rs11465804 may indicate the presence of anti-12 antibodies in Jewish individuals. See figure 12. In a particular embodiment, the presence of a haplotype in Block 3 may indicate the presence of anti-12 antibodies and/or higher serum levels of anti-12 antibodies in non-Jewish individuals. In one embodiment, the haplotype may be Block 3 H8. See figure 13.
  • the presence of anti-12 antibodies may indicate that the individual has a fibrostenotic subtype of CD; for example, as described in U.S. Patent Publication No. 2004/0203076, herein incorporated by reference as though fully set forth in its entirety.
  • the presence of anti-12 antibodies may also indicate that the individual does not have ulcerative colitis-like CD; for example, also as described in U.S. Patent Publication No. 2004/0203076.
  • the presence of Block 2 and/or Block 3 risk haplotype(s) may indicate that the individual has a fibrostenotic subtype of CD and/or does not have ulcerative colitis-like CD.
  • the presence of Block 2 H3, Block 3 H4, Block 3 H8 and/or Block 3 H10 haplotype(s) may indicate that the individual has a fibrostenotic subtype of CD and/or does not have ulcerative colitis-like CD.
  • the strength of the association between a haplotype or allele and CD may be characterized by a particular odds ratio. See Tables 1 , 3A, and 3B. Methods for determining an odds ratio are well known in the art (see, for example, Schlesselman et al., Case Control Studies: Design, Conduct and Analysis Oxford University Press, New York (1982)).
  • a variety of methods can be used to determine the presence or absence of an IL23R variant allele or haplotype.
  • enzymatic amplification of nucleic acid from an individual may be used to obtain nucleic acid for subsequent analysis.
  • the presence or absence of an IL23R variant allele or haplotype may also be determined directly from the individual's nucleic acid without enzymatic amplification.
  • nucleic acid means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA.
  • nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.
  • the presence or absence of an IL23R variant allele or haplotype may involve amplification of an individual's nucleic acid by the polymerase chain reaction.
  • Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).
  • a TaqmanB allelic discrimination assay available from Applied Biosystems may be useful for determining the presence or absence of an IL23R variant allele.
  • a TaqmanB allelic discrimination assay a specific, fluorescent, dye-labeled probe for each allele is constructed.
  • the probes contain different fluorescent reporter dyes such as FAM and VICTM to differentiate the amplification of each allele.
  • each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonant energy transfer (FRET).
  • FRET fluorescence resonant energy transfer
  • each probe anneals specifically to complementary sequences in the nucleic acid from the individual.
  • the 5' nuclease activity of Taq polymerase is used to cleave only probe that hybridize to the allele.
  • Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye.
  • the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample.
  • Mismatches between a probe and allele reduce the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescent signal.
  • Improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor grove binder (MGB) group to a DNA probe as described, for example, in Kutyavin et al., "3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature, "Nucleic Acids Research 28:655-661 (2000)).
  • MGB DNA minor grove binder
  • Minor grove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptide (DPI,). Sequence analysis also may also be useful for determining the presence or absence of an IL23R variant allele or haplotype.
  • DPI dihydrocyclopyrroloindole tripeptide
  • Restriction fragment length polymorphism (RFLP) analyse may also be useful for determining the presence or absence of a particular allele (Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York; lnnis et al.,(Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)).
  • restriction fragment length polymorphism analysis is any method for distinguishing genetic polymorphisms using a restriction enzyme, which is an endonuclease that catalyzes the degradation of nucleic acid and recognizes a specific base sequence, generally a palindrome or inverted repeat.
  • a restriction enzyme which is an endonuclease that catalyzes the degradation of nucleic acid and recognizes a specific base sequence, generally a palindrome or inverted repeat.
  • RFLP analysis depends upon an enzyme that can differentiate two alleles at a polymorphic site
  • Allele-specific oligonucleotide hybridization may also be used to detect a disease-predisposing allele. Allele-specific oligonucleotide hybridization is based on the use of a labeled oligonucleotide probe having a sequence perfectly complementary, for example, to the sequence encompassing a disease-predisposing allele. Under appropriate conditions, the allele-specific probe hybridizes to a nucleic acid containing the disease-predisposing allele but does not hybridize to the one or more other alleles, which have one or more nucleotide mismatches as compared to the probe. If desired, a second allele-specific oligonucleotide probe that matches an alternate allele also can be used.
  • the technique of allele-specific oligonucleotide amplification can be used to selectively amplify, for example, a disease-predisposing allele by using an allele- specific oligonucleotide primer that is perfectly complementary to the nucleotide sequence of the disease-predisposing allele but which has one or more mismatches as compared to other alleles (Mullis et al., supra, (1994)).
  • the one or more nucleotide mismatches that distinguish between the disease-predisposing allele and one or more other alleles are preferably located in the center of an allele-specific oligonucleotide primer to be used in allele-specific oligonucleotide hybridization.
  • an allele-specific oligonucleotide primer to be used in PCR amplification preferably contains the one or more nucleotide mismatches that distinguish between the disease-associated and other alleles at the 3' end of the primer.
  • a heteroduplex mobility assay is another well known assay that may be used to detect a SNP or a haplotype. HMA is useful for detecting the presence of a polymorphic sequence since a DNA duplex carrying a mismatch has reduced mobility in a polyacrylamide gel compared to the mobility of a perfectly base-paired duplex (Delwart et al., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306 (1992)).
  • SSCP single strand conformational, polymorphism
  • This technique can be used to detect mutations based on differences in the secondary structure of single-strand DNA that produce an altered electrophoretic mobility upon non-denaturing gel electrophoresis. Polymorphic fragments are detected by comparison of the electrophoretic pattern of the test fragment to corresponding standard fragments containing known alleles.
  • Denaturing gradient gel electrophoresis also may be used to detect a SNP and/or a haplotype.
  • DGGE Denaturing gradient gel electrophoresis
  • double-stranded DNA is electrophoresed in a gel containing an increasing concentration of denaturant; double-stranded fragments made up of mismatched alleles have segments that melt more rapidly, causing such fragments to migrate differently as compared to perfectly complementary sequences (Sheffield et al., "Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis" in lnnis et al., supra, 1990).
  • Other molecular methods useful for determining the presence or absence of a SNP and/or a haplotype are known in the art and useful in the methods of the invention.
  • Example 1 One of the study populations consisted of 567 non-Jewish, European ancestry patients with ileal CD and 571 non-Jewish controls. The inventors initially focused on ileal CD, the most common location of CD, to minimize pathogenic heterogeneity. After exclusion of study subjects with genotype completion rates less than 94%, the inventors included 547 cases and 548 controls in subsequent analyses. Single-marker allelic tests were performed using ⁇ 2 statistics for all autosomal markers. Three SNPs had nearly two orders of magnitude greater significance compared to the next most significant markers, and they are the only markers that remain significant at the 0.05 level after Bonferroni correction.
  • This gene encodes a subunit of the receptor for the pro- inflammatory cytokine, interleukin-23 (IL23), and was therefore an interesting functional candidate.
  • Table 3A Summary of markers for the non-Jewish case-control cohort corresponding to the genomic window shown in Figure 1. Shown are information about the markers (rs number and base pair position on chromosome 1 , Build 35), genotype counts for Crohn's disease (CD) cases and controls, p-values for the Hardy-Weinberg test (HW-P) and Fisher's exact test for allelic counts (AIIeIe-P). Minor allele frequencies (MAF) for cases and controls, odds ratios (ORs) and 95% confidence intervals (Cl) for the first allele are shown.
  • Example 2 The inventors next tested for association of IL23R markers in an independent, Jewish case-control cohort containing 401 patients with ileal CD and 433 controls. Significant associations were observed for several of the same markers that were associated in the non-Jewish cohort (Tables 1 and 3B). In a combined analysis of the data from the two ileal CD case-control cohorts, nine markers had highly significant association p-values ranging from 3.36 x 10 "13 to 1.60 x 10 "9 (Tables 1 and 3B).
  • Table 3B Summary of markers, data and results for the Jewish case-control cohort corresponding to the genomic window shown in Figure 1.
  • the final column lists the combined p-value (non-Jewish and Jewish cohorts) obtained using the Cochran- Mantel-Haenszel test graphed in Figure 1.
  • the inventors also performed family-based association testing (FBAT) (S. L. Lake, D. Blacker, N. M. Laird, Am J Hum Genet 67, 1515 (2000); S. Horvath, X. Xu, N. M. Laird, Eur J Hum Genet 9, 301 (2001)) of IL23R region markers in independent cohorts of nuclear families in which both parents and their CD- or UC-affected offspring were available for genotyping (Tables 2 and 4).
  • FBAT family-based association testing
  • the inventors also observed distortion of allele transmission to non-Jewish, UC-affected offspring, providing evidence for association of IL23R with non-Jewish UC. There was no evidence for association of Arg381Gln or other IL23R region markers in the Jewish UC families.
  • all ten IL23R markers in Table 2 showed significant association with IBD, even after Bonferroni correction for analyzing 308,332 SNPs in the original genome-wide association study (p-values ranged from 7 x 10 "19 to 3.55 x 10 "9 ; corrected p-values ranged from 2.16 x 10 "13 to 1.09 x 10 3 ).
  • the glutamine allele of Arg381Gln is much less common than the arginine allele, being present at an allelic frequency of 1.9% in the non-Jewish patients with ileal CD and 7.0% in non-Jewish controls.
  • the glutamine allele appears to protect against development of CD in both non-Jewish [odds ratio (OR) 0.26, 95% confidence interval (Cl) (0.15-0.43)] and Jewish [OR 0.45, 95% Cl (0.27 to 0.73)] case-control cohorts.
  • the observation of an uncommon protective allele and a common risk allele reflects an emerging theme in complex genetics; namely, that functional genetic variation exerts a continuum of susceptibility, neutral and protective effects, and alleles conferring protection against one disease may result in increased risk for another (J. H. Nadeau, E. J. Topol, Nat Genet 38, 1095 (2006)).
  • Table 4A Summary of family-based association test results in the non-Jewish CD and non-Jewish UC cohorts. Shown is information about the markers (rs number and base pair position, pos, on chromosome 1), the over-transmitted allele, counts of transmitted (T) and un-transmitted (U) alleles, and Chi square- and p-values computed using the empirical variance estimator implemented in the FBAT software package. Table 4A
  • Table 4B Summary of family-based association test results in the Jewish CD and Jewish UC cohorts. Shown is information about the markers (rs number and base pair position, pos, on chromosome 1), the over-transmitted allele, counts of transmitted (T) and un-transmitted (U) alleles, and Chi square- and p-values computed using the empirical variance estimator implemented in the FBAT software package. Table 4B
  • IL23R markers other than Arg381Gln are also associated with IBD (Tables 1 , 2, 3A, 3B, 4A and 4B).
  • other markers show evidence for association that appears to be independent of Arg381Gln.
  • the inventors performed a Cochran-Mantel-Haenszel test using the case-control data with strata given by the Arg381Gln genotypes.
  • the p-values for these conditional tests demonstrate multiple residual association signals throughout IL23R, indicating that there are multiple risk variants in the region.
  • the IL23R gene is expressed as at least six alternatively spliced mRNAs, which generate diverse isoforms of the receptor protein (X. Y. Zhang et al., lmmunogenetics 57, 934 (2006)).
  • the most common splice variants result in the deletion of exons 7 and/or 10. While not wishing to be bound by any particular theory, the inventors speculate that the multiple genetic association signals detected in the centromeric portion of IL23R (Fig. 1) could be due to differential effects of SNPs on splicing.
  • Example 8 The inventors found no evidence for association with the IL12RB1 gene, which encodes the second subunit of the IL-23 receptor heterodimer (C. Parham et al., J Immunol 168, 5699 (2002)), or the IL23A and IL12B genes, which encode the functional IL-23 heterodimer (B. Oppmann et al., Immunity 13, 715 (2000)), in our non-Jewish, ileal CD, case-control cohort.
  • transgenic expression of IL-23 results in severe systemic inflammation, including in the small and large intestine (M. T. Wiekowski et al., J Immunol 166, 7563 (2001)), highlighting this pathway's particular role in promoting strong activation of effector T cells and perpetuation of organ-specific inflammatory responses. At least part of this effect is likely mediated via inflammatory, IL-17-producing T cells (S. Aggarwal, N. Ghilardi, M. H. Xie, F. J. de Sauvage, A. L. Gurney, J Biol Chem 278, 1910 (2003); C. L. Langrish et al., J Exp Med 201 , 233 (2005); E.
  • IL-23 function may be important for proper responses to mycobacterial (F. A. Verreck et al., Proc Natl Acad Sci U S A 101 , 4560 (2004); A. M. Cooper et al., J Immunol 168, 1322 (2002)) and intestinal infections (P. R. Mangan et al., Nature 441 , 231 (2006)).
  • Example 11 Subject ascertainment and diagnostic classification In all cases, informed consent was obtained using protocols approved by the local institutional review board. Cases and geographically matched controls were ascertained through Baltimore, Chicago, Montreal, Pittsburgh, Los Angeles, and Toronto Genetics Research Centers, with additional age and ethnicity-matched controls provided by the New York Health project. The diagnosis of IBD requires a) one or more symptoms of diarrhea, rectal bleeding, abdominal pain, fever, or complicated perianal disease, b) occurring on two or more occasions separated by at least 8 weeks or ongoing symptoms of at least 6 weeks duration, and c) objective evidence of inflammation from radiologic, endoscopic, and histologic evaluation. Ileal CD involvement was defined as mucosal ulceration, cobblestoning, stricturing or bowel wall thickening from endoscopy reports, barium X-rays, operative reports and/or pathology resection specimen reports.
  • genomic DNA was used to genotype each sample on the lllumina HumanHap300 BeadChip at the Feinstein Institute for Medical Research. Samples were processed according to the lllumina lnfinium 2 assay manual. Briefly, each sample was whole-genome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared HumanHap300 beadchips for a minimum of 16 hours at 48°C. Following hybridization, the beadchips were processed for the single base extension reaction, signal amplification and imaged on an lllumina Bead Array Reader.
  • Normalized bead intensity data obtained for each sample was loaded into the lllumina Beadstudio 2.0 software which converted fluorescent intensities into SNP genotypes.
  • the IL23R region genotyping in replication cohorts was genotyped using primer extension chemistry and mass spectrometric analysis (iPlex assay) using Sequenom Genetics Services.
  • genotype data from the family-based cohorts were used to determine Mendelian inconsistencies and departures from Hardy-Weinberg equilibrium.
  • the inventors eliminated from the analysis all the families with more than two Mendelian inconsistencies and families that were not informative because a parent or the sole affected offspring failed genotyping.
  • Eight nuclear families that we analyzed were from the same extended family as another nuclear family. After zeroing genotypes that produced residual Mendelian inconsistencies, genotype completion rates for each marker in the clean family-based association data ranged from 97.2% to 99.8%.
  • the single marker analyses for the genome-wide data in each ethnic group were done using chi-square tests on allele counts; for all the markers associated at the 0.01 level, Fisher's exact tests on allelic counts were also performed and yielded the p- values reported in the manuscript.
  • the data from the Jewish and non-Jewish case- control cohorts were analyzed jointly using a Cochran-Mantel-Haenszel chi-squared test; the inventors used the test with the option that gives an exact p-value.
  • the analyses conditional on Arg381Gln were performed using the Cochran-Mantel-Haenszel test with strata defined based on Arg381Gln genotypes.

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Abstract

Les inventeurs ont exécuté une large étude d'association sur le génome, et ont identifié le récepteur de l'interleukine-23 (IL-23R) en tant que gène de maladie intestinale inflammatoire. De plus, les inventeurs ont trouvé des variantes et des haplotypes alléliques et de protection et de risque pour la maladie de Crohn. Les inventeurs ont également trouvé que chez des individus non-juifs, des haplotypes de risque sont associés à un niveau d'anticorps anti-I2 plus élevé. L'invention fournit des procédés pour identifier des individus à risque, et pour prévoir le cours de la maladie en déterminant la présence ou l'absence chez l'individu de variantes de protections et de risque dans IL-23R.
PCT/US2007/081269 2006-10-13 2007-10-12 Procédés d'utilisation de polymorphismes nucléotidiques simples dans le gène il23r pour prévoir ou diagnostiquer une maladie intestinale inflammatoire Ceased WO2008048902A2 (fr)

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US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
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US11236393B2 (en) 2008-11-26 2022-02-01 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US12084722B2 (en) 2008-11-26 2024-09-10 Cedars-Sinai Medical Center Methods of determining responsiveness to anti-TNFα therapy in inflammatory bowel disease
US10633449B2 (en) 2013-03-27 2020-04-28 Cedars-Sinai Medical Center Treatment and reversal of fibrosis and inflammation by inhibition of the TL1A-DR3 signaling pathway
US10316083B2 (en) 2013-07-19 2019-06-11 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11312768B2 (en) 2013-07-19 2022-04-26 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US12269873B2 (en) 2013-07-19 2025-04-08 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
US11186872B2 (en) 2016-03-17 2021-11-30 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2
WO2020087130A1 (fr) * 2018-10-31 2020-05-07 The Council Of The Queensland Institute Of Medical Research Pronostic et traitement de maladies inflammatoires chroniques de l'intestin
US12618111B2 (en) 2021-10-28 2026-05-05 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2

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