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WO2008112990A2 - Méthodes de diagnostic et de traitement de la maladie de crohn - Google Patents

Méthodes de diagnostic et de traitement de la maladie de crohn Download PDF

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WO2008112990A2
WO2008112990A2 PCT/US2008/057028 US2008057028W WO2008112990A2 WO 2008112990 A2 WO2008112990 A2 WO 2008112990A2 US 2008057028 W US2008057028 W US 2008057028W WO 2008112990 A2 WO2008112990 A2 WO 2008112990A2
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disease
crohn
locus
individual
atg16l1
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WO2008112990A3 (fr
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Jerome I. Rotter
Kent D. Taylor
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Cedars-Sinai Medical Center
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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

Definitions

  • NIDDK National Institute of Diabetes and Digestive and Kidney Diseases
  • IBDGC Inflammatory Bowel Disease Genetics Consortium
  • Additional U.S. Government support was provided on behalf of NIDDK through project 1 of DK46763 and DK43351.
  • Government support on behalf of the National Institute of Allergy and Infectious Diseases Extramural Activities (NIAID) was provided by grant AI062773.
  • the invention relates generally to the fields of inflammation and autoimmunity and autoimmune disease and, more specifically, to genetic methods for diagnosing Crohn's disease.
  • 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.
  • 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 variants at the ATG16L1 locus in the individual, and diagnosing susceptibility to Crohn's Disease based upon the presence of one or more risk variants at the ATG16L1 locus.
  • one of the one or more risk variants at the ATG16L1 locus further comprises SEQ. ID. NO.: 1.
  • the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.
  • Other embodiments provide methods of diagnosing susceptibility to a subtype of Crohn's Disease in an individual, comprising determining the presence or absence of abnormal autophagy processing relative to a healthy subject, and diagnosing susceptibility to the subtype of Crohn's Disease in the individual based upon the presence of the abnormal autophagy processing relative to a healthy subject.
  • inventions provide methods of treating Crohn's Disease, comprising determining the presence of one or more risk variants at the ATG16L1 locus, and treating the Crohn's Disease.
  • one of the one or more risk variants at the ATG16L1 locus further comprises SEQ. ID. NO.: 1.
  • 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 variants at the PHOX2B locus in the individual, and diagnosing susceptibility to Crohn's Disease based upon the presence of one or more risk variants at the PHOX2B locus.
  • one of the one or more risk variants at the PHOX2B locus further comprises SEQ. ID. NO.: 2.
  • the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.
  • inventions also include methods of diagnosing susceptibility to Crohn's Disease in an individual, comprising determining the presence or absence of one or more risk variants at an intergenic region on 10q21.1 in the individual, and diagnosing susceptibility to Crohn's Disease based upon the presence of one or more risk variants at the intergenic region on 10q21.1.
  • Embodiments include methods where one of the one or more risk variants at the intergenic region on 10q21.1 further comprises SEQ. ID. NO.: 3.
  • Other embodiments also comprise an ileal Crohn's Disease phenotype.
  • Embodiments also include methods of diagnosing susceptibility to Crohn's Disease in an individual, comprising determining the presence or absence of one or more risk variants at the NCF4 locus in the individual, and diagnosing susceptibility to Crohn's Disease based upon the presence of one or more risk variants at the NCF4 locus. Some embodiments also include methods where one of the one or more risk variants at the NCF4 locus further comprises SEQ. ID. NO.: 4. In other embodiments, the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.
  • 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 variants at intron 4 of the FAM92B locus in the individual, and diagnosing susceptibility to Crohn's Disease based upon the presence of one or more risk variants at intron 4 of the FAM92B locus.
  • Other embodiments include methods where one of the one or more risk variants at intron 4 of the FAM92B locus further comprises SEQ. ID. NO.: 5.
  • Other embodiments include methods where the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.
  • Various embodiments include methods of diagnosing susceptibility to a subtype of Crohn's Disease in an individual, comprising determining the presence of one or more risk variants at the ATG16L1 locus, the PHOX2B locus, an intergenic region on 10q21.1 , the NCF4 locus, and intron 4 of the FAM92B locus, and diagnosing susceptibility to the subtype of Crohn's Disease based upon the presence of one or more risk variants.
  • Other embodiments include methods where the subtype further comprises an ileal Crohn's Disease phenotype.
  • Some embodiments include methods of treating Crohn's Disease, comprising determining the presence of one or more risk variants at the ATG16L1 locus, the PHOX2B locus, an intergenic region on 10q21.1 , the NCF4 locus, and intron 4 of the FAM92B locus, and treating the Crohn's Disease.
  • Figure 1 depicts expression of autophagy components in human cell-lines and primary immune cells. Quantitative real-time PCR was used to determine the expression patterns of ATG5, 7 and 16L1 in a variety of human immune and epithelial cell lines (Panels A, B and C 1 respectively). In addition, for ATG16L1 , the inventors utilized an RNA panel from primary human immune cells (Clontech, CA) (panel D). In all cases the expression levels were normalized by comparison to GAPDH controls and arbitrary relative expression units plotted where SW480 or placental RNA is equal to 1 (for cell lines, or primary cells respectively).
  • ATG5, 7 and 16 are broadly expressed in the cell lines tested, although THP- 1 cells appear to have a very high level of ATG 7. In primary cells, it is the T cell compartment that express the most ATG16L1 , with both CD8+ and CD4+ T cells having levels approximately 15-20 times that of the control. CD19+ B cells also show moderate ATG16L1 expression. In all cases, reactions were performed in duplicate and the means plotted, error bars represent 1 standard deviation. Reactions containing no template RNA were performed to control for reaction contamination (water controls).
  • Figure 2 depicts ATG16L1 is required for autophagy of Salmonella Typhimurium.
  • Panel A Specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 is achieved in HEK293 cells within 48 hours of transfection with oligo duplexes. Protein levels of Flag-ATG16L1 were undetectable by Western blotting following treatment with specific siRNA constructs, but expression was maintained with control duplexes.
  • Panel B Endogenous ATG16L1 mRNA is efficiently knocked- down by siRNA 2 in HeLa cells within 48 hours of transfection. HeLa cells were transfected with siRNA duplexes and allowed to grow for 48 hours, RNA was then isolated and real-time quantitative RT-PCR performed.
  • FIG. 3 depicts knockdown of ATG16L1 prevents induction of autophagy by classical simuli.
  • SNP single nucleotide polymorphism
  • Haplotype refers to a set of single nucleotide polymorphisms (SNPs) on a gene or chromatid that are statistically associated.
  • “Risk variant” as used herein refers to an allele whose presence is associated with an increase in susceptibility to an inflammatory bowel disease, including but not limited to Crohn's Disease and ulcerative colitis, relative to a healthy individual.
  • biological sample means any biological material 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.
  • autophagy and “autophagic processing” refer to catabolic processes involving the degradation of a cells' own components through the lysosomal machinery. They are tightly-regulated processes that play a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products, and are specifically involved in protein degradation, antigen processing, regulation of cell signaling and many other pathways essential to the initiation and regulation of the inflammatory response.
  • abnormal autophagic processing refers to autophagic processing that is a part of, or the result of, disease pathology, such as inflammatory pathways associated with Crohn's Disease, and are no longer a constitutive process required for normal cellular homeostasis.
  • ATG16L1 is described herein as SEQ. ID. NO.: 6, and SEQ. ID. NO.: 7.
  • NCF4 An example of NCF4 is described herein as SEQ. ID. NO.: 9, and SEQ. ID. NO.: 10.
  • FAM92B is described herein as SEQ. ID. NO.: 11.
  • the inventors performed a genome-wide association study testing 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. These SNPs and haplotypes are suitable for genetic testing to identify at risk individuals and those with increased risk for complications associated with serum expression of Anti- Saccharomyces cerevisiae antibody, and antibodies to 12, OmpC, and Cbir.
  • protective and risk SNPs and/or haplotypes may be used to identify at risk individuals, predict disease course and suggest the right therapy for individual patients. Additionally, the inventors have found both protective and risk allelic variants for Crohn's Disease and Ulcerative Colitis.
  • 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 Crohn's Disease. Other embodiments provide for methods of prognosing inflammatory bowel disease including but not limited to Crohn's Disease. Other embodiments provide for methods of treating inflammatory bowel disease including but not limited to Crohn's Disease.
  • the methods may include the steps of obtaining a biological sample containing nucleic acid from the individual and determining the presence or absence of a SNP and/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 inflammatory bowel disease including but not limited to Crohn's Disease, as described herein.
  • the methods may also further include recording whether a genetic risk, susceptibility for inflammatory bowel disease including but not limited to Crohn's Disease exists in the individual.
  • the methods may also further include a prognosis of inflammatory bowel disease based upon the presence or absence of the SNP and/or haplotype.
  • the methods may also further include a treatment of inflammatory bowel disease based upon the presence or absence of the SNP and/or haplotype.
  • 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 1 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.
  • inventors performed a genome-wide association study of ileal Crohn's disease (CD) and an independent replication study that established five regions of unambiguously confirmed association of genome-wide significance.
  • CD ileal Crohn's disease
  • the inventors found three novel confirmed associations with variation in the PHOX2B and ATG16L1 genes and in an intergenic region on 10q21.1. Specifically, a non-synonymous coding change in the ATG16 autophagy related 16-like 1 (S.
  • ATG16L1 gene on chromosome 2q37.1 (rs2241880, an Ala197Thr substitution in exon 8) and a variant in the promoter region of the Paired-Like Homeobox 2B (PHOX2B) gene on chromosome 4p13 (rs16853571 , 2143 bp upstream of the first known exon and 10 bp upstream of a putative alternate first exon).
  • PHOX2B Paired-Like Homeobox 2B
  • the third is in an intergenic region on 10q21.1 (rs224136), 50 kb from the nearest annotated gene.
  • the inventors also identified a statistically significant excess of additional regions showing nominal replication of association that contain additional genes.
  • the inventors further demonstrate that the ATG16L1 gene is expressed in intestinal epithelial cells and that functional knock down of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings implicate that autophagy and other host responses to intra-cellular microbes are involved in the pathogenesis of CD.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of a risk variant at the ATG16L1 locus.
  • the risk variant at the ATG16L1 locus further comprises SEQ. ID. NO.: 1.
  • the present invention provides methods of treating Crohn's Disease by determining the presence of a risk variant at the ATG16L1 locus and administering a therapeutically effective treatment of Crohn's Disease.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of a risk variant at the PHOX2B locus.
  • the risk variant at the PHOX2B locus further comprises SEQ. ID. NO.: 2.
  • the present invention provides methods of treating Crohn's Disease by determining the presence of a risk variant at the PHOX2B locus and administering a therapeutically effective treatment of Crohn's Disease.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of a risk variant in the intergenic region on 10q21.1.
  • the risk variant in the intergenic region on 10q21.1 further comprises SEQ. ID. NO.: 3.
  • the present invention provides methods of treating Crohn's Disease by determining the presence of a risk variant in the intergenic region on 10q21.1 and administering a therapeutically effective treatment of Crohn's Disease.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of a risk variant at the NCF4 locus.
  • the risk variant at the NCF4 locus further comprises SEQ. ID. NO.: 4.
  • the present invention provides methods of treating Crohn's Disease by determining the presence of a risk variant at the NCF4 locus and administering a therapeutically effective treatment of Crohn's Disease.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of a risk variant in intron 4 of the FAM92B locus.
  • the risk variant in intron 4 of the FAM92B locus further comprises SEQ. ID. NO.: 5.
  • the present invention provides methods of treating Crohn's Disease by determining the presence of a risk variant in intron 4 of the FAM92B locus and administering a therapeutically effective treatment of Crohn's Disease.
  • the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of abnormal autophagic processing relative to a healthy individual. In another embodiment, the present invention provides methods of treatment by detecting the presence in the individual of abnormal autophagic processing relative to a healthy individual and administering a therapeutically effective amount of treatment.
  • abnormal autophagic processing might be determined by evaluating whether any of the various genes known to be involved in autophagic processing have abnormal expression profiles.
  • a variety of methods can be used to determine the presence or absence of a 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 a 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 a 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. In 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.
  • Minor grove binder 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 a PHOX2B, ATG16L1 , 10q21.1 , and/or NCF4 variant allele or haplotype.
  • Restriction fragment length polymorphism (RFLP) analysis 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 SNP and/or a haplotype.
  • DGGE 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).
  • Example 1 The inventors performed a genome-wide association study of ileal Crohn's disease (CD) and an independent replication study that established five regions of unambiguously confirmed association of genome-wide significance.
  • CD ileal Crohn's disease
  • the inventors further demonstrate that the ATG16L1 gene is expressed in intestinal epithelial cells and that functional knock down of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings implicate that autophagy and other host responses to intra-cellular microbes are involved in the pathogenesis of CD.
  • Example 2 Subject ascertainment and diagnostic classification
  • informed consent was obtained using protocols approved by the local institutional review board in all participating institutions. 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) occurrence of symptoms 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, structuring or bowel wall thickening from endoscopy reports, barium X-rays, operative reports and/or pathology resection specimen reports.
  • the inventors previously reported discovering the association between the IL23R gene and IBD from a genome-wide study of 567 CD patients and 571 controls. These samples were included in the current study as well as an additional 401 ileal CD and 433 control individuals that were ascertained in an identical manner.
  • the primary replication cohort has previously been described.
  • the second independent replication cohort that was used to further replicate the ATG16L1 Thr197Ala association to ileal CD were ascertained as part of ongoing genetic studies at the Inflammatory Bowel Center at Cedars-Sinai Medical Center, Los Angeles, California. Recruitment of subjects has been approved by the Cedars-Sinai Medical Center Institutional Review Board.
  • Example 3 Genotyping methods For the genome-wide association study, approximately 750ng of genomic DNA was used to genotype each sample on the lllumina HumanHap300 Genotyping BeadChip (lllumina, San Diego) 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, staining and imaging on an lllumina Bead Array Reader.
  • genotypes were called in this experiment; however, before analysis, quality filtering of both samples and SNPs were performed to insure robust association tests. Based on an evaluation of empirical distributions, data quality, and likely introduction of false positive associations, the inventors required that samples pass a 93% genotyping call rate threshold and SNPs pass a 95% call rate threshold in order to be included in the analysis. The data from the genome-wide association studies were used to detect possible relatedness in the case-control cohorts. The Hardy-Weinberg equilibrium (HWE) test was performed on the genotype data from controls and the inventors investigated the relationship between HWE and genotype yield (call rate).
  • HWE Hardy-Weinberg equilibrium
  • the call rate distribution suggested a 95% and 93% genotype completion threshold for inclusion of samples and SNPs, respectively in the genetic association analyses.
  • sample call rates 90% and below, there was an elevation in observed heterozygosity suggesting either a bias in missing data or the presence of false heterozygous calls.
  • the inventors selected a threshold greater than 90 at a point consistent with the tail of successful samples.
  • SNP success of 95% there was an excess of markers out of HWE (P-value ⁇ 0.001 in controls) and a significant bias in missing data between cases and controls.
  • the inventors looked at segments of the data just above and below this cutoff and observed that SNPs with a 90-95% call rate had much more substantial inflation than those in the 95-97% call rate range (genomic control correction 1.34 vs. 1.16) indicative of significant excess of false positives due to lower data quality/biases in missing data so they opted not to lower this threshold further.
  • 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 as implemented in R (The GNU Project) with the option that gives an exact p-value.
  • Example 5 Analysis of replication study data
  • the most significantly associated SNP from the top 23 ranking independently associated regions were selected. Of these, 2 failed in assay design, one failed to type, and 2 were located within previously reported associated loci, leaving 18 SNPs in the replication analysis. In addition to these, 19 additional SNPs were selected to test the variability in the ATG16L1 region.
  • This set of SNPs was evaluated for replication in a family-based ileal-CD cohort composed of 650 mother-father-affected offspring trios. The genotype data from the family-based cohorts were used in determining Mendelian inconsistencies and departures from Hardy-Weinberg equilibrium.
  • HeLa cells were obtained from ATCC and cultured in DMEM supplemented with 10% iron-supplemented calf serum (CSFe) (Hyclone) and 20 ⁇ g ml "1 gentamicin sulfate (Sigma). Salmonella ente ⁇ ca sbsp. enterica serovar Typhimurium (S.
  • strain SL1344 was transformed with a DsRed2 expression plasmid
  • the inventors obtained modified siRNA duplexes (Stealth siRNA) directed against ATG16L1 (siRNA 1 & 2) as well as a non-targeting scrambled sequence
  • siRNA control from a commercial source (Invitrogen).
  • Cells were co-transfected in the absence of antibiotics with siRNAs and plasmids using Lipofectamine (Invitrogen) according to the manufacturer's instructions. They optimized siRNA conditions using a flag-tagged overexpression ATG16L1 construct in HEK293 cells, allowing the inventors to easily assess knockdown by Western blotting.
  • HEK293 cells grown in 12 well plates were transfected with 500 ng Flag-ATG16L1 expression plasmid and 20 pmol siRNA duplex.
  • HeLa cells were plated onto 18mm glass coverslips in 12-well plates at a density of 1x10 5 well "1 and allowed to grow for 24 hours prior to transfection. Each well received 500ng of GFP- LC3 plasmid and 20 pmol siRNA duplex. Four hours later the medium was changed
  • S. Typhimurium infections were performed as previously described, with slight modifications. Briefly, S. Typhimurium SL1344 carrying a DsRed2 expression plasmid was grown overnight in LB broth containing 100 ⁇ g ml "1 ampicillin at 37 0 C with aeration and subcultured at a dilution of 1 :33 for a further 3 hours in LB.
  • This culture was further diluted in DMEM 10% CSFe without antibiotics, to yield an m.o.i of 100. Infections were allowed to proceed for 20 minutes, cells were washed once in complete medium containing 100 ⁇ g ml "1 gentamycin sulfate, and then incubated in fresh high-gentamycin medium for 1 hour.
  • the total number of bacteria per cell, and the number of LC3-GFP positive bacteria were assessed in randomly chosen fields with at least 100 cells
  • IBS identity-by-state
  • the inventors After performing these data filtering measures, the inventors had a final data set of genotypes consisting of 304,413 SNPs in 946 cases and 977 controls with an average call rate of 99.35%.
  • the baseline analysis was a Cochran-Mantel-Haenszel test using two groups (Jewish and non-Jewish) to accommodate potential subtle differences in the genetic background of the two groups.
  • the median chi-square observed was slightly inflated so the inventors corrected all statistics using a genomic control factor of 1.056.
  • Example 11 Replication study confirms three novel loci While certainly encouraging, even an overall high-quality data set and robust analyses cannot be assumed to perfectly filter out all artifactual sources of false positives.
  • the inventors therefore tested the most significantly associated SNP in the 23 independently associated regions with p ⁇ 5x10 ⁇ 5 in an independent set of 650 individuals with ileal CD and their parents, to attempt replication of these findings in a robust family-based setting and with an alternative genotyping technology. Although the inventors did not succeed in typing three of these SNPs (and the replication of CARD15 and IL23R had already been established and are not considered here) the remaining 18 SNPs had high quality genotype data that passed quality thresholds.
  • Two of the three SNPs are located in genes not previously reported as associated with IBD, while the third localizes to an intergenic region, also not previously reported.
  • PHOX2B Paired-Like Homeobox 2B
  • the third is in an intergenic region on 10q21.1 (rs224136), 50 kb from the nearest annotated gene.
  • rs224136 10q21.1
  • IL23R intergenic region on 10q21.1
  • none of these three loci appeared to have any statistically significant epistatic interactions with the CARD15 or IL23R genes (data not shown). Furthermore, none of these three appeared to be associated with UC.
  • ATG16L1 is part of a family of genes involved in autophagy - a biological process involved in protein degradation, antigen processing, regulation of cell signaling and many other pathways essential to the initiation and regulation of the inflammatory response. Since the inventors were interested in determining more about this gene's function and biological context, they examined the expression distribution of ATG16L1 and other known autophagy components by quantitative realtime RT-PCR in a variety of epithelial and immune cell lines (see Fig. 1A, B and C). ATG5, 7 and 16 were broadly expressed, with SW480 cells having the lowest overall mRNA abundance relative to GAPDH controls.
  • CD19+ cells also showed almost 5-fold greater expression of ATG16L1 than both mononuclear cells and placental control RNA.
  • ATG16L1 and its variants play roles in both the epithelial- and immune-driven aspects of CD.
  • RNA-interference directed atATG16L1 prevents autophagy
  • yeast homologue of human ATG16L1 is required for autophagy
  • the mammalian isoforms were similarly essential.
  • the inventors addressed this by utilizing oligo-based siRNA directed against ATG16L1 isoforms 1 and 2.
  • siRNA oligos Using co-transfection of a flag-tagged ATG16L1 expression plasmid and siRNA oligos the inventors established specific knockdown of ATG16L1 in HEK293 cells (see Fig. 2A).
  • the inventors confirmed efficient knockdown of endogenous ATG16L1 in HeLa cells by real-time RT-PCR (see Fig. 2B). Since siRNA 2 was the most effective, this was used in subsequent experiments, co-transfected with a plasmid expressing GFP-LC3, a marker for the autophagic compartment. It is known that a subpopulation of internalized Salmonella are targeted by autophagy in this system. Infections with S.
  • control cells targeted a mean of 17.5% (+/- 1.3 s.e.m.) of intracellular bacteria to autophagic LC3+ vacuoles within 1 hour, while siRNA 2 transfection reduced this to only 2% (+/- 0.5 s.e.m) (see Fig. 2C).
  • the autophagic targeting rates in control- treated and ATG 16L1 -knockdown cells correspond well to those seen in ATG5 +/+ and ATG5 "7' mouse embryonic fibroblasts (20% and 3%, respectively) 26 , confirming that,
  • ATG16L1 is required for autophagy. Confocal imaging of infected cells revealed a near-complete lack of LC3 localisation with intracellular bacteria in ATG 16L1 -knockdown cells, despite the presence of abundant cytoplasmic LC3-GFP and complete envelopment of targeted bacteria in control cells ( Figure 2D).
  • the inventors utilized classical autophagic stimuli to demonstrate the inhibitory effect of ATG16L1 knockdown; both serum starvation or rapamycin treatment for 24 hours induced LC3-GFP vesicles only in control siRNA transfected cells, not those with ATG16L1 knockdown. Inhibition of lysosome fusion by ammonium chloride also induced rapid accumulation of LC3+ vesicles in control cells, but these structures were not found in ATG16L1-siRNA treated cells.
  • MAF iCD minor allele frequency in ileal CD patients
  • MAF CTL, minor allele frequency in controls
  • T number of transmissions of the minor allele
  • U number of nontransmissions of the minor allele
  • NT not tested
  • 'failed ' failed genotyping
  • P. T. previously tested and having confirmed replication
  • N not done
  • Chr chromosome.

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Abstract

L'invention concerne une méthode de diagnostic ou de prédiction de la susceptibilité à la maladie de Crohn (MC) chez un individu, par détermination de la présence ou de l'absence de variants associés à un risque génétique. Dans un mode de mise en oeuvre, cette méthode consiste à déterminer en outre la présence ou l'absence d'un variant à risque au niveau du locus ATG1GL1, du locus PHOX2B, du gène NCF4, de l'intron 4 du locus FAM92B, et/ou de la région intergénique sur 10q21.1. Les méthodes décrites peuvent être utilisées pour diagnostiquer ou prédire la susceptibilité à un sous-type clinique de la maladie de Crohn, par exemple la maladie de Crohn iléale, un sous-type caractérisé par une atteinte de la région iléale de l'intestin grêle.
PCT/US2008/057028 2007-03-15 2008-03-14 Méthodes de diagnostic et de traitement de la maladie de crohn WO2008112990A2 (fr)

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Cited By (5)

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WO2010062960A3 (fr) * 2008-11-26 2010-10-14 Cedars-Sinai Medical Center Méthodes de détermination d'une réceptivité à une thérapie par anti-tnfα lors d'une maladie intestinale inflammatoire
US9580752B2 (en) 2008-12-24 2017-02-28 Cedars-Sinai Medical Center Methods of predicting medically refractive ulcerative colitis (MR-UC) requiring colectomy
US10316083B2 (en) 2013-07-19 2019-06-11 Cedars-Sinai Medical Center Signature of TL1A (TNFSF15) signaling pathway
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
US11186872B2 (en) 2016-03-17 2021-11-30 Cedars-Sinai Medical Center Methods of diagnosing inflammatory bowel disease through RNASET2

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Title
BECKER K.G. ET AL.: 'Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases' PNAS vol. 95, August 1998, pages 9979 - 9984, XP002960579 *
'Business Wire', [Online] Retrieved from the Internet: <URL:http://www.findarticles.com/p/articles/mi_m0EIN/is_2007_Jan_24/ai_n27122450> *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010062960A3 (fr) * 2008-11-26 2010-10-14 Cedars-Sinai Medical Center Méthodes de détermination d'une réceptivité à une thérapie par anti-tnfα lors d'une maladie intestinale inflammatoire
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
US9580752B2 (en) 2008-12-24 2017-02-28 Cedars-Sinai Medical Center Methods of predicting medically refractive ulcerative colitis (MR-UC) requiring colectomy
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

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