WO2008112990A2 - Methods of diagnosis and treatment of crohn's disease - Google Patents

Abstract

The invention provides a method of diagnosing or predicting susceptibility to Crohn's disease (CD) in an individual by determining the presence or absence of genetic risk variants. In one embodiment, a method of the invention is practiced by further determining the presence or absence in the individual of a risk variant at the ATG1GL1 locus, the PHOX2B locus, the NCF4 gene, intron 4 of the FAM92B locus, and/or the intergenic region on 10q21.1 The methods of the invention can be used to diagnose or predict susceptibility to a clinical subtype of Crohn's disease, for example, ileal Crohn's disease, a subtype characterized by involvment of the ileal region of the small intestine.

Description

METHODS OF DIAGNOSIS AND TREATMENT OF CROHN'S DISEASE

GOVERNMENT RIGHTS

This invention was made with U.S. Government support on behalf of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Inflammatory Bowel Disease Genetics Consortium (IBDGC), supported by the following grants: DK62431 , DK62420, DK62432, DK62423, DK62413, DK62422, and DK62429. 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.

FIELD OF THE INVENTION

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.

BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Crohn's disease (CD) and ulcerative colitis (UC), the two common forms of idiopathic inflammatory bowel disease (IBD), are chronic, relapsing inflammatory disorders of the gastrointestinal tract. Each has a peak age of onset in the second to fourth decades of life and prevalences in European ancestry populations that average approximately 100-150 per 100,000 (D. K. Podolsky, N Engl J Med 347, 417 (2002); E. V. Loftus, Jr., Gastroenterology 126, 1504 (2004)). Although the precise etiology of IBD remains to be elucidated, a widely accepted hypothesis is that ubiquitous, commensal intestinal bacteria trigger an inappropriate, overactive, and ongoing mucosal immune response that mediates intestinal tissue damage in genetically susceptible individuals (D.K. Podolsky, N Engl J Med 347, 417 (2002)). Genetic factors play an important role in IBD pathogenesis, as evidenced by the increased rates of IBD in Ashkenazi Jews, familial aggregation of IBD, and increased concordance for IBD in monozygotic compared to dizygotic twin pairs (S. Vermeire, P. Rutgeerts, Genes lmmun 6, 637 (2005)). Moreover, genetic analyses have linked IBD to specific genetic variants, especially CARD15 variants on chromosome 16q12 and the IBD5 haplotype (spanning the organic cation transporters, SLC22A4 and SLC22A5, and other genes) on chromosome 5q31 (S. Vermeire, P. Rutgeerts, Genes lmmun 6, 637 (2005); J. P. Hugot et al., Nature 411 , 599 (2001); Y. Ogura et al., Nature 411 , 603 (2001); J. D. Rioux et al., Nat Genet 29, 223 (2001); V.D. Peltekova et al., Nat Genet 36, 471 (2004)). CD and UC are thought to be related disorders that share some genetic susceptibility loci but differ at others.

The replicated associations between CD and variants in CARD15 and the IBD5 haplotype do not fully explain the genetic risk for CD. Thus, there is need in the art to determine other genes, allelic variants and/or haplotypes that may assist in explaining the genetic risk, diagnosing, and/or predicting susceptibility for or protection against inflammatory bowel disease including but not limited to CD and/or UC.

SUMMARY OF THE INVENTION

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. In another embodiment, one of the one or more risk variants at the ATG16L1 locus further comprises SEQ. ID. NO.: 1. In another embodiment the 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.

Other embodiments 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. In another embodiment, 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. In other embodiments, one of the one or more risk variants at the PHOX2B locus further comprises SEQ. ID. NO.: 2. In another embodiment, the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

Other 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 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.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawing, which illustrate, by way of example, various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. 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 C₁ 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. Data was normalized to GAPDH controls and siRNA control mRNA levels were set at 1.0. Compared to control duplex, siRNA 2 yielded an 89% reduction in mRNA transcript. RT-PCR reactions were performed in triplicate and means are plotted with error bars representing 1 standard deviation, results are representative of two independent experiments. Panel C. Knockdown of ATG16L1 prevents effective autophagy of S. Typhimurium in HeLa cells. 48 hours following co-transfection with control siRNA or duplex 2 and LC3-GFP piasmid, HeLa cells were infected for 1 hour with Salmonella Typhimurium SL1344, fixed and microscopically examined. The mean % of bacteria per cell encapsulated by LC3+ membranes and standard errors are shown. Bacterial counts were pooled from two separate experiments, each counting a minimum of 100 infected cells. Significance was assessed using a two-tailed Students T-test, assuming unequal variances. Panel D. Representative images of intracellular S. Typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells, but loss of autophagic targeting when ATG16L1 knockdown is affected. Rows 2 and 4 show magnified views of the areas marked in rows 1 and 3 respectively. Images were obtained using confocal microscopy and are flat projection of Z-stacks taken throughout the plane of the infected cell. Scale bars represent 5 μm.

Figure 3 depicts knockdown of ATG16L1 prevents induction of autophagy by classical simuli.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et a/., Dictionary of Microbiology and Molecular Biology 3^rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. "SNP" as used herein means single nucleotide polymorphism. "Haplotype" as used herein 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.

As used herein, the term "biological sample" means any biological material from which nucleic acid molecules can be prepared. As non-limiting examples, the term material encompasses whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acid.

As used herein, the terms "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.

As used herein, the term "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.

An example of ATG16L1 is described herein as SEQ. ID. NO.: 6, and SEQ. ID. NO.: 7. An example of PHOX2B is described herein as SEQ. ID. NO.: 8.

An example of NCF4 is described herein as SEQ. ID. NO.: 9, and SEQ. ID. NO.: 10.

An example of 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. The detection of 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.

Based on these findings, 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.

In one embodiment, a method of the invention is practiced with whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA₁ for example, for enzymatic amplification or automated sequencing. In another embodiment, a method of the invention is practiced with tissue obtained from an individual such as tissue obtained during surgery or biopsy procedures.

As disclosed herein, 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. In addition to CARD15 and IL23R associations, 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. cerevisiae) or 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). The third is in an intergenic region on 10q21.1 (rs224136), 50 kb from the nearest annotated gene.

As further disclosed herein, 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.

ATG16L1 In one embodiment, 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. In another embodiment, the risk variant at the ATG16L1 locus further comprises SEQ. ID. NO.: 1. In another embodiment, 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.

PHOX2B

In one embodiment, 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. In another embodiment, the risk variant at the PHOX2B locus further comprises SEQ. ID. NO.: 2.

In another embodiment, 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.

lntergenic region on 10q21.1

In one embodiment, 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. In another embodiment, the risk variant in the intergenic region on 10q21.1 further comprises SEQ. ID. NO.: 3.

In another embodiment, 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.

NCF4

In one embodiment, 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. In another embodiment, the risk variant at the NCF4 locus further comprises SEQ. ID. NO.: 4. In another embodiment, 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.

FAM92B

In one embodiment, 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. In another embodiment, the risk variant in intron 4 of the FAM92B locus further comprises SEQ. ID. NO.: 5.

In another embodiment, 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.

Autophagy

In one embodiment, 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.

There are numerous methods known to one of skill in the art for determining whether there is abnormal autophagic processing relative to a healthy individual. For example, 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.

Variety of Methods and Materials A variety of methods can be used to determine the presence or absence of a variant allele or haplotype. As an example, 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.

Analysis of the nucleic acid from an individual, whether amplified or not, may be performed using any of various techniques. Useful techniques include, without limitation, polymerase chain reaction based analysis, sequence analysis and electrophoretic analysis. As used herein, the term "nucleic acid" means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term 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. In addition, each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonant energy transfer (FRET). During PCR, 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. Thus, 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., "3¹- minor groove binder-DNA probes increase sequence specificity at PCR extension temperature, "Nucleic Acids Research 28:655-661 (2000)). 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 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)). As used herein, 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. One skilled in the art understands that the use of 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. Similarly, 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)). One skilled in the art understands that 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. In contrast, 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 (HMA) 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)).

The technique of single strand conformational, polymorphism (SSCP) also may be used to detect the presence or absence of a SNP and/or a haplotype (see Hayashi, K., Methods Applic. 1 :34-38 (1991)). 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 (DGGE) also may be used to detect a

SNP and/or a haplotype. In 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).

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. Other well-known approaches for determining the presence or absence of a SNP and/or a haplotype include automated sequencing and RNAase mismatch techniques (Winter et al., Proc. Natl. Acad. Sci. 82:7575-7579 (1985)). Furthermore, one skilled in the art understands that, where the presence or absence of multiple alleles or haplotype(s) is to be determined, individual alleles can be detected by any combination of molecular methods. See, in general, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997). In addition, one skilled in the art understands that multiple alleles can be detected in individual reactions or in a single reaction (a "multiplex" assay). In view of the above, one skilled in the art realizes that the methods of the present invention for diagnosing or predicting susceptibility to or protection against CD in an individual may be practiced using one or any combination of the well known assays described above or another art-recognized genetic assay.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

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. In addition to CARD15 and IL23R associations, the inventors found three novel confirmed associations with variation in the PHOX2B and ATG16L1 genes and in an intergenic region on 10q21.1. 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.

Example 2 Subject ascertainment and diagnostic classification In all cases, 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. 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 replication study of the all of the putative loci (Table 1), and the additional genotyping of the ATG16L1 region genotyping was performed using primer extension chemistry and mass spectrometric analysis (iPlex assay, Sequenom, San Diego) using Sequenom Genetics Services (Sequenom, San Diego). TaqMan MGB technology was used to perform the genotyping of the ATG16L1 Thr197Ala variant (rs2241880) using the design available from Applied Biosystems and following the manufacturer's recommendations (Bulletin #4317594) in the Cedars-Sinai Inflammatory Bowel Disease Center cohort. Quality of the SNP data was checked by reproducibility of 5% of samples duplicated. Rs2241880 had a 99% genotype rate across the DNA samples as arrayed for genetic studies within the Cedars-Sinai Inflammatory Bowel Disease Center. Thus the rs2241880 was found to be associated in three independent cohorts using three different technological platforms at three laboratories/genotyping centers.

Example 4

Analysis of GWA data

Overall, 98.6% of 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). The call rate distribution suggested a 95% and 93% genotype completion threshold for inclusion of samples and SNPs, respectively in the genetic association analyses. At sample call rates of 90% and below, there was an elevation in observed heterozygosity suggesting either a bias in missing data or the presence of false heterozygous calls. To avoid this, the inventors selected a threshold greater than 90 at a point consistent with the tail of successful samples. Below 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 For the replication study, the most significantly associated SNP from the top 23 ranking independently associated regions (p-value=5x10^"5 in the combined screening analyses) 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. After removal of failing SNPs (monomorphic, call rate below 75%, and causing a high number of Mendel errors), families with excess Mendelian inconsistencies and uninformative families (where a parent or the sole affected offspring had a genotyping call rate below 90%) were eliminated from the analysis, there remained 530 affected trios (from 431 independent nuclear families) with both parents and at least one affected offspring genotyped. Following the removal of additional SNPs causing excess Mendel errors, the final genotyping call rate for each of the 18 replication markers, as well as the 19 ATG16L1 tagging SNPs, ranged from 90-100%, and Mendelian inconsistencies were below 4 for all markers and less than 4 per family. Single marker association tests were performed in the family data using PLINK (Pureed, et al.; American Journal of Human Genetics; 81 (2007); Center for Human Genetic Research). The inventors then tested the replicated SNPs in the set of 530 independent IBD trios (post-QC) from the NIDDK IBDGC and in the independent Cedars-Sinai cohort of 350 patients with ileal CD and 207 controls, and one-tailed p- values are reported. The analysis of ATG16L1 SNPs conditional on Ala197Thr was performed by logistic regression in the family-based sample using the software WHAP (Purcell, et al.; Bioinformatics, 23(2): 255-256 (2007)).

Example 6

Ce// lines and bacterial strain

HeLa cells were obtained from ATCC and cultured in DMEM supplemented with 10% iron-supplemented calf serum (CSFe) (Hyclone) and 20 μg ml^"1 gentamicin sulfate (Sigma). Salmonella enteήca sbsp. enterica serovar Typhimurium (S.

Typhimurium), strain SL1344 was transformed with a DsRed2 expression plasmid

(Clontech, CA) (DsRed2 is under the control of the lac promoter, which is constitutive in the absence of lacl) to facilitate bacterial visualization. The plasmid was maintained with 100 μg ml^"1 ampicillin selection during culture in LB broth.

Example 7 siRNA directed against ATG16L1

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. For knockdown of endogenous ATG16L1 , HeLa cells were plated onto 18mm glass coverslips in 12-well plates at a density of 1x10⁵ 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

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Los Angeles again and the cells were allowed to grow for a further 48 hours. Knockdown was confirmed for each experiment using real-time quantitative RT PCR with specific primers to ATG16L1 and normalized to GAPDH controls, with RNA isolated from specimen wells.

Example 8

Induction ofautophagy and bacterial infection

Autophagy was induced in HeLa cells using serum starvation or rapamycin.

Cells were transfected as described above and after 48 hours the medium was changed to either 1% serum, or 10% serum plus 200 nM rapamycin for a further 24 hours. Ammonium chloride treatment was performed with a final concentration of 50 mM for 2 hours in 1% serum medium. 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 μg ml^"1 ampicillin at 37⁰C 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 μg ml^"1 gentamycin sulfate, and then incubated in fresh high-gentamycin medium for 1 hour.

Example 9 Microscopy

Infected, autophagy-induced and control HeLa cells were fixed in 4% formalin in PBS for 15 minutes, washed in PBS and mounted in aqueous mountant (Polysciences, PA). Slides were then either viewed under wide-field fluorescence illumination (Zeiss Axioplan) for counting, or laser scanning confocal microscopy

(BioRad Radiance 2000) was used to obtain high-resolution z-stacks, which were subsequently projected onto single images using LSM Image software (Carl Zeiss

GmbH, Germany). 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

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Los Angeles counted for each condition. The numbers of LC3-GFP positive bacteria were then calculated as a percentage of total bacteria. Significance was assessed using the two-tailed, unequal variance Student's T-test.

Example 10

Genome-wide association scan

Overall, 98.6% of 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 inventors note that so few samples had a call rate between 90 and 95% that the results are not at all dependent on these choices. As a final step, using identity-by-state (IBS) counts from this data set, they identified 8 duplicate samples and 10 additional pairs of samples that shared 10% or more of their genome by descent (first cousins through full siblings) - they eliminated one member of each of these 18 pairs of samples.

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. This GC-corrected CMH analysis produced a significant excess of positive associations - for example 27 SNPs in 9 distinct genomic regions were associated with p<10^'5, a level which the inventors would informally expect to observe roughly 3 times by chance in this scan given the number of tests performed (See Table 1). The two most significant loci, with multiple SNPs being significantly associated, corresponded to the CARD15 and IL23R genes that have previously been confirmed as IBD genes. The remaining regions identified with extreme p-values were all novel, showed no unusual pattern of missing data or departure from Hardy- Weinberg equilibrium (of the best SNPs in the top 23 regions examined further below, all had HW-p > .01 in controls and 18 were missing less than 1% genotypes), and many were supported by nearby correlated SNPs also showing association. It should be noted that the confirmed IBD5 CD risk haplotype was not among the top loci in this study of ileal CD as it only had modest evidence of association (p<0.01).

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. Subsequent quality control analysis identified DNAs with low genotype call rates and families with an excess of Mendel errors, which were excluded, such that the final analyses included 530 trios. Association testing of this final dataset identified that three of the 18 SNPs tested had significant evidence of association (p<.0005), and given the number of replications attempted were formally of genome-wide significance in the combined analysis (even when considering a conservative threshold of .05/304,413 = 1.65x10^"7), establishing these as replicated novel associations with CD.

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. Specifically, a non-synonymous coding change in the ATG16 autophagy related 16-like 1 (S. cerevisiae) or 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). The third is in an intergenic region on 10q21.1 (rs224136), 50 kb from the nearest annotated gene. Using a stratified analysis approach, 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.

In a combined analysis of the data from the GWA scan and the replication study, the ATG16L1 , PHOX2B and 10q21.1 intergenic region had highly significant association p-values of 2.98x10^"10, 1.28x10^"8, 6.82x10^"8, respectively using Fisher's method. The informal expectation that roughly three of the nine regions with p<10^'5 in the GWA study were expected by chance has been borne out (five represent true positives, three appear to represent the expected purely statistical fluctuations, and one has not yet been successfully tested in follow-up), suggesting very little of the tail of extreme p-values is due to uncharacterized artifacts in the data or analysis. In addition, three SNPs of the remaining 15 regions which were associated at a p<5x10^"5 in the GWA scan (excluding the three just defined as true associations above) show nominal replication of association (p<0.05). This by itself is in excess of the one false positive expected by chance (at alpha=0.05) suggesting that one or more of these loci will eventually be confirmed as well. The three nominal replications are on 13q22.2, 16q24.1 and 22q12.3 (replication results of p=0.037, p=0.024 and p=0.019, respectively; see Table 1), the last of which is in the first intron of NCF4; a gene of potential relevance to IBD pathogenesis.

Example 12

Mapping of the ATG16L1 causal variant

To provide further evidence for the association of the Ala197Thr variant, the inventors typed another independent cohort of 350 patients with ileal CD and 207 controls and again obtained significant association of this amino acid coding change (P-value = 1.8x10^'5; minor allele frequency in ileal CD patients of 0.353 and in controls of 0.478). Since the ATG16L1 coding variant had achieved genome-wide significance in the original scan, the inventors simultaneously generated high-density genotype data in the 650 ileal CD trios (described above), for 19 additional SNPs in the 115 kb region containing ATG16L1 , spanning 2 genes (ATG16L1 and SAG) from 233,925,244 to 234,039,454. The typing of additional SNPs confirms the association of this region and indicates that the non-synonymous coding variant identified in the scan is the most associated SNP in the region. Furthermore, logistic regression analyses conditional on the Ala197Thr in the family-based samples indicates that this coding variant can fully explain the association signal to this locus - therefore the inventors consider it is very likely to be the causal risk variant. In all of the datasets tested in this study, the Thr allele is the minor allele and has a protective effect; in the GWA dataset the estimated odds ratio is 0.692 (95% Cl of 0.608-0.788). The Thr is the ancestral allele and is the major allele in the YRI, JPT, and HCB samples typed in the International HapMap Project.

Example 13

Autophagy components are expressed broadly.

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. Apart from the markedly elevated levels of ATG7 seen in THP-1 cells, expression of each of the three components followed a similar pattern in the cells tested, as might be suggested by the stoichiometric relationship between these components in the homeostatic autophagy process. The inventors also investigated the expression of ATG16L1 in primary human immune cells, to gain a better understanding of its role in the association with CD (see Fig. 1 D). Amongst samples tested, the inventors found that ATG16L1 was most highly expressed in the T cell compartment, with both CD4+ and CD8+ cells showing high levels (approximately 13- and 10-fold higher than placental control RNA, respectively). CD19+ cells (B cells) also showed almost 5-fold greater expression of ATG16L1 than both mononuclear cells and placental control RNA. Thus it is possible that ATG16L1 and its variants play roles in both the epithelial- and immune-driven aspects of CD.

Example 14

RNA-interference directed atATG16L1 prevents autophagy Although the yeast homologue of human ATG16L1 is required for autophagy, it was not known whether the mammalian isoforms were similarly essential. The inventors addressed this by utilizing oligo-based siRNA directed against ATG16L1 isoforms 1 and 2. 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). In order to study the role of ATG16L1 in autophagy during a well-characterized host-pathogen interaction, they performed ATG16L1 knockdown in HeLa cells, followed by S. Typhimurium infection. 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. Typhimurium showed a marked difference between control and siRNA 2 -treated cells: 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,

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Los Angeles like ATG5, 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). In addition, 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.

Example 15 Table of Genome Wide Association Study (Table 1.)

Table 1 . 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. D., not done; Chr, chromosome.

While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. Furthermore, one of skill in the art would recognize that the invention can be applied to various inflammatory conditions and disorders and autoimmune diseases besides that of inflammatory bowel disease. It will also be readily apparent to one of skill in the art that the invention can be used in conjunction with a variety of phenotypes, such as serological markers, additional genetic variants, biochemical markers, abnormally expressed biological pathways, and variable clinical manifestations.

Claims

1. A method 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.

2. The method of claim 1 , wherein one of said one or more risk variants at the ATG16L1 locus further comprises SEQ. ID. NO.: 1.

3. The method of claim 1 , wherein the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

4. A method 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.

5. A method 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.

6. The method of claim 5, wherein one of said one or more risk variants at the ATG16L1 locus further comprises SEQ. ID. NO.: 1.

7. A method 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.

8. The method of claim 7, wherein one of said one or more risk variants at the PHOX2B locus further comprises SEQ. ID. NO.: 2.

9. The method of claim 7, wherein the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

10. A method 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.

11. The method of claim 10, wherein one of said one or more risk variants at the intergenic region on 10q21.1 further comprises SEQ. ID. NO.: 3.

12. The method of claim 10, wherein the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

13. A method 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.

14. The method of claim 13, wherein one of said one or more risk variants at the NCF4 locus further comprises SEQ. ID. NO.: 4.

15. The method of claim 13, wherein the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

16. A method 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.

17. The method of claim 16, wherein one of said one or more risk variants at intron 4 of the FAM92B locus further comprises SEQ. ID. NO.: 5.

18. The method of claim 16, wherein the Crohn's Disease further comprises an ileal Crohn's Disease phenotype.

19. A method 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.

20. The method of claim 19, wherein the subtype further comprises an ileal Crohn's Disease phenotype.

21. A method 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.