WO2010075584A1

WO2010075584A1 - Methods of diagnosing and predicting crohns disease from childhood hygiene and serological profiles

Info

Publication number: WO2010075584A1
Application number: PCT/US2009/069541
Authority: WO
Inventors: Jerome I. Rotter; Kent D. Taylor; Stephan R. Targan
Original assignee: Cedars Sinai Medical Center
Current assignee: Cedars Sinai Medical Center
Priority date: 2008-12-24
Filing date: 2009-12-24
Publication date: 2010-07-01
Anticipated expiration: 2011-06-24

Abstract

The present invention relates to the discovery that there is a different pathophysiology of inflammatory bowel disease in rural vs. urban areas. In one embodiment, a clean environment is positively associated with ASCA expression, but negatively associated with anti-OmpC and anti-I2 expression in subjects with Crohn's Disease. In another embodiment, the present invention provides a method of diagnosing susceptibility to Crohn's Disease in an individual by determining the presence or absence of risk factors, including serological profiles and sanitary conditions during the individual's childhood.

Description

METHODS OF DIAGNOSING AND PREDICTING CROHNS DISEASE FROM CHILDHOOD HYGIENE AND SEROLOGICAL PROFILES

FIELD OF THE INVENTION The invention relates generally to the field of inflammatory disease, specifically to

Crohn's disease and hygiene,

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 ΪBD 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 CARD 15 variants on chromosome 16ql 2 and the IBD5 haplotype (spanning the organic cation transporters, SLC22A4 and SLC22A5, and other genes) on chromosome 5q31 (S. Vermεire, P. Rutgeerts, Genes lmmun 6, 637 (2005); J.P. Hugot et al., Nature 41 1₉ 599 (2001): Y. Ogura et aL ^"Nature 41 L 603 (2001); J. D. Rioux et al., Nat Genet 29, 223 (2001): V.D. Peltekova et al., Nat Genet 36, 471 (2004)).

DWl 13734313*2 0067789-231WO0 1 CD and UC are thought to be related disorders that share some genetic susceptibility loci but differ at others.

Thus, there is a need in the art to identify environmental factors, serological profiles, genes, allelic variants and/or hapiotypes that may assist in explaining the genetic risk, diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts, in accordance with an embodiment herein, an association between environmental factors and ASCA level, where the bar indicates median ASCA level. Specifically, a) rural, b)running water, c) toilet, and d) sewer service.

Figure 2 depicts, in accordance with an embodiment herein, an association between toilet in house and anti-OmpC and anti~I2 level. Specifically, a) 12 and b) OmpC serology.

SUMMARY OF THE INVENTION Various embodiments include a method of diagnosing a Crohn's Disease subtype in an individual, comprising determining the presence or absence of a high expression of ASCA in the individual, determining whether the individual was exposed primarily to sanitary and/or urban conditions during the individual's childhood, and diagnosing the Crohn's Disease subtype in the individual based upon the presence of the high expression of ASCA in the individual and the determination of sanitary and/or urban conditions during the individual's childhood. In another embodiment, sanitary conditions comprise running water, sewer service and/or toilet. In another embodiment, the high expression of ASCA comprises approximately 1.50 EU/mL.

Other embodiments include a method of diagnosing a Crohn's Disease subtype in an individual, comprising determining the presence or absence of a high expression of anti-OmpC and/or anti-I2 in the individual, determining whether the individual was exposed primarily to unsanitary and/or rural conditions during the individual's childhood, and diagnosing the Crohn's Disease subtype in the individual based upon the presence of the high expression of anti-OmpC and/or anti-I2 in the individual and the determination of unsanitary conditions during the individual's childhood. In another embodiment, the high expression of anti-OmpC comprises approximately 26 EU/mL. In another embodiment, the high expression of anti-I2 comprises approximately 55 EU/mL.

DWT i37343I 2 Various embodiments include a method of treating Crohn's Disease in an individual, comprising determining the presence of a high expression of ASCA in the individual and the exposure primarily to sanitary and/or urban conditions during childhood, and treating the Crohn's Disease. In another embodiment, sanitary conditions comprise running water, sewer service and/or toilet.

Other embodiments include a method of treating Crohn's Disease in an individual, comprising determining the presence of a high expression of anti-OmpC and/or anli-12 in the individual and exposure to unsanitary and/or rural conditions during childhood, and treating the Crohn's Disease, Other embodiments include methods of reducing the incidence and/or development of inflammatory bowel disease (IBD) in a community, comprising improving the sanitary conditions of the community. In another embodiment, sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house. In another embodiment, inflammatory bowel disease comprises Crohn's Disease. Jn another embodiment, the community comprises a rural and/or urban community.

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

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 el al._y Dictionary of Microbiology and Molecular Biology 3^rd eά, J. WiJey & 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.

DWr 137343 Bv: 0067789-23 !WO0 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.

"!BD" as used herein is an abbreviation of inflammatory bowel disease. '"CD^*' as used herein is an abbreviation of Crohn's Disease.

''UC as used herein is an abbreviation of ulcerative colitis. "ASCA" as used herein refers to anti-saccharomyces eerevisiae antibodies. "Sanitary conditions" as used herein is meant to connote the usual meaning of that term, referring, e.g., to the state of being generally clean, such as without significant amounts of dirt or other impurities, and without the presence of an unusually high concentration of pathogens. In contrast, '"unsanitary conditions" is meant to connote the usual meaning of that term, referring, e.g., to the state of being generally unclean, such as with significant amounts of dirt or other impurities and/or with the presence of an unusually high concentration of pathogens.

As used herein, use of the criteria "toilet^"' refers to the existence of an in doors toilet in the residence.

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 disclosed herein, the inventors tested the association between childhood environmental factors with serological markers in adult CD patients in Puerto Rico. Epidemiological data from 152 subjects with CD obtained from questionnaires completed for a collaborative genetic epidemiologic study between the University of Puerto Rico and Cedars- Sinai Medical Center, as part of the NiDDK IBD GRC. The diagnosis of CD was based on established criteria that included clinical, radiological, endoscopic, and histopathologic criteria. Childhood epidemiological data included urban versus rural living, running water, toilet inside the house, public sewer, and breast feeding history. Serological markers including the level of ASCA, anti-OmpC, anti-12, anti-Cbir and ANCA were tested by FXISA.

As further disclosed herein, living in rural area had a lower ASCA level than living in urban area (median (EU): 1.26 vs. 1.54, P=O-Oi ; quarti!e(l-4): p trend=0.005); however, having running water (median: 1.48 vs. 0.88, p=0.06; quartile: p=0.04). toilet inside the house (median:

D¹A ^'I B714313v2 00677«9-231WOO 4 L5 vs. 1.2, p=0.08; quartiie: p=0.06), public sewer (median: 1.54 vs. 1.29, p=0.06; quartile: P=O.04) had a higher ASCA expression. Interestingly, having running water (median (EU): anti- 12: 26.5 vs. 59.1. p=0.07; quartϋe: p=0.04), toilet inside the house (median (EU): anti-I2: 26.1 vs. 54.8, pO.OS; quartile: p=0.06; anti OmpC: 17.7 vs. 25.9, p=0.01 ; quartile: p=0.02) was 5 associated with lower expression of anti-I2 or anli-OmpC. The inventors then put environmental score (rural living +1, running water - 1, toilet - 1, sewer service -1, range: -3 to 1) into regression analysis and found significant association with ASCA (p=0.006, R²=5%), anti -OmpC (p=0.03, R^o/o) and quartile sum of anti-12 and anti-OmpC (p=0.02, R²=3.3%). Environmental exposure in childhood was found to be associated with variation of the antibody expression in 10 CD. The 'clean^' environment is positively associated with ASCA expression but negatively associated with anti-OmpC and anti-12 in CD. The results also show that there is different pathophysiology of IBD in rural and urban areas,

In one embodiment, the present invention provides a method of diagnosing susceptibility to Crohn's Disease in an individual by determining the presence or absence of risk factors, where the presence of the risk factors is indicative of susceptibility to Crohn's Disease. In another embodiment, the risk factors include sanitary conditions during the individual's childhood in conjunction with Crohn's Disease serological profiles. In another embodiment, sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house.

In another embodiment, the present invention provides a method of diagnosing a Crohn^'s Disease subtype in an individual by determining the presence or absence of ASCA expression ! 5 and whether there were sanitary conditions during the individual's childhood, where the presence of ASCA expression and sanitary conditions during the individual's childhood is indicative of the Crohn's Disease subtype. In another embodiment, sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house.

In another embodiment, the present invention provides a method of diagnosing a Crohn's 0 Disease subtype in an individual by determining the presence or absence of anti-OmpC expression and/or anti-12 expression and whether there were unsanitary conditions during the individual^'s childhood, where the presence of anti-OmpC expression and/or anti-12 expression and unsanitary conditions during the individual's childhood is indicative of the Crohn's Disease subtype.

DWI J 37343 ! 3Λ 2 0067789-231 WOO In another embodiment, the present invention provides a method of treating Crohn's Disease in an individual by determining the presence of ASCA expression and the presence of sanitary conditions during the individual's childhood, and treating the individual. In another embodiment, sanitary conditions comprise running water, sewer service and/or existence of a to i Set in the house,

A variety of methods can be used to determine the presence or absence of a variant allele or haplotype or serological profile. 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 a 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 VlCTM 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

DWT 00ό7789-231WO0 6 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 Io no fluorescent signal. Improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor grove binder (MGB) group to a DNA probe as described, for example, in Kutyavin et al., "3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature, "Nucleic Acids Research 28:655-66] (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 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; innis 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 (MuIHs et al., supra, (1994)). One skilled in the art understands that the one or

DW r 1373431 00677K9-2Ϊ 1 W OO 7 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-specifie 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 a!., 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

DWI 137343 13V 2 0067789-231WO0 8 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 may be practiced using one or any combination of the well known assays described above or another art- recognized genetic assay.

Similarly, there are many techniques readily available in the field for detecting the presence or absence of serological markers, polypeptides or other biomarkers, including protein microarrays. For example, some of the detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy, illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).

Similarly, there are any number of techniques that may be employed to isolate and/or fractionate biomarkers. For example, a biomarker may be captured using biospecific capture reagents, such as antibodies, aptamers or antibodies that recognize the biomarker and modified forms of it. This method could also result in the capture of protein interactors that are bound to the proteins or that are otherwise recognized by antibodies and that, themselves, can be biomarkers. The biospecific capture reagents may also be bound to a solid phase. Then, the captured proteins can be detected by SELDl mass spectrometry or by eluting the proteins from the capture reagent and detecting the eluted proteins by traditional MALDI or by SELDI. One example of SELDI is called "affinity capture mass spectrometry," or "Surface-Enhanced Affinity Capture" or "SEAC," which involves the use of probes that have a material on the probe surface that captures analytes through a non-covalent affinity interaction (adsorption) between the material and the analyte. Some examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. Alternatively, for example, the presence of biomarkers such as polypeptides maybe detected using traditional immunoassay techniques. Immunoassay requires biospecific capture

DWT 137343l3v2 0067789-231WO0 9 reagents, such as antibodies, to capture the analytes. The assay may also be designed to specifically distinguish protein and modified forms of protein, which can be done by employing a sandwich assay in which one antibody captures more than one form and second, distinctly labeled antibodies, specifically bind, and provide distinct detection of, the various forms. Antibodies can be produced by immunizing animals with the biomolecules. Traditional immunoassays may also include sandwich immunoassays including ELlSA or fluorescence- based immunoassays, as well as other enzyme immunoassays,

Prior Io detection, biomarkers may also be fractionated to isolate them from other components in a solution or of blood that may interfere with detection. Fractionation may include platelet isolation from other blood components, sub-ceϋular fractionation of platelet components and/or fractionation of the desired biomarkers from other biomolecules found in platelets using techniques such as chromatography, affinity purification, ID and 2D mapping, and other methodologies for purification known to those of skill in the art. In one embodiment, a sample is analyzed by means of a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

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

DWT Ϊ 3734313v2 0067789-231 VVOO 10 Overall

The "Hygiene hypothesis" states that individuals raised in sanitary environments are more at risk to have inflammatory bowel diseases (IBD). Evidence includes the higher incidence of Crohn's disease (CD) and ulcerative colitis (UC) in Europe and Northern America after World War 11. The hypothesis suggests that modern living conditions can lead to defective maturation of regulatory T cells and antigen presenting cells, which cause aberrant immune response to commensal bacteria. However, the relationship between environmental factors and serological markers of IBD has not yet been reported. The inventors tested the association between childhood environmental factors with serological markers in adult CD patients in Puerto Rico, Epidemiological data from 152 subjects with CD obtained from questionnaires completed for a genetic epidemiologic study. The diagnosis of CD was based on established criteria that included clinical, radiological, endoscopic, and histopathologic criteria. Childhood epidemiological data included urban versus rural living, running water, toilet inside the house and connection to public sewer. Serological markers, including the level of ASCA, anit-OmpC, anti-12, anti-Cbir and ANCA, were tested by ELl SA.

The results were that living in rural area had a lower ASCA level than living in urban area (median (EU): 1 .26 vs. 1.54, p=0.01; quartile(l-4): p tτend=0.005 ); however, having running water (median: 1.48 vs. 0,88, p=O.0ό; quartile: p=0.04), toilet inside the house (median: 1.5 vs, 1.2, P=¹O₁OS; quartile: p=0.06), public sewer (median: 1.54 vs. 1.29. p=0.06; quartile: p=0.04) had a higher ASCA expression. Interestingly, having running water (median (EU): anti- 12: 26.5 vs. 59.1, p=0.07; quartϋe: p=0.04), toilet inside the house (median (EU): anti-12: 26.1 vs. 54.8, p=0.05; quartile: p=0.06; anti-OmpC: 17.7 vs. 25.9, p=0.01 ; quartile: p=0.02) was associated with lower expression of anti-12 or anti-OmpC. The inventors then put environmental score (rural living +1 , running water -1, toilet -l,sewerservice -1 , range:-3 tol) into regression analysis and found significant association with ASCA (p=0.006, Rsquare=5%), anti-OmpC (p^O.03, Rsquare =3%) and quartile sum of anti-12 and anti-OmpC (p=0.02, Rsquare =3.3%). Conclusion: environmental exposure in childhood is associated with variation of the antibody expression in CD. The 'clean' environment is positively associated with ASCA expression but negatively associated with anti-OmpC and anti-12 in CD. The results also suggest potential different pathophysiology of CD in rural and urban areas.

DW r 13734313v2 0067789-231 WOO 1 1 Example 2 Methods

• 152 subjects with CD obtained from questionnaires completed for a collaborative genetic epidemiologic study between the University of Puerto Rico and Cedars-Sinai Medical Center, as part of the NIDDK IBD GRC.

• The diagnosis of CD was based on established criteria that included clinical, radiological, endoscopic, and histopathologic criteria.

Childhood epidemiological data included urban versus rural living, running water, toilet inside the house and connection to public sewer. • Serological markers, including the level of ASCA, anit-OmpC, anti-12, anti-Cbir and

ANCA, were tested by ELISA.

Example 3 ASCA level Living in rural area had a lower ASCA level than living in urban area (median (ELi): p=0.01; quartile(l-4): p trend=0.005 ); however, having running water (median:p=0.06; quartile: p=0.04), toilet inside the house (median: p=0,08; quartile: p=0.06), public sewer (median: p=0, 06; quartile: p=0.04) had a higher ASCA expression (Figure 1).

Example 4

Anti-OmpC and anti-12

Having running water (median (EU): anti-I2: 26.5 vs. 59.1 , p^0.07; quartile: p=0.04), toilet inside the house (median (EU): anti-12: 26.1 vs. 54.8, p=0.05; quartile: p=0.06; anti-OmpC: 17.7 vs. 25.9, p=0.01 ; quartile: p=0.02) was associated with lower expression of anti-12 or anti- OmpC (Figure 2). No association between environmental factors and anti-Cbir level was identified in the study.

Example 5

Table 1 - Environmental score and serology The inventors then put environmental score (rural living +1 ,running water -1 ,toϋet - l,sewerservice -1 , range:-3 tol ) into regression analysis and found significant association with

DWT 13734333v20067789-231WOO 12 ASCA (p=Q.Q06, Rsquare=5%), anti-OmpC (p=0.03, Rsquare =3%) and quartϋe sum of anti-!2 and anti-OmpC (p=0.02, Rsquare ^3.3%)(Tabie 1).

Table 1 : association between enviromental score and antibody expression

Example 6

Conclusions

The 'clean' environment is positively associated with ASCA expression but negatively associated with anti-OmpC and anti-12 in CD.

The difference provides a possible explanation for the fact that some but not all clinical studies are consistent with 'hygiene hypothesis' .

Example 7

Childhood Hygiene Impacts Serological Profiles in Adult Crohn 's Disease The "'Hygiene hypothesis^"' states that individuals raised in sanitary environments are more at risk to have inflammatory bowe! disease (IBD), Evidence includes the higher incidence of Crohn's Disease (CD) and ulcerative colitis (UC) in Europe and Northern America after World War II. This evidence suggests that modern living conditions can lead to defective maturation of regulatory T cells and antigen presenting cells, which cause aberrant immune response to commensal bacteria. However, the relationship between environmental factors and serological markers of IBD has not been reported. The inventors tested the association between childhood environmental factors with serological markers in adult CD patients in Puerto Rico,

SJW r 137343 ϊ 3v2 0067789-231 WOO 13 Epidemiological data from 152 subjects with CD obtained from questionnaires completed for a collaborative genetic epidemiologic study between the University of Puerto Rico and Cedars-Sinai Medical Center, as part of the NlDDK IBD GRC. The diagnosis of CD was based on established criteria that included clinical, radiological, endoscopic, and histopathologic criteria. Childhood epidemiological data included urban versus rural living, running water, toilet inside the house, public sewer, and breast feeding history. Serological markers including the level of ASCA, anti-OmpC, anti-I2, anti-Cbir and ANCA were tested by ELlSA.

Living in rural area had a lower ASCA level than living in urban area (median (EU): 1.26 vs. 1.54, p=0.01 ; quartile(l-4): p trend=0,005); however, having running water (median: 1.48 vs. 0.88, p=0.06; quartile: p=0.04), toilet inside the house (median: 1.5 vs. 1.2, p=0.08; quartile: p=0.06), public sewer (median: 1.54 vs. 1.29, p=0.06; quartile: p=0.04) had a higher ASCA expression. Interestingly, having running water (median (EU)- anti-12: 26.5 vs. 59.1 , p=0.07; quartile: p=0.04), toilet inside the house (median (EU): anti-12: 26.1 vs. 54,8, p^O.OS; quartile: p=0.06; anti OmpC: 17.7 vs. 25.9, p=0.01 ; quartile: p=0.02) was associated with lower expression of anti-12 or anti-OmpC. The inventors then put environmnerttal score (rural living + 1, running water— 1 , toilet - I , sewer service -1 , range: -3 to 1) into regression analysis and found significant association with ASCA (pθ.006, R²^⁰Zo), anti-OmpC (p=0.03, R²=3%) and quartile sum of anti-12 and anti-OmpC (p=0.02, R²=3.3%).

Environmental exposure in childhood is associated with variation of the antibody expression in CD. The 'clean^" environment is positively associated with ASCA expression but negatively associated with anti-OmpC and anti-12 in CD. The results also show that there is different pathophysiology of IBD in rural and urban areas.

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.

Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of

DW r 137343 H\ 2 0067789-231 WOO 14 this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims {e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term

'"including'" should be interpreted as "including but not limited to," the term ''having'" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to,'^" etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases ^4*at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an"

DWT IS734313v2 0067789-231 WOO 1 5 (e.g., "a" and/or "an" should typically be interpreted to mean "at least one^"' or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of 'two recitations," without other modifiers, typically means at least two recitations, or two or more recitations).

Accordingly, the invention is not limited except as by the appended claims.

DWT 137343 i 3 \ 2 0067789-231 WOO 1 6

Claims

1. A method of diagnosing a Crohn's Disease subtype in an individual, comprising: determining the presence or absence of a high expression of ASCA in the individual; determining whether the individual was exposed primarily to sanitary and/or urban conditions during the individual's childhood; and diagnosing the Crohn's Disease subtype in the individual based upon the presence of the high expression of ASCA in the individual and the determination of sanitary and/or urban conditions during the individual's childhood.

2. The method of claim 1 , wherein sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house,

3. The method of claim 1 , wherein the high expression of ASCA comprises approximately 1.50 EU/mL.

4. A method of diagnosing a Crohn's Disease subtype in an individual, comprising: determining the presence or absence of a high expression of anti-OmpC and/or anti-12 in the individual; determining whether the individual was exposed primarily to unsanitary and/or rural conditions during the individual's childhood; and diagnosing the Crohn's Disease subtype in the individual based upon the presence of the high expression of anti-OmpC and/or anti-12 in the individual and the determination of unsanitary conditions during the individual's childhood.

5. The method of claim 4, wherein the high expression of anti-OmpC comprises approximately 26 EU/mL.

6. The method of claim 4, wherein the high expression of anti-12 comprises approximately

55 EU/mL.

DW r 137343 OV2 00677S9-231WO0 17

7. A method of treating Crohn^'s Disease in an individual, comprising: determining the presence of a high expression of ASCA in the individual and the exposure primarily to sanitary and/or urban conditions during childhood; and treating the Crohn's Disease.

8. The method of claim 7, wherein sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house.

9. A method of treating Crohn's Disease in an individual, comprising: determining the presence of a high expression of anti-OmpC and/or anti-I2 in the individual and exposure to unsanitary and/or rural conditions during childhood; and treating the Crohn's Disease.

10. A method of reducing the incidence and/or development of inflammatory bowel disease (IBD) in a community, comprising improving the sanitary conditions of the community.

1 1. The method of claim 10, wherein sanitary conditions comprise running water, sewer service and/or existence of a toilet in the house.

12. The method of claim 10, wherein IBD comprises Crohn's Disease.

13. The method of claim 10, wherein the community comprises a rural and/or urban community.

DWT 0067789-231WOO 18