WO2011088306A1

WO2011088306A1 - Methods of using genetic variants to diagnose crohn's disease

Info

Publication number: WO2011088306A1
Application number: PCT/US2011/021282
Authority: WO
Inventors: Jerome I. Rotter; Kent D. Taylor; Stephan R. Targan
Original assignee: Cedars-Sinai Medical Center
Priority date: 2010-01-15
Filing date: 2011-01-14
Publication date: 2011-07-21

Abstract

The present invention relates to prognosing, diagnosing, and treating of Crohn's disease. The invention also provides prognosis, diagnosis, and treatment that are based upon the presence of one or more genetic risk factors including NOD2 variants.

Description

METHODS OF USING GENETIC VARIANTS TO DIAGNOSE CROHN'S DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S. Provisional Application No. 61/295,379 filed January 15, 2010, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention relates generally to the field of inflammatory disease, specifically to Crohn's disease.

BACKGROUND

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 prevalence in European ancestry populations that average approximately 100-150 per 100,000 (1-2). 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 (1). 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 (3). Moreover, genetic analyses have linked IBD to specific genetic variants, especially NOD2 (CARD 15) variants on chromosome 16ql2 and the IBD5 haplotype (spanning the organic cation transporters, SLC22A4 and SLC22A5, and other genes) on chromosome 5q31 (3-7). 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 NOD2 (CARD 15) 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

In one embodiment, the invention provides a method of diagnosing susceptibility to Crohn's disease in an individual, comprising: obtaining a sample from the individual, assaying the sample to determine the presence or absence of a NOD2 risk variant, and diagnosing susceptibility to Crohn's disease in the individual based on the presence of the NOD2 risk variant. The NOD2 risk variant can be selected from the group consisting of rs2291263, rs2306549, rs8115510, rs7268671 , and rs7866759. The NOD2 risk variants can be located at loci including, but not limited to ITPR2 and PLCB4. Assaying of the sample comprises genotyping for one or more single nucleotide polymorphisms. The sample can be whole blood, plasma, serum, saliva, cheek swab, urine, or stool.

In a related embodiment, the invention provides a method of prognosing Crohn's disease in an individual, comprising: obtaining a sample from the individual, assaying the sample for the presence or absence of one or more genetic risk variants, and prognosing an aggressive form of Crohn's disease based on the presence of one or more NOD2 risk variants. The NOD2 risk variant can be selected from the group consisting of rs2291263, rs2306549, rs81 15510, rs7268671 , and rs7866759. The NOD2 risk variants can be located at loci including, but not limited to ITPR2 and PLCB4. Assaying of the sample comprises genotyping for one or more single nucleotide polymorphisms. The sample can be whole blood, plasma, serum, saliva, cheek swab, urine, or stool.

In a further embodiment, the invention provides method of treating an individual for Crohn's disease, comprising: prognosing an aggressive form of Crohn's disease in the individual based on the presence of one or more NOD2 risk variants, and treating the individual, wherein the one or more NOD2 risk variants are selected from rs2291263, rs2306549, rs8115510, rs7268671 , and rs7866759. The NOD2 risk variants can be located at loci including, but not limited to ITPR2 and PLCB4. Assaying of the sample comprises genotyping for one or more single nucleotide polymorphisms. The sample can be whole blood, plasma, serum, saliva, cheek swab, urine, or stool. The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.

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. Multidimensional Scaling (MDS) cluster plot used to identify Ashkenazi Jewish subjects.

Figure 2. Table of novel loci identified by GWAS and various SNPs and respective associated alleles.

DESCRIPTION OF THE INVENTION

The inventors report herein, novel CD susceptibility loci identified in a GWAS performed in Ashkenazi subjects selected by genetically derived ethnicity information. While self-declared ethnicity was utilized in order to identify the "Ashkenazi cluster" on a principal components plot, subjects were selected for inclusion in analysis solely based on their genetic information. In doing so, the inventors have replicated previously described associations between CD and NOD2 risk variants in the Ashkenazi Jewish and identified several other novel NOD2 risk variants as susceptibility genes in CD. These genes act in the intracellular calcium signaling pathway which is important for T cell receptor signaling, and further implicates adaptive immunity in the pathophysiology of CD.

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.

The term "inflammatory bowel disease" or "IBD" refers to gastrointestinal disorders including, without limitation, Crohn's disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC). Inflammatory bowel diseases such as CD, UC, and IC are distinguished from all other disorders, syndromes, and abnormalities of the gastroenterological tract, including irritable bowel syndrome (IBS).

"Risk variant" as used herein refers to genetic variants, the presence of which correlates with an increase or decrease in susceptibility to Crohn's disease. Risk variants of Crohn's disease include, but are not limited to NOD2 variants at various genetic locus, such as "haplotypes" and/or a set of single nucleotide polymorphisms (SNPs) on a gene or chromatid that are statistically associated. Risk variants can include, but are not limited to rs2291263, rs2306549, rs81 15510, rs7268671 , and rs7866759. Genetic locus can include, but are not limited to ITPR2 and PLCB4.

A NOD2 risk variant is useful in the methods of the invention. As used herein, the term "NOD2" variant means a nucleotide sequence of a NOD2 gene containing one or more changes as compared to the wild-type NOD2 gene or an amino acid sequence of a NOD2 polypeptide containing one or more changes as compared to the wild-type NOD2 polypeptide sequence. NOD2, also known as CARD 15, has been localized to the IBDl locus on chromosome 16 and identified by positional-cloning (4) as well as a positional candidate gene strategy (5).

"Treatment" or "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down and/or lessen the disease even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with Crohn's disease as well as those prone to have Crohn's disease or those in whom Crohn's disease is to be prevented. For example, in Crohn's disease treatment, a therapeutic agent may directly decrease the pathology of IBD, or render the cells of the gastroenterological tract more susceptible to treatment by other therapeutic agents.

As used herein, "diagnose" or "diagnosis" refers to determining the nature or the identity of a condition or disease. A diagnosis may be accompanied by a determination as to the severity of the disease. Diagnosis as it relates to the present invention, relates to the diagnosis of Crohn's disease.

As used herein, "prognostic" or "prognosis" refers to predicting the probable course and outcome of IBD or the likelihood of recovery from IBD. The prognosis can include the presence, the outcome, or the aggressiveness of the disease.

As used herein, the term "biological sample" means any biological material obtained from an individual from which nucleic acid molecules can be prepared. Examples of a biological sample include, but are not limited to whole blood, plasma, serum, saliva, cheek swab, urine, stool, or other bodily fluid or tissue that contains nucleic acid.

As disclosed herein, novel CD susceptibility loci were identified in a genome-wide association study (GWAS) performed in Ashkenazi subjects selected by genetically derived ethnicity information. The inventors replicated some previously described associations between CD and NOD2 risk variants in the Ashkenazi Jewish population (Figure 2) and identified several other novel NOD2 risk variants as susceptibility genes in CD. These genes act in the intracellular calcium signaling pathway which is important for T cell receptor signaling, and further implicates adaptive immunity in the pathophysiology of CD. The newly discovered NOD2 risk variants that are associated with CD include, but are not limited to rs2291263, rs2306549, rs8115510, rs7268671, and rs7866759.

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 a NOD2 risk variant, where the presence of the NOD2 risk variant is indicative of susceptibility to Crohn's disease in the individual. NOD2 risk variants can include, but are not limited to rs2291263, rs2306549, rs81 15510, rs7268671, and rs7866759. In another embodiment, the individual is Ashkenazi Jewish.

In one embodiment, the present invention provides a method of prognosing Crohn's disease in an individual by determining the presence or absence of a NOD2 risk variant, where the presence of the NOD2 risk variant is indicative of a complicated form of Crohn's disease in the individual. NOD2 risk variants can include, but are not limited to rs2291263, rs2306549, rs8115510, rs7268671, and rs7866759. In another embodiment, the individual is Ashkenazi Jewish.

In one embodiment, the present invention provides a method of treating Crohn's disease in an individual by determining the presence of a NOD2 risk variant and treating the individual. NOD2 risk variants can include, but are not limited to rs2291263, rs2306549, rs8115510, rs7268671, and rs7866759. In another embodiment, the individual is Ashkenazi Jewish.

A variety of methods can be used to determine the presence or absence of a NOD2 risk variant. 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 NOD2 risk variant 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 R A 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 NOD2 risk variant 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 (8).

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 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, previously (9). Minor grove binders include, but are not limited to, compounds such as dihydrocyclopyrrolo indole 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 (10-11). 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 (8). 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 (12-13).

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 (14). 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 (11).

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 (15). 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 (16). 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.

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

1100 CD subjects of mixed Caucasian ethnicity and 3970 controls from the Cardiovascular Health Study were geno typed, using Illumina technology (Human 610Quad, Human 317Duo or the HumanCNV370-Duo platform). 234,637 SNPs were available for analyses in all datasets. Multidimensional Scaling was used to detect population substructure. Ten principal components were calculated and principal components 1 and 2 were plotted to generate informative cluster plots for ethnicity. Self-reported ethnicity in CD cases was used to confirm the Ashkenazi Jewish "cluster" and select cases and controls ethnically matched by principal components. Association with case/control status was performed using a logistic regression model.

Example 2

The inventors identified 247 Ashkenazi Jewish CD cases and 320 ethnically matched controls by principal components. Genomic inflation was calculated at λϋΟΙ .ΟΙδ. A Q-Q plot of P values showed a modest increase of significant associations above the expected under the null hypothesis. The inventors replicated the previously reported association between CD and the NOD2 locus and identified several novel loci including ITPR2 (rs2291263; P=6.53x10-5, rs2306548; P=5.34xl0-5 and rs2306549; P=8.19x10-5) and PLCB4 (rs8115510; P=1.94xl0-4, rs6039442;P=2.69xl0-4, rs7268671 ; P=5.34xl0-5). The only locus that met with genome wide significance with Bonferroni correction (P=0.05/234,637 markers = P<2.13x10-7) was rs7866759 (P=l .33x10-7) on chromosome 9. There are no genes within lOOkb to the 5' or lOkb to the 3' of this SNP; however rs7866759 is in LD with a hypothetical transcript c9orfl53.

References

1. Podolsky, et al., N Engl J Med 347, 417 (2002).

2. Loftus, et al, Gastroenterology 126, 1504 (2004).

3. Vermeire, et al., Genes Immun 6, 637 (2005).

4. Hugot, et al, Nature 41 1 , 599 (2001).

5. Ogura, et al, Nature 411, 603 (2001).

6. Rioux, et al, Nat Genet 29, 223 (2001).

7. Peltekova, et al, Nat Genet 36, 471 (2004).

8. Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994).

9. utyavin, et al., Nucleic Acids Research 28:655-661 (2000).

10. Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York.

11. Innis et al.,(Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990).

12. Delwart, et al, Science 262: 1257-1261 (1993).

13. White, et al, Genomics 12:301-306 (1992).

14. Hayashi, K., Methods Applic. 1 :34-38 (1991).

15. Winter, et al, Proc. Natl. Acad. Sci. 82:7575-7579 (1985).

16. Birren, et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997).

Claims

1. A method of diagnosing susceptibility to Crohn's disease in an individual, comprising:

obtaining a sample from the individual;

assaying the sample to determine the presence or absence of a NOD2 risk variant; and

diagnosing susceptibility to Crohn's disease in the individual based on the presence of the risk variant,

wherein the NOD2 risk variant is selected from the group consisting of rs2291263, rs2306549, rs8115510, rs7268671, and rs7866759.

2. The method according to claim 1 , wherein the NOD2 risk variant is rs7866759.

3. The method of claim 1, wherein the individual is Ashkenazi Jewish.

4. The method of claim 1 , wherein assaying the sample comprises genotyping for one or more single nucleotide polymorphisms.

5. The method according to claim 1, wherein the sample is whole blood, plasma, serum, saliva, cheek swab, urine, or stool.

6. A method of prognosing Crohn's disease in an individual, comprising:

obtaining a sample from the individual;

assaying the sample for the presence or absence of one or more NOD2 risk variants; and

prognosing an aggressive form of Crohn's disease based on the presence of one or more NOD2 risk variants,

7. The method of claim 6, wherein assaying the sample comprises genotyping for one or more single nucleotide polymorphisms.

8. The method according to claim 6, wherein the sample is whole blood, plasma, serum, saliva, cheek swab, urine, or stool.

9. A method of treating an individual for Crohn's disease, comprising:

prognosing an aggressive form of Crohn's disease in the individual based on the presence of one or more NOD2 risk variants; and

treating the individual,

wherein the one or more risk variants are selected from rs2291263, rs2306549, rs8115510, rs7268671 , and rs7866759.

10. The method of claim 9, wherein assaying the sample comprises genotyping for one or more single nucleotide polymorphisms.

11. The method according to claim 9, wherein the sample is whole blood, plasma, serum, saliva, cheek swab, urine, or stool.

12. The method according to claim 9, where in the risk variant is rs7866759.